WO2015110659A1

WO2015110659A1 - Methods of immunization with a vaccine inducing a humoral immune response and with a vaccine inducing a cellular immune response

Info

Publication number: WO2015110659A1
Application number: PCT/EP2015/051611
Authority: WO
Inventors: Anker LUNDEMOSE; Mats ÖKVIST; Arnt Ove HOVDEN; Maja Sommerfelt GRØNVOLD
Original assignee: Bionor Immuno AS
Current assignee: Bionor Immuno AS
Priority date: 2014-01-27
Filing date: 2015-01-27
Publication date: 2015-07-30
Anticipated expiration: 2016-07-27

Abstract

The present invention relates to a novel method for immunizations against virus infections and various cancer and inflammatory diseases including infections with HCV, HIV, HPV, CMV and Influenza.

Description

METHODS OF IMMUNIZATION WITH A VACCINE INDUCING A HUMORAL IMMUNE RESPONSE AND WITH A VACCINE INDUCING A CELLULAR IMMUNE RESPONSE

FIELD OF THE INVENTION

The present invention relates to a novel method for immunizations against virus infections and various cancer and inflammatory diseases including infections with HCV, HIV, RSV, HPV, CMV and Influenza.

BACKGROUND OF THE INVENTION

Conventional approaches to vaccine development have implemented either whole replication competent virus which has been attenuated (e.g . Sabin polio vaccine, measles, mumps, rubella (MMR)) or inactivated virions that are not replication competent. On occasions, the inactivated virus vaccines may include split vaccines where the virus particles have been disrupted. Molecular techniques have also been used to develop the subunit vaccine (e.g. hepatitis B vaccine) that consists only of the surface glycoproteins of hepatitis B virus. The inactivated virus vaccines tend to induce primarily antibody responses to the viruses in question, whereas the live attenuated vaccines induce both cell-mediated immunity as well as an antibody response since the vaccine induces a transient infection.

The only disease which has been eliminated by virtue of a successful vaccination campaign is smallpox. A campaign is currently in progress to eradicate polio. Features of virus infections that can be eliminated by vaccination are infections caused by viruses with stable virus antigens (i .e. very low mutation frequency, few subtypes), that lack a reservoir in other animal species, viruses that do not persist in the body once the infection is over and where vaccination leads to long lasting immunity. Viruses such as polio and measles fulfill these criteria whereas viruses such as influenza virus (Flu), HCV, and HIV that vary their protein sequences do not. It is for this reason that new and alternate approaches are required to develop vaccines for these diseases.

Vaccination aims to stimulate the immune response to a specific pathogen in advance of infection. When an individual is exposed to that pathogen, a memory response is triggered which prevents the establishment of infection. Vaccines therefore stimulate the adaptive immune response which unlike innate immunity, is long lived and has memory. There are two major arms to the adaptive immune system . Humoral immunity, which involves the development of antibodies that can bind virus particles and certain antibodies that can neutralize infection. Cell mediated immunity that leads to the development of cytotoxic T-cells that kill infected cells exposing viral epitopes in the context of human leukocyte antigen (HLA) class I, in this way eliminating infected cells. The challenge of providing vaccines suitable for stimulation of the adaptive immune system is that peptide epitopes need to be taken up by the antigen presenting cells.

Several peptides have been demonstrated to translocate across the plasma membrane of eukaryotic cells by a seemingly energy-independent pathway. These peptides are defined as cell-penetrating peptides (CPPs) . Cellular delivery using these cell-penetrating peptides offers several advantages over conventional techniques. It is non-invasive, energy- independent, is efficient for a broad range of cell types and can be applied to cells en masse.

For humoral responses and development of antibodies it may not be needed to obtain cell-penetrating properties since stimulation of B-cells are also done by extracellular peptide antigens. Hepatitis means inflammation of the liver which can be caused by a variety of factors including toxins, certain drugs, some diseases, heavy alcohol use, and bacterial and viral infections. Hepatitis is also the name of a family of viral infections that affect the liver; the most common types in the developed world are hepatitis A, hepatitis B, and hepatitis C.

Hepatitis C is a liver disease that results from infection with the hepatitis C virus (HCV). It can range in severity from a mild illness lasting a few weeks to a serious, lifelong illness. Hepatitis C is spread via blood; the most common form of transmission is through sharing needles or other equipment used to inject drugs. The infection can be either "acute" or "chronic". Acute HCV infection is an asymptomatic, short-term illness that occurs within the first 6 months after someone is exposed to the hepatitis C virus. For most people, acute infection leads to chronic infection, which can result in long-term complications and even death.

HCV is an enveloped positive stranded ribonucleic acid (RNA) virus with a diameter of about 50nm, belonging to the genus Hepacivirus in the family Flaviviridae that replicate in the cytoplasm of infected cells. The only known reservoir for HCV is humans, although the virus has experimentally been transmitted to chimpanzees. The natural targets of HCV are hepatocytes and possibly B-lymphocytes. As of 2008, six different genotypes and more than 100 subtypes of the virus are known. Replication occurs through an RNA-dependent RNA polymerase that lacks a proofreading function, which results in a very high rate of mutations. Rapid mutations in a hypervariable region of the HCV genome coding for the envelope proteins enable the virus to escape immune surveillance by the host. As a consequence, most HCV- infected people proceed to chronic infection.

It is estimated that 170 million people are infected with HCV worldwide, equating to approximately 3% of the global population. There are also approximately 3-4 million people who are infected every year; with an estimated 80% of these newly infected patients progressing to chronic infection.

The 6 genotypes of HCV have different geographical spread. The disease in the early stages is generally asymptomatic; the majority of patients with chronic infection eventually progress to complications such as liver fibrosis and cirrhosis, and, in 1-5% of cases, hepatocellular carcinoma. HCV is the major cause of non-A, non-B hepatitis worldwide. Acute infection with HCV frequently leads to chronic hepatitis and end-stage cirrhosis. It is estimated that up to 20% of HCV chronic carriers may develop cirrhosis over a time period of about 20 years and that of those with cirrhosis between 1 to 4% is at risk to develop liver carcinoma.

The about 9.6 kb single-stranded RNA genome of the HCV virus comprises a 5'- and

3'-noncoding region (NCRs) and, in between these NCRs a single long open reading frame of about 9 kb encoding an HCV polyprotein of about 3000 amino acids.

HCV polypeptides are produced by translation from the open reading frame and cotranslational proteolytic processing. Structural proteins are derived from the amino- terminal one-fourth of the coding region and include the capsid or Core protein (about 21 kDa), the El envelope glycoprotein (about 35 kDa) and the E2 envelope glycoprotein (about 70 kDa, previously called NS1), and p7 (about 7kDa). The E2 protein can occur with or without a C-terminal fusion of the p7 protein (Shimotohno et al. 1995). An alternative open reading frame in the Core-region has been found which is encoding and expressing a protein of about 17 kDa called F (Frameshift) protein (Xu et al. 2001 ; Ou & Xu in US Patent

Application Publication No. US2002/0076415). In the same region, ORFs for other 14-17 kDa ARFPs (Alternative Reading Frame Proteins), Al to A4, were discovered and antibodies to at least Al, A2 and A3 were detected in sera of chronically infected patients (Walewski et al. 2001). From the remainder of the HCV coding region, the non-structural HCV proteins are derived which include NS2 (about 23 kDa), NS3 (about 70 kDa), NS4A (about 8 kDa), NS4B (about 27 kDa), NS5A (about 58 kDa) and NS5B (about 68 kDa) (Grakoui et al. 1993).

Influenza remains a significant cause of mortality and morbidity worldwide. The World Health Organisation (WHO) estimates that seasonal epidemics affect 3-5 million people with severe illness annually and result in 250,000 - 500,000 mortalities. Influenza is caused by viruses in the family Orthomyxoviridae which are negative stranded RNA viruses. The influenza virus exists as three types, A, B and C of which only A is associated with pandemics. Types A viruses are found in both humans and animals, particuiariy birds but also other mammals such as pigs. Type A viruses are further typed into subtypes according to different kinds and combinations of virus surface proteins. Among many subtypes in 2009 influenza A (H1N1) and A (H3N2) subtypes were circulating among humans. Influenza A and B are included in the seasonal vaccine, whereas influenza C occurs only rarely, and so it is not included in the seasonal vaccine. Type B viruses are human specific and Type C viruses cause a very mild disease. The genomes of Orthomyxoviruses are segmented. Influenza viruses Types A and B have 8 segments whereas type C has seven. Pandemics may arise as a result of re-assortment of gene segments when two different type A viruses infect the same cell. There is no immunity in the population to this novel re-assorted virus. Three pandemics occurred in the twentieth century: "Spanish influenza" in 1918, "Asian influenza" in 1957, and "Hong Kong influenza" in 1968. The 1918 pandemic killed an estimated 40-50 million people worldwide. Subsequent pandemics were much milder, with an estimated 2 million deaths in 1957 and 1 million deaths in 1968. In June 2009 the WHO declared a pandemic from influenza virus H1N1 (swine Influenza) which was declared over in August 2010.

Human papillomaviruses are made up of a group of DNA viruses in the family Papillomaviridae which infect the skin and mucous membranes. Two groups which are derived from more than 100 different identified subtypes are the main cause for clinical concern: those causing warts (both benign and genital warts), and a group of 12 "high risk" subtypes that can result in cervical cancer. This latter group has been attributed as a contributory factor in the development of nearly all types of cervical cancer. Worldwide, cervical cancer remains the second most common malignancy in women, and is a leading cause of cancer-related death for females in developing countries. HPV 16 and 18 have been mainly associated with cervical cancer, however, the virus is also a cause of throat cancer in both men and women. HPV is transmitted through contact and enters the skin through abrasions. An abortive infection, where only the early proteins are expressed is associated with cancer development.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide a method for inducing an efficient immune response in a disease subject, such as a subject infected with an infectious agent.

SUMMARY OF THE INVENTION It has been found by the present inventor(s) that there is a significantly better effect of immunisations with both a vaccine to elicit a Humoral Immune Response (HIR) and a vaccine to elicit a Cell Mediated Immune (CMI) response against a disease antigen, if the vaccine to elicit a Humoral Immune Response (HIR) is administered prior to or

simultaneously with the vaccine to elicit a Cell Mediated Immune (CMI) response against a disease antigen.

So, in a first aspect the present invention relates to a method for inducing an immune response to a disease antigen, such as an infectious agent in an subject, the method comprising:

(a) administering a first vaccine composition comprising an immunogenically effective amount of at least one first antigen derived from said disease antigen, such as an infectious agent to said subject, sufficient to elicit a Humoral Immune Response (HIR) against said first antigen; and simultaneously or followed by

(b) administering a second vaccine composition comprising an immunogenically effective amount of at least one second antigen derived from said disease antigen, such as an infectious agent to said subject, sufficient to elicit a Cell Mediated Immune (CMI) response against said second antigen.

It is to be understood that after treatment with a first vaccine composition sufficient to induce a humoral immune response, a maturation period may be needed to build up said response, such as a period of two weeks, such as a period of three weeks, such as a period of four weeks, such as a period of five weeks, such as a period of six weeks, such as a period of seven weeks, such as a period of eight weeks, before establishing by measurement the precense of said response, and/or further treatment dependent on it being established .

In a second aspect the present invention relates to a first and second vaccine composition for use in a method for inducing an immune response to a disease antigen, such as an infectious agent in an subject, the method comprising the steps of:

in a third aspect the present invention relates to a method for inducing an immune response to a disease antigen, such as an infectious agent in an subject, the method comprising :

(a) testing for and establishing the presence of a sufficient humoral immune response to a first antigen derived from said disease antigen, such as an infectious agent, in said subject; and simultaneously or followed by

(b) administering a vaccine composition comprising an immunogenically effective amount of at least one second antigen derived from said disease antigen, such as an infectious agent, to said subject, sufficient to elicit a Cell Mediated Immune (CMI) response against said second antigen. LEGENDS TO THE FIGURE

Fig . 1 Illustrates the immunisation scheme as described in example 34.

Fig . 2. Weight loss after challenge with live influenza virus. Mean per group +/- the standard error of the mean is shown. Day 7 was the last day measured . For CMI + humoral group, a last observation carried forward is used for the time points after loss of mice. DETAILED DISCLOSURE OF THE INVENTION

Definitions

When terms such as "one", "a" or "an" are used in this disclosure they mean "at least one", or "one or more" unless otherwise indicated. Further, the term "comprising" is intended to mean "including" and thus allows for the presence of other constituents, features, conditions, or steps than those explicitly recited.

As used herein, the term "immune response" refers to the reactivity of an organism's immune system in response to an antigen. In vertebrates, this may involve antibody production, induction of cell-mediated immunity, and/or complement activation (e.g., phenomena associated with the vertebrate immune system's prevention and resolution of infection by microorganisms) . In preferred embodiments, the term immune response encompasses but is not limited to one or more of a "lymphocyte proliferative response," a "cytokine response," and an "antibody response."

The term "antigen" denotes a substance of matter which is recognized by the immune system's specifically recognizing components (antibodies, T-cells) .

The term "immunogen" is in the present context intended to denote a substance of matter, which is capable of inducing an adaptive immune response in an individual, where said adaptive immune response targets the immunogen. In relation to the present invention, an immunogen will induce a humoral and/or cell-mediated immune response. In other words, an immunogen is an antigen, which is capable of inducing immunity.

The terms "epitope", "antigenic determinant" and "antigenic site" are used interchangeably herein and denotes the region in an antigen or immunogen which is recognized by antibodies (in the case of antibody binding epitopes, also known as "B-cell epitopes") or by T-cell receptors when the epitope is complexed to a Major histocompatibility complex (MHC) molecule (in the case of T-cell receptor binding epitopes, i .e. "T-cell epitopes") .

"B cell antigen" means any antigen that naturally is or could be engineered to be recognized by a B cell, and that triggers an immune response in a B cell (e.g., an antigen that is specifically recognized by a B cell receptor on a B cell) .

The term "immunogenically effective amount" has its usual meaning in the art, i. e. an amount of an immunogen, which is capable of inducing an immune response, which significantly engages pathogenic agents, which share immunological features with the immunogen.

The terms "vaccine" or "vaccine composition" is used for a composition comprising an immunogen and which is capable of inducing an immune response which is either capable of reducing the risk of developing a pathological condition or capable of inducing a therapeutically effective immune response which may aid in the cure of (or at least alleviate the symptoms of) a pathological condition.

The term "pharmaceutically acceptable" has its usual meaning in the art, i. e. it is used for a substance that can be accepted as part of a medicament for human use when treating the disease in question and thus the term effectively excludes the use of highly toxic substances that would worsen rather than improve the treated subject's condition.

A "T helper lymphocyte epitope" (a T_H epitope) is peptide, which binds an MHC Class II molecule and can be presented on the surface of an antigen presenting cell (APC) bound to the MHC Class II molecule. An "immunological carrier" is generally a substance of matter which includes one or many T_H epitopes, and which increase the immune response against an antigen to which it is coupled by ensuring that T-helper lymphocytes are activated and proliferate. Examples of known immunological carriers are the tetanus and diphtheria toxoids and keyhole limpet hemocyanin (KLH) .

The vaccine compositions according to the present invention may comprise an immunogenic peptide derived from the antigen. This sequence of amino acids derived from an antigen may herein be referred to as an epitope.

Such immunogenic peptides used in the methods according to the present invention may be a helper T lymphocyte (HTL) inducing peptide comprising HTL epitopes. A "HTL inducing peptide" is a HLA Class II binding peptide that is capable of inducing a HTL response. Also immunogenic peptides used in the methods according to the present invention may in other embodiments be CTL inducing peptides comprising CTL epitopes in addition to or as an alternative to being a HTL inducing peptide. A "CTL inducing peptide" is a HLA Class I binding peptide that is capable of inducing a CTL response.

In some embodiments the epitopes used in methods according to the present invention are CTL epitopes. A "CTL inducing peptide" is a HLA Class I binding peptide that is capable of inducing a CTL response. In other embodiments the epitopes used in the methods according to the present invention are HTL inducing peptides. A "HTL inducing peptide" is a HLA Class II binding peptide that is capable of inducing a HTL response.

The term "antibody response" refers to the production of antibodies (e.g., IgM, IgA, IgG) which bind to an antigen of interest, this response is measured for instance by assaying sera by antigen ELISA.

The term "adjuvant" as used herein refers to any compound which, when delivered together or simultaneously with an antigen, non-specifically enhances the immune response to that antigen. Exemplary adjuvants include but are not limited to oil in water and water in oil adjuvants, aluminum-based adjuvants (e.g., AIOH, AIP04, etc), and Montanide ISA 720.

In the context of the present invention, "treatment" or "treating" refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context. For example, "treatment" of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas "treatment" of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or therapy.

The terms "patient" and "subject" refer to a mammal that may be treated using the methods of the present invention.

The term "Cell Mediated Immune (CMI) response" refers to a part of an immune response that involves the activation of phagocytes, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and/or the release of various cytokines in response to an antigen .

The term "Humoral Immune Response (HIR)" refers to a part of an immune response that involves an antibody response.

prophylactic

"HIV" unless otherwise indicated generally denotes human immunodeficiency virus I . "HIV disease" is composed of several stages including the acute HIV infection which often manifests itself as a flu-like infection and the early and medium stage symptomatic disease, which has several non-characteristic symptoms such as skin rashes, fatigue, night sweats, slight weight loss, mouth ulcers, and fungal skin and nail infections. Most HIV infected will experience mild symptoms such as these before developing more serious illnesses. It is generally believed that it takes five to seven years for the first mild symptoms to appear. As HIV disease progresses, some individuals may become quite ill even if they have not yet been diagnosed with AIDS (see below), the late stage of HIV disease. Typical problems include chronic oral or vaginal thrush (a fungal rash or spots), recurrent herpes blisters on the mouth (cold sores) or genitals, ongoing fevers, persistent diarrhea, and significant weight loss. "AIDS" is the late stage HIV disease and is a condition which progressively reduces the effectiveness of the immune system and leaves individuals susceptible to opportunistic infections and tumors.

When using the term "gpl20" herein is meant the « 120 kDa N-terminal glycoprotein enzymatic cleavage product of gpl60, which in turn is the sole expression product of the HIV env gene. gpl20 forms the "spikes" on infective HIV virions and is non-covalently bound to gp41.

"gp41" denotes the «41 kDa glycoprotein C-terminal enzymatic cleavage product of gpl60. gp41 is located in the membrane of HIV infected cells or virions. gp41 has an N- terminal transmembrane domain which binds non-covalently to gpl20. This transmembrane domain is termed "the transmembrane domain of gp41" or "tm-gp41" herein. The term includes within its scope naturally occurring mutated versions of the sequence as e.g . those set forth in Formula III.

"C5" or the "C5 domain" denotes the 13 C-terminal amino acid residues of gpl20. "C2" or the "C2 domain" denotes a conserved region in gpl20. Regions in C2 form an antiparallel β-sheet with C5 in the inner proximal domain of gpl2. "Reducing and/or delaying pathological effect of HIV" is in the present context meant to denote that use of the methods of the invention provides for a statistically significant reduction and/or delay in morbidity seen in individual infected with HIV which are treated according to the present invention . That is, the time of onset of manifest disease symptoms characterizing AIDS is later compared to non-treated controls and/or the number of pathological manifestations is reduced to controls not receiving the treatment of the present invention.

The expression "association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20" means that C5 can interact non-covalently with both or one of the tm-g41 and C2. The interaction with tm-gp41 is intermolecular, whereas the interacation with C2 is intramolecular.

An "agent capable of stabilising" association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 is a composition of matter which prevents or statistically reduces release of C5 from its intermolecular binding to gp41 and/or from its intramolecular binding to C2. Generally, such an agent is any substance of matter capable of exerting this effect, but important examples are antibodies, antibody fragments, and antibody analogues. However, also other molecules having proper binding affinity for a complex between C5 on the one hand and tm-gp41 and/or C2 on the other, is an agent according the present invention - the precise molecular fomat is less important than the binding characteristics, and it is according to the invention also possible that such an agent may be a receptor or a receptor analogue, but also small molecule stabilisers are capable of functioning as an agen of the present invention.

The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g ., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity, i.e. to function as an agent described above. Various techniques relevant to the production of antibodies are provided in, e.g ., Harlow, et al ., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N .Y., ( 1988) . ) .

An "antibody fragment or antibody analogue" comprises a portion of a full-length antibody, preferably antigen-binding or variable regions thereof. Examples of antibody fragments/analogues include Fab, Fab', F(ab)₂, F(ab')₂, F(ab)₃, Fv (typically the VL and VH domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH I domain), and dAb (typically a VH domain) fragments; VH, VL, VhH, and V- NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g ., Ill et al ., Protein Eng 1997; 10: 949-57) ; camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. Various types of antibody fragments have been described or reviewed in, e.g ., Holliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136; WO2005040219, and published U.S. Patent Applications 20050238646 and 20020161201.

The term "antibody derivative", as used herein, comprises a full-length antibody or a fragment of an antibody, preferably comprising at least antigen-binding or variable regions thereof, wherein one or more of the amino acids are chemically modified, e.g. , by alkylation, PEGylation, acylation, ester formation or amide formation or the like, e.g ., for linking the antibody to a second molecule. This includes, but is not limited to, PEGylated antibodies, cysteine- PEGylated antibodies, and variants thereof.

A "conjugate" as used herein comprises an agent to be used according to the invention such as an antibody derivative associated with or linked to a second agent, such as a cytotoxic agent, a detectable agent, etc. A conjugate may be constituted of covalently linked peptides (an example of a conjugate is a fusion peptide comprising two peptides linked via peptide bonds so that the conjugate in that case may be an expression product from a nucleic acid fragment), but a conjugate can also be a combination of peptides covalent linked via chemical conjugation (a traditional example is conjugation using glutaraldehyde) . Another example of a more complex conjugation is the example where an agent or peptide multimer or other chemical substance of the present invention is linked to a carrier molecule, which in turn i coupled to other agents, peptide multimers or other chemical substances of the present invention (e.g . when such chemical substances are bound to a poly-lysine carrier (a lysine "tree")) .

A "humanized" antibody is a human/non-human chimeric antibody that contains a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR residues are those of a human immunoglobulin sequence. The humanized antibody can optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g ., Jones et al., Nature 321 : 522-525 ( 1986) ; Riechmann et al., Nature 332 : 323-329 ( 1988) ; and Presta, Curr. Op. Struct. Biol . 2 : 593-596 ( 1992), WO 92/02190, US Patent Application 20060073137, and US Patents 6,750,325, 6,632,927, 6,639,055, 6, 548,640, 6,407,213, 6, 180,370, 6,054,297, 5,929,212, 5,895,205, 5,886, 152, 5,877,293, 5,869,619, 5,821,337, 5,821, 123, 5,770, 196, 5,777,085, 5,766,886, 5,714,350, 5,693,762, 5,693,761, 5,530, 101, 5,585,089, and 5,225,539.

An antibody having a "biological characteristic" of a reference antibody, is one that possesses one or more of the biological characteristics of that antibody that distinguish it from other antibodies that bind to the same antigen .

The term "peptide" is in the present context intended to mean both short peptides of from 2 to 10 amino acid residues, oligopeptides of from 11 to 100 amino acid residues, and polypeptides of more than 100 amino acid residues. When referring to amino acids in peptides, it is intended that the amino acids are L-amino acids, unless other information is provided. Amino acids are referred to by their standard three letter or one letter designations unless otherwise stated. Some unusual amino acids referred to herein includes homoarginine usually abbreviated by Har, norleucine usually abbreviated as Nle or Nl, Ν-ε-methylated Lys usually abbreviated Lys(Me), Citrulline usually abbreviated Cit or with the single letter "B", diaminopropionic acid usually abbreviated with Dpr and serinyl diaminopropionic acid usually abbreviated Dpr(Ser) .

A "protein" is intended to denote a functional biomolecule comprising at least one peptide; when comprising at least two peptides, these may form complexes, be covalently linked, or may be non-covalently linked. The polypeptide(s) in a protein can be glycosylated and/or lipidated and/or comprise prosthetic groups.

A "peptide multimer" denotes a molecule which is constituted by at least two peptides in a non-natural configuration relative to each other. Examples are peptides from the same or from different proteins which are covalently linked via the side chains of at least one of their amino acids, or which are linked via their termini (e.g. via peptide bonds) but in a configuration which does not appear in nature. Typical examples of peptide multimers are detailed below.

A "variant" or "analogue" of a peptide refers to a peptide having an amino acid sequence that is substantially identical to a reference peptide, typically a native or "parent" polypeptide. The peptide variant may possess one or more amino acid substitutions, deletions, and/or insertions at certain positions within the native amino acid sequence.

"Conservative" amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Families of amino acid residues having similar side chains are known in the art, and include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g ., tyrosine, phenylalanine, tryptophan, histidine) . A particular form of conservative amino acid substitutions include those with amino acids, which are not among the normal 20 amino acids encoded by the genetic code. Since preferred embodiments of the present invention entail use of synthetic peptides, it is unproblematic to provide such "non-naturally occurring" amino acid residues in the peptides disclosed herein, and thereby it is possible to exchange the natural saturated carbon chains in the side chains of amino acid residues with shorter or longer saturated carbon chains - for instance, lysine may be substituted with an amino acid having an the side chain -(CH₂)_nNH₃, where n is different from 4, and arginine may be substituted with an amino acid having the side chain -(CH₂)_nNHC( = NH₂) NH₂, where n is different from 3, etc. Similarly, the acidic amino acids aspartic acid and glutamic acid may be substituted with amino acid residues having the side chains -(CH₂)_nCOOH, where n> 2.

A "retro form" of a peptide is a form of a peptide where the order of the amino acids in N- to C-terminal direction has been inverted. For instance, the retro form of ALDFR is the peptide RFDLA.

The term "substantially identical" in the context of two amino acid sequences means that the sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 95, at least about 98, or at least about 99 percent sequence identity. In one embodiment, residue positions that are not identical differ by conservative amino acid substitutions. Sequence identity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, the publicly available GCG software contains programs such as "Gap" and "BestFit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g. , GCG Version 6.1. Polypeptide sequences can also be compared using FASTA or ClustalW, applying default or recommended parameters. A program in GCG Version 6.1., FASTA (e.g. , FASTA2 and FASTA 3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences ( Pearson, Methods Enzymol . 1990; 183 : 63-98; Pearson, Methods Mol . Biol. 2000; 132: 185-219) . Another preferred algorithm when comparing a sequence to a database containing a large number of sequences from various organisms, or when deducing the is the computer program BLAST, especially blastp, using default parameters. See, e.g. , Altschul et al ., J . Mol. Biol . 1990; 215 :403-410; Altschul et al., Nucleic Acids Res.

1997; 25 : 3389-402 ( 1997) ; each herein incorporated by reference. "Corresponding" amino acid positions in two substantially identical amino acid sequences are those aligned by any of the protein analysis software mentioned herein, typically using default parameters.

The term "subsequence" in general means any consecutive stretch of at least 3 amino acids or, when relevant, of at least 3 nucleotides, derived directly from a naturally occurring amino acid sequence or nucleic acid sequence, respectively. However, when discussing peptide multimers of the present invention, the subsequence may be as short as 1 or 2 amino acids. This is because the inventive peptide multimers include amino acids from different peptide domains, where the amino acids together at least form a conformational epitope for an antibody. Hence, such a conformational epitope could be composed of 4 amino acids from C5, but only 1 or 2 from tm-gp41 - the imporant point is here that this combined epitope from 2 domains is capable of being stabilised, i.e. that antibody binding to the same epitope in vivo will stabilise the configuration between C5 and tm-gp41 and/or C2.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome-binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

An "isolated" molecule is a molecule that is the predominant species in the composition wherein it is found with respect to the class of molecules to which it belongs

(i .e., it makes up at least about 50% of the type of molecule in the composition and typically will make up at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the species of molecule, e.g ., peptide, in the

composition) . Commonly, a composition of an antibody molecule will exhibit 98% - 99% homogeneity for antibody molecules in the context of all present peptide species in the composition or at least with respect to substantially active peptide species in the context of proposed use.

In the context of the present invention, "treatment" or "treating" refers to preventing, alleviating, managing, curing or reducing one or more symptoms or clinically relevant manifestations of a disease or disorder, unless contradicted by context. For example, "treatment" of a patient in whom no symptoms or clinically relevant manifestations of a disease or disorder have been identified is preventive or prophylactic therapy, whereas "treatment" of a patient in whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventive or prophylactic therapy.

Antigens The specific natural antigen used in the peptide constructs according to the present invention may be a protein or peptide sequence derived from any B cell antigen, such as from any disease antigen, such as an infectious agent. Suitable antigens to be used according to the present invention include antigens derived from a bacteria, a mycobacterium, a virus, a parasite such as protozoa, a fungus, a cancer antigen, such as an oncogene, such as a thelomerase, a prion, an atopic disease antigen, an addictive or abused substance or a toxin or an antigen of an autoimmune disease, such as rheumatoid arthritis, insulin dependent diabetes, multiple sclerosis and the li ke.

As used herein a "disease antigen" refers to any antigen confirmed or suspected to be involved in a specific disease.

Examples of bacteria for which antigens may be provided include, but are not limited to, M . tuberculosis, Mycobacterium, mycoplasma, neisseria and legionella . Examples of parasites include, but are not limited to, rickettsia and chlamydia .

Examples of an infectious disease antigen is TbH9 (also known as Mtb 39A), a tuberculosis antigen . Other tuberculosis antigens include, but are not limited to DPV (also known as Mtb8.4), 381 , Mtb41, Mtb40, Mtb32A, ΜΛ9.9Α, Mtb9.8, Mtblo, Mtb72f, Mtb59f, Mtb88f, Mtb71f, Mtb46f and Mtb31f ("f ' indicates that it is a fusion or two or more proteins) .

