Methods of Treating and Preventing Infections

Abstract

The invention relates to composite antigens comprising a peptide with contiguous amino acid sequence derived from a plurality of antigenic epitopes of one or more pathogens that induces an immune response in a mammal that is protective against infection by the one or more pathogens. In addition, the invention relates to vaccines comprising composite antigens and to method for treating and preventing an infection.

Claims

1. A method of treating or preventing an infection of an influenza virus comprising: providing an immunogenic composition that contains a peptide which comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1-116; and a T-cell stimulating epitope; and administering the immunogenic composition to a patient to generate an immunological response to the infection.

2. The method of claim 1, wherein the influenza virus comprises one or more of H1N1, H1N7, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7 or H9N2.

3. The method of claim 1, wherein the peptide comprises the sequence of one or more of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 47, SEQ ID NO 52, SEQ ID NO 53, and SEQ ID NO 54.

4. An immunogenic composition that contains a peptide which comprises an amino acid sequence that is at least 95% identical to any one or more of SEQ ID NOs: 1-116; and a T-cell stimulating epitope.

5. The composition of claim 4, which further contains an adjuvant.

6.-45. (canceled)

Description

DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 Summary of ELISA antisera titers of peptides (or Pep) 3, 6, 9, 10 and 11 of H3N2 Influenza virus (Wuhan).

[0037] FIG. 2 Antisera mean ODs of mice immunized with different peptides of H3N2 Influenza virus (Wuhan 1:40).

[0038] FIG. 3 Mean ODs of antisera (1:100) on virus and peptide following immunization with Pep 6

[0039] FIG. 4 Antisera titers for mice immunized with Pep 9 on H1N1 (Caledonia) virus at various dilutions.

[0040] FIG. 5A Assay of Pep 9 sera on fresh Wuhan coating.

[0041] FIG. 5B Assay of Pep 9 sera on fixed Wuhan.

[0042] FIG. 5C Assay of Pep 9 sera on fresh Caledonia coating.

[0043] FIG. 5D Assay of Pep 9 sera on fixed Caledonia coating.

[0044] FIG. 6 Antisera titers from mice immunized with Pep 9 captured on fixed vs. Fresh coatings of Wuhan and Caledonia.

[0045] FIG. 7 Antisera titers for mice immunized with Pep 11 on H5N1 at various dilutions.

[0046] FIG. 8-1 Additional sequences and related information.

[0047] FIG. 8-2 Additional sequences and related information.

[0048] FIG. 8-3 Additional sequences and related information.

[0049] FIG. 8-4 Additional sequences and related information.

[0050] FIG. 8-5 Additional sequences and related information.

[0051] FIG. 8-6 Additional sequences and related information.

DESCRIPTION OF THE INVENTION

[0052] Vaccinations and vaccines are often the best mechanism for avoiding an infection and preventing the spread of debilitating and dangerous pathogens. With respect to viral infections and many bacterial infections, vaccinations are often the only effective option as treatment options are few and those that are available provide only limited effectiveness. Conventional vaccinations require a priori understanding or general identification of the existing antigenic regions of the pathogen. The pathogen itself is propagated and a suitable vaccine developed from heat-killed or otherwise attenuated microorganisms. Alternatively, an antigen or collection of antigens is identified that will generate a protective immune response upon administration. The need for a vaccine is especially urgent with respect to preventing infection by certain bacteria and viruses. Many bacteria and especially certain viruses mutate constantly often rendering the vaccine developed to the prior or originating bacteria or virus useless against the new strains that emerge. As a consequence, vaccines against infections are reformulated yearly and often administered at fairly high doses. The manufacturing costs are high and administering vaccines against pose a great many complications and associated risks to patients.

[0053] It has been surprisingly discovered that an effective vaccine can be produced from a composite antigen of the invention. Composite antigens are antigens that contains or are derived from a plurality of antigenic regions of a pathogen. Composite antigens of the invention may contain one epitope that represents a combination of conserved regions of two or more epitopes (e.g., the composite epitope as outlined herein), or a plurality of immunologically responsive regions derived from one or multiple sources (e.g., virus particles, parasites, bacteria, fungi, cells). These immunological regions are amino acid sequences or epitopes that are representative of sequences found at those antigenic regions of a pathogen or other antigen associated with an infection or a disease or, importantly, associated with stimulation of the immune system to provide protection against the pathogen.

[0054] One embodiment of the invention is directed to composite antigens. Composite antigens of the invention contain non-naturally occurring amino acid sequences that do not exist in nature and are otherwise artificially constructed. Each sequence of a composite antigen contains a plurality of immunologically responsive regions or epitopes of a pathogen, which are artificially arranged, preferably along a single amino acid sequence. The plurality may contain multiples of the same epitope, although not in a naturally occurring order, or multiples of a variety of different epitopes from one or more pathogens. Epitopes may be identical to known immunological regions of a pathogen, or entirely new constructs that have not previously existed and therefore artificially constructed. Preferably, the composite antigen of the invention induces killer T-cell (Tc or CTL) responses, helper T-cell (Tx) responses, and specific antibody production in an inoculated mammal.

[0055] A composite antigen is an engineered, artificially created antigen made from two or more constituent epitopes, such that the resulting composite antigen has physical and/or chemical properties that differ from or are additive of the individual constituent epitopes. Preferable the composite antigen, when exposed to the immune system of a mammal, is capable of simultaneously generating an immunological response to each of the constituent epitope of the composite and preferably to a greater degree (e.g., as measurable from a cellular or humoral response to an identified pathogen) than the individual constituent epitopes. In addition, the composite antigen provides the added function of generating a protective immunological response in a patient when used as a vaccine and against each of the constituent epitopes. Preferably, the composite has the additional function of providing protection against not only the pathogens from which the constituents were derived, but related pathogens as well. These related pathogenic organisms may be strains or serotypes of the same species of organism, or different species of the same genus of organism, or different organisms entirely that are only related by a common epitope.

