GB2440528A

GB2440528A - Antigen-antibody complexes

Info

Publication number: GB2440528A
Application number: GB0614962A
Authority: GB
Inventors: Emilio D Ugo; Maria Rapicetta; Roberto Giuseppetti; Claudio Argentini
Original assignee: Istituto Superiore di Sanita ISS
Current assignee: Istituto Superiore di Sanita ISS
Priority date: 2006-07-27
Filing date: 2006-07-27
Publication date: 2008-02-06
Also published as: GB0614962D0

Abstract

An antigen-antibody complex comprising at least one chronic disease antigen and at least one antibody capable of recognising at least one epitope on said antigen. The ratio of antigen bound to antibody in the complex is from 1:1 to 800:1 ug/mIU and the antigen is present in larger excess. This complex is particularly useful in the treatment or prophylaxis of patients with chronic diseases such as cancers and viral infections, in particular, hepatitis B virus, hepatitis A, hepatitis C, hepatitis D, hepatitis E, hepatitis F, hepatitis G, human papilloma virus (HPV). The antigen may also be a tumour or HIV antigen. Specific Hepatitis B antigens claimed are the Small (SEQID NO: 4). Medium (SEQQ ID NO: 3) or large preS/S (SEQ ID NO:2) proteins.

Description

M&C Folio: GBP93808 Complex for Use in Vaccines The present invention

relates to an antigen-antibody complex comprising at least one chronic disease antigen, the at least one antigen comprising at least one epitope, and at least one antibody capable of recognising the at least one epitope. This complex is useful in the treatment or prophylaxis of chronic disease conditions, particularly persistent viral infections.

The improvement of antigen presentation to the immune system relies on using molecules to drive and improve the immune response. The major aim of immunotherapy particularly in chronic infections is to improve and regulate the response against pathogens that induce tolerance and persistent infection.

The human hepatitis B virus (HBV) is a natural model to study the persistence and immunological tolerance that results from the compromising of the immune response in chronically infected patients to the viral antigens. The cell-mediated response (CD8+) both in humans, and in animal models, is weaker than in acute and resolved infections (1,2a).

The possible origin of the tolerance remains under debate and various mechanisms have been used to explain it. These include the exhaustion of competent lymphocytes, inappropriate antigenic presentation leading to an insufficient response, as well as the low affinity of the T Cell Receptor (ICR) for the antigen when presented by the Major Histocompatibility Complex (MHC).

I-IBV belongs to the family of hepadnaviruses. The HBV genome is a relaxed circular, partially double-stranded DNA of approximately 3,200 base pairs. There are four partially overlapping open reading frames encoding the envelope (pre-SIS), core (precore/core), polymerase, and X proteins.

The pre-S/S open reading frame encodes the Large, Middle, and Small surface glycoproteins. The precore/core open reading frame is translated into precore polypeptide, which is modified to a soluble protein, the hepatitis B e antigen (HBeAg), and the nucleocapsid core protein hepatitis B core antigen (I-lBcAg). The polymerase protein functions as reverse transcriptase as well as a DNA polymerase. The X protein is a potent transactivator and may play a role in hepatocarcinogenesis.

In addition to causing acute and chronic hepatitis, HBV is considered to be a major etiological factor in the development of human hepato-cellular carcinoma (HCC). HBV remains a major global concern as some 300 million people are chronic carriers of the virus and of these, a significant minority suffer severe pathologic consequences including cirrhosis and HCC, as mentioned above. In fact, HBV is second only to tobacco among the known human carcinogens and studies indicate that HCC may develop with a hundred times greater frequency amongst HBV carriers and controls. Over one million of such infected individuals die each year.

The emergence of escape mutants, able to overcome prophylactic vaccination, and the lack of effective vaccines or treatments capable of leading to viral clearance has lead to a great deal of work being undertaken in relation to HBV. Similarly, combating immunological tolerance or anergy has become a major target in the treatment of other chronic infections and conditions, such as cancer.

EP 0 913 157 Al, in the name of Shanghai Medical University, discloses a composite vaccine containing an antigen (a single polypeptide), specific antibodies raised to the antigen, and recombinant plasmid DNA harbouring encoded genes of identical or different proteins. The antigen is of a microbial source, i.e. a bacterium. Essentially, this disclosure relates to the use of antibodies or DNA as adjuvants.

US 2005/0180983 Al, in the name of Ceildex Therapeutics, Inc., discloses using an antibody to target specific receptors on Antigen Presentation Cells (APC), so as to convey the antigen to the target. This is particularly in relation to EETA-H CG, targeted at the mannose receptor, in order to induce a cytotoxic t-cell (CTL) response.

However, there is still a pressing need in the art for effective treatments, including prophylaxis, for chronic infections, diseases and conditions.

Surprisingly, the present inventors have discovered that, when administered, an antigen and antibody complex can, in fact, lead to a rapid decline of WHy viral DNA in long-term infected woodchucks, a well-established I-IBV model. What is particularly surprising is that this complex must be formed with a large excess of antigen compared to antibody.

Thus, in the first aspect, there is provided an antigen-antibody complex comprising: at least one chronic disease antigen, the at least one antigen comprising at least one epitope, and at least one antibody capable or recognising the at least one epitope, characterised in that the ratio of antigen bound to antibody in the complex is from 1:1 to 800:1.tg!m1U (micrograms! milli-Intemational Units of antibody).

It is preferred that the ratio of antigen bound to antibody in the complex is from 2,64:1 to 16:1 pg/mJU (micrograms! milli- International Units of antibody). Preferably, the antigen is present as 6,6-40 g (micrograms) of protein, relative to 2,5 mm (mull-International Units) of antibody.

The antigen may be derived from any one of a number of chronic diseases, including infections, particularly viral infections, most preferably any one of the hepatitis viruses, hepatitis A, hepatitis C, hepatitis D, hepatitis E, hepatitis F and hepatitis G. However, it most particularly preferred that the antigen is derived from hepatitis B (HBV).

The skilled person will understand that the ratio between antigen and antibodies can be altered, within the above confines, specific for each disease. The main points to be considered are the amounts of antibodies able to neutralise the viral infection (mIU, reciprocal diluition, weight, ODs) in respect of a large amount of antigen.

The ratio is expressed in micrograms/mIU, referring to milli-International Units of antibody. This is an "international" well established and standardised method of quantifying and detecting antibodies. Other ways to measure the antibodies (as for example the weight in mg of total antibodies) are though to be unreliable. It is preferred that the reading is performed by an ELISA test, a standard procedure in the art, as discussed below.

In the case of HBV, by convention, seroconversion to anti-I-lBs positivity is defined as detection of anti-HBs in an immunological assay at 2.1 standard deviations from the reading of the negative control (2b), which is usually about I mILJ/ml (2.1-9 mlU/ml).

Seroprotection against clinical disease is present when anti-HBs levels are 10 mIU/ml (2bis). For the passive immunisation between 1-5 mIU is used in transplanted patients.

