WO2008039267A2 - Inducing cellular immune responses to influenza virus using peptide and nucleic acid compositions - Google Patents

Abstract

This invention uses our knowledge of the mechanisms by which antigen is recognized by T cells to identify and prepare influenza virus epitopes, and to develop epitope-based vaccines directed towards influenza virus. More specifically, this application communicates our discovery of pharmaceutical compositions and methods of use in the prevention and treatment of influenza virus infection.

Description

INDUCING CELLULAR IMMUNE RESPONSES TO INFLUENZA VIRUS USING PEPTIDE AND NUCLEIC ACID COMPOSITIONS

BACKGROUND OF THE INVENTION

[0001] The present invention relates to influenza virus vaccine compositions and methods of treating or preventing influenza infection and disease in mammals. Influenza is caused by an RNA virus of the myxovirus group. Influenza viruses can be classified into three types (A, B and C), based on antigenic differences in the nucleoprotein and the matrix protein. Type A, which includes several subtypes, causes widespread epidemics and global pandemics. Type B causes regional epidemics. Influenza C is less severe and has been isolated from humans and pigs. Type C causes sporadic cases and minor, local outbreaks. Influenza A viruses can be further classified based on the viral surface proteins hemagglutinin (HA or H) and neuraminidase (NA or N). There are sixteen known H subtypes and nine known N subtypes of Type A viruses; while there is only one known H subtype and one N subtype of Type B viruses. Typical nomenclature identifies an influenza virus by both proteins, e.g., H3N2.

[0002] Type A and B influenza viruses each contain 8 RNA segments, while type C only has 7 RNA segments. Influenza A is most important and is very pathogenic for man, as well as for animals, for example pigs and horses. Type B influenza causes disease in humans. These virus types are distinguished in part on the basis of differences in two structural proteins, the nucleoprotein, found in the center of the virus, and the matrix protein, which forms the viral shell. The virus is transmitted through the air, mainly in droplets expelled during coughing and sneezing. The influenza viruses cause an infection of the respiratory tract, which is usually accompanied with coughing, high fever and myalgia.

[0003] Although an influenza infection does not often lead to the death of the infected individual, the morbidity can be severe. As a consequence thereof influenza epidemics may lead to substantial economic loss. Furthermore, influenza infection can be more dangerous for certain groups of individuals, such as those having suffered from a heart attack, CARA patients or the elderly. A vaccine against influenza is therefore highly desirable. Influenza Epidemiology and Virology

[0004] Pandemics of influenza A viruses continue to occur at sporadic intervals in human populations. Three have occurred in the twentieth century alone in 1918, 1957 and 1968^6*8. These worldwide pandemics are noted for their high mortality with rates approaching 30-50%⁹. For example, it is estimated that 20-40 million people died in the 1918 pandemic and at least 1.5 million people in the 1957 and 1968 outbreaks combined¹⁰. Whether a pandemic occurs from an act of nature or from the deliberate release of a novel influenza strain with pandemic potential, the extent of world travel will ensure the rapid global spread of the pandemic agent. Such an event could result in world-wide deaths totaling in the millions and severely impact health care systems such that economies and governments of smaller countries could collapse⁹'^{1 1}.

[0005] The capacity of the influenza virus to cause disease in a recurring manner is due to a complex set of factors that include: 1) the presence of an established reservoir of influenza A viruses of different subtypes in shorebirds and waterfowl; 2) the ability of avian influenza viruses to recombine with influenza viruses of other animals, most notably swine¹², a process termed 'antigenic shift'; 3) accumulation of mutations in viral gene products caused by a lack of proofreading activity of the viral RNA polymerase, a process termed 'antigenic drift'. These reassortment and mutation events combine to cause the well-characterized antigenic variability in the two surface glycoproteins of the virus, hemagglutinin (HA) and neuraminidase (NA)^13"15 which provides the virus a mechanism for escaping immune responses, particularly neutralizing antibodies, induced as the result of previous infections or vaccinations. Antigenic shift, which occurs only among influenza A viruses, results in major antigenic change introducing viruses with a new gene segment(s). Antigenic shift can occur when an animal influenza A virus is transmitted directly to humans, such as the transmission of the HlNl from swine-to- human¹⁶ or the transmission of the H5N1, H7N7 or H9N2 variants from avian to

17 18 human ' . Alternatively, a virus may acquire a new gene segment(s) as a result of genetic reassortment between animal and human influenza A viruses, the cause of the 1957 H2N2 and 1968 H3N2 pandemics¹⁹.

[0006] Since 1997, several novel avian subtypes have crossed the so-called species barrier from domestic poultry to humans and have caused a spectrum of mild to severe and even fatal human disease. In 1997, 18 cases of human infection with highly pathogenic avian H5N1 influenza viruses, including 6 deaths were documented in Hong Kong following outbreaks of disease in domestic poultry. Avian H5N1 viruses reemerged in Hong Kong and from December 30, 2003 to March 17, 2004, there were 12 human cases of confirmed H5N1 influenza in Thailand and 23 in Vietnam, including 23 deaths. As of May 2006, approximately 115 deaths have been attributed to H5N1 infection. The H5N1 strain does not jump easily from birds to humans or between humans. However since the human virus, H3N2, can coexist with avian influenza viruses and is widespread in pigs from southeast China, reassortment has the potential to occur with a highly pathogenic human-to-human transmissible H5N1 being the result. Although these wholly avian viruses were associated with only limited human-to-human transmission, their repeated emergence in humans highlights the potential for the generation of an avian-human reassortant virus with the potential for spread in the human population. Thus, the development of effective vaccines against these avian subtypes is of the highest public health priority.

[0007] Vaccine production must rely on surveillance programs to predict the influenza subtypes likely to have global impact on human health. The time required to produce subtype-matched vaccines, composed of inactivated or 'split' virions, typically requires a minimum of 6-8 months. In the face of a serious influenza virus pandemic caused by a viral subtype, this lag time could allow for national or international spread with excessive morbidity and mortality.

Virus Structures

[0008] An influenza virus is roughly spherical, but it can also be elongated or irregularly shaped. Inside the virus, eight segments of single-stranded RNA contain the genetic instructions for making the virus. The most striking feature of the virus is a layer of spikes projecting outward over its surface. There are two different types of spikes: one is composed of the molecule hemagglutinin (HA), the other of neuraminidase (NA). The HA molecule allows the virus to "stick" to a cell, initiating infection. The NA molecule allows newly formed viruses to exit their host cell without sticking to the cell surface or to each other. The viral capsid is comprised of viral ribonucleic acid and several so called "internal" proteins (polymerase proteins (PBl, PB2, and PA), matrix protein (Ml) and nucleoprotein (NP)). Because antibodies against HA and NA have traditionally proved the most effective in fighting infection, much research has focused on the structure, function, and genetic variation of those molecules. Researchers are also interested in two non-structural proteins M2 and NSl; both molecules play important roles in viral infection. [0009] Type A subtypes are described by a nomenclature system that includes the geographic site of discovery, a lab identification number, the year of discovery, and in parentheses the type of HA and NA it possesses, for example, A/Hong Kong/156/97 (H5N1). If the virus infects non-humans, the host species is included before the geographical site, as in A/Chicken/Hong Kong/G9/97 (H9N2).

[0010] Virions contain 7 segments (influenza C virus) to 8 segments (influenza A and B virus) of linear negative-sense single stranded RNA. Most of the segments of the virus genome code for a single protein. For many influenza viruses, the whole genome is now known. Genetic reassortment of the virus results from intermixing of the parental gene segments in the progeny of the viruses when a cell is co-infected by two different viruses of a given type. This phenomenon is facilitated by the segmental nature of the genome of influenza virus. Genetic reassortment is manifested as sudden changes in the viral surface antigens.

[0011] Antigenic changes in HA and NA allow the influenza virus to have tremendous variability. Antigenic drift is the term used to indicate minor antigenic variations in HA and NA of the influenza virus from the original parent virus, while major changes in HA and NA which make the new virions significantly different, are called antigenic shift. The difference between the two phenomena is a matter of degree.

[0012] Antigenic drift (minor changes) occurs due to accumulation of point mutations in the gene which results in changes in the amino acids in the proteins. Changes which are extreme, and drastic (too drastic to be explained by mutation alone) result in antigenic shift of the virus. The segmented genomes of the influenza viruses reassort readily in double infected cells. Genetic reassortment between human and non-human influenza virus has been suggested as a mechanism for antigenic shift. Influenza is a zoonotic disease, and an important pathogen in a number of animal species, including swine, horses, and birds, both wild and domestic. Influenza viruses are transferred to humans from other species.

[0013] Because of antigenic shift and antigenic drift, immunity to an influenza virus carrying a particular HA and/or NA protein does not necessarily confer protective immunity against influenza virus strains carrying variant, or different HA and/or NA proteins. Because antibodies against HA and NA have traditionally proved the most effective in fighting influenza virus infection, much research has focused on the structure, function and genetic variation of those molecules. Role of Cellular Immune Responses in Protection Against Influenza

[0014] Cellular immune responses are known to contribute to the control of viral replication in vivo and to mediate viral clearance. In murine models, influenza-specific CD8⁺ cytotoxic T-lymphocytes (CTL) limit virus replication and protect against lethal virus challenge^20"27. Recovery from infection correlated with virus-specific CD8⁺ CTL activity²² and lack of CD8⁺ CTL activity was associated with delayed viral clearance and increased mortality²⁸. Studies completed by Ulmer and Okuda using a DNA vaccine encoding the viral nucleoprotein and M gene proteins, respectively are particularly relevant. These vaccines induced influenza-specific CD8⁺ CTL that provided cross- strain protection²⁷'²⁹'³⁰. The contribution of CTL and Helper T-lymphocytes (HTL) was definitively demonstrated by adoptive transfer of CD8⁺ and CD4⁺ T-lymphocytes³¹. Similarly, Epstein and colleagues demonstrated that either CD8⁺ or CD4⁺ T-lymphocytes promoted survival in mice immunized with an experimental DNA vaccine encoding internal viral proteins³². Finally, virus-specific HTL augment the generation of CTL and size of the CTL memory pool, an effect known to be associated with long term protection³³. Cellular immune responses clearly contribute to the control and clearance of infection and reduce pathogenesis.

[0015] The exposure to an influenza virus of one subtype often induces immune responses that protect against infection or disease with another subtype, a phenomena referred to as Heterosubtypic Immunity (HSI)^34"37. The mechanisms of heterosubtypic immunity appears to involve functional activity of both CD8⁺ and CD4⁺ T- lymphocytes²³'²⁶'^38"41, although more recently antibody responses have also been implicated⁴². HSI is not only observed using the murine models; influenza virus-specific CTL appear to provide partial protection against multiple influenza A virus strains in humans. Early human studies demonstrated that cellular immune responses play a role in controlling influenza infection ' . McMichael and colleagues inoculated 63 volunteers intranasally with live unattenuated influenza A/Munich/ 1/79 virus and evaluated the protective effects of serum antibody and cytotoxic T-cell immunity against influenza.⁴³ It was found that all subjects with demonstrable T-cell responses cleared virus effectively. Sonoguchi and colleagues found that students previously infected with H3N2 virus were partially protected against subsequent infection with HlNl subtype virus suggesting cross-subtype protection in humans during sequential epidemics. Thus, the use of vaccines to induce cellular responses against pandemic influenza virus is logical and the development of suitable vaccine technologies is warranted. [0016] Immune system-mediated selection pressure on influenza virus can lead to CTL viral escape mutants^45"47. While this phenomena clearly documents the importance of virus-specific CTL it also reveals a potential limitation for vaccines designed to induce CTL responses. However, the use of carefully selected epitopes in the design of a vaccine provides a means to address this problem. Selection of epitopes that are highly conserved amongst multiple viral strains is the first step and the selection of those epitopes predicted to be capable of inducing CTL responses to the majority of related epitopes is the second step.

Role of Humoral Immune Responses in Protection Against Influenza

[0017] Influenza vaccines are formulated to include human influenza strains predicted to pose the greatest risk for infectious spread. This vaccine development process requires approximately 6-8 months using conventional strains. Neutralizing antibodies induced primarily to the surface hemagglutinin protein by the conventional vaccines are highly protective. However, due to antigenic drift of the virus, the vaccines must be reformulated on a yearly basis. The danger persists that a "new" strain will emerge by antigenic shift for which the human population has little or no pre-existing immunity. Also, since vaccine production relies on embryonated chicken eggs or potentially cells in tissue culture, there are no assurances that sufficient new virus can be produced even within the 6-8 month time frame especially if the new influenza strain is lethal to birds. Pandemic influenza vaccine development would benefit by inclusion of conserved B cell epitopes capable of inducing protective immune responses. To this end, it has been reported that the external domain of the transmembrane viral M2 protein is highly conserved and that antibodies directed to this epitope are protective in mice^48"54. The M2 protein is an integral membrane protein of influenza A virus that is expressed at the plasma membrane in virus-infected cells. Due to the low abundance of the protein in the virus, the mechanism of protection of the antibody response directed against this epitope is not mediated via viral neutralization but rather by antibody-dependent, cell-mediated cytotoxicity⁵¹.

[0018] Conserved CTL, HTL and B-cell epitopes can be used as the basis for a vaccine designed to augment and improve prototype pandemic vaccine candidates that may be poorly immunogenic or a sub-optimal match against a pandemic strain that emerges. The advantages to using defined epitopes in vaccines are many but one advantage is that many epitopes can be incorporated into a vaccine to induce a broadly specific immune response targeting numerous viral gene products. Data from natural infection studies wherein human memory CTL specific to influenza A virus were restricted by multiple HLA Class I alleles have shown that responses within a given individual were broadly directed to epitopes within the NP, NA, HA, Ml, NSl and M2 viral proteins.

Design and Testing of Vaccines to Induce Cellular and Humoral Immune Responses: [0019] The use of recombinant DNA technology to produce influenza vaccines offers several advantages: a recombinant DNA influenza vaccine can be produced under safer and more stringently controlled conditions; propagation with infectious influenza in eggs is not required; recombinant protein can be more highly purified, virtually eliminating side effects due to contaminating proteins; purification procedures for recombinant protein does not have to include virus inactivation or organic extraction of viral membrane components, therefore avoiding denaturation of antigens and additional safety concerns due to residual chemicals in the vaccine. Production of protein via recombinant DNA technology provides an opportunity to avoid the genetic heterogeneity which occurs during adaptation and passage through eggs, which should make it possible to better match vaccine strains with influenza epidemic strains, resulting in improved efficacy; and a recombinant approach may also allow for strain selection later in the year, thereby allowing time for selections based on more reliable epidemiological data. [0020] A major obstacle to the development of vaccines that induce immune responses is the selection of a suitable delivery format. DNA plasmid vaccines and viral vectors, used either alone or together, and recombinant protein or peptides are logical vaccine delivery formats; however, each format has advantages and disadvantages. For example, DNA vaccines are readily produced and safe to administer but potency has been lacking, especially in clinical trials, requiring the administration of large (milligram) doses^{5 " 5}. Studies completed in small animals have indicated increased vaccine potency^66"69. Polymer formulation technology based on polyvinylpyrrolidone (PVP) can also be utilized. PVP is a nontoxic formulation excipient used to enhance DNA plasmid uptake by muscle cells^70"73. Such vaccine design parameters can correct for at least some of the limitations of naked-DNA vaccine technology.

[0021] The use of viral vectors to deliver vaccines has raised concerns, usually related to safety and pre-existing immunity to the vector. However, AlphaVax replicons are reported to be safe, non-transmissible and there is a general lack of pre-existing immunity to the vector. Another delivery vehicle that is being evaluated is peptides in adjuvant. Generally, peptides in adjuvant have shown to be immunogenic and efficacious in humans. However, there are concerns regarding vaccine formulation wherein high numbers of peptides will need to be delivered.

[0022] Several adjuvants have been developed for the administration of influenza virus vaccines, including alum based compounds, emulsions (e.g. MF59), (lipophilic immune stimulating complexes ISCOMS) containing Quil A adjuvant) and liposomes. A development of the liposomal technique has been the use of immunopotentiating reconstituted influenza virosomes (IRIVs) as antigen delivery systems. See Mischler, R. and Metcalfe, LC, Vaccine 20: B17-B23 (2002). The IRTV vaccine delivery system is comprised of spherical unilamellar vesicles comprising naturally occurring phospholipids (PL) and phosphatidylcholine (PC) and envelope phospholipids originating from influenza virus used to provide influenza virus NA and HA glycoproteins. See id. The fusion mechanism of IRIVs enables stimulation of the MHC Class I or Class II pathway, depending upon how antigens are presented to the APCs. Virosomes are able to induce either a B- or T-cell response. See id.

[0023] The use of antigenic epitopes in vaccines has several advantages over current vaccines, particularly when compared to the use of whole antigens in vaccine compositions. There is evidence that the immune response to whole antigens is directed largely toward variable regions of the antigen, allowing for immune escape due to mutations. The epitopes for inclusion in an epitope-based vaccine may be selected from conserved regions of influenza antigens, which thereby reduces the likelihood of escape mutants. Furthermore, immunosuppressive epitopes that may be present in whole antigens can be avoided with the use of epitope-based vaccines.

An additional advantage of an epitope-based vaccine approach is the ability to combine selected epitopes {e.g., multiple CTL and/or HTL epitopes), and further, to modify the composition of the epitopes, achieving, for example, enhanced immunogenicity. Accordingly, the immune response can be modulated, as appropriate, for the target disease. Similar engineering of the response is not possible with traditional approaches.

[0024] Several groups have established the mouse model as a tool for evaluating the efficacy of influenza vaccines^26"31'⁷⁴'⁷⁵. The testing of vaccines comprised of epitopes restricted by HLA is a unique challenge, requiring the appropriate restriction elements. Specifically cell-surface expressed HLA Class I molecules for CTL epitopes and HLA Class II molecules for HTL epitopes on antigen presenting cells are required. With respect to CTL epitopes, HLA-A*0201, -A*1101 and -B*0702 transgenic mice have been developed as representative of the HLA-A2, -A3 and -B7 supertype families, respectively^76"78. The utility of these HLA transgenic mice for testing DNA, viral, protein and peptide vaccines have been validated ⁶⁸'⁷⁹. Three other transgenic lines (HLA-A*0101, HLA-A*2402 and HLA-B*4002, representing HLA-Al, -A24 and B44 supertype families, respectively) are being developed and can be utilized to evaluate the efficacy of vaccines using the established murine challenge models. With regard to evaluating Class II-restricted responses, HLA-DR4 mice are available commercially. Most HTL epitopes restricted to HLA Class II can bind murine H-2 IAb molecules and initiate a response .

[0025] Virus-specific, human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL) are known to play a major role in the prevention and clearance of virus infections in vivo (Oldstone, et al, Nature 321:239, 1989; Jamieson, et al, J. Virol. 61:3930, 1987; Yap, et al, Nature 275:238, 1978; Lukacher, et al, J. Exp. Med. 760:814, 1994; McMichael, et al, N Engl. J. Med. 309:13, 1983; Sethi, et al, J. Gen. Virol. 64:443, 1983; Watari, et al, J. Exp. Med. 165:459, 1987; Yasukawa, et al, J. Immunol. 143:2051, 1989; Tigges, et al, J. Virol. 66:1622, 1993; Reddenhase, et al, J. Virol 55:263, 1985; Quinnan, et al, N. Engl. J. Med. 307:6, 1982). HLA class I molecules are expressed on the surface of almost all nucleated cells. Following intracellular processing of antigens, epitopes from the antigens are presented as a complex with the HLA class I molecules on the surface of such cells. CTL recognize the peptide-HLA class I complex, which then results in the destruction of the cell bearing the HLA-peptide complex directly by the CTL and/or via the activation of non-destructive mechanisms e.g., the production of interferon, that inhibit viral replication.

[0026] Virus-specific T helper lymphocytes are also known to be critical for maintaining effective immunity in chronic viral infections. Historically, HTL responses were viewed as primarily supporting the expansion of specific CTL and B cell populations; however, more recent data indicate that HTL may directly contribute to the control of virus replication. For example, a decline in CD4+ T cells and a corresponding loss in HTL function characterize infection with HFV (Lane, et al, N. Engl. J. Med. 313:19, 1985). Furthermore, studies in HIV infected patients have also shown that there is an inverse relationship between virus-specific HTL responses and viral load, suggesting that HTL plays a role in controlling viremia {see, e.g., Rosenberg, et al, Science 278:1447, 1997).

[0027] The epitope approach, as we describe herein, allows the incorporation of various antibody, CTL and HTL epitopes, from various proteins, in a single vaccine composition. Such a composition may simultaneously target multiple dominant and subdominant epitopes and thereby be used to achieve effective immunization in a diverse population.

[0028] The technology relevant to multi-epitope ("minigene") vaccines is developing.

Several independent studies have established that induction of simultaneous immune responses against multiple epitopes can be achieved. For example, responses against a large number of T cell specificities can be induced and detected. In natural situations, Doolan, et al {Immunity, Vol. 7(1):97-112 (1997)) simultaneously detected recall T cell responses, against as many as 17 different P. falciparum epitopes using PBMC from a single donor. Similarly, Bertoni and colleagues (J. CHn. Invest, 700(3):503-13 (1997)) detected simultaneous CTL responses against 12 different HBV-derived epitopes in a single donor. In terms of immunization with multi-epitope nucleic acid vaccines, several examples have been reported where multiple T cell responses were induced. For example, minigene vaccines composed of approximately ten MHC Class I epitopes in which all epitopes were immunogenic and/or antigenic have been reported. Specifically, minigene vaccines composed of 9 EBV (Thomson, et al., Proc. Natl. Acad. ScL USA, P2(13):5845-49 (1995)), 7 HIV (Woodberry, et al., J. Virol, 73(7):5320-25 (1999)), 10 murine (Thomson, et al, J. Immunol, 160(4): 1717-23 (1998)) and 10 tumor-derived (Mateo, et al, J. Immunol, 7<53(7):4058-63 (1999)) epitopes have been shown to be active. It has also been shown that a multi-epitope DNA plasmid encoding nine different HLA-A2.1- and Al l -restricted epitopes derived from HBV and HIV induced CTL against all epitopes (Ishioka, et al, J. Immunol, 7<52(7):3915-25 (1999)).

[0029] Recently, several multi-epitope DNA plasmid vaccines specific for HIV have entered clinical trials (Nanke, et al, Nature Med., (5:951-55 (2000); Wilson, CC, et al, J. Immunol. 777(10):5611-23 (2003).

[0030] Thus, vaccines containing multiple MHC Class I (i.e., CTL) and Class II (i.e.,

HTL) epitopes can be designed, and presentation and recognition can be obtained for all epitopes. However, the immunogenicity of such multi-epitope constructs appears to be strongly influenced by a number of variables, a number of which have heretofore been unknown. For example, the immunogenicity (or antigenicity) of the same epitope expressed in the context of different vaccine constructs can vary over several orders of magnitude. Thus, there exists a need to identify strategies to optimize such multi-epitope containing vaccine constructs. Such optimization is important in terms of induction of potent immune responses and ultimately, for clinical efficacy. Accordingly, the present invention provides strategies to optimize antigenicity and immunogenicity of multi- epitope vaccines encompassing a certain number of epitopes. The present invention also provides optimized multi-epitope containing vaccines, particularly minigene vaccines, generated in accordance with these strategies.

[0031] The following paragraphs provide a brief review of some of the main variables potentially influencing minigene immunogenicity, epitope processing, and presentation on antigen presenting cells (APCs) in association with Class I and Class II MHC molecules.

Immunodominance

[0032] Of the many thousand possible peptides that are encoded by a complex foreign pathogen, only a small fraction ends up in a peptide form capable of binding to MHC Class I antigens and thus of being recognized by T cells. This phenomenon, of obvious potential impact on the development of a multi-epitope vaccine, is known as immunodominance (Yewdell et al., Annu Rev Immunol, 77:51-88 (1999)). Several major variables contribute to immunodominance. Herein, we describe variables affecting the generation of the appropriate peptides, both in qualitative and quantitative terms, as a result of intracellular processing.

Junctional Epitopes

[0033] A junctional epitope is defined as an epitope created due to the juxtaposition of two other epitopes. The new epitope is composed of a C-terminal section derived from a first epitope, and an N-terminal section derived from a second epitope. Creation of junctional epitopes is a potential problem in the design of multi-epitope minigene vaccines, for both Class I and Class II restricted epitopes for the following reasons. Firstly, when developing a minigene composed of, or containing, human epitopes, which are typically tested for immunogenicity in HLA transgenic laboratory animals, the creation of murine epitopes could create undesired immunodominance effects. Secondly, the creation of new, unintended epitopes for human HLA Class I or Class II molecules could elicit in vaccine recipients, new T cell specificities that are not expressed by infected cells or tumors that are the targets-induced T cell responses. These responses are by definition irrelevant and ineffective and could even be counterproductive, by creating undesired immunodominance effects. [0034] The existence of junctional epitopes has been documented in a variety of different experimental situations. Gefter and collaborators first demonstrated the effect in a system in which two different Class II restricted epitopes were juxtaposed and colinearly synthesized (Perkins et al, J Immunol, 146(7):2\37-44 (1991)). The effect was so marked that the immune system recognition of the epitopes could be completely "silenced" by these new junctional epitopes (Wang et al, Cell Immunol, 743(2):284-97 (1992)). Helper T cells directed against junctional epitopes were also observed in humans as a result of immunization with a synthetic lipopeptide, which was composed of an HLA-A2-restricted HBV-derived immunodominant CTL epitope, and a universal Tetanus Toxoid-derived HTL epitope (Livingston et al, J Immunol, 159(3): 1383-92 (1997)). Thus, the creation of junctional epitopes is a major consideration in the design of multi-epitope constructs.

[0035] The present invention provides methods of addressing this problem and avoiding or minimizing the occurrence of junctional epitopes.

Flanking Regions

[0036] Class I restricted epitopes are generated by a complex process (Yewdell et al,

Annu Rev Immunol, 77:51-88 (1999)). Limited proteolysis involving endoproteases and potential trimming by exoproteases is followed by translocation across the endoplasmic reticulum (ER) membrane by transporters associated with antigen processing (TAP) molecules. The major cytosolic protease complex involved in generation of antigenic peptides, and their precursors, is the proteosome (Niedermann et al, Immunity, 2(3):289- 99 (1995)), although ER trimming of CTL precursors has also been demonstrated (Paz et al, Immunity 77(2):241-51 (1999)). It has long been debated whether or not the residues immediately flanking the C and N terminus of the epitope, have an influence on the efficiency of epitope generation.

[0037] The yield and availability of processed epitope has been implicated as a major variable in determining imniunogenicity and could thus clearly have a major impact on overall minigene potency in that the magnitude of immune response can be directly proportional to the amount of epitope bound by MHC and displayed for T cell recognition. Several studies have provided evidence that this is indeed the case. For example, induction of virus-specific CTL that is essentially proportional to epitope density (Wherry et al, J Immunol, 163(1): 3735 -45 (1999)) has been observed. Further, recombinant minigenes, which encode a preprocessed optimal epitope, have been used to induce higher levels of epitope expression than naturally observed with full-length protein (Anton et al., J Immunol, 7J5(6):2535-42 (1997)). In general, minigene priming has been shown to be more effective than priming with the whole antigen (Restifo et al. , J Immunol, 754(9):4414-22 (1995); Ishioka et al, J Immunol, Vol. 162(7):39\5-25 (1999)), even though some exceptions have been noted (Iwasaki et al., Vaccine, 77(15- 16):2081-8 (1999)).

[0038] Early studies concluded that residues within the epitope (Hahn et al., J Exp Med,

176(5): 1335-41 (1992)) primarily regulate immunogenicity. Similar conclusions were reached by other studies, mostly based on grafting an epitope in an unrelated gene, or in the same gene, but in a different location (Chimini et al., J Exp Med, 769(7,1:297-302 (1989); Hahn et al, J Exp Med, 174{3):733-6 (1991)). Other experiments however (Del VaI et al, Cell, 66(6):U45-S3 (1991); Hahn et al, J. Exp Med, 77^:1335-41 (1992)), suggested that residues localized directly adjacent to the CTL epitope can directly influence recognition (Couillin et al, J Exp Med, 180(3): 1129-34 (1994); Bergmann et al, J Virol 68(8):530β-\Q (1994)). In the context of minigene vaccines, the controversy has been renewed. Shastri and coworkers (Shastri et al, J Immunol, 155(9):4339-46 (1995)) found that T cell responses were not significantly affected by varying the N- terminal flanking residue but were inhibited by the addition of a single C-terminal flanking residue. The most dramatic inhibition was observed with isoleucine, leucine, cysteine, and proline as the C-terminal flanking residues. In contrast, Gileadi (Gileadi et al, Eur J Immunol, 29(7):22\3-22 (1999)) reported profound effects as a function of the residues located at the N terminus of mouse influenza virus epitopes. Bergmann and coworkers found that aromatic, basic and alanine residues supported efficient epitope recognition, while G and P residues were strongly inhibitory (Bergmann et al, J Immunol, 157(8):3242-9 (1996)). In contrast, Lippolis (Lippolis et al., J Virol, Vol. 69(5):3\34-46 (1995)) concluded that substituting flanking residues did not affect recognition. However, only rather conservative substitutions which are unlikely to affect proteosome specificity, were tested.

[0039] It appears that the specificity of these effects, and in general of natural epitopes, roughly correlates with proteosome specificity. For example, proteosome specificity is partly trypsin-like (Niedermann et al, Immunity, 2(3):2S9-99 (1995)), with cleavage following basic amino acids. Nevertheless, efficient cleavage of the carboxyl side of hydrophobic and acidic residues is also possible. Consistent with these specificities are the studies of Sherman and collaborators, which found that an R to H mutation at the position following the C-terminus of a p53 epitope affects proteosome-mediated processing of the protein (Theobald et al, J Exp Med, 188(6):IO\7-2S (1998)). Several other studies (Hanke et al, J Gen Virol, 79 (Pt l):83-90 (1998); Thomson et al, Proc Natl Acad Sci USA, 92(13):5845-9 (1995)) indicated that minigenes can be constructed utilizing minimal epitopes, and that these flanking sequences appear not be required, although the potential for further optimization by the use of flanking regions was also acknowledged.

[0040] HLA Class II peptide complexes are also generated as a result of a complex series of events that is generally distinct from HLA Class I processing. The processing pathway involves association with Invariant chain (Ii), its transport to specialized compartments, the degradation of Ii to CLIP, and HLA-DM catalyzed removal of CLIP (see Blum et al, Crit Rev Immunol, 17(5-6):A\ \-1 (1997); Arndt et al, Immunol Res, 16(3):26\-72 (1997)) for review. Moreover, there is potentially crucial role of various cathepsins in general, and cathepsin S and L in particular, in Ii degradation (Nakagawa et al, Immunity, 10(2):2Q7-\7 (1999)). In terms of generation of functional epitopes however, the process appears to be somewhat less selective (Chapman H. A., Curr Opin Immunol, J0(l):93-\02 (1998)), and peptides of many sizes can bind to MHC Class I/II (Hunt et al, Science, 256(5065):l8\7-20 (1992)). Most or all of the possible peptides appear to be generated (Moudgil et al, J Immunol, 159(6): 2574-9 (1997); and Thomson et al, J Virol, 72(3):2246-52 (1998)). Thus, as compared to the issue of flanking regions, the creation of junctional epitopes can be a more serious concern in particular embodiments.