[0056] Another embodiment of the invention is directed to isolated epitopes. Isolated epitopes are regions obtained or derived from a protein or peptide of a pathogen that elicit a robust immunological response when administered to a mammal. Preferably, that robust response provides the mammal with an immunological protection against developing disease from exposure to the pathogen. A preferred example of an isolated epitope is a composite epitope, which is one artificially created from a combination of two or more highly conserved, although not identical, amino acid sequences of two or more different, but otherwise related pathogens. The pathogens may be of the same type, but of a different strain, serotype, or species or other relation. The composite epitope contains the conserved region that is in common between the related epitopes, and also contains the variable regions which differ. The sequences of a composite epitope that represents a combination of two conserved, but not identical sequences, may be illustrated as follows:

TABLE-US-00001 SequenceofEpitope1 ...AAAAABAAAAA... SequenceofEpitope2 ...AAAAACAAAAA... CompositeEpitope ...AAAAABCAAAAA...
wherein, A represents the amino acids in common between the two highly conserved epitopes, B and C represent the amino acids that differ, respectively, between two epitopes, each of A, B and C can be any amino acid and any number of amino acids. Preferably the conserved region contains about 20 or less amino acids on each side of the variable amino acids, preferably about 15 or less, preferably about 10 or less, preferably about 8 or less, preferably about 6 or less, and more preferably about 4 or less. Preferably the amino acids that vary between two similar but not identical conserved regions are 5 or less, preferably 4 or less, preferably 3 or less, preferably 2 or less, and more preferably only 1.

[0057] A composite epitope, similar to the composite antigen, is an engineered, artificially created single epitope made from two or more constituent epitopes, such that the resulting composite epitope has physical and/or chemical properties that differ from or are additive of the constituent epitopes. Preferable the composite epitope, when exposed to the immune system of a mammal, is capable of simultaneously generating an immunological response to each of the constituent epitopes of the composite and preferably to a greater degree than that achieved by either of the constituent epitopes individually. In addition, the composite epitope provides the added function of generating a protective immunological response in a patient when used as a vaccine and against each of the constituent epitopes. Preferably, the composite has the additional function of providing protection against not only the pathogens from which the constituents were derived, but related pathogens as well. These related pathogenic organisms may be strains or serotypes of the same species of organism, or different species of the same genus of organism, or different organisms entirely that are only related by a common epitope.

[0058] Composite epitopes of the invention are entirely artificial molecules that do not otherwise exist in nature and to which an immune system has not been otherwise exposed. Preferably, these conserved immunological regions that are combined as a composite epitope represent immunologically responsive regions of proteins and/or polypeptides that are highly conserved between related pathogens. Although a vaccine can be developed from a single composite epitope, in many instances the most effective vaccine may be developed from multiple, different composite epitopes.

[0059] Accordingly, composite antigens of the invention may contain one or more composite epitopes and/or one or more known epitopes to provide an effective vaccine. Although composite antigens may comprise a single composite epitope, a composite antigen would not comprise only a single known epitope. Preferably, the immunological response achieved from a vaccination with a composite antigen, or group of composite antigens, provides protection against infection caused by the original strains from which the sequence of the composite antigen was derived, and also provides immunological protection against other strains, serotypes and/or species that share one or more of the general conserved regions represented in the composite antigen. Thus, the resulting immune response achieved from a vaccination with a composite antigen is more broadly protective than can be achieved from a conventional single antigen vaccination against multiple strains, serotypes, and species or otherwise related pathogens regardless of antigenic drift that may take place in the evolution of the pathogen. Preferably, vaccines developed from composite antigens of the invention avoid any need for repeated or annual vaccinations, the associated complications and expenses of manufacture, and the elevated risks to the patient. These vaccines are useful to treat individuals and populations, thereby preventing infection, mortality and pandemics, and are useful to compliment conventional vaccines.

[0060] As discussed herein, the composite antigen preferably comprises a single chain of amino acids with a sequences derived from one or more composite eptiopes, one or more composite epitopes plus one or more known epitopes, or a plurality of known epitopes, that may be the same or different. Epitope sequences may be repeated consecutively and uninterrupted along a composite sequence or interspersed among other sequences that may be single or a few amino acids as spacers or sequences that encode peptides (collectively spacers), and may be nonimmunogenic or immunogenic and capable of inducing a cellular (T cell) or humoral (B cell) immune response in a mammal. Peptides sequence from unrelated microbes may be combined into a single composite antigen. For example, viral sequences of selected immunoresponsive peptides may be interspersed with conserved sequences or epitopes selected from other microbes, such as, for example, bacteria such as S. pneumococcus, P. auriginosa or S. aureus. Preferred viral proteins, from which preferred epitopes may be selected, include, but are not limited to the influenza virus proteins PspA, PspC, HA, NA, M2e, H. influenza protein D, and coagulase.

[0061] An epitope of the composite antigen may be of any sequence and size, but is preferable composed of natural amino acids and is more than 5 but less than 35 amino acids in length, preferably less than 30, preferably between 5 and 25 amino acids in length, preferably between 8 and 20 amino acids in length, and more preferably between 5 and 15 amino acids in length. Composite antigens preferably contain any number of composite and/or other epitopes. The most effective number of epitopes of a composite antigen against a particular pathogen, pathogen family, or group of pathogens may be determined by one skilled in the art from the disclosures of this application and using routine testing procedures. Composite antigens may be effective with one epitope, preferably with 2 or more, 3 or more 4 or more, 5 or more or greater. Optionally, composite antigens may include one or more spacers between epitopes which may be sequences of antigenic regions derived from the same or from one or more different pathogens, or sequences that serve as immunological primers or that otherwise provide a boost to the immune system. That boost may be generated from a sequence of amino acids that are known to stimulate the immune system, either directly or as an adjuvant. In one preferred embodiment, composite antigens useful to generate an immunological response against influenza virus comprise epitopes of HA and/or NA proteins, and/or new epitopes derived from similar conserved regions of different serotypes of influenza virus. Also preferred are composite antigens containing epitopes of proteins of Mycobacterium tuberculosis and Clostridium tetani, and/or new epitopes derived from similar conserved regions of different serotypes of these bacteria.

[0062] Another embodiment of the invention is directed to a contiguous sequence of one or more epitopes, which may comprise composite and/or known epitopes, from one or more pathogens in a sequence that does not exist naturally and must be artificially constructed. Preferably, a contiguous sequence of the invention contains one or more composite epitopes, which is a combination of the sequences of the conserved regions of epitopes that are common to multiple pathogens plus those amino acids that differ between the two conserved regions. For example, where two pathogens contain similar conserved regions that differ by only a single amino acid, the composite sequences would include the conserved region amino acids and each of the amino acids that differ between the two regions as discussed herein.

[0063] It is also preferable that a composite antigen of the invention contain a plurality of repeated epitopes and, optionally, epitopes conjugated with linker regions between or surrounding each epitope, and the plurality of epitopes be the same or different. Preferred linkers include amino acid sequences of antigenic regions of the same or of different pathogens, or amino acids sequences that aid in the generation of an immune response. Preferred examples include, but are not limited to any of the various antigenic regions of bacteria such as, but not limited to tuberculosis and virus such as, but not limited to influenza. It is also preferred that composite antigens contain epitopes that generate a T cell response, a B cell response, or both in conjunction with a specific response to the pathogen.