The antibody used in the experiments in the Immunogenic Complexes (2,5 mIU) will be barely detected following our standard proceedings and near to the cut off. From this evaluation it is clear that the excess of antigen in respect of antibodies is considerable.

Similarly, in the case of HCV, anti-HCV antibodies were demonstrated to neutralise the infection of the animal model (chimpanzee) at the highest dilution that gave a reading above the cut-off value specified for the kit used to detect the antibodies.

It is also preferred that, where the chronic disease is a virus, the antigen is derived from the surface or envelope of the virus. Particularly preferred is the pre-S/S protein from HBV. As mentioned above, the pre-S/S polypeptide can comprise the Small, Medium and Large proteins, and it is preferred that the antigen can be selected from any one of these proteins. An epitope from any one of these proteins is particularly preferred.

Such antigens will be readily apparent to the skilled person. However, for the avoidance of doubt, the nucleotide sequence of the pre-S/S Open Reading Frame is given in SEQ ID NO 1, (see also Genbank #AB2 19427). Pre-S antigens, and particularly pre-S, express highly immunogenic I and B cell epitopes (references 2c, 2d, 2e and 20, contained within the "L" protein (Genbank #BAE80752.l, SEQ ID NO.2), the "M" protein (Genbank #BAE80753. 1, SEQ ID NO.3) and the "S" protein (Genbank #BAE80748. 1, SEQ ID NO. 4). Thus, the antigen can be a full or partial fragment of any of these protein sequences that comprises an epitope.

Preferably, the antigen shares at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, more preferably at least 99.5%, and most preferably at least 99.9% sequence identity with any of SEQ ID NOS.2-4, which may be by substitution, insertion or deletion, provided that an epitope is presented. Dayhoff's rules may be used to provide conservative amino acid changes.

Preferably, the antigen is, therefore, encoded by the nucleotide sequence of pre-s/S, being SEQ ID NO.1 or a homologue thereof having preferably at least 80% sequence homology thereto. Preferably, the sequence has at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 99%, more preferably at least 99.5%, and most preferably at least 99.9% sequence identity with SEQ ID NO.1, which may be by substitution, insertion or deletion.

Sequence homology or identity may be identified by the BLAST program, for instance.

The antigen comprises at least one epitope recognised by the antibody. Although it is preferred that the antigen comprises only a single epitope recognised by the present antibody, it is also envisaged that the antigen may comprise multiple epitopes, all for the same antibody. In other words, the antibody may bind at a variety of places on the same antigen, or the antigen may, in fact, be a multimer, each monomer thereof displaying at least one, and preferably only one, epitope, recognised by the antibody.

The antibody is capable of recognising and specifically binding to the epitope. In other words, the three dimensional structure of the complementarity-deterrnining regions (CDR) of the antibody variable region is able to form strong interactions with the three dimensional surface structure of the epitope on the antigen, leading to the specific binding mentioned above.

Preferably, the antibody consists of two light chains and two heavy chains, together forming the Fab arid Fc fragments. However, it is also envisaged that the antibody may be formed of only a single chain.

Preferably, the immunoglobulin molecules consist of a basic unit of four polypeptide chains, two identical H chains and two identical L chains, held togheter by a number of disulfide bounds. It should be noted that papain digestion of irnmunoglobulin molecule results in cleavage N-terminally to the disulfide bridge between heavy chains a the hinge region, yielding two monovalent Fab fragments and an Fc fragment.

Alternatively, but less preferably, pepsin digestion may result in cleavage C-terminally to the disulfide bridge, resulting in a divalent fragment referred to as F(ab')2, consisting of two Fab fragments joined by the disulfide bond and several Fc subfragments (this method is used in new anti-TNF biological products, where the Fc is depleted).

In both cases the antibody should have an antigen-binding region with high specificity for the antigen, and an effector function, for instance the constant region. Preferably, the constant region is capable of binding to Complement or the FC receptor (FCR) of neutrophils or dendritic cells.

The antibodies may be polyclonal, but are preferably monoclonal antibodies.

Antibodies should be derived from humans or be humanised, when the treatment is for humans. For other animals, the Antibodies should be specific to that species or adapted in such a way that they are not detected as foreign by the host. Without being bound by theory, it is thought that only a species-specific structure of the heavy chain can, eventually, induce the proper immune response. The antigen, on the contrary, could be produced in animal, synthesised or purified from the natural host. It is particularly preferred that the antigen and the antibodies are derived from the proper species, in other words the same species as the patient. A personalisation of the protocols could be applied, in which the antigen is derived from the patients (in large excess also pooling specimen serially collected) and the antibody is produced in a standardised and controlled route, as in the Example.

As discussed above, we have surprisingly discovered that, by using a complex of antigen and antibody in a particular ratio of antigen to antibody, where the antigen is present in a large excess, administration of this complex leads to significant and long-term viral clearance, as measured by the level of HBV viral DNA.

In the formation of the Complex, it is important that the Antigen is present in a large excess compared to the Antibody. In fact, it is preferred that the quantity of Antibody is limited to the smallest possible amount of Antibody, in other words the smallest quantity of Antibody detectable by appropriate methods, such as an appropriate ELISA or Microarray. Indeed, it is even envisaged that only a single Antibody molecule is present.

Preferably, the ratio of antigen bound to antibody is from 6:1 to 60:1, preferably 16:1 to 100:1, preferably 50:1 to 700:1, preferably 100:1 to 600:1, preferably 150:1 to 550:1, preferably 200:1 to 500:1, preferably 250:1 to 450:1, preferably 300:1 to 400:1, more preferably 250:1 to 750:1, 500: to 800:1, or 500:1 to 800:1. It is also preferred that the ratio of antigen bound to antibody in the complex is from 20:1 to 250:1, preferably 50:1 to200:1 or 100: to 250:1. Particularly preferred are ratios of 16:1 to 2:1, morepreferably 2:1 to 1:1, more preferably 4:1 to 2:1, more preferably 1:1 to 10:1, more preferably 3:1 to 20:1 and mostpreferably 8:1 to 4:1.

Any of the above ranges are preferred and can be readily determined by the skilled person, based on the patient, means of administration, and nature of the chronic disease.

One of the particular advantages of the present invention its reduced toxicity, compared to previous preparations. in these previous preparations, where there is a large amount of both antigen and antibodies, this is often been found to be toxic or have severe side effects. However, this is avoided in the present invention, which does not require a high level of antibodies to be present.

Further advantages of the present invention are given below and include strong antiviral activity resulting in effective reduction of serum DNA level and anti-HBE sero-conversion, reactivation of a previously silent specific CTL cellular response and, in particular, this is the first time that this ahs been associated with a break in immune tolerance to HBV.

With regard to the ratio of antigen bound to antibody in the complex, it is particularly preferred that the amount of antibody is similar or less than that which would normally be considered sufficient to lead to neutralisation of a specified amount of antigen. In other words, it is preferred that levels of antibody, similar or less than that normally considered to lead to neutralisation, are used when forming the present antigen-antibody complex.