[0041] One of the most formidable obstacles to the development of broadly efficacious epitope-based immunotherapeutics, however, has been the extreme polymorphism of HLA molecules. To date, effective non-genetically biased coverage of a population has been a task of considerable complexity; such coverage has required that epitopes be used that are specific for HLA molecules corresponding to each individual HLA allele. Impractically large numbers of epitopes would therefore have to be used in order to cover ethnically diverse populations. Thus, there has existed a need for peptide epitopes that are bound by multiple HLA antigen molecules for use in epitope-based vaccines. The greater the number of HLA antigen molecules bound, the greater the breadth of population coverage by the vaccine.

[0042] Furthermore, as described herein in greater detail, a need has existed to modulate influenza virus peptide binding properties, e.g., so that influenza virus peptides that are able to bind to multiple HLA antigens do so with an affinity that will stimulate an immune response. Identification of influenza virus epitopes restricted by more than one HLA allele at an affinity that correlates with immunogenicity is important to provide thorough population coverage, and to allow the elicitation of responses of sufficient vigor to prevent or clear an infection in a diverse segment of the population. Such a response can also target a broad array of epitopes. In certain embodiments, the technology disclosed herein provides for such favored immune responses. Accordingly, the present invention provides multi-epitope vaccine constructs optimized for immunogenicity and antigenicity, and methods of designing such constructs.

[0043] The unpredictable emergence of novel subtypes of influenza virus exposes us to variants against which we possess little or no preexisting immunity. The resulting morbidity, mortality and economic strain on the world can be devastating. As a result of global surveillance, the human influenza strains that pose the greatest risk for infectious spread during yearly epidemics can be predicted and the annual influenza vaccines can be reformulated accordingly. The vaccine development process requires approximately 6-8 months and despite this effort, an average of 36,000 people still die annually from complications of influenza infection in the United States alone¹. In the event of a natural influenza pandemic, or an intentional introduction of a highly pathogenic subtype, it is likely that significantly higher numbers of deaths will occur. The Spanish influenza pandemic of 1918 killed 675,000 people in the USA and 20-40 million individuals worldwide². Epidemiological modeling suggests that the next pandemic will cause between 2-7.4 million deaths worldwide³. The increasing emergence of H5N1 viruses in the Far East and potential adaptation of these agents to human systems is just one example of potential influenza strains that could initiate the next pandemic. To address such a catastrophe a vaccine product capable of inducing significant levels of efficacy rapidly upon administration must be designed and manufactured prior to the beginning of a pandemic.

[0044] The present invention is thus directed to the design and production of vaccines that are capable of inducing immune responses specific for regions within viral gene products, or epitopes, that are conserved amongst the most divergent of influenza subtypes. Vaccines according to the invention are based on conserved cytotoxic T- lymphocyte (CTL), helper T-lymphocyte (HTL) and B-lymphocyte influenza-specific epitopes which can be designed and effective at rapidly inducing immune responses upon administration. While vaccine induction of cellular immunity alone will not provide optimal levels of protection, cellular immune responses may contribute to the initial control of viral replication and thus reduce disease progression in individuals and slow viral spread within a vaccinated population. A vaccine designed to induce conserved cellular and humoral responses is used to supplement conventional influenza vaccines which are designed to induce protective neutralizing antibodies. Several prototype candidate strains for conventional vaccines against a number of novel avian influenza subtypes are currently under preparation, but may be a suboptimal antigenic match when an actual pandemic strain emerges. Recent clinical trials have also demonstrated that such avian HA-based vaccines may be poorly immunogenic and additional strategies to optimize immunogenicity of these vaccines are needed ' . The induction of CTL and HTL responses using selected and highly immunogenic epitopes should augment the immunogenicity of such protein-based vaccines.

BRIEF SUMMARY OF THE INVENTION

[0045] The present invention is directed to the identification of CTL and HTL epitopes from viral gene sequences that are restricted by multiple HLA types with predictable levels of immunogenicity. The selection of epitopes restricted to multiple related HLA types, a phenomenon referred to as supertype restriction, provides a mechanism to achieve non-ethnically biased population coverage. The present invention is also directed to the development of a vaccine encompassing CTL, HTL and B-cell epitopes derived from influenza viral isolates from avian, porcine and human sources which are potential components of a pandemic influenza virus.

[0046] hi certain embodiments, the present invention is directed to the identification of conserved HLA Class I and II-restricted peptides derived from influenza subtypes that have the potential to initiate pandemic influenza disease using established motif search algorithms and HLA-peptide binding assays. In further embodiments, the invention relates to the identification of epitopes that are naturally processed and presented to the immune system using peptides identified as high affinity binders to HLA molecules and peripheral blood mononuclear cells (PBMC) from normal human donors and HLA transgenic mice. [0047] In other embodiments, the present invention is directed to the design and optimization of an influenza virus vaccine for immunogenicity using nucleic acids or peptides, including, e.g., DNA plasmids, AlphaVax replicons, liposomes, virosomes and peptide vaccines.

[0048] In further embodiments, the present invention is directed to evaluating the efficacy of the experimental vaccines alone and in combination with recombinant HA protein using HLA transgenic mice and infectious challenges and the identification of an effective rapid vaccination schedule.

[0049] The present invention is also directed to the development of a single epitope- based vaccine, delivered using a DNA plasmid, viral vector and/or peptides suitable for preclinical development, e.g., as liposomes, virosomes, or other pharmaceutically acceptable carriers. In certain embodiments, this vaccine product will not induce neutralizing antibody responses and may therefore be designed for use in combination with protein or inactivated viral vaccines. The unique advantage to this approach is that an epitope-based vaccine can be produced prospectively for administration to at-risk populations while the more conventional vaccines are being produced.

[0050] The present invention is further directed to enhancing the immune response of a vertebrate in need of protection against influenza virus infection by administering in vivo, into a tissue of the vertebrate, at least one multi-epitope construct, wherein the multi-epitope construct comprises an influenza virus CTL and/or HTL epitope, and wherein the multi-epitope construct is capable of eliciting an immune response.

[0051] In certain embodiments, the invention is directed to a polynucleotide selected from the group consisting of:

(a) a multi-epitope construct comprising between five and fifty nucleic acids, each encoding an influenza virus cytotoxic T lymphocyte (CTL) epitope, wherein the CTL epitope is any one of the epitopes listed in Tables 1-17, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(b) a multi-epitope construct comprising between five and fifty nucleic acids, each encoding an influenza virus cytotoxic T lymphocyte (CTL) epitope, wherein the CTL epitope is any one of the epitopes listed in Tables 3, 6, 8, 11, 14 and 17, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(c) a multi-epitope construct comprising between five and fifty nucleic acids, each encoding an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 18-49, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(d) a multi-epitope construct comprising between five and fifty nucleic acids, each encoding an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 20, 22, 24, 26, 28, 30, 32, 35, 37, 39, 41, 44 _; 46, and 49, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(e) a multi-epitope construct comprising between five and fifty nucleic acids, each encoding an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 18, 33, 42 and 47, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(f) the multi-epitope construct of (a) or (b), further comprising any of said nucleic acids of (c), (d), or (e), directly or indirectly joined in the same reading frame to said CTL epitope nucleic acids of (a) or (b);

(g) the multi-epitope construct of (c), (d), or (e), further comprising any of said nucleic acids of (a) or (b), directly or indirectly joined in the same reading frame to said HTL epitope nucleic acids of (c), (d) or (e);

(h) the multi-epitope construct of (a) or (b) or (c) or (d) or (e) or (f) or (g), further comprising one or more spacer nucleic acids, directly or indirectly joined in the same reading frame to said CTL and/or HTL epitope nucleic acids;

(i) the multi-epitope construct of (h), wherein said one or more spacer nucleic acids are positioned between the CTL epitope nucleic acids of (a) or (b), between the HTL epitope nucleic acids of (c) or (d) or (e), or between the CTL and/or HTL epitope nucleic acids of (f) or (g);

(j) the multi-epitope construct of (h) or (i), wherein said one or more spacer nucleic acids each encode 1 to 8 amino acids;

(k) the multi-epitope construct of any one of (h) to (j), wherein one or more of said spacer amino acid residues are selected from the group consisting of : K, R, N, Q, G, A,

S, C, and T at a C+l position of one of said CTL epitopes;

(1) the multi-epitope construct of any of (h) to (k), wherein two or more of said spacer nucleic acids encode different (i.e., non- identical) amino acid sequences;

(m) the multi-epitope construct of any of (h) to (1), wherein two or more of said spacer nucleic acids encode an amino acid sequence different from an amino acid sequence encoded by one or more other spacer nucleic acids; (n) the multi-epitope construct of any of (h) to (m), wherein two or more of the spacer nucleic acids encodes the identical amino acid sequence;

(o) the multi-epitope construct of any of (h) to (n), wherein one or more of said spacer nucleic acids encode an amino acid sequence comprising or consisting of three consecutive alanine (Ala) residues;

(p) the multi-epitope construct of (h) to (o), wherein one or more of said spacer nucleic acid encodes an amino acid sequence selected from the group consisting of: an amino acid sequence comprising or consisting of GPGPG (SEQ E) NO: ), an amino acid sequence comprising or consisting of PGPGP (SEQ BD NO: ), an amino acid sequence comprising or consisting of (GP)n, an amino acid sequence comprising or consisting of (PG)n, an amino acid sequence comprising or consisting of (GP)nG, and an amino acid sequence comprising or consisting of (PG)nP, where n is an integer between zero and eleven;

(q) the multi-epitope construct of any of (a) to (p), further comprising one or more nucleic acids encoding one or more HTL epitopes, directly or indirectly joined in the same reading frame to said CTL and/or HTL epitope nucleic acids and/or said spacer nucleic acids;

(r) the multi-epitope construct of (q), wherein said one or more HTL epitopes comprises a pan-DR binding epitope;

(s) the multi-epitope construct of any of (a) to (r), further comprising one or more

MHC Class I and/or MHC Class II targeting nucleic acids;

(t) the multi-epitope construct of (s), wherein said one or more targeting nucleic acids encode one or more targeting sequences selected from the group consisting of : an

Ig kappa signal sequence, a tissue plasminogen activator signal sequence, an insulin signal sequence, an endoplasmic reticulum signal sequence, a LAMP-I lysosomal targeting sequence, a LAMP-2 lysosomal targeting sequence, an HLA-DM lysosomal targeting sequence, an HLA-DM-association sequence of HLA-DO, an Ig-a cytoplasmic domain, Ig-ss cytoplasmic domain, a Ii protein, an influenza matrix protein, an HCV antigen, and a yeast Ty protein;

(u) the multi-epitope construct of any of (a) to (t), which is optimized for CTL and/or

HTL epitope processing;

(v) the multi-epitope construct of any of (a) to (u), wherein said CTL and/or HTL nucleic acids are sorted to minimize the number of CTL and/or HTL junctional epitopes encoded therein; (w) the multi-epitope construct of any of (a)-(v), wherein said influenza virus CTL and/or HTL epitopes are selected from the epitopes listed in Table 50; and (x) the multi-epitope construct of any of (c), (d) or (e)-(v) wherein said influenza virus CTL and/or HTL epitopes are directly or indirectly joined in the order shown in Figure 6.

[0052] In certain embodiments, the multi-epitope construct comprises between 10 and 50 nucleic acids, each encoding influenza virus CTL and/or HTL epitopes.

[0053] hi certain other embodiments, the polynucleotide or peptide of the present invention comprises a CTL epitope, where the CTL epitope is from about 8 to about 13 amino acids in length. In further embodiments, the CTL epitope is from about 8 to about 11 amino acids in length, about 9 to about 11 amino acids in length, or about 9 to about 10 amino acids in length.

[0054] In certain other embodiments, the polynucleotide or peptide of the present invention comprises an HTL epitope, where the HTL epitope is from about 6 to about 30 amino acids in length. In further embodiments, the HTL epitope is from about 8 to about 20 amino acids in length, or from about 10 to about 18 amino acids in length.

[0055] hi other embodiments, the influenza virus CTL and/or HTL epitope is from a polypeptide at least 90% identical to an influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), RNA polymerase subunit PA, RNA polymerase basic protein 1 (PBl), RNA polymerase basic protein 2 (PB2), nonstructural gene 1 (NSl), nonstructural gene 2 (NS2), matrix protein 1 (Ml) or matrix protein 2 (M2) polypeptide.

[0056] hi additional embodiments, the influenza virus CTL and/or HTL epitope is from an influenza strain selected from the group consisting of: Human A/Viet Nam/1203/2004 (H5N1), Human A/Hong Kong/156/97 (H5N1), Human A/Hong Kong/483/97 (H5N1), Human A/Hong Kong/1073/99 (H9N2), Avian A/Chicken/HK/G9/97 (H9N2), Swine A/Swine/Hong Kong/10/98 (H9N2), Avian A/FPV/Rostock/34 (H7N1), Avian A/Turkey/Italy/4620/99 (H7N1), Avian A/FPV/Weybridge/34 (H7N7 ), Human A/New Caledonia/20/99 (HlNl), Human A/Hong Kong/1/68 (H3N2), Human A/Shiga/25/97 (H3N2), Human A/Singapore/1/57 (H2N2), Human A/Leningrad/I 34/57 (H2N2), Human A/Ann Arbor/6/60 (H2N2), Human A/Brevig Mission/1/18 (HlNl), Swine A/Swine/Wisconsin/464/98 (HlNl), Human A/Netherlands/219/03 (H7N7).

[0057] In certain embodiments, the CTL epitope comprises a Class I HLA motif selected from the group consisting of HLA-Al, HLA- A2, HLA- A3, HLA- A24, HLA-B7 and HLA-B44. In further embodiments, the polynucleotide of the present invention comprises at least one HLA-Al epitope, at least one HLA-A2 epitope, at least one HLA- A3/A11 epitope, at least one HLA- A24 epitope, at least one HLA-B7 epitope, or at least one HLA-B44 epitope; or any combinations thereof.

[0058] In certain embodiments, the CTL epitope is any one of the HLA- A3 epitopes listed in Tables 1-3, any one of the HLA-A24 epitopes listed in Tables 4-6, any one of the HLA-B7 epitopes listed in Tables 7-8, any one of the HLA-B44 epitopes listed in Tables 9-11, any one of the HLA-Al epitopes listed in Tables 12-14, or any one of the HLA- A2 epitopes listed in Tables 15-17.

[0059] In other embodiments, the HTL epitope comprises a Class II HLA motif selected from the group consisting of HLA-DRl and HLA-DR3.

[0060] In certain embodiments, the HTL epitope is any of the DR epitopes listed in

Tables 48-49.

[0061] In other embodiments, the HTL epitope is any one of the DRl epitopes listed in

Tables 18-39. In further embodiments, the HTL DRl epitope is from an influenza virus protein NA, NP, NSl, NS2, PA, PBl, PB2, HA, Ml, or M2 sequence. For example, the HTL epitope is any one of the NA DRl epitopes listed in Table 18, any one of the NP DRl epitopes listed in Tables 19-20, any one of the NSl DRl epitopes listed in Tables 21-22, any one of the NS2 DRl epitopes listed in Tables 23-24, any one of the PA DRl epitopes listed in Tables 25-26, any one of the PBl DRl epitopes listed in Tables 27-28, any one of the PB2 DRl epitopes listed in Tables 29-30, any one of the HA DRl epitopes listed in Tables 31-33, any one of the Ml DRl epitopes listed in Tables 34-35, any one of the M2 DRl epitopes listed in Tables 36-37, or any one of the NA DRl epitopes listed in Tables 38-39.

[0062] In other embodiments, the HTL epitope is any one of the DR3 epitopes listed in

Tables 40-47. In further embodiments, the HTL DR3 epitope is from an influenza virus protein NA, NP, NSl, NS2, PA, PBl, PB2, HA, Ml or M2 sequence. For example, the HTL epitope any one of the NA DR3 epitopes listed in Tables 40-42, or any one of the HA DR3 epitopes listed in Tables 45-47.

[0063] In certain embodiments, the polynucleotide of the invention further comprises a polynucleotide encoding a polypeptide at least 90% identical to an influenza virus NA, NP, NSl, NS2, PA, PBl, PB2, Ml, M2 or HA sequence, or fragment, variant, or derivative thereof. The HA sequence can be a wild-type HA sequence from any of the influenza virus strains set forth above. The M2 sequence can be an M2e sequence, where the M2e sequence is selected from the sequences listed in Table 51.

[0064] hi certain embodiments, the polynucleotide of the present invention comprises a nucleic acid sequence encoding a pan-DR binding epitope, where the pan-DR binding epitope comprises the amino acid sequence AFKV AA WTLKAAA (SEQ ID NO: ).

[0065] In additional embodiments, the polynucleotide of the invention further comprises one or more regulatory sequences, where the one or more regulatory sequences comprises an IRES element or a promoter.

[0066] The present invention is also directed to a polypeptide encoded by a polynucleotide of the present invention, or a synthetic polypeptide. hi further embodiments, the polypeptide further comprises a pan-DR binding epitope, hi further embodiments, the pan-DR binding epitope comprises the amino acid sequence ajKXV AAWTLKAAa₂, where "X" is selected from the group consisting of cyclohexylalanine, phenylalanine, and tyrosine; and "ai" is either D-alanine or L-alanine; and "a₂" is either D-alanine or L-alanine; or if encoded by a nucleic acid the pan-DR binding epitope comprises the amino acid sequence AFKV AA WTLKAAA. hi further embodiments, the polypeptide of the present invention is from about 10 to about 2000 amino acids in length.

[0067] The present is also directed to a vector comprising the polynucleotide of the present invention, hi further embodiments, the vector is an expression vector.

[0068] The present invention is also directed to a composition comprising the polynucleotide of the present invention, a polypeptide of the present invention, or the vector of the present invention. In further embodiments, the composition of the present invention further comprises an influenza HA or NA polypeptide, wherein said HA polypeptide is encoded by a sequence 90% identical to a wild-type HA sequence from an influenza strain selected from the group consisting of: Human A/Viet Nam/1203/2004 (H5N1), Human A/Hong Kong/156/97 (H5N1), Human A/Hong Kong/483/97 (H5N1), Human A/Hong Kong/1073/99 (H9N2), Avian A/Chicken/HK/G9/97 (H9N2), Swine A/Swine/Hong Kong/10/98 (H9N2), Avian A/FPV/Rostock/34 (H7N1), Avian A/Turkey/Italy/4620/99 (H7N1), Avian A/FPV/Weybridge/34 (H7N7 ), Human A/New Caledonia/20/99 (HlNl), Human A/Hong Kong/1/68 (H3N2), Human A/Shiga/25/97 (H3N2), Human A/Singapore/ 1/57 (H2N2), Human A/Leningrad/ 134/57 (H2N2), Human A/Ann Arbor/6/60 (H2N2), Human A/Brevig Mission/1/18 (HlNl), Swine A/Swine/Wisconsin/464/98 (HlNl), Human A/Netherlands/219/03 (H7N7). [0069] In certain embodiments, the polynucleotide further comprises a nucleic acid encoding a targeting sequence located at the N-terminus of said construct. In further embodiments, the targeting sequence is selected from the group consisting of: an Ig kappa signal sequence, a tissue plasminogen activator signal sequence, an insulin signal sequence, an endoplasmic reticulum signal sequence, a LAMP-I lysosomal targeting sequence, a LAMP-2 lysosomal targeting sequence, an HLA-DM lysosomal targeting sequence, an HLA-DM-association sequence of HLA-DO, an Ig-a cytoplasmic domain, Ig-ss cytoplasmic domain, a Ii protein, an influenza matrix protein, an HCV antigen, and a yeast Ty protein, a baculovirus signal sequence, or a prokaryotic signal sequence.

[0070] Polypeptides of the present invention, for example an influenza virus HA or M2e polypeptide, can be altered from their native state in one or more of the following ways. An influenza virus polypeptide can be mutated so as to, for example, remove from the encoded polypeptide non-desired protein motifs present in the encoded polypeptide or virulence factors associated with the encoded polypeptide. For example, the polypeptide sequence could be mutated so as not to encode a membrane anchoring region that would prevent release of the polypeptide from the cell as with, e.g., M2e. Upon delivery, the polynucleotide of the invention is incorporated into the cells of the vertebrate in vivo, and a prophylactically or therapeutically effective amount of an immunologic epitope of an influenza virus is produced in vivo. Additionally, epitopes may be modified (to create analogs thereof) to increase their immunogenicity as compared to native epitopes.

[0071] The present invention further provides polypeptides encoded by the polynucleotides described above, a vector comprising the polynucleotides described above as well as immunogenic compositions comprising the polynucleotides and/or polypeptides described above. In certain other embodiments, the present invention is directed to a cell comprising polynucleotides, polypeptides, or immunogenic compositions as described above. In certain other embodiments, a composition comprises two or more polypeptides as described above, where the polypeptides are different from each other.

[0072] In certain embodiments, immunogenic compositions can further comprise, for example, carriers, excipients, transfection facilitating agents, lipids, liposomes and/or adjuvants as described herein. In certain other embodiments, immunogenic compositions can further comprise a virosome. In further embodiments, the virosome is an immunopotentiating reconstituted influenza virosome (IRIV). [0073] The compositions of the invention can be univalent, bivalent, trivalent or multivalent. A univalent composition will comprise only one polynucleotide of the present invention, or a polypeptide encoding the polynucleotide of the present invention, where the polynucleotide comprises between 10 and 100 nucleic acids encoding an influenza virus CTL and/or HTL multi-epitope and a second influenza virus polypeptide or a fragment, variant, or derivative thereof. A bivalent composition will comprise, either in polynucleotide or polypeptide form, two different influenza virus-polypeptides, each capable of eliciting an immune response. The polynucleotide(s) of the composition can encode two influenza virus polypeptides or alternatively, the polynucleotide can encode only one influenza virus polypeptide and the second influenza virus polypeptide would be provided by an isolated influenza virus polypeptide of the invention as in, for example, a single formulation heterologous prime-boost vaccine composition. In the case where both influenza virus polypeptides of a bivalent composition are delivered in polynucleotide form, the nucleic acid operably encoding those influenza virus multi- epitope constructs need not be on the same polynucleotide, but can be on two different polynucleotides. A trivalent or further multivalent composition will comprise three influenza virus polypeptides or fragments, variants or derivatives thereof, either in isolated form or encoded by one or more polynucleotides of the invention.

[0074] In one embodiment, a multivalent composition comprises a single multi-epitope polynucleotide construct, e.g., plasmid, comprising one or more CTL and/or HTL influenza virus epitopes. Reducing the number of polynucleotides, e.g., plasmids, in the compositions of the invention can have significant impacts on the manufacture and release of product, thereby reducing the costs associated with manufacturing the compositions. There are a number of approaches to include more than one expressed antigen coding sequence on a single plasmid. These include, for example, the use of Internal Ribosome Entry Site (IRES) sequences, dual promoters/expression cassettes, and fusion proteins.

[0075] The present invention is further directed to enhancing the immune response of a vertebrate in need of protection against influenza virus infection by administering, in vivo, into a tissue of the vertebrate, a polynucleotide, a polypeptide, or a composition as described above. The isolated polypeptide can be, for example, a purified subunit, a recombinant protein, a viral vector expressing an isolated influenza virus polypeptide, or can be an inactivated or attenuated influenza virus, such as those present in conventional influenza virus vaccines. According to either method, the polynucleotide is incorporated into the cells of the vertebrate in vivo, and an immunologically effective amount of an immunogenic influenza virus multi-epitope construct is produced in vivo. When utilized, an isolated influenza virus polypeptide or a fragment, variant, or derivative thereof is also administered in an immunologically effective amount.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA-D. Influenza peptide specific responses obtained from Human

Donors. Peripheral blood samples from 3 HLA A2.1 positive healthy volunteers were obtained by leukopheresis and PBMC isolated by Ficoll gradient separation. HLA typing was performed by Terasaki First HLA-ABC well Tray analysis. Cryopreserved PBMC were thawed, resuspended in 1 ml of 5% HS RPMI and plated at 4 x 10⁶ cells per well in 24-well plates. The PBMC were stimulated with a pool (9-10) peptides at a final concentration of 2 μg/ml of each peptide. Cell cultures were supplemented on days 1, 3 and 6 with a final concentration of 10 U/ml IL2. After 7 days in culture, CD8⁺ cells were purified using MACS Miltenyi Microbeads for use in IFN-γ ELISPOT assays. Membrane-based 96-well Millipore plates were coated overnight at 4⁰C with the Mabtech murine mAb specific for human IFN- γ. In triplicate wells, two concentrations of PBMC (25,000 and 5,000), A2.1 transfected .221 target cells (10,000) and peptide at a final concentration of 10 μg/ml were added. The assay plates were incubated at 37⁰C for 20 h, after which they were washed with PBS + 0.05% Tween 20. To each well, 100 μl of biotinylated mAb specific for human IFN-γ (Mabtech) at the concentration of 2 μg/ml was added and plates were incubated at 37°C for 2 h. The plates were again washed, avidin-peroxidase complex (Vectastain) was added to each well, and the plates were incubated at room temperature for 1 h. The plates were developed using 3-amino-9- ethyl-carbazole (Sigma), washed and dried. Spots were counted using an AID ELISPOT reader. Triplicate well experimental values are expressed as the mean net spots/10⁶ CD8+ lymphocytes ± SEM for each peptide. Responses were determined for positive (EBV bmlfl 259, Flu Ml 58, CMV pp65 495) and negative (HBVenv 183, HBVcore 18, Plasmodium falciparum (Pf) expl 83, Pf expl 2, Pf expl 91) control peptides. All three donors exhibited responses in the range of 200-10,000 SFC specific for the positive control peptides. Responses to the negative control peptides were generally < 10 SFC except for Donor 638 which responded to 4 of 7 peptides in the range of 40-2,000 SFC. [0077] Figure 2. Broadly specific CTL responses induced by vaccination with the multi-peptide epitope vaccine. CTL responses were measured against each vaccine epitope from weeks 9 and 18 peripheral blood samples from colon cancer patient #604. The two post-vaccination samples were collected after the patient had received 3 and all 6 treatments with vaccine, respectively. Post-vaccination samples were tested in separate experiments together with a pre-vaccination sample. CTL responses were measured using an IFN- γ ELISPOT assay following short-term in vitro expansion of PBMCs with each vaccine peptide. Effector cells (5 x 10⁴ and 1.25 x 10⁴ cells/well) and peptide- treated irradiated autologous PBMCs as APCs (1 x 10⁵/well) were plated into nitrocellulose wells pre-coated with an α-human IFN-γ mAb. An irrelevant HLA- A2- binding HBV core antigen peptide was used as a negative control. Eighteen hours later, assay wells were developed to detect spot-forming cells which were counted by a computer-assisted ELISPOT reader (Carl Zeiss Inc.). Data are expressed as net spot- forming cells (SFC) per 5 x 10⁴ effector cells induces by the vaccine peptide after subtracting background (SFC induced by the irrelevant HBV core antigen peptide). A positive vaccine response must meet 3 criteria: 1) The net spots of post-vaccination sample (after subtracting background spots induced by irrelevant peptide) must be >5; 2) The response must exceed the background and account for its variability such that net spots, post-vaccination> background spots + (2 x SD); 3) The response must exceed the pre-vaccination response and account for its variability such that net spots, specific peptide, post-vaccination > (2 x net spots, specific peptide, pre-vaccination) + (2 x SD).

[0078] Figures 3A-D. Broadly specific CTL responses induced by vaccination with a

DNA plasmid vaccine encoding SIV-derived CTL and HTL epitopes. Peripheral blood samples from 8 Mamu A*01 positive rhesus macaques were obtained by venipuncture and PBMC isolated by Ficoll gradient separation following an overnight shipment of blood. Results are depicted from PBMC obtained 2 weeks prior, 2 weeks post and 14 weeks post SFV infection. The 6 immunized macaques received 4 DNA immunizations (4 mg/animal formulated in polyvinylpyrollidone, PVP) on a monthly basis. Following a 5 month rest period, 3 animals (ID, 2D, 3D, depicted as grey bars) received 2 additional DNA immunizations on a monthly basis. The remaining 3 animals (4DP, 5DP, 6DP, depicted as black bars) received 2 polyepitope, 100 μg/animal formulated in Al (OH)3 (same order of epitopes and spacers as DNA vaccine) immunizations on a monthly basis. The polyepitope protein vaccine was obtained from a baculovirus system. Two animals (7N, 8N, depicted as white bars) were naive or non- immunized animals. The PBMCs were depleted of CD4⁺ cells by Dynal Microbeads for use in IFN-γ ELISPOT assays. Membrane-based 96-well Millipore plates were coated overnight at 4°C with the Mabtech murine mAb specific for monkey IFN-γ. In triplicate wells, CD4⁺ depleted PBMC (200,000/well), and peptide at a final concentration of 10 μg/ml were added. The assay plates were incubated at 37°C for 20 h, after which they were washed with PBS + 0.05% Tween 20. To each well, 100 μl of biotinylated mAb specific for monkey EFN-γ (Mabtech) at the concentration of 2 μg/ml was added and plates were incubated at room temperature for 2 h. The plates were again washed, avidin-peroxidase complex (Vectastain) was added to each well, and the plates were incubated at room temperature for 1 h. The plates were developed using 3-amino-9- ethyl-carbazole (Sigma), washed and dried. Spots were counted using an AID ELISPOT reader. Triplicate well experimental values are expressed as the mean net spots/10 CD8+ lymphocytes ± SEM for each peptide. To determine the level of significance, a Student's t test was performed in which p < 0.05 using the mean of triplicate values of immunized animals (peptide response-no peptide response) versus non-immunized animals (peptide response-no peptide response). Only responses with values of p < 0.05 are shown. Plasma viral loads were determined following SIV infection. Macaques were challenged intravenously 8 months following the last immunization with SrVmac239. Quantitation of virion-associated RNA in plasma was performed by real time PCR. Figures 4A-D. Induction of CTL epitope-specific responses following virus infection was similar in humans and mice. Groups of 10 HLA-A2 transgenic mice were infected by intranasal route using 600 and 1,200 HA units of virus. After 1 week of infection, the group receiving the higher dose exhibited signs of illness (weight loss, immobilization) and were sacrificed to obtain splenocytes. The lower dose group showed no signs of illness and the mice were sacrificed at 2 weeks following infection to obtain splenocytes. The CD8⁺ cells were purified using MACS Miltenyi Microbeads for use in IFN- γ ELISPOT assays. Membrane-based 96-well Millipore plates were coated overnight at 4°C with the Mabtech mAb specific for mouse IFN-γ. In triplicate wells, CD8+ cells (200,000/well), A2.1/K^b transfected Jurkat target cells (100,000/well) and peptide at a final concentration of 10 μg/ml were added. The assay plates were incubated at 37°C for 20 h, after which they were washed with PBS + 0.05% Tween 20. To each well, 100 μl of biotinylated mAb specific for mouse IFN-γ (Mabtech) at the concentration of 2 μg/ml was added and plates were incubated at 37°C for 4 h. The plates were again washed, avidin-peroxidase complex (Vectastain) was added to each well, and the plates were incubated at room temperature for 1 h. The plates were developed using 3-amino-9-ethyl-carbazole (Sigma), washed and dried. Spots were counted using an AID ELISPOT reader. Triplicate well experimental values are expressed as the mean net spots/10⁶ CD8+ lymphocytes ± SEM for each peptide. To determine the level of significance, a Student's t test was performed in which p < 0.05 using the mean of triplicate values of immunized mice (relevant peptide response- irrelevant peptide response) versus non-immunized mice (relevant peptide response- irrelevant peptide response). Only responses with values of p < 0.05 are shown.