[0064] Another embodiment of the invention is directed to immunizing animals with the composite antigens of the invention. Antisera obtained from the immunized animals are reactive against the pathogens from which the composite antigen was derived. Another embodiment of the invention is therefore antisera obtained from the immunized animals, which may be further purified for testing or utilized therapeutically for administration to another mammal and thereby provides protection against infection from the one or more pathogens. It is also preferred that the antisera obtained provide a broad protection, not just against the pathogens from which the composite antigen was derived, but also from additional pathogens that may differ by strain, serotype, or even species.

[0065] Another embodiment of the invention is a vaccine composed of the composite antigen or antisera developed from the composite antigen of the invention. Preferably, the vaccines of the invention are less susceptible to variation of antigenicity due to antigenic shift of pathogens which reduces or eliminates the need for annual or repeated vaccination to maintain protection of patient populations against potential outbreaks of infection from new viral isolates. In addition, the vaccines of the invention generally and advantageously provide increased safety considerations, both in their manufacture and administration (due in part to a substantially decreased need for repeated administration), a relatively long shelf life in part due to minimized need to reformulate due to strain-specific shift and drift, an ability to target immune responses with high specificity for particular microbial epitopes, and an ability to prepare a single vaccine that is effective against multiple pathogens, each of which may be a different but know strain or species of the same pathogen. The invention encompasses antigenic and antibody compositions, methods of making such compositions, and methods for their use in the prevention, treatment, management, and/or prophylaxis of an infection. The compositions disclosed herein, as well as methods employing them, find particular use in the treatment or prevention of viral, bacterial, parasitic and/or fungal pathogenesis and infection using immunogenic compositions and methods superior to conventional treatments presently available in the art.

[0066] Another embodiment of the invention is directed to methods of preventing or controlling infection, such as, for example, an outbreak of viral, parasitic, fungal or bacterial infection, preferably but not limited to an influenza virus and/or a tuberculosis bacterial infection, in a selected mammalian population. The method includes at least the step of providing an immunologically effective amount of one or more of the disclosed immunogenic or vaccine compositions to a susceptible or an at-risk member of the population, for a time sufficient to prevent, reduce, lessen, alleviate, control, or delay the outbreak of such an infection in the general population.

[0067] Another embodiment of the invention is directed to methods for producing a protective immune response against infection, for example by influenza virus, in a mammal in need thereof. Such a method generally includes a step of providing to a mammal in need thereof, an immunologically-effective amount of one or more of the immunogenic compositions disclosed herein under conditions and for a time sufficient to produce such a protective immune response against one or more species, strains, or serotypes of an infectious organism. Additionally, the invention also provides a method for administering a prophylactic antiviral or antimicrobial composition to at least a first cell, tissue, organ, or organ system in a mammal that generally involves providing to such a mammal a prophylactically-effective amount of at least a first immunogenic composition as disclosed herein.

[0068] Another embodiment of the invention is directed to an immunogenic composition comprising the composite antigens of the invention having one or more repeated peptide sequences, or fragments, variants, or derivatives of such peptide sequences that are conserved across a plurality of proteins in the same or different pathogen. The conserved regions from which the composite sequence is derived may be conserved within subtypes of the same pathogen or different pathogens. Preferred pathogens include, but are not limited to bacteria, viruses, parasites, fungi and viruses.

[0069] Composite antigens of the invention may also be obtained or derived from the sequences of bacteria such as, for example, multiple or combined epitopes of the proteins and/or polypeptides of, for example, but not limited to Streptococcus, Pseudomonas, Mycobacterium such as M. tuberculosis, Shigella, Campylobacter, Salmonella, Haemophilus influenza, Chlamydophila pneumonia, Corynebacterium diphtheriae, Clostridium tetani, Mycoplasma pneumonia, Staphylococcus aureus, Moraxella catarrhalis, Legionella pneumophila, Bordetella pertussis, Escherichia coli, such as E. coli 0157, and multiple or combined epitomes of conserved regions of any of the foregoing. Exemplary parasites from which sequences may be obtained or derived include but are not limited to Plasmodium such as Plasmodium falciparum and Trypanosoma. Exemplary fungi include, but are not limited to Aspergillus fumigatus or Aspergillus flavus. Exemplary viruses include, but are not limited to arena viruses, bunyaviruses, coronaviruses, filoviruses, hepadna viruses, herpes viruses, orthomyxoviruses, parvoviruses, picornaviruses, papillomaviruses, reoviruses, retroviruses, rhabdoviruses, and togaviruses. Preferably, the virus epitopes are obtained or derived from sequences of Influenza viruses (e.g., the paramyxoviruses).

[0070] In another preferred embodiment, the composite antigens contain a conserved region derived from an influenza virus subtypes (e.g., influenza viruses with varying HA or NA compositions, such as H1N1, H5N1, H3N2, and H2N2). Epitopes of conserved regions on NA or HA may also confer cross-subtype immunity. As an example, conserved epitopes on NA(N1) may confer enhanced immunity to H5N1 and H1N1. With respect to similar or homologous chemical compounds among influenza A subtypes and/or strains within a subtype, preferably these are at least about 80 percent, more preferably at least about 90 percent, more preferably at least about 95 percent identical, more preferably at least about 96 percent identical, more preferably at least about 97 percent identical, more preferably at least about 98 percent identical, more preferably at least about 99 percent identical, and even more preferably 100 percent identical (invariant). Preferably, at least one peptide sequence within the composite antigen is also conserved on homologous proteins (e.g., protein subunits) of at least two viral particles, preferably influenza particles. Proteins of influenza virus include, for example, expressed proteins in the virus structure, such as HA, NA, protein polymerases (PB1, PB2, PA), matrix proteins (M1, M2), and nucleoprotein (NP). Preferably, the conserved peptide sequences are conserved on at least two or more of the M1, M2, HA, NA, or one or more polymerase proteins.

[0071] In a preferred example, a selected sequence in the M1 and M2 proteins of the H5N1 influenza virus corresponds to the M1 and M2 proteins found in other H5N1 particles, and to the same sequence in the M1 and M2 proteins of the H3N2 influenza virus. In addition, while HA and NA proteins have highly variable regions, conserved sequences from HA and NA are found across many influenza strains and many subtypes (e.g., HA and NA sequences are conserved across H5N1 and H1N1). In a preferred embodiment of the invention, the composite sequences is derived from a conserved sequence present within variants or strains (viral isolates expressing substantially the same HA and NA proteins, but wherein the HA and NA protein amino acid sequences show some minor drift), of a single influenzavirus subtype and more preferably across at least two influenzavirus subtypes, e.g., subtypes of influenza A virus.