The main point remains the large excess of antigen that must be always preferred.

With regard to the antigen-antibody complex itself, this may consist of a single antibody bound to a single antigen, or may comprise a number of antibodies bound to the same antigen or a number of antibodies bound to a number of antigens and linked together in some way. In particular, either the antigen or the antibody, or both, may be multivalent or formed from multimers.

The antibody may be selected from any of a range of isotypes. The isotype is preferably IgG, as IgG levels often remain elevated for long periods of time following primary and, indeed, further challenge. However, 1gM is also particularly preferred as 1gM was effective in the control of virus replication in the present case.

Polyclonal and degenerate antibodies are also preferred. An immune secondary response could also be based on polyclonal antibodies with low affinity and/or avidity. In the case of pathologies in which tolerance or anergy are implied, degenerate antibodies could be functional in reactivating the impaired immune responses. Each protocol could be assessed specifically.

The antigen is preferably in the form of a particle, such as an entire virus particle, active or inactivated, a virusome or virus-like particle (VLP). In this way, multiple antigens can be presented on the viral envelope, or its equivalent, and the antibodies of the present invention can bind thereto, provided that they are in the appropriate ratio to the antigens.

A virusome is a unilamellar phospholipid bilayer visicle presenting virus antigens for use in vaccination. These are semi- synthetic complexes derived from nucleic acid-free viral particles. Essentially, the virusome is a reconstituted viral coat, wherein the infectious nucleocapsid is replaced by a compound of choice, in this case the antigen according to the present invention. Virusomes retain their fusogenic activity and, therefore, deliver any incorporated compounds such as antigens, drugs, or genes inside the target cell, for instance.

Thus, where the antigen is presented on, or forms part of, a virusome or VLP, it is preferred that the virusome or VLP further comprises additional antigens. Preferably, these antigens may be derived from a heterologous chronic disease, so that the complex may be used as part of a broad-spectrum vaccination protocol effective against a range of conditions. However, it is particularly preferred that the additional antigens comprised within the virusome or VLP are derived from the same chronic disease condition as the antigen according to the present invention. This is particularly preferred in co-infection (for instance HBV plus HCV, HBV plus MDV, HBV plus HIV, HCV plus WV, triple or quadruple infections) when a secondary antigen could be inserted in the presented particle.

These additional antigens may be presented on the viral envelope, or its equivalent, or may be physically contained within the envelope. Alternatively, the antigens may be encoded by polynucleotides, such as DNA or RNA, contained within the envelope.

With respect to HBV, it is particularly preferred that the antigen which forms the antigen-antibody complex is derived from pre S/S and that the additional antigens are derived from the HBV core, polymerase or X proteins. It is particularly preferred that these additional antigens are derived from precore polypeptide and, most preferably, from the HBe antigen and/or the I-lBc antigen.

The antigen may be derived from a broad range of chronic disease-causing agents.

Preferably, these include HIV, HCV, I-IDV, I-lerpesviridae and Human Papilloma Virus (HPV). Viral and bacterial infections that induce tolerance/anergy are candidates for this approach.

Anti-cancer treatments are also envisaged. It may be necessary to use an appropriate tumour marker to target the immune response. The Antibody-Antigen complex of the present invention is likely to play a role in DC maturation in vivo and in vitro, which is clearly advantageous in anti-cancer treatments.

It will be understood that the term "derived from" relates to any antigenic fragment, comprising at least one epitope, obtained from, or including all of, said protein or polypeptide, provided that it displays at least one eptiope capable to be recognised or bound specifically by an antibody.

The additional antigens comprised within the viral envelope or its equivalent may also include, for instance, the hepatitis D antigen and DNA encoding antigenic polypeptides or fragments thereof from hepatitis D and, most preferably hepatitis B, HCV Core antigen, HTV core or TAT proteins or peptides in case of co-infection.

The present complex may be administered in any of a number of forms, and is preferably administered in a pharmaceutically acceptable formulation. Thus, in a further aspect, there is provided pharmaceutically acceptable formulation comprising the complex. The complex may be administered in any of a number of ways, including transdermally, orally, nasally or via any other mucus membrane of the body, via means of a patch, pill, spray, solution or capsule, for instance. However, it is particularly preferred that a complex is delivered intravenously, preferably by injection.

Alternative and more direct routes of administration are also preferred, such as APC cells. The plasmacytiod Dendritic Cells (pDCs), myeloid Dendritc Cells (mDCs) or any kind of bone marrow-derived cells of myelbid typ are extremely efficient antigen-presenting cells (APCs). Because antigen can enter the body via several different routes, specialized APC are found at all these distinct antigen entry sites. The function of these APCs is to take up antigen (or an antigen-antibody complex), process it, and present it to T cells. This frequently involves the APC transporting processed antigen from the site at which it interacts with antigen via lympho vessels or endothelial venules to the T cell area of the closest (draining) lymph node.

Antigen uptake and migration results in the differentiation of the APC to a cell known as the mature dendritic cell in the lympho node. In the T cell area of nodes, the antigen-bearing mature dendritic cells (pDC or DC originating from Lymph Nodes) interact with a I cell which express the appropriate antigen recepror specificity, and initiates the cascade of events involved in T and B cell activation.

Activate plasmacytoid DCs, for example, are known to migrate to inflammed lymph nodes to create local immune fields that generate antiviral Cli response in association with Lymph Nodes Dendritic Cells, in a model of simplex virus infection (HSV).

The direct administration (charge) may play a pivotal role in pDCs or any kind of DCs activation. The migration to the lymph nodes and the interaction with Lymph Node Dendritic Cells (LNDC) could be able to induce an antiviral immune response, breaking the tolerogenic mechanism or balancing the immune response in tolerogenic host.

Depending on the means of administration, the complex is preferably formed in vitro, particularly if it is to be injected.

However, it is also envisaged that complex could be formed in situ. This may be by means of molecular biology techniques, where expression of the antigen and antibody are induced, for instance by means of suitable viral vectors comprising polynucleotides encoding these proteins under the control of suitable promoters, in a tempospatial manner, as required. However, it will be appreciated that the levels of expression of the antigen and the antibody need to be tightly controlled in order to ensure that the correct ratio of these two is attained. However, this may be managed by use of suitable promoters andior enhancers, leading to differential levels of expression of these proteins which can be individually controlled by additional or external factors.

In a further aspect, there is also provided the use of the complex in the manufacture of a medicament or prophylaxis of a chronic disease.

In a still further aspect, there is also provided a method for the treatment or prophylaxis of a chronic disease condition, comprising forming the antigen-antibody complex, preferably in vitro, and administering this to the patient in an appropriate form, preferably in a pharmaceutically acceptable dose, preferably administered intravenously.

Furthermore, there is also provided a method of HBV viral clearance, comprising administering the antigen-antibody complex, as discussed above, for instance.