Examples of cancer antigens may be a tumor associated antigen such as H ER2, H ER3 or H ER4 receptor or one or more tumor-associated antigens or cell-surface receptors disclosed in US Publication No. 20080171040 or US Publication No. 20080305044 and are incorporated in their entirety by reference.

Other suitable cancer antigens that may be used by the present invention include CD proteins such as CD2, CD3, CD4, CD5, CD6, CD8, CD11 , CD 14, CD18, CD 19, CD20, CD21 , CD22, CD25, CD26, CD27, CD28, CD30, CD33, CD36, CD37, CD38, CD40, CD44, CD52, CD55, CD56, CD70, CD79, CD80, CD81, CD103, CD105, CD 134, CD137, CD138, and CD152 ; members of the ErbB receptor family such as the EGF receptor, H ER2, H ER3 or H ER4 receptor; cell adhesion molecules such as LFA-I, Macl , pi 50.95, VLA-4, ICAM-I, VCAM, EpCAM, alpha4/beta7 integrin, and alpha v/beta3 integrin including either alpha or beta subunits thereof (e.g . anti -CDl la, anti-CD 18 or anti-CD l lb antibodies) ; growth factors such as VEGF; tissue factor (TF) ; TGF-β. ; alpha interferon (alpha-IFN ) ; an interleukin, such as IL- 8; IgE; blood group antigens Apo2, death receptor; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C etc. In some embodiment the antigen is selected from IGF- IR, CanAg, EphA2, MUC1 , MUC16, VEGF, TF, CD 19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD56, CD 138, CA6, Her2/neu, EpCAM, CRIPTO (a protein produced at elevated levels in a majority of human breast cancer cells) , darpins, alpha_v/beta₃ integrin, alpha_v/beta₅ integrin , alpha y/beta integrin, TGF- β, CD l la, CD18, Apo2 and C242. In some embodiment the antigen is selected from a CD proteins such as CD3, CD4, CD8, CD 19, CD20, CD27, CD34, CD37, CD38, CD46, CD56, CD70 and CD 138; members of the ErbB receptor family such as the EGF receptor, H ER2, HER3 or H ER4 receptor; cell adhesion molecules such as LFA-I, Macl, pl50.95, VLA-4, ICAM-I, VCAM, EpCAM, alpha4/beta7 integrin, and alpha v/beta3 integrin including either alpha or beta subunits thereof (e.g. anti-CDlla, anti-CD18 or anti-CDl lb antibodies); growth factors such as VEGF; tissue factor (TF); TGF-β.; alpha interferon (alpha-IFN); an interleukin, such as IL-8; IgE; blood group antigens Apo2, death receptor; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, etc. The most preferred targets herein are IGF-IR, CanAg, EGF-R, EGF-RvIII, EphA2, MUC1, MUC16, VEGF, TF, CD19, CD20, CD22, CD27, CD33, CD37, CD38, CD40, CD44, CD56, CD70, CD138, CA6, Her2/neu, CRIPTO (a protein produced at elevated levels in a majority of human breast cancer cells), alpha_v/beta₃ integrin, alpha_v/beta₅ integrin, TGF- β, CDlla, CD18, Apo2, EpCAM and C242. In some embodiment the antigen is selected from a cellular oncogene, such as ras or myc.

Examples of viral antigens for use with the present invention include, but are not limited to, e.g., HIV, HCV, CMV, HPV, Influenza, adenoviruses, retroviruses, picornaviruses, etc. Non- limiting example of retroviral antigens such as retroviral antigens from the human immunodeficiency virus (HIV) antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV components; hepatitis viral antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA; influenza viral antigens such as hemagglutinin and neuraminidase and other influenza viral components; measles viral antigens such as the measles virus fusion protein and other measles virus components; rubella viral antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components;

cytomegaloviral antigens such as envelope glycoprotein B and other cytomegaloviral antigen components; respiratory syncytial viral antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components; herpes simplex viral antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components; varicella zoster viral antigens such as gpl, gpll, and other varicella zoster viral antigen components; Japanese encephalitis viral antigens such as proteins E, M- E, M-E-NSI, NSI, NS1-NS2A, 80% E, and other Japanese encephalitis viral antigen

components; rabies viral antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens.

The epitopes and antigens to be used in the methods according to the present invention may be derived from an adenovirus, retrovirus, picornavirus, herpesvirus, rotavirus, hantavirus, coronavirus, togavirus, flavirvirus, rhabdovirus, paramyxovirus, orthomyxovirus, bunyavirus, arenavirus, reovirus, papilomavirus, parvovirus, poxvirus, hepadnavirus, degngue virus, or spongiform virus. In certain specific, non-limiting examples, the viral antigen are peptides obtained from at least one of HIV, CMV, hepatitis A, B, and C, influenza, measles, polio, smallpox, rubella; respiratory syncytial, herpes simplex, varicella zoster, Epstein-Barr, Japanese encephalitis, rabies, Influenza, and/or cold viruses.

Peptides that stimulate cell-mediated immunity (CMI)

Vaccination aims to stimulate the immune response to a specific pathogen in advance of infection. When an individual is exposed to that pathogen, a memory response is triggered which prevents the establishment of infection. Vaccines therefore stimulate the adaptive immune response which unlike innate immunity, is long lived and has memory.

There are two major arms to the adaptive immune system. Humoral immunity which involves the development of antibodies that can bind virus particles and certain antibodies that can neutralize infection. Cell mediated immunity that leads to the development of cytotoxic T-cells that kill infected cells exposing viral epitopes in the context of human leukocyte antigen (HLA) class I, in this way eliminating infected cells.

As used herein a peptide that elicits a cell-mediated immune response refers to any peptide that elicits an activation of antigen-specific cytotoxic T-lymphocytes. These peptides elicit a Cytotoxic T-lymphocyte immune (CTL) response that leads to the development of cytotoxic T-cells that kill infected cells exposing viral epitopes in the context of human leukocyte antigen (HLA) class I, in this way eliminating infected cells. Cell-mediated immunity (CMI) may also involve the activation of phagocytes, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.

These peptides may be a helper T lymphocyte (HTL) inducing peptide comprising HTL epitopes. A "HTL inducing peptide" is a HLA Class II binding peptide that is capable of inducing a HTL response. Also the peptides may in other embodiments be CTL inducing peptides comprising CTL epitopes in addition to or as an alternative to being a HTL inducing peptide. A "CTL inducing peptide" is a HLA Class I binding peptide that is capable of inducing a CTL response.

A peptide that elicit a cell-mediated immune response as used according to the present invention includes but is not limited to any peptide described in any one of international patent applications WO0052040, WO2013/182661, WO 2012/092934 or WO 2012/072088, which patent applications are hereby incorporated by reference.

HIV-specific peptides

In some aspects, the compositions according to the present invention comprise one or more peptide that elicits a cell-mediated immune response. In some embodiments, this peptide is at least one HIV-specific peptide.

One aspect of the peptides that elicits a cell-mediated immune response relates to HIV-specific peptides based on conserved regions of HIV gag p24, antigens in free or carrier- bound form comprising at least one of the said peptides. The HIV-specific peptides to be used according to the invention may originate from conserved areas of the HIV-1 core protein p24, having the properties of maintaining the uniqueness (sensitivity and specificity) of the HIV-l-epitope. Further the new peptides to be used according to the invention possess no recognized cytotoxic T lymphocyte (CTL) antagonistic effect and shall have at least one potential CTL epitope.

In some embodiments, the HIV-specific peptides, to be used according to the present invention, and which have met the above criteria are selected from the following groups;

Xaaj Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa₉ Gin Thr Pro Trp Xaa₁₄ Xaa₁₅ Xaa₁₆ Xaa_{i 7} Xaa₁₈ Val Xaa₂₀ (SEQ ID NO:47) ;

Wherein Xaa in position 1 of the peptide derivate is Lys or Arg,

Xaa in position 2 is Ala, Gly, Ser or Arg,

Xaa in position 3 is Leu or Met,

Xaa in position 4 is Gly or Arg,

Xaa in position 5 is Pro, Thr, Val, Ser, Gin or Ala,

Xaa in position 6 is Gly, Ala, Lys, Arg, Gin or Glu,

Xaa in position 8 is Thr or Ser,

Xaa in position 9 is Leu or He ,

Xaa in position 14 is Thr, Ser or Val,

Xaa in position 15 is Ala or Ser,

Xaa in position 16 is Cys or Ser,

Xaa in position 17 is Gin or Leu

Xaa in position 18 is Gly, Glu or Arg, and

Xaa in position 20 is Gly or Arg;

Xaai Xaa₂ Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val [Gly],, Xaa_i2 Xaa₁₃ Tyr Xaa-_{i 5} Pro Xaa-_{i 7} Xaa_i8 He Leu Xaa₂₁ Xaa₂₂ (SEQ ID NO: 50) wherein Xaa in position 1 is Arg, Lys, Asp or none

Xaa in position 2 is Trp, Gly, Lys or Arg,

Xaa in position 3 is He, Leu, Val or Met

Xaa in position 4 is He, Val or Leu

Xaa in position 5 Leu, Met, Val or Pro

Xaa in position 12 is Arg, Lys

Xaa in postion 13 is Met or Leu,

Xaa in position 15 is Ser, Cys or Gin,

Xaa in position 17 is Thr, Val, He, Ser or Ala,

Xaa in position 18 is Ser, Gly or Thr, Xaa in position 21 is Asp, Glu, Cys or Gly,

Xaa in position 22 is Gly or none, and

n = 0, 1,2 or 3; Xaai Xaa₂ Xaa₃ Pro He Pro Xaa₇ Xaa₈ Xaa₉ Xaaio Xaa Xaa_i2 [Gly]_n Xaa_{i 3} Xaa_i4 Xaais Xaa^ Xaa, ; Xaa is Xaa_i9 Xaa₂o Xaa₂i Xaa₂₂ Xaa₂₃ Xaa₂₄ (SEQ ID NO: 55) wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or none

Xaa in position 2 is Asn, Ala or Lys

Xaa in position 3 is Pro, Gin, Gly, He or Leu

Xaa in position 7 is Val or Ala

Xaa in position 8 is Gly or Lys

Xaa in position 9 is Glu, Asp, Lys, Phe or Thr

Xaa in position 10 is He, Met, Val or Leu

Xaa in position 11 is Tyr, Leu or none

Xaa in position 12 is Ser or none

Xaa in position 13 is Arg or none

Xaa in position 14 is Asp, Arg, Trp, Ala or none

Xaa in position 15 is He or none

Xaa in position 16 is Tyr or none

Xaa in position 17 is Lys or Arg

Xaa in position 18 is Arg, Lys or Asp

Xaa in position 19 is Trp or Gly

Xaa in position 20 is He, Met, Val, Gin or Ala

Xaa in position 21 is He, Val or Ala

Xaa in position 22 is Leu, Met or Val

Xaa in position 23 is Gly or Cys

Xaa in position 24 is Leu or none,

n = 1,2 or 3, and Xaai Xaa₂ He He Xaa_s Xaa₆ Xaa₇ Xaa₈ Xaa₉ Leu Xaau [Gly]_n [Arg]_m Xaa_{i 2} Xaa_{i 3} Xaa_i Xaa-_{l 5}

Xaaie Xaa i 7 Xaa_i8 Xaai₉ Xaa₂₀ Xaa_2i Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ (SEQ ID NO: 61) wherein the Xaa in position 1 is Pro, Lys, Arg or none

Xaa in position 2 is Glu, Arg, Phe or Lys

Xaa in position 5 is Pro or Thr

Xaa in position 6 is Met, Thr or Nleu

Xaa in position 7 is Phe or Leu

Xaa in position 8 is Ser, Thr, Ala or Met Xaa in position 9 is Ala, Glu or Leu

Xaa in position 11 is Ser or none

Xaa in position 12 is Ala, Arg or none

Xaa in position 13 is He, Leu or none

Xaa in position 14 is Ser, Ala, Leu or none

Xaa in position 15 is Tyr, Glu or Asp

Xaa in position 16 is Gly or Asp

Xaa in position 17 is Ala or Leu

Xaa in position 18 is Thr, He, Val, Leu or Asn,

Xaa in position 19 is Pro, Thr or Ser

Xaa in position 20 is Tyr, Phe, Nleu, His or Gin

Xaa in position 21 is Asp, Asn, Leu or Ala

Xaa in position 22 is Leu, He, Val or Asn

Xaa in position 23 is Asn, Tyr, Cys or Gly

Xaa in position 24 is Thr, Met, He, Ala, Val or none

Xaa in postion 25 is Giy or none

n = 1, 2 or 3 and m= 0, 1, 2 or 3 independent of each other the terminal ends of each HIV specific peptide may be free carboxyl- or amino groups, amides, acyls, acetyls or salts thereof.

The HIV-specific peptide sequences have the potential to serve as a good antigen wherein the antigen comprises at least one peptide selected from the group of sequences of SEQ ID NO:47, SEQ ID NO : 50, SEQ ID NO : 55 or SEQ ID NO: 61. The antigenicity may be adapted through adjusting the ratio or concentration of different peptides or size of the peptides by for instance dimerisation or polymerisation and/or immobilisation to a solid phase. The antigen comprises two or more polypeptide sequences, to be used according to the invention, which are either linked by a bridge for instance a disulphide bridge between the Cys residues of the chains or bridges like Ci -C₈ alkylen possibly intervened by one or more heteroatoms like O, S, or N or preferably they are unlinked. The chains may be immobilized to a solid phase in monomeric, dimeric or oligomeric forms. Further amino acids may be added to the ends in order to achieve an «arm» to facilitate immobilization.

All amino acids in the HIV-specific peptides to be used according to the invention can be in both D- or L-form, although the naturally occurring L- form is preferred.

The C- and N-terminal ends of the HIV-specific peptide sequences could deviate from the natural sequences by modification of the terminal NH₂-group and/or COOH-group, they may for instance be acylated, acetylated, amidated or modified to provide a binding site for a carrier or another molecule. The HIV-specific peptides to be used according to the invention are consisting of 6 to 50 amino acids, preferably between 10 and 30 amino acids. They are covering all natural variation of amino acids in the identified positions.

The polypeptide antigen to be used according to the invention is either in a free or in a carrier-bound form . The carrier or solid phase to which the peptide is optionally bound can be selected from a wide variety of known carriers. It should be selected with regard to the intended use of the immobilized polypeptide as a diagnostic antigen or as an immunizing component in a vaccine.

Examples of carriers that can be used for e.g . diagnostic purposes are magnetic beads or latex of co-polymers such as styrene-divinyl benzene, hydroxylated styrene-divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy-activated porous magnetic glass , gelatine or polysaccharide particles or other protein particles, red blood cells, mono- or polyclonal antibodies or fab fragments of such antibodies. In one embodiment of the vaccine according to the present invention comprises antigens containing the peptides of the SEQ ID NO:47, 50, 55 and 61, more preferred the peptides occur in the ratio 1 : 1 : 1 : 1.

In a further preferred embodiment the vaccine composition contains the antigens ;

R A LG PA AT LQT P WT AS LGVG - N H ₂ (SEQ ID NO:49)

RWLLLGLNPLVGGGRLYSPTSILG-NH₂ (SEQ ID NO: 52)

RAIPIPAGTLLSGGGRAIYKRTAILG-NH₂ (SEQ ID NO: 57)

and

RFIIPNIFTALSGGRRALLYGATPYAIG-NH2 (SEQ ID NO: 64) . (Nl in position 6 is Norleucine) . One of the sequences contains in addition to CTL epitopes a B-cell epitope and will also activate the humoral immune system - necessary for helper activity, whereas the other sequences contribute with CTL-epitopes and the amino acid changes implemented within the frame of the CTL-epitope are designed to achieve enhanced binding. Other amino acid changes have been conducted in order to facilitate the synthesis of the peptide and/or increase the solubility of the peptide.

Compound to elicit a humoral response in a subject, such as C5 related compounds:

The present invention also relates to the use of compounds that stimulate the humoral immunity in a subject. Humoral immunity involves the development of antibodies that can bind virus particles and certain antibodies that can neutralize infection.

A peptide that stimulate the humoral immunity in a subject as used according to the present invention includes but is not limited to any peptide described in any one of international patent applications WO2011/000962, WO0052040, WO 2012/092934 or WO 2012/072088, which patent applications are hereby incorporated by reference.

In some embodiments these compounds are agents capable of stabilising the association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20. In other embodiments these compounds are immunogens, which induces antibodies that stabilise association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.

One aspect of the invention relates to compositions and method for reducing and/or delaying pathological effects of human retrovirus infection, such as immunodeficiency virus I (HIV) in a human infected with such virus, such as HIV, the method including administering an effective amount of an agent capable stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.

Another aspect is much similar, but relates to compositions and methods of reducing the risk of developing acquired immunodeficiency syndrome (AIDS), the method including administering an effective amount of an agent capable of stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.

These aspects primarily aim at treating retrovirus infections, such as HIV infected individuals with agents, which can mimic the antibodies which according to the present invention are characteristic for HIV infected long-term non-progressors - this is the most straightforward therapeutic utilisation of the findings underlying the present invention. Where the one aspect aims at reducing pathological effects of retrovirus infections, such as HIV or prolonging the time it takes to develop manifest AIDS, the other aspect aims at reducing the risk of developing AIDS altogether and may therefore be used in individuals which are currently treated prophylactically with anti retroviral therapy.

In one embodiment, the agent in these first aspects of the invention is a molecule comprising at least one amino acid sequence selected independently from an amino acid sequence derived from the transmembrane domain of gp41 and an amino acid sequence derived from the C2 domain, wherein the at least one amino acid sequence binds the C5 domain and optionally comprises at least one D-amino acid ; in certain embodiments all the amino acids in the amino acid sequence are D-amino acids. The molecule is preferably a peptide, and in certain embodiments this peptide comprises the at least one amino acid sequence. The amino acid sequences typically include at most 10 amino acid residues, such as at most 9, at most 8, at most 7, at most 6, and at most 5 amino acid residues. Preferred molecules are therefore peptides having at least 4, 5, 6, 7, 8, 9, or 10 amino acid residues. Specific embodiments of the at least one molecule are therefore the peptides having or comprising SEQ ID NO: 34, 35, 36, 37, 39, 40, 42, 43 and 45, which may all be composed partly or entirely of D-amino acids. Also molecules comprising peptides having Formula III are interesting embodiments of the at least one molecule.

In one embodiment, the agent in these first aspects of the invention is selected from an antibody, an antibody fragment or an antibody analogue. The antibody may be a fully human antibody, a humanized antibody, or a chimeric antibody, or a derivative thereof. Typically, the antibody is an IgA, an IgD, an IgG, an IgE or an IgM - the antibody may be both monoclonal and polyclonal . The antibody fragment is typically selected from a Fab fragment, a Fab' fragment, a Fab'-SH fragment, a F(ab)2 fragment, a F(ab')2 fragment, an Fv fragment, a Heavy chain Ig (a llama or camel Ig), a V_HH fragment, a single domain FV, and a single-chain antibody fragment, and the antibody analogue is typically selected from a scFV, a dsFV, a minibody, a diabody, a triabody, a kappa body, an IgNAR, a tandAb, a BiTE, and a multispecific antibody.

In one embodiment of these first aspects of the invention, the agent binds to and stabilises association between one or more amino acid residues in the amino acid stretch TZ¹AKRRVVZ²REKR, where Z¹ is K, R or E and where Z² is Q or E, and one or more amino acid residues in an amino acid stretch in the transmembrane domain of gp41 and/or in the constant C2 domain of gpl20. This amino acid stretch from C5 is highly conserved across the multiple HIV clades known and effective interaction with this stretch by the agent is therefore believed to be highly advantageous.

A further aspect of the invention relates to a method for reducing the risk of or reducing and/or delaying pathological effects of human immunodeficiency virus I (HIV) in a human infected with HIV, the method including administering an effective amount of an immunogen, which induces antibodies that stabilise association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20, whereas other aspects relates to a prophylactic method using the same means. In other words, one aspect relates to therapeutic active immunotherapy, whereas another aspect relates to prophylactic immunotherapy of HIV disease, including AIDS. This also entails prophylaxis of HIV infection.

These particular aspects are based on the realisation that it is feasible to induce the same type of antibody repertoire in the average HIV infected individual as the one that is found in the HIV LTNP individuals. By carefully selecting peptide regions in both C5 and in tm-gp41 and/or C2 in order to prepare peptide multimers that mimic the antibody binding epitopes present in HIV composed of these regions, it becomes possible to prepare vaccines which will induce the desired immunity - interestingly, this approach does not aim at vaccinating so as to obtain neutralizing antibodies in the classical sense.

In one embodiment the immunogen is selected from a peptide multimer detailed below when discussing these aspects of the invention, a composition detailed below, a nucleic acid fragment discussed in relation to other aspects, a virus or plasmid vector compositions discussed elsewhere. In common for the first aspects is that they all include embodiments where the targeted association between the C5 domain and C2 and/or the transmembrane domain of gp41 involves at least one amino acid in the sequence TZ^!AKRRVVZ²REKR, where Z¹ is K, R or E and where Z² is Q or E and an amino acid and involves at least one amino acid in the transmembrane domain of gp41 or at least one amino acid in the constant C2 domain of gpl20. As explained above, this particular sequence is extremely well-conserved across known HIV ciades, and therefore it is the interaction between this sequence and tm-gp41 or C2 it is most feasible to target.

Another aspect relates to a composition comprising ( 1) a peptide muitimer, said muitimer comprising

a first peptide comprising the amino acid sequence of the 13 amino acid residue amino acid sequence of the C5 domain of HIV gpl20 including between 0 and 4 amino acid substitutions, a subsequence thereof, or an amino acid sequence comprising the inverso-, retro- or retro-inverso form of said amino acid sequence or subsequence, and at least one second peptide comprising an amino acid stretch present in the transmembrane domain of gp41 or present in the constant C2 domain of gpl20 or comprising an amino acid stretch present in any one of SEQ ID NOs. 6- 13 or comprising an inverso-, retro- or retro-inverso form of an amino acid stretch present in the transmembrane domain of gp41 or present in the constant C2 domain of gpl20, wherein said peptide muitimer is capable of inducing an antibody which can bind and stabilise the association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20, and wherein said peptide muitimer lacks amino acids N-terminal of C5 in gpl20.

In other words, this aspect relates to peptide multimers which have a resemblance in 3 dimensions with the epitopes which characterise the interacting areas in C5 on the one hand and tm-gp41 and/or C2 on the other. The peptide multimers to be used according to the invention are useful immunogens that can induce antibodies having the same

characteristics as the antibodies found in HIV LT P individuals, but the peptide multimers also are promissing as diagnostic/prognostic tools. The inclusion of retro-, inverso-, and retro-inverso peptides i .a. enables production of proteolytically stable peptides as well as peptides are truly foreign compared to the HIV counterpart.

In one embodiment of the peptide muitimer, said first peptide comprises the amino acid sequence having formula I:

X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³ (I) wherein X¹ is Thr, X² is selected from Lys, Arg, Har and Glu, X³ is selected from Ala and Val, X⁴ is selected from Arg, Har, Lys and Cit (citrulline), X^s is selected from Arg, Har, Lys and Cit, X⁶ is selected from Arg, Har, Lys and Cit, X⁷ is selected from Val, Leu, He and NIe (norleucin), X^s is selected from Val, Leu He and NIe, X⁹ is selected from Gin, Glu, Asn and Asp, X¹⁰ is selected from Arg, Har and Cit, X¹¹ is selected from Glu and Asp, X¹² is Lys, and X¹³ is selected from Arg, Har and Cit,

or comprises a subsequence the amino acid sequence of formula I, or comprising the inverso-, retro- or retro- inverso form of said amino acid sequence or subsequence. The first peptide may further comprise the dipeptide Ala-Pro linked to the N-terminus of the amino acid sequence having formula I and/or the first peptide may further comprise the dipeptide χΐ4_χΐ5 ijnkgfj to the C-terminus of the amino acid sequence having formula I, wherein X¹⁴ is selected from Ala and Val, and wherein X¹⁵ is selected from Val, Leu and NIe.

Particularly interesting peptides derived from C5 are set forth in the preamble to the

Examples and constitute embodiments of a first peptide of the peptide multimers to be used according to the invention .

A number of naturally occurring mutants of gp41 and gpl20 has been observed, so when stating that the second peptide comprises an amino acid stretch present in the transmembrane domain of gp41 or present in the constant C2 domain of gpl20, this is intended to denote that the amino acid stretch is present in any such naturally occurring form. So, the at least second peptide, when derived from gp41, is in certain embodiments one which includes the amino acid sequence having the formula :

Z¹-Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-Z¹¹-Z¹²-Z¹³-Z¹⁴-Z¹⁵-Z¹⁵-Z¹⁷ (III)

-wherein Z¹ is Asp, Z² is Arg, Z³ is Pro, Z⁴ is Glu or Gly, Z⁵ is Gly or Arg, Z⁶ is He, Z⁷ is Glu, Z⁸ is Glu, Z⁹ is Glu, Z¹⁰ is Gly, Z¹¹ is Gly, Z¹² is Glu or is absent, Z¹³ is Arg or Gin, Z¹⁴ is Asp or Gly, Z¹⁵ is Arg or Lys, Z¹⁶ is Asp or Gly and Z¹⁷ is Arg,

or includes a subsequence of formula (III), such as a subsequence having at least 5 amino acid residues (such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, and at least 16 amino acid residues) . Further, this embodiment of the second peptide may contain amino acid substitutions which result in a sequence identity of at least 80% with a corresponding amino acid sequence found in gp41.

Particularly interesting peptides derived from C2 and gp41, and gpl20 are set forth in the preamble to the Examples and constitute embodiments of a second peptide of the peptide multimers to be used according to the invention.

In certain embodiments of the peptide multimer, the first peptide and the at least one second peptide are associated via a linker; the linker can be any peptide linker, such as a glycine, a lysine or an arginine linker, a polyhistidinyl tag, Protein G, and Protein A but it is also possible to use a bis-maleimide linker, a disulfide linker, or a polyethylene glycol (PEG) linker. In practice, any linker found useful in peptide chemistry is also useful as a linker according to the present invention. Thus, the invention contemplates the use of "simple" linear peptides which are conjugated or fused to each other, but also peptide multimers where the individual peptides derived from C5 and other regions of gpl20 or gp41 are linked via non-peptide linkers e.g. complementary nucleic acids, nucleic acid derivatives or analogues e.g . PNA, LNA. Use of multiple linker types are also within the scope of the present invention, and it is e.g . also a part to be used according to the invention to utilise linear peptides which include intrachain disulphide linkers.

Particularly interesting peptide multimers to be used according to the invention are set forth in the preamble to the examples.

In certain embodiments, at least one of the first and at least one second peptides in the peptide multimer comprises an N- or C-terminal modification, such as an amidation, acylation, or acetylation .

Since the peptide multimers are contemplated as vaccine agents or diagnostic agents, they are in certain embodiments coupled to a carrier molecule, such as an immunogenic carrier. The peptides of the peptide multimers may thus be linked to other molecules either as recombinant fusions (e.g. via CLIP technology) or through chemical linkages in an oriented (e.g. using heterobifunctional cross-linkers) or nonoriented fashion. Linking to carrier molecules such as for example diphtheria toxin, latex beads (convenient in diagnostic and prognostic embodiments), and magnetic beads (also convenient in diagnostic and prognostic embodiments), polylysine constructs etc, are all possible carrier molecules to be used according to the invention .

The immunogenic carrier is conveniently selected from carrier proteins such as those conventionally used in the art (e.g. diphtheria or tetanus toxoid, KLH etc.), but it is also possible to use shorter peptides (T-helper epitopes) which can induce T-cell immunity in larger proportions of a population. Details about such T-helper epitopes can e.g . be found in WO 00/20027, which is hereby incorporated by reference herein - all immunolgic carriers and "promiscuous" (i .e. universal) T-helper epitopes discussed therein are useful as immunogenic carriers in the present invention.

In certain embodiments, the carrier is a virus like particle, i .e. a particle sharing properties with virions without being infectious. Such virus-like particles may be provided chemically (e.g . Jennings and Bachmann Ann Rev Pharmacol . Toxicol . 2009. 49: 303-26 Immunodrugs: Therapeutic VLP-based vaccines for chronic diseases) or using cloning techniques to generate fusion proteins (e.g. Peabody et al . J . Mol . Biol. 2008; 380: 252-63. Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2) . Another example is "Remune", an HIV vaccine originally made by Immune Response Corporation, which consists of formalin inactivated HIV that has been irradiated to destroy the viral genome. The company was started by Jonas Salk who used the same technique to generate the killed polio vaccine in widespread use today. However, on fixation of HIV, gpl20 fell off leaving only gp41 on the virion surface. This opens for the possibility of directly admixing C5- derived peptides disclosed herein with Remune particles, because it should still be possible to obtain the binding between C5 and gp41 on a Remune particle.

Embodiments of the aspect related to peptide multimers also include those wherein the first peptide is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, and 5 or a fragment thereof, or the inverso-, retro- or retro- in verso form of a peptides selected from SEQ ID NO: 1 , 2, 3, 4, and 5 or a fragment thereof, and wherein the second peptide is selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 46 or a fragment thereof or the inverso-, retro- or retro-inverso form of a peptides selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, or 46 or a fragment thereof. As mentioned above, in such a case the fragment may be very short, as long as the peptide muitimer provides for the ability to induce antibodies which will stabilise association between C5 and gp41 and/or C2. A number of interesting peptide multimers of the present invention are listed in the Preamble to the Examples.