[0072] In another embodiment, the invention provides a composite peptide or polypeptide that includes at least one epitopic antigen, which comprises one or more repeatedly occurring epitope sequences, each of which is conserved across a plurality of homologous proteins that is conserved in a population of influenzavirus strains or serotypes, and a pharmaceutically acceptable carrier. In exemplary composite antigens, at least one epitopic sequence is repeated at least once, preferably at least twice times, more preferably at least three times. In other embodiments, the at least one epitopic sequence is repeated four or more times. Preferably, the composite sequences are identical with the sequences in the homologous protein subunits of at least two circulating viral isolates. In each embodiment, the compositions may include a pharmaceutically acceptable carrier.

[0073] In additional preferred embodiments, the composite peptide sequences include sequences derived from genome (i.e., RNA) segment 7 of the influenza virus, while in a more preferred embodiment, the sequences include at least portions of the M1 and M2 proteins. In other preferred embodiments, the composite sequences include sequences expressed from genome segments encoding the HA or NA proteins. Such sequences are less affected by subtype drift and more broadly protective against infections.

[0074] In additional embodiments, the composite antigen includes one or more T-cell stimulating epitopes, such as diphtheria toxoid, tetanus toxoid, a polysaccharide, a lipoprotein, or a derivative or any combination thereof (including fragments or variants thereof). Typically, the at least one repeated sequence of the composite antigen is contained within the same molecule as the T-cell stimulating epitopes. In the case of protein-based T-cell stimulating epitopes, the at least one repeated sequence of the composite antigen may be contained within the same polypeptide as the T-cell stimulating epitopes, may be conjugated thereto, or may be associated in other ways. Preferably, at least one repeated sequence is incorporated within or alongside the one or more T-cell stimulating epitopes in a composite antigen of the invention.

[0075] In additional embodiments, the composite antigens, with or without associated T-cell stimulating epitopes may include one or more polysaccharides or portions thereof. In preferred embodiments, at least one sequence of a composite antigen is conjugated to one or more polysaccharides. In other embodiments, one or more polysaccharides are conjugated to other portions of the composite antigen. Certain embodiments of the present invention are selected from polysaccharide vaccines, protein-polysaccharide conjugate vaccines, or combinations thereof.

[0076] Composite antigens of the invention may be synthesizing by in vitro chemical synthesis, solid-phase protein synthesis, and in vitro (cell-free) protein translation, or recombinantly engineered and expressed in bacterial cells, fungi, insect cells, mammalian cells, virus particles, yeast, and the like.

[0077] A preferred composite antigen includes at least one of the following elements: at least one repeated composite sequence; at least one T-cell epitope; at least one polysaccharide; at least one polynucleotide; at least one structural component; or a combination thereof. The at least one structural component may include one or more of: at least one linker segment; at least one sugar-binding moiety; at least one nucleotide-binding moiety; at least one protein-binding moiety; at least one enzymatic moiety; or a combination thereof. The invention encompasses methods of preparing an immunogenic composition, preferably a pharmaceutical composition, more preferably a vaccine, wherein a target antigen of the present invention is associated with a pharmaceutically acceptable diluent, excipient, or carrier, and may be used with most any adjuvant.

[0078] Within the context of the present invention, that a relatively small number of conservative or neutral substitutions (e.g., 1 or 2) may be made within the sequence of the composite antigen or epitope sequences disclosed herein, without substantially altering the immunological response to the peptide. In some cases, the substitution of one or more amino acids in a particular peptide may in fact serve to enhance or otherwise improve the ability of the peptide to elicit an immune or T-cell response in an animal that has been provided with a composition that comprises the modified peptide, or a polynucleotide that encodes the peptide. Suitable substitutions may generally be identified using computer programs and the effect of such substitutions may be confirmed based on the reactivity of the modified peptide with antisera and/or T-cells. Accordingly, within certain preferred embodiments, a peptide for use in the disclosed diagnostic and therapeutic methods may comprise a primary amino acid sequence in which one or more amino acid residues are substituted by one or more replacement amino acids, such that the ability of the modified peptide to react with antigen-specific antisera and/or T-cell lines or clones is not significantly less than that for the unmodified peptide.

[0079] As described above, preferred peptide variants are those that contain one or more conservative substitutions. A conservative substitution is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the peptide to be substantially unchanged. Amino acid substitutions may generally be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Examples of amino acid substitutions that represent a conservative change include: (1) replacement of one or more Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, or Thr; residues with one or more residues from the same group; (2) replacement of one or more Cys, Ser, Tyr, or Thr residues with one or more residues from the same group; (3) replacement of one or more Val, Ile, Leu, Met, Ala, or Phe residues with one or more residues from the same group; (4) replacement of one or more Lys, Arg, or His residues with one or more residues from the same group; and (5) replacement of one or more Phe, Tyr, Trp, or His residues with one or more residues from the same group. A variant may also, or alternatively, contain non-conservative changes, for example, by substituting one of the amino acid residues from group (1) with an amino acid residue from group (2), group (3), group (4), or group (5). Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the peptide.

[0080] Epitopes may be arranged in any order relative to one another in the composite sequence. The number of spacer amino acids between two or more of the epitopic sequences can be of any practical range, including, for example, from 1 or 2 amino acids to 3, 4, 5, 6, 7, 8, 9, or even 10 or more amino acids between adjacent epitopes.

[0081] Another embodiment of the invention is directed to polynucleotides including DNA, RNA and PNA constructs that encode the composite sequences of the invention. These polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials. As is appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a given primary amino acid sequence. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Polynucleotides that encode an immunogenic peptide may generally be used for production of the peptide, in vitro or in vivo. Any polynucleotide may be further modified to increase stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 and/or 3-ends; the use of phosphorothioate or 2-o-methyl rather than phosphodiesterase linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queosine and wybutosine, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine.

[0082] Another embodiment of the invention encompasses methods of vaccinating a subject against Influenza that includes administering to a patient in need of influenza vaccination a therapeutically or prophylactically effective amount of an influenza vaccine, which influenza vaccine includes a composite antigen comprising one or more repeatedly occurring composite or other sequences, each of which is conserved across a plurality of homologous proteins in a plurality of influenza virus particles, and a pharmaceutically acceptable carrier, to provide a detectable immune response in the patient against influenza.