Also provided is a method for reducing the immunotolerance of a chronically infected patient, wherein the chronic disease is preferably HBV.

There is also provided a method of immunopotentiation comprising forming or administering said complex.

The present invention will now be described in detail in the following Examples, which are non limiting. Any references disclosed herein are hereby incorporated by reference.

Example

Preliminary Assessment of Protocol Therapy against Hepadnavirus Chronic Inftction based on Anti-preS/S-Vi ral particles immunogenic complexes

Introduction

The improvement of antigen presentation to the immune system presume of using molecules able to drive and improve the immune response. The fine regulation and improvement of the response against pathogens that induce tolerance and persistent infections is the major aim of the immuno-therapy.

The human hepatitis B virus is a natural model to study the persistence and the immunological tolerance based on the strong compromising of the response to the viral antigens in the chronically infected. The cell-mediated response (CD8+ ) both in humans and in the animal models is weaker than in acutely and resolved infections (1, 2a).

On the possible origin of the tolerance remains under debate and various mechanisms could explain it: 1) exhaustion of competent lymphocytes; 2) inappropriate antigenic presentation and consequently insufficient response; 3) low affinity of the I Cell Receptor (ICR) through the antigen presentation Major Histo-compatibil ity Complex (MHC).

Experimentation of new vaccine formulations and adjuvanted antigens able to produce an improved cellular response or to interfere to break tolerance could be based, also facilitating the recognizing of the viral antigens (3).

The emergence of escaping mutants, able to overcome the prophylactic vaccination, and the existence of no-response vaccines requested the development of newly designed vaccine formulation based on the presentation of small S antigen (already used in the commercially available vaccines) in association with middle and long S antigens. These formulations, mostly targeting the hepadnavirus particles, appeared able to give better protection than the yeast derived small S vaccine and induce antibodies also in those populations known to be less responsive to small S preparations.

The aim of the present work is to investigate the role of the adjuvancy of the anti-PreS antibodies (against the HBV virus) able to sterilize the infection in our previous Woodchuck study (4). The study represents a preliminary trail based on the recognized animal model of the HBV, the Woodchuck (Marmoia Monax) infected from Woodchuck Hepatitis Virus (W1-IV). This model is widely known for the relevant similarity with the human infection.

The protocol that we are presenting is based on the "in vitro" formation of a complex we refer to as the "Immunogenic Complex" (IGC), defined as a complex with a very low ratio of antibody/antigen, as distinct from the immunocomplex (usually called IC) formed by a large amount of both antibodies and antigens. The amount of protein complex in IC is so high that is related to pathologies.

Interestingly, the therapeutic protocol has shown a strong antiviral activity that resulted in effective reduction of serum DNA level and Anti-HBe sero-conversion (the anti-HBe are antibodies raised against the E antigen of HBV an early viral protein possibly involved in the establishment of the persistent infection).

The results has been achieved after just a single inoculation of the IGC preparation in chronically infected animal w3953. The viremic DNA reduction and the disappearance of WHeAg are followed by the reactivation of previously silent cellular response CTL driven through viral antigens. To date, this is the first time that tolerance break is achieved and

this is related to the reduced viral load after administration of the IGC. This newly designed and original experimental protocol will be the basis for further developments. The analysis of these study will shed light on the vaccine formulation efficacy, on the improvement of antigenic adjuvancy and, finally, on the role of immune response regulation during the chronic infections.

Malerials and Methods

I

Woodchucks Adult WHV-negative woodchucks trapped in the state of New York were purchased from North Eastern Wildlife (Ithaca, N.Y.). Animals were kept in captivity and maintained, in ISS animal House, following ethical conditions established by European Community and controlled by "Servizio Qualita e Sicurezza della Sperimentazione Animate" Istituto Superiore di Sanità.

Wif V neutralization (Immunogenic Complex IGC) Antisera pool from woodchucks (w22 11 and w2 197), vaccinated against HBV envelope proteins preS/S was quantified and 100 jil corresponding to 2.5 mlii (milli International Units) were incubated I h to 37 C with 50i1 of WHy challenge, harbouring three different type of particles containing an amount of long (preS I -S2-L protein), middle (preS2-L protein), and short (L protein). The antigens HBsAg is calculated between 50 and 300 microg/mI. In other words, we have a large excess of antigen (ranging between 2 and 15 microg) complexed with a small amount of specific antibodies (2,5 mIU). In the final tube, 150 p.1 contains a ratio of I a 6 p.g/ mUl.

The WHV challenge from pooi sera of woodchuck w197 was previously 1:5 diluted and I 6 genome equivalents (g.eq.) were incubated for neutralization.

The incubation mixture ratio is designed to obtain a large excess of viral particle for more efficient ingestion and in order to avoid generation of immune complex.

The neutralization mixture was intravenously administered and the sera was collected weekly.

WHVseroogy and nucleic acid detection Detection of antibodies to WHy core antigen (WHcAb), antibodies to WHV e antigen (WHeAb), WHV e antigen (WFIeAg) and antibodies to HBV s and pre/S antigen ( HBsAb) in woodchuck serum were determined by electro-chemiluminescence immunoassay (ECLIA, Roche).

Serum WHV DNA was determined by PCR by using the following reaction mixture: 1X PCR buffer II (Perkin Elmer), 2.5mM MgCI2, 0.5pM primers, 0.2mM dNTPs and 2.5U Taq Polymerase (Perkin Elmer). The selected region (nucleotides 2490-419), which encodes a portion of P, Pre-S and the amino-terminal part of the S protein, was amplified from 0.25.d of plasma with primers 02490 (5'- CTTCTAGGTCCCCCAG4JGACGCACTCCC..3I) and A419 (5'-CCCCTGGA}L&ACTGAGAGGTCCACCACCA.3') PCR was performed in a Thermal Cycler (Gene Amp PCR System 9600 Perkin Elmer) with a preliminary denaturation at 92 C for 3 mm followed by 35 cycles of three steps: denaturation at 94 C for 15s, annealing at 65 C for 15s and polymerization at 72 C for 90s.

Analysis of T-cell-mediaied WHV specific response Peripheral blood cells (PBMC) were collected as above, separated by Ficoll-Hypaque gradient centrifugation, washed twice in PBS, and then re-suspended in AIM-V medium (Gibco Lab., Grand Island NY), supplemented with 2tM L-glutamine (Sigma-Aldrich), 1% non essential amino acids ( Sigma-Aldrich), lj.i.M sodium pyruvate ( Sigma-Aldrich ), 100 U/mI penicillin (Sigma-Aldrich), 100tg1ml streptomycin (Sigma-Aldrich) and 10% fetal calf serum (FCS, Gibco) at 106 cells/mI.

The proliferation assay was performed seeding cells per well in 96-well bottomed plates (Falcon, Becton Dickinson) in triplicate and incubating the cells with WHV specific antigen (see below). In all experiments, PBMC were also stimulated with 0.5/ltg per ml of Phytoemoagglutinin (PHA) as positive control. Five days after incubation at 37 C in a humidified atmosphere containing 5% C02, l.tl of tritiate Adenine (Amersham, Little Chalfont, UK) was added: after an additional 12h, the cells were harvested, and the DNA-incorporated radioactivity was measured by liquid scintillation counting.