[0080] Figures 5A-B. HTL Human Recall Responses in Donor X753. Immune responses in Donor X753 using a panel of negative control HTL epitope-containing peptides and a panel of HTL epitope-containing peptides derived from internal flu proteins, NSl, NS2, PBl, PB2, PA, NP, Ml and M2. The sequences of the HTL epitopes used in the experiment correspond to the nomenclature of the influenza HTL candidates in Tables 48 and 49.

[0081] Figure 6. Influenza virus multi-epitope construct. An influenza virus multi- epitope construct is illustrated showing influenza virus CTL and HTL epitopes linked or joined by spacer sequences. The sequences of the CTL and HTL epitopes shown correspond to the nomenclature of the influenza candidate CTL and HTL epitopes shown in Table 50. Table 50 also provides binding data for each of the epitopes within the multi-epitope construct.

[0082] Figures 7A-D. HLA-Al Influenza-Specific Recall Responses for Humans.

Immune responses in Donors X6018, X1257, X757 and X716 using a panel of influenza- derived HLA-Al peptides and a panel of HLA-Al and -A24 negative control peptides. The sequences of the influenza derived HLA-Al peptides used in the experiment correspond to the nomenclature of the influenza peptides in Tables 12 and 13.

[0083] Figures 8A-D. HLA-A2 Influenza-Specific Recall Responses for Humans.

Immune responses in Donors X1211, X716, AC08 and AC04 using a panel of influenza- derived HLA-A2 peptides and a panel of HLA-A2 negative control peptides. The sequences of the influenza derived HLA- A2 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 16.

[0084] Figure 9. HLA-A2 Influenza-Specific Recall Responses for Mice. Immune responses in HLA-A2 transgenic mice using a panel of influenza-derived HLA-A2 peptides. The sequences of the influenza derived HLA- A2 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 16.

[0085] Figures 10A-D. HLA-A3/A11 Influenza-Specific Recall Responses for

Humans. Immune responses in Donors X709 and X3501 using a panel of influenza- derived HLA-A3/A11 peptides and a panel of HLA-A3/A11 negative control peptides. The sequences of the influenza derived HLA-A3/A11 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 2.

[0086] Figures HA-C. HLA- A3/A11 Influenza-Specific Recall Responses for Mice.

Immune responses in HLA-AI l transgenic mice using a panel of influenza-derived HLA-A3/A11 peptides. The sequences of the influenza derived HLA-A3/A11 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 2.

[0087] Figures 12A-D. HLA-A24 Influenza-Specific Recall Responses for Humans.

Immune responses in Donors 1257, X759, X716 and XBB24 using a panel of influenza- derived HLA-A24 peptides and a panel of HLA-A24 negative control peptides. The sequences of the influenza derived HLA- A24 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 5.

[0088] Figures 13A-B. HLA-A24 Influenza-Specific Recall Responses for Mice.

Immune responses in HLA-AI l transgenic mice using a panel of influenza-derived HLA- A24 peptides. The sequences of the influenza derived HLA- A24 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 5.

[0089] Figures 14A-C. HLA-B7 Influenza-Specific Recall Responses for Humans.

Immune responses in Donors X685, 7357, and X3501 using a panel of influenza-derived HLA-B7 peptides and a panel of HLA-B7 negative control peptides. The sequences of the influenza derived HLA-B7 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 7.

[0090] Figure 15. HLA-B7 Influenza-Specific Recall Responses for Mice. Immune responses in HLA-B7 transgenic mice using a panel of influenza-derived HLA-B7 peptides. The sequences of the influenza derived HLA-B7 peptides used in the experiment correspond to the nomenclature of the influenza peptides in Table 7.

[0091] Figures 16A-H. HLA-DR Influenza-Specific Recall Responses for Humans.

Immune responses in Donors X753, X6018,X3501 and X709 using a panel of influenza- derived HLA-DR peptides and a panel of negative control peptides. The sequences of the influenza derived HLA-DR peptides used in the experiment correspond to the nomenclature of the influenza peptides in Tables 19, 21, 23, 25, 27, 29, 35, 37, and 38.

[0092] Figure 17. HLA-DR Influenza-Specific Recall Responses for DR4

Transgenic Mice. Immune responses in DR4 transgenic mice using a panel of influenza-derived HLA-DR peptides. The sequences of the influenza derived HLA-DR peptides used in the experiment correspond to the nomenclature of the influenza peptides in Tables 19, 21, 23, 25, 27, 29, 35, 37, and 38.

[0093] Figure 18. HLA-DR Influenza-Specific Recall Responses for b x d

Haplotype Mice. Immune responses in b x d haplotype mice using a panel of influenza- derived HLA-DR peptides. The sequences of the influenza derived HLA-DR peptides used in the experiment correspond to the nomenclature of the influenza peptides in Tables 19, 21, 23, 25, 27, 29, 35, 37, and 38.

DETAILED DESCRIPTION OF THE INVENTION

[0094] The present invention is directed to compositions and methods for enhancing the immune response of a vertebrate in need of protection against influenza virus infection by administering in vivo, into a tissue of a vertebrate, at least one multi-epitope polynucleotide construct, at least one polypeptide encoded by such a multi-epitope polynucleotide construct, or at least one synthetic peptide, where the multi-epitope polynucleotide construct, polypeptide, or synthetic peptide comprises one or more CTL and/or HTL epitopes, where each CTL and/or HTL epitope is identified from an influenza virus polypeptide, in cells of the vertebrate in need of protection. The polynucleotide or polypeptide can also comprise a nucleic acid sequence encoding a pan- DR binding epitope (e.g. PADRE ) or the peptide encoded therein.

[0095] The invention also relates to a method of designing and constructing a multi- epitope influenza virus vaccine construct with optimized immunogenicity and comprising influenza virus CTL and/or HTL epitopes. A multi-epitope influenza virus vaccine construct in accordance with the invention allows for significant, non-ethnically biased population coverage, and can preferably focus on epitopes conserved amongst different viral or other antigenic isolates. Through the method and system disclosed herein, a multi-epitope influenza virus vaccine construct can be optimized with regard to the magnitude and breadth of responses, and can allow for the simplest epitope configuration. [0096] In one aspect, the present invention provides for simultaneous induction of responses against specific influenza virus CTL and/or HTL epitopes, using single promoter multi-epitope constructs. Such constructs can contain many different epitopes, between 5 and 50, preferably greater than 10, often greater than 20, 25, 30, 25, 40, or 45.

[0097] Non-limiting examples of influenza virus CTL and/or HTL epitopes within the scope of the invention include, but are not limited to, CTL and/or HTL epitopes from the influenza HA, NA, NP, PA, PBl, PB2, NSl, NS2, Ml or M2 polypeptides, and fragments, e.g., M2e derivatives, and variants thereof. Nucleotide and amino acid sequences of influenza polypeptides from a wide variety of influenza types and subtypes are known in the art.

[0098] Epitopes for inclusion in the multi-epitope constructs typically bear HLA Class I or Class II binding motifs as described, for example, in PCT applications PCT/USOO/27766, or PCT/USOO/19774. Multi-epitope constructs can be prepared according to the methods set forth in Ishioka, et al, J Immunol 162(7):39\5-3925 (1999), for example, the disclosure of which is herein incorporated by reference.

[0099] Multiple HLA class II or class I epitopes present in a multi-epitope construct can be derived from the same antigen, or from different antigens. For example, a multi- epitope construct can contain one or more HLA epitopes that can be derived from two different antigens of the same virus or from two different antigens of different viruses. Epitopes for inclusion in a multi-epitope construct can be selected by one of skill in the art, e.g., by using a computer to select epitopes that contain HLA allele-specific motifs or supermotifs. The multi-epitope constructs of the invention also encode one or more broadly cross-reactive binding, or universal, HLA class II epitopes, i.e., pan-DR binding epitopes, e.g., PADRE^®. (Epimmune, San Diego, Calif), (described, for example, in U.S. Pat. No. 5,736,142) or a PADRE^® family molecule.

[00100] Universal HLA Class II epitopes can be advantageously combined with other

HLA Class I and Class II epitopes to increase the number of cells that are activated in response to a given antigen and provide broader population coverage of HLA-reactive alleles. Thus, the multi-epitope constructs of the invention can include HLA epitopes specific for an antigen, universal HLA class II epitopes, or a combination of specific HLA epitopes and at least one universal HLA class II epitope.

[00101] HLA Class I epitopes, referred to as "CTL epitopes" are peptides of defined length that can be from about 8 to about 13 amino acids in length, from about 8 to about 11 amino acids in length, from about 9 to about 11 amino acids in length, or about 9 to about 10 amino acids in length. HLA Class II epitopes, referred to as "HTL epitopes" as peptides of defined length that can be from about 6 to about 30 amino acids in length, from about 8 to about 30 amino acids in length, from about 10 to about 30 amino acids, from about 12 to about 30 amino acids in length, from about 6 to about 25 amino acids in length, from about 8 to about 25 amino acids in length, from about 10 to about 25 amino acids, from about 12 to about 25 amino acids in length, from about 6 to about 18 amino acids in length, from about 8 to about 18 amino acids in length, from about 10 to about 18 amino acids, or from about 12 to about 18 amino acids in length, which is recognized by a particular HLA molecule. An HLA Class I or II epitope can be derived from any desired antigen of interest. The antigen of interest can be any protein from an influenza virus for which an immune response is desired. Epitopes can be selected based on their ability to bind one or multiple HLA alleles. Epitopes that are analogs of naturally occurring sequences can also be included in the multi-epitope constructs described herein. Such analog peptides are described, for example, in PCT applications PCT/US97/03778, PCT/USOO/19774, and co-pending U.S. Ser. No. 09/260,714 filed Mar. 1, 1999.

[00102] Initially, influenza CTL epitopes of the present invention were obtained from the influenza virus HA, NA, NP, PA, PBl, PB2, NSl, NS2, Ml or M2 protein sequences which were scanned for HLA- A3, -A24, -B7, B44, -Al and -A2 motifs using computer algorithm analysis as previously described. Approximately 450 sequences bearing the appropriate motifs were identified. In order to select potential epitopes that would be cross-reactive amongst a variety of influenza strains, these sequences were compared to other viral strains, typically 11 to 20, and conserved sequences were selected for peptide synthesis. Peptide binding assays were performed using peptide and purified HLA molecules.

[00103] As used herein, the phrases "sequence conservancy", "strain conservancy", or

"strain sequence conservancy" reflect the results of an amino acid sequence comparison among a plurality of influenza strains to determine the degree of homology between amino acid sequences of the same protein of various strains. Typically, the following influenza strains were compared to determine the percentage sequence conservancy:

[00104] Binding analyses of 119 conserved HLA- A3 peptides are provided in Table 1. In order to select epitopes that would be cross-reactive amongst various humans to obtain maximal population coverage, the number of vaccine candidate peptides was subsequently reduced to 77 by selecting only degenerate binding peptides demonstrating binding at < 500 nM to at least A*0301 or A*1101, and a strain sequence conservancy equal to or greater than 30%, provided in Table 2. These 77 candidate peptides were again reduced to 25 peptides demonstrating binding at < 500 nM to at least A*0301 or A* 1101, and a strain sequence conservancy equal to or greater than 38%, provided in Table 3. The most preferred HLA- A3 candidate epitopes demonstrate binding at < 500 nM to at least A*0301 or A* 1101, have a strain sequence conservancy equal to or greater than 30% and are positive in human and/or mouse influenza recall responses. These most preferred HLA-A3 candidate epitopes are listed in Table 52.

[00105] Binding analyses of 50 conserved HLA-A24 peptides are provided in Table 4.

These candidate peptides were reduced to 32 peptides demonstrating binding at < 500 nM to A*2402, and a strain sequence conservancy equal to or greater than 30%, provided in Table 5. These candidate peptides were again reduced to 20 peptides by limiting to 3 peptides at most per influenza virus protein, provided in Table 6. The most preferred HLA-A24 candidate epitopes demonstrate binding at < 500 nM to A*2402, have a strain sequence conservancy equal to or greater than 30% and are positive in human and/or mouse influenza recall responses. These most preferred HLA-A3 candidate epitopes are listed in Table 53. [00106] Binding analyses of 30 conserved HLA-B7 peptides are provided in Table 7.

These candidate peptides were reduced to 16 peptides demonstrating binding at < 500 nM to B*0702 and a strain sequence conservancy equal to or greater than 38%, and a total limit of 3 peptides at most per influenza virus protein, provided in Table 8. The most preferred HLA-B07 candidate epitopes demonstrate binding at < 500 nM to at least A*B702 or B*3501, have a strain sequence conservancy equal to or greater than 30% and are positive in human and/or mouse influenza recall responses. These most preferred HLA-B07 candidate epitopes are listed in Table 54.

[00107] Binding analyses of 131 conserved HLA-B44 peptides are provided in Table 9.

These candidate peptides were reduced to 36 peptides demonstrating binding at < 500 nM to at least two of B*4001, B*4402 or B*4403, and a strain sequence conservancy equal to or greater than 30%, provided in Table 10. These candidate peptides were again reduced to 24 peptides by limiting to 3 peptides at most per influenza virus protein, provided in Table 11.

[00108] Binding analyses of 46 conserved HLA-Al peptides are provided in Table 12.

These candidate peptides were reduced to 33 peptides demonstrating binding at < 500 nM to at least A*0101 or A*3002, and a strain sequence conservancy equal to or greater than 38%, provided in Table 13. These candidate peptides were again reduced to 20 peptides by limiting to 3 peptides at most per influenza virus protein, provided in Table 14. The most preferred HLA-AOl candidate epitopes demonstrate binding at < 500 nM to at least A*0101 or A*3002, have a strain sequence conservancy equal to or greater than 38% and are positive in human and/or mouse influenza recall responses. These most preferred HLA-Al candidate epitopes are listed in Table 55.

[00109] Binding analyses of 68 conserved HLA-A2 peptides are provided in Table 15.

These candidate peptides were reduced to 40 peptides demonstrating binding at < 500 nM to A*0201 and 2 additional alleles, and a strain sequence conservancy equal to or greater than 30%, provided in Table 16. These candidate peptides were again reduced to 26 peptides by limiting to 3 peptides at most per influenza virus protein, provided in Table 17. The most preferred HLA- A2 candidate epitopes demonstrate binding at < 500 nM to A*0201 and two additional alleles, have a strain sequence conservancy equal to or greater than 30% and are positive in human and/or mouse influenza recall responses. These most preferred HLA-A2 candidate epitopes are listed in Table 56.

[00110] Influenza HTL epitopes of the present invention were obtained from the H5N1

(AF036362) and H2N2 (M25924) viral protein sequences which were scanned for HLA- DRl and -DR3 motifs using computer algorithm analysis as described above. Approximately 1,500 sequences bearing the appropriate motifs were identified. Conserved sequences were selected and peptide binding assays were performed as described above. Binding analyses of 157 conserved DR peptides are provided in Table 48. In order to select epitopes that would be cross-reactive amongst various humans to obtain maximal population coverage, the number of vaccine candidate peptides was subsequently reduced to 53 by selecting only degenerate binding peptides demonstrating at least high to intermediate binding and high strain conservancy, provided in Table 49. Binding analyses of 163 conserved DR3 peptides are provided in Table 43. These 163 candidate DR3 peptides was subsequently reduced to 67 peptides demonstrating binding at < 1100 nM to DRB 1*0301, and a strain sequence conservancy of equal to or greater than 30% provided in Table 44.

[00111] Analyses of 133 conserved NP HLA-DRl peptides are provided in Table 19.

These candidate peptides were reduced to 40 peptides predicted to bind to at least DRBl*0101 at < 100 nM, and a strain sequence conservancy >35%, provided in Table 20.

[00112] Analyses of 72 conserved NSl HLA-DRl peptides are provided in Table 21.

These candidate peptides were reduced to 25 peptides predicted to bind to at least DRBl*0101 at < 100 nM, and a strain sequence conservancy >30%, provided in Table 22.

[00113] Analyses of 27 conserved NS2 HLA-DRl peptides are provided in Table 23.

These candidate peptides were reduced to 15 peptides predicted to bind to at least DRBl*0101 at < 200 nM, and a strain sequence conservancy >35%, provided in Table 24.

[00114] Analyses of 185 conserved PA HLA-DRl peptides are provided in Table 25.

These candidate peptides were reduced to 58 peptides predicted to bind to at least DRBl*0101 at < 100 nM, and a- strain sequence conservancy >35%, provided in Table 26.

[00115] Analyses of 239 conserved PBl HLA-DRl peptides are provided in Table 27.

These candidate peptides were reduced to 81 peptides predicted to bind to at least DRBl*0101 at < 100 nM, and a strain sequence conservancy >35%, provided in Table 28.

[00116] Analyses of 223 conserved PB2 HLA-DRl peptides are provided in Table 29.

These candidate peptides were reduced to 78 peptides predicted to bind to at least DRB 1*0101 at < 100 nM, and a strain sequence conservancy >35%, provided in Table

30. [00117] Analyses of 118 conserved HA HLA-DRl peptides are provided in Table 31.

These candidate peptides were reduced to 59 peptides predicted to bind to at least

DRB 1*0101 at < 100 nM, and a strain sequence conservancy >35%, provided in Table

32. [00118] Analyses of 80 conserved Ml HLA-DRl peptides are provided in Table 34.

These candidate peptides were reduced to 33 peptides predicted to bind to at least

DRBl*0101 at < 100 nM, and a strain sequence conservancy >35%, provided in Table

35. [00119] Analyses of 23 conserved M2 HLA-DRl peptides are provided in Table 36.

These candidate peptides were reduced to 7 peptides predicted to bind to at least

DRBl*0101 at < 300 nM, and a strain sequence conservancy >35%, provided in Table

37. [00120] Analyses of 145 conserved NA HLA-DRl peptides are provided in Table 38.

These candidate peptides were reduced to 79 peptides predicted to bind to at least

DRBl*0101 at < 100 nM, and a strain sequence conservancy >35%, provided in Table

39. [00121] Analyses of 39 conserved NA HLA-DR3 peptides are provided in Table 40.

These candidate peptides were reduced to 29 peptides demonstrating anchor conservancy

>40%, provided in Table 41. [00122] Analyses of 28 conserved HA HLA-DR3 peptides are provided in Table 45.

These candidate peptides were reduced to 19 peptides demonstrating anchor conservancy

>30%, provided in Table 46. [00123] Preferred HLA-DR candidate epitopes are listed in Table 57. The most preferred

HLA-DR candidate epitopes demonstrate binding to at least five of the thirteen common

DR alleles listed and are positive in human and/or mouse influenza recall responses.

These most preferred HLA- A3 candidate epitopes are listed in Table 58. [00124] The influenza hemagglutinin (HA) and neuraminidase (NA) are highly variable sequences. Because of the high variability of these sequences, subsets of HLA-DRl and

HLA-DR3 epitopes from the HA and NA sequences specific to influenza strain Human

A/Viet Nam/1203/2004 (H5N1) were identified and provided in Tables 18, 33, 42 and

47. [00125] The number of candidate HTL epitopes with increased binding characteristics and/or having an increased percentage of conservancy are again reduced to 1-10 HTL peptides for inclusion in an influenza virus vaccine. The selection of these 1-10 HTL peptides is based on obtaining positive immune responses in human and mouse recall assays. A preference is also given for inclusion of peptides representing each of the 10 influenza proteins.

[00126] The amino acid sequence of a representative influenza virus multi-epitope construct has the following sequence, referred to herein as SEQ ED NO: .

AKFVAAWTLKAAAKAAAGEISPLPSLKMPAHGPAKSMKAAAMEVASQARQMNAPIEHIA SMNRLFFKCIYRGAANMDRAVKLYNAAAFYRYGFVANFGAAALPFERATIMKAAAMQAL QLLLEVGAAAILGFVFTLTVNAMLASIDLKYGAAALMEWLKTRGAAAGLFGAIAGFINA AAFYIQMCTELKFAAICTHLNAAAFEDLRVSSFKAAASYINRTGTFKAAAMVLSAFDER NARMGTVTTEVNLTIGECPKYNAAMGTVTTEVALGLVCAGPGPGFEQITFMQALQLLLE GPGPGIRPLLVEGTASLSPGGPGPGVGTMVMELIRMIKRGGPGPGLRHFQKDAKVLFQN WGPGPGEYIMKGVYINTALLNGPGPGLIFLARSALILRGSVGPGPGIRWLIEEVRHRLR ITGPGPGISSMVEAMVSRARIDGPGPGNPLIRHENRMVLASTGPGPGDLIFLARSALIL RGSGPGPGARILTSESQLTITKEGPGPGDFALIVNAPNHEGIQGPGPGITFMQALQLLL EVEQGPGPGLFTIRQEMASRGLWDGPGPGQNSITIERMVLSAFDGPGPGPTLLFLKVPA

QNAIST

A diagram of the multi-epitope construct shown above is illustrated in Figure 6. Binding data of the individual CTL and HTL epitopes of the multi-epitope construct shown above is presented in Table 50.

[00127] In certain embodiments, the composition according to the invention comprises a multi-epitope polynucleotide influenza virus vaccine construct or a polypeptide encoded by such a polynucleotide, and further comprises an influenza virus polypeptide. The influenza virus polypeptide can be an HA, NA, NP, PA, PBl, PB2, NSl, NS2, Ml or M2 polypeptide, or fragment, variant, or derivative thereof. For example, the influenza virus polypeptide can correspond to the mature HA protein of Influenza A/Vietnam/I 203/2004 (H5N1) which is available in GenBank (Accession Number AAT73274), and has the following sequence, referred to herein as SEQ ID NO: :

DQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPLILRDCSVAG WLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIP KSSWSSHEASLGVSSACPYQGKSSFFRNVWLIKKNSTYPTIKRSYNNTNQEDLLVLWG IHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPN DAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIH PLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGY HHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKK MEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYH KCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAI MVAGLSLWMCSNGSLQCR

[00128] Additional HA sequences of the present invention correspond to isolated wild- type HA sequences from influenza A and influenza B strains as are known in the art. For example, wild-type HA sequences from influenza strains can also be found at http://www.flu.lanl. gov/search/index.html?form_page=search.

[00129] The influenza virus polypeptide can correspond to an M2e sequence. Examples of M2e sequences are set forth in Table 51.

[00130] Multi-epitope constructs can be generated using methodology well known in the art. For example, polypeptides comprising the multi-epitope constructs can be synthesized and linked. Typically, multi-epitope constructs are constructed using recombinant DNA technology.

[00131] The present invention is also directed to administering in vivo, into a tissue of the vertebrate the above described polynucleotide and/or at least one isolated influenza polypeptide, or a fragment, variant, or derivative thereof. The isolated influenza polypeptide or fragment, variant, or derivative thereof can be, for example, a recombinant protein, a purified subunit protein, a protein expressed and carried by a heterologous live or inactivated or attenuated viral vector expressing the protein, or can be an inactivated influenza, such as those present in conventional, commercially available, inactivated influenza vaccines. According to either method, the polynucleotide is incorporated into the cells of the vertebrate in vivo, and an immunologically effective amount of the influenza protein, or fragment or variant encoded by the polynucleotide is produced in vivo. The isolated protein or fragment, variant, or derivative thereof is also administered in an immunologically effective amount. The polynucleotide can be administered to the vertebrate in need thereof either prior to, at the same time (simultaneously), or subsequent to the administration of the isolated influenza polypeptide or fragment, variant, or derivative thereof.

Expression Vectors and Construction of a Multi-Epitope Constructs [00132] The multi-epitope constructs of the invention are typically provided as an expression vector comprising a nucleic acid encoding the multi-epitope polypeptide. Construction of such expression vectors is described, for example in PCT/US99/ 10646, the disclosure of which is herein incorporated by reference. The expression vectors contain at least one promoter element that is capable of expressing a transcription unit encoding the nucleic acid in the appropriate cells of an organism so that the antigen is expressed and targeted to the appropriate HLA molecule. For example, for administration to a human, a promoter element that functions in a human cell is incorporated into the expression vector.

[00133] In preferred embodiments, the invention utilizes routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, eds., 1994); Oligonucleotide Synthesis. A Practical Approach (Gait, ed., 1984); Kuijpers, Nucleic Acids Research 18(17):5\91 (1994); Dueholm, J. Org. Chem. 59:5767-5773 (1994); Methods in Molecular Biology, volume 20 (Agrawal, ed.); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, e.g., Part I, chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" (1993)).

[00134] The nucleic acids encoding the epitopes are assembled in a construct according to standard techniques, hi general, the nucleic acid sequences encoding multi-epitope polypeptides are isolated using amplification techniques with oligonucleotide primers, or are chemically synthesized. Recombinant cloning techniques can also be used when appropriate. Oligonucleotide sequences are selected which either amplify (when using PCR to assemble the construct) or encode (when using synthetic oligonucleotides to assemble the construct) the desired epitopes.

[00135] Amplification techniques using primers are typically used to amplify and isolate sequences encoding the epitopes of choice from DNA or RNA (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Methods such as polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used to amplify epitope nucleic acid sequences directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. Restriction endonuclease sites can be incorporated into the primers. Multi-epitope constructs amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector.

[00136] Synthetic oligonucleotides can also be used to construct multi-epitope constructs.

This method is performed using a series of overlapping oligonucleotides, representing both the sense and non-sense strands of the gene. These DNA fragments are then annealed, ligated and cloned. Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et al., Nucleic Acids Res., 72:6159-6168 (1984). Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, J Chrom. 255:137-149 (1983).

[00137] The epitopes of the multi-epitope constructs are typically subcloned into an expression vector that contains a strong promoter to direct transcription, as well as other regulatory sequences such as enhancers and polyadenylation sites. Suitable promoters are well known in the art and described, e.g., in Sambrook et al. and Ausubel et al. Eukaryotic expression systems for mammalian cells are well known in the art and are commercially available. Such promoter elements include, for example, cytomegalovirus (CMV), Rous sarcoma virus LTR and SV40.

[00138] The expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the multi- epitope construct in host cells. A typical expression cassette thus contains a promoter operably linked to the multi-epitope construct and signals required for efficient polyadenylation of the transcript. Additional elements of the cassette may include enhancers and introns with functional splice donor and acceptor sites.

[00139] In addition to a promoter sequence, the expression cassette can also contain a transcription termination region downstream of the structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.

[00140] The particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic cells may be used. Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, e.g., SV40 vectors, CMV vectors, papilloma virus vectors, and vectors derived from Epstein Bar virus.

[00141] The multi-epitope constructs of the invention can be expressed from a variety of vectors including plasmid vectors as well as viral or bacterial vectors. Examples of viral expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. As an example of this approach, vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host bearing a tumor, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.

[00142] A wide variety of other vectors useful for therapeutic administration or immunization, e.g. adeno and adeno-associated virus vectors, retroviral vectors, non- viral vectors such as BCG (Bacille Calmette Guerin), Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art.

[00143] Immunogenicity and antigenicity of the multi-epitope constructs are evaluated as described herein.

Targeting Sequences

[00144] The expression vectors of the invention may encode one or more MHC epitopes operably linked to a MHC targeting sequence, and are referred to herein as "targeting nucleic acids" or "targeting sequences." The use of a MHC targeting sequence enhances the immune response to an antigen, relative to delivery of antigen alone, by directing the peptide epitope to the site of MHC molecule assembly and transport to the cell surface, thereby providing an increased number of MHC molecule-peptide epitope complexes available for binding to and activation of T cells.

[00145] MHC Class I targeting sequences can be used in the present invention, e.g., those sequences that target an MHC Class I epitope peptide to a cytosolic pathway or to the endoplasmic reticulum (see, e.g., Rammensee et al, Inmunogenetics 47:178-228 (1995)). For example, the cytosolic pathway processes endogenous antigens that are expressed inside the cell. Although not wishing to be bound by any particular theory, cytosolic proteins are thought to be at least partially degraded by an endopeptidase activity of a proteosome and then transported to the endoplasmic reticulum by the TAP molecule (transporter associated with processing). In the endoplasmic reticulum, the antigen binds to MHC Class I molecules. Endoplasmic reticulum signal sequences bypass the cytosolic processing pathway and directly target endogenous antigens to the endoplasmic reticulum, where proteolytic degradation into peptide fragments occurs. Such MHC Class I targeting sequences are well known in the art, and include, e.g., signal sequences such as those from Ig kappa, tissue plasminogen activator or insulin. A preferred signal peptide is the human. Ig kappa chain sequence. Endoplasmic reticulum signal sequences can also be used to target MHC Class II epitopes to the endoplasmic reticulum, the site of MHC Class I molecule assembly. MHC Class II targeting sequences can also be used in the invention, e.g., those that target a peptide to the endocytic pathway. These targeting sequences typically direct extracellular antigens to enter the endocytic pathway, which results in the antigen being transferred to the lysosomal compartment where the antigen is proteolytically cleaved into antigen peptides for binding to MHC Class π molecules. As with the normal processing of exogenous antigen, a sequence that directs a MHC Class II epitope to the endosomes of the endocytic pathway and/or subsequently to lysosomes, where the MHC Class II epitope can bind to a MHC Class II molecule, is a MHC Class II targeting sequence. For example, group of MHC Class II targeting sequences useful in the invention are lysosomal targeting sequences, which localize polypeptides to lysosomes. Since MHC Class II molecules typically bind to antigen peptides derived from proteolytic processing of endocytosed antigens in lysosomes, a lysosomal targeting sequence can function as a MHC Class II targeting sequence. Lysosomal targeting sequences are well known in the art and include sequences found in the lysosomal proteins LAMP-I and LAMP-2 as described by August et al. U.S. Pat. No. 5,633,234, issued May 27, 1997), which is incorporated herein by reference.