In an embodiment, the peptide muitimer to be used according to the invention comprises at most 70 amino acids, such as the most 69, at most 68, at most 67, at most 66, at most 65, at most 64, at most 63, at most 62, at most 61, at most 60, at most 59, at most 58, at most 57, at most 56, at most 55, at most 54, at most 53, at most 52, at most 51, at most 50, at most 49, at most 48, at most 47, at most 46, at most 45, at most 44, at most 43, at most 42, at most 41, at most 40, at most 39, at most 38, at most 37, at most 36, at most 35, at most 34, at most 33, at most 32, at most 31, at most 30, at most 29, at most 28, at most 27, at most 26, at most 25, at most 24, at most 23, at most 22, at most 21, at most 20, at most 19, at most 18, at most 17, at most 16, at most 15, at most 14, at most 13, at most 12, at most 11 , at most 10, at most 9, at most 8, and at most 7 amino acids.

In an embodiment, the peptide muitimer to be used according to the invention comprises at least 6 amino acid residues, such as at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 51, at least 52, at least 53, at least 54, at least 55, at least 56, at least 57, at least 58, at least 59, at least 60, at least 61, at least 62, at least 63, at least 64, at least 65, at least 66, at least 67, at least 68, and at least 69 amino acid residues.

In one embodiment, the peptide muitimer to be used according to the invention consists of 6 amino acid residues or 7 amino acid residues or 8 amino acid residues or 9 amino acid residues or 10 amino acid residues or 11 amino acid residues or 12 amino acid residues or 13 amino acid residues or 14 amino acid residues or 15 amino acid residues or 16 amino acid residues or 17 amino acid residues or 18 amino acid residues or 19 amino acid residues or 20 amino acid residues or 21 amino acid residues or 22 amino acid residues or 23 amino acid residues or 24 amino acid residues or 25 amino acid residues or 26 amino acid residues or 27 amino acid residues or 28 amino acid residues or 29 amino acid residues or 30 amino acid residues or 31 amino acid residues or 32 amino acid residues or 33 amino acid residues or 34 amino acid residues or 35 amino acid residues or 36 amino acid residues or 37 amino acid residues or 38 amino acid residues or 39 amino acid residues or 40 amino acid residues or 41 amino acid residues or 42 amino acid residues or 43 amino acid residues or 44 amino acid residues or 45 amino acid residues or 46 amino acid residues or 47 amino acid residues or 48 amino acid residues or 49 amino acid residues or 50 amino acid residues or 51 amino acid residues or 52 amino acid residues or 53 amino acid residues or 54 amino acid residues or 55 amino acid residues or 56 amino acid residues or 57 amino acid residues or 58 amino acid residues or 59 amino acid residues or 60 amino acid residues or 61 amino acid residues or 62 amino acid residues or 63 amino acid residues or 64 amino acid residues or 65 amino acid residues or 66 amino acid residues or 67 amino acid residues or 68 amino acid residues or 69 amino acid residues or 70 amino acid residues.

In one embodiment, the peptide multimer to be used according to the invention is selected from the group consisting of disulphide linked peptides between SEQ ID NO: 28 and any one of SEQ ID NOs: 29, 31, and 33, between SEQ ID NO: 30, and any one of SEQ ID NO: 29, 31, and 33, or between SEQ ID NO: 32 and any one of SEQ ID NO: 29, 31, and 33; or selected from the group consisting of cysteine-lysine linked peptides between SEQ ID NO : 38 and any one of SEQ ID NO: 39, SEQ ID NO:40; SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO: 68, or between SEQ ID NO:41 and any one of SEQ ID NO: 39, SEQ ID NO:40; SEQ ID NO:42, and SEQ ID NO:43.

In one embodiment, the peptide multimer to be used according to the invention is selected from the group consisting of:

CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 28)

I

CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO: 29),

CGGAKRRVVGGAKRRVVGGQREKR (SEQ ID NO: 30)

I

CGGGDQQLLGGAEEEIVGGIEEEGG (SEQ ID NO: 31),

CGGAEEEVVGGDQQLL (SEQ ID NO : 32)

I GCGGAKRRVVGGAKRRVV (SEQ ID NO: 33),

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO : 38)

I

GKGGIEEEGGRDRDRGGEQDRDR (SEQ ID NO: 39) , GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)

I

GKGGIEEEGGERDRDRGGQDRDR (SEQ ID NO:40),

GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO:41)

I

G GGIEEEGGQDRDRGGRDRDR (SEQ ID NO:42),

GAKRRVVGGCGGA RRVVEREKRAGQRE RA (SEQ ID NO :41)

I

G GGIEEEGGEQDRDRGGERDRD (SEQ ID NO:43) and GAKRRVVGGCGGAKRRVVQRE RAGERE RA (SEQ ID NO: 38)

I

GKGGIEEEGGRDRDRGGQDRDR (SEQ ID NO: 68) .

In one embodiment, the peptide multimer to be used according to the invention is (H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-ethyl)-Gly-Giy-Aia-Lys-Arg-Arg-Val-Val- Gin-Arg-Glu-Lys-Arg-Aia-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH2)_A (H-Gly-Lys-Gly-Gly-Ile-Glu-Glu- Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gin-Asp-Arg-Asp-Arg-NH2)_B, acetate salt (amide bond between Cys(2-oxo-ethyl) 10 (A-chain) and Lys2 (B-chain)) . Another aspect relates to the use of an immunogenic composition (such as a vaccine composition) comprising a composition described herein in combination with a

pharmaceutically acceptable diluent or vehicle and optionally one or more immunological adjuvant.

In some aspects the present invention relates to the use of one or more peptide that stimulate a cell-mediated immune response, such as with at least one HIV-specific peptide selected from the group of amino acid sequences:

Xaa_x Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa_g Gin Thr Pro Trp Xaa₁ Xaa_{i 5} Xaa₁₅ Xaa₁₇ Xaa_iS Val Xaa₂₀ (SEQ ID NO:47) ;

Wherein Xaa in position 1 of the peptide derivate is Lys or Arg,

Xaa in position 2 is Ala, Gly, Ser or Arg,

Xaa in position 3 is Leu or Met,

Xaa in position 4 is Gly or Arg,

Xaa in position 5 is Pro, Thr, Val, Ser, Gin or Ala,

Xaa in position 6 is Gly, Ala, Lys, Arg, Gin or Glu,

Xaa in position 8 is Thr or Ser,

Xaa in position 9 is Leu or He ,

Xaa in position 14 is Thr, Ser or Val,

Xaa in position 15 is Ala or Ser, Xaa in position 16 is Cys or Ser,

Xaa in position 17 is Gin or Leu

Xaa in position 18 is Gly, Glu or Arg, and

Xaa in position 20 is Gly or Arg;

Xaai Xaa₂ Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val [Gly]_n Xaa_i2 Xaa_{i 3} Tyr Xaa_{i 5} Pro Xaa^ Xaais He Leu Xaa₂₁ Xaa₂₂ (SEQ ID NO: 50) wherein Xaa in position 1 is Arg, Lys, Asp or none

Xaa in position 2 is Trp, Gly, Lys or Arg,

Xaa in position 3 is He, Leu, Val or Met

Xaa in position 4 is He, Val or Leu

Xaa in position 5 Leu, Met, Val or Pro

Xaa in position 12 is Arg, Lys

Xaa in postion 13 is Met or Leu,

Xaa in position 15 is Ser, Cys or Gin,

Xaa in position 17 is Thr, Val, He, Ser or Ala,

Xaa in position 18 is Ser, Gly or Thr,

Xaa in position 21 is Asp, Glu, Cys or Gly,

Xaa in position 22 is Gly or none, and

n = 0, 1,2 or 3;

Xaai Xaa₂ Xaa₃ Pro He Pro Xaa₇ Xaa₈ Xaa₉ Xaa_i0 Xaa Xaa-_{i 2} [Gly],, Xaa-_{l 3} Xaa_i4 Xaa_{i 5} Xaa_i6 Xaa_{i 7} Xaa is Xaa_{: g} Xaa₂₀ Xaa_2i Xaa₂₂ Xaa₂₃ Xaa₂₄ (SEQ ID NO: 55) wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or none

Xaa in position 2 is Asn, Ala or Lys

Xaa in position 3 is Pro, Gin, Gly, He or Leu

Xaa in position 7 is Val or Ala

Xaa in position 8 is Gly or Lys

Xaa in position 9 is Glu, Asp, Lys, Phe or Thr

Xaa in position 10 is He, Met, Val or Leu

Xaa in position 11 is Tyr, Leu or none

Xaa in position 12 is Ser or none

Xaa in position 13 is Arg or none

Xaa in position 14 is Asp, Arg, Trp, Ala or none

Xaa in position 15 is He or none

Xaa in position 16 is Tyr or none

Xaa in position 17 is Lys or Arg Xaa in position 18 is Arg, Lys or Asp

Xaa in position 19 is Trp or Gly

Xaa in position 20 is He, Met, Val, Gin or Ala

Xaa in position 21 is He, Val or Ala

Xaa in position 22 is Leu, Met or Val

Xaa in position 23 is Gly or Cys

Xaa in position 24 is Leu or none,

n = 1,2 or 3, and Xaa₅ Xaa₂ He He Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa₉ Leu Xaa_n [Gly]_n [Arg]_m Xaa_i2 Xaa_{i 3} Xaa_i4 Xaa_{i 5} Xaa_{i 6} Xaa I _/ Xaa_{i S} Xaa_i9 Xaa₂₀ Xaa_2i Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa_2s (SEQ ID NO: 61) wherein the Xaa in position 1 is Pro, Lys, Arg or none

Xaa in position 2 is Glu, Arg, Phe or Lys

Xaa in position 5 is Pro or Thr

Xaa in position 6 is Met, Thr or Nleu

Xaa in position 7 is Phe or Leu

Xaa in position 8 is Ser, Thr, Ala or Met

Xaa in position 9 is Ala, Glu or Leu

Xaa in position 11 is Ser or none

Xaa in position 12 is Ala, Arg or none

Xaa in position 13 is He, Leu or none

Xaa in position 14 is Ser, Ala, Leu or none

Xaa in position 15 is Tyr, Glu or Asp

Xaa in position 16 is Gly or Asp

Xaa in position 17 is Ala or Leu

Xaa in position 18 is Thr, He, Val, Leu or Asn,

Xaa in position 19 is Pro, Thr or Ser

Xaa in position 20 is Tyr, Phe, Nleu, His or Gin

Xaa in position 21 is Asp, Asn, Leu or Ala

Xaa in position 22 is Leu, He, Val or Asn

Xaa in position 23 is Asn, Tyr, Cys or Gly

Xaa in position 24 is Thr, Met, He, Ala, Val or none

Xaa in postion 25 is Gly or none

n = 1, 2 or 3 and m= 0, 1, 2 or 3 independent of each other, the terminal ends of each HIV specific peptide may be free carboxyl- or amino groups, amides, acyls, acetyls or salts thereof. In some embodiments two or more of the Cys residues of said HIV-specific peptide may form part of an intrachain- or interchain disulphide binding, a -S-(CH₂)_P-S- or a - (CH₂)_P- bridge wherein p = 1-8 optionally intervened by one or more heteroatoms such as O, N and S and/or the said peptide sequences are immobilized to a solid support.

In some embodiments the amino acid sequence of SEQ ID NO:47 is selected from the groups of SEQ ID NO:48 and SEQ ID NO:49.

In some embodiments the amino acid sequence of SEQ ID NO: 50 is selected from the groups of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54.

In some embodiments the amino acid sequence of SEQ ID NO: 55 is selected from the groups of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60.

In some embodiments the amino acid sequence of SEQ ID NO: 61 is selected from the groups of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.

In some embodiments the at least one HIV-specific peptide comprises at least , two, three, or four peptides selected from each of the groups of SEQ ID NO :47, SEQ ID NO: 50, SEQ ID NO: 55 and SEQ ID NO: 61.

In some embodiments the at least one HIV-specific peptide consist of or comprises the peptides of the SEQ ID NO:49, SEQ ID NO: 52, SEQ ID NO: 57 and SEQ ID NO: 64.

In some embodiments the one or more peptide is in the form of an acetate salt.

In some embodiments one, two, three or four peptides are used in the therapeutic HIV- 1 immunization phase.

In some embodiments the all four peptide as acetate salts are used in the therapeutic HIV-1 immunization phase.

In some embodiments the peptides have amide C-terminal ends of formula -

C(0)NH2, or acetate salts thereof.

In some embodiments all four peptide are used in the ratio of 1 : 1 : 1 : 1 w/w.

In some embodiments one, two, three or four peptide acetate salts are in a disolved liquid state. In some embodiments this liquid is water.

HCV:

For antigens derived from HCV these antigens may be derived from the Core, El, E2, P7, NS2, NS3, NS4 (NS4A and NS4B) and NS5 (NS5A and NS5B) protein of the Hepatitis C Virus (HCV) . The epitopes are those which elicit a HLA class I and/or class II restricted T lymphocyte response in an immunized host. More specific, the HLA class I restricted peptides of the present invention may bind to at least one HLA molecule of the following HLA class I groups: HLA-A*01, HLA-A*02, HLA-A*03, HLA-A*11, HLA-A*24, HLA-B*07, HLA-B*08, HLA- B*35, HLA-B O, HLA-B 4, HLA-Cw3, HLA-Cw4, HLA-Cw6 or HLA-Cw7. The HLA class II restricted peptides of the present invention may bind to at least one HLA molecule of the following HLA class II groups: HLA-DRB1, -DRB2, -DRB3, -DRB4, -DRB5, -DRB6, -DRB7, - DRB8 or -DRB9.

MHC binding HCV peptides that may be used according to the present invention as epitopes are disclosed in e.g . WO02/34770 (Imperial College Innovations Ltd), WO01/21189 and WO02/20035 (Epimmune), WO04/024182 (Intercell), W095/25122 (The Scripps

Research Institute), W095/27733 (Government of the USA, Department of Health and Human Services), EP 0935662 (Chiron), WO02/26785 (Immusystems GmbH), W095/12677 (Innogenetics N.V), W097/34621 (Cytel Corp), and EP 1652858 (Innogenetics N .V.) .

The vaccine composition according to the present invention may comprise a PADRE peptide, such as the universal T cell epitope called PADRE as disclosed in WO95/07707 (Epimmune) the content of which are enclosed herein by reference. A 'PanDR binding peptide or PADRE peptide" is a member of a family of molecules that binds more that one HLA class II DR molecule. PADRE binds to most HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses. Alternatively T-help epitopes can be used from universally used vaccines such as tetanos toxoid .

The vaccine composition according to the present invention may comprise a polyepitopic peptide derived from a HCV protein, or more specifically from at least one of the following HCV regions selected from the group consisting of Core, El, E2/NS1, NS2, NS3,

NS4A, NS4B, NS5A and NS5B. Even more preferred is that peptides are characterized in that they are present in the HCV consensus sequence of genotype la, lb and/or 3 a.

Other HLA class I and II binding peptides that may be used according to the invention may be identified by the method as described in WO03/105058 -Algonomics, by the method as described by Epimmune in WO01/21189 and/or by three public database prediction servers, respectively Syfpeithi, BIMAS and nHLAPred . It is also an aspect of this present invention that each peptide may be used within the scaffold design of the invention in combination with the same peptide as multiple repeats, or with any other peptide(s) or epitope(s) .

CMV:

The vaccine composition according to the present invention may comprise epitopes derived from cytomegalovirus (CMV) including CMV glycoproteins gB and gH .

Influenza :

The vaccine composition according to the present invention may comprise epitopes derived from fragments or portions of Influenza hemagglutinin (HA) or Influenza

neuraminidase (NA), nucleoprotein (NP), M l, M2, NSl, NEP, PA, PBl, PB1-F2, PB2 for each of the subgroups, such as H1N 1, H2N2 og H3N2. Suitable epitopes may be derived from an HA protein of one, or more than one subtype, including H I, H2, H3, H4, H5, H6, H7, H8, H9, H 10, H l l, H 12, H 13, H14, H 15 or H 16 or fragment or portion thereof. Examples of subtypes comprising such HA proteins include A/ ew Caledonia/20/99 (H 1N1 ) A/Indonesia/5/2006 (H5N 1), A/chicken/New

York/1995, A/herring gull/DE/677/88 (H2N8), A/Texas/32/2003, A/mallard/MN/33/00, A/duck/Shanghai/1/2000, A/northern pintail/TX/828189/02, A/Turkey/Ontario/6118/68 (H8N4), A/shoveler/Iran/G54/03, A/chicken/Germany/N/1949 (H10N7), A/duck/England/56 (H 11 N6), A/duck/Alberta/60/76 (H 12N5), A/Gull/Maryland/704/77 (H13N6),

A/Mallard/Gurjev/263/82, A/duck/Australia/341/83 (H 15N8), A/black-headed

gull/Sweden/5/99 (H 16N3), B/Lee/40, C/Johannesburg/66, A/PuertoRico/8/34 (H 1 N1 ), A/Brisbane/59/2007 (H 1N 1 ), A/Solomon Islands 3/2006 (H 1 N 1), A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2), B/Malaysia/2506/2004, B/Florida/4/2006,

A/Singapore/1/57 (H2N2), A/Anhui/1/2005 (H5N 1), A/Vietnam/1194/2004 (H5N 1 ),

A/Teal/HongKong/W312/97 (H6N1), A/Equine/Prague/56 (H7N7), A/Hong ong/1073/99 (H9N2)) .

In some embodiments of the invention, the HA protein may be an H I, H2, H3, H5, H6, H7 or H9 subtype. In other embodiments, the HI protein may be from the A/ New Caledonia/20/99 (H1 N1), A/PuertoRico/8/34 (H 1N1), A/Brisbane/59/2007 (H 1N 1), or A/Solomon Islands 3/2006 (H 1N 1) strain. The H3 protein may also be from the A/Brisbane 10/2007 (H3N2) or A/Wisconsin/67/2005 (H3N2) strain. In other embodiments, the H2 protein may be from the A/Singapore/1/57 (H2N2) strain. The H5 protein may be from the A/Anhui/1/2005 (H5N 1), A/Vietnam/1194/2004 (H5N 1), or A/Indonesia/5/2005 strain. In other embodiments, the H6 protein may be from the A/Teal/Hong Kong/W312/97 (H6N 1 ) strain. The H7 protein may be from the A/Equine/Prague/56 (H7N7) strain . In other embodiments, the H9 protein is from the A/HongKong/1073/99 (H9N2) strain. In other embodiments, the HA protein may be from an influenza virus may be a type B virus, including B/Malaysia/2506/2004 or

B/Florida/4/2006. The influenza virus HA protein may be H5 Indonesia. Human immunodeficiency virus (HIV) :

For HIV, the epitopes to be incorporated into the vaccine composition according to the present invention may be derived from the group consisting of gpl20, gpl60, gp41, p24gag or p55gag derived from HIV, including members of the various genetic subtypes. Human papillomavirus (HPV) :

For HPV, the epitopes to be incorporated into the vaccine composition according to the present invention may be derived from the group consisting El, E2, E3, E4, E6 and E7, LI and L2 proteins. The epitopes may be derived from any type including types 8, 11 , 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. Carriers, adjuvants and vehicles - delivery

The peptides used in the methods according to the invention may be delivered by various means and within various compositions, herein referred to as "compositions", "vaccine compositions" or "pharmaceutical compositions". The peptides of the present invention and pharmaceutical and vaccine compositions of the invention are useful for administration to mammals, particularly humans, to treat and/or prevent virus infection . Vaccine compositions containing the peptides of the invention are administered to a patient infected with the virus in question or to an individual susceptible to, or otherwise at risk for, virus infection to elicit an immune response against the specific antigens and thus enhance the patient's own immune response capabilities.

Various art-recognized delivery systems may be used to deliver the peptides, into appropriate cells. The peptides can be delivered in a pharmaceutically acceptable carrier or as colloidal suspensions, or as powders, with or without diluents. They can be "naked" or associated with delivery vehicles and delivered using delivery systems known in the art.

A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" or just "adjuvant" is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen. Suitable carriers or adjuvant typically comprise one or more of the compounds included in the following non-exhaustive list: large slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles; aluminium hydroxide, aluminium phosphate (see International Patent Application Publication No. W093/24148), alum (KAI(S04)2.12H20), or one of these in combination with 3-0-deacylated monophosphoryl lipid A (see International Patent Application Publication No. WO93/19780) ; N-acetyl-muramyl-L-threonyl-D-isoglutamine (see U.S. Patent No.

4,606,918), N-acetyl-normuramyl-L-alanyl-D-isoglutamine, N-acetylmuramyl-L-alanyl-D- isoglutamyl-L-alanine2-( l',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) ethylamine; RIBI (ImmunoChem Research Inc., Hamilton, MT, USA) which contains monophosphoryl lipid A (i .e., a detoxified endotoxin), trehalose-6,6-dimycolate, and cell wall skeleton (MPL + TDM + CWS) in a 2% squalene/Tween 80 emulsion . Any of the three components MPL, TDM or CWS may also be used alone or combined 2 by 2; adjuvants such as Stimulon (Cambridge Bioscience, Worcester, MA, USA) , SAF-1 (Syntex) ; adjuvants such as combinations between QS21 and 3-de-O-acetylated monophosphoryl lipid A (see International Application No.

WO94/00153) which may be further supplemented with an oil-in-water emulsion (see, e.g ., International Application Nos. WO95/17210, WO97/01640 and W09856414) in which the oil- in-water emulsion comprises a metabolisable oil and a saponin, or a metabolisable oil, a saponin, and a sterol, or which may be further supplemented with a cytokine (see

International Application No. W098/57659) ; adjuvants such as MF-59 (Chiron), or poly[di(carboxylatophenoxy) phosphazene] based adjuvants (Virus Research Institute); blockcopolymer based adjuvants such as Optivax (Vaxcei, Cytrx) or inulin-based adjuvants, such as Algammulin and Gammalnulin (Anutech); Complete or Incomplete Freund's Adjuvant (CFA or I FA, respectively) or Gerbu preparations (Gerbu Biotechnik); a saponin such as QuilA, a purified saponin such as QS21, QS7 or QS17, -escin or digitonin; immunostimulatory oligonucleotides comprising unmethylated CpG dinucleotides such as [purine-purine-CG- pyrimidine-pyrimidine] oligonucleotides. These immunostimulatory oligonucleotides include CpG class A, B, and C molecules (Coley Pharmaceuticals), ISS (Dynavax), Immunomers (Hybridon). Immunostimulatory oligonucleotides may also be combined with cationic peptides as described, e.g., by Riedl et al. (2002); Immune Stimulating Complexes comprising saponins, for example Quil A (ISCOMS); excipients and diluents, which are inherently nontoxic and non-therapeutic, such as water, saline, glycerol, ethanol, isopropyl alcohol, DMSO, wetting or emulsifying agents, pH buffering substances, preservatives, and the like; a biodegradable and/or biocompatible oil such as squalane, squalene, eicosane,

tetratetracontane, glycerol, peanut oil, vegetable oil, in a concentration of, e.g., 1 to 10% or 2.5 to 5%; vitamins such as vitamin C (ascorbic acid or its salts or esters), vitamin E (tocopherol), or vitamin A; carotenoids, or natural or synthetic flavanoids; trace elements, such as selenium; any Toll-like receptor ligand as reviewed in Barton and Medzhitov (2002).

For a further enhancement of the vaccine antigenic properties, one possibility could be to combine a well known adjuvant with an oral immune modulant, such as IMID or adjuvant such as a Cox-2 inhibitor or a immunomodulating compound.

A further aspect of the invention is the use of the vaccine combined with adjuvant, with an (oral) immunemodulating agent and a reservoir purging agent.

Other suitable adjuvants includes response-selective C5a agonists, such as EP54 and EP67 described in Hung CY et al. An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection. Vaccine (2012) and Kollessery G et al.

Tumor-specific peptide based vaccines containing the conformationally biased, response- selective C5a agonists EP54 and EP67 protect against aggressive large B cell lymphoma in a syngeneic murine model. Vaccine (2011) 29: 5904-10.

Other suitable adjuvants include an oil-in-water emulsion containing a stabilizing detergent, a micelle-forming agent and a biodegradable oil, such as Provax described in e.g. US 5,585, 103.

Any of the afore-mentioned adjuvants comprising 3-de-O-acetylated

monophosphoryl lipid A, said 3-de-O-acetylated monophosphoryl lipid A may be forming a small particle (see International Application No. W094/21292).

In any of the aforementioned adjuvants MPL or 3-de-O-acetylated monophosphoryl lipid A can be replaced by a synthetic analogue referred to as RC-529 or by any other amino- alkyl glucosaminide 4-phosphate (Johnson et al. 1999, Persing et al. 2002). Alternatively it can be replaced by other lipid A analogues such as O -197 (Byl et al. 2003). A "pharmaceutically acceptable vehicle" includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles. Delivery systems known in the art are e.g. lipopeptides, peptide compositions encapsulated in poly-DL-lactide-co-glycolide ("PLG"), microspheres, peptide compositions contained in immune stimulating complexes (ISCOMS), multiple antigen peptide systems (MAPs), viral delivery vectors, particles of viral or synthetic origin, adjuvants, liposomes, lipids, microparticles or microcapsules, gold particles, nanoparticles, polymers, condensing agents, polysaccharides, polyamino acids, dendrimers, saponins, QS21, adsorption enhancing materials, fatty acids or, naked or particle absorbed cDNA.

The peptides may be delivered in oils such as Endocine™ and Montanide™

(Eurocine) - Montanide™ ISA 51 VG or Montanide™ ISA 720 VG (Seppic) .

The adjuvant may be stimulators of the innate immune system that can be given separately from the peptide such as Leukotriene B4 (LTB4) and granulocyte macrophage colony stimulating factor (GM-CSF), such as Sargramostim/Leukine (glycosylated GM-CSF) and Molgramostim (nonglycosylated GM-CSF) .

Typically, a vaccine or vaccine composition is prepared as an injectable, either as a liquid solution or suspension. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intradermal, or intraepidermal . Other types of administration comprise electroporation, implantation, suppositories, oral ingestion, enteric application, inhalation, aerosolization or nasal spray or drops. Solid forms, suitable for dissolving in, or suspension in, liquid vehicles prior to injection may also be prepared . The preparation may also be emulsified or encapsulated in liposomes for enhancing adjuvant effect.

A liquid formulation may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Preferably carbohydrates include sugar or sugar alcohols such as mono-, di-, tri-, oligo- or polysaccharides, or water- soluble glucans. The saccharides or glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and beta cyclodextrin, soluble starch, hydroxethyl starch and carboxymethylcellulose, or mixtures thereof. Sucrose is most preferred. "Sugar alcohol" is defined as a C4 to C8 hydrocarbon having an -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol . Mannitol is most preferred. These sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used as long as the sugar or sugar alcohol is soluble in the aqueous preparation. Preferably, the sugar or sugar alcohol concentration is between 1.0 % (w/v) and 7.0 % (w/v), more preferable between 2.0 and 6.0 % (w/v) . Preferably amino acids include levorotary (L) forms of carnitine, arginine, and betaine; however, other amino acids may be added . Preferred polymers include polyvinylpyrrolidone (PVP) with an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer in the composition to minimize pH changes in the solution before lyophilization or after reconstitution. Any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer. Preferably, the concentration is from 0.01 to 0.3 molar.

Surfactants that can be added to the formulation are shown in EP patent applications No. EP 0 270 799 and EP 0 268 110.

Additionally, the peptides used in the methods according to the present invention may be chemically modified by covalent conjugation to a polymer to increase their circulating half-life, for example. Preferred polymers, and methods to attach them to peptides, are shown in U.S. Patent Nos. 4,766,106; 4,179,337; 4,495,285; and 4,609,546. Preferred polymers are polyoxyethylated polyols and polyethylene glycol (PEG). PEG is soluble in water at room temperature and has the general formula:

R(0-CH2-CH2)nO-R where R can be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, the protective group has between 1 and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1.000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40.000, more preferably between 2000 and 20.000, most preferably between 3.000 and 12.000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated.

However, it will be understood that the type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/polypeptide of the present invention.

Water soluble polyoxyethylated polyols are also useful in the present invention. They include polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylated glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of polyoxyethylated glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, triglycerides. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG has a preferred molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, and a discussion of POG/IL-2 conjugates is found in U.S. Patent No. 4,766,106.

Another drug delivery system for increasing circulatory half-life is the liposome. The peptides and nucleic acids of the invention may also be administered via liposomes, which serve to target a particular tissue, such as lymphoid tissue, or to target selectively infected cells, as well as to increase the half-life of the peptide and nucleic acids composition.

Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide or nucleic acids to be delivered is incorporated as part of a liposome or embedded, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide or nucleic acids of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide and nucleic acids compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g ., liposome size, acid lability and stability of the liposomes in the blood stream . A variety of methods are available for preparing liposomes, as described in, e.g ., Szoka et al, 1980, and U.S. Patent Nos. 4,235,871, 4, 501,728, 4,837,028, and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated. For example, liposomes carrying either immunogenic polypeptides are known to elicit CTL responses in vivo (Reddy et al . , 1992; Collins et al ., 1992; Fries et al ., 1992; Mabel et al ., 1992) .

After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art.

Another aspect of the present invention relates to the use of conjugates of the peptides or isolated multimeric peptides in the methods according to the present invention. Accordingly, the peptides or isolated multimeric peptides used in the methods according to the present invention may be an amino acid sequence conjugated at any amino acid sidechain or within the amino acid sequence with any chemical moiety, such as any therapeutic agent, such as any immunomodulating compound .

The terms "therapeutic agent", such as "immunomodulating agent" or

"immunomodulatory compound" or "virus reservoir purging agent" as used herein, includes but is not limited to cytokines, such as interferons, monoclonal antibodies, such as ant-PDl antibodies, cyclophosphamide, Thalidomide, Levamisole, and Lenalidomide.