PBMC stimulations were performed with different antigenic preparations: WHy serum particles corresponding to inocula (j)ool w197) were diluted and i05 genome equivalents were added to each well (lg.eq./cell).

WHy particles were complexed with antibodies as above described and I 0 complexes were added to well at ratio of I antigen-antibody ( IGC) per cell.

WFIV proteins were extracted by chloroform and precipitated by 10% acetone and the pellet re-suspended in PBSA. i05 genome equivalents particles were added to each well (1 g.eq./cell).

lj.tl of pool sera from vaccinated woodchucks w221 I and 2197, corrisponding to 0.0025 mIU of HBsAb was added to each well in PBMC proliferation test.

Results The liglA shows the WHy DNA detection results by PCR in WHy chronic carrier, woodchuck w3953. The chronicity was established monthly by WHY DNA and WHeAg and WhcAb from 24 weeks before administration (-24, figlA).

Dramatically decreasing of WHy DNA level was observed at I week of immunogenic complex ( IGC) administration and negative/borderline results were observed until 12 weeks after administration.

The fig.IB depicts the pattern of seroconversion WHeAg/WHeAb in woodchuck w3953.

The level of WHeAg decreased dramatically respect to beginning of study (24 and 0 week) reaching undetectable level iweek after IGC administration. The levels of WHeAg remain undetectable until end of antigen sera detection (week 17). In the contrast the level of of WHeAb rapidly increased.

The limphoproliferation assay was performed and PBMC activity measured against Concavanavalin A (Co-A), WHy extracted antigens, WI-IV virus, woodchucks antisera from w2211 and 2197, IGC and WHsAg (FigiC). The stimulation index from the beginning week of experimentation (week 0) until last week (week 13) showed a hardly increase of activity towards antigenic stimulation. The weak responses against virus (WHV) and WHy proteins were evident in time 0, in the contrast antisera woodchuck from w221 I and w2197 and IGC showed similar stimulation activity upper S.1.

The following weeks a constant increasing of Con-A, immune (antisera), IGC and virus specific (WHy, W1-JV proteins, WHsAg) PBMC response could be described. The increasing of mitogenic activity towards antigenic components at week 13, reaches a value ratio of 50/1 00 fold respect to week 0 (see Fig 1 C).

Discussion The results obtained applying our newly designed protocol clearly indicate a rapid anti-viral action as demonstrated from the dynamics of DNA disappearance in sera of the treated animal. DNA levels declined rapidly under the in-house PCR detection limits (102 genome equivalents) in the animal and remained at least at this level successively. In the animal, the viremia before of the treatment was between 108 and l0 g.e./mI.

Observations indicate clearly that the viremia declined persistently.

In our knowledge, this is the first time in which it was described the viremia control by means of vaccine therapy without combination with antivirals or cytokines. It must be considered that the antivirals administration in clinics and in experimental procedures never achieved a so deeply rapid decrement of replication, with the resulting seroconversion (5).

Inhibition of viral replication are observed with long treatment and repeated administrations that achieved only a partial control of replication. Furthermore, the emergence of resistant mutants during antivirals treatments (eg lamivudina) limits the wide application of these compound and pushes to newly developed antivirals and therapeutic protocols (6).

The detection of HBeAg and anti-I-lBe in sera is summarised in figure Ic. HBeAg values strongly declined in animal w3953 immediately after the beginning of treatment and disappearance of HBeAg is clearly related to the development of a specific humoral anti-HBe response.

The kinetics of 1-IBeAg disappearance and the anti-I-ffie response is overlapping the decrement of serum DNA. The Anti-HBe levels and the absence of HBeAg and viremia suggest, as widely believed, the control of infection and the strong antiviral activity induced by our therapeutic control. In other words this new approach has achieved the following results: decline of serum DNA, production of anti-HBe antibodies associated to the control of infection. This antigenic disappearance was rarely found in parallel to DNA decline. These results demonstrate that our protocol has achieved a difficult goal in HBV therapy.

The 1-IBeAg could directly be involved in the induction of tolerance both through it self than through Core protein (the primary protein structure is partially overlapping, 7.). In the specific case of infections caused by resistant mutants, the majority of the associated immunopathology to liver demonstrates that the induced tolerance at the beginning of the chronicity can be broken.

In figure Ic we have shown the kinetics of lympho-proliferation with viral antigens, entire virus or the IGC used in the vaccination. The results can dramatically change in a week time. In the week of the inoculation, well before that that our preparation was exposed to the immune system, we can describe the typical pattern of the chronic cells response. The lymph cells did not recognize the viral components or, at least, only weakly respond. After a week, the antigenic stimulation, the immune system presented a strong response, completely changing the pattern of the host response. The reactivation of lymphocytes clearly evidence the breaking of tolerance and it is overlapping with the antiviral activity.

The kinetics replicates that of the antiviral inhibition demonstrating the radical changing in the host surveillance to the virus. The aim of the immune-system is, effectively, this: control infection avoiding liver damage and to invert the pathways that have induced the tolerance in the persisting hepatitis viruses (especially I-IBV, HCV and HDV, 8).

Another point of our therapeutic protocol is the importance given to the ratio in which antigens and antibodies are mixed to achieve the complexes. We have used 2,5 mIU are below the level of the neutralisation. In our protocol when we mixed antibodies and antigens, the IGC presents higher ratio of antibodies/HBsAg (although comparable) than the ratio present in the vaccinated subject. Naturally the calculation can be considered as true for the IGC but are only virtual for the vaccinee, because we do not know the amount of HBsAg introduced during the infection of the vaccinated host.

The ratio of antibodies/antigen range for a single mUl will be 0,15 to 0,025, whereas the ratio in neutralisation would be (by definition in case of infection with 1 ml of HBV positive serum) between 0,2 to 0,033. If the relation was considered from the point of view of the protein excess, the standard neutralisation is based on a range between 5 and p.g/mUI, whereas the protocol range is between 6,66 and 40 tgImUI. In such ratio, the IGC are structured as immunogenic complexes and not as immune complex (IC).

These latter are characterised by an excess of antibodies in respect of the antigen and are known to be generated during the pathogenic process of type III hyper-sensibility with very low immunogenicity.

The mechanism of action, that justifies and clarify the effect of this therapeutic protocol, is certainly related to the presentation of the antigen-antibodies complexes via Dendritic cells or complement cascade (9, 10). Analysing the protocol, two major factor could be underlined: firstly, our protocol is inducing is a kind of "secondary" immunological response. When IGC are exposed to the immune system in the chronic carrier, the anergy (or tolerance) of the immune cells is broken. A role in this response could be played by the ratio in which IGC are composed. Secondly, the intravenous inoculation of the IGC, with a probably early involvement of the lymph nodes in which the cascade could be activated by the specialised cells (as Antigen Presenting Cells, ref 11).

