HK1178062A - Immunogenic influenza composition - Google Patents

Description

Immune influenza component

Technical Field

The present invention relates to an immune influenza component.

Background

Existing approved vaccines in the human and livestock fields are generally effective against so-called "Class One" pathogens. Generally, a group of pathogens (e.g., measles, mumps, and rubella virus) refers to the group of pathogens: (1) infections or serious illnesses in infants, young children, children and adolescents; (2) carrying a relatively stable microbial genome; (3) have a natural medical history that can lead to spontaneous recovery; and (4) induce long-term memory associated with polyclonal (polyclonal) and polyepitopic antigen recognition.

In contrast, the characteristics of two types of pathogens, such as influenza virus, HIV-1, Plasmodium, Mycoplasma (such as those responsible for tuberculosis), Trypanosoma, Schistosoma, Leishmania, Bordetella (Anaplasma), Enterovirus, Star virus, rhinovirus, Norwalk virus, virulent/pathogenic E.coli, Neisseria, Streptomyces, Haemophilus immigosae influenza virus, hepatitis C virus, cancer cells, and the like, are in opposition. For example, two types of pathogens: (1) tend to infect and spread over a considerable range of host ages, with infections and relapses occurring from childhood to the elderly; (2) exhibit microbial genetic instability in a designated region of its genome (a marker for successful evolution of such pathogens); (3) in some cases, including the spontaneous recovery of the disease, the host is still frequently susceptible to repeated infections and/or to a definitive diagnosis of a chronic/active or chronic/latent infection state; (4) inducing an oligoclonal early immune response that directly results in a very limited set of immunodominant epitopes that provide narrow species-specific protection, no protection and/or enhanced infection; and (5) immune disorders caused by infection or vaccination, such as epitope-blocking antibodies (epitope-blocking antibodies), atypical primary immune responses of the Ig subclasses, memory cross-reactive recall (anamnestic cross-reactive recalls) and inappropriate T_H1And/or T_H2Cytokine metabolism.

At the immunological level, widely divergent pathogens produce different pathogenesis and disease outcomes, such as HIV-1 versus the human rhinovirus. Highly successful immune system evasion strategies, such as "false Imprinting" (de-rive Imprinting) have evolved and are selected and maintained across host and microorganism populations. Thus, the failure of the vertebrate immune system to function (e.g., due to pseudoimprinting by a pathogen) is essentially the same as whether the average year would be infected with HIV-1 or the common cold virus 2-6 times per year over 60 years.

Although some advances in antigen delivery and expression have improved the immunogenicity of some of these two types of pathogenic microorganisms, the existing vaccine technology has not yet quickly translated into a new and widely effective safety-approved vaccine for use in humans. This is probably largely due to the lack of rationale understanding governing vertebrate quality defense system origin, spectrum development (regression evolution), maintenance, activation, aging, and co-evolution in similar and dissimilar environments.

What is currently lacking in human influenza vaccine development is an ingredient that elicits immunity and protection that is less isotype and subtype dependent and therefore does not require the annual mixing and production of multiple subtypes in current egg-based technical production schemes. One suitable new product is a recombinant HA or NA subunit influenza vaccine that can elicit an immune response that cross neutralizes antigenic variants within subtypes and influenza viruses of different subtypes.

Influenza is a NIAID C pathogen that causes 36,000 deaths and 220,000 hospitalizations each year in the united states. As a respiratory disease, influenza is transmitted by droplets and/or contaminated infectious agents from coughing or sneezing of infected persons. High risk populations, including children and the elderly, develop secondary complications of influenza-associated pneumonia, upper respiratory complications (otitis media in children), and other systemic diseases (e.g., cardiovascular disease, etc.). Influenza is the root of the largest pandemic in history; spanish influenza in 1918 caused more than 4000 million deaths worldwide. The direct medical costs (hospitalization, medical visits, medications, etc.) incurred by influenza annually are projected to be $ 46 million in the united states. In addition, the U.S. has a working day of 1.11 billion annual influenza losses, with associated costs for U.S. commerce exceeding $ 70 billion per year due to morbidity and productivity losses. The total direct and indirect costs (weekday losses, day of school losses, etc.) for a severe influenza epidemic is at least $ 120 billion per year.

Influenza virus, and when accompanied by secondary bacterial infections, has long been recognized as a cause of significant morbidity and mortality. Complications include pneumonia, bronchitis, congestive heart failure, myocarditis, meningitis, encephalitis, and myositis. Some of the high risk groups of complications are those suffering from chronic lung or cardiovascular disease, hospitalized in chronic (illness) nursing facilities (including nursing homes), and elderly people aged 85 years and older. (recommendations of the Association for the Administration of influenza prevention and control immunological practices (ACIP.) MMWR, 1996, Vol.45; and Thompson et al, JAMA 2003; 289: 179-186). The number of elderly people in the united states has doubled between 1976 and 1999, and is expected to increase in the next few years as the peak of fertility of the second generation after two war. People of this age group die 16 times more frequently than people of 65-69 ages due to influenza-related illness. Another important factor in the increased number of influenza-related deaths in the 1990's was rampant influenza a (H3N 2), a more pathogenic, recently pandemic influenza virus.

Influenza is a single-stranded ribonucleic acid (RNA) virus that rapidly mutates to become a new virulent bacterium. Such strains fall into three categories, namely influenza A, B and C. The virus is further divided into at least 16 HA and at least 9 NA subtypes based on two surface glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA). Recent genome-wide analysis of human influenza viruses initiated by NIAID/NIH (collected in 1996-2004 from New York) revealed that, despite the same HA, multiple phylogenetically distinct lineages co-propagate (co-circular) in the same population, resulting in reassortment and generation of new clades of antigens. While antigenic variation of HA remains a major selective pressure on human influenza a virus evolution, the discovery that new clades of antigens emerge through reassortment between persistent viral lineages rather than through antigenic drift is significant to current, past annual approaches to influenza vaccine strain selection and production (Holmes et al, PLoS biol.20053 (9): e 300). Influenza can be obtained from pigs, birds, horses, dogs and other mammals.

A central problem in annual global virus tracking programs and in the subsequent production of "reactive" vaccines is antigenic variation. Antigenic variation is an evolutionary mechanism that ensures rapid sequence variation of specific pathogen genes that encode homologous chromosomes of a single protein antigen, often involving multiple related gene copies, resulting in a structural change of an antigen on the surface of the pathogen. Thus, the host immune system has a reduced ability to recognize the pathogen during infection or reinfection, and new antibodies must be made to recognize the altered antigen before the host can continue to fight the disease. As a result, the host does not maintain intact immunity to this viral disease. This phenomenon represents one of the greatest difficulties faced in modern vaccine development, or the greatest difficulty.

As expected, the immune response generated after infection or vaccination with all approved vaccines is highly specific to subtype and strain. In practice, this means that antibodies induced in nature, experimental infections and vaccination can only neutralize the homotype of virus. The humoral immune response of a particular subtype/strain appears to be related to the relative immunological dominance of the various antigenic sites found on the globular head of hemagglutinin molecules (Wiley et al, Nature, 1981; 289: 373-378). In particular, antibody responses have been mapped to five major antigenic sites within the globular head of HA. Of these five HA epitopes (A-E), the two sites A and B are most immunodominant and associated with the highest number of amino acid hypermutability (hypermutability), due in part to recurrent gene-site mutations, deletions and occasional introduction of N-linked glycosylation sites, collectively referred to as "antigenic drift" of the virus (Cox & Bender, Semin. Virol.1995; 6: 359-70; Busch et al, Sci.286:1921,1999; Plotkin & Dushoff, PNAS 100:7152,2003; and Munoz & Deem, Vaccine,23,1144,2005).

The original antigen trace (originalantibigenic sin) first described by Francis (ann. int. med.,1953,399: 203) in 1953 was a primary immune response that when enhanced not by homology but by cross-reactive vaccines or input virus subtypes/strains, resulted in the newly formed antibody reacting more optimally with the previous antigen than with the input antigen.

Loss of immune specificity guided by the messy memories (oral recollections) presents a real problem to the host immune system of an effective humoral response to the balance of viral changes during and between infections. Thus, it is not surprising that natural infection and vaccination do not produce a more effective cross-reaction between primary and memory immunity, as the lineage develops against poorly immunogenic epitopes (probably the most conserved and cross-strain) immunity, which are lower at the antigenic level. Immunodominant epitopes mislead the immune response away from more conserved and weaker immune regions on an antigen, an immunological phenomenon initially referred to as "clonal dominance" (Kohler et al, J Acquir. Immune Defic. Syndr.1992;5: 1158-68), and later on as "pseudo-imprinting" (Kohler et al, Immunol. today, 1994 (10): 475-8).

The immunological mechanism of immunodominance behind pseudoimprinting is not fully understood, and there is no mechanism that fully explains how and why certain epitopes have evolved into immunomodulation and immunodominance. The range of immune responses observed in this phenomenon includes the induction of highly strain/isolate-specific neutralizing antibodies that can elicit passive protection in laboratory animal model-virus challenge systems (experimental animal model-viral challenge systems) up to the induction of a binding non-protective/non-neutralizing blocking or even pathogen-enhancing antibody that in some cases can prevent the host immune system from recognizing nearby epitopes in order to interfere with CD 4T cell help. Immune responses induced by immunodominance are also observed, leading to a more narrowly focused set of epitopes, as well as T cell and cytotoxic cell-mediated immunity in host helpers (Gzyl et al, Virology 2004; 318 (2): 493-506; Kiszka et al, J.Virol.200276 (9): 4222-32; and Goulder et al, J.Virol.2000; 74 (12): 5679-90).

Vaccination is the best way to prevent disease, and current trivalent inactivated virus vaccines and live attenuated (attenuated) influenza vaccines are developed each year based on epidemiological monitoring of active virus strains worldwide. Both vaccines comprise influenza a and influenza B subtypes. Approved influenza vaccines are made from inactivated whole or chemically split subunits from two influenza a subtypes (H1N 1 and H3N 2) and one influenza B subtype. Production of influenza vaccines involves adaptation of selected variants to high production in ovo by serial passage or reassortment with other high producing strains. Selected influenza viruses are grown in eggs and influenza virus particles are purified from allantoic fluid. And then prepared by inactivating whole or split virus by treatment with an inactivating agent such as formalin. More than 90% of the U.S. vaccine market is occupied by two companies, namely Aventis Pasteur (market share more than 50%) and chiron (powerderj) (uk). Intranasal vaccineApproved and first marketed in 2003.

Limitations of existing influenza vaccines include:

(1) efficacy is reduced in the elderly population. In the elderly, the rate of disease prevention is low, particularly those who have developed a lifestyle habit (Gorse et al, infec. dis,190:11-19, 2004). Significant antibody responses to the trivalent subviral particle influenza vaccine were observed in less than 30% of the elderly population over 65 or 65 years of age (Powers & Belshe, J.Infec. Dis,167:584 592, 1993);

(2) is produced by eggs. The existing manufacturing process relies on eggs. Influenza virus strains must replicate well in eggs, thus requiring a large supply of eggs each year. Production is worried every year due to the need to find the appropriate virus combinations;

(3) failure to cope with the recently emerging strains and drifted strains, such as A/Sydney/5/97 at the end of the 90 s, or with potentially pandemic strains, such as the hong Kong H5N1 virus that emerged in 1997;

(4) protection with existing whole or split influenza vaccines has a short life span and the effect is diminished as genetic changes occur in influenza strains due to antigenic variation. Ideally, the vaccine strain is paired with the influenza virus that causes the disease. Alterations that may occur in the hemagglutinin of egg-cultured influenza viruses when compared to the original isolate from the infected individual (Oxfordet al, J.Gen.Virol.72:185-189, 1989; and Rocha et al, J.Gen.Virol.74:2513-2518, 1993) attenuate the potential effects of such vaccines;

(5) those who are egg sensitive have side effects with vaccines produced in eggs; and

(6) currently approved production systems produce one vaccine per egg infected with influenza virus, with a production time of about 24 weeks.