"A virus reservoir purging agent", or just "reservoir purging agent" includes but is not limited to auranofin, IL-7, prostratin, bryostatin, HDAC inhibitors, such as vorinostat, and Disulfiram .

Peptides used in vaccine compositions as vaccine agents or diagnostic agents, are in certain embodiments coupled to a carrier molecule, such as an immunogenic carrier. Peptides of peptide combinations may thus be linked to other molecules either as recombinant fusions (e.g. via CLIP technology) or through chemical linkages in an oriented (e.g. using

heterobifunctional cross-linkers) or nonoriented fashion. Linking to carrier molecules such as for example diphtheria toxin, latex beads (convenient in diagnostic and prognostic embodiments), and magnetic beads (also convenient in diagnostic and prognostic

embodiments), polylysine constructs etc, are all possible according to the invention.

In certain embodiments, the carrier is a virus like particle, i .e. a particle sharing properties with virions without being infectious. Such virus-like particles may be provided chemically (e.g . Jennings and Bachmann Ann Rev Pharmacol . Toxicol. 2009. 49: 303-26 Immunodrugs: Therapeutic VLP-based vaccines for chronic diseases) or using cloning techniques to generate fusion proteins (e.g . Peabody et al . J . Mol . Biol . 2008; 380: 252-63. Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2) . Another example is "Remune", an HIV vaccine originally made by Immune Response Corporation, which consists of formalin inactivated HIV that has been irradiated to destroy the viral genome.

One aspect of the present invention relates to the use of one or more HIV-specific peptide as defined above.

In certain embodiments, peptides comprise an N- or C-terminal modification, such as an amidation, acylation, or acetylation . When the C-terminal end of a peptide is an amide, suitable amides included those having the formula -C(0)-NR^xR^Y, wherein R^x and R^Y are independently selected from hydrogen and Ci-₆ alkyl, which alkyl group may be substituted with one of more fluoro atoms, for example -CH₃, -CH₂CH₃ and -CF₃, a particular amide group which may be mentioned is -C(0)NH₂. When the N-terminal end of the peptide is acetylated, suitable acetylated N-terminal ends include those of formula -NH-C(0)R^z, wherein R^z is hydrogen, C< ₅ alkyl, which alkyl group may be substituted with one of more fluoro atoms, for example -CH₃, -CH₂CH₃ and -CF₃, or phenyl .

Since the peptides are contemplated as vaccine agents or diagnostic agents, they are in certain embodiments coupled to a carrier molecule, such as an immunogenic carrier. The peptides may thus be linked to other molecules either as recombinant fusions (e.g . via CLIP technology) or through chemical linkages in an oriented (e.g . using heterobifunctional cross-linkers) or nonoriented fashion. Linking to carrier molecules such as for example diphtheria toxin, latex beads (convenient in diagnostic and prognostic embodiments), and magnetic beads (also convenient in diagnostic and prognostic embodiments), polylysine constructs etc, are all possible according to the invention .

In certain embodiments, the carrier is a virus like particle, i .e. a particle sharing properties with virions without being infectious. Such virus-like particles may be provided chemically (e.g . Jennings and Bachmann Ann. Rev. Pharmacol . Toxicol . 2009. 49: 303-26 Immunodrugs: Therapeutic VLP-based vaccines for chronic diseases) or using cloning techniques to generate fusion proteins (e.g. Peabody et al . J . Mol . Biol. 2008; 380: 252-63. Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2) . Another example is "Remune", an HIV vaccine originally made by Immune Response Corporation, which consists of formalin inactivated HIV that has been irradiated to destroy the viral genome. The company was started by Jonas Salk who used the same technique to generate the killed polio vaccine in widespread use today.

Preparation of immunogenic compositions includes the use of state-of-the-art constituents such as immunological adjuvants. Apart from these adjuvants, which are detailed, by way of example, below, immunogenic compositions are prepared as generally taught in the art:

The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251 ; 4,601,903; 4,599,231 ; 4,599,230; 4,596,792; and 4, 578, 770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared . The preparation may also be emulsified . The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccines; cf. the detailed discussion of adjuvants below.

The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously, intracutaneously, intradermally, subdermally or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral, nasal, buccal, sublinqual, intraperitoneal, intravaginal, anal, epidural, spinal, and intracranial formulations. For suppositories, traditional binders and carriers may include, for example, polyalkalene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10% (w/w), preferably 1-2% (w/w). Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and may contain 10-95% (w/w) of active ingredient, preferably 25-70% (w/w).

The peptides may be formulated into a vaccine as neutral or salt forms.

Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the individual's immune system to mount an immune response, and the degree of immunity desired. Suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination with a preferred range from about 0.1 pg to 2,000 pg (even though higher amounts in the 1-10 mg range are contemplated), such as in the range from about 0.5 pg to 1,000 pg, preferably in the range from 1 pg to 1500 pg and especially in the range from about 100 pg to 1200pg. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by subsequent inoculations or other administrations.

Some of the peptides are sufficiently immunogenic in a vaccine, but for some of the others the immune response will be enhanced if the vaccine further comprises an adjuvant substance. The immunogenic molecules described herein can therefore be formulated with adjuvants:

The adjuvants to be combined are known to induce humoral responses and include: i) Salt suspensions (e.g. varieties of salts containing aluminum ions or calcium ions), ii) Oil - in-water emulsions (e.g. varieties of squalane-based or squalene-based emulsions), iii) Water-in-oil emulsions (e.g. Montanide ISA51 or ISA720), iv) Neutral liposomes, v) Cationic liposomes, vi) Microspheres, vii) Immunostimulating complexes (e.g. ISCOMs or

ISCOMATRIX), viii) Pattern-recognition receptor agonists (e.g. agonists for C-type lectin receptors (CLRs), NOD-like receptors (NLRs), RIG-like helicases (RLHs), Triggering receptor expressed on myeloid cells (TREMs) and Toll-like receptors (TLRs)), ix) Saponins (i.e. Any saponin derived from Quillaja saponaria or Platycodon grandiflorum), x) Virosomes/Virus-like particles, xi) Enterotoxins (i.e. Cholera toxin, CTA1-DD or Esherichia coli heat-labile enterotoxin), and combinations thereof.

A further aspect of the invention is the use of the vaccine combined with adjuvant, with one or more further therapeutic agents, such as an (oral) immunemodulating agent and/or a second reservoir purging agent .

The terms "therapeutic agent", such as "immunomodulating agent" or virus reservoir purging agent as used herein, includes but is not limited to cytokines, such as interferons, monoclonal antibodies, such as anti-PDl antibodies, cyclophosphamide, Thalidomide, Levamisole, and Lenalidomide.

"A virus reservoir purging agent", includes but is not limited to auranofin, IL-7, prostratin, bryostatin, HDAC inhibitors, such as vorinostat, Disulfiram and any suitable agent disclosed in any one of WO2013050422, WO2012051492 A3 and in Barton et al., Clinical

Pharmacology & Therapeutics (2013); 93 1, 46-561, including but not limited to a NF-kappa- B-inducer selected from the group comprising : PMA, prostratin, bryostatin and TNF-alpha, and/or b) a histone deacetylase inhibitor selected from the different families (hydroxamates, cyclic peptides, aliphatic acids, and benzamides) including: TSA, SAHA, MS-275,

aminosuberoyl hydroxamic acids, M-Carboxycinnamic acid bishydroxamate, LAQ-824, LBH- 589, belinostat (PXD-101 ), Panobinostat (LBH-589), a cinnamic hydroxamic acid analogue of M-carboxycinnamic acid bishydroxamate, IF2357, aryloxyalkanoic acid hydroxamides, depsipeptide, apicidin, cyclic hydroxamic acid- containing peptide group of molecules, FK- 228, red FK, cyclic peptide mimic linked by an aliphatic chain to a hydroxamic acid, butyrate, phenylbutyrate, sodium butyrate, valproic acid, pivaloyloxymethyl butyrate, 5 NOX-275, and MGCD0103. Any of the above virus reservoir purging agents may be used alone or in combination with any one other suitable virus reservoir purging agent, such as with another class of HIV inducers.

DNA methylation, probably together with repressive histone modifications, may also contribute to a "lock" in a silent state of the provirus and makes its return to an active state difficult. These observations suggest that HDAC or HMT or DNA methylation inhibitors together with efficient cART constitute good anti-latency drug candidates aimed at reducing/eliminating the pool of latent reservoirs to a level bearable by the host immune system .

Accordingly suitable immunomodulatory compounds or purging agents may be DNA methylation inhibitors selected from the two classes (non-nucleoside and nucleoside demethylating agents) including : 5-azacytidine (azacitidine), Sinefungin, 5-aza-2'- deoxycytidine (5-aza-CdR, decitabine), l-3-Darabinofuranosyl-5-azacytosine (fazarabine) and dihydro-5-azacytidine (DHAC), 5-fluorodeoxycytidine (FdC), oligodeoxynucleotide duplexes containing 2-H pyrimidinone, zebularine, antisense oligodeoxynucleotides (ODNs), MG98, (-)-epigallocatechin-3-gallate, hydralazine, procaine and procainamide.

Other suitable immunomodulatory compounds or purging agents to be used according to the present invention includes histone deacetylase inhibitor selected from the different families of HDACI (hydroxamates, cyclic peptides, aliphatic acids, and benzamides) including TSA, SAHA, MS-275, aminosuberoyl hydroxamic acids, M-Carboxycinnamic acid bishydroxamate, LAQ-824, LBH-589, belinostat (PXD-101 ), Panobinostat (LBH-589), a cinnamic hydroxamic acid analogue of M-carboxycinnamic acid bishydroxamate, IF2357, aryloxyalkanoic acid hydroxamides, depsipeptide, apicidin, cyclic hydroxamic acid-containing peptide group of molecules, FK-228, red FK, cyclic peptide mimic linked by an aliphatic chain to a hydroxamic acid, butyrate, phenylbutyrate, sodium butyrate, valproic acid,

pivaloyloxymethyl butyrate, 5 NOX-275, and MGCD0103.

Other suitable immunomodulatory compounds or purging agents to be used according to the present invention includes histone methyltransferase inhibitors (chaetocin and BIX-01294) ; Inhibitors of Enhances of Zeste 2 (EZH2) - such as 3-deazaneplanocin A (DZNep) used alone or in combination with other classes of immunomodulatory compounds or purging agents.

Other suitable adjuvants include response-selective C5a agonists, such as EP54 and EP67 described in Hung CY et al . An agonist of human complement fragment C5a enhances vaccine immunity against Coccidioides infection. Vaccine (2012) and Kollessery G et al . Tumor-specific peptide based vaccines containing the conformationally biased, response- selective C5a agonists EP54 and EP67 protect against aggressive large B cell lymphoma in a syngeneic murine model . Vaccine (2011 ) 29: 5904-10.

Various methods of achieving adjuvant effect for the vaccine are thus known.

General principles and methods are detailed in "The Theory and Practical Application of Adjuvants", 1995, Duncan E.S. Stewart-Tull (ed. ), John Wiley & Sons Ltd, ISBN 0-471 - 95170-6, and also in "Vaccines: New Generationn Immunological Adjuvants", 1995,

Gregoriadis G et a/, (eds.), Plenum Press, New York, ISBN 0-306-45283-9, both of which are hereby incorporated by reference herein, but a number of later publications also deal with the technology of incorporating adjuvants: Roestenberg M et a/. , PLoS One. 2008;3( 12) : e3960. Epub 2008 Dec 18; Relyveld E and Chermann JC, Biomed Pharmacother. 1994;48(2) : 79-83; Hsu FJ et a/. , Blood . 1997 May l ;89(9) : 3129-35 ; Galli G et a/. , Proc Natl Acad Sci U S A. 2009 May 12; 106( 19) : 7962-7. Epub 2009 Apr 27; Bojang KA et a/. , Lancet. 2001 Dec 8; 358(9297) : 1927-34; Odunsi K et a/. , Proc Natl Acad Sci U S A. 2007 Jul

31 ; 104(31 ) : 12837-42. Epub 2007 Jul 25 ; Patel GB and Sprott GD; Crit Rev Biotechnol .

1999; 19(4) : 317-57. Review; Agger EM et a/. , PLoS One. 2008 Sep 8; 3(9) : e3116; Kirby DJ et a/. J Drug Target. 2008 May; 16(4) : 282-93; Florindo HF et a/. , Vaccine. 2008 Aug

5; 26(33) :4168-77. Epub 2008 Jun 17; Sun HX et a/. , Vaccine. 2009 May 28; Guy B, Nat Rev Microbiol . 2007 Jul ; 5(7) : 505-17. Review. ; Vandepapeliere P et a/. , Vaccine. 2008 Mar 4; 26(10) : 1375-86. Epub 2008 Jan 14; Ghochikyan A et al. Vaccine. 2006 Mar 20; 24( 13) : 2275-82. Epub 2005 Dec 5; Xie Y et al. , Vaccine. 2008 Jun 25; 26(27-28) : 3452- 60. Epub 2008 May 1 ; Chung YC et at. , Vaccine. 2008 Mar 28; 26( 15) : 1855-62. Epub 2008 Feb 25; Maier M et at. , Vaccine. 2005 Oct 25; 23(44) : 5149-59; Sundiing C et al. , J Gen Virol . 2008 Dec;89(Pt 12) : 2954-64.

In the methods of the invention the one or more peptide to stimulate a cell- mediated immune response and/or a compound to elicit a humoral response in a human, may be administered in combination with one or more further immunomodulatory compound and/or a reservoir purging agent, such as a histone deacetylase (HDAC) inhibitor.

The term "immunomodulating compound" or "reservoir purging agent" as used herein, includes but is not limited to cytokines, such as interferons, monoclonal antibodies, such as ant-PDl antibodies, cyclophosphamide, Thalidomide, Levamisole, and Lenalidomide.

"A reservoir purging agent", includes but is not limited to auranofin, IL-7, prostratin, bryostatin, HDAC inhibitors, such as vorinostat, and Disulfiram, and the further agents described herein.

The failure of antiretroviral therapy (ART) to eradicate HIV-1 infection lies in the observation that HIV- 1 remains quiescent in latent reservoirs. Latently infected resting CD4+ cells (either naive or long lived memory cells) carry transcriptionally silent HIV- 1 and represent the predominant reservoir of HIV- 1 infection. Other cells may also act as reservoirs (Reviewed in Alexaki et al., 2008, Curr. HIV Res. 6: 388-400), such as macrophages, dendritic cells and astrocytes (where HIV-1 infection occurs via a CD4-independent mechanism) . It is these latent reservoirs that represent the major challenge to eradication of HIV-1 infection. Approaches towards eradication include attempts to purge reservoirs by selective activation of latently infected cells (such as memory cells) in the presence of ART such that released virus may not infect and replicate in neighbouring cells (Richman et al., 2009, Science 323 : 1304-1307) . Agents include histone deacetylase inhibitors, cytokines, such as IL-2 and IL-7, as well as bryostatin, the protein kinase C activator (Kovochich et al ., 2011, PLoS ONE 6 (4) : el8270) . Therapeutic vaccines have the advantage of being able to penetrate sanctuary sites less well accessed by ART such as lymphoid tissue (Pantaleo et al ., 1991, Proc. Natl . Acad. Sci . USA 88: 9838-42; Fox et al ., 1991, J . Infect. Dis. 164: 1051-57) and the central nervous system (Alexaki et al ., 2008, Curr. HIV Res. 6 : 388-400), that represent regions for viral persistence. This relates to therapeutic interventions targeting both the virus itself as well as HIV-associated immune activation.

A number of studies have been conducted with the aim of providing compounds that can safely and effectively be used to treat diseases associated with abnormal production of TNF-a. See, e.g ., Marriott, J . B., et al, Expert Opin. Biol. Ther. (4) : 1-8 (2001 ) ; G.W. Muller, et al, Journal of Medicinal Chemistry, 39( 17) : 3238-3240 ( 1996) ; and G.W. Muller, et al, Bioorganic & Medicinal Chemistry Letters, 8: 2669-2674 ( 1998) . Some studies have focused on a group of compounds selected for their capacity to potently inhibit TNF-a production by LPS stimulated PBMC. L.G. Corral, et al, Ann . Rheum. Dis., 58 (suppl I) : 1107-1113 ( 1999) . These compounds, often referred to as immunomodulatory compounds, show not only potent inhibition of TNF-a, but also marked inhibition of LPS induced monocyte IL1B and IL12 production. LPS induced IL6 is also inhibited by immunomodulatory compounds, albeit partially. These compounds are potent stimulators of LPS induced IL10. Particular examples include, but are not limited to, the substituted 2-(2,6-dioxopiperidin-3-yl)phthalimides and substituted 2-(2,6-dioxopiperidin-3-yl)-l-oxoisoindoles as described in US 6281230 and US 6316471. Monocyte/macrophage function is part of the Innate Immune System that serves as a first line of defense against an infection. By modulating the host's monocytes and macrophages, immunomodulatory compounds can change the dynamics of the response to a viral infection, such as influenza.

Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups from N-acetylated lysines amino acid on histone proteins. Currently 18 HDACs have been identified in mammals. They have been divided into four classes based on cellular localization, function, and sequence similarity. Class I includes HDACs 1, 2, 3, and 8 which are found primarily in the nucleus. Class II HDACs (HDACs 4, 5, 6, 7 9, and 10) are found primarily in the cytoplasm but may be able to shuttle between the nucleus and the cytoplasm ; class Ila comprises four HDACs (HDACs 4, 5, 7 and 9) while class lib comprises two HDACs (HDACs 6 and 10) which are expressed only in the cytoplasm . HDAC11, which is ubiquitously expressed, shares sequence similarities with both class I and class II HDACs and represents Class IV. Class III (also called "sirtuin family") groups NAD+-dependent proteins which do not act primarily on histones.

The immunomodulatory compounds may be selected from anti-PDl antibodies, such as MDX-1106 (Merck), THALOMID ^* (thalidomide), anti-PDl antibodies, cyclophosphamide, Levamisole, lenalidomide, CC-4047 (pomalidomide), CC-11006 (Celgene), and CC-10015 (Celgene), and immunomodulatory compound described in any one of WO2007028047, WO2002059106, and WO2002094180. The immunomodulatory compound may be selected from 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-l,3-dione and 3-(4-amino-l-oxo-l,3- dihydro-isoindol-2-yl)-piperidine-2,6-dione. In particular the immunomodulatory compound is lenalidomide. The immunomodulatory compound may be enantiomerically pure.

The second reservoir purging agent , such as a histone deacetylase (HDAC) inhibitor, may be selected from M344 (4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide), chidamide (CS055/HBI-800) , 4SC-202, (4SC), Resminostat (4SC), hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824, trichostatin A and panobinostat (LBH589) ; benzamides such as entinostat (MS-275), CI994, and mocetinostat (MGCD0103), cyclic tetrapeptides (such as trapoxin, such as trapoxin B), and the depsipeptides, such as romidepsin (Istodax® (Celgene)), electrophilic ketones, and the aliphatic acid compounds such as phenylbutyrate, valproic acid, Oxamflatin, ITF2357 (generic givinostat), Apicidin, MC1293, CG05, and CG06; compounds that activate transcription factors including NF- KappaB, Prostratin, auranofin, bryostatin, a nontumorigenic phorbol ester, DPP (12- deoxyphorbol-13-phenylacetate), PMA, and Phorbol 12-myristate 13-acetate (PMA);

Compounds that activate HIV mRNA elongation including P-TEF-b kinase and

hexamethylbisacetamide (HMBA); IL-7; T-cell stimulating factors including anti-CD3/CD28 - T-cell stimulating Ab's; Kinase inhibitors including Tyrphostin A, Tyrphostin B, and Tyrphostin C;PTEN (phosphatase and tensin homologue) gene inhibitors including SF1670 (Echelon Bioscience), Disulfiram (DSF), an inhibitor of acetaldehyde dehydrogenase, Protein Tyrosine Phosphatase Inhibitors including bpV(HOpic), bpV(phen), and bpV(pic) (Calbiochem; EMD Millipore), Toll-like receptors agonists including Toll-like receptor-9 (TLR9) and Toll-like receptor- 7 (TLR9) agonists, quercetin, lipoic acid, sodium butyrate, TNF-alpha, PHA, Tat.

In the methods of the invention the components of the at least one HIV-specific peptide and/or the one or more further thereapeutically active agents, may be administered simultaneously, sequentially or separately in any order.

Thus the invention provides a pharmaceutical composition comprising one, two or more components of the at least one HIV-specific peptide and/or the one or more further thereapeutically active agents optionally in combination with one or more pharmaceutically acceptable adjuvants, diluents or carriers.

Similarly, the invention also provides a combination product comprising of components of the at least one HIV-specific peptide and/or the one or more further thereapeutically active agents, wherein each of component may be formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier. In this aspect of the invention, the combination product may be either a single (combination) pharmaceutical formulation or a kit-of-parts. In a kit-of-parts some or all of the components may be formulated separately and may each be provided in a form that is suitable for administration in conjunction with the other(s).

The component(s) may also be provided for use, e.g. with instructions for use, in combination with one or more further component(s) as defined above.

The peptides for use in the invention may be produced synthetically using art recognised methods. Further details for the synthetic production of such peptides are found in the Examples. Alternatively the peptides may be produced recombinantly. When recombinantly producing the peptides for use in the invention by means of transformed cells, it is convenient, although far from essential, that the expression product is either exported out into the culture medium or carried on the surface of the transformed cell.

When an effective producer cell has been identified it is preferred, on the basis thereof, to establish a stable cell line which carries the vector of the invention and which expresses the nucleic acid fragment of the invention. Preferably, this stable cell line secretes or carries the peptide expression product, thereby facilitating purification thereof.

In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with the hosts. The vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (see, e.g ., Bolivar et a/. , 1977). The pBR322 plasmid contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, promoters which can be used by the prokaryotic microorganism for expression.

Those promoters most commonly used in recombinant DNA construction include the β-lactamase (penicillinase) and lactose promoter systems (Chang et a/. , 1978; Itakura et a/. , 1977; Goeddel et a/. , 1979) and a tryptophan (trp) promoter system (Goeddel et a/. , 1979; EP-A-0 036 776) . While these are the most commonly used, other microbial promoters have been discovered and utilized, and details concerning their nucleotide sequences have been published .

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures may also be used, and also here the promoter should be capable of driving expression. Saccharomyces cerevisiase, or common baker's yeast is the most commonly used among eukaryotic microorganisms, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et a/. , 1979; ingsman et a/. , 1979; Tschemper et a/. , 1980) .

Suitable promoting sequences in yeast vectors include the promoters for 3- phosphoglycerate kinase (Hitzman et a/. , 1980) or other glycolytic enzymes (Hess et a/. , 1968; Holland et a/. , 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In constructing suitable expression plasmids, the termination sequences associated with these genes are also incorporated into the expression vector 3' of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination .

Other promoters, which have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing a yeast-compatible promoter, origin of replication and termination sequences is suitable.

In addition to microorganisms, cultures of cells derived from multicellular organisms may also be used as hosts. In principle, any such cell culture is workable, whether from vertebrate or invertebrate culture. Examples of such useful host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, and W138, PER.C6, BHK, COS-7 293, Spodoptera frugiperda (SF) cells, Drosophila melanogaster cell lines (such as Schneider 2 (S₂)), and MDCK cell lines.

Expression vectors for such cells ordinarily include (if necessary) an origin of replication, a promoter located in front of the gene to be expressed, along with any necessary ribosome binding sites, RNA splice sites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expression vectors are often provided by viral material . For example, commonly used promoters are derived from polyoma, Adenovirus 2, and most frequently Simian Virus 40 (SV40) . The early and late promoters of SV40 virus are particularly useful because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of replication (Fiers et al. ,

1978) . Smaller or larger SV40 fragments may also be used, provided there is included the approximately 250 bp sequence extending from the Hlndlll site toward the Bgll site located in the viral origin of replication. Further, it is also possible, and often desirable, to utilize promoter or control sequences normally associated with the desired gene sequence, provided such control sequences are compatible with the host cell systems.

An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral (e.g ., other Polyoma viruses, Adeno, VSV, BPV) or may be provided by the host cell chromosomal re- plication mechanism. If the vector is integrated into the host cell chromosome, the latter is often sufficient.

As for routes of administration and administration schemes of polypeptide based vaccines which have been detailed above, these are also applicable for the nucleic acid vaccines of the invention and all discussions above pertaining to routes of administration and administration schemes for polypeptides apply mutatis mutandis to nucleic acids. To this should be added that nucleic acid vaccines can also be administered intraveneously and intraarterially. Furthermore, it is well-known in the art that nucleic acid vaccines can be administered by use of a so-called gene gun and/or by use of electroporation, and hence also these and equivalent modes of administration are regarded as part of the present invention.

Under normal circumstances, the nucleic acid fragment is introduced in the form of a vector wherein expression is under control of a viral promoter. For more detailed discussions of vectors according to the invention, cf. the discussion above. Also, detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, cf. Donnelly JJ et a/, 1997, Annu. Rev. Immunol . 15: 617-648 and Donnelly JJ et a/. , 1997, Life Sciences 60: 163- 172. Both of these references are incorporated by reference herein.

An alternative of using peptide immunogens or nucleic acid immunogens is the use of live immunogen technology. This entails administering a non-pathogenic microorganism which has been transformed with a nucleic acid fragment or a vector of the present invention. The non-pathogenic microorganism can be any suitable attenuated bacterial strain (atten- uated by means of passaging or by means of removal of pathogenic expression products by recombinant DNA technology), e.g. Mycobacterium bovis BCG., non-pathogenic

Streptococcus spp., E. coli, Salmonella spp., Vibrio cholerae, Shigella, etc. Reviews dealing with preparation of state-of-the-art live vaccines can e.g . be found in Saliou P, 1995, Rev. Prat. 45: 1492-1496 and Walker PD, 1992, Vaccine 10: 977-990, both incorporated by reference herein. For details about the nucleic acid fragments and vectors used in such live vaccines, cf. the discussion below.

As an alternative to bacterial live immunogens, the nucleic acid fragment of the invention can be incorporated in a non-virulent viral vaccine vector such as a vaccinia strain or any other suitable poxvirus.

Normally, the non-pathogenic microorganism or virus is administered only once to a subject, but in certain cases it may be necessary to administer the microorganism/virus more than once in a lifetime in order to maintain protective immunity. It is even contemplated that immunization schemes as those detailed above for polypeptide vaccination will be useful when using live or virus vaccines.

Alternatively, live or virus immunization is combined with previous or subsequent polypeptide and/or nucleic acid immunization. For instance, it is possible to effect primary immunization with a live or virus vaccine followed by subsequent booster immunizations using the polypeptide or nucleic acid approach.

Specific embodiments of the invention

The present invention relates in a broad aspect to a method for inducing an immune response to a disease antigen, such as an infectious agent in an subject, the method comprising :

In some embodiments the second vaccine is administered after the administering of said first vaccine. In some embodiments the second vaccine is administered simultaneously with the administering of said first vaccine.

In some embodiments the first vaccine composition comprises an adjuvant, such as recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) or such as Alhydrogel .

In some embodiments the second vaccine composition comprises an adjuvant, such as recombinant human granulocyte- macrophage colony-stimulating factor (rhuGM-CSF) or such as or ISA51.

In some embodiments the first and second antigen is derived from the same compound, such as a protein of said infectious agent, such as a virus.

In some embodiments the first and/or second disease antigen is an immunogen. In some embodiments the method is a treatment.

In some embodiments the first and/or second antigen is protein, DNA, or RNA.

In some embodiments the first and/or said second antigen is derived from a peptide, said peptide comprising a specific natural antigen of a protein or peptide sequence derived from a disease antigen, such as an infectious agent, such as bacteria, virus, parasite, fungus, or cancer antigens such as oncogene (lung, stomach, breast cancer) or an antigen causing an autoimmune disease such as diabetes, multiple sclerosis (MS), celiac disease, Myalgic Encephalomyelitis (ME), psoriasis, and/or Crohn's Disease.

In some embodiments the specific natural antigen is a viral protein, such as a structural protein, such as a capsid protein, a regulatory protein, an enzymatic protein, and a proteolytic protein .

In some embodiments the viral protein is selected from a core protein or an envelope protein, of a virus selected from the Hepatitis C virus, influenza virus human immunodeficiency virus (HIV), cytomegalovirus (CMV), Respiratory syncytial virus (RSV), and Human papillomavirus (HPV) .

In some embodiments the viral protein is a viral protein of Hepatitis C virus selected from any one HCV consensus sequence of a specific genotype, such as 1, such as subtypes la and lb, genotype 2, such as 2a and 2b, genotype 3, such as 3a, genotype 4, genotype 5, and genotype 6.

In some embodiments the first and second antigens are derived from HIV.

EXAMPLES

PREAMBLE TO EXAMPLES Overview sequences and abbreviations: C5-sequences:

APTKAKRRVVQREKRAV (SEQ ID NO: 1)

APTKAKRRVVEREKRAV (SEQ ID NO: 2)

APTRAKRRVVQREKRAV (SEQ ID NO: 3)

APTRAKRRVVEREKRAV (SEQ ID NO: 4)

APTEAKRRVVEREKRAV (SEQ ID NO: 5)

WWGCAKRRVCGGAKRRVVQREKRA (SEQ ID NO: 44)

(underlined amino acid residues in SEQ ID NO: 44 are linked via a disulphide linker; the N- terminal W is preferably a D-amino acid and the C-terminal A may be amidated; the peptide is termed BI450-AdjBT 1, when having these two modifications).