The involvement of follicular Dendritic cells should involve an early activation of the Fc7RJ/Fc7R.J1I receptors and the processing of the antigen/antibodies complex with (Major Histo-compatibility Complex) MIHC II mediated presentation directed to I Cell Receptors (TCR) of T helper (Th) cells. This drives the CD4+ mediated response.

Alternatively, the mechanisms of cross-presentation could direct the IGC through the chain of MHC I and activate the CD8+ cell response (12). As previously recalled, the intravenous inoculation could induce an high concentration of IGC in the reticular endothelium of the lymph nodes with a parallel increased probability of binding and internalization in APCs and consequent amplification of the signal.

The activation of the complement cascade is far more complicated with at least 30 proteins involved, able to activate a wide range response based on both innate and acquired immune responses (9).

In this contest the antibodies mediated response and the complement cascade could collaborate in a synergic action able to activate the Fc binding dendritic cells and macrophages trafficking in the follicular region of the lymph nodes with a rapid activation of the cascade reaction similar to a secondary response (11).

In conclusion the strong biological activities (humoral response activation and cellular stimulation) has allowed the control of the hepadnavirus infection in the woodchuck model.

Future development of the protocol will be addressed on the use of standardised product.

Further improvements could involve monoclonal or humanised antibodies, identification of particles to use in the preparation of the calculation, as inactivated virus particles, virosomes, virus-like-particles, nano and micro-particles.

References 1) Barbara Rehermann and Michelina Nascimbeni Nature Reviews, Volume 5 March 2005; 2 15-229 Immunology of Hepatitis B Virus and Hepatitis.C infection.

2a) Fabien Zoulim Journal of Hepatology, 2005, 42 302-308 New insight hepatitis B virus persistence from the study of intrahepatic viral cccDNA 2b) Oon CJ, Lim GK, Ye Z, Goh KT, Tan KL, Yo SL et. al. Vaccine 1995, 13:699-702.

Molecular epidemiology of I-IBV vaccine variants in Singapore.

2c) Neurath AR, Kent SBH.

Elsevier Science Publishers BV 1985, pp 325-365.

Antigenic structure of human hepatitis viruses. In: MHV van Regeninantel and AR Neurath, eds. Immunochemistry of viruses: The basis for serodiagnosis and vaccines.

2d) Neurath AR, Kent SBH, Strick N, Taylor P, Stevens CE. Hepatitis B virus contains pre-S gene-encoded domains. Nature 1985 3 15:154-156.

2e) Neurath AR, Kent SB, Parker K et al. Vaccine 1986, 4:35-37.

Antibodies to a synthetic peptide from the pre-S 120-145 region of the hepatitis B virus envelope are virus neutralizing.

Gerlich WE, Deepen R, Heermann KR, Krone B, LU XY, Seifer M, Thomssen R. Vaccine 1990, 8:S63-S68 Protective potential of hepatitis B virus antigens other than the S gene.

3) Dennis R.Burton Nature Reviews, Volume 2 September 2002, 706-7 13 Antibodies, viruses and vaccines 4) Claudio Argentini, Roberto Giuseppetti, Emilio D'Ugo, Valentina La Sorsa, Elena Tritarelli, Sara Orobello, Andrea Canitano, Reinhard Gluckk and Maria Rapicetta.

Vaccine: Volume 23, 25 May 2005, p 3649-3656 A pre-S/S CHO-derived hepatitis B virus vaccine protects woodchucks from WHV productive infection 5) Avidan U.Neumann Hepatology, August 2005 Vol.42 Number 2, 249-254 Hepatitis B Viral Kinetics, A Dynamic Puzzle Still to Be Resolved 6) Jordan Feld, Jia-yee Lee, and Stephen Locamini Hepatology, September 2003, 545-553 New Targets and Possible New Therapeutic Approach in the Chemotherapy of Chronic Hepatitis B 7) Margaret Chen, Matti Sallberg, Janice Hughes, Joyce Jones, Luca G. Guidotti, Francis V. Chisari, Jean-Noel Billaud, and David R. Milich Journal of Virology, March 2005; 3016-3027 Immune Tolerance Split between Hepatitis B Virus Precore and Core Proteins 8) Paul Klenerman and Ann Hill Nature Immunology, volume 6 September 2005; 873-879 I cells and viral persistence: lessons from diverse infection.

9) Michael C.Carrol Nature Immnunology, volume 5 Number 10 October 2004; 98 1-986 The Complement system in regulation of adaptive immunity 10) B.Heyman Annu.Rev.Immunol. 2000, 18.709-737 Regulation of antibody responses via antibodies, complement and Fc recertors 11) T.Diaz De Stahl and B.Heyman Scand.J.Immunol. 2001, 54, 495-500 IG2A-Mediated Enhancement of Antibody Response is dependent on FcR7 Bone Marrow-Derived Cells 12) William R.Heath and Francis R. Carbone Nature Immunology, Volume I November 2001, 126-135 Cross-Presentation in Viral immunity and self-tolerance Sequences: SEQ ID NOS: 1-4 Genbank # AB219427 and translation products thereof LOCUS AB219427 3215 bp DNA circular VRL 19-MAY-2006 DEFINITION Hepatitis B virus DNA, complete genome, isolate:Patient #4075, PNN2.

ACCESSION AB21 9427 VERSION AB219427.1 GI:89274072

KEYWORDS

SOURCE Hepatitis B virus ORGANISM Hepatitis B virus Viruses; Retro-transcnbing viruses; Hepadnaviridae; Orthohepadnavirus.

REFERENCE I

AUTHORS Nagasaki,F., Niitsuma,H., Cervantes,J.G., Chiba,M., Hong,S., Ojima,T., Ueno,Y., Bondoc,E., Kobayashi,K, Ishii,M. and Shimosegawa,T.

TITLE Analysis of the entire nucleotide sequence of hepatitis B virus genotype B in the Philippines reveals a new subgenotype of genotype

B

JOURNAL J. Gen. Virol. 87 (PT 5), 1175-1180 (2006) PUBMED 16603518 REFERENCE 2 (bases I to 3215) AUTHORS Nagasaki,F. and Niitsuma,H.