[0083] Another embodiment of the invention is directed to nucleotide or DNA vaccines encoding composite antigens of the invention. A DNA vaccine of the invention contains the genetic sequence of a composite antigen, plus other necessary sequences that provide for the expression of the composite antigen in cells. By injecting the mammal with the genetically engineered DNA, the composite antigen is produced in or preferably on cells, which the mammal's immune system recognizes and thereby generates a humoral or cellular response to the composite antigen, and therefore the pathogen. DNA vaccines have a number of advantages over conventional vaccines, including the ability to induce a more general and complete immune response in the mammal. Accordingly, DNA vaccines can be used to protect a mammal against disease caused from many different pathogenic organisms of viral, bacterial, and parasitic origin as well as certain tumors.

[0084] DNA vaccines typically comprise a bacterial DNA contained that encodes the composite antigen contained in vectors or plasmids that have been genetically modified to transcribe and translate the composite antigenic sequences into specific protein sequences derived from a pathogen. By way of example, the vaccine DNA is injected into the cells of the body, where the cellular machinery transcribed and translates the DNA into the composite antigen. Composite antigens, being non-natural and unrecognized by the mammalian immune system, are processed by cells and the processed proteins, preferably the epitopes, displayed on cell surfaces. Upon recognition of these composite antigens as foreign, the mammal's immune system generates an appropriate immune response that protects the mammal from infection. In addition, DNA vaccine of the invention are preferably codon optimized for expression in the mammalian cells of interest, such as but not limited to mouse or human cells. In a preferred embodiment, codon optimization involves selecting a desired codon usage bias (the frequency of occurrence of synonymous codons in coding DNA) for the particular cell type so that the desired peptide sequence is expressed.

[0085] Another embodiment of the invention is directed to therapeutic and prophylactic agents in a pharmaceutically acceptable composition for administration to a cell or an animal, either alone, or in combination with one or more other modalities of prophylaxis and/or therapy. Therapeutic and prophylactic agents of the invention include composite antigens, composite epitopes, compositions containing composite antigens and epitopes, composite sequences, DNA vaccines of the invention, antibodies of the invention, and/or T cells primed or exposed to composite antigens of the invention. The formulation of pharmaceutically-acceptable excipients and carrier solutions is well known to those of ordinary skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens.

[0086] The amount of immunogenic composition(s) and the time needed for the administration of such immunogenic composition(s) will be within the purview of the ordinary-skilled artisan having benefit of the present teachings. The administration of a therapeutically-effective, pharmaceutically-effective, and/or prophylactically-effective amount of the disclosed immunogenic compositions may be achieved by a single administration, such as for example, a single injection of a sufficient quantity of the delivered agent to provide the desired benefit to the patient undergoing such a procedure. Alternatively, in some circumstances, it may be desirable to provide multiple, or successive administrations of the immunogenic compositions, either over a relatively short, or even a relatively prolonged period of time, as may be determined by the medical practitioner overseeing the administration of such compositions to the selected individual.

[0087] The immunogenic compositions and vaccines of the present invention are preferably administered in a manner compatible with the dosage formulation, and in such an amount as will be prophylactically or therapeutically effective and preferably immunogenic. The quantity to be administered depends on the subject to be treated, including, e.g., the capacity of the patient's immune system to mount an immune response, and the degree of protection desired. Suitable dosage ranges may be on the order of several hundred micrograms (g) of active ingredient per vaccination with a preferred range from about 0.1 g to 2000 g (even though higher amounts, such as, e.g., in the range of about 1 to about 10 mg are also contemplated), such as in the range from about 0.5 g to 1000 g, preferably in the range from about 1 g to about 500 g and especially in the range from about 10 g to about 100 g. Suitable regimens for initial administration and booster shots are also variable but are typified by an initial administration followed by optional but preferred subsequent inoculations or other periodic administrations.

[0088] In certain embodiments, the dose would consist of the range of about 1 g to about 1 mg total protein or target antigen. In one exemplary embodiment, the vaccine dosage range is about 0.1 g to about 10 mg. However, one may prefer to adjust dosage based on the amount of peptide delivered. In either case, these ranges are merely guidelines from which one of ordinary skill in the art may deviate according to conventional dosing techniques. Precise dosages may be determined by assessing the immunogenicity of the conjugate produced in the appropriate host so that an immunologically effective dose is delivered. An immunologically effective dose is one that stimulates the immune system of the patient to establish an immune response to the immunogenic composition or vaccine. Preferably, a level of immunological memory sufficient to provide long-term protection against disease caused by microbial infection is obtained. The immunogenic compositions or vaccines of the invention may be preferably formulated with an adjuvant. By long-term it is preferably meant over a period of time of at least about 6 months, over at least about 1 year, over at least about 2 to 5 or even at least about 2 to about 10 years or longer.

[0089] Another embodiment of the invention is directed to antibodies that are specific for the composite antigens as described here and conservative variants thereof. Antibodies specific for these polypeptides are useful, e.g., in both diagnostic and therapeutic purposes, e.g., related to the activity, distribution, and expression of target polypeptides. Antibodies of the invention may be classes IgG, IgM, IgA, IgD or IgE and include, but are not limited to, polyclonal antibodies, monoclonal antibodies, multiple or single chain antibodies, including single chain Fv (sFv or scFv) antibodies in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide, and humanized or chimeric antibodies.

[0090] Antibodies specific for the composite peptides of the invention can be generated by methods well known in the art. Such antibodies can include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Numerous methods for producing polyclonal and monoclonal antibodies are known to those of skill in the art, and can be adapted to produce antibodies specific for the polypeptides of the invention, and/or encoded by the polynucleotide sequences of the invention (see, e.g., Coligan Current Protocols in Immunology Wiley/Greene, NY; Paul (ed.) (1991); (1998) Fundamental Immunology Fourth Edition, Lippincott-Raven, Lippincott Williams & Wilkins; Harlow and Lane (1989) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY, USA; Stites et al. (Eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif., USA and references cited therein; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York, N.Y., USA; 1986; and Kohler and Milstein (1975).

[0091] The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention.