Thus, the existing approved influenza vaccines: (1) does not elicit antibodies that neutralize common antigen variants (which recur during an epidemic every year) as well as subtype and reassortant viruses; (2) does not generate strong immune response in the elderly; and (3) do not find broader applicability due to the presence of side effects (e.g., some vaccines are not available for children).

Disclosure of Invention

The present disclosure relates in part to novel influenza antigens with enhanced or up-to-date immunogenicity. An influenza component of interest may be modified to provide a virus or virus subunit antigen with a different array and/or newly recognizable epitopes as an improved vaccine.

The more efficient and rapid use of recombinant technology coupled with the latest immune refocusing technology that results in the subunits HA, NA (or both) and/or components containing HA, NA (or both) greatly changes current vaccine development practices by producing an influenza vaccine with improved cross-strain effects, thereby breaking the need for current practice of tracking viruses every year around the world, saving significant capital, offloading medical resources, including increasing time and labor to produce and manufacture vaccines annually from eggs, and human life.

Other features and advantages are described herein, and will be apparent from, the following drawings and detailed description.

Drawings

The following drawings are provided for purposes of illustrating various aspects of the present disclosure and are in no way to be construed as limiting the scope of the present disclosure.

FIG. 1 shows Table 1 relating to the results of an immuno-refocusing HA antigen assay;

FIG. 2 shows Table 2 relating to specific mutations and their sequences;

FIG. 3 shows the approximate positions of a set of instances of immunorefocusing mutations on H1N1 HA. Group A shows the structure of an HA trimer comprising three HA-1 and HA-2 chains. Panels B, C and D represent HA monomers, showing the amino acids in the approximate positions of the selected mutations. These figures are modified from the structural file lru7.pdb of influenza H1N 1HA (a/PR 8/1934, Gamblin et al, Science,303: 1838-;

FIG. 4 shows Table 3 which provides the combinatorial mutations in epitopes 2 and 3;

figure 5 shows table 4 providing combinatorial mutations in epitope 5.

Detailed Description

Influenza is defined herein as comprising A, B, C types of virus. This virus is present in birds and various mammals, such as cats, dogs, horses, pigs, etc. Type a is the most pathogenic in humans, resulting in both seasonal epidemics and occasionally, very rarely, more fatal national infections. These types are defined by several serotypes, which reflect the host immune response to antigens expressed on the surface of viral particles. Two structures on the surface of the virus that carry most epitopes relevant for vaccine protection are hemagglutinin (HA or H) and neuraminidase (NA or N). There are at least 16 known subtypes of H and at least 9 known subtypes of N. HA mediates adsorption and fusion of the virus. NA has sialidase activity.

"wild-type" refers to a naturally occurring organism or portion thereof. This reference also relates to nucleic acids and proteins produced by natural processes that naturally occur in the natural organisms of the human population, as seen in polymorphisms that arise as a result of natural mutations and are maintained by gene drift, natural selection, etc., and does not include nucleic acids or proteins having sequences obtained, for example, by reassortment means.

"immunogen" and "antigen" are used interchangeably herein to mean a molecule that elicits a specific immune response in the body fluid (mediated) and/or T cell origin (mediated by cells), e.g., comprising a peptide that binds to the molecule or a CD₄ ⁺Or CD₈ ⁺T cell-bound antibody, wherein, CD₄ ⁺Or CD₈ ⁺T cells recognize cells that express this molecule (e.g., cells infected with a virus). This molecule may contain one or several sites for specific antibodies or T-cell binding. It is well known that such sites are called epitopes or determinants. The antigen can be polypeptide, polynucleotide, polysaccharide, lipid, etc., and their combination, such as glycoproteinOr a lipoprotein. An immunogenic compound or product, or antigenic compound or product, refers to a compound or product that is capable of eliciting a specific immune response, which may be humoral, cellular, or both.

Vaccines are immunogens or antigens used to generate an immune protective response, i.e., a response such as an antibody, that mitigates the negative effects of the immunogen or antigen or the entity expressing them in the host. It is well known that amounts are derived, inferred and/or determined from preclinical and clinical studies. Multiple doses may be administered as needed to ensure prolonged prophylactic or anergic status. For purposes of this disclosure, a successful endpoint (vaccine end point) for use with a vaccine is the presence of an induced immune response (e.g., humoral and/or T cell mediated) in a host that results, for example, in the production of serum antibodies, or antibodies produced by the host in any tissue or organ that bind to the antigen or immunogen of interest. In some embodiments, the induced antibodies bind in some manner to a compound, molecule, etc. that carries a cognate antigen or immunogen, or direct the host to neutralize, reduce, protect against, and/or eliminate a pathogen that infects and/or causes a clinical disease. This immune response can be monitored by methods known in the art, such as enzyme linked immunosorbent assay (ELISA), Western blot (Western blot), and the like. For the purposes of this disclosure, immunoprotection is the presence of such an anti-pathogenic, anti-immunogenic, anti-viral, etc. immune response (e.g., antibodies and/or T cells that bind to the immunogen or infected cells) in the exposed host. This can be determined using any known immunoassay, such as an ELISA and/or hemagglutinin inhibition assay. Alternatively, one can use a virus neutralization assay to determine, for example, whether circulating neutralizing anti-viral antibodies are present. For the purposes of this disclosure, the observation of immunoprotection in a host, i.e., the presence of circulating anti-influenza antibodies, for at least seven days, at least fourteen days, at least twenty-one days, at least thirty days, or longer, is evidence that the vaccine is of interest for efficacy. Furthermore, in general, the production of a Hemagglutination Inhibition (HI) titer of about 1:40 for homologous single influenza strains that were used to make the vaccine can be used as an endpoint, indicating the availability of a candidate vaccine. For the purposes of the present disclosure, any delay in death following exposure in animal models can be taken as evidence of protection. Thus, in the case of mice exposed to pathogenic influenza strains, the first mouse typically dies about 10 days after exposure. Thus, for the purposes of this disclosure, protection is considered to occur if the first day of death of the exposed mouse is delayed by at least one, at least two, at least three, or more days. The period of immunoprotection can be at least 14 days, at least 21 days, at least 28 days, at least 35 days, at least 45 days, at least 60 days, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 1 year, at least 2 years, or longer. The preferred observation population for immune protection is an outcrossing population, which observes different types, subtypes, virus strains, variant strains, alleles and the like of pathogens. Interesting components may include viral particles, inactivated or attenuated (live attenuated) viruses, or viral subunits such as NA or HA. Its constituents may also include virus-like particles (VLPs), structures known in the art to express viral proteins and antigens, which are found in fully replicating virions, but in the present disclosure, an immunodominant epitope of reduced immunity (immunodampened) is expressed. Normally, virus-like particles lack viral nucleic acid or a portion that allows its nucleic acid to replicate, and thus are not infectious.

In another embodiment, the antigen, determinant or portion thereof may be cloned into the genome of a wild-type virus, replacing its cognate wild-type gene. The recombinant virus can preserve the immunity-reducing molecule in the same way as the wild-type virus. The virus may be influenza virus or another viral vector. Thus, for example, an influenza virus strain that can be well propagated in chicken eggs is manipulated to express an immunity-reducing molecule, and the recombinant virus can be propagated using existing materials and methods for producing vaccines (using chicken eggs to produce immunity-reducing vaccines). Vaccines can be tailored to produce an immune response to one, two or more antigens; for a single virus, an immune response to two or more separate virus lines (lines), virus strains, clades (clades), etc.; (ii) an immune response to one, two or more epitopes; (ii) an immune response to one, two or more serotypes; and so on. This may be achieved by comprising a plurality of components, elements, viruses, virus-like particles, etc., wherein one of the above components, complexes, viruses, virus-like particles, etc., expresses an immunity-attenuating antigen of interest that is responsive to a plurality of HA types, NA types (or a mixture of both). In addition, multiple components may be incorporated into a single multi-functional composition (composition).

An "immunodominant epitope" is an epitope on an antigen that selectively elicits a host immune response and effectively or functionally excludes other epitopes on or in the antigen, which exclusion may be partial or complete.

By "reduced epitope immunity" is meant that the epitope is modified to significantly suppress the host immune system from producing antibodies, helper or cytotoxic T cells directed against the epitope. However, a decrease in immunity does not necessarily result in complete clearance of the epitope or a response to the epitope.

Immune Refocusing (IR) or Immune Refocusing Techniques (IRT) can be used to make effective vaccines against pathogens that express immunodominant epitopes. The organisms to which this technique is most applicable are characterized in that: this organism evolved a strategy called "pseudo-imprinting" to evade the host immune response, such as by revealing high levels of immunodominant epitopes that are antigenic shifted. Such immunodominant epitopes are typically represented as multiple amino acids that can be altered without affecting the viability of the pathogenic microorganism. Immune refocusing is synonymous with immune attenuation.

The reduced immunity to immunodominant epitopes of an antigen can result in the host organism producing high titers of antibodies or T cells in response to non-dominant epitopes on the antigen, and/or new antibody titers or T cell responses to other relatively immunosilent epitopes. Such reduced immunity antigens are useful as vaccines against microorganisms having moderately or highly variant and/or conserved immunodominant epitope antigens. The immunodominant epitope or antigen-induced antibody can be used as an effective vaccine against homologous microorganisms.

Immunodominant epitopes can be identified by examining serum or T cell reactivity in host organisms infected with pathogenic microorganisms. Serum is used to assess the amount of antibody bound to an identified antigen that may elicit an immune response in the host organism. If an immunodominant epitope is present, a significant amount of antibodies in the serum will bind to the immunodominant epitope, while little or no binding will occur to other epitopes present on or within the antigen.

Following determination of immunodominant epitopes, immunodominant epitopes are immunologically attenuated according to the materials and methods described herein and are a design choice in the art. Such manipulations can be performed at the nucleic acid level, protein level, carbohydrate level, etc., or in combinations at each level, and the methods of manipulation are described herein and known in the art.

For example, the presence of an N-linked sugar (CHO) can be determined from the primary amino acid sequence of the polypeptide. The composition of the tripartite amino acid sequence, which is a target for the addition of N-linked CHO, is asparagine, followed by any amino acid, and finally by serine or threonine (N-X-S/T), where X is any amino acid except proline and aspartic acid. N-linked glycosylation sites can be added or removed from the epitope, and methods and materials of operation are known in the art.

For example, recombinant gpl20 from HIV appears to introduce N-linked sequences at the molecular level (NXT/S), which can lead to the excessive addition of N-linked glycans in the immunodominant V3 region, showing new antigenic properties, such as the inability to bind antibodies that recognize the wild-type V3 epitope, while inducing antibody responses against other epitopes that were previously silent or less immunogenic. The presence of excessive carbohydrate moieties does not affect the infectivity of the HIV-1 recombinant virus. Experimental animals immunized with recombinant glycoproteins exhibit moderate to high titers of antibodies that neutralize infection with both homologous and heterologous wild-type HIV-1 in vitro. Thus, immunological attenuation of immunodominant epitopes within the gp 120/160V 3 region may lead to refocusing of the immune response to other neutralizing epitopes on the same antigen, see US 5,585,250 and US 5,853,724.