C5-complex forming sequences:

DRPEGIEEEGGERDR (where amino acid 4 can be G and/or where amino acid 5 can be R and/or where amino acid 13 can be Q and/or where amino acid 14 can be G and/or where amino acid 15 can be K; SEQ ID NO: 6);

DRPEGIENNGGERDR (SEQ ID NO: 7 where amino acid 4 can be G and/or where amino acid 5 can be R and/or where amino acid 13 can be Q and/or where amino acid 14 can be G and/or where amino acid 15 can be K);

DRPEGIENNGGERDRDR (where amino acid 4 can be G and/or where amino acid 5 can be R and/or where amino acid 13 can be Q and/or where amino acid 14 can be G and/or where amino acid 15 can be K and/or where amino acid 16 can be G); SEQ ID NO: 46).

VERYLKDQQLLG (SEQ ID NO: 8);

VERYLKDEELLG (SEQ ID NO: 9);

VERYLKDNNLLG (SEQ ID NO: 10);

QLLLNGSLAEEEIVI (SEQ ID NO: 11 )

QLLLNGSLAEEEVVIV (SEQ ID NO: 12)

QLLLNSLAEEEVVI (SEQ ID NO: 13)

GGAIVNGSLADDDIVI (SEQ ID NO: 37, also termed 204d herein)

WWGCJEEEGCGGIEEEGGERDR (SEQ ID NO: 45: underlined amino acid residues are linked via a disulphide linker; the N-terminal W is preferably a D-amino acid and the C-terminal R may be amidated; the peptide is termed BI450-AdjBT 2, when having these two

modifications).

Polypeptides I: (Z-SEQ _c5-Z-SEQ_c5)_n

n= l, 2,3,4

Polypeptides II:

(Z-SEQ _cx-Z-SEQ_cx)_n

n = l, 2,3,4

Peptide complexes:

(Z-SEQ_c5-Z-SEQ_c5)_n

I

Bis-maleimide linker

I

(Z-SEQ_cx-Z-SEQ_cx)_n

n = l, 2,3,4

(Z-SEQ_c5-Z-SEQ_c5)_n

I

(Z - SEQ_cx-Z-SEQ_cx)_n

n = l, 2,3,4

Examples of polypeptides I can be, but are not restricted to, the following sequences:

APTKAKRGGGAPTRAKRGGGAPTEAKPv (SEQ ID NO: 14)

RVVEREKGGGAKRRVVGGGRVVQREK (SEQ ID NO: 15)

GGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 16)

CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 17)

GGAKRRVVGGAKRRVVGGQREKR (SEQ ID NO: 18)

CGGAKRRVVGGAKRRVVGGQREKR (SEQ ID NO: 19)

GGAKRRWGGAKRRVV (SEQ ID NO: 20)

GCGAKRRVVGGAKRRW (SEQ ID NO: 21)

Examples of polypeptides II can be, but are not restricted to, the following sequences:

GGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO: 22)

CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO: 23)

GGDQQLLGGAEEEIVGGGERDR (SEQ ID NO: 24)

CGGGDQQLLGGAEEEIVGGIEEEGG (SEQ ID NO: 25)

GGAEEEVVGGDQQLL (SEQ ID NO: 26) CGGAEEEVVGGDQQLL (SEQ ID NO: 27)

Examples of disulfide linked constructs can be, but are not restricted to, the following linked peptide sequences:

CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 28)

I

CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO : 29)

CGGAKRRVVGGAKRRVVGGQREKR (SEQ ID NO: 30)

I

CGGGDQQLLGGAEEEIVGGIEEEGG (SEQ ID NO: 31) CGGAEEEVVGGDQQLL (SEQ ID NO: 32)

I

GCGGAKRRVVGGAKRRVV (SEQ ID NO: 33)

The above disulfide linked constructs may e.g . be synthesised by titration of 2- pyridinesulfenyl (SPyr)-protected cysteine-containing peptides with thiol-unprotected peptides. This has proven to be a superior procedure to selectively generate disulfide-linked peptide heterodimers preventing the formation of homodimers (Schutz A et al. , Tetrahedron, Volume 56, Issue 24, 9 June 2000, Pages 3889-3891) . Similar constructs where SEQ ID NO: 28 is disulphide linked to SEQ ID NOs 31 or 33, or where SEQ ID NO: 30 is disulphide linked to SEQ ID NOs: 29 or 33, or where SEQ ID NO: 32 is disulphide linked to SEQ ID NOs: 29 or 31 are also within the scope of the present invention .

Examples of other linked constructs can be, but are not restricted to, the following linked peptide sequences, which have all been obtained from Bachem (UK) Ltd :

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)

I GKGGIEEEGGRDRDRGGEQDRDR (SEQ ID NO: 39)

(the peptides are linked via the underlined Cys and Lys residues; the entire construct is termed BI400-B herein) .

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)

I GKGGIEEEGGERDRDRGGQDRDR (SEQ ID NO: 40)

(the peptides are linked via the underlined Cys and Lys residues; the entire construct is termed BI400-Bul herein) .

GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO: 41 ) GKGGIEEEGGQDRDRGGRDRDR (SEQ ID NO: 42)

(the peptides are linked via the underlined Cys and Lys residues; the entire construct is termed BI400-Bu2 herein) . GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO: 41)

GKGGIEEEGGEQDRDRGGERDRD (SEQ ID NO: 43)

(the peptides are linked via the underlined Cys and Lys residues; the entire construct is termed BI400-Bu3 herein) . The Cys-Lys linker is typically established in the form of an amide bond between (2-oxo- ethyl) derivatized cysteine in one peptide and lysine in the other peptide.

Similar constructs where SEQ ID NO: 38 is Cys-Lys linked to SEQ ID NOs 42 or 43, or where SEQ ID NO: 41 is Cys-Lys linked to SEQ ID NOs: 39 or 40 are also within the scope of the present invention. Small molecule inhibitors:

DQQLL (SEQ ID NO: 34)

AKRRVV (SEQ ID NO: 35)

AEEEVV (SEQ ID NO: 36)

SEQ ID NOs 34-36 are preferably composed partly or completely of D-amino acids. One preferred agent capable of stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 is a compound of the following structure:

(H-Glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-glycyl-glycyl-L-cysteinyl( 2- oxo-ethyl)-glycyl-glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-L-glutaminyl-L- arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-glycyl-L-glutamyl-L-arginyl-L-glutamyl-L-lysyl-L- arginyl-L-alanyl-NH₂) (H-Glycyl-L-lysyl-glycyl-glycyl-L-isoleucyl-L-glutamyl-L-glutamyl-L- glutamyl-glycyl-glycyl-L-arginyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-glycyl-glycyl-L- glutaminyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-NH₂), acetate salt (amide bond between Cys(2-oxo-ethyl) ⁱ⁰ (A-chain) and Lys² (B-chain)) This compound may also be referred to as Vacc-C5 with the following formula: (H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys( 2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val- Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH₂) (H-Gly-Lys-Gly-Gly-Ile-Glu-Glu- Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH₂), acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) and Lys² (B-chain))

This preferred C5 compound consists of two linear peptide amide chains with 31 amino acids (A-chain) and 22 amino acids (B-chain) . Each chain has a free amino group at the

N-terminus and an amide group at the C-terminus. The chains are covalently linked via an amide bond between Cys(2-oxo-ethyl)¹⁰ of the A-chain and Lys² of the B-chain. All amino acid residues except the achiral Gly are in the L-configuration.

H-Gly-Ala-Lys-Arg-Arg-Val-Va!-G!y-G!y-Cys-Gly-Gly-Ala-Lys-Arg-Arg-Val-Va!-G!n-Arg-G!u ys-Arg-A!a-G!y-G!u-A

H-Giy-Lys-Gly-Gly-!le-G!u-Glu-Glu-G!y-G!y-Arg-Asp-Arg-Asp-Arg-Gly-Gly-G]n-Asp-Arg-Asp-Arg-NH₂

The preferred C5 compound may be provided as an acetate salt. The counter ion acetate is bound in ionic form to basic groups of the peptide molecule.

HIV-SPECIFIC PEPTIDES ACCORDING TO THE INVENTION

The present invention relates to agents capable of stabilising the association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 in combination with peptides based on conserved regions of HIV gag p24, antigens in free or carrier-bound form comprising at least one of the said peptides.

The HIV-specific peptides according to the invention are originating from the four different conserved areas of the HIV-1 core protein p24 which are described above, having the properties of maintaining the uniqueness (sensitivity and specificity) of the HIV-l-epitope. Further the new peptides according to the invention possess no recognized cytotoxic T lymphocyte (CTL) antagonistic effect and shall have at least one potential CTL epitope. The HIV-specific peptides, according to the invention, which have met the above criteria are selected from the following groups;

Xaai Xaa₂ Xaa₃ Xaa₄ Xaa_s Xaa₆ Ala Xaa₈ Xaa₉ Gin Thr Pro Trp Xaa_i4 Xaa, ₅ Xaa_i6 Xaa_{i 7} Xaa₁₈ Val Xaa₂₀ (SEQ ID NO:47) ; Wherein Xaa in position 1 of the peptide derivate is Lys or Arg,

Xaa in position 2 is Ala, Gly, Ser or Arg,

Xaa in position 3 is Leu or Met,

Xaa in position 4 is Gly or Arg,

Xaa in position 5 is Pro, Thr, Val, Ser, Gin or Ala,

Xaa in position 6 is Gly, Ala, Lys, Arg, Gin or Glu,

Xaa in position 8 is Thr or Ser,

Xaa in position 9 is Leu or He ,

Xaa in position 14 is Thr, Ser or Val,

Xaa in position 15 is Ala or Ser,

Xaa in position 16 is Cys or Ser,

Xaa in position 17 is Gin or Leu

Xaa in position 18 is Gly, Glu or Arg, and

Xaa in position 20 is Gly or Arg; Xaa_: Xaa₂ Xaa₃ Xaa₄ Xaa₅ Gly Leu Asn Pro Leu Val [Gly]., Xaa-_{i 2} Xaa_{i 3} Tyr Xaa_{i 5} Pro Xaa_{i 7} Xaa_i8 He Leu Xaa₂₁ Xaa₂₂ (SEQ ID NO: 50) wherein Xaa in position 1 is Arg, Lys, Asp or none

Xaa in position 2 is Trp, Gly, Lys or Arg,

Xaa in position 3 is He, Leu, Val or Met

Xaa in position 4 is He, Val or Leu

Xaa in position 5 Leu, Met, Val or Pro

Xaa in position 12 is Arg, Lys

Xaa in postion 13 is Met or Leu,

Xaa in position 15 is Ser, Cys or Gin,

Xaa in position 17 is Thr, Val, He, Ser or Ala,

Xaa in position 18 is Ser, Gly or Thr,

Xaa in position 21 is Asp, Glu, Cys or Gly,

Xaa in position 22 is Gly or none, and

n = 0, 1,2 or 3; Xaai Xaa₂ Xaa₃ Pro He Pro Xaa₇ Xaa₈ Xaa₉ Xaaic Xaau Xaai ₂ [Gly]_n Xaa_{i 3} Xaa_i4 Xaa^ Xaaie Xaa, 7 Xaais Xaa_i9 Xaa₂₀ Xaa₂i Xaa₂₂ Xaa₂₃ Xaa₂₄ (SEQ ID NO: 55) wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or none

Xaa in position 2 is Asn, Ala or Lys

Xaa in position 3 is Pro, Gin, Gly, He or Leu

Xaa in position 7 is Vai or Ala

Xaa in position 8 is Gly or Lys

Xaa in position 9 is Glu, Asp, Lys, Phe or Thr

Xaa in position 10 is He, Met, Val or Leu

Xaa in position 11 is Tyr, Leu or none

Xaa in position 12 is Ser or none

Xaa in position 13 is Arg or none

Xaa in position 14 is Asp, Arg, Trp, Ala or none

Xaa in position 15 is He or none

Xaa in position 16 is Tyr or none

Xaa in position 17 is Lys or Arg

Xaa in position 18 is Arg, Lys or Asp

Xaa in position 19 is Trp or Gly

Xaa in position 20 is He, Met, Val, Gin or Ala

Xaa in position 21 is He, Val or Ala

Xaa in position 22 is Leu, Met or Val

Xaa in position 23 is Gly or Cys

Xaa in position 24 is Leu or none,

n = 1,2 or 3, and

Xaaj Xaa₂ He He Xaa₅ Xaa₆ Xaa₇ Xaa_s Xaa₉ Leu Xaa_n [Gly]_n [Arg],,, Xaa₁₂ Xaa₁₃ Xaa_i Xaa₁₅ Xaa_{i 6} Xaa _/ Xaa, ₈ Xaa_{i 9} Xaa₂₀ Xaa_2i Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ (SEQ ID NO: 61) wherein the Xaa in position 1 is Pro, Lys, Arg or none

Xaa in position 2 is Glu, Arg, Phe or Lys

Xaa in position 5 is Pro or Thr

Xaa in position 6 is Met, Thr or Nleu

Xaa in position 7 is Phe or Leu

Xaa in position 8 is Ser, Thr, Ala or Met

Xaa in position 9 is Ala, Glu or Leu

Xaa in position 11 is Ser or none

Xaa in position 12 is Ala, Arg or none Xaa in position 13 is He, Leu or none

Xaa in position 14 is Ser, Ala, Leu or none

Xaa in position 15 is Tyr, Glu or Asp

Xaa in position 16 is Gly or Asp

Xaa in position 17 is Ala or Leu

Xaa in position 18 is Thr, He, Val, Leu or Asn,

Xaa in position 19 is Pro, Thr or Ser

Xaa in position 20 is Tyr, Phe, Nleu, His or Gin

Xaa in position 21 is Asp, Asn, Leu or Ala

Xaa in position 22 is Leu, He, Val or Asn

Xaa in position 23 is Asn, Tyr, Cys or Gly

Xaa in position 24 is Thr, Met, He, Ala, Val or none

Xaa in postion 25 is Gly or none

The HIV-specific peptide sequences have the potential to serve as a good antigen wherein the antigen comprises at least one peptide selected from the group of sequences of SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 55 or SEQ ID NO: 61 . The antigenicity may be adapted through adjusting the ratio or concentration of different peptides or size of the peptides by for instance dimerisation or polymerisation and/or immobilisation to a solid phase. The antigen comprises two or more polypeptide sequences, according to the invention, which are either linked by a bridge for instance a disulphide bridge between the Cys residues of the chains or bridges like Q-Cs alkylen possibly intervened by one or more heteroatoms like O, S, or N or preferably they are unlinked . The chains may be immobilized to a solid phase in monomeric, dimeric or oligomeric forms. Further amino acids may be added to the ends in order to achieve an «arm» to facilitate immobilization.

All amino acids in the HIV-specific peptides of the invention can be in both D- or L-form, although the naturally occurring L- form is preferred .

The C- and N-terminal ends of the HIV-specific peptide sequences could deviate from the natural sequences by modification of the terminal NH₂-group and/or COOH-group, they may for instance be acylated, acetylated, amidated or modified to provide a binding site for a carrier or another molecule. The HIV-specific peptides according to the invention are consisting of 6 to 50 amino acids, preferably between 10 and 30 amino acids. They are covering all natural variation of amino acids in the identified positions.

The polypeptide antigen according to the invention is either in a free or in a carrier-bound form. The carrier or solid phase to which the peptide is optionally bound can be selected from a wide variety of known carriers. It should be selected with regard to the intended use of the immobilized polypeptide as a diagnostic antigen or as an immunizing component in a vaccine.

Examples of carriers that can be used for e.g. diagnostic purposes are magnetic beads or latex of co-polymers such as styrene-divinyl benzene, hydroxylated styrene-divinyl benzene, polystyrene, carboxylated polystyrene, beads of carbon black, non-activated or polystyrene or polyvinyl chloride activated glass, epoxy-activated porous magnetic glass , gelatine or polysaccharide particles or other protein particles, red blood cells, mono- or polyclonal antibodies or fab fragments of such antibodies.

In a preferred embodiment of the vaccine according to the present invention it comprises antigens containing the peptides of the SEQ ID NO : 1, 4, 9 and 15, more preferred the peptides occur in the ratio 1 : 1 : 1 : 1 w/w.

RALG PAATLQTPWTAS LG VG - H ₂ (SEQ ID NO: 49)

RWLLLGLNPLVGGGRLYSPTSILG-NH₂ (SEQ ID NO: 52)

RAIPIPAGTLLSGGGRAIYKRTAILG-NH2 (SEQ ID NO: 57)

and

RFIIPNIFTALSGGRRALLYGATPYAIG- NH2 (SEQ ID NO: 64). (Nl in position 6 is Norleucine).

One of the sequences contains a B-cell epitope and will activate the humoral immune system, whereas the other sequences contribute with CTL-epitopes and the amino acid changes implemented within the frame of the CTL-epitope are designed to achieve enhanced binding. Other amino acid changes have been conducted in order to facilitate the synthesis of the peptide and/or increase the solubility of the peptide.

EXAMPLE 1

Synthesis of peptides using conventional techniques for linear sequences Preparation of APTKAKRRVVQREKR

The peptide was synthesized in amide form, from the corresponding starting point according to the general description of F-moc synthesis (Atherton et al . 1978 J . Chem. Soc. Chem Commun 539), which is beiow termed "the general description of synthesis.

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1822.2

Experimental molecular weight: 1823.0 ES+

Preparation of APTKAKR

The peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 769.6

Experimental molecular weight: 760.7 ES+

Preparation of APTRAKR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 797.6

Experimental molecular weight: 797.6 ES+ Preparation of APTEAKR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 770.9

Experimental molecular weight: 770.9 ES+

Preparation of RVVEREK

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 914.1

Experimental molecular weight: 913.9 ES+

Preparation of RVVQREK

The peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis. Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 913.1

Experimental molecular weight: 913.0 ES+

Preparation of AKRRVV

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 726.9

Experimental molecular weight: 726.9 ES+ Preparation of DRPEGIEEEGGERDR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1742.1

Experimental molecular weight: 1742.8

Preparation of VERYLKDQQLLG

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1460.7

Experimental molecular weight: 1460.1

Preparation of VERYLKDEELLG

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1462.6

Experimental molecular weight: 1463.0

Preparation of VERYLKDNNLLG

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1432.6

Preparation of QLLLNGSLAEEEIVI The peptide was synthesized in amide form, from the corresponding starting point according to the general description of synthesis.

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1639.9 Preparation of QLLLNGSLAEEEWI

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1625.9 Preparation of QLLLNSLAEEEVVI

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1568.8 Preparation of APTKAKRGGGAPTRAKRGGGAPTEAKR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2647.0

Experimental molecular weight: 2646.3 ES+

Preparation of RVVEREKGGGAKRRVVGGGRVVQREK

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2862.3

Experimental molecular weight: 2863.3 ES+

Preparation of GGAKRRVVGGAKRRVVGQREKRAV

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2590.1 Preparation of CGGAKRRVVGGAKRRVVGQREKRAV

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2693.2

Preparation of GGAKRRVVGGAKRRVVGGQREKR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2476.9

Preparation of CGGAKRRVVGGAKRRVVGGQREKR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2580.0

Preparation of GGAKRRVVGGAKRRVV

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1665.0

Preparation of GCGAKRRVVGGAKRRVV

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1768.1

Preparation of GGGDQQLLGGAEEEIVGGIEEEGGERDRDR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 3127.2

Preparation of CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 3230.4

Preparation of GGDQQLLGGAEEEIVGGGERD

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2242.4

Preparation of CGGGDQQLLGGAEEEIVGGIEEEGG

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 2402.5

Preparation of GGAEEEVVGGDQQLL

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1499.6

Preparation of CGGAEEEVVGGDQQLL

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 1602.7 EXAMPLE 2: Synthesis of complexed peptides

Preparation of CGGAKRRVVGGA RRVVGQREKRAV

I

CGGGDQQLLGGAEEEIVGGIEEEGGERDRD

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 5750.4

Experimental molecular weight: Preparation of CGGAKRRVVGGAKRRVVGGQREKR

I

CGGGDQQLLGGAEEEIVGGIEEEGG Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 4965.6

Experimental molecular weight:

Preparation of CGGAEEEVVGGDQQLL

I

GCGGAKRRVVGGAKRRVV

Purity (HPLC) : more than 90 %.

Mass spectral analysis: Theoretical molecular weight: 3410.9

Experimental molecular weight:

EXAMPLE 3

Recognition of SEQ ID NO: 1 alone and in combination with SEQ ID NOs: 6, 8 and 9 by pooled human sera from HIV chronically infected individuals, LTNP and non-infected blood donors

Seroreactivity to SEQ ID NO: 1 alone or in combination with SEQ ID NOs. 6 (with the sequence DRPEGIEEEGGERDR), 8 and 9 was determined according to a general ELISA principle either using magnetic particles as a solid support or attachment of peptides to a 96- well tray.

Methods:

In the system described below, peptide was coated on to magnetic particles using generally accepted techniques. 300 pg was coated onto particles for all peptides with the exception of SEQl where 600 \iq was used. SEQ ID NO: 1 (from C5) and SEQ ID NO: 6, 8 and 9 (from gp41) were preincubated overnight at 4 degrees C to allow interactions to form between between C5 and gp41 sequences respectively and all combined . Sera were then incubated with the peptide coated beads according to established protocols. Visualisation of antibody binding to C5 peptides was achieved using protein G that can bind immunoglobulins from different species coupled to alkaline phosphatase. The positive control was commercially available serum from a sheep immunised with the C5 derived sequence APTKAKRRVVQREKR (SEQ ID NO: 1 ). Pooled sera from 25 LTNP were tested for seroreactivity to SEQ ID NO: 1 alone and SEQ ID NO : 1 when in combination with SEQ ID NOs: 6 (DRPEGIEEEGGERDR), 8 and 9 respectively and all combined. Pooled sera were also tested from 12 HIV positive, chronically infected individuals and 20 sera from blood donors. The results are shown in Table A: Table A: Results of seroreactivity of pooled sera to SEQ ID NO: 1 and SEQ ID NO: 1 combined with sequences to gp41. Positivity is determined visually.

Results/ Discussion points: The results in Table A show that pooled LTNP sera generally provide strong reactivity to SEQ ID NO: 1 from HIV-1 when compared to pooled sera from patients chronically infected with HIV - this has been reported previously. However, combining SEQ ID NO: 1 with other peptides derived from gp41 (e.g . all combined or only SEQ ID NO: 8) reduced the level of background observed in blood donors as well as responses in pooled sera from chronically infected individuals. The response in LTNP remains strong . EXAMPLE 4

Recognition of SEQ ID NO: 1 and SEQ ID NO: 1 in combination with SEQ ID NO: 6 by individual human sera from HIV chronically infected, LTNP, Blood donors

Seroreactivities of individual LTNP patient sera to SEQ ID NO: 1 ( 16 pg) alone and in combination with SEQ ID NO: 6 ( 16 pg ) were determined using an ELISA plate as a solid support. Sheep anti-C5 antibodies were used as a positive control . Optical density (OD) at 280 nm was used as a read out following the enzymatic reaction from protein G coupled to alkaline phosphatase.

Results have shown that a greater proportion of LTNP sera (n=8, 2 most right-hand, grey bars) have reactivity to SEQ ID NO: 1 when it is in combination (i .e. >80% ratio) with SEQ ID NO: 6 (DRPEGIEEEGGERDR) when compared to the reactivity against SEQ ID NO: 1 alone (n =6, most left-hand hatched bar, <0% ratio) . For LTNP sera that only had a low response to SEQ ID NO: 1, this effect was enhanced when SEQ ID NOs: 1 and 6 were combined. This demonstrates that the C5 : gp41 complex has the ability to capture and increase the response dramatically, even when the response from C5 alone is low; Results have shown that the OD was high in the serum samples that only showed binding to SEQ ID NO: 1. However, when combined with the gp41 sequence, the responses to C5 alone were reduced since antibodies now preferably bound the combination.

Data has been made showing the magnitude of responses in individual sera from LTNP, chronically infected patients and blood donors to SEQ ID NO: 1 alone and SEQ ID NO: 1 in combination with SEQ ID NO: 6 (DRPEGIEEEGGERDR) . There was a greater response to SEQ ID NO: 1 alone and SEQ ID NO: 1 combined with SEQ ID NO: 6 (DRPEGIEEEGGERDR) .

among LTNP patients compared to patients chronically infected with HIV. The median OD value for binding to SEQ ID NO: 1 and SEQ ID NO: 6 in combination is higher than binding to SEQ ID NO: 1 alone for both LTNP and patients chronically infected with HIV, showing that combination with SEQ ID NO: 6 improved seroreactivity. Responses in blood donors are consistently low, there is a very tight interquartile range and no difference in seroreactivity to C5 alone or in combination with SEQ ID NO: 6 (DRPEGIEEEGGERDR) in this negative control .

A Wilcoxon rank-test performed on the OD-values derived from SEQ ID NOs: 1 and 6 combined on LTNP-sera and the OD-values derived from SEQ ID NOs: 1 and 6 combined on HIV-sera, gives that the true median differs within a 25 % confidence-interval . EXAMPLE 5

Immunological studies Rabbit immunizations

New Zealand White female rabbits (n=3) were immunized intradermally at weeks 0, 2 & 6 with 1 ml of BI400-B vaccine consisting of 500 pg BI400-B in 50% V/V Freund ^' s adjuvant (i.e. Complete Freund ^' s adjuvant used for priming, followed by boostings with Incomplete Freund ^' s adjuvant). Individual blood serum was isolated for ELISA.

Direct ELISA for human sera

50-100 μΙ of a mixture of BI400-015 and -201 (pre-incubated in Coating buffer - 0.05M Na₂C0₃ pH9.6; denoted CB - in cold at 16 pg/ml for each peptide 1-3 days prior to coating) or just CB (background control) was used for coating wells in microtiter plates at 4°C overnight. The microtiter plates were then washed 3x with washing buffer (PBS + 1% v/v Triton-X100; denoted WB), followed by 2h blocking at room temperature (RT) with 200 μΙ/well of blocking buffer (PBS + 1% w/v BSA). Plates were then washed 3x with WB, followed by lh incubation at 37°C with 50-70 ul/well of added human (serial dilutions ranging from 1 : 1 - 1 : 250 in dilution buffer (PBS + 1% v/v Triton-X100 + 1% w/v BSA; denoted DB)). Plates were then washed 6x with WB, followed by lh incubation at RT with 70 μΙ/well of Alkaline Phosphatase- conjugated Protein G (3pg/ml in DB; Calbiochem 539305). Plates were then washed 6x with WB, followed by 10-60 min incubation at room temperature with 100 μΙ/well of 0.3% w/v of Phenophtalein monophosphate (Sigma P-5758). Plates were finally quenched by adding 100 μΙ/well of Quench solution (0.1M TRIS + 0.1M EDTA + 0.5M NaOH + 0.01% w/v NaN₃;

pH14), followed by ELISA reader (ASYS UVM 340) at 550 nm.

Competitive ELISA for rabbit sera after immunization with BI400-B

50-100 μΙ of a mixture of BI400-015 and -201 (pre-incubated in Coating buffer - 0.05M Na₂C0₃ pH9.6; denoted CB - in cold at 16 pg/ml for each peptide 1-3 days prior to coating) or just CB (background control) was used for coating wells in microtiter plates at 4°C overnight. Plates were then washed 3x with washing buffer (PBS + 1% v/v Triton-X100; denoted WB), followed by 2h blocking at room temperature (RT) with 200 μΙ/well of blocking buffer (PBS + 1% w/v BSA). Plates were then washed 3x with WB, followed by lh incubation at 37°C with 60-100 μΙ/well of added rabbit serum samples (diluted 1 : 10 - 1 : 250 final concentration) preincubated together (4°C overnight) with serial dilutions (ranging from 10-1000 μΜ final concentration) of 400-SEQ.B, BI400-015, BI400-201, BI400-204d, recombinant gp41 (Shin- Won Scientific, SWO 102 gp41), BI301-23 (irrelevant protein; control), no peptide (i.e. PBS; control), LTNP-sera pools (diluted 1 : 10 final concentration), or Blood donor sera-pools (diluted 1 : 10 final concentration; control). Plates were then washed 6x with WB, followed by lh incubation at RT with 70 μΙ/well of Alkaline Phosphatase-conjugated Goat-anti-Rabbit-Ig (6pg/ml ; Dako D0487) . Plates were then washed 6x with WB, followed by 10-60 min incubation at RT with 100 μΙ/well of 0.3% w/v of Phenoftalein monophosphate (Sigma P- 5758) . Plates were finally quenched by adding 100 μΙ/well of Quench solution (0.1M TRIS + 0.1 M EDTA + 0.5M NaOH + 0.01% w/v NaN₃; pH14), followed by ELISA reader (ASYS UVM 340) at 550 nm .

Results

Data have been made that demonstrates that sera from rabbits immunized with the vaccine antigen 400 SEQ-B bound to peptides corresponding to C5/gp41 (015/201) in the presence of PBS. This binding could be inhibited by recombinant gp41 as well as by peptides derived from C5 (015), gp41 (201), and C2 (204d) as well as by 400-SEQ-B itself. The binding could not be inhibited using an irrelevant peptide (B301-23) .

Anti-C5/gp41 sera from BI400-B immunized rabbits is competitively inhibited by LT P-sera pools, but not with BD control sera. Also, antibodies against C5/gp41 were observed in 26/43 natural virus suppressor HIV patients with viral loads < 15000 copies/ml) and in 4/15 HIV patients with viral loads above 15000 copies/ml . Furthermore, significantly (p=0.018 when using a Mann-Whitney test) higher anti-C5 IgG responses (i .e. grouped with respect to OD-value measured at same serum dilution) were observed in HIV-1 patients with viral load below 15000 copies/ml (n=43) compared to patients with viral load above 15000 copies/ml (n = 15) .