[00146] Other lysosomal proteins that contain lysosomal targeting sequences include

HLA-DM. HLA-DM is an endosomal/lysosomal protein that functions in facilitating binding of antigen peptides to MHC Class II molecules. Since it is located in the lysosome, HLA-DM has a lysosomal targeting sequence that can function as a MHC Class II molecule targeting sequence (Copier et al, J. Immunol. 757:1017-1027 (1996), which is incorporated herein by reference).

[00147] The resident lysosomal protein HLA-DO can also function as a lysosomal targeting sequence. In contrast to the above described resident lysosomal proteins LAMP-I and HLA-DM, which encode specific Tyr-containing motifs that target proteins to lysosomes, HLA-DO is targeted to lysosomes by association with HLA-DM (Liljedahl et al, EMBO J, 75:4817-4824 (1996)), which is incorporated herein by reference. Therefore, the sequences of HLA-DO that cause association with HLA-DM and, consequently, translocation of HLA-DO to lysosomes can be used as MHC Class II targeting sequences. Similarly, the murine homolog of HLA-DO, H2-D0, can be used to derive a MHC Class II targeting sequence. A MHC Class II epitope can be fused to HLA-DO or H2-D0 and targeted to lysosomes.

[00148] In another example, the cytoplasmic domains of B cell receptor subunits Ig-α and

Ig-β mediate antigen internalization and increase the efficiency of antigen presentation as described in, for example, Bonnerot et al, Immunity, 5:335-347 (1995). Therefore, the cytoplasmic domains of the Ig-α and Ig-β proteins can function as MHC Class II targeting sequences that target a MHC Class II epitope to the endocytic pathway for processing and binding to MHC Class II molecules.

[00149] Another example of a MHC Class II targeting sequence that directs MHC Class II epitopes to the endocytic pathway is a sequence that directs polypeptides to be secreted, where the polypeptide can enter the endosomal pathway. These MHC Class II targeting sequences that direct polypeptides to be secreted mimic the normal pathway by which exogenous, extracellular antigens are processed into peptides that bind to MHC Class II molecules. Any signal sequence that functions to direct a polypeptide through the endoplasmic reticulum and ultimately to be secreted can function as a MHC Class II targeting sequence so long as the secreted polypeptide can enter the endosomal/lysosomal pathway and be cleaved into peptides that can bind to MHC Class II molecules.

[00150] In another example, the Ii protein binds to MHC Class II molecules in the endoplasmic reticulum, where it functions to prevent peptides present in the endoplasmic reticulum from binding to the MHC Class II molecules. Therefore, fusion of a MHC Class II epitope to the Ii protein targets the MHC Class II epitope to the endoplasmic reticulum and a MHC Class II molecule. For example, the CLIP sequence of the Ii protein can be removed and replaced with a MHC Class II epitope sequence so that the MHC Class II epitope is directed to the endoplasmic reticulum, where the epitope binds to a MHC Class II molecule.

[00151] In some cases, antigens themselves can serve as MHC Class II or I targeting sequences and can be fused to a universal MHC Class II epitope to stimulate an immune response. Although cytoplasmic viral antigens are generally processed and presented as complexes with MHC Class I molecules, long-lived cytoplasmic proteins such as the influenza matrix protein can enter the MHC Class MHC Class II molecule processing pathway as described in, for example, Gueguen & Long, Proc. Natl. Acad. ScL USA, 93:14692-14697 (1996). Therefore, long-lived cytoplasmic proteins can function as a MHC Class MHC Class II targeting sequence. For example, an expression vector encoding influenza matrix protein fused to a universal MHC Class I/MHC Class II epitope can be advantageously used to target influenza antigen and the universal MHC Class I/MHC Class II epitope to the MHC Class I/MHC Class II pathway for stimulating an immune response to influenza.

[00152] Other examples of antigens functioning as MHC Class I/MHC Class II targeting sequences include polypeptides that spontaneously form particles. The polypeptides are secreted from the cell that produces them and spontaneously form particles, which are taken up into an antigen-presenting cell by endocytosis such as receptor-mediated endocytosis or are engulfed by phagocytosis. The particles are proteolytically cleaved into antigen peptides after entering the endosomal/lysosomal pathway.

[00153] One such polypeptide that spontaneously forms particles is HBV surface antigen

(HBV-S) as described in, for example, Diminsky et al, Vaccine 75:637-647 (1997) or Le Borgne et al, Virology, 240:304-315 (1998). Another polypeptide that spontaneously forms particles is HBV core antigen as described in, for example, Kuhrober et al, International Immunol, P: 1203-1212 (1997). Still another polypeptide that spontaneously forms particles is the yeast Ty protein as described in, for example, Weber et al, Vaccine, 73:831-834 (1995). For example, an expression vector containing HBV-S antigen fused to a universal MHC Class II epitope can be advantageously used to target HBV-S antigen and the universal MHC Class II epitope to the MHC Class II pathway for stimulating an immune response to HBV.

[00154] Methods of designing and selecting CTL and/or HTL epitopes having an HLA-

DR binding motif according to the present invention are described in Rammensee et al., "MHC ligands and peptide motifs: first listing," Immunogenetics 41:178-228 (1995) and Sette et al, "Prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis," Proc. Natl. Acad. ScL 86: 3296-3300 (1989), the disclosure of each which is incorporated herein by reference in its entirety.

[00155] Methods of designing and generating a multi-epitope construct comprising an influenza virus CTL and/or HTL epitope are performed according to methods of designing and using multi-epitope constructs as described in WO 01/47541, WO 02/083714 and US 2004/0248113 Al, the disclosure of each which is incorporated herein by reference in its entirety. The minimization of junctional motifs, the influence of flanking regions on CTL and HTL epitope immunogenicity within a multi-epitope construct, and the correlation between epitope immunogenicity and levels of epitope presentation in transfected cell lines are also described in WO 01/47541, WO 02/083714 and US 2004/0248113 Al, the disclosure of each which is incorporated herein by reference in its entirety.

[00156] The present invention also provides vaccine compositions and methods for delivery of influenza virus multi-epitope constructs to a vertebrate with optimal expression and safety. These vaccine compositions are prepared and administered in such a manner that the encoded gene products are optimally expressed in the vertebrate of interest. As a result, these compositions and methods are useful in stimulating an immune response against influenza virus infection. Also included in the invention are expression systems and delivery systems.

[00157] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a polynucleotide," is understood to represent one or more polynucleotides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[00158] The term "polynucleotide" is intended to encompass a singular nucleic acid or nucleic acid fragment as well as plural nucleic acids or nucleic acid fragments, and refers to an isolated molecule or construct, e.g., a virus genome (e.g., a non-infectious viral genome), messenger RNA (mRNA), plasmid DNA (pDNA), or derivatives of pDNA (e.g., minicircles as described in (Darquet, A-M et ai, Gene Therapy 4:1341-1349 (1997)) comprising a polynucleotide. A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).

[00159] The terms "nucleic acid" or "nucleic acid fragment" refer to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide or construct. A nucleic acid or fragment thereof may be provided in linear (e.g., mRNA) or circular (e.g., plasmid) form as well as double-stranded or single-stranded forms. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically.

[00160] As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, and the like, are not part of a coding region. Two or more nucleic acids or nucleic acid fragments of the present invention can be present in a single polynucleotide construct, e.g., on a single plasmid, or in separate polynucleotide constructs, e.g., on separate (different) plasmids. Furthermore, any nucleic acid or nucleic acid fragment may encode a single influenza polypeptide or fragment, derivative, or variant thereof, e.g., or may encode more than one polypeptide, e.g., a nucleic acid may encode two or more polypeptides. In addition, a nucleic acid may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator, or may encode heterologous coding regions fused to the influenza coding region, e.g., specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

[00161] The terms "fragment," "variant," "derivative" and "analog" when referring to influenza virus polypeptides of the present invention include any polypeptides which retain at least some of the immunogenicity or antigenicity of the corresponding native polypeptide. Fragments of influenza virus polypeptides of the present invention include proteolytic fragments, deletion fragments and in particular, fragments of influenza virus polypeptides which exhibit increased secretion from the cell or higher immunogenicity or reduced pathogenicity when delivered to an animal, such as deletion of signal sequences or one or more domains. Polypeptide fragments further include any portion of the polypeptide which comprises an antigenic or immunogenic epitope of the native polypeptide, including linear as well as three-dimensional epitopes. Variants of influenza virus polypeptides of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally, such as an allelic variant. By an "allelic variant" is intended alternate forms of a gene occupying a given locus on a chromosome or genome of an organism or virus. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985), which is incorporated herein by reference. For example, as used herein, variations in a given gene product. When referring to influenza virus NA or HA proteins, each such protein is a "variant," in that native influenza virus strains are distinguished by the type of NA and HA proteins encoded by the virus. However, within a single HA or NA variant type, further naturally or non-naturally occurring variations such as amino acid deletions, insertions or substitutions may occur. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of influenza virus polypeptides of the present invention are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. An analog is another form of an influenza virus polypeptide of the present invention. An example is a proprotein which can be activated by cleavage of the proprotein to produce an active mature polypeptide.

[00162] The terms "infectious polynucleotide" or "infectious nucleic acid" are intended to encompass isolated viral polynucleotides and/or nucleic acids which are solely sufficient to mediate the synthesis of complete infectious virus particles upon uptake by permissive cells. Thus, "infectious nucleic acids" do not require pre-synthesized copies of any of the polypeptides it encodes, e.g., viral replicases, in order to initiate its replication cycle in a permissive host cell.

[00163] The terms "non-infectious polynucleotide" or "non-infectious nucleic acid" as defined herein are polynucleotides or nucleic acids which cannot, without additional added materials, e.g, polypeptides, mediate the synthesis of complete infectious virus particles upon uptake by permissive cells. An infectious polynucleotide or nucleic acid is not made "non-infectious" simply because it is taken up by a non-permissive cell. For example, an infectious viral polynucleotide from a virus with limited host range is infectious if it is capable of mediating the synthesis of complete infectious virus particles when taken up by cells derived from a permissive host (i.e., a host permissive for the virus itself). The fact that uptake by cells derived from a non-permissive host does not result in the synthesis of complete infectious virus particles does not make the nucleic acid "non-infectious." In other words, the term is not qualified by the nature of the host cell, the tissue type, or the species taking up the polynucleotide or nucleic acid fragment.

[00164] In some cases, an isolated infectious polynucleotide or nucleic acid may produce fully-infectious virus particles in a host cell population which lacks receptors for the virus particles, i.e., is non-permissive for virus entry. Thus viruses produced will not infect surrounding cells. However, if the supernatant containing the virus particles is transferred to cells which are permissive for the virus, infection will take place.

[00165] The terms "replicating polynucleotide" or "replicating nucleic acid" are meant to encompass those polynucleotides and/or nucleic acids which, upon being taken up by a permissive host cell, are capable of producing multiple, e.g., one or more copies of the same polynucleotide or nucleic acid. Infectious polynucleotides and nucleic acids are a subset of replicating polynucleotides and nucleic acids; the terms are not synonymous. For example, a defective virus genome lacking the genes for virus coat proteins may replicate, e.g., produce multiple copies of itself, but is NOT infectious because it is incapable of mediating the synthesis of complete infectious virus particles unless the coat proteins, or another nucleic acid encoding the coat proteins, are exogenously provided.

[00166] In certain embodiments, the polynucleotide, nucleic acid, or nucleic acid fragment is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid which encodes a polypeptide normally also comprises a promoter and/or other transcription or translation control elements operably associated with the polypeptide-encoding nucleic acid fragment. An operable association is when a nucleic acid fragment encoding a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide-encoding nucleic acid fragment and a promoter associated with the 5' end of the nucleic acid fragment) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the gene product, or (3) interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid fragment encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid fragment. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein. [00167] A variety of transcription control regions are known to those skilled in the art.

These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

[00168] Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, elements from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

[00169] A DNA polynucleotide of the present invention may be a circular or linearized plasmid or vector, or other linear DNA which may also be non-infectious and nonintegrating (i.e., does not integrate into the genome of vertebrate cells). A linearized plasmid is a plasmid that was previously circular but has been linearized, for example, by digestion with a restriction endonuclease. Linear DNA may be advantageous in certain situations as discussed, e.g., in Cherng, J. Y., et ah, J. Control. Release 60:343-53 (1999), and Chen, Z. Y., et al. MoI. Ther. 3:403-10 (2001), both of which are incorporated herein by reference. As used herein, the terms plasmid and vector can be used interchangeably.

[00170] Alternatively, DNA virus genomes may be used to administer DNA polynucleotides into vertebrate cells. In certain embodiments, a DNA virus genome of the present invention is nonreplicative, noninfectious, and/or nonintegrating. Suitable DNA virus genomes include without limitation, herpesvirus genomes, adenovirus genomes, adeno-associated virus genomes, and poxvirus genomes. References citing methods for the in vivo introduction of non-infectious virus genomes to vertebrate tissues are well known to those of ordinary skill in the art, and are cited supra.

[00171] In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). Methods for introducing RNA sequences into vertebrate cells are described in U.S. Patent No. 5,580,859, the disclosure of which is incorporated herein by reference in its entirety. [00172] Polynucleotides, nucleic acids, and nucleic acid fragments of the present invention may be associated with additional nucleic acids which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a nucleic acid fragment or polynucleotide of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have, a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native leader sequence is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian leader sequence, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

[00173] As used herein, the term "plasmid" refers to a construct made up of genetic material (i.e., nucleic acids). Typically a plasmid contains an origin of replication which is functional in bacterial host cells, e.g., Escherichia coli, and selectable markers for detecting bacterial host cells comprising the plasmid. Plasmids of the present invention may include genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in eukaryotic cells. Also, the plasmid may include a sequence from a viral nucleic acid. However, such viral sequences normally are not sufficient to direct or allow the incorporation of the plasmid into a viral particle, and the plasmid is therefore a non-viral vector. In certain embodiments described herein, a plasmid is a closed circular DNA molecule.

[00174] The term "expression" refers to the biological production of a product encoded by a coding sequence. In most cases a DNA sequence, including the coding sequence, is transcribed to form a messenger-RNA (mRNA). The messenger-RNA is then translated to form a polypeptide product which has a relevant biological activity. Also, the process of expression may involve further processing steps to the RNA product of transcription, such as splicing to remove introns, and/or post-translational processing of a polypeptide product. [00175] As used herein, the term "polypeptide" is intended to encompass a singular

"polypeptide" as well as plural "polypeptides," and comprises any chain or chains of two or more amino acids. Thus, as used herein, terms including, but not limited to "peptide," "dipeptide," "tripeptide," "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included in the definition of a "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term further includes polypeptides which have undergone post-translational modifications, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.

[00176] Also included as polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. Polypeptides, and fragments, derivatives, analogs, or variants thereof of the present invention can be antigenic and immunogenic polypeptides related to influenza virus polypeptides, which are used to prevent or treat, i.e., cure, ameliorate, lessen the severity of, or prevent or reduce contagion of infectious disease caused by the influenza virus.

[00177] As used herein, an "antigenic polypeptide" or an "immunogenic polypeptide" is a polypeptide which, when introduced into a vertebrate, interacts with the vertebrate's immune system molecules, i.e., is antigenic, and/or induces an immune response in the vertebrate, i.e., is immunogenic.

[00178] An affinity threshold associated with immunogenicity in the context of HLA class II DR molecules has been delineated {see, e.g., Southwood et al. J. Immunology 160:3363-3373,1998, and U.S.S.N. 60/087192 filed 5/29/98). In order to define a biologically significant threshold of DR binding affinity, a database of the binding affinities of 32 DR-restricted epitopes for their restricting element {i.e., the HLA molecule that binds the motif) was compiled, hi approximately half of the cases (15 of 32 epitopes), DR restriction was associated with high binding affinities, i.e. binding affinity values of 100 nM or less. In the other half of the cases (16 of 32), DR restriction was associated with intermediate affinity (binding affinity values in the 100-1000 nM range). In only one of 32 cases was DR restriction associated with an IC₅₀ of 1000 nM or greater. Thus, 1000 nM can be defined as an affinity threshold associated with immunogenicity in the context of DR molecules.

[00179] By an "isolated" influenza virus polypeptide or a fragment, variant, or derivative thereof is intended an influenza virus polypeptide or protein that is not in its natural form. No particular level of purification is required. For example, an isolated influenza virus polypeptide can be removed from its native or natural environment. Recombinantly produced influenza virus polypeptides and proteins expressed in host cells are considered isolated for purposes of the invention, as are native or recombinant influenza virus polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique, including the separation of influenza virus virions from eggs or culture cells in which they have been propagated. In addition, an isolated influenza virus polypeptide or protein can be provided as a live or inactivated viral vector expressing an isolated influenza virus polypeptide and can include those found in inactivated influenza virus vaccine compositions. Thus, isolated influenza virus polypeptides and proteins can be provided as, for example, recombinant influenza virus polypeptides, a purified subunit of influenza virus, a viral vector expressing an isolated influenza virus polypeptide, or in the form of an inactivated or attenuated influenza virus vaccine.

[00180] The term "immunogenic carrier" as used herein refers to a first polypeptide or fragment, variant, or derivative thereof which enhances the immunogenicity of a second polypeptide or fragment, variant, or derivative thereof. Typically, an "immunogenic carrier" is fused to or conjugated to the desired polypeptide or fragment thereof. An example of an "immunogenic carrier" is a recombinant hepatitis B core antigen expressing, as a surface epitope, an immunogenic epitope of interest. See, e.g., European Patent No. EP 0385610 Bl, which is incorporated herein by reference in its entirety.

[00181] In the present invention, antigenic epitopes preferably contain a sequence of at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, or between about 8 to about 30 amino acids contained within the amino acid sequence of an influenza virus polypeptide of the invention, e.g., an NP polypeptide, an Ml polypeptide or an M2 polypeptide. Certain peptides comprising immunogenic or antigenic epitopes are at least 8, 9, 10, 11, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenic as well as immunogenic epitopes may be linear, i.e., be comprised of contiguous amino acids in a polypeptide, or may be three dimensional or conformational, i.e., where an epitope is comprised of non-contiguous amino acids which come together due to the secondary or tertiary structure of the polypeptide, thereby forming an epitope.

[00182] As to the selection of peptides or polypeptides bearing an antigenic epitope (e.g., that contain a region of a protein molecule to which an antibody or T cell receptor can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, e.g., Sutcliffe, J. G., et ah, Science 279:660-666 (1983), which is herein incorporated by reference.

[00183] Peptides capable of eliciting an immunogenic response are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins nor to the amino or carboxyl terminals. Peptides that are extremely hydrophobic and those of six or fewer residues generally are ineffective at inducing antibodies that bind to the mimicked protein; longer peptides, especially those containing proline residues, usually are effective. Sutcliffe et ah, supra, at 661. For instance, 18 of 20 peptides designed according to these guidelines, containing 8-39 residues covering 75% of the sequence of the influenza virus hemagglutinin HAl polypeptide chain, induced antibodies that reacted with the HAl protein or intact virus; and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies glycoprotein induced antibodies that precipitated the respective proteins.

[00184] Throughout this disclosure, "binding data" results are often expressed in terms of

"IC₅₀." IC₅₀ is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Given the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values approximate K_D values. Assays for determining binding are described in detail, e.g., in PCT publications WO 94/20127 and WO 94/03205, the disclosure of each which is herein incorporated by reference. It should be noted that IC_5O values can change, often dramatically, if the assay conditions are varied, and depending on the particular reagents used (e.g., HLA preparation, etc.). For example, excessive concentrations of HLA molecules will increase the apparent measured IC₅₀ of a given ligand. Alternatively, binding is expressed relative to a reference peptide. Although as a particular assay x becomes more, or less, sensitive, the IC₅₀ ⁵S of the peptides tested may change somewhat, the binding relative to the reference peptide will not significantly change. For example, in an assay run under conditions such that the IC₅₀ of the reference peptide increases 10- fold, the IC₅₀ values of the test peptides will also shift approximately 10-fold. Therefore, to avoid ambiguities, the assessment of whether a peptide is a good, intermediate, weak, or negative binder is generally based on its IC₅₀, relative to the IC₅₀ of a standard peptide. Binding may also be determined using other assay systems including those using: live cells (e.g., Ceppellini et al, Nature 339:392, 1989; Christnick et al^ Nature 352:67, 1991; Busch et al, Int. Immunol. 2:443, 19990; Hill et al, J. Immunol. 747:189, 1991; del Guercio et al., J. Immunol. 754:685, 1995), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 27:2069, 1991), immobilized purified MHC (e.g., Hill et al, J. Immunol. 152, 2890, 1994; Marshall et al, J. Immunol. 752:4946, 1994), ELISA systems (e.g., Reay et al, EMBO J. 77:2829, 1992), surface plasmon resonance (e.g., Khilko et al, J. Biol. Chem. 2(55:15425, 1993); high flux soluble phase assays (Hammer et al, J. Exp. Med. 180:2353, 1994), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al, Nature 346:476, 1990; Schumacher et al, Cell 62:563, 1990; Townsend et al, Cell (52:285, 1990; Parker et al, J. Immunol. 149:1896, 1992).

[00185] The designation of a residue position in an epitope as the "carboxyl terminus" or the "carboxyl terminal position" refers to the residue position at the end of the epitope that is nearest to the carboxyl terminus of a peptide, which is designated using conventional nomenclature as defined below. "C+l" refers to the residue or position immediately following the C-terminal residue of the epitope, i.e., refers to the residue flanking the C-terminus of the epitope. The "carboxyl terminal position" of the epitope occurring at the carboxyl end of the multi-epitope construct may or may not actually correspond to the carboxyl terminal end of polypeptide. In preferred embodiments, the epitopes employed in the optimized multi-epitope constructs are motif-bearing epitopes and the carboxyl terminus of the epitope is defined with respect to primary anchor residues corresponding to a particular motif.

[00186] The designation of a residue position in an epitope as "amino terminus" or

"amino-terminal position" refers to the residue position at the end of the epitope which is nearest to the amino terminus of a peptide, which is designated using conventional nomenclature as defined below. "N-I" refers to the residue or position immediately adjacent to the epitope at the amino terminal end (position number 1) of an epitope. The "amino terminal position" of the epitope occurring at the amino terminal end of the multi-epitope construct may or may not actually correspond to the amino terminal end of the polypeptide. In preferred embodiments, the epitopes employed in the optimized multi-epitope constructs are motif-bearing epitopes and the amino terminus of the epitope is defined with respect to primary anchor residues corresponding to a particular motif. [00187] A "construct" as used herein generally denotes a composition that does not occur in nature. A construct can be produced by synthetic technologies, e.g., recombinant DNA preparation and expression or chemical synthetic techniques for nucleic or amino acids. A construct can also be produced by the addition or affiliation of one material with another such that the result is not found in nature in that form. A "multi-epitope construct" can be used interchangeably with the term "minigene" or "multi-epitope nucleic acid vaccine," and comprises multiple epitope nucleic acids that encode peptide epitopes of any length that can bind to a molecule functioning in the immune system, preferably a class I HLA and a T-cell receptor or a class II HLA and a T-cell receptor. All of the epitope nucleic acids in a multi-epitope construct can encode class I HLA epitopes or class II HLA epitopes. Class I HLA-encoding epitope nucleic acids are referred to as CTL epitope nucleic acids, and class II HLA-encoding epitope nucleic acids are referred to as HTL epitope nucleic acids. Some multi-epitope constructs can have a subset of the multi-epitope nucleic acids encoding class I HLA epitopes and another subset of the multi-epitope nucleic acids encoding class II HLA epitopes. The CTL epitope nucleic acids preferably encode an epitope peptide of about eight to about thirteen amino acids in length, more preferably about eight to about eleven amino acids in length, and most preferably about nine amino acids in length. The HTL epitope nucleic acids can encode an epitope peptide of about six to about thirty, preferably seven to about twenty three, preferably about seven to about seventeen, and even more preferably about eleven to about fifteen, and most preferably about thirteen amino acids in length. The multi-epitope constructs described herein preferably include five or more, ten or more, fifteen or more, twenty or more, or twenty-five or more epitope nucleic acids. All of the epitope nucleic acids in a multi-epitope construct may be from one organism (e.g., the nucleotide sequence of every epitope nucleic acid may be present in HIV strains), or the multi-epitope construct may include epitope nucleic acids present in two or more different organisms (e.g., some epitopes from HIV and some from HCV). As described hereafter, one or more epitope nucleic acids in the multi-epitope construct may be flanked by a spacer nucleic acid.

[00188] A "multi-epitope vaccine," which is synonymous with a "polyepitopic vaccine," or a "multi-epitope construct" or "minigene" is a vaccine comprising multiple epitopes.

[00189] "Cross-reactive binding" indicates that a peptide is bound by more than one HLA molecule; a synonym is "degenerate binding." [00190] A "cryptic epitope" elicits a response by immunization with an isolated peptide, but the response is not cross-reactive in vitro when intact whole protein that comprises the epitope is used as an antigen.

[00191] A "dominant epitope" is an epitope that induces an immune response upon immunization with a whole native antigen (see, e.g., Sercarz, et a\., Annu. Rev. Immunol. 77:729-766, 1993). Such a response is cross-reactive in vitro with an isolated peptide epitope.

[00192] A "subdominant epitope" is an epitope which evokes little or no response upon immunization with whole antigens which comprise the epitope, but for which a response can be obtained by immunization with an isolated epitope, and this response (unlike the case of cryptic epitopes) is detected when whole protein is used to recall the response in vitro or in vivo.

[00193] With regard to a particular amino acid sequence, an "epitope" is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) receptors. In an immune system setting, in vitro or in vivo, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by an immunoglobulin, T cell receptor or HLA molecule. Throughout this disclosure epitope and peptide are often used interchangeably. It is to be appreciated, however, that isolated or purified protein or peptide molecules larger than and comprising an epitope of the invention are still within the bounds of the invention.

[00194] A "flanking residue" is a residue that is positioned next to an epitope. A flanking residue can be introduced or inserted at a position adjacent to the N-terminus or the C- terminus of an epitope.

[00195] An "immunogenic peptide" or "peptide epitope" or "epitope" is a peptide that comprises an allele-specific motif or supermotif such that the peptide will bind an HLA molecule and induce a CTL and/or HTL response. Thus, immunogenic peptides of the invention are capable of binding to an appropriate HLA molecule and thereafter inducing a cytotoxic T cell response, or a helper T cell response, to the antigen from which the immunogenic peptide is derived.

[00196] "Heteroclitic analogs" are defined herein as a peptide with increased potency for a specific T cell, as measured by increased responses to a given dose, or by a requirement of lesser amounts to achieve the same response. Advantages of heteroclitic analogs s include that the epitopes can be more potent, or more economical (since a lower amount is required to achieve the same effect). In addition, modified epitopes might overcome antigen-specific T cell unresponsiveness (T cell tolerance).

[00197] "Human Leukocyte Antigen" or "HLA" is a human class I or class II Major

Histocompatibility Complex (MHC) protein {see, e.g., Stites, et al., Immunology, 8th ed., Lange Publishing, Los Altos, Calif. (1994)).

[00198] An "HLA supertype or HLA family," as used herein, describes sets of HLA molecules grouped based on shared peptide-binding specificities. HLA class I molecules that share similar binding affinity for peptides bearing certain amino acid motifs are grouped into such HLA supertypes. The terms HLA superfamily, HLA supertype family, HLA family, and HLA xx-like molecules (where xx denotes a particular HLA type), are synonyms. HLA types, include, for example, HLA-Al, -A2, A3/A11, -A24, - B7, B44.

[00199] As used herein, "high affinity" with respect to HLA class I molecules is defined as binding with an IC_5O, or K_D value, of 50 nM or less; "intermediate affinity" with respect to HLA class I molecules is defined as binding with an IC₅₀ or K_D value of between about 50 and about 500 nM. "High affinity" with respect to binding to HLA class II molecules is defined as binding with an IC₅₀ or KD value of 100 nM or less; "intermediate affinity" with respect to binding to HLA class II molecules is defined as binding with an IC₅₀ or K_D value of between about 100 and about 1000 nM.

[00200] An " IC₅₀" is the concentration of peptide in a binding assay at which 50% inhibition of binding of a reference peptide is observed. Depending on the conditions in which the assays are run (i.e., limiting HLA proteins and labeled peptide concentrations), these values may approximate K_D values.

[00201] The terms "identical" or percent "identity," in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.

[00202] "Introducing" an amino acid residue at a particular position in a multi-epitope construct, e.g., adjacent, at the C-terminal side, to the C-terminus of the epitope, encompasses configuring multiple epitopes such that a desired residue is at a particular position, e.g., adjacent to the epitope, or such that a deleterious residue is not adjacent to the C-terminus of the epitope. The term also includes inserting an amino acid residue, preferably a preferred or intermediate amino acid residue, at a particular position. An amino acid residue can also be introduced into a sequence by substituting one amino acid residue for another. Preferably, such a substitution is made in accordance with analoging principles set forth, e.g., in PCT application number PCT/USOO/19774.

[00203] The phrases "isolated" or "biologically pure" refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment.

[00204] "Link" or "join" refers to any method known in the art for functionally connecting peptides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, and electrostatic bonding.

[00205] The term "directly joined" refers to being joined without anything intervening.

For example, in the case of two peptides being directly joined, one peptide would be joined or bonded to another peptide, as described above, without any sequence, molecule, spacer, linker, etc. intervening between the two peptides.

[00206] The term "indirectly joined" refers to being joined with something intervening.

For example, in the case of two peptides being indirectly joined, one peptide would be joined or bonded to another peptide, as described above, with a sequence, molecule, spacer, linker, etc. intervening between the two peptides.

[00207] "Major Histocompatibility Complex" or "MHC" is a cluster of genes that plays a role in control of the cellular interactions responsible for physiologic immune responses. In humans, the MHC complex is also known as the HLA complex. For a detailed description of the MHC and HLA complexes, see, Paul, Fundamental Immunology, 3rd ed., Raven Press, New York, 1993.

[00208] As used herein, "middle of the peptide" is a position in a peptide that is neither an amino nor a carboxyl terminus.

[00209] A "minimal number of junctional epitopes" as used herein refers to a number of junctional epitopes that is lower than what would be created using random selection criteria.

[00210] The term "motif refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

[00211] A "supermotif" is an amino acid sequence for a peptide that provides binding specificity shared by HLA -molecules encoded by two or more HLA alleles. Preferably, a supermotif-bearing peptide is recognized with high or intermediate affinity (as defined herein) by two or more HLA antigens.

[00212] The term "peptide" is used interchangeably with "oligopeptide" in the present specification to designate a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. The preferred CTL-inducing peptides of the invention are 13 residues or less in length and usually consist of between about 8 and about 11 residues, preferably 9 or 10 residues. The preferred HTL-inducing peptides are less than about 50 residues in length and usually consist of between about 6 and about 30 residues, more usually between about 12 and 25, and often between about 15 and 20 residues.