In addition, a particular amino acid on an immunodominant epitope may be substituted, replaced or deleted to reduce its immunogenicity. The reduction of immunity can occur by substitution, or deletion of one, two, three, or more amino acids from the immunodominant epitope, e.g., by site-directed mutagenesis of the nucleic acid encoding the antigen. Methods of altering nucleic acids/polypeptides are provided herein and are known in the art.

Various techniques can affect the attenuation of immunity, such as altering, replacing, or deleting specific amino acids of an epitope, or, for example, adding glycosylation sites at or near an epitope. The alteration may be effected at the polypeptide level or the polynucleotide level, as described herein, and methods of operation are known in the art. Thus, a polypeptide can be altered by the addition, deletion, or replacement of one or more molecules, groups, compounds, etc., at an epitope or at an internal target site. For example, a particular amino acid may be chemically derivatized, or modified to carry an additional group, such as a polysaccharide, e.g., polyethylene glycol.

Following manipulation of the immunogenic structure, a screening assay for the binding of the mutein (i.e.the manipulated antigen of interest), i.e.the antigen of reduced immunity of interest, to a defined known antibody can be used to determine whether reduced immunity has occurred; the antibody can bind to one or more immunodominant epitopes of influenza virus. For example, a synthetic polypeptide may contain one or more alterations in the primary amino acid sequence of its immunodominant epitope. Alternatively, the nucleic acid sequence of the immunodominant epitope is modified to express an immunity-attenuating epitope. For example, the nucleic acid sequence may thus be modified by site-directed mutagenesis, expressing amino acid substitutions, insertions, deletions and the like, some of which may introduce further modifications at or near the immunodominant epitope, e.g., introduction of a glycosylation site, e.g., introduction of a mutation that results in N-glycosylation or O-glycosylation at or near the immunodominant epitope, and the like.

Thus, a nucleic acid of interest encoding one or more immunity-attenuating epitopes of a polypeptide of interest can be placed in a cloning and/or expression vector, and the materials and methods of manipulation are known in the art and are generally commercially available as a design choice. For example, plasmids, cosmids, viruses, and replicons may be used, many of which are commercially available. Sources of viral vectors include adenovirus, adeno-associated virus, alphavirus, phage, and the like. Influenza viruses may also be used. Suitable host cells known for propagation of vectors of interest include prokaryotic cells, eukaryotic cells, bacteria, insect cells, mammalian cells, and the like. Cells can be constructed to secrete recombinant proteins of interest. Such that the polypeptide can be purified and used as an immunogen.

Alternatively, the polypeptide can be inserted into a vector comprising the genome of an influenza virus or an influenza virus, typically in place of the homologous coding sequence from which it is produced, to enable expression of the immuno-refocusing polypeptide in the influenza virus, virus-like particle or structure, viral surface structural component, subunit, or the like.

Thus, the immunological refocusing technique is not dependent on vector technology and can be applied to multiple expression systems. Thus, immune refocusing antigens can be incorporated into a number of novel vaccine vectors currently in use, including but not limited to: 1) recombinant influenza viruses produced using currently acceptable techniques (e.g., culture in mammalian cells or chicken embryos); 2) recombinant temperature sensitive influenza viruses, such as Flu-Mist; 3) a split vaccine; 4) subunit vaccines expressed in insect, mammalian, yeast, bacterial or other cells; 5) a DNA expression plasmid; and 6) heterologous viral expression systems, such as recombinant vaccinia virus, adenovirus, lentivirus, and the like.

Various purification methods for immunogens comprising immunorefocusing HA antigens are known in the art. Recombinant viruses incorporating immunorefocused HA antigens are purified using established and currently approved techniques for preparing licensed vaccines.

Subunit vaccines produced in insect, mammalian or other in vitro expression systems can be purified by conventional chromatography methods, and ion exchange, lectin binding resins, size fractions, antibody affinity, and the like can be used for isolation and purification. In addition, it is also contemplated that the immunogen may be expressed to contain a fusion domain to facilitate purification. For example, the HA antigen can be expressed as a poly-histidine fusion, allowing for rapid and simple purification on metal-activated resins. Such fusion partners can be cleaved off by specific proteolysis, if desired, using methods known in the art.

One procedure and its analogous method for obtaining epitope muteins (mutant epitopes different from the wild type) is "alanine scanning mutagenesis" (Cunningham & Wells, Science 244: 1081-645 (1989); and Cunningham & Wells, Proc. Natl. Acad. Sci. USA 84:6434-6437 (1991)). One or more residues are replaced by alanine (Ala) or polyalanine (polyalanine) residues. These residues exhibiting functional sensitivity to their substitution can then be refined by introducing further or other mutations at the substitution sites. Thus, when the site of introducing an amino acid sequence variation is specified in advance, the nature of the mutation itself is not necessarily specified in advance. Similar substitutions may be attempted for other amino acids, depending on the desired characteristics of the scanned residues. In addition, the choice of substituted amino acid can be based on, for example, the size, shape, or other parameters of the substituted amino acid. For example, glutamine can replace glutamate to reduce the charge of an epitope, while its size and shape changes to a very slight degree.

One method of more systematically identifying amino acid residues in need of modification includes identifying residues involved in immune system stimulation or immunodominant antibody recognition, as well as those residues that are involved little or not in immune system stimulation or immunodominant antibody recognition. Alanine scans of the residues involved were performed and each alanine mutation should be examined for reduced immune system stimulation or immunodominant antibody recognition of immunodominant epitopes. In another embodiment, residues are selected for modification that are involved in little or no stimulation of the immune system. Modifications may involve deletion of one or more residues, substitution of one residue, or insertion of one or more residues adjacent to the residue of interest. In one embodiment, the modification involves the substitution of the residue with another amino acid. The first substitution may be a conservative substitution. If such a substitution induces stimulation of the immune system or increased reactivity with known immunodominant antibodies, another conservative substitution may be made to determine if a more significant change is available.

By selecting amino acids that differ in their properties more than the residues normally located at this site, more pronounced changes in the ability of the immune system to respond away from immunodominant epitopes can be achieved. Thus, this replacement is performed while maintaining: (a) the backbone structure of the polypeptide chain of the replacement region, such as a folded (sheet) or helical (helical) conformation; (b) charge or hydrophobicity of the target site molecule, or (c) size of the side chain.

For example, naturally occurring amino acids can be divided into, based on the properties of the general side chains:

(1) hydrophobicity: methionine (M or Met), alanine (a or Ala), valine (V or Val), leucine (L or Leu) and isoleucine (I or lie);

(2) neutral, hydrophilic: cysteine (C or Cys), serine (S or Ser), threonine (T or Thr), asparagine (N or Asn), and glutamine (Q or Gin);

(3) acidity: aspartic acid (D or Asp) and glutamic acid (E or Glu);

(4) alkalinity: histidine (H or His), lysine (K or Lys) and arginine (R and Arg);

(5) residues affecting side chain orientation: glycine (G or Gly) and proline (P or Pro), and

(6) aromatic, tryptophan (W or Trp), tyrosine (Y or Tyr) and phenylalanine (F or Phe).

Non-conservative substitutions are exchanges of one amino acid for an amino acid in another class. Conservative substitutions refer to the exchange of one amino acid for another within the same class.

Preferred amino acid substitutions are those that attenuate immunodominant epitopes, but may also include, for example: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter immune system stimulatory activity, and/or (4) confer or modify other physicochemical or functional properties of these analogs. Analogs can include a variety of muteins of a sequence other than the naturally occurring peptide chain sequence. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the sequence of a naturally occurring or mutant protein. Generally, conservative amino acid substitutions do not significantly alter the structural characteristics of the parent sequence (parentsequence) (e.g., the tendency of the substituted amino acid not to break a helix in the parent or mutein sequence, or disrupt other types of characteristic secondary structure in the parent sequence), unless the size or conformation of the R group or side chain is altered (protein, structure and molecular principles (Creighton, ed., W.H.Freeman and Company, New York (1984); protein structural profiles, Branden & Tooze, eds, Garland Publishing, New York, NY (1991)); and Thorne et al, Nature 354:105 (1991)).

Typically, the epitope with altered biological properties is mutated to have an amino acid sequence which is at least 75% identical or similar, at least 80%, 85% and 90%, and usually at least 95% identical to the amino acid sequence of the parent molecule. Identical or similar to a parent amino acid sequence is defined herein as the proportion of amino acid residues in a candidate sequence that, when necessary, align or fill in gaps, are identical (i.e., identical residues) or similar (i.e., amino acid residues from the same class, supra, based on common side chain properties) to the parent molecular residues, achieving maximum uniformity of sequence proportions.

Covalent modifications to the molecule of interest are included within the scope of the disclosure, e.g., immunological attenuation of the molecule or obtaining a functional equivalent, which may or may not carry other enhanced, additional, or different functions or characteristics in addition to immunological attenuation. This can be prepared by chemical synthesis or by enzymatic or chemical cleavage of the molecule, if applicable. Other types of covalent modifications of the molecule can be introduced by reacting an organic derivatizing agent with targeted amino acid residues of the molecule, such derivatizing agent being capable of reacting selectively with side chains, N-terminal residues, or C-terminal residues.

Also, a variety of organic chemical materials and methods can be used to modify the components of the epitope. For example, WO 05/35726 teaches various methods for introducing, modifying, altering, replacing, etc. substituents on biomolecules.

For example, a cysteinyl residue can be reacted with an α -haloacetate (and its corresponding amine), such as chloroacetic acid or chloroacetamide, to form a carboxymethyl or ureidomethyl derivative. Cysteinyl residues can also be derivatized by reaction with bromotrifluoroacetone (bromotrifluoroacetone), α -bromo- β - (5-imidazolyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide (3-nitro-2-pyridyl disulfide), methyl 2-pyridyl disulfide (methyl 2-pyridyl disulfide), p-chloromercuryl benzoate, 2-chloromercuryl-4-nitrophenol, and chloro-7-nitrobenzo-2-oxa-1, 3-diazole, and the like.

At pH 5.5-7.0, histidyl residues may be derivatized by reaction with diethylpyrocarbonate. Para-bromoacetophenone bromide may also be used, preferably in 0.1M sodium cacodylate at pH 6.0.

The lysyl and alpha amino terminal residues can be reacted with succinic acid or other carboxylic acid anhydrides to charge the residues oppositely. Other suitable reagents for derivatizing alpha amino-containing residues include imidoesters such as methyl picolinate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, and 2, 4-pentanedione, as well as amino acids that can be transaminated with glyoxylic acid.

Modification of arginyl residues can be carried out by reaction with one or several conventional reagents, such as phenylglyoxal, 2, 3-butanedione, 1, 2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues typically requires basic reaction conditions. In addition, these reagents can also react with the epsilon amino groups of lysine as well as arginine.

Specific modifications of tyrosyl residues may be made by aromatic diazo compounds or tetranitromethane. For example, 1-acetylimidazole and tetranitromethane can be used to form O-acetyl tyrosinyl (O-acetyl tyrosyl species) and 3-nitro derivatives, respectively.

The carboxyl side chain group (aspartyl or glutamyl) may be modified by reaction with carbodiimide (R-N = C-R '), where R and R' may be different alkyl groups, such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylpentyl) carbodiimide (1-ethyl-3- (4-azonia-4, 4-dimethylpenyl) carbodiimide). In addition, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are generally deamidated under neutral or basic conditions to yield the corresponding glutamyl and aspartyl residues, respectively. Deamidated forms of these residues are within the scope of this disclosure.

Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups on serine or threonyl residues, methylation of lysine, arginine and histidine alpha amino groups (Creighton, protein: structural and molecular characteristics, W.H.Freeman & Co., San Francisco, pp.79-86 (1983)), and acetylation of the N-terminal amino group and amidation of any C-terminal carboxyl group.