To conclude, the results from the immunization studies with BI400-B demonstrate that it is possible to generate peptides that elicit antibody responses to C5 and gp41/C2 not only as individual components but also as complexes. The specificity of these antibody responses is confirmed in blocking studies using specific peptide antigens. Furthermore, antibodies generated to these peptides in animal models are comparable with antibodies elicited in natural HIV infection and associated with longterm nonprogression. These results show that these peptides are suitable for diagnostics as well as the development of a vaccine targeting HIV-induced immune activation. The finding that BI400-B elicits antibodies that bind to the complex between gp41 and C5, and that these antibodies compete with antisera against the same complex epitopes in LTNP HIV patients indicates that it is possible to stimulate immune responses against these epitopes and thereby induce an LT P-like condition in patients which do not themselves raise antibodies of this type against HIV. EXAMPLE 6

In the following, a summary of the procedures for the synthesis and purification of C5- Peptide is given. Experience is still limited which may eventually lead to improvements in the manufacture and quality of this product. The SPPS synthesis was started with 15 mmoles (A-chain) and 30 mmoles (B-chain) of the resin. After purification of a part of the crude C5-Peptide, 16.6 g of final product were obtained.

Stage 1: Solid phase peptide synthesis

The amino acid sequences of the A-chain (H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys-Gly- G!y-Ala-Lys-Arg-Arg-Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH₂) and the B-chain (H-Gly-Lys-Gly-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-GIn- Asp-Arg-Asp-Arg- NH₂) are assembled by standard solid phase peptide synthesis (SPPS) using the Fmoc-strategy. (W.C. Chan and P.D. White; Solid Phase Peptide Synthesis - A Practical Approach, Oxford University Press Inc., New York, 2000, ISBN 978-0-19-963724-9) The solid phase is transferred into an SPPS reactor and the synthesis cycle is started with the Fmoc-deprotection. Following the deprotection step, the peptide chain is elongated by the coupling of the following N-a-protected AA derivative or the dipeptide according to the amino acid sequence in the presence of suitable activating agents. To avoid the formation of long peptide sequences as by-products, a systemic acetylation of unreacted peptide chains (capping procedure) can be performed after every coupling step.

For each single step, the solvents and/or reagents are added, and the reaction mixture is stirred and then filtered to remove solvents and/or reagents from the resin. Single steps of the SPPS cycle may be repeated in case the reaction is incomplete. The SPPS cycle is repeated until the solid phase carries the complete amino acid sequence of the A-chain or the B-chain.

For the A-chain, a final Fmoc-deprotection is performed and the SPPS is completed by drying the peptide resin under reduced pressure.

The B-chain is further modified with a bromoacetyl linker at Lys². This procedure consists of selectively cleaving the side-chain protecting group of Lys² and coupling bromoacetic acid to Lys² in the presence of a suitable activating agent. If the coupling reaction is incomplete, recoupling procedures can be performed. The SPPS is then completed by drying the peptide resin under reduced pressure.

Stage 2: Cleavage from the resin including cleavage of the acid labile protecting groups

Cleavage of the peptides from the resin and concomitant cleavage of the acid labile protecting groups is accomplished by treatment with TFA in the presence of water.

Scavengers are added as needed to trap reactive cations and to avoid alkylation of side-chain functions. After filtering off and washing the resin with TFA, the products are precipitated in IPE. They are filtered off, washed with IPE, and dried under reduced pressure.

Stage 3: Purification of the intermediates by preparative HPLC (TFA system)

The A-chain and the B-chain obtained in the previous stage are purified by preparative HPLC on reversed phase columns with AC gradient elution (TFA system) and UV detection at λ =

220 nm.

Portions of the peptides are dissolved in water or a mixture of water and acetic acid and loaded onto the column. Subsequently, the ACN gradient of the TFA system is started. The collected fractions are checked by analytical HPLC and pooled accordingly.

Side fractions can be repurified with the TFA system. Finally, the pooled fractions with adequate purity are lyophilized.

Stage 4: Coupling of A-chain and B-chain

The coupling of the two peptide chains is performed by the addition of a solution of the B- chain (1 equivalent) in aqueous TFA to a solution of the A-chain (1 equivalent) in TRIS buffer (adjusted to pH 8.5 by the addition of hydrochloric acid). Additional TRIS buffer is added to maintain a pH > 8 in the reaction mixture. The reaction mixture is then stirred and the reaction progress is monitored by analytical HPLC. Upon completion, the pH of the reaction mixture is lowered to approx. pH 3 by the addition of TFA. Stage 5: Purification by preparative HPLC (TFA system)

The C5-peptide obtained in the previous stage is purified by preparative HPLC on reversed phase columns with ACN gradient elution (TFA system) and UV detection at λ = 220 nm.

Portions of the C5-peptide are directly loaded onto the column. Subsequently, the ACN gradient of the TFA system is started. The collected fractions are checked by analytical HPLC and pooled accordingly. Side fractions can be repurified with the TFA system. Finally, the pooled fractions with adequate purity are lyophilized .

Stage 6: Ion exchange, microfiltration, and lyophilization

The last stage of the manufacture of C5-Peptide is the ion exchange from the TFA salt, obtained in the previous stage, into the acetate salt.

The lyophilized material from the TFA purification is dissolved in 5% acetic acid and the solution loaded onto the ion exchange resin (acetate form) . The elution is performed with 5% acetic acid and checked by TLC. The product solution is filtered through a 0.2 pm membrane filter and lyophilized to yield the final product as a white to off-white material . EXAMPLE 7

PREPARATION OF HIV-SPECIFIC PEPTIDES ACCORDING TO THE INVENTION DESCRIPTION OF THE PREPARATION OF THE PEPTIDES

The peptides of the invention can be produced by any known method of producing a linear amino acid sequence, such as recombinant DNA techniques. A nucleic acid sequence which encodes a peptide of the invention or a multimer of the said peptides, is introduced into an expression vector. Suitable expression vectors are for instance plasmids, cosmids, viruses and YAC (yeast artifical chromosome) which comprise necessary control regions for replication and expression. The expression vector may be stimulated to expression in a host cell . Suitable host cells are for example bacteria, yeast cells and mammal cells. Such techniques are well known in the art and described for instance by Sambrook et al ., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989. Other well-known techniques are degradation or synthesis by coupling of one amino acid residue to the next one in liquid phase or preferably on a solid phase (resin) for instance by the so-called Merrifield synthesis. See for instance Barany and Merri field in the Peptides, Analysis, Synthesis, Biology, Vol .2, E. Gross and Meinhofer, Ed. (Acad . Press, N .Y., 1980), Kneib-Coronier and Mullen Int. J . Peptide Protein Res. ,30, p.705-739 ( 1987) and Fields and Noble Int.] . Peptide Protein Res., 35, p.161 -214 ( 1990) .

In case a linked or cyclic peptide is desired, the amino acid sequence is subjected to a chemical oxidation step in order to cyclize or link the two cysteine residues within one or between two peptide sequences, when the appropriate linear amino acid sequences are synthesized, see Akaji et al ., Tetrahedron Letter, 33, 8, p.1073- 1076, 1992. GENERAL DESCRIPTION OF SYNTHESIS

Ail peptide derivatives prepared in the Examples given below were synthesized on a Milligen 9050 Peptide Synthesizer using a standard program. The resin used was Tenta Gel P RAM with a theoretical loading of 0,20 meq/g (RAPP POLYMERE GmbH, Tubingen). The final product of the synthesis was dried In vacuo overnight. The peptide was then cleaved from the resin by treatment with 90% trifluoroacetic acid in the presence of ethandithiol (5%) and water (5%) as scavengers (1,5 hours at RT). Then the resin was filtered and washed on filter with additional trifluoroacetic acid (100%) (2 x 20 ml). The combined filtrates were evaporated In vacuo (water bath at RT) and the residue was triturated with ethyl ether (200 ml) and the precipitated product filtered off. The solid was promptly dissolved on filter with glacial acetic acid (100 ml) and added to 1,5 I of 20% acetic acid in methanol and treated with 0,1 M solution of iodine in methanol until a faint brown colour remained. Then Dowex 1 x 8 ion exchange in acetate form (15g) (Bio-Rad, Richmond, CA) was added and the mixture filtered. The filtrate was evaporated and the residue freeze-dried from acetic acid. The product was then purified by reversed phase liquid chromatography on a column filled with

Kromasil® 100 - 5 C8 (EKA Nobel, Surte, Sweden) in a suitable system containing acetonitrile in 0,1 % trifluoroacetic acid water solution. The samples collected from the column were analyzed by analytical high performance liquid chromatography (HPLC) (Beckman System Gold, USA) equipped with a Kromasil® 100 - 5 C8 Column (EKA Nobel, Surte, Sweden). Fractions containing pure substance were pooled, the solvent was evaporated and the product freeze-dried from acetic acid. The final HPLC analysis was performed on final product, and the structure of the peptide was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

All amino acids used during the synthesis were L-amino acids and they were protected with a fluorenylmethoxy-carbonyl group at the (/-amino function. The side chains were protected as follows :

Cys (Trt), Gln(Trt), Glu(OtBu), Thr(tBu).

The abbreviations, within the brackets are :

Trt = triphenylmethyl

t-Bu = tert. Butyl

OtBu = tert. Butylester

The amino acid derivatives was supplied by Bachem AG, Switzerland.

EXAMPLE 8 Preparation of KALG PGATLQTPWTACQGVG - NH₂ (SEQ ID NO : 48).

The peptide was synthesized in amide form, from corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS). Purity (HPLC): 87 %

EXAMPLE 9

Preparation of RALG PAATLQTPWTAS LGVG (SEQ ID NO : 49).

The peptide was synthesized in amide form, from corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC): more than 95%

Molecular weight (free base): 1966

Molecular formula : CssHn^s^

EXAMPLE 10 Preparation ofWI I PG LN PLVGGG K LYS PTSI LCG - NH₂ (SEQ ID NO : 51).

The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS). Purity (HPLC) : 95%

Mass spectral analysis : Theoretical molecular weight : 2454.9

Experimental molecular weight : 2454.8 ES+

EXAMPLE 11 Preparation of R W L LLG L N P LVGG G LY S PT S I LG (SEQ ID NO : 52).

The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 95 %

Molecular weight (free base) : 2552

Molecular formula : C_!19H₁₉₅029N

EXAMPLE 12 Preparation of K I LLG L N P LVGGG R LY S PT S I LG (SEQ ID NO : 53) , R L L L G L N P LVG G G R LY S PTT I LG (SEQ ID NO : 54) and N I P I PVG D I YG G G D I Y K R W Q A L C L (SEQ ID NO : 70). The peptides are synthesized in amide form, from the

corresponding starting materials according to the general description of synthesis. The purity are determined by HPLC analysis and the structures are confirmed by amino acid analysis and mass spectrometry (LDI-MS).

EXAMPLE 13

Preparation of R N I PI PVG D IYG G G D I Y K RW QA LC L (SEQ ID NO : 56).

Purity (HPLC) : 85 %

Mass spectral analysis : Theoretical molecular weight : 2817.3 Experimental molecular weight : 2813.7 ES+

EXAMPLE 14 Preparation of RAI PI PAGTLLSGGG RAIY K RWAI LG (SEQ ID NO : 57).

Purity (HPLC) : more than 95 %

Molecular weight (free base) : 2707

Molecular formula :

EXAMPLE 15 Preparation ofALPI PAG FIYGGG RIYK RWQALG (SEQ ID NO : 58), K I P I P V G FIGGGWIYKRWAI LG (SEQ ID NO : 59) and KI PI PVGTLLSGGG RIYKRW A I L G ( SEQ ID NO : 60). The peptides are synthesized in amide form, from the

EXAMPLE 16

Preparation of K FI I PNI FSALGGAISYD LNTNI LNC I (SEQ ID NO : 62).

The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. Nl in the sequence is Norleucine. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 80 %

Mass spectral analysis : Theoretical molecular weight : 2783.3 Experimental molecular weight : 2783.3 ES+

EXAMPLE 17 Preparation of K F I I P NI F SA LS G G GA I SY D L N T F L N C I G (SEQ ID NO : 63).

The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. NI in the sequence is Norleucine. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS).

Purity (HPLC) : more than 80 %

Mass spectral analysis : Theoretical molecular weight : 2932.4 Experimental molecular weight : 2931.8 ES+ EXAMPLE 18 Preparation of R F I I P NI FTA LS G G R RA L LYG AT PYA I G (SEQ ID NO : 64).

The peptide was synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. NI in the sequence is Norleucine. The purity was determined by HPLC analysis and the structure was confirmed by amino acid analysis and mass spectrometry (LDI-MS). Purity (HPLC) : more than 95 %

Molecular weight (free base) : 2894

Molecular formula : C137H217O32 37

EXAMPLE 19

Preparation of K I I P NI F SA LG G G R L LYGAT PYA I G (SEQ ID NO : 65), R I I P NI F TA LS G G G R L LYG AT PYA I G (SEQ ID NO : 66) and W I I P NI F S A L G G A I S Y D L N T NI L N C I (SEQ ID NO : 71). The peptides are synthesized in amide form, from the corresponding starting materials according to the general description of synthesis. The purity are determined by HPLC analysis and the structures are confirmed by amino acid analysis and mass spectrometry (LDI-MS). EXAMPLE 20 Dimerisation via disulphide bridge.

The peptide sequences of the Examples 8 and 10 were linked via an oxidation step to form a dipeptide wherein the cysteine residues formed a disulphide bridge. The bridge was formed in either ways; A) Oxidation with I_2. Equal amounts of the peptides were dissolved in acetic acid/methanol (1 :4) and 0.1 M I₂ in methanol was added yielding a mixture of the dimer. or

B) Oxidation via [Cys(Spy)¹⁶]-SEQ ID NO:48. 2,3mM of the peptide of SEQ ID NO:48 dissolved in 2 M AcOH (aq) and 2-propanol (1 : 1) was treated with 2,2 dithiodipyridin (3 eqv) to yield [Cys(Spy)¹⁵]-SEQ ID NO:48. Equal amounts of [Cys(Spy)ⁱ⁶]-SEQ ID NO:48 and peptide of SEQ ID NO: 51 were dissolved in 10 mM NH₄Oac (aq pH=6, 5) and methanol (5: 2) to yield the dimer of SEQ ID NO:67.

The purity of the peptide was determined by HPLC analysis and the peptide structure was confirmed by amino acid analysis. The peptide content (aminoacid free base ) was 80%, Purity (HPLC) : 92%.

EXAMPLE 21

A vaccine comprising the peptides of the SEQ ID NO: 49, 52, 57 and 64 was prepared. The freeze-dried peptides were dissolved in sterile water at a final concentration of 4 mg/ml. The final salt concentration was 0,9 %. A preparation of a granulocyte-macrophage-colony stimulating factor (GM-CSF) was also prepared, according to the manufacturer's directions for use, to a final concentration of 0.3 mg/ml. The two solutions are administered

intracutaneously. A typical injection dose is 100 μΙ.

EXAMPLE 22

An antigen solution or suspension is mixed with equal parts of Freund ^' s adjuvant of

Behring, complete or incomplete, and is then finely emulsified by being drawn up into, and vigorously pressed out of, an injection syringe, or with a homogenisator. The emulsion should remain stable for at least 30 minutes. The antigen-adjuvant emulsions is best injected subcutaneously as a depot. EXAMPLE 23

Toxicity studies were performed in mice and rats on the peptide composition of the vaccine in Example 22. The mouse was selected for the study to provide comparative data from a second commonly used rodent species. The test substance was a mixture of four peptides supplied as one vial containing lyophilised material for reconstitution with physiological saline, and dose levels were expressed in terms of total peptide load. The individual peptides was present in ratio 1 : 1 : 1 : 1 w/w giving dose levels of each peptide of 0.0075 mg/kg body weight, 0.075 mg/kg body weight and 0.75 mg/kg body weight, which are up to 500 fold the intended human dose. The test animals were divided into four groups of ten animals each (five males and five females); a saline control group and groups for low, intermediate and high doses. The test composition was administered once, by intravenous infusion into a tail vein at a dose rate of 3 ml/minute. The animals were killed at day 15 and 16 by intraperitoneal injection of sodium pentobarbitone.

The results of these studies indicated that the dose levels administered to the mice and rats elicited no adverse reactions and that the no effect level was in excess of 3 mg/kg.

EXAMPLE 24

Immunoassay for detection of antibodies induced by HIV-1.

The magnetic particle reagents are to be prepared according to the manufacturers recommended protocol. Dynal AS, is the manufacturer of the Dynabeads, which are employed. The magnetic particles coated with ligand are called Reagent 1. A peptide according to the invention is covalently coupled to the pre-activated surface of the magnetic particles. It is also possible to physically absorb the peptide to the surface of the magnetic particles. The concentration of particles in Reagent 1 is within the range from 1 mg/ml to 15 mg/ml. The particle size varies between 0.2 um to 15 μΠΊ . The concentration of peptides is within the range from 0.01 mg/mg particle to 1 mg/mg particle.

The anti human Ig Alkaline Phosphatase (AP) conjugated antibody reagent is prepared according to the recommended protocol of Dako AS. This protocol is a standard procedure in this field. This reagent is called Reagent 2.

The substrate solution phenolphtalein-monophosphate is to be prepared according to the recommended protocol of Fluka AG. This protocol is a standard procedure in this field. The substrate solution is called Reagent 3.

The washing and incubation buffer which is used is standard 0,05M tris-base buffer with the following additional compounds; Tween 20 (0.01% to 0.1%), glycerol (0.1% to 10%) and sodium chloride (0.2% to 0.1%).

The assay procedure comprises an incubation step wherein 1 drop of Reagent 1 is mixed with 2 drops of washing buffer in each well. After mixing, 30 μΙ of sample is added and the solution is incubated for 5 minutes. The magnetic particles can be trapped by a magnet and the liquid removed, before the magnet is separated . Then the wells are washed twice in 4 drops of washing solution, before incubation with Reagent 2. 1 drop of Reagent 2 is added with 2 drops of washing buffer and the solution is incubated for 5 minutes. The magnetic particles can be trapped by a magnet and the liquid removed, before the magnet is separated . Then the washing step is repeated before incubation with Reagent 3. 2 drops of Reagent 3 is added to each well and the solution is incubated for 3 minutes. The results can be read against a white background . Positive results are red (3+ = strong red) whereas negative results are clearly light yellow/brown solutions as obtained in the negative control.

The immunoassay kit could be used in detection of antibodies, induced either by HIV virus or HIV-specific peptides or proteins, for instance the peptides of the present invention.

The above Examples are only meant as illustrating the invention. It must be understood that a person skilled in the art can modify the peptides, antigens and vaccines herein described without deviating from the concept and scope of this invention as set forth in the claims.

The polypeptides of the invention can be used in a combination of at least one peptide selected from each group of sequences, SEQ ID NOs: 47, 50, 55 and 61 to form antigens and the active principle of a prophylactic or therapeutic vaccine intended to provide protection against the human immunodeficiency virus type 1 (HIV-1) . The vaccine may include compounds having beneficial effects in protecting or stimulating the host^' s immune system (human being or vertebrate animal) for instance interleukins, interferons, granulocyte macrophage growth factors, haematopoietic growth factors or similar. Preferably the vaccine composition further contain an adjuvant or vehicle, more preferably the adjuvant or vehicle is Monophosphoryl Lipid A (MPL ®) possibly with alum, Freund ^' s adjuvant (complete or incomplete) or aluminum hydroxide. The optimal amount of adjuvant/vehicle will depend on the type(s) which is chosen.

The peptide or vaccine formulation can be freeze-dried prior to storage. The vaccine may be stored preferably at low temperature, in ampoules containing one or more dosage units, ready for use. Persons skilled in the art will appreciate that a suitable dose can depend on the body weight of the patient, the type of disease, severity of condition, administration route and several other factors. The vaccine might be administered up to twelve times and through injection, typically it will be administered about three times. In preparation of an injection solution the peptides are dissolved in sterile water or sodium chloride solution at a final concentration of 1-12 mg/ml per peptide and 0 to 0,9% sodium chloride. Typically an injection volume is 100 μΙ to 200 μΙ (2 x 100 μΙ) . The peptide is preferably co-administered with a suitable adjuvant and/or a granulocyte-macrophage growth factor for instance Leucomax© «Shering Plough* . Suitable administration may be intracutane, subcutane, intravenous, peroral, intramuscular, intranasal, mucosal or any other suitable route. Booster administrations may be required in order to maintain protection. EXAMPLE 25

The anti-HIV p24 immune response resulting from Vacc-4x immunization could in combination with ART potentially improve immune reconstitution in patients who have not fully regained a healthy CD4 level (>600xl0⁶/L). Potential benefits of Vacc-4x in subjects with incomplete immune reconstitution include a possible sustained improvement in the immune response to p24 and HIV.

Potential risks include the discomfort and inconvenience associated with the immunizations and the risk of known or unknown side effects of exposure to Vacc-4x and Leukine (rhu-GM-CSF) including, most commonly, local reactions at the site of injections and fatigue (likelihood not yet determined).

The results of non-clinical single-dose studies in mice and rats indicate that the dose levels of intravenous Vacc-4x elicited no adverse reactions and that the no effect level was in excess of 3 mg/kg, which constitutes a 500 fold safety margin over the planned human dose level.

In a rabbit study the effect of Vacc-4x was evaluated in the presence of concomitant

GM-CSF, the adjuvant used in the clinical program. Local intradermal reactions such as erythema and edema were noted, however, similar effects were noted in control animals both macroscopically and histological. These local reactions were slightly more pronounced in the Vacc-4x treated animals. There were no systemic reactions in this study. These data indicate that Vacc-4x has no limiting toxicology in a model that is relevant to the proposed clinical study.

The therapeutic vaccine candidate Vacc-4x, has been studies in a Phase I and three Phase II clinical studies. The Phase I study enrolled 11 HIV-positive subjects, including nine subjects on ART. Subjects were maintained on ART (if entered on ART); all subjects were treated with 12 immunizations of Vacc-4x at a dose of 0.4 mg/injection over a period of 26 weeks. Immunizations were performed following injection of rhu-GM-CSF (Leucomax®

[molgramostim]) as adjuvant. All subjects experienced one or more adverse events (AEs); nine subjects experienced events judged related to treatment. The adverse reactions reported were mild or moderate in severity except for severe local reactions in one subject. No subjects were withdrawn due to treatment-related AEs or toxicological reactions; no serious adverse events (SAEs) occurred. Treatment related events observed in more than one subject included painful injection (seven subjects), fatigue-vertigo (four subjects), influenza-like symptoms (two subjects), and irritated skin at injection site (two subjects).

All subjects experienced a cell-mediated immune response, measured by delayed- type hypersensitivity (DTH) skin reaction. Some cell-mediated immune response, measured by γ IFN release using enzyme-linked immunosorbent spot assay (ELISPOT), was reported for 45% of the subjects; no antibody response to Vacc-4x peptides was observed.

The Phase II dose-finding study (CTN B-HIV 2/2001) enrolled 40 HIV positive subjects, of which 38 completed the trial. Subjects were maintained on ART and treated with 10 immunizations at a dose of 0.4 mg (20 subjects) or 1.2 mg (20 subjects) per Vacc 4x injection, over a period of 26 weeks. Immunizations with Vacc 4x were performed following injection of rhu-GM-CSF (Leucomax [molgramostim]) as a local adjuvant. ART was interrupted from Week 26 to Week 30 to allow exposure to the subject's own virus

(autologous immunization). ART was resumed from Week 30 to Week 38 to allow maturation of immune responses to the Vacc 4x peptides and to the subject's own virus. ART was discontinued from Week 38 to Week 52 when the study was formally concluded. Treatment- related AEs were observed in 20 subjects (8 subjects in the 0.4mg group and 12 subjects in the 1.2mg group). No SAEs were reported during the period of immunization. One subject experienced a transient vasovagal reaction in conjunction with immunization and the DTH test at Week 26 and Week 38. A second subject experienced a vasovagal reaction in conjunction with the DTH test at Week 52. For the laboratory parameters, vital signs, and performance status, no changes attributable to immunization were observed. Changes in HIV RNA, CD4 cell counts, and CD8 cell counts showed no safety concerns related to

immunization.

Immunological responses reported as DTH positive reactions were observed for all subjects. Overall, positive responses both for induration and erythema were statistically significantly higher in the high dose (HD, 1.2mg Vacc-4x) group compared to the low dose (LD, 0.4 mg Vacc 4x) group. The dose-dependent differences in DTH reactions were maintained throughout the study. T-cell proliferation appeared stable after Week 12 and demonstrated an HD advantage, consistent with the DTH results. ART was interrupted at Week 38 with planned restart when CD4 counts fell to less than 200/μΙ_ or when AIDS- or HIV related events were observed (i.e. clinical practice). DTH responses to Vacc-4x (high versus low response determined at Week 38) were associated with reduced viral loads and correspondingly improved CD4 counts at the end of the study (Week 52).

During the immunization period, CD4 counts were stable or increased. Interruption of ART resulted in reduction of CD4 counts. However, 14 weeks after the last interruption of ART (Week 52), the mean CD4 counts were still above 200 x 106 cells/L No difference between the LD and the HD groups was observed. The majority of subjects remained off ART following completion of the study (Week 52); permission was given to follow the subjects until they resumed ART. The duration of treatment interruption was linked to immune responsiveness to the peptides. When subjects were compared to similar subjects in the Netherlands that had stopped treatment without Vacc-4x administration, a significantly slower decline in CD4 cells was noted for the Vacc-4x subjects. The median treatment interruption achieved for all the subjects that participated in the Vacc-4x Phase II clinical study was 31 months.

CTN BI Vacc-4x/2009/l was an open-label follow-up of study CTN B-HIV-2/2001 to detemine whether a re-boost with Vacc-4x could reactivate or increase the immune response obtained during the immunization performed in the CTN B-HIV-2/2001 study. The secondary objectives were to evaluate: the in vivo immunogenicity of Vacc-4x by evaluation of DTH and to compare the DTH response to DTH in the initial study; the effect of Vacc-4x on CD4 counts, CD8 counts and HIV viral RNA; and the safety and tolerability of Vacc-4x. All 26 subjects included in the study received two booster administrations of Vacc-4x.

A total of 74 AEs were reported by 23 subjects. Most adverse events (n = 60) were scored as possibly/probably related to the study treatment. The majority (98%) of the related adverse events were mild. Two adverse events related to study treatment, one headache and one injection site indurations, were scored as moderate intensity. Itching (injection site pruritus) was the most frequent reported adverse event related to the study treatment. Nineteen patients (73%) reported this adverse event at least once. Ten of these patients reported itching related to both immunizations, while for the other nine patients it was only reported once. Five patients reported swelling related to the immunization. For three of these patients swelling was reported after both immunizations. No patient died during the study. No patient reported serious adverse events and no clinically relevant changes were recorded.

The study demonstrated that Vacc-4x peptides induced T cell responses lasting up to seven years. By re-boosting it was possible to increase killing markers, this again indicates that T cells had increased their potential to kill HIV-infected cells. Before re-boosting, all the patients had returned to CD4, CD8 and viral load levels that were similar to those before ART was stopped in the main study. Re- boosting had no negative effect on the CD4, CD8 and viral load of the patients. No safety concern was reported as a result of the re-boost of these patients.

The Phase II Study CT-BI Vacc-4x 2007/1 (EudraCT Number 2007-006302-13) was performed in US and Europe (UK, Germany, Spain and Italy). The study was a randomized, double-blind, multicenter, immunogenicity study of Vacc-4x versus placebo in patients infected with HIV-1 who have maintained an adequate response to ART. The primary objective was to evaluate the effect of Vacc-4x immunizations versus placebo on CD4 counts, T-cell function (ELISPOT, T-cell proliferative responses and intracellular cytokine staining) and the response to interruption of ART. The necessity to resume ART between the interruption of ART at Week 28 and the end of the study at Week 52, due to decreased CD4 count or increased viral loads, was monitored as one of the primary efficacy endpoints.

In the ITT analysis population, it was concluded that Vacc-4x did not reduce the proportion of subjects requiring resumption of ART after ART cessation at Week 28 in comparison with placebo. There was also no effect compared with placebo on the percentage change in CD4 count between Week 28 and the last CD4 assessment before resumption of ART. The time to restarting ART was similar in Vacc-4x and placebo-treated subjects.

The viral load results after ART cessation varied between subjects with evidence of favourable effects of Vacc-4x immunization over placebo. There were no significant differences in the repeated measures AN OVA for viral load over Weeks 4 to 52 when data included all evaluable subjects, irrespective of whether they were or were not taking ART. In the subgroup of subjects who remained off ART until Week 52, the average viral load was lower in the Vacc-4x-treated subjects than the placebo group. A post-hoc analysis showed the Week 52 (Last Observation Carried Forward [LOCF]) viral load to be statistically significantly lower in the Vacc-4x group than the placebo group.

The analysis of change in HIV-1 RNA from Week 28 through to Week 52 revealed a statistically significant difference between groups in favour of Vacc-4x. The AUC in those who remained off ART at Week 52 was lower in the Vacc-4x group than in the placebo group. A post-hoc analysis showed this difference in AUC to be statistically significant.

No safety concern was raised during this study. The study was supervised by a Data

Safety Monitoring Board (DSMB).

EXAMPLE 26

Test of peptides together with IMiDs for increased proliferation, polyfunctionality, IL- 2 secretion and IFN-γ production.

Expansion of polyfunctional HIV-specific T-cells upon stimulation with Dendritic Cells, pre-incubated with peptides to be used according to the invention, may be studied by methods described by Keersmaecker et al. (J . Virol., 2012 86: 9351-9360) and referenced therein, HIV proteins Gag or Nef, they are incubated with peptides to be used according to the invention , before they are used to stimulate T-cells in a co-culture.