TITLE Direct Submission JOURNAL Submitted (28-JUN-2005) Futoshi Nagasaki, Tohoku University; 1-1 Seiryoumachi, Aoba-ku, Seridai City, Miyagi 980-8574, Japan (E-maiI:fnagasakiint3.med.tohoku.ac.jp, TeI:81 -22-717-7171, Fax:81 -22-717-7177) FEATURES Location/Qualifiers source 1..3215 /organism="Hepatitls B virus" /moLtype="genomic DNA Iisolate="Patjent #4075, PNN2" /db_xref="taxon: 10407" CDS join(2307..3215,1..1623) Icodon_start= 1 /product=DNA polymerase" /protein_id='BAE 80751.1' /db_xref"G I:89274076" /tranSlatiOri='MPLSYPHFRKLLLLDEEAGPLEEELPRLADEGLNRRVAEDLNLG NLNVSIPWTHKVGNFTGLYSSWPGFNPDWQTPSFPNIHLQEDIVDRCKQpJGpLN ENRRLKLI MPARFYPNVTKYLPLDK3I KPYYPEHWNHYFQTRHYLHTLWKAGILYKR

ETTRSASFCGSPYSWEQELQHGRLVFQTSKRHGDES FCPQSAG I LSRSPVGPCI QSQL

RQSRLGPQPTQGLLAGLQQGGSGSIRAGIHSTPWGTVGVESSSSGHTHNCASSSSSCL

HQSAGSKAAYSPVSTAKGHSSSGHAVELHHVPPNSSRSQSQGSVLSCWWLQFRNSEPC

SEHCLFHIVNLI EDWGPCTEHGEHRIRTPRTPARVTGGVFLVDKNPHNTTESRLWDF

SQFSRGNTRVSWPKFAVPNLQSLTNLLSSDLSWLSLDVSAAFYHIPLHPIAAMPHLLVG

SSGLSRYVARLSSNSRIINHQHRTMQNLHDSCSRNLWSLLLLYKTYGRKLHLYSHPI

I LGFRKIPMGVGLSPFLLAQFTSAICSWRRAFPHCLAFSYMDDWLGTKSVQHLESV

YAAVTNFLLSLGIHLNPHKTKRWGYSLNFMGYI IGSWGTLPQEHI VLKI KQCFRKLPV

NRPIDWKVCQRIVGLLGFAAPFTQCGYPALMPLYACIQAKQAFTFSP1-YFLNKQYL

NLYPVARQRPGLCQVFADATPTGWGLAIGHQRMRGTFVSPLPIHTAELLCFARSRS

GARLIGTDN SWLSRKYTSFPWLLGCAANWILRGTS FVYVPSALNPADDPSRGRLGLY

RPLLRLPYQPTTGRTSLYADSPSVPSHLPDRVHFASPLHVAWRPP.' CDS join(2848..3215,1..835) /codon_start= 1 /product="Iarge surface protein" (SEQ ID NO. 2) /protein_id="BAE80752. 1" /db_xrefr"GI:89274077" ItranSIatiOn=MGGWSSKPRKGMGTNLSVPNPLGFFPDHQLDPAFNSDNPDWD LNPHKDYWPDSNKVGVGAFGPGFTPPHGGLLGWSPQAQGI L1TVPAAPPPASTNRQVA

RPPTPLSPPLRDTHPQAMQWNSTTFHQTLQDPRVRALYFPAGGSSSGWNPVQNTASS

TPVCLGQNSQSQISSHSPTCCPPICPGYRWMCLRRSIIFLCILLLCLIFLLVLLDYQG

MLPVCPLI PGSSTTSTGPCRTCTTPAQGTSMFPSCCCTKPTEGNCTCI P1 PSSWAFAK

YLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWFWGPSLYN ILSPFMPLLPI

FFCLWVYI" CDS join(3205..3215,1..835) /codon_start= 1 /product="middle surface protein" (SEQ ID NO. 3) /protein_id="BAE80753. I" /db_xref&'GI:89274078" /translation="MQWNSTTF}-IQTLQDPRVRALYFPAGGSSSGTVNPVQNTASSISS

ILSKTGDPVP NMENIASGLLGPLLVLQAGFFLLTKILTI PQSLDSWWTSLSFLGGTPV

CLGQNSQSQISSHSPTCCPPICPGYRWMCLRRSIIFLCILLLCUFLLVLLDYQGMLP

VCPLIPGSSTTSTGPCRTCTTPAQGTSMFPSCCCTKPTEGNCTCI PIPSSWAFAKYLW

EWASVRFSWLSLLVPFVQWF'VGLSPTVWLSVIWMMWFWG PSLYN I LSPFMPLLPIFFC LWVY" CDS 155..835 /codon_start= I /product= small surface protein' (SEQ ID NO. 4) /protein_id="BAE80748. I' /db_xref"GI:89274073" /translation="MENIASGLLGPLLVLQAGFFLLTKILTIPQSLDSWWTSLSFLGG

TPVCLGQNSQSQISSHSPTCCPPICPGYRWMCLRRSI I FLCI LLLCLIFLLVLLDYQG

MLPVCPLI PGSSTTSTGPCRTCTTPAQGTSMFPSCCCTKPTEGNCTCIPI PSSWAFAK

YLWEWASVRFSWLSLLVPFVQWFVGLSPTVWLSVIWMMWFWGPSLYN ILSPFM PLLPI

FFCLWVYI" CDS 1374..1838 fcodon_start=1 /product="X protein" /protein_id="BAE80749. I" /db_xref="GI:89274074" /translation="MAARLCCQLDPARDVLCLRPLSAESRGRPLPGPLGALPPASpSA

VPTDHGAHLSLRGLPVCAFSSAGPCALRFTSARRMETWNAHRNLPKVLHKRTLGLLA

MSTTDLEAYFKDCVFTEWEELGAEARLKVFVLGGCRHKLVCSPAPCNFFTSA" CDS 1955..2452 /codon_start=1 /product"HBc antigen" /protein_id="BAE80750. 1" /db_xref="GI:89274075" /translation="MPSDFFPSIRDLLDTASALYREALESPEHCTP HHTALRQAI LCW G ELMNLATWVGSNLEDPASRELWGVVNVNMGLKLRQLLwFH LSCLTFGRETVLEYLV