EXAMPLES

Sequences

[0092] The following is a list of exemplary sequence. These sequences include composite sequences as well as sequences of interest that can be combined to form composite sequences of the invention:

TABLE-US-00002 SEQIDNO1 DWSGYSGSFVQHPELTGLD(N1sequence;H1N5 SEQIDNO2 ETPIRNE(N1epitope) SEQIDNO3 FVIREPFISCSHLEC(Pep5) SEQIDNO4 GNFIAP(HAepitope;Pep1) SEQIDNO5 GNLIAP(HAepitope;Pep2) SEQIDNO6 GNLFIAP(compositesequenceofSEQIDNOs4and5;Pep3) SEQIDNO7 GNLIFAP(compositesequenceofSEQIDNOs4and5) SEQIDNO8 HYEECSCY(NAepitope;Pep10) SEQIDNO9 LLTEVETPIR SEQIDNO10 LLTEVETPIRN SEQIDNO11 LLTEVETPIRNE SEQIDNO12 DWSGYSGSFVQHPELTGL(N1sequence;H1N5) SEQIDNO13 EVETPIRNE SEQIDNO14 FLLPEDETPIRNEWGLLTDDETPIRYIKANSKFIGITE SEQIDNO15 GNLFIAPGNLFIAPHYEECSCYHYEECSCYQYIKANSKFIGITEHYEECSCYTPIRNETPIRNE SEQIDNO16 GNLFIAPGNLFIAPQYIKANSKFIGITEGNLFIAP(compositeofSEQ IDNO6,SEQIDNO6,SEQIDNO60,andSEQIDNO6) SEQIDNO17 HYEECSCYDWSGYSGSFVQHPELTGLHYEECSCYQYIKANSKFIGITE SEQIDNO18 ITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDP SEQIDNO19 IWGIHHP(HAepitope) SEQIDNO20 IWGVHHP(HAepitope) SEQIDNO21 IWGVIHHP(compositeofSEQIDNOs.19and20) SEQIDNO22 IWGIVHHP(compositeofSEQIDNOs.19and20) SEQIDNO23 KSCINRCFYVELIRGR SEQIDNO24 LLTEVETPIRNESLLTEVETPIRNEWG(M2eepitope) SEQIDNO25 LLTEVETPIRNEW(M2eepitope) SEQIDNO26 LLTEVETPIRNEWG(M2eepitope) SEQIDNO27 LTEVETPIRNE(M2eepitope) SEQIDNO28 LTEVETPIRNEW(M2eepitope) SEQIDNO29 LTEVETPIRNEWG(M2eepitope) SEQIDNO30 MSLLTEVET(M2eepitope) SEQIDNO31 MSLLTEVETP(M2eepitope) SEQIDNO32 MSLLTEVETPI(M2eepitope) SEQIDNO33 MSLLTEVETPIR(M2eepitope) SEQIDNO34 MSLLTEVETPIRN(M2eepitope) SEQIDNO35 MSLLTEVETPIRNE(M2eepitopes) SEQIDNO36 MSLLTEVETPIRNETPIRNE(M2eepitope) SEQIDNO37 MSLLTEVETPIRNEW(M2eepitope) SEQIDNO38 MSLLTEVETPIRNEWG(M2eepitope) SEQIDNO39 MSLLTEVETPIRNEWGCRCNDSSD(M2eepitope) SEQIDNO40 SLLTEVET(M2eepitope) SEQIDNO41 SLLTEVETPIRNE(M2eepitope) SEQIDNO42 SLLTEVETPIRNEW(M2eepitope) SEQIDNO43 SLLTEVETPIRNEWG(M2eepitope) SEQIDNO44 SLLTEVETPIRNEWGTPIRNE(M2eepitope) SEQIDNO45 SLLTEVETPIRNEWGTPIRNETPIRNE(M2eepitope) SEQIDNO46 SLLTEVETPIRNEWGTPIRNETPIRNETPIRNE(M2eepitopes) SEQIDNO47 SLLTEVETPIRNEWGLLTEVETPIRQYIKANSKFIGITE(M2eepitope) SEQIDNO48 TEVETPIRNE(M2eepitope) SEQIDNO49 TPIRNE SEQIDNO50 VETPIRNE SEQIDNO51 VTREPYVSCDPKSCINRCFYVELIRGRVTREPYVSCDPWYIKANSKFIGITE SEQIDNO52 WGIHHP(HAconservedregion;Pep5) SEQIDNO53 WGVHHP(HAconservedregion;Pep4) SEQIDNO54 WGVIHHP(compositeofSEQIDNOs52and53;Pep6) SEQIDNO55 WGIVHHP(compositeofSEQIDNOs52and53;Pep7) SEQIDNO56 YIWGIHHP SEQIDNO57 YIWGVHHP SEQIDNO58 YIWGVIHHP(compositeofSEQIDNOs56and57) SEQIDNO59 YIWGIVHHP(compositeofSEQIDNOs56and57) SEQIDNO60 QYIKANSKFIGITE SEQIDNO61 PIRNEWGCRCNDSSD SEQIDNO65 SEYAYGSFVRTVSLPVGADEGNLFIAPWGVIHHPHYEECSCYGLPVEYLQVPSPSMGRDI KVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYS DWYQPACRKAGCQTYKWETFLTSELPGWLQANRHVQPTGSAVVGLSMAASSALTLAI YHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDP LLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGHNG VFDFPDSGTHSWEYWGAQLNAMKPDLQRHWVPRPTPGPPQGAFDFPDSGTHSWEYWG AQLNAMKPDLQRHWVPRPTPGPPQGA(SequenceforDNAvaccinedevelopment373 aminoacids;hasaTBconservedregionsoneachsideofPep11) SEQIDNO66 GNLFIAPWGVIHHPHYEECSCY(SEQIDNOs6,54,and8;Pep11) SEQIDNO67 WGVIHHPGNLFIAPHYEECSCY(SEQIDNOs54,6,and8) SEQIDNO68 SRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEW YDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKP TGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGY KASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLE GFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPN TGPAPQGA(TBcodingregionsequenceof85a) SEQIDNO69 TCCCGGCCGGGCTTGCCGGTGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGT GACATCAAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTC GACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACACCCCGGCGTT CGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGGCCAGTCAA GCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCCGGTTGCCAGACTTACA AGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGGGTGGCTGCAGGCCAACAGGCAC GTCAAGCCCACCGGAAGCGCCGTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTG ACGCTGGCGATCTATCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTG TTGGACCCCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCT GGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCG CAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCGCGTCTGGG TGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAACCTGCCGGCCAAG TTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTTCCAAGACGCCTACAACGCC GGTGGCGGCCACAACGGCGTGTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGA GTACTGGGGCGCGCAGCTCAACGCTATGAAGCCCGACCTGCAACGGGCACTGGGTG CCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCC(nucleotidesequencecorresponding