Another class of covalent modifications involves coupling of glycosides, sugars, saccharides, etc. to molecules of interest by chemical or enzymatic reactions. Depending on the coupling scheme used, the saccharide may be attached to: (a) arginine and histidine; (b) a free carboxyl group; (c) free sulfhydryl groups, such as cysteine sulfhydryl groups; (d) free hydroxyl groups, such as the hydroxyl groups of serine, threonine, or hydroxyproline; (e) aromatic residues, such as residues of phenylalanine, tyrosine or tryptophan; or (f) the amide group of glutamine. These methods are described in WO 87/05330 and Aplin & Wriston, CRC Crit. Rev. biochem., pp.259-306 (1981). Enzymatic methods can also be used to add sugar groups, such as glucosyltransferases, sialyltransferases, galactosyltransferases, and the like.

Removal of the glycosyl moieties present on the molecule of interest can be achieved by chemical or enzymatic means. For example, chemical deglycosylation requires exposure of the molecule to triflic acid or its equivalent, resulting in the dissociation of most or all of the sugar groups except the linking sugar (linkingsugar) (N-acetylglucosamine or N-acetylgalactosamine) while maintaining the integrity of the rest of the molecule. Chemical deglycosylation is described, for example, in Hakimuddin et al, Arch.biochem.Biophys.259:52 (1987) and Edge et al, anal.biochem.118:131 (1981). Enzymatic cleavage of the glycosyl moieties on the molecule can be achieved by any of a variety of endoglycosidases and exoglycosidases, see Thotakura et al, meth.enzymol.138:350 (1987). Thus, mannosidase, fucosidase, glucosaminidase, galatosidase, and the like can be used.

RNA or DNA encoding influenza HA, NA, etc. can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes capable of specifically binding the gene of interest, Innis et al, see PCR handbook: instructions for methods and applications, Academic (1990) and Sanger et al, Proc. Natl. Acad. Sci.74:5463 (1977)). After isolation, the DNA may be placed into an expression vector, which is then placed into a host cell, such as an E.coli cell, NS0 cell, COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell, to obtain a synthetic protein of interest in the recombinant host cell. RNA or DNA may also be modified, for example, by base substitutions to optimize codon usage in a particular host cell, or by covalent attachment of a coding sequence for a heterologous polypeptide.

Thus, as described herein, a polynucleotide of interest encoding and expressing one or more immunity-attenuating epitopes can be expressed with reference to molecular biology and recombinant materials and methods known in the art to obtain a source of the recombinant polypeptide of interest.

In another embodiment, a polynucleotide of interest encoding one or more immune attenuating epitopes can replace a homologous wild-type sequence in the influenza virus genome resulting in a virus, virus-like structure, or subunit expressing the one or more immune attenuating epitopes. Thus, the virus strain may be propagated using standard techniques, such as currently used egg-based techniques, to produce immunogens which may be used alone as vaccines or in combination with other active agents, such as unmodified viruses or multiple viruses (e.g., to mimic current bivalent and trivalent vaccines).

These phrases, terms and combinations of "functional fragments, portions, variants, derivatives or analogs" and combinations thereof of influenza virus, antigens, components, subunits, HA, NA, etc., relate to an element whose biological activity is qualitatively the same as that of the wild-type, parent element or original non-immunologically reduced antigen, which is the source of the variant, derivative, analog, etc. For example, a functional portion, fragment or analog of HA may stimulate an immune response as does native HA, although the response may be to a different epitope on HA.

Thus, included within the scope of this disclosure are functional equivalents of the virus, or portions or derivatives of interest. The term "functional equivalent" includes viruses and moieties capable of stimulating an immune response against influenza virus.

Thus, once candidate immunity-attenuating polypeptides have been identified, those skilled in the art are well able to modify the amino acid sequence while maintaining the goal of immunity attenuation and exposure of the originally silenced epitope. Thus, for example, the encoding nucleic acid may be modified to encode amino acid insertions, deletions or substitutions under conditions that do not adversely affect both of these objectives. In another embodiment, amino acids may be modified, derivatized, substituted, etc., by reference to procedures and materials known in the art, so long as both objectives are not adversely affected. As described herein, these changes and modifications to the original immunity-attenuating polypeptide are considered functionally equivalent. The degree of attenuation of immunity can be quantified in vitro or in animal assays as described herein or as known in the art. Similarly, the extent of response to the originally silent epitope can be quantified in vitro or in animal assays as described herein or as known in the art. The degree of reduction that one of the two objectives described herein is acceptable to a degree when modifying the original immunity-attenuating sequence is a design choice. In general, a reduction in reactivity of no more than 25% can be tolerated, since normally the response to immunodominant epitopes is required to be as low as possible. Thus, if a modification to the original immune attenuating sequence shows that e.g. 25% reactivity is restored as determined by ELISA using immunodominant epitope antibodies, compared to non-reactivity in the original immune attenuating epitope, it is considered that a reduction in reactivity is acceptable for functional equivalents. Functional equivalents to an immunologically attenuated antigen may be considered to be acceptably reduced by no more than 20%, 15%, 10%, 5% or no reduction. Similarly, a reduction of no more than 25% is tolerable for the current reactivity against the original silent epitope, since normally the highest possible reactivity against the original silent epitope is required. Thus, if a modification to the original immunity-attenuating sequence shows a loss of, for example, 25% of reactivity, as determined, for example, by ELISA using antibodies against the original silent epitope, as compared to the maximum reactivity in the original immunity-attenuating epitope, it is considered that a reduction in reactivity is acceptable for functional equivalents. For antigenic equivalents where the silencing epitope was originally expressed, it is considered acceptable to reduce by no more than 20%, 15%, 10%, 5% or not.

Interesting parts of influenza viruses, such as enveloped or non-enveloped preparations carrying HA, NA, M2 and combinations thereof, and any other influenza virus antigenic preparation, can be obtained by methods known in the art. When one or more non-protective immunodominant epitopes (IDNPEs), including epitopes that stimulate virus-specific, but narrow-range immunity, are removed or attenuated, for example, by intramolecular modifications (e.g., deletions, charge changes, addition of one or more N-linked sequences, etc.) and injected as antigens into the non-immunized animal, the alteration of IDNPE induces a new level of immune response at the B-and/or T-cell level against the subdominant or previously silenced epitope (Garrity et al, j.immunol. (1997) 159 (1): 279-89). The technique described herein is referred to as "immune refocusing".

Once altered, whether or not the alteration reduces the reactivity of, for example, a currently modified immunodominant epitope, attenuated epitopes, antigens, etc., are identified herein or as known in the art. The antigen may be tested in vitro by determining the reactivity of the attenuated antigen with an anti-toxin serum reactive with the dominant epitope, for example using an ELISA or Western blot. Candidates showing reduced reactivity are selected for in vivo testing with defined anti-virus sera to determine whether these attenuated antigens are immunogenic and whether the host is immune-responsive thereto. Thus, for example, mice are immunized to attenuate the antigen as known in the art, serum is harvested and immediately tested for reactivity in vitro. The anti-virus sera were then tested against wild-type virus to determine whether the antibody still recognized the wild-type epitope or wild-type antigen. This can be done by, for example, ELISA or Western blot. The latter is informative and shows whether a particular immunodominant epitope is bound, and whether the antisera is still reactive with influenza virus, carries the size of the epitope molecule reactive with mouse antisera, and possibly its identity.

Immune refocusing vaccine candidates are designed to fold exactly and adopt conformational epitopes similar to native for stimulation of appropriate immune responses. Prior to antigen testing, purified HA antigens are typically evaluated for their ability to bind to conformation-dependent antibodies and viral receptors on cell surfaces. Methods for assessing this interaction are well known in the art and widely employed.

Non-clinical trials are often required before development of immune refocusing antigens into commercial vaccines. One method is to inject or immunize an animal, such as a mouse, guinea pig, rabbit, chicken, rat, ferret, etc., with an immune refocusing candidate vaccine. Following immunization, the plates of immune sera were evaluated for various activities including reactivity to purified antigens or viruses (binding activity), hemagglutination inhibition (inhibition of virus-receptor action, HI) and virus neutralization (inhibition of infection of cultured cells and/or inhibition of replication in cultured cells, VN). Methods for assessing serum reactivity are well known in the art.

Sera from animals vaccinated with immune refocusing antigens were compared to the heterologous HI and VN activities of sera from animals vaccinated with non-mutated antigens for evaluation of broadening (broadening) and improvement of protective immunity. Thus, HI and VN of the influenza virus H1N1 or other serotype of heterologous virus strains were tested with a variety of immune sera generated in antigenic studies. The rise in HI or VN titres in the test sera relative to sera produced by unmodified antigen vaccination indicates that the antigen used to produce the test sera described above may stimulate a broadening and an improvement in immunity.

To further illustrate an example of analysis of candidate vaccine selection of interest, a hypothetical data set is provided in table 1 of fig. 1. The immuno-refocusing HA antigens from the A/California/04/2009 strain were tested (Mut HI, Mut H2, Mut H3 and Mut H4) and tested for their antiviral activity against both homologous and heterologous viruses. The heterologous viruses (strains 2-4) are described in the order of increasing d from the antigenic divergence (antigenic divergence) of the parental California/04/2009 (Cal/04/09) strain. All immunogen stimulated HI and VN titers were equivalent to those induced by unmodified antigen (WT-HA). The serum from Mut-Hl statistically contained levels of antiviral activity against virus strains 2-4 higher than the WT-HA titer, as determined by both assays. Sera derived from Mut-H2 and Mut-H3 had 2-fold titers of HI and VN activity against strain 2, but may not be statistically significant if the sera were diluted in a 2-fold gradient. However, sera derived from Mut-H2 and Mut-H3 had significantly higher HI and VN titers against the more divergent strains 3 and 4. Analysis of these data in Table 1 shows that Mut-Hl or Mut-H2 stimulates the highest level of antiviral activity, normally measured as a correlation in influenza preclinical testing.

To further evaluate the protective immunity of the immune refocused candidate antigen, more animal model studies can be performed. In one such experimental example, ferret groups were immunized with unmodified antigen or the preferred immunological refocusing candidate, respectively (e.g., Mut-H2 listed in Table 1). After immunization, the two groups of ferrets were divided into subgroups and each subgroup was vaccinated (challenged) with homologous or heterologous strains of influenza, as shown in table 1. Within two weeks after challenge, measures of pathogenesis, and viral replication were collected. Analysis of the measurements showed that ferrets immunized with Mut-H2 and challenged with a heterologous strain had reduced morbidity and viral replication compared to the WT-HA immunized group, indicating that Mut-H2 stimulated a broader range of immunity and could be an improved vaccine candidate.

Candidate immune-diminished antigens that react little or no longer with the candidate immune-diminished antigen with known anti-viral sera that bind to the maternal immunodominant antigen, but which remain immunogenic in the host, are selected as candidate vaccines for further testing. The candidate vaccines can also stimulate and strengthen the reactivity to maternal immunodominant antigens while being used for immune recognition against immune refocusing epitopes. For example, in a standardized antiviral-based assay, multiple influenza virus strains can be used to test the reactivity of mouse anti-virus sera thereto, to determine the versatility of the antibody response, i.e., whether the newly recognized epitope on the attenuated antigen is universal for a wider range of influenza virus strains, and whether the antibody has broad-spectrum antiviral activity.