[00213] The term "CTL epitope" refer to a peptide of defined length that can be from about 8 to about 13 amino acids in length, from about 9 to about 11 amino acids in length, or from about 9 to about 10 amino acids in length, which is recognized by a particular HLA class I molecule.

[00214] The term "HTL epitope" refers to a peptide of defined length that can be from about 6 to about 30 amino acids in length, from about 8 to about 30 amino acids in length, from about 10 to about 30 amino acids, from about 12 to about 30 amino acids in length, from about 6 to about 25 amino acids in length, from about 8 to about 25 amino acids in length, from about 10 to about 25 amino acids, from about 12 to about 25 amino acids in length, from about 6 to about 18 amino acids in length, from about 8 to about 18 amino acids in length, from about 10 to about 18 amino acids, or from about 12 to about 18 amino acids in length, which is recognized by a particular HLA class II molecule.

[00215] A "PanDR binding peptide or pan-DR binding epitope" is a member of a family of molecules that binds more than one HLA class II DR molecule. The pattern that defines this family of molecules can be thought of as an HLA Class II supermotif. For example, PADRE^® binds to most HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses.

[00216] A "negative binding residue" or "deleterious residue" is an amino acid which, if present at certain positions (typically not primary anchor positions) in a peptide epitope, results in decreased binding affinity of the peptide for the peptide's corresponding HLA molecule.

[00217] "Optimizing" refers to increasing the immunogenicity or antigenicity of a multi- epitope construct having at least one epitope pair by sorting epitopes to minimize the occurrence of junctional epitopes, inserting flanking residues that flank the C-terminus or N-terminus of an epitope, and inserting spacer residue to further prevent the occurrence of junctional epitopes or to provide a flanking residue. An increase in immunogenicity or antigenicity of an optimized multi-epitope construct is measured relative to a multi- epitope construct that has not been constructed based on the optimization parameters and is using assays known to those of skill in the art, e.g., assessment of immunogenicity in HLA transgenic mice, ELISPOT, inteferon-gamma release assays, tetramer staining, chromium release assays, and presentation on dendritic cells.

[00218] "Pathogenic virus strain" is used herein to refer to any virus strain that is capable of causing disease; preferably, the virus is on the current World Health Organization (WHO), Centers for Disease Control and Prevention (CDC), Food and Drug Administration (FDA) or other public health authority list of likely circulating viruses; more preferably, the virus has been indicated as one of the three annual viral strains for inclusion in an influenza annual vaccine (i.e., "seasonal strains"). This information is readily available from these agencies, e.g., at http://www.fda.gov/cber/flu/flu.htm or at http://www.who.int/csr/disease/influenza/vaccinerecommendationsl/en/index.html.

[00219] "Pharmaceutically acceptable" refers to a generally non-toxic, inert, and/or physiologically compatible composition.

[00220] "Presented to an HLA Class I processing pathway" means that the multi-epitope constructs are introduced into a cell such that they are largely processed by an HLA Class I processing pathway. Typically, multi-epitope constructs are introduced into the cells using expression vectors that encode the multi-epitope constructs. HLA Class II epitopes that are encoded by such a multi-epitope construct are also presented on Class II molecules, although the mechanism of entry of the epitopes into the Class II processing pathway is not defined.

[00221] A "primary anchor residue" or a "primary MHC anchor" is an amino acid at a specific position along a peptide sequence that is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a "motif for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding grooves of an HLA molecule, with their side chains buried in specific pockets of the binding grooves themselves. In one embodiment, for example, the primary anchor residues of an HLA class I epitope are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 9-residue peptide epitope in accordance with the invention. The primary anchor positions for each motif and supermotif are described, for example, in Tables I and III of PCT/USOO/27766, or PCT/USOO/19774, the disclosure of each which is herein incorporated by reference. Preferred amino acids that can serve as in the anchors for most Class II epitopes consist of M and F in position one and V, M, S, T, A and C in position six. Tolerated amino acids that can occupy these positions for most Class II epitopes consist of L, I, V, W, and Y in position one and P, L and I in position six. The presence of these amino acids in positions one and six in Class II epitopes defines the HLA-DRl, 4, 7 supermotif. The HLA-DR3 binding motif is defined by preferred amino acids from the group of L, I, V, M, F, Y and A in position one and D, E, N, Q, S and T in position four and K, R and H in position six. Other amino acids may be tolerated in these positions but they are not preferred.

[00222] Furthermore, analog peptides can be created by altering the presence or absence of particular residues in these primary anchor positions. Such analogs are used to modulate the binding affinity of a peptide comprising a particular motif or supermotif

[00223] "Promiscuous recognition" occurs where a distinct peptide is recognized by the same T cell clone in the context of various HLA molecules. Promiscuous recognition or binding is synonymous with cross-reactive binding.

[00224] A "protective immune response" refers to a CTL and/or an HTL response to an antigen derived from an infectious agent, which in some way prevents or at least partially arrests disease symptoms, side effects or progression, and clears the infectious agent. The immune response may also include an antibody response that has been facilitated by the stimulation of helper T cells.

[00225] The term "residue" refers to an amino acid or amino acid mimetic incorporated into a peptide or protein by an amide bond or amide bond mimetic.

[00226] A "secondary anchor residue" is an amino acid at a position other than a primary anchor position in a peptide that may influence peptide binding. A secondary anchor residue occurs at a significantly higher frequency amongst bound peptides than would be expected by random distribution of amino acids at one position. The secondary anchor residues are said to occur at "secondary anchor positions." A secondary anchor residue can be identified as a residue which is present at a higher frequency among high or intermediate affinity binding peptides, or a residue otherwise associated with high or intermediate affinity binding. For example, analog peptides can be created by altering the presence or absence of particular residues in these secondary anchor positions. Such analogs are used to finely modulate the binding affinity of a peptide comprising a particular motif or supermotif. The terminology "fixed peptide" is sometimes used to refer to an analog peptide.

[00227] "Sorting epitopes" refers to determining or designing an order of the epitopes in a multi-epitope construct.

[00228] A "spacer" refers to a sequence that is inserted between two epitopes in a multi- epitope construct to prevent the occurrence of junctional epitopes and/or to increase the efficiency of processing. A multi-epitope construct may have one or more spacer nucleic acids. A spacer nucleic acid may flank each epitope nucleic acid in a construct, or the spacer nucleic acid to epitope nucleic acid ratio may be about 2 to 10, about 5 to 10, about 6 to 10, about 7 to 10, about 8 to 10, or about 9 to 10, where a ratio of about 8 to 10 has been determined to yield favorable results for some constructs.

[00229] The spacer nucleic acid may encode one or more amino acids. A spacer nucleic acid flanking a class I HLA epitope in a multi-epitope construct is preferably between one and about eight amino acids in length, between two and eight amino acids in length, between three and eight amino acids in length, between four and eight amino acids in length, between five and eight amino acids in length, between six and eight amino acids in length, or between seven and eight amino acids in length. A spacer nucleic acid flanking a class II HLA epitope in a multi-epitope construct is preferably greater than five, six, seven, or more amino acids in length, and more preferably five or six amino acids in length.

[00230] The number of spacers in a construct, the number of amino acids in a spacer, and the amino acid composition of a spacer can be selected to optimize epitope processing and/or minimize junctional epitopes. It is preferred that spacers are selected by concomitantly optimizing epitope processing and junctional motifs. Suitable amino acids for optimizing epitope processing are described herein. Also, suitable amino acid spacing for minimizing the number of junctional epitopes in a construct are described herein for class I and class II HLAs. For example, spacers flanking class II HLA epitopes preferably include G, P, and/or N residues as these are not generally known to be primary anchor residues (see, e.g., PCT/USOO/19774). A particularly preferred spacer for flanking a class II HLA epitope includes alternating G and P residues, for example, (GP)_n, (PG)_n, (GP)_nG, (PG)_nP, and so forth, where n is an integer between one and ten, preferably two or about two, and where a specific example of such a spacer is GPGPG or PGPGP. A preferred spacer, particularly for class I HLA epitopes, comprises one, two, three or more consecutive alanine (A) residues, optionally preceded by K, N or G.

[00231] In some multi-epitope constructs, it is sufficient that each spacer nucleic acid encodes the same amino acid sequence. In multi-epitope constructs having two spacer nucleic acids encoding the same amino acid sequence, the spacer nucleic acids encoding those spacers may have the same or different nucleotide sequences, where different nucleotide sequences may be preferred to decrease the likelihood of unintended recombination events when the multi-epitope construct is inserted into cells.

[00232] In other multi-epitope constructs, one or more of the spacer nucleic acids may encode different amino acid sequences. While many of the spacer nucleic acids may encode the same amino acid sequence in a multi-epitope construct, one, two, three, four, five or more spacer nucleic acids may encode different amino acid sequences, and it is possible that all of the spacer nucleic acids in a multi-epitope construct encode different amino acid sequences. Spacer nucleic acids may be optimized with respect to the epitope nucleic acids they flank by determining whether a spacer sequence will maximize epitope processing and/or minimize junctional epitopes, as described herein.

[00233] Multi-epitope constructs may be distinguished from one another according to whether the spacers in one construct optimize epitope processing or minimize junctional epitopes over another construct, and preferably, constructs may be distinguished where one construct is concomitantly optimized for epitope processing and junctional epitopes over the other. Computer assisted methods and in vitro and in vivo laboratory methods for determining whether a construct is optimized for epitope processing and junctional motifs are described herein.

[00234] "Synthetic peptide" refers to a peptide that is not naturally occurring, but is man- made using such methods as chemical synthesis or recombinant DNA technology.

[00235] A "TCR contact residue" or "T cell receptor contact residue" is an amino acid residue in an epitope that is understood to be bound by a T cell receptor; these are defined herein as not being any primary MHC anchor. T cell receptor contact residues are defined as the position/positions in the peptide where all analogs tested induce T-cell recognition relative to that induced with a wild type peptide. [00236] The term "homology," as used herein, refers to a degree of complementarity between two nucleotide sequences. The word "identity" may substitute for the word "homology" when a nucleic acid has the same nucleotide sequence as another nucleic acid. Sequence homology and sequence identity can also be determined by hybridization studies under high stringency and/or low stringency, and disclosed herein are nucleic acids that hybridize to the multi-epitope constructs under low stringency or under high stringency. Also, sequence homology and sequence identity can be determined by analyzing sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a nucleic acid is identical or homologous to the multi-epitope constructs disclosed herein. The invention pertains in part to nucleotide sequences having 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the nucleotide sequence of a multi-epitope construct disclosed herein.

[00237] As used herein, the term "stringent conditions" refers to conditions which permit hybridization between nucleotide sequences and the nucleotide sequences of the disclosed multi-epitope constructs. Suitable stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature. For example, hybridization under high stringency conditions could occur in about 50% formamide at about 37°C to 42°C. In particular, hybridization could occur under high stringency conditions at 42°C in 50% formamide, 5 X SSPE, 0.3% SDS, and 200 μg/ml sheared and denatured salmon sperm DNA or at 42⁰C in a solution comprising 50% formamide, 5 X SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at about 65 °C. Hybridization could occur under reduced stringency conditions in about 35% to 25% formamide at about 30°C to 35°C. For example, reduced stringency conditions could occur at 35°C in 35% formamide, 5 X SSPE, 0.3% SDS, and 200 μg/ml sheared and denatured salmon sperm DNA. The temperature range corresponding to a particular level of stringency can be further narrowed by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. Variations on the above ranges and conditions are well known in the art. [00238] In addition -to utilizing hybridization studies to assess sequence identity or sequence homology, known computer programs may be used to determine whether a particular nucleic acid is homologous to a multi-epitope construct disclosed herein. An example of such a program is the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711), and other sequence alignment programs are known in the art and may be utilized for determining whether two or more nucleotide sequences are homologous. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters may be set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[00239] The nomenclature used to describe peptide compounds follows the conventional practice wherein the amino group is presented to the left (the N-terminus) and the carboxyl group to the right (the C-terminus) of each amino acid residue. When amino acid residue positions are referred to in an epitope, they are numbered in an amino to carboxyl direction with position one being the position closest to the amino terminal end of the epitope, or the peptide or protein of which it may be a part. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three-letter or single-letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form for those amino acids having D-forms is represented by a lower case single letter or a lower case three letter symbol. Glycine has no asymmetric carbon atom and is simply referred to as "GIy" or G.

[00240] Symbols for the amino acids are shown below.

[00241] Amino acid "chemical characteristics" are defined as: Aromatic (F, W, Y);

Aliphatic-hydrophobic (L, I, V, M); Small polar (S, T, C); Large polar (Q, N); Acidic (D,

E); Basic (R, H, K); Proline; Alanine; and Glycine. [00242] Acronyms used herein are as follows:

APC: Antigen presenting cell

CD3: Pan T cell marker

CD4: Helper T lymphocyte marker

CD8: Cytotoxic T lymphocyte marker

CFA: Complete Freund's Adjuvant

CTL: Cytotoxic T lymphocytes

DC: Dendritic cells. DC functioned as potent antigen presenting cells by stimulating cytokine release from CTL lines that were specific for a model peptide derived from hepatitis B virus (HBV). In vitro experiments using DC pulsed ex vivo with an HBV peptide epitope have stimulated CTL immune responses in vitro following delivery to naive mice.

DMSO: Dimethylsulfoxide

ELISA: Enzyme-linked immunosorbant assay

E:T: Effectoπtarget ratio

FCS: Fetal calf serum G-CSF: Granulocyte colony-stimulating factor

GM-CSF: Granulocyte-macrophage (monocyte)-colony stimulating factor

HBV: Hepatitis B virus

HLA: Human leukocyte antigen

HLA-DR: Human leukocyte antigen class II

HPLC: High Performance Liquid Chromatography

HTC: Helper T cells

HTL: Helper T Lymphocyte

ID: Identity

IFA: Incomplete Freund's Adjuvant

IFNγ: Interferon gamma

IL-4: Interleukin-4 cytokine

IV: Intravenous

LU₃₀o_/o: Cytotoxic activity required to achieve 30% lysis at a 100:1 (E:T) ratio

MAb: Monoclonal antibody

MLR: Mixed lymphocyte reaction

MNC: Mononuclear cells

PB: Peripheral blood

PBMC: Peripheral blood mononuclear cell

SC: Subcutaneous

S.E.M.: Standard error of the mean

QD: Once a day dosing

TCR: T cell receptor

WBC: White blood cells [00243] In particular embodiments to prevent HTL junctional epitopes, a spacer composed of amino acid residues that do not correspond to any known HLA Class II anchor residue, are used, e.g, alternating G and P residues (a GP spacer) is included between two HTL epitopes. [00244] Another aspect of the invention, (consideration (ii) above) involves the introduction or substitution of particular amino acid residues at positions that flank epitopes, e.g., a position immediately adjacent to the C-terminus of the epitope, thereby generating multi-epitope constructs with enhanced antigenicity and immunogenicity compared to constructs that do not contain the particular residue introduced or substituted at that site, i.e., non-optimized multi-epitope constructs. The methods of optimizing multi-epitope constructs comprise a step of introducing a flanking residue, preferably K, N, G, R, or A at the C+l position of the epitope, i.e., the position immediately adjacent to the C-terminus of the epitope. In an alternative embodiment, residues that contribute to decreased immunogenicity, i.e., negatively charged residues, e.g., D, aliphatic residues (I, L, M, V) or aromatic non-tryptophan residues, are replaced. The flanking residue can be introduced by positioning appropriate epitopes to provide the favorable flanking residue, or by inserting a specific residue.

Eliminating Class I and Class II Junctional Epitopes and Testing for Class II Restricted Responses In Vivo

[00245] As a further element in eliminating junctional epitopes, spacer sequences can be inserted between two epitopes that create a junctional epitope when juxtaposed.

[00246] In one embodiment, to correct the problem of junctional epitopes for HTL epitopes, a spacer of, for example, five amino acids in length is inserted between the two epitopes. The amino acid residues incorporated into such a spacer are preferably those amino acid residues that are not known to be primary anchor residues for any of the HLA Class II binding motifs. Such residues include G, P, and N. hi a preferred embodiment, a spacer with the sequence GPGPG is inserted between two epitopes. Previous work has demonstrated that the GP spacer is particularly effective in disrupting Class II binding interactions (Sette et al, J. Immunol, 143:1268-73 (1989)). All known human Class II binding motifs and the mouse IA^b (the Class II expressed by HLA transgenic mice) do not tolerate either G or P at these main anchor positions, which are spaced four residues apart. This approach virtually guarantees that no Class II restricted epitopes can be formed as junctional epitopes.

[00247] Polypeptides are synthesized incorporating influenza-derived HTL epitopes.

These epitopes are broadly cross-reactive HLA DR binding epitopes. These epitopes will also efficiently bind the murine IA^b Class II molecule.

[00248] Responses against multiple influenza-derived Class II epitopes can be simultaneously induced, and IA^b/DR crossreactivity can be utilized to investigate the immunogenicity of various constructs incorporating HTL epitope candidates. Finally, appropriate spacers can be employed to effectively disrupt Class II junctional epitopes that would otherwise interfere with effective vaccine immunogenicity.

[00249] In the case of Class I restricted responses, one case of a naturally occurring junctional epitope and the consequent inhibition of epitope specific responses has been presented by McMichael and coworkers (Tussey et al, Immunity, 3(l):65-77 (1995)). To address the problem of junctional epitopes for Class I, similar analyses can be performed. For example, a specific computer program is employed to identify potential Class I restricted junctional epitopes, by screening for selected murine motifs and for the most common human Class I HLA A and B motifs.

[00250] Spacer sequences can also similarly be employed to prevent CTL junctional epitopes. Often, very small residues such as A or G are preferred spacer residues. G also occurs relatively infrequently as a preferred primary anchor residue (see, e.g., PCT/USOO/24802) of an HLA Class I binding motif. These spacers can vary in length, e.g., spacer sequences can typically be 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues in length and are sometimes longer. Smaller lengths are often preferred because of physical constraints in producing the multi-epitope construct.

Sorting and Optimization of Multi-Epitope Constructs

[00251] To develop multi-epitope constructs using the invention, the epitopes for inclusion in the multi-epitope construct are sorted and optimized using the parameters defined herein. Sorting and optimization can be performed using a computer or, for fewer numbers of epitopes, not using a computer. Methods of sorting and optimization and disclosed in WO 02/083714, the disclosure of which is herein incorporated by reference.

[00252] Multi-epitope constructs can also be optimized by determining the structure of each construct to be considered. Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al, Molecular Biology of the Cell (3^rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. [00253] Structural predictions such as charge distribution, hydrophobic/hydrophilic region analysis, or folding predictions can be performed using sequence analysis programs known to those of skill in the art, for example, hydrophobic and hydrophilic domains can be identified (see, e.g., Kyte & Doolittle, J. MoI. Biol. 757:105-132 (1982) and Stryer, Biochemistry (3^rd ed. 1988); see also any of a number of Internet based sequence analysis programs, such as those found at dot.imgen.bcm.tmc.edu.

[00254] A three-dimensional structural model of a multi-epitope construct can also be generated. This is generally performed by entering amino acid sequence to be analyzed into the computer system. The amino acid sequence represents the primary sequence or subsequence of the protein, which encodes the structural information of the protein. The three-dimensional structural model of the protein is then generated by the interaction of the computer system, using software known to those of skill in the art.

[00255] The amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the protein of interest. The software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms," and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion. The computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model. The tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure. The user can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g., cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure, hi modeling the tertiary structure, the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like. Those multi-epitope constructs that are most readily accessible to the HLA processing apparatus are then selected.

Assessment of Immunogenicity of Multi-Epitope Vaccines

[00256] The development of multi-epitope constructs represents a unique challenge, because the species-specificity of the peptide binding to MHC. Different MHC types from different species tend to bind different sets of peptides (Rammensee et al, Immunogenetics, 41(4):\18-228 (1995)). As a result, it is not possible to test in regular laboratory animals a construct composed of human epitopes. Alternatives to overcome this limitation are generally available. They include: 1) testing analogous constructs incorporating epitopes restricted by non-human MHC; 2) reliance on control epitopes restricted by non human MHC; 3) reliance on crossreactivity between human and non- human MHC; 4) the use of HLA transgenic animals; and 5) antigenicity assays utilizing human cells in vivo. The following is a brief overview of the development of the technology for analyzing antigenicity and immunogenicity.

Class I HLA Transgenics

[00257] The utility of HLA transgenic mice for the purpose of epitope identification

(Sette et al, J Immunol, 153(12):5586-92 (1994); Wentworth et al, Int Immunol, 8(5):65l-9 (1996); Engelhard et al., J Immunol, 146(4) Λ226-32 (1991); Man et al, Int Immunol, 7(^:597-605 (1995); Shirai et al, J Immunol, 154(6) :2133 -42 (1995)), and vaccine development (ϊshioka et al, J Immunol, 162(7):39l5-25 (1999)) has been established. Most of the published reports have investigated the use of HLA A2.1K^b mice but it should be noted that B*27, and B*3501 mice are also available. Furthermore, HLA A*l l/K^b mice (Alexander et al, J Immunol, 159(10)Al 53-61 (1997)), HLA A24/K^b, HLA B7/K^b and HLA Al/K^b mice have also been generated.

[00258] Data from 38 different potential epitopes was analyzed to determine the level of overlap between the A2.1 -restricted CTL repertoire of A2.1/K^b-transgenic mice and A2.1+humans (Wentworth et al, Eur J Immunol, 26(l):91-\0\ (1996)). In both humans and mice, an MHC peptide binding affinity threshold of approximately 500 nM correlates with the capacity of a peptide to elicit a CTL response in vivo. A high level of concordance between the human data in vivo and mouse data in vivo was observed for 85% of the high-binding peptides, 58% of the intermediate binders, and 83% of the low/negative binders. Similar results were also obtained with HLA Al 1 and HLA B7 transgenic mice (Alexander et al, J Immunol, 159(10) Al 53-βX (1997)). Thus, because of the extensive overlap that exists between T cell receptor repertoires of HLA transgenic mouse and human CTLs, transgenic mice are valuable for assessing immunogenicity of the multi-epitope constructs described herein.

[00259] The different specificities of TAP transport as it relates to HLA Al l mice does not prevent the use of HLA-Al 1 transgenic mice of evaluation of immunogenicity. While both murine and human TAP efficiently transport peptides with an hydrophobic end, only human TAP has been reported to efficiently transport peptides with positively charged C terminal ends, such as the ones bound by A3, Al l and other members of the A3 supertype. This concern does not apply to A2, Al or B7 because both murine and human TAP should be equally capable of transporting peptides bound by A2, B7 or Al . Consistent with this understanding, Vitiello (Vitiello et al, J Exp Med, 173(4): 1007-15 (1991)) and Rotzschke (Rotzschke O, FaIk K., Curr Opin Immunol, 6(l):45-5l (1994)) suggested that processing is similar in mouse and human cells, while Cerundolo (Rotzschke O, FaIk K., Curr Opin Immunol, 6(l):45-5l (1994)) suggested differences in murine versus human cells, both expressing HLA A3 molecules. However, using HLA Al l transgenics, expression of HLA molecules on T and B cells in vivo has been observed, suggesting that the reported unfavorable specificity of murine TAP did not prevent stabilization and transport of Al 1/K^b molecules in vivo (Alexander et al, J Immunol, 159(10)Al 53-61 (1997)). These data are in agreement with the previous observation that peptides with charged C termini could be eluted from murine cells transfected with Al l molecules (Maier et al., Immunogenetics; 40(4):306-& (1994)). Responses in HLA Al 1 mice to complex antigens, such as influenza, and most importantly to All restricted epitopes encoded by multi-epitope constructs (Ishioka et al., J Immunol, 162(7):39\5-25 (1999)) has also been detected. Thus, the TAP issue appears to be of minor concern with transgenic mice. Another issue of potential relevance in the use of HLA transgenic mice is the possible influence of β2 microglobulin on HLA expression and binding specificity. It is well known that human β2 binds both human and mouse MHC with higher affinity and stability than mouse β2 microglobulin (Shields et al, MoI Immunol, 35(14-15):9\9-2% (1998)). It is also well known that more stable complexes of MHC heavy chain and β2 facilitate exogenous loading of MHC Class I (Vitiello et al, Science, 250(4986):\423-6 (1990)). We have examined the potential effect of this variable by generating mice that are double transgenics for HLA/K^b and humanβ2. Expression of human β2 was beneficial in the case HLA B7/K mice, and was beneficial to achieve good expression levels in the case of HLA Al transgenic mice. Accordingly, HLA/K^b and β2 double transgenic mice are currently and routinely bred and utilized by the present inventors. Thus, HLA transgenic mice can be used to model HLA-restricted recognition of five major HLA specificities (namely A2, All, B7, A24 and Al) and transgenic mice for other HLA specificities can be developed as suitable models for evaluation of immunogenicity.

Antigenicity Testing for Class I Epitopes

[00261] Several independent lines of experimentation indicate that the density of Class

I/peptide complexes on the cell surface may correlate with the level of T cell priming. Thus, measuring the levels at which an epitope is generated and presented on an APCs surface provides an avenue to indirectly evaluate the potency of multi-epitope nucleic acid vaccines in human cells in vitro. As a complement to the use of HLA Class I transgenic mice, this approach has the advantage of examining processing in human cells. (Ishioka et al, J Immunol, 162(7):39\5-25 (1999)).

[00262] Several possible approaches to experimentally quantitate processed peptides are available. The amount of peptide on the cell surface can be quantitated by measuring the amount of peptide eluted from the APC surface (Sijts et al, J Immunol, 156(2):6S3-92 (1996); Demotz et al., Nature, 342(6250):6S2-4 (1989)). Alternatively, the number of peptide-MHC complexes can be estimated by measuring the amount of lysis or lymphokine release induced by infected or transfected target cells, and then determining the concentration of peptide necessary to obtain equivalent levels of lysis or lymphokine release (Kageyama et al., J Immunol, 154(2):561-16 (1995)).

[00263] A similar approach has also been used to measure epitope presentation in multi- epitope nucleic acid-transfected cell lines. Specifically, multi-epitope constructs that are immunogenic in HLA transgenic mice are also processed into optimal epitopes by human cells transfected with the same constructs, and the magnitude of the response observed in transgenic mice correlates with the antigenicity observed with the transfected human target cells (Ishioka et al, J Immunol, 162 (7) :39l 5 -25 (1999)).

[00264] Using antigenicity assays, a number of related constructs differing in epitope order or flanking residues can be transfected into APCs, and the impact of the aforementioned variables on epitope presentation can be evaluated. This can be a preferred system for testing where a relatively large number of different constructs need to be evaluated. Although it requires large numbers of epitope-specific CTLs, protocols that allow for the generation of highly sensitive CTL lines (Alexander-Miller et al, Proc Natl Acad Sci USA, 93(9):4lO2-7 (1996)) and also for their expansion to large numbers (Greenberg P. D., Riddell S. R., Science, 285(5427):546-5\ (1999)) have been developed to address this potential problem. [00265] It should also be kept in mind that, if the cell selected for the transfection is not reflective of the cell performing APC function in vivo, misleading results could be obtained. Cells of the B cell lineage, which are known "professional" APCs, are typically employed as transfection recipients. The use of transfected B cells of this type is an accepted practice in the field. Furthermore, a good correlation has already been noted between in vitro data utilizing transfected human B cells and in vivo results utilizing HLA transgenic mice, as described in more detail herein.

Measuring HTL Responses

[00266] In preferred embodiments, vaccine constructs are optimized to induce Class II restricted immune responses. One method of evaluating multi-epitope constructs including Class II epitopes, is to use HLA-DR transgenic mice. Several groups have produced and characterized HLA-DR transgenic mice (Taneja V., David C. S., Immunol Rev, 169:61-19 (1999)).

[00267] An alternative also exists which relies on crossreactivity between certain human

MHC molecules and particular MHC molecules expressed by laboratory animals. Bertoni and colleagues (Bertoni et al, J Immunol, 161 (8) KAAAl '-55 (1998)) have noted that appreciable crossreactivity can be demonstrated between certain HLA Class I supertypes and certain PATR molecules expressed by chimpanzees. Crossreactivity between human and macaques at the level of Class II (Geluk et al., J Exp Med, /77(^:979-87 (1993)) and Class I molecules (Dzuris, et al, J. Immunol, July 1999) has also been noted. Finally, it can also be noted that the motif recognized by human HLA B7 supertype is essentially the same as the one recognized by the murine Class I L^d (Rammensee et al, Immunogenetics, 41(4):\lS-228 (1995)). Of relevance to testing HLA DR restricted epitopes in mice, it has been shown by Wall et al (Wall et al, J. Immunol, 752:4526-36 (1994)) that similarities exist in the motif of DRl and IA^b. We routinely breed our transgenic mice to take advantage of this fortuitous similarity. Furthermore, we have also shown that most of our peptides bind to IA^b, so that we use these mice for the study of CTL and HTL immunogenicity.

Measuring and Quantitating Immune Responses from Clinical Samples

[00268] A crucial element to assess vaccine performance is to evaluate its capacity to induce immune responses in vivo. Analyses of CTL and HTL responses against the immunogen, as well as against common recall antigens are commonly used and are known in the art. Assays employed included chromium release, lymphokine secretion and lymphoproliferation assays.

[00269] More sensitive techniques such as the ELISPOT assay, intracellular cytokine staining, and tetramer staining have become available in the art. It is estimated that these newer methods are 10- to 100-fold more sensitive than the common CTL and HTL assays (Murali-Krishna et al, Immunity, 8(2): 177-87 (1998)), because the traditional methods measure only the subset of T cells that can proliferate in vitro, and may, in fact, be representative of only a fraction of the memory T cell compartment (Ogg G. S., McMichael A. J., Curr Opin Immunol, I0(4):393-6 (1998)). Specifically in the case of HIV, these techniques have been used to measure antigen-specific CTL responses from patients that would have been undetectable with previous techniques (Ogg et al, Science, 279(5359):2\03-6 (1998); Gray et al, J Immunol, 162(3)Λ780-8 (1999); Ogg et al, J Virol, 73(11):9153-6O (1999); Kalams et al, J Viro; 73(8):672\-S (1999); Larsson et al, _v AIDS, 13(7):767-77 (1999); Come et al, J Acquir Immune Defic Syndr Hum Retrovirol, 20(5):442-7 (1999)).