toTBsequence85aorSEQIDNO64) SEQIDNO70 SEFAYGSFVRTVSLPVGADEGNLFIAPWGVIHHPHYEECSCYSRPGLPVEYLQVPSPSMG RDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSS FYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALT LAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQR NDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGG GHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA(336aminoacid sequencecomprisingHSPx,Pep11andTB85a) SEQIDNO71 TTTGGGCCCATTATGTCGGAATTCGCGTACGGTTCCTTCGTTCGCACGGTGTCGCTGC CGGTAGGTGCTGACGAGGGGAATCTAttcATTGCTCCTTGGGGGGTTattCA CCACCCGCATTATGAGGAATGTTCCTGTTACTCCCGGCCGGGCTTGCCGG TGGAGTACCTGCAGGTGCCGTCGCCGTCGATGGGCCGTGACATCAAGGTCCAATTCC AAAGTGGTGGTGCCAACTCGCCCGCCCTGTACCTGCTCGACGGCCTGCGCGCGCAG GACGACTTCAGCGGCTGGGACATCAACACCCCGGCGTTCGAGTGGTACGACCAGTC GGGCCTGTCGGTGGTCATGCCGGTGGGTGGCCAGTCAAGCTTCTACTCCGACTGGTA CCAGCCCGCCTGCGGCAAGGCCGGTTGCCAGACTTACAAGTGGGAGACCTTCCTGA CCAGCGAGCTGCCGGGGTGGCTGCAGGCCAACAGGCACGTCAAGCCCACCGGAAGC GCCGTCGTCGGTCTTTCGATGGCTGCTTCTTCGGCGCTGACGCTGGCGATCTATCACC CCCAGCAGTTCGTCTACGCGGGAGCGATGTCGGGCCTGTTGGACCCCTCCCAGGCGA TGGGTCCCACCCTGATCGGCCTGGCGATGGGTGACGCTGGCGGCTACAAGGCCTCC GACATGTGGGGCCCGAAGGAGGACCCGGCGTGGCAGCGCAACGACCCGCTGTTGAA CGTCGGGAAGCTGATCGCCAACAACACCCGCGTCTGGGTGTACTGCGGCAACGGCA AGCCGTCGGATCTGGGTGGCAACAACCTGCCGGCCAAGTTCCTCGAGGGCTTCGTGC GGACCAGCAACATCAAGTTCCAAGACGCCTACAACGCCGGTGGCGGCCACAACGGC GTGTTCGACTTCCCGGACAGCGGTACGCACAGCTGGGAGTACTGGGGCGCGCAGCT CAACGCTATGAAGCCCGACCTGCAACGGGCACTGGGTGCCACGCCCAACACCGGGC CCGCGCCCCAGGGCGCCTAGTTTCTTAAGTTT Underlinedsequences: Startandstopcodons AflIIRestrictionSite(NEBBuffer4) Note:multiTSpacerbetweenstopandAflIIRE ApaIRestrictionSite(NEBBuffer4) Note:SpacerbetweenstartandApaIREisKozakminimaltranslationinitiationsite Boldsequences: HA1sequenceboldedandunderlined(SEQIDNO72) HA2sequencebolded(SEQIDNO73 NA1sequenceboldedandunderlined(SEQIDNO74) SEQIDNO72 GGGAATCTAttcATTGCTCCT SEQIDNO73 TGGGGGGTTattCACCACCCG SEQIDNO74 CATTATGAGGAATGTTCCTGTTAC SEQIDNO75 SEFAYGSFVRTVSLPVGADE (HeatShockproteinsequenceofepitopeHSPxderivedfrom MycobacteriumtuberculosisH37Rv(NC_000962.2) SEQIDNO76 TCGGAATTCGCGTACGGTTCCTTCGTTCGCACGGTGTCGCTGC CGGTAGGTGCTGACGAG(nucleotidesequencecorrespondingto HSPx;SEQIDNO66) SEQIDNO77 GNLFIAPWGVIHHPHYEECSCY(underlinedsequencesareepitopes HA{composite}andNA,respectively,ofInfluenzaA;Pep11) SEQIDNO78 GGGAATCTAttcATTGCTCCTTGGGGGGTTattCACCACCCG CATTATGAGGAATGTTCCTGTTAC(Pep11-SEQIDNO68) SEQIDNO79 TCGGAATTCGCGTACGGTTCCTTCGTTCGCACGGTGTCGCTGCCGGTAGGTGCTGAC GAGGGGAATCTAttcATTGCTCCTTGGGGGGTTattCACCACCCGCATTATGAGGAATGT TCCTGTTACTCCCGGCCGGGCTTGCCGGTGGAGTACCTGCAGGTGCCGTCGCCGTCG ATGGGCCGTGACATCAAGGTCCAATTCCAAAGTGGTGGTGCCAACTCGCCCGCCCTG TACCTGCTCGACGGCCTGCGCGCGCAGGACGACTTCAGCGGCTGGGACATCAACAC CCCGGCGTTCGAGTGGTACGACCAGTCGGGCCTGTCGGTGGTCATGCCGGTGGGTGG CCAGTCAAGCTTCTACTCCGACTGGTACCAGCCCGCCTGCGGCAAGGCCGGTTGCCA GACTTACAAGTGGGAGACCTTCCTGACCAGCGAGCTGCCGGGGTGGCTGCAGGCCA ACAGGCACGTCAAGCCCACCGGAAGCGCCGTCGTCGGTCTTTCGATGGCTGCTTCTT CGGCGCTGACGCTGGCGATCTATCACCCCCAGCAGTTCGTCTACGCGGGAGCGATGT CGGGCCTGTTGGACCCCTCCCAGGCGATGGGTCCCACCCTGATCGGCCTGGCGATGG GTGACGCTGGCGGCTACAAGGCCTCCGACATGTGGGGCCCGAAGGAGGACCCGGCG TGGCAGCGCAACGACCCGCTGTTGAACGTCGGGAAGCTGATCGCCAACAACACCCG CGTCTGGGTGTACTGCGGCAACGGCAAGCCGTCGGATCTGGGTGGCAACAACCTGC CGGCCAAGTTCCTCGAGGGCTTCGTGCGGACCAGCAACATCAAGTTCCAAGACGCC TACAACGCCGGTGGCGGCCACAACGGCGTGTTCGACTTCCCGGACAGCGGTACGCA CAGCTGGGAGTACTGGGGCGCGCAGCTCAACGCTATGAAGCCCGACCTGCAACGGG CACTGGGTGCCACGCCCAACACCGGGCCCGCGCCCCAGGGCGCC(1008nucleotide DNAconstructofcompositepeptidecomprisingTBepitopesateachendwithaninfluenza sequenceinthemiddlewhichiscomposedofthreeepitopes-2HAcomposites{underlined} withanNAepitopebetweenthem). SEQIDNO80 CAGAGNFIAP SEQIDNO81 CAGAGNLIAP SEQIDNO82 CAGAGNLFIAP SEQIDNO83 CAGAWGVHHP SEQIDNO84 CAGAWGIHHP SEQIDNO85 CAGAWGVIHHP SEQIDNO86 CAGAWGIVHHP SEQIDNO87 GNLIAPWGVIHHP SEQIDNO88 CAGAGNLIAPWGVIHHP SEQIDNO89 GNLFIAPWGVIHHP SEQIDNO90 CAGAGNLFIAPWGVIHHP SEQIDNO91 HYEECSCY SEQIDNO92 CAGAHYEECSCY SEQIDNO93 GNLFIAPWGVIHHPHYEECSCY SEQIDNO94 CAGAGNLFIAPWGVIHHPHYEECSCY SEQIDNO95 GNLFIAPWGVIHHPGNLFIAPWGVIHHP SEQIDNO96 CAGAGNLFIAPWGVIHHPGNLFIAPWGVIHHP SEQIDNO97 HYEECSCYGNLFIAPWGVIHHP SEQIDNO98 GNLFIAPHYEECSCYWGVIHHP SEQIDNO99 SLLTEVETPIRNEWGLLTEVETPIRQYIKANSKFIGITE SEQIDNO100 GNLFIAPGNLFIAPQYIKANSKFIGITEGNLFIAP SEQIDNO101 HYEECSCYDWSGYSGSFVQHPELTGLHYEECSCYQYIKANSKFIGITE SEQIDNO102 VTREPYVSCDPKSCINRCFYVELIRGRVTREPYVSCDPQYIKANSKFIGITE SEQIDNO103 DWSGYSGSFVQHPELTGL SEQIDNO104 ITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDP SEQIDNO105 KSCINRCFYVELIRGR SEQIDNO106 GNLFIAPRYAFA SEQIDNO107 CAGAGNLFIAPRYAFA SEQIDNO108 GNLVVPRYAFA SEQIDNO109 CAGAGNLVVPRYAFA SEQIDNO110 GNLIAPRYAFA SEQIDNO111 CAGAGNLIAPRYAFA SEQIDNO112 GNLVVP SEQIDNO113 CAGAGNLVVP SEQIDNO114 FVIREPFISCSHLEC SEQIDNO115 CAGAFVIREPFISCSHLEC