Thus, the recombinant ha (rha) subunit protein vaccine is sufficient to protect against challenge by homologous strains of influenza virus. rHA is also used as an immunogen in the elderly. As described herein, second generation immune refocused HA subunit vaccines can also induce protective immunity against heterologous virus strains (Treanor et al, j. infection Diseases 2006;193: 1223-8). Thus, for example, a candidate vaccine of interest may confer protection not only against homologous antigens, such as H1, but also against different lines or isolates of HI, such as H2, H3, H4, and the like.

In one embodiment, the HA and NA of the influenza virus are selected as refocusing targets for other non-dominant sites on the HA and NA of the host immune response, as targets for a new immunoprotective response, preferably one of a broad range and spectrum of responses, active against a variety of viral strains, and the like.

For example, HA HAs 5 immunodominant sites or epitopes, designated A-E. Site A includes amino acids at position 140-146 of the HA-type virus strain, and the sequence is KRRSNKS (SEQ ID NO: l). In contrast to the hong kong virus strain, the wyoming virus strain (wyoming strain) has3 glycosylation sites associated with it at this site. Thus, one approach is to remove the loop structure defined by site A, e.g., replacing the KRRSNKS (SEQ ID NO: 1) sequence with GG.

Site B includes amino acids 189-197 of HA, with sequence SDQISLYAQ (SEQ ID NO: 2). It forms a helix and interacts with the amino acid with the sequence KYKY at position 158-161 (SEQ ID NO: 3). The B site can be subject to many possible variations, such as replacing the QIS with NAS; replacement of SLY with NIT; replacing KYK with NST at position 158; and replacement of YKY with NAS at position 159, all of these changes at these 4 sites introduced an N-glycosylation.

The site C comprises 276-278 amino acids, and the sequence is KCN. NCT can replace KCN.

Site D comprises a large antiparallel loop of amino acids 201 and 220. The entire ring may be deleted. Furthermore, the glycosylation site NIT can replace the RIT at position 201-203.

Position E includes amino acids 79-82, and the sequence is FQNK (SEQ ID NO: 4). The glycosylation site NET may replace QNK.

The above changes may be combined, for example, changes in the B site NST and NTS may both be combined with the proposed exemplary changes in the C and/or E sites.

As described herein, and as is known in the art, such immunodominant site changes can be obtained by cloning, site-directed mutagenesis, gene synthesis amplification, and the like.

Thus, the above A site variations can be obtained by site-directed mutagenesis using the following primers: top A: GGAACAAGCTCTGCTTGCggcggtTTCTTTAGTAGATTGAATTGG (SEQ ID NO: 5) and bottom A: CCAATTCAATCTACTAAAGAAaccgccGCAAGCAGAGCTTGTTCC (SEQ ID NO: 6), was used to obtain the sequence GTSSACGGFFSRLN (SEQ ID NO: 7) containing the above deletion and to insert the GG dipeptide at the deletion site.

The change in B site can be obtained using the following primers, top of B1: CAAATCAGCCTATATGCTaatGCATCAGGAAGAATCAC (SEQ ID NO: 8) and B1 bottom: GTGATTCTTCCTGATGCattAGCATATAGGCTGATTTG (SEQ ID NO: 9), resulting in a sequence QISLYANASGRI (SEQ ID N0: 10); primer B2 top: CACCACCCGGTTACGGACaatGACacAATCAGCCTATATGCTCAAGC (SEQ ID NO: 11) and B2 bottom: GCTTGAGCATATAGGCTGATTgtGTCattGTCCGTAACCGGGTGGTG (SEQ ID NO: 12), resulting in a sequence HHPVTDNDTISLYAQ (SEQ ID NO: 13); primer B3 top: CGGACAGTGACCAAATCAatCTAtcTGCTCAAGCATCAGGAAG (SEQ ID NO: 14) and B3 bottom: CTTCCTGATGCTTGAGCAgaTAGatTGATTTGGTCACTGTCCG (SEQ ID N0: 15), resulting in a sequence of DSDQINLSAQASG (SEQ ID NO: 16); primer B4 top: GAATTGGTTGACCCACTTAAAtTACAcATACCCAGCATTGAACGTGAC (SEQ ID NO: 17) and B4 bottom: GTCACGTTCAATGCTGGGTATgTGTAaTTTAAGTGGGTCAACCAATTC (SEQ ID NO: 18), resulting in a sequence NWLTHLNYTYPALNV (SEQ ID NO: 19); primer B5 top: GAATTGGTTGACCCACTTAAAAaACAAAacCCCAGCATTGAACGTGACTATG (SEQ ID NO 0: 20) and B5 bottom: CATAGTCACGTTCAATGCTGGGgtTTTGTtTTTTAAGTGGGTCAACCAATTC (SEQ ID NO: 21), the resulting sequence was NWLTHLKNKTPALNVTM (SEQ ID NO: 22).

The change in C-site can be obtained using the following primers, top of C1: GATCAGATGCACCCATTGGCAAtTGCAgTTCTGAATGCATCACTCC (SEQ ID NO: 23) and C1 base: GGAGTGATGCATTCAGAAcTGCAaTTGCCAATGGGTGCATCTGATC (SEQ ID NO: 24), the resulting sequence was SDAPIGNCSSECIT (SEQ ID NO: 25).

The D site changes can be obtained using the following primers, top of D1: CTATATGCTCAAGCATCAGGAAatATCACAGTCTCTACCAAAAG (SEQ ID NO: 26) and D1 bottom: CTTTTGGTAGAGACTGTGATatTTCCTGATGCTTGAGCATATAG (SEQ ID NO: 27), the resulting sequence was LYAQASGNITVSTKRS (SEQ ID NO: 28).

The change in E-site can be obtained using the following primers, top of E1: GATGGCTTCCAAAATAAGAcATGGGACCTTTTTGTTGAAC (SEQ ID NO: 29) and E1 bottom: GTTCAACAAAAAGGTCCCATgTCTTATTTTGGAAGCCATC (SEQ ID N0: 30), the resulting sequence is DGFQNKTWDLFVE (SEQ ID NO: 31).

HAS 1: CAGTCCTCATCAGATCCTTG (SEQ ID NO: 32) and HAS2: GGTAAGGGATATCTCCAGCAG (SEQ ID NO: 33) primers were used for sequencing, HAS3: cgcgattgcgccaaatatgcc (SEQ ID NO: 34) as a negative control.

The plurality of antigenic sites are enriched in charged amino acid residues. Another approach is to replace those charged residues with alanine residues. Examples of such changes include substitution of AGA for KRR at the A site; the B site was substituted with AYKY (SEQ ID NO: 35) for KY (SEQ ID NO: 3) and SAQI (SEQ ID NO: 36) for SDQI; the C site replaces KCN with ACN; the D site replaced RIT with AIT.

Furthermore, mutations at the B site to assess the function of hydrophobic tyrosine residues can be obtained by replacing SLY with SLT.

In addition to B cell epitopes, T cell epitopes may also undergo immunological attenuation. Major CD in the 177-199 residue region₄Epitopes include an MHC class II binding epitope outside of the targeted B site. The LYIWGVHHP (SEQ ID NO: 37) residue mutation that attenuates T cell responses includes VYIW (SEQ ID NO: 38) or VTIW (SEQ ID NO: 39) in place of LYIW; and IHAG (SEQ ID N0: 40) instead of VHHP.

In another embodiment, a recombinant polypeptide of interest comprising one or more immunologically reduced epitopes, or a vector comprising the same epitope, such as a virus-like particle, a subunit component of a virus, can be used as an immunogen to obtain specific antibodies or anti-viral sera that recognize epitopes that are abnormal but immunogenic due to the reduced immunity of the immunodominant epitope. The antibody may be prepared using materials and methods known in the art and thus may be polyclonal, obtained by immunizing an animal and collecting serum, or obtained from a human exposed to the same virus, similar to immunoglobulins currently used, or may be monoclonal, obtained according to known materials and methods. It would be beneficial to have procedures for obtaining human or human antibodies, and to engineer such antibodies to minimize immunogenicity, such as the addition of sugar groups (e.g., PEG molecules and other substituents) to mask immunogenic sites of the influenza antibody that are not normally immunodominant epitopes, as is known in the art, see, for example, U.S. Pat. nos. 5,821,337 and WO 09/32661. The antibody can be used as a passive vaccine (passive vaccine) for collecting and managing.

In testing, for example, an immunogen of interest is injected into a non-human mammal to obtain preclinical data. Typical non-human mammals include non-human primates, canines, felines, rodents, and other mammals. These mammals can be established animal models, animal models for formulating a treatment for a disease, or for studying the toxicity of an immunogen of interest. In these examples, dose escalation studies can be performed in mammals.

To obtain approval for human products by regulatory agencies such as the U.S. food and drug administration or the european drug administration, biopharmaceuticals must meet purity, safety and performance standards as specified by the relevant regulatory agencies. To produce a vaccine meeting these criteria, the recombinant microorganism may be stored in a culture medium, such as a certified transmissible spongiform encephalopathy (hereinafter "TSE") free medium.

For example, plasmids containing the vaccine coding sequence may carry a non-antibiotic selectable marker, as it is not always desirable to use an antibiotic resistance marker to select and maintain plasmids within bacteria designed for use in humans, although the preferred embodiments are related to the use of recombinant subunit vaccines. Thus, in one embodiment, the present disclosure provides a selection strategy in which, for example, a catabolic enzyme is used as a selectable marker to enable the growth of bacteria on a medium containing a substrate for the catabolic enzyme as described above as a carbon source. Examples of such lytic enzymes include, but are not limited to, lacYZ encoding lactose uptake and β -galactosyl glycinase (GenBank Nos. J01636, J01637, K01483or K01793). Other selectable markers that provide metabolic advantage in defined media include, but are not limited to, galTK with galactose (GenBank No. X02306), sacPA with sucrose (GenBank No. J03006), trePAR with trehalose (GenBank No. Z54245), xy1AB with xylose (GenBank No. CAB 13644 and AAB 41094), and the like. Alternatively, its selection may also involve the use of antisense mRNA to suppress a toxic allele, such as the sacB allele (GenBank No. np 391325).

The particular method used to formulate the novel vaccine and the above formulations is not critical to the present disclosure and may be selected from or may include: a physiological buffer (Feigner et al, U.S. Pat.No.5,589,466 (1996)); aluminum phosphate or hydroxyphosphate (e.g., Ulmer et al, Vaccine,18:18 (2000)); monophosphoryl lipid A (also known as MPL or MPLA; Schnerson et al, J.Immunol.,147: 2136-; QS-21 saponins (e.g., Sasaki et al, J.Virol.,72:4931 (1998)); dexamethasone (e.g., Malone et al, j.biol. chem.269:29903 (1994)); CpG DNA sequences (Davis et al, j.immunol.,15:870 (1998)); interferon- α (Mohanty et al, J.Chemother.14 (2): 194-197, (2002)); lipopolysaccharide (LPS) antagonists (Hone et al, J.HumanVirol, 1: 251-. The use of commercial adjuvants is also a design choice, as is known in the art.

Since the immunological refocusing techniques of interest are independent of adjuvants and immunomodulators, the antigens produced by the methods described herein or antibodies reactive therewith are compatible with adjuvants such AS alumina gel and novel compositions developed by Novartis and GlaxoSmithKline et al (e.g., MF59 and AS 03), AS well AS modulators and enhancers such AS CpG and cytokines.

The formulations herein may also contain more than one active compound necessary to treat a particular indication (indication), preferably those compounds that have complementary activities without adversely affecting each other. For example, it may be desirable to further provide an adjuvant. These molecules may suitably be present in combination in an amount to achieve the desired purpose. The adjuvants may be administered sequentially, either before or after administration of the antigen.