SFGVWIRTPPAYRPQNAP ILSTLPETTVIRRRGRSPRRRTPSPRRRRSQSPRRRRSQS

REPQC" ORIGIN (SEQ ID NO. 1) 1 ctccaccacg ttccaccaaa ctcttcaaga tcccagagtc agggctctgt actttcctgc 61 tggtggctcc agttcaggaa cagtgaaccc tgttcagaac actgcctctt ccatatcgtc 121 aatcttatcg aagactgggg accctgtacc gaacatggag aacatcgcat caggactcct 181 aggacccctg ctcgtgttac aggcggggtt tttcttgttg acaaaaatcc tcacaatacc 241 acagagtcta gactcgtggt ggacttctct cagttttcta gggggaacac ccgtgtgtct 301 tggccaaaat tcgcagtccc aaatctccag tcactcacca acctgttgtc ctccgatttg 361 tcctggctat cgctggatgt gtctgcggcg ttctatcata ttcctctgca tcctgctgct 421 atgcctcatc ttcttgttgg ttcttctgga ctatcaaggt atgttgcccg tttgtcctct 481 aattccagga tcatcaacca ccagcaccgg accatgcaga acctgcacga ctcctgctca 541 aggaacctct atgtttccct cctgttgctg tacaaaacct acggaaggaa actgcacctg 601 tattcccatc ccatcatctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 661 tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 721 tgtctggctt tcagttatat ggatgatgtg gttttgggga ccaagtctgt acaacatctt 781 gagtccgttt atgccgctgt taccaatttt cttttgtctt tgggtataca tctaaaccct 841 cataaaacaa aaagatgggg ctattccctg aacttcatgg gatatataat tggaagttgg 901 ggcactttgc cccaggaaca tattgtgttg aaaatcaaac aatgttttcg aaaacttcct 961 gtaaacaggc ctattgattg gaaagtgtgt caacgaattg tgggtctttt gggttttgct 1021 gctcctttca cacaatgtgg ttatcctgct ttaatgcctt tatatgcatg tatacaagct 1081 aaacaagctt ttactttctc gccaacttat aaggcttttc taaacaaaca gtatctgaac 1141 ctttaccccg ttgcccggca acggccaggt ctgtgccaag tgtttgctga cgcaaccccc 1201 actggctggg gcttggccat aggccatcag cgcatgcgtg gaacctttgt gtctcctctg 1261 ccgatccata ctgcggaact cctagcagct tgttttgctc gcagcaggtc tggagcaaga 1321 cttatcggga ccgacaattc tgtcgtcctg tcccgtaaat atacatcatt tccatggctg 1381 ctcggctgtg ctgccaactg gatcctgcgc gggacgtcct ttgtttacgt cccctcagcg 1441 ctgaatcccg cggacgaccc ctcccggggc cgcttggggc tctaccgccc gcttctccgt 1501 ctgccgtacc aaccgaccac ggggcgcacc tctctctacg cggactcccc gtctgtgcct 1561 tctcatctgc cggaccgtgt gcacttcgct tcacctctgc acgtcgcatg gagaccaccg 1621 tgaacgccca ccggaacctg cccaaggtct tgcataagag gactcttgga cttttagcaa 1681 tgtcaacgac cgaccttgag gcatacttca aagactgtgt gtttactgag tgggaggagt 1741 tgggggcgga ggctaggtta aaggtctttg tattaggagg ctgtaggcat aaattggtct 1801 gttcaccagc accatgcaac tttttcacct ctgcctaatc atctcatgtt catgtcctac 1861 tattcaagcc tccaagctgt gccttgggtg gctttagggc atggacattg acccgtataa 1921 agaatttgga gcttctgtgg agttactctc ttttttgcct tctgacttct ttccttctat 1981 tcgagatctt ctcgacaccg cctctgctct gtatcgggag gccttagagt ctccggaaca 2041 ctgtacacct caccatacgg cactcaggca agctattctg tgttggggtg agttgatgaa 2101 tctagccacc tgggtgggaa gtaatttgga agatccagca tccagggaat tagtagtcgg 2161 ctatgtcaat gttaatatgg gcctaaaact cagacaacta ttgtggtttc acctttcctg 2221 tcttactttt ggaagagaaa ctgttcttga atatttggtg tcttttggag tgtggattcg 2281 cactcctcct gcatatagac cacaaaatgc ccctatctta tccacacttc cggaaactac 2341 tgttattaga cgaagaggca ggtcccctag aagaagaact ccctcgcctc gcagacgaag 2401 gtctcaatcg ccgcgtcgca gaagatctca atctcgggaa cctcaatgtt agtattcctt 2461 ggactcataa ggtgggaaac tttacggggc tttattcttc tacggtaccg ggctttaatc 2521 ctgattggca aactccttct tttcctaaca ttcatttgca ggaggatatt gttgataggt 2581 gtaaacaatt tgtgggaccc ctcacagtaa atgaaaacag gagactaaaa ttgatcatgc 2641 ctgctaggtt ctatcctaat gttaccaaat atttgccctt agataaagga attaaacctt 2701 attatccaga gcatgtagtt aatcattact tccagacaag acattattta catactcttt 2761 ggaaggcggg tatcttatat aaaagagaga caacacgtag cgcctcattt tgcgggtcac 2821 catattcttg ggaacaagag ctacagcatg ggaggttggt cttccaaacc tcgaaaaggc 2881 atggggacga atctttctgt ccccaatccg ctgggattct ttcccgatca ccagttggac 2941 cctgcattca aagccaactc cgacaatccc gattgggacc tcaacccaca caaggactac 3001 tggccggact ccaacaaggt gggagtggga gcattcgggc cgggattcac tccaccccat 3061 gggggactgt tggggtggag tcctcaagct cagggcatac tcacaactgt gccagcagct 3121 cctcctcctg cctccaccaa tcggcaggta gcaaggccgc ctactcccct gtctccaccg 3181 ctaagggaca ctcatcctca ggccatgcag tggaa

Claims

CLAIMS: 1. An Antigen-Antibody complex comprising: at least 1 chronic

disease Antigen, the at least one Antigen comprising at least one epitope, and at least one Antibody capable of recognising the at least 1 epitope, characterised in that the ratio of Antigen bound to Antibody in the complex is from 1:1 to 800:1 g/mIU.

2. A complex according to claim 1, wherein the Antigen is present in a large excess.

3. A complex according to claim 1 or 2, wherein the ratio of Antigen bound to Antibody in the complex is selected from the group consisting of from 6:1 to 60:1, 16:1 to 2:1, and 6:1 to 2:1 pg/mIU.

4. A complex according to claim I or 2, wherein the ratio of Antigen bound to Antibody in the complex is from 4:1 to 8:1 pg/mIU.

5. A complex according to claim 1 or 2, wherein the ratio of Antigen bound to Antibody in the complex is from 2,64:1 to 16:1 p.g/mIU.

6. A complex according to claim 1 or 2, wherein the antigen is present as 6,6-40 p.g (micrograms) of protein, relative to 2,5 mIU of antibody.

7. A complex according to any preceding claim, wherein the antigen is derived from any one of hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F hepatitis G, and Human Papilloma Virus (HPV).

8. A complex according to claim 7, wherein the antigen is derived from hepatitis B (HBV).

9. A complex according to any of claims 1 to 6, wherein the antigen is derived from a tumour.

10. A complex according to any of claims I to 6, wherein the antigen is derived from H1V.

11. A complex according to claim 8, wherein the antigen is derived from any one of the group consisiting of the Small (SEQ ID NO. 4), Medium (SEQ ID NO. 3) and Large (SEQ ID NO. 2) pre-S/S proteins.

12. A complex according to claim 8, wherein the antigen is derived from a polypeptide encoded by the nucleotide sequence of SEQ ID NO. 1.

13. A method for the treatment or prophylaxis of a chronic disease condition, comprising forming the antigen-antibody complex according to any preceding claim, and administering this to the patient.

14. A method for reducing the immunotolerance of a chronically infected patient, comprising forming the antigen-antibody complex according to any of claims 1-9.

15. A method of immunopotentiation, comprising administering or forming the antigen-antibody complex according to any of claims 1-12.

16. A method according to any of claims 13-15, wherein the chronic disease is HBV.