[0093] FIG. 1 shows titers as determined by ELISA of mice vaccinated with Pep 6, Pep 9, Pep 10 or Pep 11. As can be seen, vaccinations with Pep 9, Pep 10 and Pep 11 provided a generally strong repose to the native antigen and the highest titers in mice.

[0094] FIG. 2 shows mean OD values of sera from mice immunized peptides derived from the Wuhan strain of Influenza (H3N2). Results indicate that Pep 10 and Pep 11 provide a significant immune response as compared to unvaccinated mice (the Balb/c pool) and mice vaccinated with Pep 3.

[0095] FIG. 3 shows mean OD values of antisera (titered at 1:100) following immunization with Pep 6. As can be seen, Pep 6 the antisera reacted strongly with viruses of both H1N1 and H3N2.

[0096] FIG. 4 shows antisera titers of mice immunized with Pep 9 as reacted with Influenza strain Caledonia virus (H1N1) at various dilutions. As seen, Pep 9 reacts strongly with whole virus.

[0097] FIG. 5 shows four graphs (A-D), each depicting the absorbance of Pep 9 sera to one of four substrates: (A) fresh Wuhan virus; (B) fixed Wuhan virus; (C) fresh Caledonia virus; and (D) fixed Caledonia virus. As shown, Pep 9 sera reacting strongly in with all substrates tested.

[0098] FIG. 6 shows the titers from mice immunized with Pep 9 as captured with substrates of fresh or fixed Wuhan or Caledonia virus substrates. Again, Pep 9 antisera were strongly reactive with each and the amount of binding observed for many of the antisera tested was similar between fresh and fixed.

[0099] FIG. 7 shows antisera titers of mice immunized with Pep 11 as captured with various dilutions of substrates of H5N1. As can be seen, binding was observed with both sera tested.

[0100] A composite antigen that contains previously determined conserved surface protein epitopes of hemagglutinin and neuraminidase from Influenza A viruses (several subtypes including human H1, H3 and high-path H5) was constructed. Also included were proteins segments from HspX and 85a from Mycobacterium tuberculosis. The expressed protein hybrid construct included the following: NH2+----HspX-HA1-HA2-NA1-85a-----COO wherein, HspX is 20 amino acids from Mycobacterium tuberculosis HspX protein; HA1 is a 7 amino acid highly conserved hybrid region influenza A hemagglutinin protein; HA2 is a 7 amino acid highly conserved hybrid region of influenza A hemagglutinin protein; NA1 is an 8 amino acid highly conserve hybrid region of influenza A neuraminidase protein; and 85a is 294 amino acids from the 85 kD 85a protein of Mycobacterium tuberculosis. The mature corresponding protein sequence is the 336 amino acid sequence of SEQ ID NO 70.

[0101] The composite antigen vaccine is constructed using the pVAX1 (Invitrogen Inc, Cat # V260-20) by recombinant methods. Briefly, a single stranded (ss) DNA polymer corresponding to SEQ ID 70 is synthesized. The ssDNA sequence also include: 1) a 5 ApaI restriction endonuclease recognition site and a 3 Afl II restriction site for directional insertion into pVAX1 cloning vector, 2) a ATT minimal Kozak translation initiation sequence at the 5 end, 3) a ATG start codon, and 4) a 3 TAG termination (stop) codon. The nucleotide sequence in its entirety will consist of 1038 nucleotides which is SEQ ID NO 71.

[0102] This single stranded nucleotide sequence is used as a template to generate double stranded amplicon by polymerase chain reaction. Following PCR amplification the amplicon is subjected to restriction endonuclease digestion, ligated into pVAX1 and transformed into E. coli bacteria using standard transformation and screening methods with Kanamycin selection. Plasmids containing recombinant insert are grown in overnight culture and purified using known plasmid extraction methods. Concentrations of recombinant pVAX1 plasmid are subjected to DNA sequencing and utilized in downstream transfection experiments in mice.

[0103] FIGS. 8-1 to 8-6 list a large number of sequences that include conserved or otherwise targeted regions of various genetic sequence of interest, and composite sequences of the invention.

[0104] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.