Immunogens of interest may be used with a second component, such as a therapeutic moiety linked or mixed to the same molecule, administered as a conjugate (conjugate) as a therapeutic agent, administered separately as two components of a therapeutic agent, mixed as a therapeutic agent prior to administration, and the like, see, for example, New generation vaccine by Levine et al, 2nd Marcel Dekker, inc. The other therapeutic agent may be any drug, vaccine, etc. useful for achieving the intended purpose. Thus, the therapeutic agent may be a small biological molecule or the like. The immunogen of interest may be administered simultaneously with the second influenza immunogenic component, the third second influenza immunogenic component, etc., or sequentially, e.g., may or may not be attenuated. Thus, immunity-reducing antigens of interest can be combined with existing vaccines to form bivalent vaccines, trivalent vaccines, and the like, but such methods should be avoided when the existing vaccines are produced in eggs. Alternatively, an immunity-attenuating antigen of interest can be combined with a variety of other antigens, subunits, components, virus-like particles, etc., to form a multivalent vaccine.

The term "small molecule" and similar terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, carbohydrates, lipids, nucleotides, nucleoside analogs, organic or inorganic compounds having a molecular weight of less than 10,000g/mol (i.e., including heteroatomic organic compounds (heteroorganic) and/or metalorganic compounds), organic or inorganic compounds having a molecular weight of less than 5,000g/mol, organic or inorganic compounds having a molecular weight of less than 1,000g/mol, organic or inorganic compounds having a molecular weight of less than 500g/mol, salts, esters, and combinations thereof, as well as other pharmaceutically acceptable forms of such compounds that can stimulate an immune response or be immunogenic, or have a desired pharmacological activity.

Thus, the immunogens of this disclosure may be administered alone, or in combination with other types of therapies, including secondary immunizations or treatments to treat the disease. The second component may be an immunostimulant.

Furthermore, the immunogens disclosed in the present invention may be linked to a variety of effector molecules, such as heterologous polypeptides, drugs, etc., see WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EPO 396,387. The immunogen may also be linked to a therapeutic moiety, such as an antibiotic (a therapeutic agent) or an adjuvant.

The therapeutic compounds of this disclosure reduce at least one influenza-associated symptom. The products of this disclosure may be provided as pharmaceutically acceptable ingredients, as in the present process or as described herein. The terms "physiologically acceptable", "pharmaceutically acceptable", and the like, mean approved by a regulatory agency of the federal or a state government for use in a human body, or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.

The product of interest may be administered to a mammal in any acceptable manner. Modes of introduction include, but are not limited to, parenteral, subcutaneous, transdermal, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation, and oral routes, and if immunosuppressive therapy is desired, intralesional administration. Parenteral infusion includes intramuscular, intradermal, intravenous, intraarterial, or intraperitoneal administration. The product or composition can be administered by any convenient route, such as infusion or intravenous bolus injection (bolus injection), absorbed through epithelial or skin mucosal linings (such as oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other bioactive agents. Can be administered systemically or locally. In addition, it may be desirable to introduce the therapeutic products or compositions of the present disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; for example, intraventricular injection may be assisted by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Furthermore, the product may suitably be administered by pulse infusion, particularly where the dose of the product of interest is reduced. The preferred method of administration is by injection, preferably intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic.

In the current disclosure, a variety of other delivery systems are known for the administration of products, including encapsulation in Liposomes, microparticles, or microcapsules (see Langer, Science 249:1527 (1990); Liposomes in the Therapy of Infectious diseases and Cancer, Lopez-Beresein et al, (1989)).

Microcapsules, such as hydroxymethylcellulose or gelatin-microcapsules and polymethacrylate microcapsules, are prepared by coacervation techniques (coascervation techniques) or interfacial polymerization, and the active ingredient is encapsulated therein for use in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. These techniques are disclosed in Remington's pharmaceutical Sciences,16th edition, A.Osal, Ed. (1980).

Pulmonary administration can also be employed, such as by use of an inhaler or nebulizer, and formulation with a nebulizer. The ingredients of interest may also be administered to the lungs of a patient in the form of dry powdered ingredients, see U.S. Pat. No. 6,514,496.

It may be desirable to topically apply the therapeutic products or ingredients of the present disclosure to the area in need of treatment. This may be accomplished, for example, by local infusion, local application, by injection, by use of a catheter, suppository, or implant, but is not limited to such, which may be a porous, non-porous, or gelatinous material (including hydrogels or films such as silicone rubber films or fibers). Preferably, when applying the products of the present disclosure, special attention needs to be paid to which materials are protein-absorbing and which are non-absorbing.

In yet another embodiment, the product may be delivered by a controlled release system. In one embodiment, pumps may be used (see Langer, Science 249:1527 (1990); Sefton, CRC crit. Ref. biomed. Eng.14:201 (1987); Buchwald et al, Surgery 88:507 (1980); and Saudek et al, NEJM 321:574 (1989)). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer et al, eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al, eds., Wiley (1984); Ranger et al, J.Macromol.Sci.Rev.Macromol.Chem.23:61 (1983); also see Levy et al, Science 228:190 (1985); During et al, Ann.Neurol.25:351 (1989); and Howard et al, J.Neurog.71: 105 (1989)). In yet another embodiment, the controlled release system may be placed in proximity to a treatment target.

Sustained release formulations are prepared for use with the product of interest. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the immunogen, which matrices are in the form of shaped articles, e.g. films, or matrices. Suitable examples of such sustained release formulation matrices include polyesters, hydrogels (e.g., poly (methyl 2-hydroxyethyl acrylate), polyvinyl alcohol), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid (ethyl-L-glutamate), non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., injectable microspheres of lactic acid-glycolic acid copolymers), and poly-D- (-) -3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid may allow release of molecules for periods of time in excess of 100 days, certain hydrogels may release cells, proteins, and products in a shorter period of time. Depending on the mechanism involved, a reasonable strategy can be devised for stability.

The form of the ingredient (compositions) may be solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, depot preparations (depots), and the like. The composition can be formulated into suppository with conventional binder (binder) and carrier such as triglyceride. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described by Martin Remington's Pharmaceutical Sciences. These components contain an effective amount of the immunogen, preferably in pure form, in combination with an appropriate amount of carrier to provide the appropriate form of administration to the patient. The formulation will be configured according to the appropriate mode of administration, as is known in the art.

Therapeutic formulations of the product may be prepared by mixing the product of the desired purity with optional pharmaceutically acceptable carriers, diluents, excipients or stabilizers, for storage as lyophilized formulations and aqueous solutions. The process generally employs buffers, stabilizers, preservatives, isotonicity agents (isotonicifiers), nonionic detergents, antioxidants and various other additives, as described in Remington pharmaceutical sciences 16 edition (1980) by Osal. The additives are generally used in dosages and concentrations that are non-toxic to the recipient and, therefore, excipients, diluents, carriers, and the like, are pharmaceutically acceptable or generally recognized as safe.

The resulting or produced immuno-refocusing polypeptide (which includes an antigen, a portion of an antigen, an epitope, a determinant, etc., which can be produced in subunits, is substantially free of contaminating proteins (which includes other influenza proteins, in combination with other viral or non-viral polypeptides), as an IR polypeptide of interest, which can be expressed or produced in a recombinant virus, in a virus-like particle, or in combination with one or more proteins of viral or cellular origin, as an IR polypeptide, which can be expressed or produced as an isolated molecule and then in combination with one or more proteins of viral or cellular origin, etc., or antibodies thereto) is in a substantially purified form. An "isolated" or "purified" immunogen or vaccine is substantially free of contaminating proteins from the culture medium from which the immunogen or vaccine was obtained, or substantially free of chemical precursors and other chemicals in the culture medium, which comprise chemically synthesized components. The term "substantially free of subcellular material" includes preparations of cells in which the cells are destroyed to form a fraction which can be separated from the subcellular fraction of the cells, including dead cells and cell fractions such as cell membranes, debris cells, etc., within which the immunogen or vaccine can be isolated or recombinantly produced. Thus, an immunogen or vaccine that is substantially free of subcellular material includes an immunogen or vaccine that is less than 30%, 25%, 20%, 15%, 10%, 5%, 2.5% or 1% (dry weight) of subcellular contaminants or any other element(s) different from the product of interest.

The terms "stability" and "stable" as used herein in the context of a liquid formulation containing an immunogen or vaccine refer to the resistance of the immunogen or vaccine in the formulation to heat and chemical aggregation, degradation or fragmentation under given manufacturing, preparation, transport and storage conditions, such as 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or more. In this disclosure, a "stable" formulation maintains a biological activity equal to or greater than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% under given manufacturing, preparation, shipping and storage conditions. The stability of the immunogen or vaccine formulation can be assessed by methods known to those skilled in the art, including but not limited to physical observation against a reference, such as by microscopy, particle size and counting, and the like.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is co-administered. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical agent is administered intravenously, water is a suitable carrier. Saline solutions, dextrose solutions, and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, ethanol and the like. The ingredients may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. The carrier may include a salt and/or buffer.

Buffering agents help to maintain the pH in a range near physiological conditions. The optimal concentration range for buffering is about 2mM to 50 mM. Suitable buffering agents for use in the present disclosure include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Phosphate buffers, carbonate buffers, histidine buffers, trimethylamine salt buffers such as Tris, HEPES and other known buffers may also be used.

The addition of the preservative can delay the growth of microorganisms, and the addition amount of the preservative is in a range of 0.2-1% (w/v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, m-cresol, octadecyl dimethyl benzyl ammonium chloride, benzalkonium halides (e.g., chlorides, bromides, and iodides), hexabasic ammonium chloride, alkyl parabens, such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, and 3-pentanol.

Isotonic agents, which ensure the physiological isotonicity of the liquid components of the present disclosure, include polyhydric sugar alcohols, particularly ternary or higher sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. The polyol may be present in an amount of about 0.1% to 25% by weight, with relative amounts of about 1% to 5% other ingredients being considered preferred.

Stabilizers refer to a broad class of excipients that can range in function from fillers to dissolved therapeutic agents, or additives that help prevent denaturation or adherence to the container walls. Typical stabilizers may be polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol, and the like, including cyclitols such as inositol; polyethylene glycol; an amino acid polymer; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, alpha-thioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins, such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, saccharides, monosaccharides such as xylose, mannose, fructose or glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides, such as raffinose; polysaccharides such as dextran, etc. The stabilizer is present in the range of 0.1 to 10,000w/w per fraction of immunogen.

Other excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, or vitamin E), and solubilizing agents.

As used herein, the term "surfactant" refers to an organic substance having an amphiphilic structure, i.e. consisting of groups of opposite solubility tendencies (solubility into the polar groups), typically oil-soluble hydrocarbon chains and water-soluble ionic groups. Surfactants can be classified into anionic, cationic and nonionic surfactants, depending on the charge of the surface active group. Surfactants are commonly used as wetting, emulsifying, solubilizing and dispersing agents in the formulation of various pharmaceutical and biological materials.

The addition of nonionic surfactants or detergents (also known as "wetting agents") aids in the dissolution of the therapeutic drug and protects against agitation-induced aggregation of the therapeutic protein, and also allows the formulation to be exposed to shear face pressure without causing denaturation of the protein. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.),Polyhydric alcohol and polyoxyethylene sorbitan monoether ((C))Etc.). The nonionic surfactant is present in the range of about 0.05mg/ml to 1.0mg/ml, preferably 0.07mg/ml to 0.2 mg/ml.

As used herein, the term "inorganic salt" refers to any compound that does not contain carbon, resulting from substitution of part or all of the hydrogen atoms of an acid by a metal or metal-like acting group, commonly used as osmolyte regulating compounds in pharmaceutical compositions and biomaterial formulations. The most common inorganic salts are NaCl, KCl, NaH₂PO₄And the like.