Keersmaecker et al. found that the presence of IMiDs (Lenalidomide (IMiD3; CC- 5013) and pomalidomide (IMiDl ; CC-4047) during in vitro T-cell stimulation with dendritic cells presenting Gag- or Nef-specific peptides, resulted in a number of improvements in the function of the T-cells. Among these were; polyfunctional HIV specific CD8+ T cells with enhanced lytic capacity, more Gag antigen epitopes recognized and at lower antigen peptide concentrations, reduced proliferation of CD4+ T cells with increased number of polyfunctional CD4+ T-cells , increased IL-2 production by CD8 T-cells, detectable IFN-γ production by CD8+ T-cells and CD4 T-cells after antigen stimulation.

"Expansion of Polyfunctional HIV-Specific T Cells upon Stimulation with mRNA Electroporated Dendritic Cells in the Presence of Immunomodulatory Drugs"

Brenda De Keersmaecker, Sabine D. Allard, Patrick Lacor, Rik Schots, Kris Thielemans, and Joeri L. Aerts

J. Virol. September 2012 86: 9351-9360; published ahead of print 20 June 2012,

doi : 10.1128/JVI.00472-12

EXAMPLE 27

Suggested clinical study protocol for the test of Peptide composition comprising 4 peptides in combination with Lenalidomide and HDAC inhibitor Immunizations (four primary immunizations and two booster immunizations) at Weeks 1, 2, 3 and 4, and booster immunizations at Weeks 12 and 13 with either:

1) Peptide composition with GM-CSF as adjuvant and Lenalidomide (CC-5013), or

2) Peptide composition with GM-CSF as adjuvant and Placebo for Lenalidomide (CC-5013) .

3) Placebo

Suggested doses:

Peptide composition : 0.6, 0.9, 1.2 and 1.5 mg (Equal amount of each peptide, w/w)

Lenalidomide: 5, 10, and 25 mg .

Subjects randomized to the Lenalidomide (CC-5013) arm will take a single oral dose of Lenalidomide (CC-5013) daily the two preceding days before immunization with the Peptide composition and on the day of each immunization.

The Peptide composition used according to this clinical trial setup consists of SEQ ID NO:49, SEQ ID NO: 52, SEQ ID NO : 57, and SEQ ID NO: 64.

At week 20 subjects in all study arms will receive 20 mg panobinostat (LBH 589) orally on days 1, 3, and 5 (i.e. 3 times a week) every other week for a period of 8 weeks (up to week 28) while maintaining background ART. This will be followed by a 24 week follow up period (up to week 52) . Upon completion of the study, subjects may be invited to participate in an additional observational study in which ART will be interrupted to evaluate the effect of study treatment on virological control . Enrolment into this part of the study will be optional and determined by the effect of study treatments on the latent HIV- 1 reservoir. (Maximum duration of treatment interruption : 16 weeks) .

In summary :

Study arm 1 : Peptide composition + IM iD + HDAC (panobinostat)

Study arm 2 : Peptide composition + HDAC (panobinostat)

Study arm 3 : HDAC (panobinostat)

Depletion of the viral reservoir as a result of the combination treatments according to the present invention may be quantified by for instance following the procedures set forth in Lehrman et al . (The Lancet (366), 2005, pp. 549-555) and references there in. In brief, this includes measuring in samples of patient blood obtained before, during and after treatment; p24 expression from stimulated latently infected cells, plasma HIV RNA concentration (viral load), and integrated HIV DNA by realtime PGR analysis.

EXAMPLE 28

DC/ T-CELL PROLIFERATION ASSAY

Dendritic cells (DC) were generated from monocytes isolated from buffy coat preparations from healthy blood donors. Briefly, peripheral blood mononuclear cells were separated by a density gradient centrifugation and the monocytes were then negatively isolated using the Dynabeads Untouched Human Monocytes (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. The monocytes were cultured with IL-4 (20 ng/ml ; Immunotools, Friesoythe; Germany) and GM-CSF ( 100 ng/ml ; Immunotools) in X-VIV015 medium (Lonza, Basel, Switerland) for 5-6 days to generate immature DC. Cytokines were replenished every 2-3 days. The maturation of the cells was performed for 24 hours with IFN-Y ( 1000 IU/ml), TNF-a (50 ng/ml), IL-Ιβ (25 ng/ml) IFN-a (3000 IU/ml) . After maturation, the DC were pulsed for 2 hours at 37°C with peptides at ΙΟμς/ιηΙ, before extensive washing and co-culture with Peripheral blood mononuclear cells (PBMC) labelled with a fluorescent dye (VPD450, BD biosciences, Sam Jose, CA) . Various ratios with DC:T cell were tested alongside with appropriate controls. IL-2 (50U/ml) and IL-7 (50ng/mL) (Both, Immunotools) and wells with or without IMiDs were added at the start of co-culture. At day 6-10, the level of T cell proliferation was analysed by flow cytometry. The supernatants from the co-culture wells were investigated with Luminex technology to establish any suppressor activity.

EXAMPLE 29

The peptides according to the invention used in the following examples were synthesized by Schafer-N as c-terminal amides using the Fmoc-strategy of Sheppard, ( 1978) J .Chem.Soc, Chem . Commun., 539.

Cell penetration assay

Intracellular staining for biotinylated peptides

96-well U-bottom polystyrene plates ( NUNC, cat no: 163320) were used for staining of human PBMCs. Briefly, 8ul of N- or C-terminally biotinylated peptides according to the invention (i .e. 5mM, 2.5mM & 1.25mM tested for each peptide) were incubated at 37°C for 2h with 40ul of PBMC ( 12.5 x 106 cells/ml) from blood donors. Cells were then washed 3x with 150ul of Cellwash (BD, cat no: 349524), followed by resuspension of each cell pellet with lOOul of Trypsin-EDTA (Sigma, cat no: T4424), then incubated at 37°C for 5 min.

Trypsinated cells were then washed 3x with 150ul of Cellwash (BD, cat no: 349524), followed by resuspension with BD Cytofix/Cytoperm™ plus (BD, cat no: 554715), then incubated at 4°C for 20 min according to manufacturer. Cells were then washed 2x with 150ul PermWash (BD, cat no: 554715) . Cells were then stained with Streptavidin-APC (BD, cat no: 554067) & Anti-hCDl lc (eBioscience, cat no: 12-0116) according to manufacturer at 4°C for 30 min aiming to visualize biotinylated peptides & dendritic cells, respectively. Cells were then washed 3x with 150ul PermWash, followed by resuspension in staining buffer (BD, cat no: 554656) before flow cytometry. Dendritic cells were gated as CDl lc+ events outside lymphocyte region (i.e. higher FSC & SSC signals than lymphocytes) . 200 000 total cells were acquired on a FACSCanto II flow cytometer with HTS loader, and histograms for both total cells & dendritic cells with respect to peptide-fluorescence (i .e. GeoMean) were prepared . Extracellular staining for biotinylated peptides

96-well U-bottom polystyrene plates ( NUNC, cat no: 163320) were used for staining of human PBMCs. Briefly, 8ul of N- or C-terminally biotinylated peptides according to table 1 or table 2 (i .e. 5mM, 2.5mM & 1.25mM tested for each peptide; all peptides manufactured by solid phase synthesis by commercial suppliers) were incubated at 37°C for 2h with 40ul of PBMC ( 12.5 x 106 cells/ml) from blood donors. Cells were then washed 3x with 150ul of Cellwash (BD, cat no: 349524), then stained with Streptavidin-APC (BD, cat no: 554067) & Anti-hCDl lc (eBioscience, cat no: 12-0116) according to manufacturer at 4°C for 30 min aiming to visualize biotinylated peptides & dendritic cells, respectively. Cells were then washed 3x with 150ul of Cellwash (BD, cat no: 349524), followed by resuspension in staining buffer (BD, cat no: 554656) before flow cytometry. Dendritic cells were gated as CDl lc+ events outside lymphocyte region (i .e. higher FSC & SSC signals than lymphocytes) . 200 000 total cells were acquired on a FACSCanto II flow cytometer with HTS loader, and histograms for both total cells & dendritic cells with respect to peptide-fluorescence (i .e. GeoMean) were prepared.

It was clearly seen that the CMI peptides according to the invention had improved ability to enter the cell compared to its native counterparts

The data are geomean-value of each testet peptide, as calculated by the FACS Duva software. The Geomean values by trypsinating/Cytofix/Cytoperm. :

EXAMPLE 30

Positive CTL response may alternatively be assayed by ELISPOT assay.

Human IFN-gamma cytotoxic T-cell (CTL) response by ELISPOT assay

Briefly, at day 1, PBMC samples from HCV patients were incubated in flasks

(430 000 PBMCs/cm2) for 2h at 37°C, 5% C02 in covering amount of culture media (RPMI

1640 Fisher Scientific; Cat No. PAAE15-039 supplemented with L- Glutamine, (MedProbe Cat. No. 13E17-605E, 10% Foetal Bovine serum (FBS), Fisher Scientific Cat. No. A15-101 ) and Penicillin/Streptomycin, (Fisher Acientific Cat. No. Pl l-010) in order to allow adherence of monocytes. Non-adherent cells were isolated, washed, and frozen in 10% V/V DMSO in FBS until further usage. Adherent cells were carefully washed with culture media, followed by incubation at 37°C until day 3 in culture media containing 2ug/ml final concentration of hrGM-CSF (Xiamen amoytop biotech co, cat no: 3004.9090.90) & lug/ml hrIL-4 (Invitrogen, Cat no: PHC0043) and optionally an immunomodulationg agent (IMiD), and this procedure was then repeated at day 6. At day 7, cultured dendritic cells (5 000-10 000 per well) were added to ELISPOT (Millipore multiscreen HTS) plates coated with 0.5,ug/well anti-human γ Interferon together with thawed autologous non-adherent cells (200 000 per well), antigen samples ( l-8ug/ml final concentration for peptide antigens; 5ug/ml final concentration for Concanavalin A (Sigma, Cat no: C7275) or PHA (Sigma, Cat no: L2769)) & anti-Anergy antibodies (0.03-0.05ug/ml final concentration for both anti-PD-1 (eBioscience, cat no: 16- 9989-82) & anti-PD-Ll (eBioscience, cat no : 16-5983-82) ) . Plates were incubated overnight and spots were developed according to manufacturer. Spots were read on ELI SPOT reader

(CTL-ImmunoSpot® S5 UV Analyzer) . EXAMPLE 31

ELI SPOT assay

At day one, PBMC samples from blood donors were thawed, washed with warm medium and incubated in flasks (250000PBMCs/cm2) for 24 hours at 37°C, 5% C02 in covering amount of culture media (RPMI 1640 with ultra-glutamine, Lonza, BE12-702F701 ; 10% Foetal Bovine serum ( FBS), Fisher Scientific Cat. No. A15 - 101 ; Penicillin/Streptomycin, Fisher Scientific Cat. No. Pl l-010) to allow the cells to recover after thawing . At day two, the cells were added to a Falcon Microtest Tissue Culture plate, 96well flat bottom, at 500 000 cells per well in a volume of 200μΙ total medium . Parallel wells were added the indicated stimuli in duplicate and optionally an immunomodulationg agent (IMi D), or left with medium as a control for 6 days at 37°C, 5% C0₂. After the six days of incubation, ΙΟΟμΙ of the cell suspension were transferred to an ELI SPOT ( Millipore multiscreen HTS) plate coated with Ιμς/ιηΙ native influenza M2e protein . After a 24 hour incubation, the plate was washed four times with PBS + 0,05% Tween20, and a fifth time with PBS, 200μ l/well . A mouse Anti- human IgG or Ig M biotin (Southern Biotech 9040- 08 and 9020-08) was diluted in PBS with 0.5% FBS and incubated for 90 minutes at 37°C. The washing was repeated as described, before 80μΙ Streptavidin-Al kaline- Phosphatase (Sigma Aldrich, S289) was added each well and incubated at 60 minutes in the dark, at room temperature. The wells were then washed 2 times with PBS + 0.05% Tween20 and 4 times with PBS, 200μΙ/ννθΙΙ, before the substrate, Vector Blue Al kaline Phosphatase Substrate kit III (Vector Blue, SK-5300 ) was added and let to develop for 7 minutes at room temperature. The reaction was stopped with running water, the plates let dry and the sport enumerated by an ELISPOT reader (CTL-ImmunoSpot® S5 UV Analyzer) .

ELISA

100 μΙ of antigen as indicated (pre-incubated in Coating buffer - 0.05M Na₂C0₃ pH9.6; denoted CB - in cold at 8pg/ml 1-3 days) or just CB (background control ) was used for coating wells in microtiter plates at 4°C. The microtiter plates are then washed 3x with washing buffer ( PBS + 1 % v/v Triton-X100; denoted WB), followed by 2h blocking at room temperature (RT) with 200 μΙ/well of blocking buffer ( PBS + 1% w/v BSA) . Plates are then washed 3x with WB, followed by lh incubation at 37°C with 50-70 μΙ/well of added human (or rabbit or sheep) sera (serial dilutions ranging from 1 : 5 - 1 : 250 in dilution buffer ( PBS + 1 % v/v Triton-Xl OO + 1 % w/v BSA; denoted DB) ) . Plates are then washed 6x with WB, followed by l h incubation at RT with 70 μΙ/well of Al kaline Phosphatase-conjugated Protein G (3pg/ml in DB; Calbiochem 539305) or goat anti -mouse IgG biotin

Southern Biotech, 1030-08. In case of the goat anti-mouse IgG biotin, the plates were washed one extra step as described, before addition of ΙΟΟμΙ Streptavidin-Alkaline-Phosphatase (lpg/ml, Sigma Aidrich, S289) and incubated 1 hour at RT. Plates are then washed 6x with WB, followed by 10-60 min incubation at room temperature with 100 μΙ/well of 0.3% w/v of Phenophtalein monophosphate (Sigma P-5758). Plates are finally quenched by adding 100 μΙ/well of Quench solution (0.1M TRIS + 0.1M EDTA + 0.5M NaOH + 0.01% w/v NaN₃;

pH14), followed by a measurement with a ELISA reader (ASYS UVM 340) at 550 nm. The strength of the sera, i.e. the magnitude of the humoral immune response, is then reported as the dilution of sera that result in the described Optical Density (OD) value, or the OD value at the indicated dilution of sera.

EXAMPLE 32

CLINICAL TRIAL PROTOCOL - Phase I/IIa Study to Evaluate the Effect of Therapeutic HIV-1 Immunization using Vacc-4x + rhuGM-CSF, and HIV-1 Reactivation using Romidepsin, on the Viral Reservoir in Virologically Suppressed HIV-1 Infected Adults on cART.

The primary objective is to measure the effect of treatment with Vacc-4x + rhuGM-CSF and cyclic romidepsin treatment on the HIV-1 latent reservoir in HIV-infected patients

virologically suppressed on cART.

Endpoints:

Primary Endpoints:

1) Safety and tolerability evaluation as measured by adverse events (AE), adverse reactions (AR), serious adverse events (SAE), serious adverse reactions (SAR), serious unexpected adverse reactions (SUSAR)

2) Latent reservoir size measured in CD4+ T cells by:

a) HIV-1 viral outgrowth assay (HIV-1 RNA per 106 in resting memory CD4+ T cells (RUPM)) b) Integrated HIV-1 DNA (copies per 106 CD4+ T cells)

c) Total HIV-1 DNA (copies per 106 CD4+ T cells)

Secondary Endpoints PART B

1) Time to re-initiation of cART

2) Time to detectable viremia during cessation of cART

3) HIV transcription measured as cell associated unspliced HIV-1 RNA (copies per 10⁶ CD4+ T cells)

4) HIV-specific T-cell responses as measured by ELISpot, proliferation and/or intracellular cytokine staining

5) Plasma HIV-1 viral load

6) Histone H3 acetylation as measured in lymphocytes

7) T cell count and phenotype 8) Antibody titer to Vacc-4x peptides and to p24 as measured by ELISA.

An Open Phase I/IIa Study to Evaluate the Effect of Therapeutic HIV-1 Immunization using Vacc-4x + rhuGM-CSF, and HIV-1 Reactivation using Romidepsin, on the Viral Reservoir in Virologically Suppressed HIV-1 Infected Adults on cART. The study is conducted to evaluate the safety/tolerability of Vacc-4x + rhuGM-CSF as adjunctive therapy to Romidepsin and to assess the impact on the latent HIV reservoir and the ability to control viral load during an Analytical Treatment Interruption (n=20, ie. 20 patients).

Target Population: Virologically suppressed (pVL < 50copies/mL) HIV-1 infected adults currently on cART.

Study Procedures/frequency:

1. A pre-treatment phase of 4 weeks (visit 1 to visit 2) to confirm the stability of the latent HIV-1 reservoir and determine baseline HIV-1 T lymphocyte specific immunity.

2. A therapeutic HIV-1 immunization phase of 12 weeks (from visit 2 to visit 7) in which

Vacc-4x will be administered together with rhuGM-CSF at visit 2, 3, 4, 5, 6 and 7 follow by a follow-up period of 2 weeks (visit 8- visit 9).

3. A viral reactivation phase of 3 weeks (visit 10-visit 12) consisting of one cycle of

Romidepsin infusions at a dosing of 5 mg/m2.

4. A post-treatment observation phase of ~8 weeks (visit 13-visit 14) to assess the effect of the investigational treatment on the size of the latent HIV-1 reservoir.

5. An Analytical Treatment Interruption phase of 16 weeks (from after visit 15-34).

Investigational Medicinal Products:

Vacc-4x: 1.2mg administered intradermally at day 0, 7, 14, 21, 77 and 84 (visit 2, 3, 4, 5, 6 and 7)

rhuGM-CSF: Leukine® (Sanofi) 0.06mg administered intradermally, 10 min prior to Vacc-4x administration, at day 0, 7, 14, 21, 77 and 84 (visit 2, 3, 4, 5, 6 and 7)

Romidepsin: Istodax® (Celgene) 5mg/m2 administered by 3 intravenous infusion in three consecutive weeks (day 105, 112 and 119) (visit 10, lib and 12) (corresponding to one 28 day cycle).

Trial Design:

2. A therapeutic HIV-1 immunization phase of 12 weeks (2to visit 7) in which Vacc-4x will be administered together with rhuGM-CSF at visit 2, 3, 4, 5, 6 and 7 followed by a follow-up period of 2 weeks (visit 8 to visit 9) . 3. A viral reactivation phase of 3 weeks (visit 10 to visit 12) consisting of one cycle of romidepsin infusions at a dosing of 5 mg/m2.

4. A post-treatment observation phase of ~8 weeks (visit 13to visit 14) to assess the effect of the romidepsin on the size of the latent HIV-1 reservoir.

5. An Analytical Treatment Interruption phase of 16 weeks (visit 15-34).

Treatment

Vacc-4x

Vacc-4x, consists of four synthetic peptides (Vacc-10 acetate, Vacc-11 acetate, Vacc-12 acetate, and Vacc-13 acetate), each corresponding to conserved domains on the HIV-1 p24 capsid protein representing the native Gag regions with residues 166-185, 252-269, 264- 284, and 335-354, respectively.

Vacc-4x is manufactured in accordance with Good Manufacturing Practice (GMP) (ref. Vacc-4x IMPD). Vacc-4x is supplied as sterile vials of freeze-dried white powder. There is no additional ingredient in the product.

RhuGM-CSF (sargramostim, Leukine®, Sanofi)

Leukine® is manufactured by Sanofi and supplied by Genzyme. It is a glycoprotein of 127 amino acids characterized by three primary molecular species having molecular masses of 19,500, 16,800 and 15,500 daltons. The liquid Leukine® presentation is formulated as a sterile, preserved (1.1% benzyl alcohol), injectable solution (500mcg/mL) in a vial.

Lyophilized Leukine® is a sterile, white, preservative-free powder (250mcg) that requires reconstitution with 1 mL Sterile Water for Injection, USP or 1 mL Bacteriostatic Water for Injection, USP. Liquid Leukine® has a pH range of 6.7 - 7.7 and lyophilized Leukine® has a pH range of 7.1 - 7.7.

For further information refer to IB (Leukine® prescribing information).

Romidepsin (Istodax®, Celgene)

Istodax® is manufactured by Celgene Corporation. This histone deacetylase (HDAC) inhibitor is a bicyclic depsipeptide. At room temperature, romidepsin is a white powder and is described chemically as (lS,4S,7Z,10S, 16E,21R)-7-ethylidene-4,21-bis(l-methylethyl)-2- oxa-12, 13-dithia-5,8,20,23-tetraazabicyclo[8.7.6] tricos-16-ene-3,6,9, 19,22-pentone. The empirical formula is C24H36N406S2. Istodax® is supplied as a kit containing two vials.

Istodax® (romidepsin) for injection is a sterile lyophilized white powder and is supplied in a single-use vial containing lOmg romidepsin and 20mg povidone, USP. Diluent for Istodax® is a sterile clear solution and is supplied in a single-use vial containing a 2-mL deliverable volume. Diluent for Istodax® contains 80% (v/v) propylene glycol, USP and 20% (v/v) dehydrated alcohol, USP. For further information refer to IB for romidepsin .

Vacc-4x

Each dose of Vacc-4x (O. lmL of a 12mg/mL solution), will be administered by intradermal injections following the intradermal administration of rhuG -CSF (Leukine®) as adjuvant. A total of 6 Vacc-4x/rhuGM-CSF immunizations (visit 3, 4, 5, 6, 7 and 8) are planned in the HIV-1 therapeutic vaccination phase.

Approximately 10 minutes before each administration of Vacc-4x, rhuGM-CSF will be administered intradermally as an adjuvant. Vacc-4x must be administered intradermally at the same site as rhuGM-CSF, superficial to the deltoid muscle and in the same arm during the course of the study.

When administering the intradermal injection, utmost care must be taken so that no material is injected subcutaneously. If administered correctly, after puncture of the skin a small bleb should appear following the injection of only a small amount of product. An injection that is too superficial should be avoided as this will result in loss of the sample volume from the injection site during injection or after withdrawal of the needle.

RhuGM-CSF

Each dose of rhuGM-CSF (O. lmL of 0.60mg/mL solution) will be administered as an adjuvant by intradermal injection 10 minutes prior to the intradermal administration of Vacc-4x immunizations (visit 3, 4, 5, 6, 7 and 8) during the HIV- 1 therapeutic vaccination phase. rhuGM-CSF must be administered intradermally at the same site as Vacc-4x, superficial to the deltoid muscle and in the same arm during the entire course of the study.

When administering the intradermal injection, utmost care must be taken so that no material is injected subcutaneously. If administered correctly, after puncture of the skin a small bleb should appear following the injection of only a small amount of product. An injection that is too superficial should be avoided as this will result in loss of the sample volume from the injection site during injection or after withdrawal of the needle. Romidepsin

The dose is 5mg/m2 administered intravenously over a 4 hour period on Days 1, 8, and 15 of a 28-day cycle (visit 10, 11 and 12) .

Trial Assessment:

Laboratory Assessment

Biochemistry:

Routine biochemistry includes haematology parameters (haemoglobin, total and differential leukocyte count, platelet count), ALAT, bilirubin, alkaline phosphatase, creatinine, sodium, potassium, phosphorus, magnesium, calcium, urea, albumin and GRP. HIV Virology:

HIV- 1 viral outgrowth (HIV-1 RNA per 10⁵ resting memory CD4+ T cells (RUPM)) : The gold standard assay used to measure the frequency of resting CD4+ T cells carrying latent but replication competent virus is based on co-culture of highly purified resting CD4+ T cells from the patient together with PBMCs from an HIV-negative donor and is measured as infectious units per million cells (IUPM) [Finzi 1999, Chun 2007] .

Integrated HIV-1 DNA (copies per 10⁶ CD4+ T cells) : Within infected cells, HIV DNA can exist as linear non-integrated forms, circular forms and as an integrated provirus. In patients receiving effective cART, the majority of HIV DNA is integrated in resting latently infected CD4+T cells. The most widely used technique to quantify the number of cells that contain integrated virus is the Alu-LTR PGR assay [Sonza 1996] .

Total HIV-1 DNA (copies per 10⁶ CD4+ T cells) : Total HIV DNA quantifies integrated and non- integrated DNA as well as latent and defective virus. There is a strong correlation between total HIV DNA and integrated HIV DNA in patients on cART and therefore cell-associated HIV DNA is likely to be a good surrogate marker of the total number of latently infected cells [Koelsch 2008] .

Unspliced HIV-1 RNA (copies per 10⁶ CD4+ T cells) : HIV transcription is measured as copies of cell-associated unspliced HIV- 1 RNA/106 CD4+ T cells using digital droplet PGR

Plasma HIV-1 RNA detection by NAT screen : Measured by a transcription mediated amplification (TMA)-based methodology, usually referred to as a nucleic acid test (NAT)- screen (PROCLEIX ULTRIO Plus, Genprobe) .

Plasma HIV RNA, quantitative viral load : Measured by Roche VL (routine clinical assay) Histone H3 acetylation : Measured in lymphocytes using flow cytometry with intracellular cytokine stain on fresh isolated PBMCs.

T Cell count (CD4 and CD8)

Phylogenetic analysis

Immunology:

HIV-specific T cell response as measured by ELISpot, proliferation and/or intracellular cytokine staining

EXAMPLE 33

ELISA assay to determine levels of antibodies against HIV envelope glycoprotein gpl20 and/or gp41 Buffers

Coating buffer

3.18 g Na₂C0₃

5.88 g NaHCOs

Purified water to 1 litre, pH 7.5

Buffer 1

8.18 g NaCI

0.201 g KCI

2.12 g Na₂HP0₄ x 7H₂0

0.204 g KH₂P0₄

Purified water to 1 litre

Blocking buffer

2 L Buffer 1

20g Bovine serum albumin (BSA)

19 mL 1% NaN₃

0.2 mL Antifoam

pH 7.5

Washing buffer

7.948 g NaCI

0.201 g KCI

2.1 g Na₂HP0₄ x 7H₂0

0.2 g KH₂P0₄

9.5 mL 1% NaN₃

10.0 mL Triton-X-100

0.1 mL Antifoam

Purified water to 1 litre, pH 7.5

Dilution buffer

20g BSA

19 mL 1% NaN₃

0.2 mL Antifoam

20 mL Triton-X-100

Buffer 1 to 2 L

Stop-dilution solution

24.23 g Trizma Base

58.45 g EDTA

40.00 g NaOH

19 mL 1% NaN₃

Purified water to 2 L Vacc-C5:

Dimeric peptide, which consists of C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 with the following structure:

(H-Glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-glycyl-glycyl-L-cysteinyl(2- oxo-ethyl)-glycyl-glycyl-L-alanyl-L-lysyl-L-arginyl-L-arginyl-L-valyl-L-valyl-L-glutaminyl-L- arginyl-L-glutamyl-L-lysyl-L-arginyl-L-alanyl-glycyl-L-glutamyl-L-arginyl-L-glutamyl-L-lysyl-L- arginyl-L-alanyl-NH₂) (H-Glycyl-L-lysyl-glycyl-glycyl-L-isoleucyl-L-glutamyl-L-glutamyl-L- glutamyl-glycyl-glycyl-L-arginyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-glycyl-glycyl-L- glutaminyl-L-aspartyl-L-arginyl-L-aspartyl-L-arginyl-NH₂), acetate salt (amide bond between Cys(2-oxo-ethyl)ⁱ⁰ (A-chain) and Lys² (B-chain))

This compound may also be referred to as:

(H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg-Val-Val- Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH₂) (H-Gly-Lys-Gly-Gly-Ile-Glu-Glu- Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH₂), acetate salt (amide bond between Cys(2-oxo-ethyl)¹⁰ (A-chain) and Lys² (B-chain))

Vacc-C5 consists of two linear peptide amide chains with 31 amino acids (A-chain) and 22 amino acids (B-chain). Each chain has a free amino group at the N-terminus and an amide group at the C-terminus. The chains are covalently linked via an amide bond between Cys(2- oxo-ethyl)¹⁰ of the A-chain and Lys² of the B-chain. All amino acid residues except the achiral Gly are in the L-configuration.

H-G!y-Ala-Lys ly-Gty

u-Giu-Gly-G!y-Arg-Asp-Arg-Asp-Arg-G!y-G!y-Gln-Asp-Arg-Asp-Arg-NH₂

Method

ELISA plates (Nunc 249946, conical bottomed) were coated with Vacc-C5 at a final concentration of 16 pg/mL in coating buffer for 4 days at 4°C, washed three times with washing buffer (WB) and stored dried at 4°C until required. Blocking buffer (200 pL per well) was added and the plates were incubated at room temperature for 2 hours before washing three times with WB. Plasma samples were diluted using dilution buffer and stored at 2-8°C for up to a maximum of 24 hours. Samples (100 μΙ_) were added to the appropriate wells of the ELISA plate(s) and incubated at 37°C for 2 hours. After washing the plates six times with WB, the bound antibody was detected using a Protein G-alkaline phosphatase conjugate (Calbiochem 539305), which binds immunoglobulin, and the substrate phenolphtalein monophosphate. The conjugate was diluted and 100 μΙ_ added to each well at a concentration of 500 pg/mL The plates were incubated for 1 hour at room temperature before washing six times with WB. The substrate was added (100 μΙ_ per well) and incubated for 15 minutes at room temperature before colour development was stopped by the addition of stop-dilution solution. Colour changes of the substrate occur in proportion to the amount of bound serum antibody and values are reported as optical density (OD) readings taken at 550nm/620nm.

Each assay included a sheep anti-HIV-1 positive control serum (D7324; Aalto, Dublin, Ireland) raised against the C5 region of gpl20. The concentration of anti-C5 antibody in the test samples was determined by comparison to a standard curve prepared using dilutions of the positive control. As the positive control anti-C5 antibodies will not be identical to the anti- C5 antibodies present in HIV-infected individuals the ELISA is regarded a semi-quantitative immunoassay.