[00270] With relatively few exceptions, direct activity of freshly isolated cells has been difficult to demonstrate by the means of traditional assays (Ogg G. S., McMichael A. J., Curr Opin Immunol, 10(4):393-6 (1998)). However, the increased sensitivity of the newer techniques has allowed investigators to detect responses from cells freshly isolated from infected humans or experimental animals (Murali-Krishna et al, Immunity, 8(2):\77-S7 (1998); Ogg G. S., McMichael A. J., Curr Opin Immunol, 10(4):393-6 (1998)). The availability of these sensitive assays, which do not depend on an in vitro restimulation step, has greatly facilitated the study of CTL function in natural infection and cancer. In contrast, assays utilized as an endpoint to judge effectiveness of experimental vaccines are usually performed in conjunction with one or more in vitro restimulation steps (Ogg G. S., McMichael A. J., Curr Opin Immunol, 10(4):393-6 (1998)). In fact, with few exceptions (Hanke et al, Vaccine, 16(4):426-35 (1998)), freshly isolated Class I-restricted CD8+ T cells have been difficult to demonstrate in response to immunization with experimental vaccines designed to elicit CTL responses. The use of sensitive assays, such as ELISPOT or in situ IFNγ ELISA, has been combined with a restimulation step to achieve maximum sensitivity; MHC tetramers are also used for this purpose.

[00271] MHC tetramers were first described in 1996 by Altaian and colleagues. They produced soluble HLA-A2 Class I molecules which were folded with HlV-specific peptides containing a CTL epitope complexed together into tetramers tagged with fluorescent markers. These are used to label populations of T cells from HIV-infected individuals that recognize the epitope (Ogg G. S., McMichael A. J., Curr Opin Immunol, 10(4):393-6 (1998)). These cells were then quantified by flow cytometry, providing a frequency measurement for the T cells that are specific for the epitope. This technique has become very popular in HFV research as well as in other infectious diseases (Ogg G. S., McMichael A. J., Curr Opin Immunol, J0(4):393-6 (1998); Ogg et al, Science, 279(5359):2\03-6 (1998); Gray et al, J Immunol, 162(3): 1780-8 (1999); Ogg et al, J Virol, 7J(77J:9153-60 (1999); Kalams et al, J Virol, 73(8):6721-8 (1999)). However, HLA polymorphism can limit the general applicability of this technique, in that the tetramer technology relies on defined HLA/peptide combinations. However, it has been shown that a variety of peptides, including HIV-derived peptides, are recognized by peptide-specific CTL lines in the context of different members of the A2, A3 and B7 supertypes (Threlkeld et al, J Immunol, 759(4J: 1648-57 (1997); Bertoni et al, J Clin Invest, 100(3) :503-13 (1997)). Taken together these observations demonstrate that a T cell receptor (TCR) for a given MHC/peptide combination can have detectable affinity for the same peptide presented by a different MHC molecule from the same supertype. [00272] In circumstances in which efficacy of a prophylactic vaccine is primarily correlated with the induction of a long-lasting memory response, restimulation assays can be the most appropriate and sensitive measures to monitor vaccine-induced immunological responses. Conversely, in the case of therapeutic vaccines, the main immunological correlate of activity can be the induction of effector T cell function, most aptly measured by primary assays. Thus, the use of sensitive assays allows for the most appropriate testing strategy for immunological monitoring of vaccine efficacy.

Antigenicity of Multi-Epitope Constructs in Transfected Human APCs

[00273] Antigenicity assays are performed to evaluate epitope processing and presentation in human cells. An episomal vector to efficiently transfect human target cells with multi-epitope nucleic acid vaccines is used to perform such an analysis.

[00274] For example, 221 A2K^b target cells were transfected with an HIV multi-epitope vaccine. The 221 A2K^b target cell expresses the A2K^b gene that is expressed in HLA transgenic mice, but expresses no endogenous Class I (Shimizu Y, DeMars R., J Immunol, 142(9):3320-8 (1989)). These transfected cells are assayed for their capacity to present antigen to CTL lines derived from HLA transgenic mice and specific for various HIV-derived CTL epitopes. To correct for differences in antigen sensitivity of different CTL lines, peptide dose titrations, using untransfected cells as APC, are run in parallel. [00275] These data have several important implications. First, they suggest that different epitopes contained within a given construct may be processed and presented with differential efficiency. Second, they suggest that immunogenicity is proportional to the amount of processed epitope generated. Finally, these results provide an important validation of the use of transgenic mice for the purpose of optimization of multi-epitope vaccines destined for human use.

Methods of Administration

[00276] The invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an expression vector of the invention or a polypeptide derived therefrom. Pharmaceutically acceptable carriers are well known in the art and include aqueous or non-aqueous solutions, suspensions and emulsions, including physiologically buffered saline, alcohol/aqueous solutions or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters, lipids or liposomes.

[00277] A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize the expression vector or increase the absorption of the expression vector. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight polypeptides, antimicrobial agents, inert gases or other stabilizers or excipients. Expression vectors can additionally be complexed with other components such as peptides, polypeptides and carbohydrates. Expression vectors can also be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

[00278] The invention further relates to methods of administering a pharmaceutical composition comprising an expression vector of the invention or a polypeptide derived therefrom to stimulate an immune response. The expression vectors are administered by methods well known in the art as described in, for example, Donnelly et al. {Ann. Rev. Immunol, 75:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997). In one embodiment, the multi-epitope construct is administered as naked nucleic acid.

[00279] A pharmaceutical composition comprising an expression vector of the invention or a polypeptide derived therefrom can be administered to stimulate an immune response in a subject by various routes including, for example, orally, intravaginally, rectally, or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intracisteraally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. Furthermore, the composition can be administered by injection, intubation or topically, the latter of which can be passive, for example, by direct application of an ointment or powder, or active, for example, using a nasal spray or inhalant. An expression vector also can be administered as a topical spray, in which case one component of the composition is an appropriate propellant. The pharmaceutical composition also can be incorporated, if desired, into liposomes, microspheres or other polymer matrices as described in, for example, Feigner et al, U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, VoIs. I to III (2nd ed. 1993). Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[00280] The expression vectors of the invention or a polypeptide derived therefrom can be delivered to the interstitial spaces of tissues of an animal body as described in, for example, Feigner et al, U.S. Pat. Nos. 5,580,859 and 5,703,055. Administration of expression vectors of the invention to muscle is a particularly effective method of administration, including intradermal and subcutaneous injections and transdermal administration. Transdermal administration, such as by iontophoresis, is also an effective method to deliver expression vectors of the invention to muscle. Epidermal administration of expression vectors of the invention can also be employed. Epidermal administration involves mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al, U.S. Pat. No. 5,679,647).

[00281] Other effective methods of administering an expression vector of the invention or a polypeptide derived therefrom to stimulate an immune response include mucosal administration as described in, for example, Carson et al, U.S. Pat. No. 5,679,647. For mucosal administration, the most effective method of administration includes intranasal administration of an appropriate aerosol containing the expression vector and a pharmaceutical composition. Suppositories and topical preparations are also effective for delivery of expression vectors to mucosal tissues of genital, vaginal and ocular sites. Additionally, expression vectors can be complexed to particles and administered by a vaccine gun.

[00282] The dosage to be administered is dependent on the method of administration and will generally be between about 0.1 μg up to about 200 μg. For example, the dosage can be from about 0.05 μg/kg to about 50 mg/kg, in particular about 0.005-5 mg/kg. An effective dose can be determined, for example, by measuring the immune response after administration of an expression vector. For example, the production of antibodies specific for the MHC Class II epitopes or MHC Class I epitopes encoded by the expression vector can be measured by methods well known in the art, including ELISA or other immunological assays, hi addition, the activation of T helper cells or a CTL response can be measured by methods well known in the art including, for example, the uptake of ³H-thymidine to measure T cell activation and the release Of ⁵¹Cr to measure CTL activity.

[00283] The pharmaceutical compositions comprising an expression vector of the invention or a polypeptide derived therefrom can be administered to mammals, particularly humans, for prophylactic or therapeutic purposes. Diseases related to influenza virus infection can be treated or prevented using the expression vectors of the invention.

[00284] In therapeutic applications, the expression vectors of the invention or a polypeptide derived therefrom are administered to an individual already suffering from influenza virus infection or a related disease. Those in the incubation phase or acute phase of the disease can be treated with expression vectors of the invention, including those expressing all universal MHC Class II epitopes, separately or in conjunction with other treatments, as appropriate.

[00285] In therapeutic and prophylactic applications, pharmaceutical compositions comprising expression vectors of the invention or a polypeptide derived therefrom are administered to a patient in an amount sufficient to elicit an effective immune response to an antigen and to ameliorate the signs or symptoms of a disease. The amount of expression vector to administer that is sufficient to ameliorate the signs or symptoms of a disease is termed a therapeutically effective dose. The amount of expression vector sufficient to achieve a therapeutically effective dose will depend on the pharmaceutical composition comprising an expression vector of the invention, the manner of administration, the state and severity of the disease being treated, the weight and general state of health of the patient and the judgment of the prescribing physician.

[00286] The present invention also provides methods for delivering an influenza polypeptide or a fragment, variant, or derivative thereof to a human, which comprise administering to a human one or more of the compositions described herein; such that upon administration of compositions such as those described herein, an influenza polypeptide, or fragment, variant, or derivative thereof is expressed in human cells, in an amount sufficient to generate an immune response to the influenza virus or administering the influenza virus polypeptide or a fragment, variant, or derivative thereof itself to the human in an amount sufficient to generate an immune response.

[00287] The present invention further provides methods for delivering an influenza virus polypeptide or a fragment, variant, or derivative thereof to a human, which comprise administering to a vertebrate one or more of the compositions described herein; such that upon administration of compositions such as those described herein, an immune response is generated in the vertebrate.

[00288] The term "vertebrate" is intended to encompass a singular "vertebrate" as well as plural "vertebrates" and comprises mammals and birds, as well as fish, reptiles, and amphibians.

[00289] The term "mammal" is intended to encompass a singular "mammal" and plural

"mammals," and includes, but is not limited to humans; primates such as apes, monkeys (e.g., owl, squirrel, cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), orangutans, baboons, gibbons, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equines such as horses, donkeys, and zebras, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; ursids such as bears; and others such as rabbits, mice, ferrets, seals, whales, hi particular, the mammal can be a human subject, a food animal or a companion animal.

[00290] The term "bird" is intended to encompass a singular "bird" and plural "birds," and includes, but is not limited to feral water birds such as ducks, geese, terns, shearwaters, and gulls; as well as domestic avian species such as turkeys, chickens, quail, pheasants, geese, and ducks. The term "bird" also encompasses passerine birds such as starlings and budgerigars.

[00291] The present invention further provides a method for generating, enhancing or modulating an immune response to an influenza virus comprising administering to a vertebrate one or more of the compositions described herein. In this method, the compositions may include one or more isolated polynucleotides comprising at least one nucleic acid fragment where the nucleic acid fragment is optionally a fragment of a coding region encoding an influenza virus polypeptide, or a fragment, variant, or derivative thereof. In another embodiment, the compositions may include both a polynucleotide as described above, and also an isolated influenza virus polypeptide, or a fragment, variant, or derivative thereof, wherein the protein is provided as a recombinant protein, in particular, a fusion protein, a purified subunit, a chemically synthesized peptide, viral vector expressing the protein, or in the form of an inactivated influenza virus vaccine. Thus, the latter compositions include both a polynucleotide encoding an influenza virus polypeptide or a fragment, variant, or derivative thereof and an isolated influenza virus polypeptide or a fragment, variant, or derivative thereof. The influenza virus polypeptide or a fragment, variant, or derivative thereof encoded by the polynucleotide of the compositions need not be the same as the isolated influenza virus polypeptide or a fragment, variant, or derivative thereof of the compositions. Compositions to be used according to this method may be univalent, bivalent, trivalent or multivalent.

[00292] The polynucleotides of the compositions may comprise a fragment of a human

(or other vertebrate) coding region encoding a protein of the influenza virus, or a fragment, variant, or derivative thereof. The polynucleotides are incorporated into the cells of the vertebrate in vivo, and an antigenic amount of the influenza virus polypeptide, or fragment, variant, or derivative thereof, is produced in vivo. Upon administration of the composition according to this method, the influenza virus polypeptide or a fragment, variant, or derivative thereof is expressed in the vertebrate in an amount sufficient to elicit an immune response. Such an immune response might be used, for example, to generate antibodies to the influenza virus for use in diagnostic assays or as laboratory reagents, or as therapeutic or preventative vaccines as described herein.

[00293] The present invention further provides a method for generating, enhancing, or modulating a protective and/or therapeutic immune response to influenza virus in a vertebrate, comprising administering to a vertebrate in need of therapeutic and/or preventative immunity one or more of the compositions described herein. In this method, the compositions include one or more polynucleotides comprising at least one nucleic acid fragment, where the nucleic acid fragment is optionally a fragment of a coding region encoding an influenza virus polypeptide, or a fragment, variant, or derivative thereof. In a further embodiment, the composition used in this method includes both an isolated polynucleotide comprising at least one nucleic acid fragment, where the nucleic acid fragment is optionally a fragment of a coding region encoding an influenza virus polypeptide, or a fragment, variant, or derivative thereof; and at least one isolated influenza virus polypeptide, or a fragment, variant, or derivative thereof. Thus, the latter composition includes both an isolated polynucleotide encoding an influenza virus polypeptide or a fragment, variant, or derivative thereof and an isolated influenza virus polypeptide or a fragment, variant, or derivative thereof, for example, a recombinant protein, a purified subunit, viral vector expressing the protein, or an inactivated virus vaccine. Upon administration of the composition according to this method, the influenza virus polypeptide or a fragment, variant, or derivative thereof is expressed in the human in a therapeutically or prophylactically effective amount.

[00294] As used herein, an "immune response" refers to the ability of a vertebrate to elicit an immune reaction to a composition delivered to that vertebrate. Examples of immune responses include an antibody response or a cellular, e.g., cytotoxic T-cell, response. One or more compositions of the present invention may be used to prevent influenza infection in vertebrates, e.g., as a prophylactic vaccine, to establish or enhance immunity to influenza virus in a healthy individual prior to exposure to influenza or contraction of influenza disease, thus preventing the disease or reducing the severity of disease symptoms.

[00295] As mentioned above, compositions of the present invention can be used to prevent influenza virus infection. The term "prevention" refers to the use of one or more compositions of the present invention to generate immunity in a vertebrate which has not yet been exposed to a particular strain of influenza virus, thereby preventing or reducing disease symptoms and death if the vertebrate is later exposed to the particular strain of influenza virus. The methods of the present invention therefore may be referred to as a preventative or prophylactic vaccination. It is not required that any composition of the present invention provide total immunity to influenza or totally cure or eliminate all influenza disease symptoms. As used herein, a "vertebrate in need of preventative immunity" refers to an individual for whom it is desirable to treat, i.e., to prevent, cure, retard, or reduce the severity of influenza disease symptoms, and/or result in no worsening of influenza disease over a specified period of time. Vertebrates to treat and/or vaccinate include humans, apes, monkeys (e.g., owl, squirrel, cebus, rhesus, African green, patas, cynomolgus, and cercopithecus), orangutans, baboons, gibbons, and chimpanzees, dogs, wolves, cats, lions, and tigers, horses, donkeys, zebras, cows, pigs, sheep, deer, giraffes, bears, rabbits, mice, ferrets, seals, whales, ducks, geese, terns, shearwaters, gulls, turkeys, chickens, quail, pheasants, geese, starlings and budgerigars.

[00296] One or more compositions of the present invention are utilized in a "prime boost" regimen. An example of a "prime boost" regimen may be found in Yang, Z. et al. J. Virol. 77:799-803 (2002), which is incorporated herein by reference in its entirety. In these embodiments, one or more polynucleotide vaccine compositions of the present invention are delivered to a vertebrate, thereby priming the immune response of the vertebrate to an influenza virus, and then a second immunogenic composition is utilized as a boost vaccination. One or more compositions of the present invention are used to prime immunity, and then a second immunogenic composition, e.g., a recombinant viral vaccine or vaccines, a different polynucleotide vaccine, or one or more purified subunit isolated influenza virus polypeptides or fragments, variants or derivatives thereof is used to boost the anti-influenza virus immune response.

[00297] In one embodiment, a priming composition and a boosting composition are combined in a single composition or single formulation. For example, a single composition may comprise an isolated influenza virus polypeptide or a fragment, variant, or derivative thereof as the priming component and a polynucleotide encoding an influenza protein as the boosting component. In this embodiment, the compositions may be contained in a single vial where the priming component and boosting component are mixed together. In general, because the peak levels of expression of protein from the polynucleotide does not occur until later {e.g., 7-10 days) after administration, the polynucleotide component may provide a boost to the isolated protein component. Compositions comprising both a priming component and a boosting component are referred to herein as "combinatorial vaccine compositions" or "single formulation heterologous prime-boost vaccine compositions." In addition, the priming composition may be administered before the boosting composition, or even after the boosting composition, if the boosting composition is expected to take longer to act.

[00298] In another embodiment, the priming composition may be administered simultaneously with the boosting composition, but in separate formulations where the priming component and the boosting component are separated.

[00299] The terms "priming" or "primary" and "boost" or "boosting" as used herein may refer to the initial and subsequent immunizations, respectively, i.e., in accordance with the definitions these terms normally have in immunology. However, in certain embodiments, e.g., where the priming component and boosting component are in a single formulation, initial and subsequent immunizations may not be necessary as both the "prime" and the "boost" compositions are administered simultaneously.

[00300] In certain embodiments, one or more compositions of the present invention are delivered to a vertebrate by methods described herein, thereby achieving an effective therapeutic and/or an effective preventative immune response. More specifically, the compositions of the present invention may be administered to any tissue of a vertebrate, including, but not limited to, muscle, skin, brain tissue, lung tissue, liver tissue, spleen tissue, bone marrow tissue, thymus tissue, heart tissue, e.g., myocardium, endocardium, and pericardium, lymph tissue, blood tissue, bone tissue, pancreas tissue, kidney tissue, gall bladder tissue, stomach tissue, intestinal tissue, testicular tissue, ovarian tissue, uterine tissue, vaginal tissue, rectal tissue, nervous system tissue, eye tissue, glandular tissue, tongue tissue, and connective tissue, e.g., cartilage.

[00301] Furthermore, the compositions of the present invention may be administered to any internal cavity of a vertebrate, including, but not limited to, the lungs, the mouth, the nasal cavity, the stomach, the peritoneal cavity, the intestine, any heart chamber, veins, arteries, capillaries, lymphatic cavities, the uterine cavity, the vaginal cavity, the rectal cavity, joint cavities, ventricles in brain, spinal canal in spinal cord, the ocular cavities, the lumen of a duct of a salivary gland or a liver. When the compositions of the present invention is administered to the lumen of a duct of a salivary gland or liver, the desired polypeptide is expressed in the salivary gland and the liver such that the polypeptide is delivered into the blood stream of the vertebrate from each of the salivary gland or the liver. Certain modes for administration to secretory organs of a gastrointestinal system using the salivary gland, liver and pancreas to release a desired polypeptide into the bloodstream is disclosed in U.S. Patent Nos. 5,837,693 and 6,004,944, both of which are incorporated herein by reference in their entireties.

[00302] In certain embodiments, the compositions are administered into embryonated chicken eggs or by intra-muscular injection into the defeathered breast area of chicks as described in Kodihalli S. et al., Vaccine 18:2592-9 (2000), which is incorpρrated herein by reference in its entirety.

[00303] In certain embodiments, the compositions are administered to muscle, either skeletal muscle or cardiac muscle, or to lung tissue. Specific, but non-limiting modes for administration to lung tissue are disclosed in Wheeler, CJ. , et al, Proc. Natl. Acad. Sci. USA 95:11454-11459 (1996), which is incorporated herein by reference in its entirety.

[00304] According to the disclosed methods, compositions of the present invention can be administered by intramuscular (i.m.), subcutaneous (s.c), or intrapulmonary routes. Other suitable routes of administration include, but are not limited to intratracheal, transdermal, intraocular, intranasal, inhalation, intracavity, intravenous (i.v.), intraductal (e.g., into the pancreas) and intraparenchymal (i.e., into any tissue) administration. Transdermal delivery includes, but not limited to intradermal (e.g., into the dermis or epidermis), transdermal (e.g., percutaneous) and transmucosal administration (i.e., into or through skin or mucosal tissue). Intracavity administration includes, but not limited to administration into oral, vaginal, rectal, nasal, peritoneal, or intestinal cavities as well as, intrathecal (i.e., into spinal canal), intraventricular (i.e., into the brain ventricles or the heart ventricles), inraatrial (i.e., into the heart atrium) and sub arachnoid (i.e., into the sub arachnoid spaces of the brain) administration.

[00305] Any mode of administration can be used so long as the mode results in the expression of the desired peptide or protein, in the desired tissue, in an amount sufficient to generate an immune response to influenza virus and/or to generate a prophylactically or therapeutically effective immune response to influenza virus in a human in need of such response. Administration means of the present invention include needle injection, catheter infusion, biolistic injectors, particle accelerators (e.g., "gene guns" or pneumatic "needleless" injectors) Med-E-Jet (Vahlsing, H., et al., J. Immunol. Methods 171:11-22 (1994)), Pigjet (Schrijver, R., et al., Vaccine 15: 1908-1916 (1997)), Biojector (Davis, H., et al, Vaccine 12: 1503-1509 (1994); Gramzinski, R., et al, MoI. Med. 4: 109-118 (1998)), AdvantaJet (Linmayer, L, et al., Diabetes Care 9:294-297 (1986)), Medi-jector (Martins, J., and Roedl, E. J. Occup. Med. 21:821-824 (1979)), gelfoam sponge depots, other commercially available depot materials (e.g., hydrogels), osmotic pumps (e.g., Alza minipumps), oral or suppositorial solid (tablet or pill) pharmaceutical formulations, topical skin creams, and decanting, use of polynucleotide coated suture (Qin, Y., et al., Life Sciences 65: 2193-2203 (1999)) or topical applications during surgery. Certain modes of administration are intramuscular needle-based injection and pulmonary application via catheter infusion. Energy-assisted plasmid delivery (EAPD) methods may also be employed to administer the compositions of the invention. One such method involves the application of brief electrical pulses to injected tissues, a procedure commonly known as electroporation. See generally Mir, L.M. et al., Proc. Natl. Acad. Sci USA 96:4262-7 (1999); Hartikka, J. et al, MoI. Ther. 4:407-15 (2001); Mathiesen, L, Gene Ther. 6:508-14(1999); Rizzuto G. et al., Hum. Gen. Ther. 11:1891-900 (2000). Each of the references cited in this paragraph is incorporated herein by reference in its entirety.

[00306] Determining an effective amount of one or more compositions of the present invention depends upon a number of factors including, for example, the antigen being expressed or administered directly, e.g., HA, NA, NP, Ml or M2, or fragments, e.g., M2e, variants, or derivatives thereof, the age and weight of the subject, the precise condition requiring treatment and its severity, and the route of administration. Based on the above factors, determining the precise amount, number of doses, and timing of doses are within the ordinary skill in the art and will be readily determined by the attending physician or veterinarian.

[00307] Compositions of the present invention may include various salts, excipients, delivery vehicles and/or auxiliary agents as are disclosed, e.g., in U.S. Patent Application Publication No. 2002/0019358, published February 14, 2002, which is incorporated herein by reference in its entirety.

[00308] Furthermore, compositions of the present invention may include one or more transfection facilitating compounds that facilitate delivery of polynucleotides to the interior of a cell, and/or to a desired location within a cell. As used herein, the terms "transfection facilitating compound," "transfection facilitating agent," and "transfection facilitating material" are synonymous, and may be used interchangeably. It should be noted that certain transfection facilitating compounds may also be "adjuvants" as described infra, i.e., in addition to facilitating delivery of polynucleotides to the interior of a cell, the compound acts to alter or increase the immune response to the antigen encoded by that polynucleotide. Examples of the transfection facilitating compounds include, but are not limited to inorganic materials such as calcium phosphate, alum (aluminum sulfate), and gold particles (e.g., "powder" type delivery vehicles); peptides that are, for example, cationic, intercell targeting (for selective delivery to certain cell types), intracell targeting (for nuclear localization or endosomal escape), and ampipathic (helix forming or pore forming); proteins that are, for example, basic (e.g., positively charged) such as histones, targeting (e.g., asialoprotein), viral (e.g., Sendai virus coat protein), and pore-forming; lipids that are, for example, cationic (e.g., DMPJE, DOSPA, DC-Choi), basic (e.g., steryl amine), neutral (e.g., cholesterol), anionic (e.g., phosphatidyl serine), and zwitterionic (e.g., DOPE, DOPC); and polymers such as dendrimers, star-polymers, "homogenous" poly-amino acids (e.g., poly-lysine, poly- arginine), "heterogeneous" poly-amino acids (e.g., mixtures of lysine & glycine), copolymers, polyvinylpyrrolidinone (PVP), poloxamers (e.g. CRL 1005) and polyethylene glycol (PEG). A transfection facilitating material can be used alone or in combination with one or more other transfection facilitating materials. Two or more transfection facilitating materials can be combined by chemical bonding (e.g., covalent and ionic such as in lipidated polylysine, PEGylated polylysine) (Toncheva, et al., Biochim. Biophys. Acta 1380(3):354-368 (1988)), mechanical mixing (e.g., free moving materials in liquid or solid phase such as "polylysine + cationic lipids") (Gao and Huang, Biochemistry 35:1027-1036 (1996); Trubetskoy, et al., Biochem. Biophys. Acta 1131 :311-313 (1992)), and aggregation (e.g., co-precipitation, gel forming such as in cationic lipids + poly-lactide, and polylysine + gelatin). Each of the references cited in this paragraph is incorporated herein by reference in its entirety.

EXAMPLES

Materials and Methods

[00309] The following materials and methods apply generally to all the examples disclosed herein. Specific materials and methods are disclosed in each example, as necessary.

[00310] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology (including PCR), vaccinology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al, ed., Cold Spring Harbor Laboratory Press: (1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N. Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, VoIs. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); and in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989). Each of the references cited in this paragraph is incorporated herein by reference in its entirety.

In situ IFNγ ELISA

[00311] An in situ IFNγ ELISA assay has been developed and optimized for both freshly isolated and peptide-restimulated splenocytes {see, e.g., McKinney et al., J. Immunol. Meth. 237 (l-2):105-l 17 (2000)). This assay is based on the ELISPOT assay, but utilizes a soluble chromagen, making it readily adaptable to high-throughput analysis. In both the primary and restimulation assays, this technique is more sensitive than either a traditional supernatant ELISA or the 51 ^r-release assay, in that responses are observed in the in situ ELISA that are not detectable in these other assays. On a per cell basis, the sensitivity of the in situ ELISA is approximately one IFNγ secreting cell/ 10⁴ plated cells.

[00312] 96-well ELISA plates are coated with anti-IFNγ (rat anti-mouse IFN-α-MAb,

Clone R4-6A2, Pharmingen) overnight at 4 °C, and then blocked for 2 hours at room temperature with 10% FBS in PBS. Serially diluted primary splenocytes or CTLs are cultured for 20 hours with peptide and 10⁵ Jurkat A2.1/Kb cells/well at 37° C with 5% CO2. The following day, the cells are washed out and the amount of IFNγ that had been secreted into the wells is detected in a sandwich ELISA, using biotinylated α-IFNγ (rat anti-mouse IFNγ mAb, Clone XMG 1.2, Pharmingen) to detect the secreted IFNγ. HRP- coupled streptavidin (Zymed) and TMB (ImmunoPure™ TMB Substrate Kit, Pierce) are used according to the manufacturer's directions for color development. The absorbance is read at 450 nm on a Labsystems Multiskan RC ELISA plate reader. In situ IFNγ ELISA data is evaluated in secretory units (SU), based on the number of cells that secrete 100 pg of IFNγ in response to a particular peptide, corrected for the background amount of EFN in the absence of peptide.

ELISPOT assay

[00313] The ELISPOT assay quantifies the frequency of T cells specific for a given peptide by measuring the capacity of individual cells to be induced to produce and release specific lymphokines, usually IFNγ. The increased sensitivity of the ELISPOT assay has allowed investigators to detect responses from cells freshly isolated from infected humans or experimental animals (Murali-Krishna et al., Immunity, 5(2): 177-87 (1998); Ogg et al., Science, 279(5359):2103-6 (1998)). The ELISPOT assays are conducted as described above for the IFNγ ELISA until the final steps, where ExtrAvidin-AP (Sigma, 1:500. dilution) is used in place of HRP-streptavidin. Color is developed using the substrate 5 -BCIP (BioRad) according to the manufacturer's directions. Spots are counted using a phase contrast microscope. Alternatively, spots are counted utilizing the Zeiss KS ELISPOT reader. In this case the BCIP/NBT substrate is used.

[00314] The ELISPOT assay is routinely utilized to quantitate immune responses. The spots can be manually counted, however, in a preferred mode, a KS ELISPOT reader from Zeiss, a microscope-based system with software specifically designed to recognize and count spots is used.

Hemagglutination Inhibition (HAI) Assays

[00315] Preimmune and postimmune mouse sera were treated with receptor-destroying enzyme (RDE). HAI antibodies were measured against influenza rgA/Vietnam/1203/2004 x A/PR/8/34 influenza (H5N1) vaccine virus. Four HA units of virus were incubated with serial dilutions of RDE-treated mouse sera for at least 30 minutes at room temperature followed by a 30 minute incubation with 0.5% horse erythrocytes. The HAI titer was recorded as the reciprocal of the highest dilution of antisera which inhibits the agglutination of horse erythrocytes.

Viral Micro Neutralization Assays

[00316] Influenza vaccine virus rgA/Vietnam/1203/2004 x A/PR/8/34 (H5N1) and diluted RDE-treated mouse sera were incubated together at room temperature for 1 hour. The mixture was titrated on monolayers of Madin-Darby canine kidney (MDCK) cells grown in 96-well tissue culture plates. Plates were incubated for 3 days at 37°C in 5% CO₂. At the end of 3 days, the presence of cytopathic effects on cell monolayers was evaluated. Neutralization titers were expressed as the reciprocal of the antibody dilution that completely inhibited virus infectivity in 50% of quadruplicate cultures Mice, Immunizations and Cell Cultures

[00317] The derivation of the HLA-A2.1/K^b (Vitiello et al, J Exp Med, 173(4): 1007-15

(1991)) and Al lK^b (Alexander et al, J Immunol, 75P(10):4753-61 (1997)) transgenic mice used in this study has been described. HLA B7 K^b, HLA Al/K^b and HLA A24/K^b transgenic mice are available. HLA DR4 transgenic mice are obtained from C. David (Mayo Clinic) or purchased from Taconic. Non-transgenic H-2 mice are purchased from Charles River Laboratories or other commercial vendors. Immunizations are performed as described in (Ishioka et al., J Immunol, 162(i):39l5-25 (1999)). All cells are grown in culture medium consisting of RPMI 1640 medium with HEPES (Gibco Life Technologies) supplemented with 10% FBS, 4 mM L-glutamine, 50 μM 2-ME, 0.5 mM sodium pyruvate, 100 μg/ml streptomycin and 100 U/ml penicillin.