The present disclosure provides liquid formulations of immunogens or vaccines that provide a pH in the range of about 5.0-7.0, 5.5-6.5, 5.8-6.2, 6.0-7.5, or 6.5-7.0.

Formulations contemplated by the present disclosure, such as liquid formulations, are stable at temperatures of-20 ℃ to 5 ℃ in commercial refrigerators and doctor's office or laboratory freezers. For storage purposes, the stability was assessed by microscopic analysis for 60 days, 120 days, 180 days, 1 year, 2 years or more. The liquid formulations of the present disclosure also exhibit stability by particle analysis, at room temperature, for at least several hours, such as 1 hour, 2 hours, or about 3 hours prior to use.

Examples of diluents include phosphate buffered saline, buffers that buffer stomach acid in the bladder (doubler), such as citrate buffer with sucrose (pH 7.4), bicarbonate buffer alone (pH 7.4), or bicarbonate buffer with ascorbic acid, lactose or saccharin (pH 7.4). Examples of carriers include proteins, such as those found in skim milk, sugars, such as sucrose, or polyvinylpyrrolidone. These carriers are used in concentrations of usually 0.1 to 90% (w/v), but preferably in the range of 1 to 10% (w/v).

Formulations for in vivo administration must be sterile. This can be achieved by filtration through sterile filtration membranes. For example, the subcellular preparations of the present disclosure may be sterilized by filtration.

In the case of oral preparations, the preparations of interest may include one or more flavorants, flavoring agents, scents, coloring agents, surfactants, binding agents, and the like, to provide a more palatable form for ingestion.

The formulation, dispensed dose and administration form of the immunogen or vaccine component should be consistent with good medical practice. Factors to be considered include the severity of the disease, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to physicians. The amount of a "therapeutically effective amount" of an immunogen or vaccine administered will be guided by such considerations and may be the minimum dose necessary to prevent, ameliorate or treat the target disease, condition or disorder.

The amount of antigen or vaccine is an amount sufficient to induce the desired humoral and/or cell-mediated immune response or to produce protection in the target host. The amount of immunogen or vaccine administered in the present disclosure will depend on the subject species, host physical characteristics such as age, weight, etc., preferred mode of delivery, etc. In general, the dosage employed is approximately 10-1500. mu.g per dose. In contrast, current subunit preparations contain elements from three virus subtypes. Their trivalent vaccines typically contain about 7-25 μ g of HA from each of the three constituent virus strains. This can serve as a starting point for titration of vaccine components of interest.

As used herein, the term "effective amount" refers to a therapeutic amount sufficient to reduce the severity and/or duration of a target disease, ameliorate one or more symptoms thereof, prevent progression of a target disease, or cause regression of a target disease, or to prevent progression, recurrence, onset, or progression of a target disease or one or more symptoms thereof. For example, based on baseline or normal levels, a treatment of interest can increase the viability of the host or reduce the severity of the disease by at least 5%, preferably by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In another embodiment, an effective amount of a therapeutic or prophylactic agent can reduce the symptoms of a target disease, such as reducing influenza symptoms or the duration of the disease by at least 5%, preferably by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. Its equivalent term "therapeutically effective amount" is also used herein.

Where necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine or other "caine" terminated anesthetic, to reduce pain at the injection site.

Typically, the components are provided separately or mixed in unit dosage form, such as a lyophilized powder or a water-free concentrate in a sealed container such as an ampoule or sachet, indicating the amount of active agent. In the case of administration by infusion, the ingredient may be dispersed by infusion bottle containing pharmaceutical grade sterile water or saline. In the case where the components are administered by injection, for example, an ampoule containing sterile water for injection or saline may be provided in the kit, and the components may be mixed before administration. Alternatively, the ampoule may contain a liquid containing the active agent of interest, e.g., as a concentrate for dilution prior to use, or in a form ready for administration. In another embodiment, the formulation of interest is provided in a suitable dosage and volume for ease of single use.

Provided are articles of manufacture containing materials useful in the treatment of the above-mentioned diseases. Articles of manufacture thereof may include containers and labels. Suitable containers include bottles, vials, syringes, and test tubes. The container may be made of a variety of materials, such as glass or plastic. The container may contain the compound of interest and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial containing a stopper that can be pierced by a hypodermic needle). The label on or associated with the container indicates that the composition is for use in treating influenza. The article of manufacture may further comprise a secondary container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and packaging for insertion of instructions for use.

The present disclosure also includes kits, e.g., containing immunogenic or vaccine components of interest and homologs, derivatives, etc., e.g., useful as active or passive vaccines, with instructions for use being equivalent. The instructions may include directions for compounding ingredients, derivatives, and the like. The composition may be in liquid form or in solid form, typically dried or lyophilized. The kit may contain suitable additional reagents, such as buffers, resuspension solution (resuspension solution) and other components necessary for the intended use. Predetermined amounts of the combination of agents may be considered for packaging, e.g., for treatment, along with instructions for use thereof. In addition, other additives, such as stabilizers, buffers, and the like, may also be included. The relative amounts of the various reagents may vary, providing a concentrate of the reagent solution, providing flexibility for the user, saving space and reagents, etc. The kit may comprise a delivery device, such as a needle-containing device, e.g. a syringe, optionally pre-filled with the component of interest for delivery when desired.

Citation of any reference discussed above shall not be construed as an admission that such reference is prior art to the presently disclosed invention. All references cited herein are incorporated by reference in their entirety.

The present disclosure will now be illustrated by the following non-limiting examples.

Example 1

8 of the immuno-attenuated and refocused hemagglutinin genes from the wyoming strain (H3N 2) were designed and processed as described above. For example, substitution of nucleotides by site-directed mutagenesis, introduction of N-linked sequences, resulting in complex glycosyl modifications, and/or deletion of amino acids and/or alteration of the charge of amino acids at the 5 major immunogenic and hypervariable sites comprising a strain-specific epitope.

With each change, the introduction of an N-linked sequence serves to maximize the scale of attenuation of immunity, particularly at larger antigenic sites, while minimizing any impact on the conformational complexity of the glycoprotein and receptor binding regions while reducing the number of wild-type amino acid changes required for attenuation. In some cases, only 3 amino acid changes are required. Antigenic site B (187-196) was directed against both B cell and CD4 helper T cell IDNPs.

To speed up the study, both DNA and protein subunit vaccines were designed. For DNA immunization, the full-length hemagglutinin gene was cloned into the pTriEx vector (Invitrogen) behind the Cytomegalovirus (CMV) promoter. Transient transfection of mammalian cells with pTriEx-HA constructs (constructs) showed full-length hemagglutinin gene expression as a trimer similar to the native viral proteins and soluble in plasma membrane extracts.

Groups 9 of outcrossed mice were immunized with DNA constructs containing 8 mutations and 1 unmodified full-length wild-type HA glycoprotein. Group 10 was immunized with empty pTriEx vector as a negative control.

In addition to DNA expression vectors, recombinant proteins should also be made for immunization. The extracellular domain of HA contains regions of trimeric glycoprotein peak assembly and regions that bind host cell receptors. In addition, removal of its transmembrane and cytoplasmic domains can cause the recombinant HA trimer to be released into the culture supernatant. Thus, each mutant HA gene was truncated at the end of the ectodomain and cloned into a vector containing the bacteriophage T7 promoter. The ectodomain vector is transfected into cells infected with a recombinant vaccinia virus that expresses bacteriophage T7RNA polymerase, resulting in the production of HA trimers that are secreted into the culture medium. Purifying the trimer of the ectodomain as a protein immunogen.

The mice were bled beforehand. One group of mice served as negative controls, the other group was immunized with unmodified (wild-type) antigen.

In another set of experiments, mice from the main group (primary groups) were immunized by injection of 10 micrograms of mutant HA glycoprotein DNA per quadriceps muscle (in 0.1mL of sterile water). After 5 weeks of rest, the mice were injected with a second DNA immunization. After a further 4-5 weeks, mice were again injected with purified ectodomain glycoprotein and immunized subcutaneously in two sessions, each at 10 μ g. The first protein immunization was formulated in Freund's complete adjuvant and the second in Freund's incomplete adjuvant. Two weeks after the final immunization, mice were euthanized and blood was collected to extract serum.

The following properties of the serum were tested: 1) their reactivity towards mutant and wild-type HA proteins was determined using Western blot and ELISA formats, 2) their recognition of linear epitopes was determined by peptide chain ELISA, 3) their protection against conformational epitope degradation was determined by proteases, and 4) functional assays were performed by hemagglutinin inhibition and virus neutralization of both homologous and heterologous influenza virus strains.

Sera from mice immunized with immune refocused HA subunit engineered antigens resulted in the production of high titers of anti-toxin sera as measured by HA-specific ELISA. Each group of mice showed titers for wild type HA in the range of 1:100-300,000. The various mutant HA glycoproteins were down-selected based on the ability of the anti-virus sera to display cross-subtype HI antibodies in a standard HI assay.

Mutants a2, B1, B2, B3, CE, CEB4, CEB5 and D1 of H3N2A/Wyoming/03/2003 were equal or higher in cross-subtype HI and/or virus neutralization titer for a set of heterologous virus subtypes used in the assay. Thus, as measured in vitro by standardized and accepted surrogate (surrogate) HI and virus neutralization tests, immune attenuation and refocusing led to the generation of HA glycoprotein subunit candidate vaccines, capable of inducing significantly enhanced cross-subtype antiviral protection.

Mutant A2 is a mutation in the A epitope of HA where KRRSNKS (SEQ ID NO: 1) is replaced by GG. B1 is a mutation in the B epitope of HA, with a single glycosylation site introduced at amino acid 197 (QIS replaced by NAS). B2 is a mutation in the B epitope of HA that introduces a glycosylation site at amino acid 189 (SDQ to NYT). B3 is a mutation in the B epitope of HA, introducing a glycosylation site at amino acid 193 (SLY to NIT). CE contains two mutations, one glycosylation site introduced into the C epitope at position 276 (KCN to NCT substitution), and one glycosylation site introduced into the E epitope at position 83 (NKK to NKT substitution). CEB4 is an additional mutation in the B epitope of CE, adding a glycosylation site at position 158 (KYK to NST). CEB5 is an additional mutation in the B epitope of CE, adding a glycosylation site at position 159. D1 is a mutation in the D epitope of HA to introduce a glycosylation site at amino acid 201 (replacement of RIT for NIT).

In another set of experiments, hemagglutinin inhibition titers and serum neutralization titers of refocusing polypeptide antigens were measured, as compared to a different strain of H3N2 virus. The mutant is derived from A/Wyoming/2003 virus strain. M3 has the B2 epitope; m5 has a CE epitope; m6 has the B4CE epitope, as described above. Mice were exposed to multiple muteins, a/Wyoming/2003 strain virus as a wild-type positive control, and vector alone as a negative control. The sera of the mice were then tested for their hemagglutinin inhibitory titers against the three viruses of the homologous Wyoming strain, Panama/1999 strain and Wellington/2004 strain. Other mouse sera were used to detect serum neutralization titers against homologous Wyoming strain, Korea/2003 strain, Korea/2002 strain, Fujian/2002 strain, Brisbane/09/2006 strain, and Brisbane/10/2007 strain. Control sera of mice exposed to vehicle alone did not produce specific hemagglutinin inhibitory antibodies reactive with Wyoming, Panama and Wellington strains (titer = 10).