The samples were initially screened in duplicate at a 5x dilution and if the OD value was outside the linear part of the standard curve (i.e., OD>0.8) the plasma samples were diluted further and assayed again until an OD value within the linear range was obtained. If more than one value was within the linear range, the lowest dilution of sample (normally with an OD value of around 0.5) was used to calculate the concentration of anti-C5 antibodies in the sample under test.

In order to determine the region(s) of the Vacc-C5 sequence recognised by antibodies in the plasma samples, blocking experiments were conducted. Plasma samples were diluted in dilution buffer and incubated overnight at 2-8°C with either 100 or 500 μΜ of peptide. The dilution factor used for each plasma sample was calculated to give a value in the linear range for the assay. The blocking peptides used were either complete Vacc-C5 or shorter peptide sequences from within the C5 region of gpl20 (peptides 105, 104 and 015) or within the adjacent gp41 region (peptide 201). The amino acid sequences for the shorter peptide sequences were derived from the HIV-1 wild-type consensus sequence (www.hiv.Ianl.gov) The sequences of the peptides used in the blocking assays are shown in Error! Reference source not found, below and the location of the peptides within the Vacc-C5 antigen is shown diagrammatically in Error! Reference source not found..

OD values in the absence of peptide (phosphate-buffered saline (PBS) control) were taken as 100% and the percentage of blocking by each peptide calculated against this value. Subtraction from 100 gave the % inhibition for each peptide in the sample. Negative values were set to zero.

105

104

015

201 CZZZ^_J peptides within the Vacc-C5 antigen .

Table 1. Amino acid sequences of Vacc-C5 and overlapping peptides.

¹ The two peptides contained within Vacc-C5 are linked by a thioether bond between the side- chains of the marked cysteine and lysine residues.

Results:

Patients in the Phase II Study CT-BI Vacc-4x 2007/1 with high levels of pre-existing antibodies against HIV envelope glycoprotein gpl20 and/or gp41 as measured be the ELISA assay described in this example (33) and subsequently treated with Vacc-4X had a significant lower viral load (VL) set-point as compared to patients treated with placebo. The data indicates that the higher amount of antibodies against HIV envelope glycoprotein gpl20 and/or gp41, the lower was the viral load set-point and the larger the difference to the group treated with placebo.

Serum samples from patients in the Phase II Study CT-BI Vacc-4x 2007/1 (N = 115) at different time points (week 1, 28 and 52) were analysed for antibodies against Vacc-C5. The effect of the level of pre-existing antibodies against Vacc-C5 at Week 1 (prior to Vacc-4x treatment and ART interruption) on Viral Load (VL) was analyzed for 'Subgroup D' of the clinical study (LOCF population, patients that resumed ART after Week 40 that in addition had a pre-ART viral load value available) . The results are summarized in Table 2.(N = 71) . Table 2:

Throughout the specification and the claims which follow, unless the context requires otherwise, the word 'comprise', and variations such as 'comprises' and 'comprising', will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

All patents and patent applications referred to herein are incorporated by reference in their entirety.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the claims.

EXAMPLE 34 Objective.

To investigate the morbidity and immune response after challenge in mice vaccinated with a humoral vaccine, adjuvanted with Alhydrogel or CMI vaccines, adjuvanted with ISA51, in an order of immunisation experiment.

Design.

BPs candidate humoral influenza vaccine peptide is based on the external domain of influenza's M2 protein, which is expressed in the viral membrane and function as an ion channel protein. The M2e vaccine (BI150-2mod) consists of two monomers linked with a oxime linker. The CMI peptides (BI100-190, BI100-260b) are directed against NP and the M2 protein. NP and M2's properties and function are crucial in the virus' lifecycle and therefore to a large degree conserved between influenza A subtypes and strains. Peptides are disclosed in patent applications WO 2012/092934 and WO 2012/072088, A transgenic strain (C57BL/6- Tg(HLA-A2.1)) was bred at NIPH, Oslo, Norway. This mouse strain carry one allele

(heterozygote) of the human HLA A2 gene (45 percent of Caucasians are A2^* ) .

Table 1. Overview of planned vaccine groups

Immunisations, Number of

Group Vaccine Dose Adjuvant

week mice

50μς each CMI

ISA51 0, 2, 6, 5

CMI then peptide

1

humoral 50pg humoral

Alhydrogel 10, 12, 16

peptide

50μς humoral

Alhydrogel 0, 2, 6 5

Humoral peptide

2

then CM I 50pg each CMI

ISA51 10, 12, 16

peptide

Inactivated

3 0.07μςΗΑ - 10, 12, 16 5

PR8 No

4 Naive mice 5

immunisations

Mice were vaccinated a total of six times (PR8 group three times, conventional inactivated vaccine), three times with CMI peptides with ISA51 and three times with a humoral peptide adjuvanted with Aihydrogel . The mice were infected with a titrated dose of live influenza virus (identical to the inactivated virus used in the PR8 group) . To measure the weight after challenge is an established method of determining the morbidity of the animals. In figure 2 the weight loss is presented as percent of weight pre-challenge for each individual animal . The figure shows that in this homologue influenza challenge, mice vaccinated with inactivated PR8 had very little loss of weight, whereas the weight losses in the other groups were substantial .

The immune response in serum (Table 2) . The results show that only in the group that got the humoral peptide first (BI150-2mod), there was a marked IgG response towards the BI150-2mod at all time-points, also taken into account that the CMI+humoral group had not got any humoral peptide at the week 7 time point. It is also noteworthy that the PR8 group did not raise any antibodies cross-reactive towards BI150-2mod (comparable levels to naive controls) throughout the study. The IgG response towards the native sequence (BI100-CG2, Table 3) was however, lower in comparison, with the exception of the naive group that showed a clear IgG response towards BI100-CG2 after challenge. The IgG response in the Humoral+CMI group had a low, but detectable response at week 7 (after three doses of humoral peptide) .

It is noteworthy that there was 5 week lag period from last immunisation at week 6 to the next immunisation at week 11 to allow the immune response after the first immunisation to mature before the next vaccination.

Table 2. Serum IgG response against BI150-2mod (OP values, sample dilution)

Week 18 | 0.046 (ΙΟχ) | 0.727 (50x) | 0.029 (ΙΟχ) | 0.039 (lOx)

Table 3. Serum IqG response against BI 100-CG2 (OP values)

Baseline 0.101 (lOx) 0.102 (lOx) 0.062 (lOx) 0.114 (lOx) Week 7 0.093 (lOx) 0.154 (lOx) ND (lOx) ND (lOx) Week 17 0.121 (lOx) 0.117 (lOx) 0.108 (lOx) ND (lOx) Week 18 0.090 (lOx) 0.126 (lOx) 0.083 (lOx) 0.216 (lOx)

Claims

1. A method for inducing an immune response to a disease antigen, such as an infectious agent in an subject, the method comprising :

2. The method according to claim 1, wherein said second vaccine is administered after the administering of said first vaccine, optionally separated by a maturation period, such as a period of up to 4 weeks.

3. The method according to claim 1, wherein said second vaccine is administered simultaneously with the administering of said first vaccine.

4. The method according to any one of claims 1-3, wherein said first vaccine composition comprises an adjuvant, such as recombinant human granulocyte-macrophage colony- stimulating factor (rhuGM-CSF),or such as Alhydrogel.

5. The method according to any one of claims 1-4, wherein said second vaccine composition comprises an adjuvant, such as recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF), or such as ISA51.

6. The method according to any one of claims 1-5, wherein said first and second antigen is derived from the same infectious agent, such as a virus.

7. The method according to any one of claims 1-6, wherein said first and/or second disease antigen is an immunogen.

8. The method according to any one of claims 1-7, wherein said method is a treatment.

9. The method according to any one of claims 1-8, wherein said first and/or second antigen is protein, DNA, or RNA.

10. The method according to any one of claims 1-9, wherein said first and/or said second antigen is derived from a peptide, said peptide comprising a specific natural antigen of a protein or peptide sequence derived from a disease antigen, such as an infectious agent, such as bacteria, virus, parasite, fungus, or cancer antigens such as oncogene (lung, stomach, breast cancer) or an antigen causing an autoimmune disease such as diabetes, multiple sclerosis (MS), celiac disease, Myalgic Encephalomyelitis (ME), psoriasis, and/or Crohn's Disease.

11. The method according to claim 10, wherein said specific natural antigen is a viral protein, such as a structural protein, such as a capsid protein, a regulatory protein, an enzymatic protein, or a proteolytic protein.

12. The method according to claim 11, wherein said viral protein is selected from a core protein or an envelope protein, of a virus selected from the Hepatitis C virus, influenza virus, such as an M2 protein, human immunodeficiency virus (HIV), cytomegalovirus (CMV), Respiratory syncytial virus (RSV), and Human papillomavirus (HPV) .

13. The method according to claim 12, wherein said viral protein is a viral protein of Hepatitis C virus selected from any one HCV consensus sequence of a specific genotype, such as 1, such as subtypes la and lb, genotype 2, such as 2a and 2b, genotype 3, such as 3a, genotype 4, genotype 5, and genotype 6.

14. The method according to any one of claims 1 -13, wherein said first and second antigens are derived from HIV.

15. The method according to claim 14, wherein said first antigen induces antibodies that stabilise association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20.

16. The method according to claim 15, wherein said first antigen inducing antibodies stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 is a molecule comprising at least one amino acid sequence selected independently from an amino acid sequence derived from the transmembrane domain of gp41 and an amino acid sequence derived from the C2 domain, wherein the at least one amino acid sequence binds the C5 domain, optionally comprising at least one D-amino acid .

17. The method according to claim 16, wherein the amino acid sequence derived from the transmembrane domain of gp41 has an amino acid sequence of at most 10 amino acid residues.

18. The method according to any one of claims 15-16, wherein said first antigen stabilising association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20 binds to and stabilises association between one or more amino acid residues in the amino acid stretch TZ¹AKRRVVZ²REKR, where Z¹ is K, R or E and where Z² is Q or E, and one or more amino acid residues in an amino acid stretch in the transmembrane domain of gp41 and/or in the constant C2 domain of gpl20.

19. The method according to claim 18, wherein said first antigen is a peptide muitimer comprising a first peptide comprising the amino acid sequence of the 13 amino acid residue amino acid sequence of the C5 domain of HIV gpl20 including between 0 and 4 amino acid substitutions, a subsequence thereof, or an amino acid sequence comprising the inverso-, retro- or retro-inverso form of said amino acid sequence or subsequence, and at least one second peptide comprising an amino acid stretch present in the transmembrane domain of gp41 or present in the constant C2 domain of gpl20 or comprising an amino acid stretch present in any one of SEQ ID NOs. 6- 13 or comprising a inverso-, retro- or retro-inverso form of an amino acid stretch present in the transmembrane domain of gp41 or present in the constant C2 domain of gpl20, wherein said peptide muitimer is capable of inducing an antibody which can bind and stabilise the association of the C5 domain of HIV gpl20 with the transmembrane domain of gp41 and/or with the constant C2 domain of gpl20, and wherein said peptide muitimer lacks amino acids N-terminal of C5 in gpl20.

20. The method according to claim 19, wherein said first peptide comprises the amino acid sequence having formula I :

X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13 (I) wherein XI is Thr, X2 is selected from Lys, Arg, Har and Glu, X3 is selected from Ala and Val, X4 is selected from Arg, Har, Lys and Cit (citrulline), X5 is selected from Arg, Har, Lys and Cit, X6 is selected from Arg, Har, Lys and Cit, X7 is selected from Val, Leu, He and Nle (norleucin), X8 is selected from Val, Leu, He and Nle, X9 is selected from Gin, Glu, Asn and Asp, XIO is selected from Arg, Har and Cit, XI 1 is selected from Glu and Asp, X12 is Lys, and X13 is selected from Arg, Har and Cit,

or comprises a subsequence the amino acid sequence of formula I, or comprising the inverso-, retro- or retro-inverso form of said amino acid sequence or subsequence.

21. The method according to any one of claims 19-20, wherein the first peptide further comprises the dipeptide Ala-Pro linked to the N-terminus of the amino acid sequence having formula I.

22. The method according to any one of claims 19-21, wherein the first peptide further comprises the dipeptide X14-X15 linked to the C-terminus of the amino acid sequence having formula I, wherein X14 is selected from Ala and Val, and wherein X15 is selected from Val, Leu and Nle.

23. The method according to any one of claims 19-22, wherein the at least one second

peptide includes an amino acid sequence having the formula:

Z¹-Z²-Z³-Z⁴-Z⁵-Z⁶-Z⁷-Z⁸-Z⁹-Z¹⁰-Z¹¹-Z¹²-Z¹³-Z¹⁴-Z¹⁵-Z¹⁶-Z¹⁷ (III) wherein Z¹ is Asp, Z² is Arg, Z³ is Pro, Z⁴ is Glu or Gly, Z⁵ is Gly or Arg, Z⁶ is lie, Z⁷ is Glu, Z⁸ is Glu, Z⁹ is Glu, Z¹⁰ is Gly, Z¹¹ is Gly, Z¹² is Glu or is absent, Z¹³ is Arg or Gin, Z¹⁴ is Asp or Gly, Z¹⁵ is Arg or Lys, Z¹⁵ is Asp or Gly and Z¹⁷ is Arg,

or includes a subsequence of formula (III).

24. The method according to any one of claims 19-23, wherein the second peptide includes at least 5 consecutive amino acid residues from formula III.

25. The method according to any one of claims 19-24, wherein the first peptide and the at least one second peptide are associated via a linker.

26. The method according to claim 25, wherein the linker is selected from the group consisting of a bis-maleimide linker, a disulfide linker, a polyethylene glycol (PEG) linker, a glycine linker, a lysine linker, and an arginine linker.

27. The method according to any one of claims 19-26, where at least one of the first and at least one second peptides comprises an N- or C-terminal modification, such as an amidation, acylation, or acetylation.

28. The method according to any one of claims 19-27, wherein said peptide multimer is coupled to a carrier molecule, such as an immunogenic carrier.

29. The method according claim 28, wherein the carrier is a virus like particle.

30. The method according to any one of claims 19-29, wherein the first peptide is selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 38, 41 and 44 or a fragment thereof, or the inverso-, retro- or retro-inverso form of a peptides selected from SEQ ID NO: 1, 2, 3, 4, 5, 38, 41 and 44 or a fragment thereof, and wherein the second peptide is selected from the group consisting of SEQ ID NO: 6, 7, 8, 9, 10, 11 , 12, 13, 37, 39, 40, 42, 43, 45, 46 or a fragment thereof, or the inverso-, retro- or retro-inverso form of a peptides selected from SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 37, 39, 40, 42, 43, 45, 46 or a fragment thereof, and/or wherein the peptide multimer is selected from the peptides having SEQ ID NOs: 1 -46.

31. The method according to any one of claims 19-30, wherein said peptide multimer comprises at most 70 amino acids.

32. The method according to any one of claims 19-31, wherein said peptide multimer comprises at least 6 amino acid residues.

33. The method according to any one of claims 19-32, wherein said peptide multimer consist of a number of amino acid residues selected from the group consisting of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 amino acid residues.

34. The method according to any one of claims 19-33, wherein said peptide multimer is selected from the group consisting of disulphide linked peptides between SEQ ID NO: 28 and any one of SEQ ID NOs: 29, 31, and 33, between SEQ ID NO: 30, and any one of SEQ ID NO: 29, 31, and 33, or between SEQ ID NO: 32 and any one of SEQ ID NO: 29, 31, and 33; or selected from the group consisting of cysteine-lysine linked peptides between SEQ ID NO : 38 and any one of SEQ ID NO: 39, SEQ ID NO:40; SEQ ID NO: 42, SEQ ID NO:43, and SEQ ID NO: 68, or between SEQ ID NO:41 and any one of SEQ ID NO: 39, SEQ ID NO: 40; SEQ ID NO:42, and SEQ ID NO:43.

35. The method according to any one of claims 19-34, wherein said peptide multimer is selected from the group consisting of: CGGAKRRVVGGAKRRVVGQREKRAV (SEQ ID NO: 28)

I

CGGGDQQLLGGAEEEIVGGIEEEGGERDRDR (SEQ ID NO: 29),

CGGAKRRVVGGAKRRVVGGQREKR (SEQ ID NO: 30)

I

CGGGDQQLLGGAEEEIVGGIEEEGG (SEQ ID NO : 31),

CGGAEEEVVGGDQQLL (SEQ ID NO: 32)

I

GCGGAKRRVVGGAKRRW (SEQ ID NO:33),

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)

I

G GGIEEEGGRDRDRGGEQDRDR (SEQ ID NO: 39),

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)

I

GKGGIEEEGGERDRDRGGQDRDR (SEQ ID NO:40),

GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO:41)

I

G GGIEEEGGQDRDRGGRDRDR (SEQ ID NO:42), GAKRRVVGGCGGAKRRVVEREKRAGQREKRA (SEQ ID NO:41)

G GGIEEEGGEQDRDRGGERDRD (SEQ ID NO:43) and

GAKRRVVGGCGGAKRRVVQREKRAGEREKRA (SEQ ID NO: 38)

GKGGIEEEGGRDRDRGGQDRDR (SEQ ID NO: 68) .

36. The method according to any one of claims 19-35, wherein said peptide multimer is selected (H-Gly-Ala-Lys-Arg-Arg-Val-Val-Gly-Gly-Cys(2-oxo-ethyl)-Gly-Gly-Ala-Lys-Arg-Arg- Val-Val-Gln-Arg-Glu-Lys-Arg-Ala-Gly-Glu-Arg-Glu-Lys-Arg-Ala-NH2) (H-Gly-Lys-Gly-Gly-Ile- Glu-Glu-Glu-Gly-Gly-Arg-Asp-Arg-Asp-Arg-Gly-Gly-Gln-Asp-Arg-Asp-Arg-NH2), acetate salt (amide bond between Cys(2-oxo-ethyl) 10 (A-chain) and Lys2 (B-chain)) .

37. The method according to any one of claims 14-36, wherein said second antigen is at least one HIV-specific peptide selected from the group of amino acid sequences:

Xaai Xaa₂ Xaa₃ Xaa₄ Xaa₅ Xaa₆ Ala Xaa₈ Xaa_g Gin Thr Pro Trp Xaa₅₄ Xaa-_{i 5} Xaa_i6 Xaa_{i 7} Xaa_i8 Val Xaa₂₀ (SEQ ID NO : 47) ;

wherein Xaa in position 1 is Lys or Arg, Xaa in position 2 is Ala, Gly, Ser or Arg,

Xaa in position 3 is Leu or Met,

Xaa in position 4 is Gly or Arg,

Xaa in position 5 is Pro, Thr, Val, Ser, Gin or Ala,

Xaa in position 6 is Gly, Ala, Lys, Arg, Gin or Glu,

Xaa in position 8 is Thr or Ser,

Xaa in position 9 is Leu or He,

Xaa in position 14 is Thr, Ser or Val,

Xaa in position 15 is Ala or Ser,

Xaa in position 16 is Cys or Ser,

Xaa in position 17 is Gin or Leu,

Xaa in position 18 is Gly, Glu or Arg, and

Xaa in position 20 is Gly or Arg; Xaa, Xaa₂ Xaa₃ Xaa Xaa_s Gly Leu Asn Pro Leu Val [Gly]_n Xaa_i2 Xaa_i3 Tyr Xaa₁₅ Pro Xaa₁₇ Xaa₁₈

He Leu Xaa₂₁ Xaa₂₂ (SEQ ID NO: 50) ;

wherein Xaa in position 1 is Arg, Lys, Asp or none,

Xaa in position 2 is Trp, Gly, Lys or Arg,

Xaa in position 3 is He, Leu, Val or Met,

Xaa in position 4 is He, Val or Leu,

Xaa in position 5 Leu, Met, Val or Pro,

Xaa in position 12 is Arg or Lys,

Xaa in position 13 is Met or Leu,

Xaa in position 15 is Ser, Cys or Gin,

Xaa in position 17 is Thr, Val, He, Ser or Ala,

Xaa in position 18 is Ser, Gly or Thr,

Xaa in position 21 is Asp, Glu, Cys or Gly,

Xaa in position 22 is Gly or none, and

n = 0, 1, 2 or 3;

Xaa, Xaa₂ Xaa₃ Pro He Pro Xaa₇ Xaa₈ Xaa₉ Xaa_i0 Xaa Xaa_i2 [Gly]_n Xaa-_{l 3} Xaa_i Xaa_{i 5} Xaa_i6 Xaa_{i 7} Xaais Xaa_i9 Xaa₂₀ Xaa₂₁ Xaa₂₂ Xaa₂₃ Xaa₂₄ (SEQ ID NO : 55) ;

wherein Xaa in position 1 is Asn, Ser, Gly, His, Ala, Pro, Arg or none,

Xaa in position 2 is Asn, Ala or Lys,

Xaa in position 3 is Pro, Gin, Gly, He or Leu,

Xaa in position 7 is Val or Ala,

Xaa in position 8 is Gly or Lys,

Xaa in position 9 is Glu, Asp, Lys, Phe or Thr,

Xaa in position 10 is He, Met, Val or Leu, Xaa in position 11 is Tyr, Leu or none,

Xaa in position 12 is Ser or none,

Xaa in position 13 is Arg or none,

Xaa in position 14 is Asp, Arg, Trp, Ala or none

Xaa in position 15 is He or none,

Xaa in position 16 is Tyr or none,

Xaa in position 17 is Lys or Arg,

Xaa in position 18 is Arg, Lys or Asp,

Xaa in position 19 is Trp or Gly,

Xaa in position 20 is He, Met, Val, Gin or Ala,

Xaa in position 21 is He, Val or Ala,

Xaa in position 22 is Leu, Met or Val,

Xaa in position 23 is Gly or Cys,

Xaa in position 24 is Leu or none,

n = 1, 2 or 3; and

Xaai Xaa₂ He He Xaa₅ Xaa₆ Xaa₇ Xaa₈ Xaa_g Leu Xaau [Gly]_n [Arg]_m Xaai ₂ Xaa_{i 3} Xaa^ Xaa^

Xaa_i6 Xaa i 7 Xaa_i8 Xaa_i9 Xaa₂o Xaa₂i Xaa₂₂ Xaa₂₃ Xaa₂₄ Xaa₂₅ (SEQ ID NO : 61) ;

wherein Xaa in position 1 is Pro, Lys, Arg or none,

Xaa in position 2 is Glu, Arg, Phe or Lys,

Xaa in position 5 is Pro or Thr,

Xaa in position 6 is Met, Thr or Nleu,

Xaa in position 7 is Phe or Leu,

Xaa in position 8 is Ser, Thr, Ala or Met,

Xaa in position 9 is Ala, Glu or Leu,

Xaa in position 11 is Ser or none,

Xaa in position 12 is Ala, Arg or none,

Xaa in position 13 is He, Leu or none,

Xaa in position 14 is Ser, Ala, Leu or none,

Xaa in position 15 is Tyr, Glu or Asp,

Xaa in position 16 is Gly or Asp,

Xaa in position 17 is Ala or Leu,

Xaa in position 18 is Thr, He, Val, Leu or Asn,

Xaa in position 19 is Pro, Thr or Ser,

Xaa in position 20 is Tyr, Phe, Nleu, His or Gin,

Xaa in position 21 is Asp, Asn, Leu or Ala,

Xaa in position 22 is Leu, He, Val or Asn,

Xaa in position 23 is Asn, Tyr, Cys or Gly,

Xaa in position 24 is Thr, Met, He, Ala, Val or none, Xaa in postion 25 is Gly or none,

n = 1, 2 or 3 and m= 0, 1, 2 or 3 independent of each other; wherein the terminal ends of each HIV specific peptide may be free carboxyl- or amino- groups, amides, acyls or acetyls or salts thereof, such as wherein each peptide is in the form of an acetate salt.

38. The method according to claim 37, wherein the amino acid sequence of SEQ ID NO:47 is selected from the groups of SEQ ID NO:48 and SEQ ID NO:49.

39. The method according to any one of claims 37 or 38, wherein the amino acid sequence of SEQ ID NO: 50 is selected from the groups of SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO : 54.

40. The method according to any one of claims 37-39, wherein the amino acid sequence of SEQ ID NO: 55 is selected from the groups of SEQ ID NO: 56 SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60.

41. The method according to any one of claims 37-40, wherein the amino acid sequence of SEQ ID NO: 61 is selected from the groups of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ ID NO: 66.

42. The method according to any one of claims 37-41, wherein at least one HIV-specific peptide comprises at least , two, three, or four peptides selected from each of the groups of SEQ ID NO:47, SEQ ID NO : 50, SEQ ID NO: 55 and SEQ ID NO: 61.

43. The method according to any one of claims 37-42, wherein at least one HIV-specific peptide consist of or comprises the peptides of the SEQ ID NO:49, SEQ ID NO: 52, SEQ ID NO: 57 and SEQ ID NO: 64.

44. The method according to any one of claims 37-43, wherein said one or more peptide is in the form of an acetate salt.

45. The method according to any one of claims 37-44, wherein one, two, three or four peptides are used in the therapeutic HIV-1 immunization phase.

46. The method according to any one of claims 37-45, wherein all four peptide as acetate salts are used in the therapeutic HIV-1 immunization phase.

47. The method according to any one of claims 37-46, wherein the peptides have amide C- terminal ends of formula -C(0)NH2, or acetate salts thereof.

48. The method according to any one of claims 37-47, wherein all four peptide are used in the ratio of 1 : 1 : 1 : 1 w/w.

49. The method according to any one of claims 37-48, wherein said one, two, three or four peptide acetate salts are in a dissolved liquid state.

50. The method according to claim 49, wherein said liquid is water.

51. The method according to any one of claims 1-50, which method further comprises the administration of an immunomodulatory compound and/or a reservoir purging agent, such as a histone deacetylase (HDAC) inhibitor.

52. The method according to claims 51, which method further comprises the subsequent administering of an effective amount of a reservoir purging agent.

53. The method according to claims 51 , which method further comprises the administering of an immunomodulatory compound.

54. The method according to claim 53, wherein said immunomodulatory compounds is selected from anti-PDl antibodies, such as MDX- 1106 (Merck), THALOMID " (thalidomide), anti-PDl antibodies, cyclophosphamide, Levamisole, lenalidomide, CC-4047 (pomalidomide), CC-11006 (Celgene), and CC-10015 (Celgene), and immunomodulatory compound described in any one of WO2007028047, WO2002059106, and WO2002094180.

55. The method according to claim 53, wherein said immunomodulatory compound is selected from a 4-(amino)-2-(2,6-dioxo(3-piperidyl))-isoindoline-l , 3-dione and a 3-(4- amino-l -oxo-l, 3-dihydro-isoindol-2-yl)-piperidine-2,6-dione.

56. The method according to any one of claims 54-55, wherein said immunomodulatory compound is enantiomerically pure.

57. The method according to claims 52, which method further comprises the administering of a reservoir purging agent, such as a Histone deacetylase (HDAC) inhibitor, such as romidepsin or panobinostat.

58. The method according to claim 57, wherein said reservoir purging agent, such as a Histone deacetylase (HDAC) inhibitor is selected from M344 (4-(dimethylamino)-N- [7- (hydroxyamino)-7-oxoheptyl]benzamide), chidamide (CS055/HBI-800), 4SC-202, (4SC), Resminostat (4SC), hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101 ), LAQ824, trichostatin A and panobinostat (LBH589) ; benzamides such as entinostat (MS-275), CI994, and mocetinostat (MGCD0103), cyclic tetrapeptides (such as trapoxin, such as trapoxin B), and the depsipeptides, such as romidepsin (ISTODAX), electrophiiic ketones, and the aliphatic acid compounds such as phenylbutyrate, valproic acid, Oxamflatin, ITF2357 (generic givinostat), Apicidin, MC1293, CG05, and CG06; compounds that activate transcription factors including NF-KappaB, Prostratin, auranofin, bryostatin, a nontumorigenic phorbol ester, DPP ( 12-deoxyphorbol-13-phenylacetate), PMA, and Phorbol 12-myristate 13- acetate (PMA) ; Compounds that activate HIV mRNA elongation including P-TEF-b kinase and hexamethylbisacetamide (HMBA) ; IL-7; T-cell stimulating factors including anti-CD3/CD28 - T-cell stimulating Ab's; Kinase inhibitors including Tyrphostin A, Tyrphostin B, and Tyrphostin C; PTEN (phosphatase and tensin homologue) gene inhibitors including SF1670 (Echelon

Bioscience), Disulfiram (DSF), an inhibitor of acetaldehyde dehydrogenase, Protein Tyrosine Phosphatase Inhibitors including bpV( HOpic), bpV(phen), and bpV(pic) (Calbiochem; EMD Millipore), Toll-like receptors agonists including Toll-like receptor-9 (TLR9) and Toll-like receptor- 7 (TLR9) agonists, quercetin, lipoic acid, sodium butyrate, TNF-alpha, PHA, Tat.

59. A first and second vaccine composition for use in a method for inducing an immune response to a disease antigen, such as an infectious agent in an subject, the method comprising the steps of:

60. The first and second vaccine composition according to claim 59, wherein said method is as defined in any one of claims 1-58.

61. A method for inducing an immune response to a disease antigen, such as an infectious agent in an subject, the method comprising : (a) testing for and establishing the presence of a sufficient humoral immune response to a first antigen derived from said disease antigen, such as an infectious agent, in said subject; and simultaneously or followed by

(b) administering a vaccine composition comprising an immunogenically effective amount of at least one second antigen derived from said disease antigen, such as an infectious agent, to said subject, sufficient to elicit a Cell Mediated Immune (CMI) response against said second antigen.

62. The method according to claim 61, wherein said vaccine composition comprising an immunogenically effective amount of at least one second antigen derived from said disease antigen is a second vaccine composition as defined in claim 1 and as defined in any one of claims 5-14, 37-50.