[00318] The natural crossreactivity between HLA-DR and IA^b can also be exploited to test HTL responses. This evaluation provides an assessment of the antigenicity and immunogenicity of multi-epitope constructs.

Example 1 : Identification of conserved HLA Class I- and Class II- restricted peptides derived from influenza subtypes using established motif search algorithms and

HLA-peptide binding assays

[00319] To identify epitopes useful for vaccine design, a multidisciplinary approach was used based initially on amino acid motif searching of viral sequences to identify potential HLA Class I and Class PI motifs (see Tables 1-49). This was followed by high throughput synthetic peptide binding assays using purified HLA molecules to determine affinity and breadth of epitope peptide binding.

[00320] Selection of influenza virus strains with potential to initiate pandemics: Influenza virus strains for this study were selected on the basis of host diversity (avian, swine, human), agents of past pandemics (HlNl, H2N2, H3N2) and capacity to cause zoonotic influenza infections of man (H5N1, HlNl, H7N7, H9N2). Examples of selected strains are shown below.

[00321] Algorithm motif searches: Motif search algorithms were validated for the most common HLA Class I alleles and HLA Class II alleles and were focused on the HLA- Al, -A2, -A3/11, -A24, -B7, -B44, -DRl and -DR3 supertypes in order to attain virtually 100% population coverage. The selected influenza viral sequences were scanned for motif positive amino acid sequences using the motif definitions. The peptides specific for HLA-Al, -A2, -A3/11, -A24, -B7, -B44, -DRl and -DR3 supertypes are produced as synthetic peptides.

(a) Presence of this symbol (■) indicates that the gene sequence is available; (b) numerous cases of avian-to-human transmission and fatalities caused by H5N1; (c) The 1968 pandemic was due to a H3N2 virus; (d) The 1957 pandemic was due to H2N2 virus; (e) Classical swine HlNl virus strain; (f) Isolated from a fatal human case.

[00322] Peptide synthesis: The Class I and Class II peptides were synthesized initially as crude peptides from Mimotopes (Minneapolis, MN/Clayton, Victoria, Australia) or Pepscan Systems B.V. (Lelystad, Netherlands). These peptides were supplied in small amounts and were typically only 50-70% pure. Larger quantities of selected peptides were synthesized, when needed, using an Applied Biosystems (Foster City, CA) 430A peptide synthesizer and fluronylmethyloxy carbonyl (F-moc) solid phase methods. Peptides synthesized were typically purified to >95% homogeneity by reverse phase HPLC.

[00323] In vitro HLA-peptide epitope binding assays: High affinity binding of epitope peptides to HLA molecules is required for immune recognition and has proved to be one of the most highly predictive approaches for identifying epitopes. Capture assays based on the use of the TopCount benchtop microplate scintillation counter (Packard Instruments) allow the high throughput, sensitivity and compatibility with data automation platforms. [00324] HLA Class I and II purification: The binding assay requires the use of purified

HLA Class I and II molecules. A large number of different types of cells are available including EBV-transformed homozygous human B cell lines, mouse B cell lymphomas or mastocytomas, transfected fibroblasts or single MHC allele transfected 721.221 lines. HLA molecules were purified from cell lysates using monoclonal antibody-based affinity chromatography.

[00325] Measurement of peptide binding to HLA molecules and data analysis: The binding assay utilized is a competitive system that is based on the use of known ¹²⁵I radiolabeled peptide ligands¹¹². To determine the IC₅₀ of peptide binding, the concentration of test peptide yielding 50% inhibition of the binding of the radiolabeled peptide was calculated. Typical test concentrations ranged from 120 μg/ml-120 pg/ml. Under the conditions utilized, the measured IC₅₀ values were reasonable approximations of the Kd values.

[00326] Epitopes that are naturally processed and presented to the immune system using peptides were identified as high affinity binders to HLA molecules and peripheral blood mononuclear cells (PBMC) from normal human donors and HLA transgenic mice. It was necessary to address epitope immunogenicity because not all motif positive peptides are immunogenic nor is it likely that all epitopes are generated equally during infection. Two methods to document epitope immunogenicity and utility were used; 1) in vitro assays using PBMC from normal donors and 2) immunization studies with HLA transgenic mice. Recognition of epitope peptides by human PBMC in a recall assay was the most direct method to verify the authenticity of an epitope because responses demonstrated that the epitope was generated as the course of natural infection and that the needed T-cell receptor (TCR) repertoire exists. Finally, the HLA transgenic mouse is ' well suited for testing vaccine constructs because the proteosome processing preferences and TCR repertoires of mice overlap significantly with humans.

[00327] Assay for recall memory influenza responses using human PBMC: Based on preliminary data presented, past studies⁴⁴, and those of others ^42>43>45 ₅ responses to multiple epitopes were expected because the selection process was for immunologically conserved epitopes. The assays detecting IFN-γ were performed as described for Figure 1. Since CTL contribute to influenza viral clearance by releasing perforin and granzymes from cytoplasmic granules, measurement of these factors may also be performed by ELISPOT analysis. Manufacture's (Mabtech) instructions are followed to perform these additional assays. The assays are specifically based on detection of perforin and granzymes from human PBMC.

[00328] It has been demonstrated that CD4⁺ cells can promote survival to a lethal dose of influenza infection. The mechanisms that may be involved are several including their classic contribution as helpers during the generation of flu-specific CD8⁺ CTL and antibody producing B cells. Potentially, CD4⁺ cells following influenza infection may have an effector function and directly mediate viral clearance by EFN-γ-dependent mechanisms and/or by direct cytolytic effects on infected cells. Accordingly, HTL activity was measured as a function of IFN-γ secretion by CD4⁺ T-lymphocytes, again using an ELISPOT assay as described. Depending on the results obtained using IFN-γ as a readout, IL-2 or TNF-α may also be assayed using an ELISPOT format.

[00329] A collection of positive and control peptides for each supertype was required to ensure the specificity of the influenza-specific responses. Defined epitopes from various pathogens, generally HIV, HBV, HCV and Plasmodium falciparum were used as negative controls when the donors had not been exposed. Positive control peptides were usually derived from HCMV, EBV, and influenza. Negative and positive control peptides for each supertype were identified from previous studies and the literature.

[00330] Immunogenicity testing of CTL, HTL and B cell epitopes in HLA transgenic and non transgenic mice: Large colonies of HLA-A2, Al 1 and B7 transgenic mice have been established and HLA-A24 and Al mice have been generated^76"78. HLA-DR4 transgenic mice from Taconic, a commercial source were also utilized. Additionally, mice of the b haplotype, e.g., C57B1/6 were utilized to evaluate the immunogenicity of HLA-DR-restricted peptides⁶⁷'¹³⁹. The rationale for using b haplotype mice was based on the observation that the motifs recognized by DR alleles often cross-react on murine class II alleles. Immunogenicity of test epitopes were generally accomplished by immunizing mice with pools of peptides (5-10) emulsified in IFA (for CTL) and CFA (for HTL) followed by in vitro testing of splenocytes 14 days later for epitope-specific T lymphocyte responses.

Example 2: Identification of CTL and HTL epitopes for influenza virus vaccine inclusion

[00331] A clear understanding of how T-lymphocytes recognize antigen has emerged over the past decade. It is now well established that small fragments of protein antigens are generated, defined as peptide epitopes, which bind to MHC molecules expressed on the cell surface. These epitope-MHC complexes represent the ligands recognized by T- lymphocytes through the function of T-cell receptors (TCR)⁸¹'⁸². The main anchor residues of peptides that bind to HLA Class I molecules typically occur at position two and the carboxyl terminus of peptides 8-11 amino acids in length^82"93. Amino acids at other positions can contribute to peptide-MHC binding affinity; these sites are commonly referred to as secondary anchors⁹⁴. The analysis of data on both primary and secondary anchors led to the definition of statistically based algorithms, generally referred to as polynomial algorithms, for estimating the likelihood that peptides can bind to HLA Class I molecules. This approach is referred to as a matrix-based method. In addition to the matrix-based method, other computer-based approaches for predicting epitopes have been developed. These include variations of the motif scanning and matrix approaches ^{5" 98} neural networks⁹⁹ and threading algorithms¹⁰⁰. All of these methods function comparably¹⁰¹'¹⁰² and regardless of the method used to predict epitopes, laboratory studies to document epitope peptide binding to HLA molecules and immunogenicity studies are needed to document the authenticity of predicted epitopes.

[00332] Motifs for different HLA molecules were found to be similar and this lead to the definition of HLA supertypes¹⁰³. The biological effect of this supertype relationship was first demonstrated for HIV-I epitopes in a study where the HLA- A3 and -Al 1 repertoires were demonstrated to be overlapping, not only with each other but also with HLA- A31 , - A33 and -A*6801¹⁰⁴'¹⁰⁵. This binding specificity was defined as the HLA- A3 supertype. A significant overlap in peptide binding repertoires was also demonstrated amongst several serologically distant HLA-B alleles¹⁰⁶'¹⁰⁷, and multiple HLA-A2 alleles¹⁰⁸'¹⁰⁹, resulting in the definition of the HLA-B7 and HLA-A2 supertype families. A large fraction of HLA Class I molecules can be classified into a relatively few supertypes, each characterized by largely overlapping peptide binding repertoires and consensus structures of the main peptide binding pockets. Recognition of epitopes by CTL in supertype manner has been demonstrated to occur naturally in infectious diseases and cancer¹⁰⁴'^{110" 115}. Each of the known HLA Class I supertypes includes a relatively common set of HLA alleles and due to this, the use of these supertype relationships when identifying epitopes allows for the selection of those most likely to be targets for the immune system in a genetically diverse population.

[00333] A similar approach is utilized to identify potential HTL epitopes, focusing on the identification of peptides that bind to Class II MHC molecules. There exists a significantly higher level of variation in the motif definition and peptide length, since binding to Class II molecules is generally more promiscuous. However, highly predictive peptide binding motifs for the major supertypes, HLA-DRl and HLA-DR3, which include most of the common HLA-DR types have been identified¹¹⁶. These motifs have been used to identify HLA-DR-restricted epitopes from several viruses.

[00334] Epitope predictions are useful but a significant number of motif-positive peptides identified using predictive algorithms will fail to bind with high affinity to MHC molecules. Thus, it is logical to increase the accuracy of the identification process using laboratory assays to directly measure the affinity of the binding between peptides and MHC molecules '' . Epitope peptide binding assays are based on the use of purified HLA-A, -B or -DR molecules and radio-labeled peptides with known binding affinity. A library of > 100 different HLA specificities, in the purified form, has been developed and can be utilized. To determine the binding affinity of an unknown peptide, increasing concentrations are allowed to compete with the known radio-labeled peptide for binding to the HLA molecule. The assay endpoint is based on the ratio of bound and unbound radio-labeled peptide and actual binding affinities can be calculated. Binding affinity threshold values, based on IC₅₀ values, of < 500 nM for CTL epitopes and <1 μM for HTL epitopes are routinely utilized, for initial selection of epitopes, since peptides binding with these affinity levels are most likely to be immunogenic ^66>117.

[00335] The goal of epitope identification is to assemble sufficient numbers of epitopes for vaccine development based on a need for sufficient population coverage. Potential population coverage was calculated using gene frequencies for HLA-A and -B alleles. Total potential supertype and/or population coverage was calculated from the sum gene frequencies of corresponding alleles and subsequently converted to phenotypic frequencies. In summary, population coverage is estimated using the distribution data on all HLA types (generally 5 for each supertype) in the world. HLA- A2, -A3/A11, and - B7 are very common so using these supertypes dictates high population coverage of > 90%. 100% population coverage can be approximated by adding HLA-Al and -A24 restricted epitopes. In the case of class II, > 95% population coverage can be achieved by considering epitopes representative of the DRl, and DR3 supermotifs.

[00336] Epitopes from several disease pathogens including Plasmodium falciparium,

HIV, HBV, HCV and HPV for the goal of vaccine development have been identified. Epitope identification is presented herewith for the influenza virus. Viral protein amino acid sequences were scanned using algorithm-based computer programs for the presence of class I-restricted HLA-Al, -A2 -A3/A11, -A24, -B7 -B44 and class II-restricted DRl and DR3 supermotif viral conserved peptide supertype sequences. Critical parameters used to identify the peptide sequences were conservancy of peptide sequence amongst divergent influenza subtypes and a predicted binding affinity IC^5OnM of <100 to the prototypic MHC allele representing the supertype, i.e., HLA-A*0101 (Al), HLA- A*0201 (A2), HLA-A* 1101 (A3/A11), HLA-A*2402 (A24), HLA-B*0702 (B7) and HLA-B*4002 (B44). For example, in the case of peptides restricted by the A2 supertype, 68 motif positive peptides were identified according to the methods described in Example 1 (see Table 15). However, only about 40 peptides were predicted to have strain sequence conservancy and to bind with high affinity (see Table 16). This number of peptides was further reduced by limiting to 3 peptides at most per influenza virus protein, (see Table 17).

[00337] Peptides restricted by the other Class I supertypes HLA-A3/A11, -A24, -B7, -

B44, and -Al, identified according to methods described above, are listed in Tables 1, 4, 7, 9, and 12, respectively. HLA-A3/A11, -A24, -B7, -B44, and -Al peptides predicted to be conserved and to bind with high affinity are listed in Tables 2, 5, 8, 10, and 13, respectively. In the case of HLA-A3/A11, -A24, -B44, and -Al, further restrictions limiting to a specific number of peptides at most per influenza virus protein, reduced the number of peptides to those listed in Tables, 3, 6, 11 and 14. The number of peptides identified for each supertype and protein are shown below.

[00338] PIC analysis of conserved influenza sequences:

[00339] A total of about 450 Class I-restricted peptide sequences were identified with a relatively high number selected from NP, PA, PBl and PB2 proteins. An intermediate number of sequences were identified from Ml, NSl, NS2 and the least number of sequences were identified from the HA and M2 proteins.

[00340] A total of about 1500 Class II-restricted peptide sequences were identified that were specific for the DRl and DR3 supertypes (see Tables 18-49). The DRl and DR3 peptides in Tables 18-49 are organized based on the influenza virus proteins from which each peptide is derived. Tables 20, 22, 24, 26, 28, 30, 32, 35, 37, 39, 41, and 46 list those DRl and DR3 peptides predicted to be conserved and to bind with high affinity from influenza virus proteins NP, NSl, NS2, PA, PBl, PB2, HA, Ml, M2, NA and NA, respectively. There are two main contributors to the number of peptides identified, size of the protein and amino acid mutations occurring within the protein. As might be expected, numerous DRl supertype peptide sequences were identified from the HA and NA proteins that were subtype specific. However, relatively very little sequence conservancy was observed amongst the subtypes. DRl peptides from the NA and HA protein specific to influenza strain A/Viet Nam/ 1203/04 were identified and are listed in Tables 18 and 33, respectively. DR3 peptides from the NA and HA protein specific to influenza strain A/Viet Nam/1203/04 were identified and are listed in Tables 42 and 47, respectively. Finally, a set of DR3 peptides was identified and is listed in Table 43 with the corresponding preferred subset listed in Table 44. Additionally a set of DR peptides is listed in Table 48, with the corresponding preferred subset of these peptides listed in Table 49.

[00341] 40 HLA-A2-restricted peptide sequences were synthesized and were evaluated for their binding capacity to purified MHC molecules. The 40 peptides were highly degenerate with exhibited binding of high (<50 IC₅₀I1M) or intermediate (50-500 IC_5OnM) affinity to multiple MHC alleles within the supertype. As expected, the 2 identified HA- and NA-derived peptides had relatively lower sequence conservancy amongst influenza subtypes, in the range of 38-50%. However, the majority of the other identified peptide sequences were highly conserved.

[00342] The 40 peptides exhibiting high binding capacity and sequence conservancy were next evaluated for their capacity to induce influenza-specific recall responses. Human donor PBMCs were cultured in the presence of a pool of peptides, generally 9-10 peptides per pool. Following expansion of the T cells for 1 week, CD8+ cells were purified and influenza peptide-specific responses were measured using peptide coated HLA- A2.1 transfected target cells and an IFN-γ ELISPOT assay. As shown in Figure 1, 19 of the 40 peptides induced measurable responses in the range of 10-6,000 spot forming cells (SFC) per 1 x 10⁶ CD8⁺ cells. Two of the peptides, NS2 173 and PB2 193, were relatively less immunogenic inducing SFC of only 10. However, the remaining 17 peptides induced responses greater than 100 SFC. Four of the peptides (Ml 3, NP 458, PBl 87, and PB2 446 version 1-histidine at position 8) were active in 3 donors, 9 peptides (NA 360, HA 447, Ml 58, NSl 14, PA 283, PBl 83, PBl 413, PBl 501, and PB2 446 version 2-proline at position 8) were active in 2 donors and the remaining 7 peptides were recognized by 1 donor (NA 128, NP 275, NS2 173, PA70, PA 335, PB2 193, and PB2 630). Control peptides were also used to validate the specificity of influenza responses (data not shown). Three positive control peptides were used in each assay, EBV bmlfl 259, CMV pp65 495 and influenza Ml 58 which induced recall responses in all cases in the range of 200-10,000 SFCl 18. Known HLA-A2.1 -restricted epitopes were used as negative control peptides, HBVenv 183, HBVcore 18, HIV env 134, Plasmodium falciparium (Pf)expl 83, Pfexpl 2 and Pfexpl 91. It was assumed that there would be absence of recall responses specific for the Hepatitis, HIV and malaria- derived peptides. However, it was observed that Donor 638 responded to 4 of the 7 peptides with responses in the range of 40-100 SFC for 3 peptides. Therefore, additional donors are included in the analysis to be confident that the final selection of vaccine candidate epitopes is based on a sufficient number of donors. At least 5 donors are being utilized to identify epitopes that are immunogenic. It should be noted that two of the 19 immunogenic influenza peptides have been previously described to induce recall responses in humans, Ml 58 and PBl 413. The 19 epitope sequences were aligned with a collection, 12 to 16, of various influenza strains which have the potential to initiate pandemics. Eight epitopes were a perfect match, HA 360, Ml 3, PA 283, PBl 83, PBl 87, PBl 413, PBl 501, and therefore would be considered as potential vaccine epitope candidates. Seven epitopes, HA 447, Ml 58, NP 458, PA 70, PA 335, PB2 630, PB2 446, have conservative amino acid changes at either MHC anchor positions or at TCR contact positions. These epitopes are also considered to be potential vaccine candidates. However, addition evaluation would be required to determine whether an immune responses specific for the vaccine epitope would also recognize target cells presenting the variant sequence. Four epitopes, NA 128, NP 275, PB2 193, have mutations considered to diminish binding to the MHC molecule and are not viable vaccine candidate epitopes. Examples of epitope sequence alignments are shown below. The PB2 630 epitope is considered a good vaccine candidate since its sequence is altered in only 1 of 13 virus strains, A/Swine/Wisconsin/464/98, with a conservative threonine to alanine substitution at a non-MHC anchor position. Similarly, PB2 446 has substitutions at position 8, a non- MHC anchor, of either serine or histidine for proline. In the PB2 446 case, epitopes with proline and histidine at position 8 were evaluated for the capacity to induce a recall response. As shown in Figure 1, both versions induced a significant recall response suggesting that PB2 446 would be a good vaccine candidate. However, it should be noted that the virus strain A/Chicken/Hong Kong/G9/97 has a non-preferred lysine at a primary MHC anchor position. An immune response would not be generated against this strain. In the case of PB2 193, lysine or arginine at position 2 would abrogate peptide binding to MHC and render this vaccine epitope ineffective against 5 of the 13 strains depicted. Therefore, PB2 193 would not be considered a vaccine candidate.

[00344] In summary, 15 HLA- A2 -restricted epitopes, based on the studies described above, have been identified for potential vaccine inclusion.

[00345] The same process is applied to the collection of the additional Class I and II- restricted peptides

[00346] Following candidate epitope selection, the design of multi-epitope constructs is undertaken. A representative multi-epitope construct is shown in Figure 6. The order of epitopes and amino acid spacers used between epitopes was determined to promote optimum processing and presentation of the vaccine epitopes. The peptide binding data of each of the epitopes within this representative construct is shown in Table 50.

Example 3: Design and development of multi-epitope vaccines

[00347] Immunogenicity of multi-peptide epitope in adjuvant vaccines: Several vaccine delivery methods amenable for use with epitopes. Synthetic peptides representing CTL or HTL epitopes derived from HIV-I have been tested in clinical trials delivered in the high quality Incomplete Freund's Adjuvant¹¹⁹'¹²⁰ or as lipidated peptides¹²¹. Phase I cancer clinical trial with 16 patients suffering from non-small cell lung carcinoma (stage Ilb/IIIa) and colon (stage III) cancer have been initiated. The cancer vaccine is based on 9 CTL epitopes derived from carcinoembryonic antigen (CEA), MAGE 2/3, p53 and HER-2/neu tumor-associated antigens (TAA). All 9 epitopes displayed high HLA-A2 supertype binding affinity and immunogenicity in human primary in vitro induction assay and in in vivo HLA-A2 transgenic mice. The CTL epitopes together with a previously described universal HTL epitope, PADRE, were emulsified in Montanide ISA51 adjuvant. Patients received 6 vaccine treatments at 3 week intervals, at a dose of 0.5 mg/epitope. CTL responses in the peripheral blood of patients were measured using a validated IFN-γ ELISPOT assay. Fifty percent of the patients treated with the vaccine demonstrated CTL responses to at least 5 of the vaccine epitopes at the week 9 and/or 18 week time-points. As an example, shown in Figure 2 patient 604, significant responses were induced in the range of 50-100 SFC/5 x 10⁴ cells for 6 of the 9 vaccine CTL epitopes at the 9 week time-point. These responses were subsequently boosted with responses measured in the range of 200-300 SFC at the 18 week time-point. No responses specific for the vaccine were measured pre-vaccination.

[00348] The influenza virus multi-epitope vaccine is formulated in various test adjuvants as described above. Other vaccine delivery formats are also utilized including DNA, AlphaVax viral vaccines and virosomes, and in particular IRIVs.

[00349] Immunogenicity of multi-epitope based DNA vaccines: Efficient delivery of multiple CTL and HTL epitopes encoded in a DNA plasmid or viral vector cannot be accomplished by simply aligning epitopes in a 'string-of-beads' format. At least three factors contribute to significant variation of the cellular immune responses induced using epitope-based vaccines: 1) the efficiency with which an epitope is generated through intracellular processing and then presented bound to MHC molecules; 2) the binding affinity of the epitope to MHC molecules and 3) the existence of a suitable TCR repertoire. /

[00350] The influence that amino acids flanking CTL epitopes have on the efficiency of processing and presentation can be significant, particularly for C-terminal flanking amino acids¹²²'¹²³. Immunogenicity data obtained from HLA- A2, -Al l and -B7 transgenic mice immunized with a number of unrelated experimental multi-epitope DNA vaccine constructs have been analyzed. A total of 94 different epitope/flanking residue combinations were analyzed. Significant effects of the C-terminus flanking amino acids, the Cl residue, were identified. Positively charged amino acids, such as K or R , were most frequently associated with optimal CTL responses and amino acids, such as N and Q, or small amino acids, such as C, G, A, T, and S were also associated with moderate epitope immunogenicity.

[00351] The design process and evaluation of HTL epitope-based vaccines includes different features to address the properties of HTL epitopes, including the highly promiscuous manner in which they bind to MHC Class II molecules and the properties of antigen processing pathways most commonly utilized. To address both of these issues, universal spacers, such as one consisting of GPGPG are utilized. Neither G or P in the GPGPG spacer are routinely used as primary anchors, at positions one or six in the core region of an HTL peptide epitope, by any know murine or human MHC Class II molecule. The gap of five amino acids introduced by this spacer separates adjacent epitopes so the amino acids of two epitopes cannot physically serve as anchors in the 1 and 6 positions⁶⁷. This type of spacer is also predicted to introduce a β-turn, which should enhance processing between epitopes¹²⁴.

[00352] hi summary, the use of appropriate spacers to promote efficient CTL and HTL epitope processing is an important strategy to use in vaccine design.

[00353] Epitope-based vaccines optimized for antigen processing also addresses the question of competition or immunological dominance between CTL epitopes, which would effectively reduce the breath of the total response induced by vaccination. Approximately 20 CTL and HTL DNA plasmid constructs for HBV, HIV, HPV, and malaria indications have been generated and tested. The HBV-derived DNA vaccine based on CTL and HTL epitopes is currently in Phase I clinical testing and the HIV DNA vaccine construct is slated to begin clinical trials within a year. As an example, as shown in Figure 3, the results obtained following immunization of rhesus macaques with a DNA construct encoding 12 Mamu A*01 -restricted SlV-derived CTL epitopes, 4 Mamu class II-restricted SlV-derived epitopes and the universal HTL epitope PADRE is given. The epitope order and spacers used between epitopes were chosen to maximize epitope processing and presentation. The model also provides an opportunity to evaluate the efficacy of the vaccine by performing a viral challenge. Six macaques were immunized on a monthly basis for 4 months with the DNA encoding the CTL and HTL epitopes formulated in polyvinylpyrollidone (PVP). PVP is thought to protect against degradation and promote distribute of the DNA following intramuscular injection. Three of the macaques received an additional 2 DNA immunizations following a rest period of 5 months. The other 3 monkeys, following 4 DNA immunizations, received 2 immunizations with a polyepitope protein containing the same epitopes order and spacers used in the DNA construct. Vaccine induced immunogenicity was measured 2 weeks prior, 2 weeks post and 14 weeks post-SIVmac239 infection. Significant vaccine induced EFN-γ responses were observed for all 12 CTL epitopes following immunizations.

[00354] The response pattern with regard to consistency and magnitude fell into 2 groups.

Responses specific for Tat 28, Gag 181, Env 235 and Env 622 following immunizations and prior to SIV infection were in the 50-300 SFC/106 CD8+ range. Responses induced specific for the other 8 epitopes, Gag 340, Gag 372, Vpx 39, Pol 359, Pol 143, Vif 144, Pol 147 and Pol 588 were typically more variable and in the 10-50 SFC range. Responses specific for Pol 359, Pol 143, Vif 144, Pol 147 and Pol 588 are not depicted in the graphs. Following SIV infection, the CTL responses were dramatically increased to values generally in the 200-3,000 SFC range. The epitopes Tat 28 and Gag 181 have been previously described as inducing dominant responses following viral infection, hi this study, responses in the 200-1,000 SFC range were measured using PBMC from the non-immunized animals confirming their dominant role. Responses to epitopes Env 235 and Env 622 were not observed 2 weeks following infection in non-immunized animals and are considered subdominant epitopes. Responses have persisted out to the 14 week post-infection time point but are typically reduced in magnitude, 20-1,000 SFC.

[00355] The immunogenicity observed in this study suggests two important considerations for vaccine development. First, multiple CTL responses can be induced following DNA immunization specific for dominant and subdominant epitopes using an epitope-based vaccine. Second, these responses were boosted following infection suggesting that vaccine immunization resulted in priming for these responses. To establish whether vaccine induced immunity had an influence on viral levels, plasma virus loads were measured following infection. As shown in Figure 3, virus numbers peaked at 2 weeks following infection with levels measured in the 5 x 10⁵ to 1 x 10⁷range. Five of the six immunized animals controlled infection with an average decrease in viral load of 2.0 logs at the 8 week time point. Control of viral infection is still evident although decreases to an average of 1.4 log reduction of viral load of the 6 immunized animals versus un-immunized animals. These results indicate that the vaccine induced immune response can initially control viral infection. In the case of influenza infection, it is anticipated that cellular immunity would be required to control infection for a much shorter time relative to control of persistent viral infections such as HBV and HIV.

[00356] SrV-specific HTL responses were also measured following immunization and post-infection, data not shown. Responses were induced specific for the 4 SlV-derived HTL epitopes, Rev 9, Rev 40, Nef 210, Gag 260 and the universal helper epitope PADRE in the 20-200 SFC range prior to infection. By week 14 following infection, responses were maintained in the immunized animals.

[00357] Identification, characterization and use of CTL and HTL epitopes in DNA vaccines are in Phase 1 clinical testing for HIV in both infected and uninfected volunteers.

[00358] Delivery of epitope-based vaccines using AlphaVax vector technology: The utility of self-replicating RNA (replicon) vector technology to induce protective antiviral, antibacterial and antitumor cellular and humoral immune responses in several animal models including guinea pig, mouse, Cynomolgus Monkey and Rhesus macaques has been established^125'130. Based on these studies, a clade C HIV vaccine based on the AlphaVax replicon vector is being tested in a dose-escalation, placebo-controlled trial under the NTH VTN at 4 sites in the U.S. and 2 sites in South Africa. Specifically, the AlphaVax vector system is genetically derived from an avirulent form of Venezuelan equine encephalitis virus (VEE) virus. Alphaviruses such as VEE are positive-strand RNA viruses that can mediate efficient cytoplasmic gene expression in mammalian cells. Since an RNA virus vector cannot integrate into chromosomal DNA, concerns about cell transformation are reduced. At least two immunological mechanisms may explain the enhanced immunogenicity of this vector; 1) the spike glycoproteins target the vector to dendritic cells in the draining lymph node and 2) cells transfected with the vector activate the innate pathways via double-stranded RNA recognition and interferon action^131"132. Several characteristics of AlphaVax replicon make it competitive relative to other viral vector systems such as vaccinia and adenovirus. The vector has been proven to be safe and non-transmissible with the potential for multiple delivery routes, nasal, mucosal, subcutaneous and intramuscular. In contrast to the vaccinia and Adenovirus vectors, there is a lack of pre-existing immunity to the replicon vector.

[00359] With regard to an influenza virus vaccine, the replicon vector containing the VEE nonstructural genes with the structural genes of the virus may be replaced by the minigene encoding the influenza-derived CTL, HTL and B cell epitopes. The replicon RNA is packaged into VEE replicon particles by supplying the structural genes in trans via split structural protein gene helpers. The AlphaVax replicon delivers multiple influenza-derived CTL and HTL epitopes.

[00360] A crucial step in the process of evaluating immunogenicity of epitope-based vaccines delivered by DNA plasmid or viral vector is use of the HLA transgenic mouse. Development of a successful vaccine would require that the epitopes encoded by the vaccine are correctly processed and presented to the immune system following immunization. To establish whether antigen processing and T cell repertoire are similar in man and mouse, HLA -A2 transgenic mice were infected with the influenza A/Puerto Rico/8/34 (PR8) strain and IFN-γ responses specific for the HLA-A2 -restricted peptides were evaluated. As shown in Figure 4, significant responses were measured for a majority of the peptides tested inducing responses specific for a majority of the peptides in the 20-100 SFC range. The most dominate response in the mouse, specific for Ml 3, was also the most dominant response observed in humans. Of the 19 epitopes that generated recall responses in humans, 18 induced responses in mice following infection. A specific response for PBl 501 was not generated in HLA- A2 transgenic mice following infection although a recall response specific for this peptide was demonstrated in humans. These results show that the HLA transgenic mouse is a tool for evaluating epitope processing and presentation from DNA or viral epitope-based vaccines. These attributes also suggest that the mouse model can be used in influenza challenge studies following vaccination.