Mice exposed to wild-type Wyoming virus produced anti-virus sera reactive with Wyoming and Wellington strains (titer = 1280), but slightly reactive with Panama strain (titer = 226). The M5 mutant produced an anti-virus serum that reacted 2-fold more strongly with the Wyoming and Wellington strains than the wild type (titer = 2560), but only slightly less strongly than the Panama strain (titer = 1920). The M6 mutant produced anti-virus sera that reacted equally well (2560 and 1280 titers, respectively) to the M5 mutant and the Panama and Wyoming strains. However, when the M6 mutant was exposed to Wellington, it produced inhibitory anti-virus serum titers that were higher than 4-fold higher than other titers (titer = 10240).

Thus, immunological refocusing resulted in an expanded (broadenered) response to two strains other than the homologous strain, and a very high response against the Wellington strain when triple modified muteins were used. In the neutralization study, mice exposed to the vehicle did not produce specific antibodies. Mice exposed to Wyoming produced antitoxic sera that reacted strongly with Wyoming (titer = 640); titers of Korea and Brisbane 2006 were one quarter of Wyoming (titer = 160); there was little reaction with Brisbane 2007 (titer = 20). The M3 mutant protein produced in mouse antisera was 4-fold more reactive than the wild type to Wyoming, Brisbane 2006 and Brisbane 2007 (titer = 2560). The anti-virus sera did not react with Korea (titer = 3). Mice exposed to M5 produced anti-virus sera that were 2-fold more reactive (titer = 80) than Brisbane 2006 (titer = 160) and 30-fold more reactive (titer = 2560) than Korea with Wyoming. The anti-virus serum was essentially unreactive with Brisbane 2007 (titer = 20). Thus, an amplified response to the other 3 strains was obtained from the immune refocusing antigen of interest.

Thus, the components of interest may stimulate protective immunity against viruses present in subsequently related parent strains, as well as protective immunity against more common viruses in the past. Thus, Brisbane/07 based immune refocusing H3N2 vaccine would stimulate a protective response against viruses that have not evolved yet, avoiding the necessity of reformulating in the next few years. Immune refocusing vaccines based on Wyoming or Brisbane can be developed for protection against all strains of H3N2 in the past and in the future.

Example 2

Based on a variety of considerations, including analysis of immunodominant epitopes, evolution, sequence diversity and structural data, an immunodocusing hemagglutinin antigen of the porcine-like strain HINLVA/California/04/2009 was designed. The following mutations were designed to refocus the immune response away from the highly variant immunodominant site and towards a wider range of protective epitopes.

As described herein, immune refocusing mutations can take a variety of forms, including deletion, addition, or reduction of glycosylation sites, as well as substitution of epitopes that affect charge, hydrophobicity, or some other chemical property. The addition of glycosylation has the advantage of shielding a relatively large fraction of epitopes, whereas charge changes can be concentrated at specific sites of antibody-antigen interaction. Due to the high similarity of the protein structures of various influenza strains with serotype HA, 3-D structural analysis of related A/PR8/34 can be used to determine putative loops and other active sites in the HA sequence. The HA of A/California/04/2009 was aligned with the HA of A/PR8/34, transferring the identified residues of PR8 to Cal/04/09.

Table 2 in figure 2 represents single point mutations aimed at refocusing the immunity to produce a broader protective response. The mutations originally proposed consisted largely of amino acid substitutions, which affected glycosylation patterns and/or local charge elements. The mutations are selected based on available sequence and structural data that are used to identify immunodominant epitopes that can facilitate a strain-specific response.

The sequences and mutations associated with the epitope sites along the HA glycoprotein that begin at the N-terminus are listed in Table 2. Residues in the C-terminal part (starting at position 214) and the fusion region (starting at position 314) containing the D site have been eliminated to save space in the table.

Mutations were designed as follows:

(i) the M1 and M3 mutations of epitope C and the M6 and M7 mutations of epitope E were selected as being involved in binding to the site of the monoclonal antibody or its immediate vicinity. Since it is located in highly flexible loops and regions of high sequence variability, we can predict that it contributes to virus strain-specific immunity;

(ii) mutations M1, M2, M5, M6, M8, M9, M10, M14, M16, M18, M20, M22, M23, M25 achieve the dual purpose when a virus strain-specific immunodominant site is introduced into a glycosylation site, while changing the charge of the relevant amino acids within the epitope;

(iii) mutations M3, M4, M7, M11, M12, M13, M15, M17, Ml9, M21, M24, and M26 replace charged residues with uncharged amino acid residues;

(iv) mutation M5 accomplishes the special purpose of adding glycosylation at the E site, to refocus the immunity towards conserved elements within the fusion region or HA-1HA-2 fusion site (e.g., C and E mutations); and

(v) the targeted positions of mutations M25 and M26 were immunodominant sites located near the HA1-HA2 cleavage site and fusion region (indicated as site "f" in Table 1), refocusing the immunity towards these highly conserved loci.

FIG. 3 identifies a subset of single point mutations overlapping the A/PR8/34HA structure. FIG. 1 shows that most mutations were inserted into highly flexible loop sites to mask epitopes without disrupting the native-like folding of the recombinant protein. Mutations are inserted into a diverse loop, ensuring that most conformational epitopes can be formed.

Example 3

HA mutations can be engineered using known methods, such as site-directed mutagenesis (quick-change) or gene synthesis. In general, mutations that are rapidly variable can be used to induce single or double mutations, while gene synthesis can be used for more complex combinations.

Single point mutations are most helpful in evaluating immune refocusing techniques directed to specific residues of each epitope. However, since pathogens utilize more than one immunodominant variable epitope (decoy epitope) to escape long-term immune pressure, improved immune refocusing antigens can incorporate mutations into multiple antigenic sites. Examples of immunorefocusing HA antigens comprising mutations in 2 or 3 epitopes are shown in figure 4, which is listed in table 3. Examples of immunorefocusing HA antigens containing mutations in all 5 epitopes are shown in figure 5, which is listed in table 4. In tables 3 and 4, the mutations consisted of combinations of single point mutations shown in fig. 2. Structural analysis as shown in FIG. 3 is very useful in the design of combinatorial mutations, allowing selection of mutations that reduce the likelihood of interference with each other in terms of charge, steric hindrance, or other adverse effects.

To evaluate candidate immune refocused HA, it is necessary to detect combinations of mutations, some of which consist of glycosylation insertions, while most others consist of substitutions that merely reduce the electrostatic charge in the epitope.

The list of mutations is exemplary of training requirements in the art, and once training is initiated, additional mutations can be designed at similar sites or to achieve similar goals according to the materials and methods provided herein.

The antigen based on the H1N 1HA sequence described in example 3 can stimulate broad immunity against all H1N1 variants as a vaccine. Additional mutations were made as described herein to allow the H1N 1-based antigen to stimulate protective immunity against other serotypes of influenza virus strains, e.g., H2, H5, etc.

The antigen design methods described herein can be used to make vaccines or antibodies based on other influenza serotypes, such as H2N2, H5N1, H7, H9, and the like.

These mutations are used to stimulate immunity to produce broadly reactive antibodies that can be used for therapeutic, diagnostic, or other purposes.

The use of these mutations in combination with adjuvants can lead to additional improvements in the breadth of cross-reactive immunity.

Example 4

The safety, toxicity and efficacy of recombinant immunogens were evaluated according to the guidelines of 21CFR 610, including: (i) general safety tests, and acute and chronic toxicity tests.

Immunogenicity data was derived from well-established animal models (e.g., guinea pigs, mice, ferrets or cotton rats) that respond well to human influenza vaccines. The investigation involved the assessment of the immune response using a vaccine containing the expected viral strain of the final product, based on dose and dosing interval. Immunogenicity studies in relevant animal models are used to document product consistency, particularly during the validation phase of the production process for novel influenza vaccines. Appropriate non-clinical endpoints selected for animal studies include death, weight loss, rate of viral excretion (excretion), clinical signs such as fever, ocular-nasal secretions, and the like.

Groups of ferrets or other suitable animals are inoculated intraperitoneally with 100 μ l of immunogen containing 300 μ g of the immunogen of interest. Appropriate negative and positive controls were used.

Animals were monitored for general health and body weight within 14 days after injection. Animals receiving both placebo and immunogen remained healthy with no weight loss and no obvious signs of disease during the observation period.

For a more stringent safety test, each group of animals was injected with 300. mu.g of immunogen.

The first day after inoculation, 3 animals in each group were euthanized and spleen, lung and liver homogenates were analyzed for immunogens. At weeks 4, 8, 12 and 16 post-injection, 3 animals in each group were individually euthanized and spleen, lung and liver homogenates were harvested for analysis to assess the presence of immunogen.

The immunogen is considered safe if no adverse health reactions are observed and the body weight increases at a normal rate compared to animals vaccinated with placebo as an internal control.

To evaluate the acute and chronic toxicity of the immunogen, groups of ferrets were inoculated intradermally with 300 μ g of immunogen at graded doses (graded spots) or in saline.

3 days after inoculation, 8 animals in each group were euthanized to obtain an acute effect of the immunogen on the animals. The remaining 8 animals in each group were euthanized 28 days after inoculation to assess any chronic effects on the animals. At both time points, the body weight of each animal was taken. In addition, the general pathology and appearance of the injection site should also be examined. At each time point, blood was drawn for blood chemistry examinations and histopathological examination of the internal organs and injection site was performed.

Other animals were given 3 doses of vaccine on days 0, 14 and 60, and their immune response to hemagglutinin was measured by ELISA using sera collected from the animals every 10 days. For example, the influenza virus neutralization rate in collected sera was measured 80 days after the first vaccination.

It should be understood that variations and modifications to the preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be included within the scope of the appended claims.

All references cited herein are incorporated by reference in their entirety.

Reference to the literature

Thomas Francis,Jr.in Proceedings of the American Philosophical Society，Vol.104,No.6（Dec.15,1960）,pp.572-578,according to The Swine Flu Episode and the Fog of Epidemics byRichard Krause in DCD's Emerging Infectious Diseases Journal,Vol.12,No.1,January 2006,published December 20,2005.

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Garrity，R.R.,G.Rimmelzwaan,A.Minassian,W.P.Tsai,G.Lin,J.J.de Jong,J.Goudsmit & P.L.Nara.1997.Refocusing neutralizing antibody response by targeted dampening of animmunodominant epitope.J.Immunol.159:279-89.

Kohler H,Goudsmit,J.&Nara P.Clonal dominance:cause for a limited and failing immuneresponse to HIV-1 infection and vaccination.J.Acquir.Immune Defic.Syndr.1992.5（11）:1158-68.

Andreansky，S.S.,John Stambas,Paul G.Thomas,Weidong Xie,Richard J.Webby，&Peter C.Doherty.Consequences of immunodominant epitope deletion for minor influenza virus-specificCD8T cell responses.J.Virol.2005Apr,79（7）:4329-39.

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Claims (11)

1. An isolated component comprising an influenza immunogenic epitope that is not immunodominant in a wild-type virus.

2. The composition of claim 1, comprising a hemagglutinin.

3. The composition of claim 1, wherein the composition comprises a neuraminidase.

4. The composition of claim 1, wherein the composition comprises influenza virus particles.

5. The composition of claim 4, wherein the particle is inactivated, attenuated, or non-infectious.

6. The moiety of claim 1, wherein said moiety comprises the addition or removal of glycosylation sites.

7. The composition of claim 1, wherein the composition comprises an addition, substitution, or deletion of an amino acid.

8. The composition of claim 1, comprising a virus-like particle.

9. The composition of claim 1, wherein the composition is expressed on the surface of an influenza virus.

10. The composition of claim 1, further comprising a pharmaceutically acceptable carrier, diluent or excipient.

11. The composition of claim 1, wherein the influenza is human influenza, avian influenza, swine influenza, canine influenza, or equine influenza.