Example 4: Design and optimization of genetic DNA plasmid and viral vectored vaccines

[00361] Constructs are designed based on computer programs to optimize proteosomal processing and minimize junctional epitopes: Strategies have been developed to optimize epitope processing efficiency from multi-epitope genetic constructs and to minimize the generation of neo-epitopes generated at the junction of epitopes which may divert the immune responses from the specified desired epitopes⁶⁷'⁶⁹. The incorporation of preferred flanking amino acids to optimize proteosomal processing and a motif searching function is performed using a computer program.

[00362] DNA Vaccine production: DNA vaccine production is performed using routine methods based on primer extension with overlapping oligonucleotide PCR primers, averaging 70 nucleotides in length with 15 nucleotide overlaps . The synthetic gene encoding the epitopes is cloned into the clinically accepted pMB75.6 vaccine backbone¹⁴⁵.

[00363] Assessment of vaccine immunoRenicity: Immunogenicity testing is performed primarily using the HLA-DR4 transgenic mice from Taconic and CB6F1 (b x d haplotype) mice to measure responses specific for the influenza-derived HTL epitopes and HA-specific antibodies. Immunogenicity evaluation in mice is a useful tool to assess efficient antigen processing and epitope presentation specifically for the vaccine construct. The spacers adjacent to epitopes that are found to be suboptimally immunogenic in a vaccine construct can be modified, through site-directed mutagenesis, in one or more cycles of secondary optimization.

[00364] Immunization of mice: HLA transgenic or normal inbred strains of mice, in groups of 10, will be injected with 1-100 μg of DNA vaccine using the tibialis anterior muscle as the injection site. When the AlphaVax replicon is used, mice will be immunized with 1 x 10⁴- 5 x 10⁶ infectious units of the virus, s.c. Ten to 14 days later, the mice are sacrificed, a single-cell suspension of splenocytes prepared for ELISPOT assay purposes. When heterologous prime:boost experiments are run, DNA vaccine immunization will precede the AlphaVax replicon and peptides in adjuvant immunizations by 2-4 weeks. Alternative vaccine immunization schedules are also evaluated. For example, repeat administration of DNA vaccines daily or twice weekly are evaluated as a way to better prime CTL and HTL responses prior to AlphaVax boost.

[00365] In the case of delivery of DNA and peptides in adjuvant, novel adjuvant systems are evaluated for ease of formulation and immunogenicity of the formulated vaccine. IC31 is mixed with the antigen and delivered by either a s.c. or i.m. route. Specifically, the peptide solution (KLK) and oligodeoxynucleotide solution (ODNIa) are prepared and sterile filtered separately before mixing. The optimal concentration of IC31 is evaluated for each antigen system using a dose range of 100-1,000 mmol KLK/ml + 4-40 nmol/ml ODNl a/ml). A dose range of peptide (0.1, 1, 10 μg) of each peptide per mouse is typically used. In the case of the TLR7/8 agonists, a dose range of the adjuvant (1, 10, 100 μg/mouse) is evaluated to determine optimal dose. DNA in adjuvant is administered i.m. and peptides in adjuvant s.c. Initially, a single immunization is evaluated followed by administration of booster immunizations.

[00366] Murine CTL and HTL assays: An IFN-γ-based ELISPOT assay is utilized to measure CTL and HTL activity. The assay is performed essentially as described in the legend to Figure 4. The ELISPOT is performed using an 18 hour culture step with the peptide epitope (10 μg/ml), A2.1/K^b transfected .221 target cells (or other supertype transfected target cells) and purified CD8+ and CD4+ T lymphocytes (200,000/well). [00367] Augmentation of HA-derived HTL and antibody responses using DNA vaccines followed by HA protein immunization: Prior immunization with conserved influenza virus HTL epitopes will augment HTL and antibody responses induced using protein- based or inactivated virus-based vaccines. HLA transgenic mice are initially immunized separately or in a prime-boost format using the DNA, and peptides in adjuvant vaccines. These immunizations are followed by inoculation with various HA proteins (0.1, 1, 10 μg/mouse). The HTL and antibody responses are measured (as described above) and directly compared to mice receiving only the conventional HA vaccines. Purified baculovirus-expressed recombinant HA proteins (Protein Sciences, Inc, Meriden, CT) corresponding to A/Hong Kong/156/97 (H5) and A/Hong/Kong/I 073/99 (H9) are used. The rationale for using H5 and H9 proteins is due to their pandemic potential as observed by transmission of these variants from avian to human ¹⁸'¹⁴⁶.

Example 5. Evaluation of efficacy of the experimental vaccines alone and in combination with recombinant HA protein using HLA transgenic mice and infectious challenges

[00368] The efficacy of vaccines composed of conserved influenza HTL and B cell epitopes are evaluated in an influenza viral challenge mouse model. For example, peptides are formulated in various adjuvants and tested for immunogenicity. If a particular adjuvant is superior in augmenting cellular and humoral responses then this adjuvant is used in the challenge studies. Initially, protection against various divergent influenza subtypes is determined by immunizing mice separately with selected DNA, peptides in adjuvant, HA proteins, inactivated and live attenuated vaccines. Doses and immunization schedules are determined according to the immunogenicity studies described above. The capacity of the influenza HTL and B cell epitope-based vaccines to afford protection is compared to the HA protein, inactivated and live attenuated vaccines. Finally, the HA protein combined with the DNA, and peptides in adjuvant vaccines using heterologous prime boost immunization schemes are evaluated for protection. Additionally, emphasis is placed on validating an immunization strategy that induces a protective immune response in the shortest amount of time which is likely an important factor to consider in the event of a pandemic influenza occurrence. [00369] Murine influenza challenge models: Viral challenge studies are performed as previously described ⁷⁵'¹⁴⁷-¹⁴⁸. Initially, mice are immunized with selected vaccines or combinations using doses and immunization schedules that are most immunogenic. To determine the level of protection afforded by the various immunization strategies, immunized mice are challenged with various subtypes of influenza viruses that differ in virulence for mice including human viruses as well as avian and viruses with pandemic potential. Using a number of different subtypes the level of protective broadly cross- reactive immunity induced by immunization of mice with the various vaccines expressing conserved HTL epitopes are evaluated. The following are examples of subtypes for challenge studies: mouse adapted A/Taiwan/1/86 (HlNl); mouse-adapted A/Ann Arbor/6/60 (H2N2); mouse-adapted A/Philippines/ 1/82 (H3N2); highly pathogenic avian A/Hong Kong/483 (H5N1); a recent human isolate A/Hong Kong/213/03 (H5N1); A/Hong Kong/1073/99 (H9N2); and an H7N7 strain.

[00370] The 50% mouse infectious dose (MID50) and 50% lethal dose (LD50) titers are determined for the C57B1/6 mouse strain. Groups of 10-20 mice are lightly anesthetized and infected intranasally (i.n.) with approximately 100-1,000 MID50 of virus. Three and six days post-infection, 5 mice per group are sacrificed and multiple organs including nasal turbinates, lungs and brains are collected and titered in embryonated eggs or MDCK cells for the presence of infectious virus. For viruses that cause lethal disease, and additional group of ten mice are monitored for weight loss and survival over a period of 14 days post-infection.

[00371] The use of conserved HTL epitopes delivered by peptides in adjuvant and DNA viral vehicles are used to generate a protective vaccine against influenza.

Example 6: Human Recall Responses in Donor X753

[00372] Primary interferon-gamma (IFN-γ) ELISPOT (enzyme linked immunospot) assay was used to identify candidate vaccine epitopes. Peripheral blood mononuclear cells (PBMCs) were collected by leukapheresis from healthy human donors. The PBMCs were purified using standard Ficoll-Paque (Amersham) density gradient centrifugation and subsequently frozen at 5x10⁷ cells per ml. PBMCs were thawed and were either rested for 5 days (no peptide) or stimulated for 7 days with the appropriate peptides at 37°C in media at 2.5 x 10⁶ cells per mL. Elispot plates (Millipore IP plate) were coated with anti-human IFN-γ antibody clone 1-DlK (Mabtech, Cat# 3420-3, 1 mg/niL) and incubated overnight at 4°C. The following day, PBMCs were depleted of CD8⁺ cells using human DYNAbeads (DYNAL Biotec Cat# 111.47, OSLO, Norway). The depleted PBMCs with enriched CD4⁺ cells were then plated onto ELISPOT plates previously blocked with RPMI 1640 containing 10% FCS. Irradiated PBMCs coated with peptide were added to the plated PBMCs and the plates were incubated at 37⁰C for 20 hours. The next day the plates were incubated with biotinylated mouse anti-human IFN- γ antibody and developed with Vectastain Elite Vector Cat# PK-6100 according to manufacturer's instructions. The spots were counted on an ELISPOT counter (AID). Donors were considered positive for a peptide if the number of spots was over 3 times background as determined by responses to irrelevant peptides (non influenza). Representative results are shown in Figure 5. Five peptides induced significant immune responses, PB2.438, PB1.94, Ml.173, NP.189 and PA.178

Example 7: Influenza-Specific Recall Responses for Humans

[00373] Primary interferon-gamma (IFN-γ) ELISPOT (enzyme linked immunospot) assay was performed to identify candidate HLA-Al, HLA- A2, HLA-A3/A11, HLA- A24, HLA- B7 and HLA-DR vaccine epitopes. Representative results are shown in Figures 7, 8, 10, 12, 14 and 16 respectively. Assays to identify CTL epitopes were performed essentially as described in Example 2 and the legend to Figure 1. Assays to identify HTL epitopes were performed essentially as described in Example 6.

Example 8: Influenza-Specific Recall Responses for Mice

[00374] Primary interferon-gamma (IFN-γ) ELISPOT (enzyme linked immunospot) assay was performed using mice carrying an HLA- A2, HLA-AI l, HLA- A24, HLA-B7 or HLA-DR4 transgene, and results are shown in Figures 9, 11, 13, 15 and 17 respectively. In addition, HLA-DR4 candidate vaccine epitopes were tested in b x d haplotype mice, and results are shown in Figure 18. Assays were performed essentially as described in Example 4 and the legend to Figure 4. Literature Cited

1. Thompson,W.W., D.K. Shay, E.Weintraub, L. Brammer, N. Cox, LJ. Anderson, and K. Fukuda, 2003, "Mortality associated with influenza and respiratory syncytial virus in the United States", JAMA 259:179-186.

2. Nguyen-Van-Tam, J.S., and A.W.Hampson, 2003, "The epidemiology and clinical impact of pandemic influenza", Vaccine. 27:1762-1768.

3. "The Lancet Infectious Diseases", 4:595, 2004.

4. Nicholson, K.G., A.E. Colegate, A. Podda, I. Stephenson, J. Wood, E. Ypma, and M.C. Zambon, 2001, "Safety and antigenicity of non-adjuvanted and MF59- adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza", Lancet 557:1937-1943.

5. Treanor, JJ., B. E. Wilkinson, F. Masseoud, J. Hu-Primmer, R. Battaglia, D. O'Brien, M. Wolff, G. Rabinovich, W. Blackwelder, and J.M. Katz, 2001, "Safety and immunogenicity of a recombinant hemagglutinin vaccine for H5 influenza in humans", Vaccine 19: 1732- 1737.

6. Potter, C.W., 2001, "A history of influenza", J Appl. Microbiol, 97:572- 579.

7. Hilleman, M.R., 2002, "Realities and enigmas of human viral influenza: pathogenesis, epidemiology and control", Vaccine 20:3068-3087.

8. Beveridge, W.I., 1991, "The chronicle of influenza epidemics", Hist. Philos. Life Sci. 73:223-234.

9. Gust, I.D., A. W. Hampson, and D. Lavanchy, 2001, "Planning for the next pandemic of influenza" Rev. Med. Virol. 11:59-70.

10. Oxford, J.S., 2000, "Influenza A pandemics of the 20th century with special reference to 1918: virology, pathology and epidemiology", Rev.Med. Virol. 70:119-133.

11. Laver, G. and E. Garman, 2002, "Pandemic influenza: its origin and control" Microbes.Infect. 4:1309-1316.

12. Tollis, M. and L. Di Trani, 2002, "Recent developments in avian influenza research: epidemiology and immunoprophylaxis", Vet. J. 764:202-215.

13. Palese, P. and J.F. Young, 1982, "Variation of influenza A, B, and C viruses", Science 275:1468-1474. 14. Gorman, O.T., W.J. Bean, and R.G. Webster, 1992, "Evolutionary processes in influenza viruses: divergence, rapid evolution, and stasis", Curr. Top. Microbiol. Immunol 176:15-91.

15. Yewdell, J.W., R.G.Webster, and W.U. Gerhard, 1979, "Antigenic variation in three distinct determinants of an influenza type A haemagglutinin molecule", Nature 279:246-248.

16. Top, F.H., Jr. and P.K. Russell, 1977, "Swine influenza A at Fort Dix, New Jersey (January-February 1976). IV, Summary and speculation. J. Infect. Dis. 136 Suppl:S376-S380.

17. Kurtz, J., RJ. Manvell, and J.Banks, 1996, "Avian influenza virus isolated from a woman with conjunctivitis", Lancet 345:901-902.

18. Peiris, M., K.Y. Yuen, CW. Leung, K.H. Chan, P.L. Ip, R.W. Lai, W.K. Orr, and K.F. Shortridge, 1999, "Human infection with influenza H9N2", Lancet 554:916-917.

19. Scholtissek, C, W. Rohde, H. Von, V, and R. Rott, 1978, "On the origin of the human influenza virus subtypes H2N2 and H3N2", Virology 57:13-20.

20. Wells, M. A., F. A. Ennis, and P. Albrecht, 1981, "Recovery from a viral respiratory infection, II. Passive transfer of immune spleen cells to mice with influenza pneumonia", J. Immunol. 72(5:1042-1046.

21. Yap, K.L. and G.L. Ada, 1978, "Cytotoxic T cells in the lungs of mice infected with an influenza A virus", Scand. J. Immunol. 7:73-80.

22. Mackenzie, CD., P.M. Taylor, and B. A. Askonas, 1989, "Rapid recovery of lung histology correlates with clearance of influenza virus by specific CD8+ cytotoxic T cells", Immunology (57:375-381.

23. Taylor, P.M. and B. A. Askonas, 1986, "Influenza nucleoprotein-specific cytotoxic T-cell clones are protective in vivo", Immunology 55:417-420.

24. Kuwano, K., M. Scott, J.F. Young, and F.A.Ennis, 1988, "HA2 subunit of influenza A Hl and H2 subtype viruses induces a protective cross-reactive cytotoxic T lymphocyte response", J. Immunol. 140:1264-1268.

25. Kuwano, K., M. Tamura, and F.A. Ennis, 1990, "Cross-reactive protection against influenza A virus infections by an NSl -specific CTL clone", Virology 775:174-179.

26. Sambhara, S., A. Kurichh, R. Miranda, T. Tumpey, T. Rowe, M. Renshaw, R. Arpino, A. Tamane, A. Kandil, O. James, B. Underdo wn, M. Klein, J. Katz, and D. Burt, 2001, "Heterosubtypic immunity against human influenza A viruses, including recently emerged avian H5 and H9 viruses, induced by FLU-ISCOM vaccine in mice requires both cytotoxic T-lymphocyte and macrophage function", Cell Immunol. 277:143-153.

27. Okuda, K., A. Ihata, S. Watabe, E. Okada, T. Yamakawa, K. Hamajima, J. Yang, N. Ishii, M. Nakazawa, K. Okuda, K. Ohnari, K. Nakajima, and K.Q. Xin, 2001, "Protective immunity against influenza A virus induced by immunization with DNA plasmid containing influenza M gene", Vaccine 79:3681-3691.

28. Bender, B.S., T. Croghan, L. Zhang, and P.A. Small, Jr., 1992, "Transgenic mice lacking class I major histocompatibility complex-restricted T cells have delayed viral clearance and increased mortality after influenza virus challenge", J. Exp. Med. 775:1143-1145.

29. Ulmer, J.B., JJ. Donnelly, S.E. Parker, G.H. Rhodes, P.L. Feigner, VJ. Dwarki, S.H. Gromkowski, R.R. Deck, CM. DeWitt, A. Friedman, et al, 1993, "Heterologous protection against influenza by injection of DNA encoding a viral protein", Science 259: 1745-1749.

30. Fu, T.M., L. Guan, A. Friedman, T.L. Schofield, J.B. Ulmer, M.A. Liu, and JJ. Donnelly, 1999, "Dose dependence of CTL precursor frequency induced by a DNA vaccine and correlation with protective immunity against influenza virus challenge", J. Immunol. 7(52:4163-4170.

31. Ulmer, J.B., T.M. Fu, R.R. Deck, A. Friedman, L. Guan, C. DeWitt, X. Liu, S. Wang, M.A. Liu, JJ. Donnelly, and MJ. Caulfield, 1998, "Protective CD4+ and CD8+ T cells against influenza virus induced by vaccination with nucleoprotein DNA", J. Virol. 72:5648-5653.

32. Epstein, S.L., A. Stack, J. A. Misplon, CY. Lo, H. Mostowski, J. Bennink, and K. Subbarao,. 2000, "Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge", Int. Immunol 72:91-101.

33. Langlade-Demoyen, P., F. Garcia-Pons, P. Castiglioni, Z. Garcia, S. Cardinaud, S. Xiong, M. Gerloni, and M. Zanetti, 2003, "Role of T cell help and endoplasmic reticulum targeting in protective CTL response against influenza virus", Eur. J. Immunol. JJ:720-728. 34. Anker, WJ. , A. K. Bakker, and N. Masurel. 1978, "Cross-protection in mice after immunization with H2N2, H3N2, and H2N2 influenza virus strains". Infect. Immun. 27:96-101.

35. Schulman, J. L. and E.D. Kilbourne, 1965, "Induction of partial specific heterotypic immunity in mice by a single infection with influenza A virus", J. Bacterid. 89: 170- 174.

36. Benton, K.A., J.A. Misplon, CY. Lo, R.R. Brutkiewicz, S.A. Prasad, and S. L. Epstein, 2001, "Heterosubtypic immunity to influenza A virus in mice lacking IgA, all Ig, NKT cells, or gamma delta T cells", J. Immunol. 166: 7437-7445.

37. O'Neill, E., S.L. Krauss, J.M. Riberdy, R.G. Webster, and D.L. Woodland, 2000, "Heterologous protection against lethal A/HongKong/156/97 (H5N1) influenza virus infection in C57BL/6 mice", J. Gen. Virol. 57:2689-2696.

38. Epstein, S. L., T.M. Tumpey, J.A. Misplon, CY. Lo, L.A. Cooper, K. Subbarao, M. Renshaw, S. Sambhara, and J.M. Katz, 2002, "DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice", Emerg. Infect. Dis. 5:796-801.

39. Nguyen, H.H., Z. Moldoveanu, MJ. Novak, F.W. Van Ginkel, E. Ban, H. Kiyono, J.R. McGhee, and J. Mestecky, 1999, "Heterosubtypic immunity to lethal influenza A virus infection is associated with virus-specific CD8(+) cytotoxic T lymphocyte responses induced in mucosa-associated tissues", Virology 254:50-60.

40. Lukacher, A.E., V. L. Braciale, and TJ. Braciale, 1984, "In vivo effector function of influenza virus-specific cytotoxic T lymphocyte clones is highly specific", J. Exp. Med. 760:814-826.

41. Liang, S., K. Mozdzanowska, G. Palladino, and W. Gerhard, 1994, "Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity", J. Immunol. 752:1653-1661.

42. Tumpey, T.M., M. Renshaw, J.D. Clements, and J.M. Katz, 2001, "Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection", J. Virol. 75:5141-5150.

43. McMichael, AJ., F.M. Gotch, G.R. Noble, and P.A.S. Beare, 1983, "Cytotoxic T-cell immunity to influenza", N. Engl. J. Med. 309:13-17. 44. Sonoguchi, T., H. Naito, M. Hara, Y. Takeuchi, and H. Fukumi, 1985, "Cross-subtype protection in humans during sequential, overlapping, and/or concurrent epidemics caused by H3N2 and HlNl influenza viruses", J. Infect. Dis. 757:81-88.

45. Voeten, J. T., T.M. Bestebroer, NJ. Nieuwkoop, R. A. Fouchier, A.D. Osterhaus, and G.F. Rimmelzwaan, 2000, "Antigenic drift in the influenza A virus (H3N2) nucleoprotein and escape from recognition by cytotoxic T lymphocytes", J. Virol. 74:6800-6807.

46. Kashiwagi, T., N. Hamada, J. Iwahashi, K. Hara, T. Ueda, H. Noguchi, and T. Toyoda, 2000, "Emergence of new influenza A viruses which carry an escape mutation of the HLA-B27-restricted CTL epitope of NP in Japan", Microbiol. Immunol 44:867-870.

47. Boon, A.C., G.de Mutsert, Y.M. Graus, R.A. Fouchier, K. Sintnicolaas, A.D. Osterhaus, and G.F. Rimmelzwaan, 2002, "Sequence variation in a newly identified HLA-B35-restricted epitope in the influenza A virus nucleoprotein associated with escape from cytotoxic T lymphocytes", J. Virol. 76:2567-2572.

48. Treanor, JJ. , E. L. Tierney, S. L. Zebedee, R.A. Lamb, and B.R. Murphy, 1990, "Passively transferred monoclonal antibody to the M2 protein inhibits influenza A virus replication in mice", J. Virol. 64:1375-1377.

49. Frace, A.M., A.I. Klimov, T. Rowe, R.A. Black, and J.M. Katz, 1999, "Modified M2 proteins produce heterotypic immunity against influenza A virus", Vaccine 77:2237-2244.

50. Neirynck, S., T. Deroo, X. Saelens, P. Vanlandschoot, W.M. Jou, and W. Fiers, 1999, "A universal influenza A vaccine based on the extracellular domain of the M2 protein", Nat. Med. 5:1157-1163.

51. Jegerlehner, A., N. Schmitz T. Storni, and M.F. Bachmann, 2004, "Influenza A vaccine based on the extracellular domain of M2: Weak protection mediated via antibody-dependent NK cell activity", J Immunol. 772:5598-5605.

52. Fiers, W., M. DeFilette, A. Birkett, S. Neirynck, and W.M. Jou, 2004, "A "universal' human influenza A vaccine", Virus Res. 703:173-176.

53. Wanli, L., P. Aou, Y.-H. Chen, 2004, "Monoclonal antibodies recognizing EVETPIRN epitope of influenza A virus M2 protein could protect mice from lethal influenza A virus challenge", Immunol. Lett. 93:131-136.

54. Fan, J., X. Liang, M.S. Horton, H.C. Perry, M.P. Citron, GJ. Heidecker, T.M. Fu, J. Joyce, CT. Przysiecki, P.M. Keller, V.M. Garsky, R. Ionescu, Y. Rippeon, L. Shi, M.A. Chastain, J.H. Condra, M. E. Davies, J. Liao, E. A. Emini, and J.W. Shiver, 2004, "Preclinical study of influenza virus A M2 peptide conjugate vaccines in mice, ferrets, and rhesus monkeys", Vaccine 22:2993-3003.

55. Jameson, J., J. Cruz, M. Terajima, and F.A. Ennis, 1999, "Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species", J. Immunol. 762:7578-7583.

56. Jameson, J., J. Cruz, and F.A. Ennis, 1998, "Human cytotoxic T- lymphocyte repertoire to influenza A viruses", J. Virol. 72:8682-8689.

57. Gianfrani, C, C. Oseroff, J. Sidney, R. W. Chesnut, and A. Sette, 2000, "Human memory CTL response specific for influenza A virus is broad and multispecific", Hum. Immunol. (57:438-452.

58. Boon, A.C., G.de Mutsert, Y.M. Graus, R.A. Fouchier, K. Sintnicolaas, A.D. Osterhaus, and G.F. Rimmelzwaan, 2002, "The magnitude and specificity of influenza A virus-specific cytotoxic T-lymphocyte responses in humans is related to HLA-A and -B phenotype", J. Virol. 76:582-590.

59. Liu, M.A, 2003, "DNA vaccines: a review", J. Intern. Med. 253:402-410.

60. Gurunathan, S., D.M. Klinman, and R.A. Seder, 2000, "DNA vaccines: immunology, application, and optimization", Annu. Rev. Immunol 75:927-974.

61. Calarota, S. A., A.C. Leandersson, G. Bratt, J. Hinkula, D.M. Klinman, KJ. Weinhold, E. Sandstrom, and B. Wahren, 1999, "Immune responses in asymptomatic HIV-I -infected patients after HIV-DNA immunization followed by highly active antiretroviral treatment", J. Immunol. 765:2330-2338.

62. Roy, M.J., M.S. Wu, LJ. Barr, J.T. Fuller, L.G. Tussey, S. Speller, J. CuIp, J.K. Burkholder, W.F. Swain, R.M. Dixon, G. Widera, R. Vessey, A. King, G. Ogg, A. Gallimore, J.R. Haynes, and F.D. Heydenburg, 2000, "Induction of antigen- specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine", Vaccine 79:764- 778.

63. Ugen, K.E., S.B. Nyland, J.D. Boyer, C. Vidal, L. Lera, S. Rasheid, M. Chattergoon, M.L. Bagarazzi, R. Ciccarelli, T. Higgins, Y. Baine, R. Ginsberg, R.R. MacGregor, and D. B. Weiner, 1998, "DNA vaccination with HIV-I expressing constructs elicits immune responses in humans", Vaccine 76:1818-1821. 64. Calarota, S. A., A. Kjerrstrom, K.B. Islam, and B. Wahren, 2001, "Gene combination raises broad human immunodeficiency virus-specific cytotoxicity", Hum. Gene Ther. 72:1623-1637.

65. Wang, R., J. Epstein, F.M. Baraceros, EJ. Gorak, Y. Charoenvit, DJ. Carucci, R.C. Hedstrom, N. Rahardjo, T. Gay, P. Hobart, R. Stout, T.R. Jones, T.L. Richie, S. E. Parker, D. L. Doolan, J. Norman, and S. L. Hoffman, 2001, "Induction of CD4(+) T cell-dependent CD8(+) type 1 responses in humans by a malaria DNA vaccine", Proc. Natl. Acad. Sci. USA 95:10817-10822.

66. Sette, A., M. Newman, B. Livingston, D. McKinney, J. Sidney, G. Ishioka, S. Tangri, J. Alexander, J. Fikes, and R. Chesnut, 2002, "Optimizing vaccine design for cellular processing, MHC binding and TCR recognition", Tissue Antigens 59:443-451.

67. Livingston, B., C. Crimi, M. Newman, Y. Higashimoto, E. Appella, J. Sidney, and A. Sette, 2002, "A Rational Strategy to Design Multiepitope Immunogenes Based on Multiple TH Lymphocyte Epitopes", J. Immunol. 7(55:5499-5506.

68. Ishioka, G.Y., J. Fikes, G. Hermanson, B. Livingston, C. Crimi, M. Qin, M.F. del Guercio, C. Oseroff, C. Dahlberg, J. Alexander, R. W. Chesnut, and A. Sette, 1999, "Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes", J. Immunol. 762:3915-3925.

69. Livingston, B.D., M. Newman, C. Crimi, D. McKinney, R. Chesnut, and A. Sette, 2001, "Optimization of epitope processing enhances immunogenicity of multiepitope DNA vaccines", Vaccine 79:4652-4660.

70. Alila, H., M.E. Coleman, H. Nitta, M. French, K. Anwer, Q. Liu, T. Meyer, J. Wang, RJ. Mumper, D. Oubari, S.D. Long, J. L. Nordstrom, and A.P. Rolland, 1997, "Expression of biologically active human insulin-like growth factor-I following intramuscular injection of a formulated plasmid in rats", Human Gene Therapy 5:1785- 1795.

71. Anwer, K., K.A. Earle, M. Shi, J. Wang, RJ. Mumper, B. Proctor, K. Jansa, H.C. Ledebur, S. S. Davis, W. Eaglstein, and A.P. Rolland, 1999, "Synergistic Effect of Formulated Plasmid and Needle-Free Injection for Genetic Vaccines", Pharm. Res. 7(5:889-95.

72. Mumper, RJ., J.G. Duguid, K. Anwer, M.K. Barron, H. Nitta, and A.P. Rolland, 1996, "Polyvinyl derivatives as novel interactive polymers for controlled gene delivery to muscle", Pharm. Res. 75:701-709. 73. Mumper, RJ., J. Wang, S. L. Klakamp, H. Nitta, K. Anwer, F. Tagliaferri, and A.P. Rolland, 1998, "Protective interactive noncondensing (PINC) polymers for enhanced plasmid distribution and expression in rat skeletal muscle", J. Contr. ReI. 52:191-203.

74. Epstein, S. L., A. Stack, J.A. Misplon, CY. Lo, H. Mostowski, J. Bennink, and K. Subbarao, 2000, "Vaccination with DNA encoding internal proteins of influenza virus does not require CD8(+) cytotoxic T lymphocytes: either CD4(+) or CD8(+) T cells can promote survival and recovery after challenge", Int. Immunol 72:91-101.

75. Epstein, S.L., T.M. Tumpey, J.A. Misplon, CY. Lo, L.A. Cooper, K. Subbarao, M. Renshaw, S. Sambhara, and J.M. Katz, 2002, "DNA vaccine expressing conserved influenza virus proteins protective against H5N1 challenge infection in mice", Emerg. Infect. Dis. 5:796-801.

76. Vitiello, A., D. Marchesini, J. Furze, L.A. Sherman, and R.C Chesnut, 1991, "Analysis of the HLA-restricted influenza-specific cytotoxic T lymphocyte response in transgenic mice carrying a chimeric human-mouse class I major histocompatibility complex", J. Exp. Med. 773:1007-15.

77. Alexander, J., C Oseroff, J. Sidney, P. Wentworth, E. Keogh, G. Hermanson, F. V. Chisari, R.T. Kubo, H.M. Grey, and A. Sette, 1997, "Derivation of HLA-Al 1/K^b transgenic mice: functional CTL repertoire and recognition of human Al 1- restricted CTL epitopes", J. Immunol. 759:4753-4761.

78. Alexander, J., C. Oseroff, J. Sidney, and A. Sette, 2003, "Derivation of HLA-B*0702 transgenic mice: functional CTL repertoire and recognition of human B*0702-restricted CTL epitopes", Hum. Immunol (54:211-223.

79. Alexander, J., C. Oseroff, C. Dahlberg, M. Qin, G. Ishioka, M. Beebe, J. Fikes, M. Newman, R.W. Chesnut, P.A. Morton, K. Fok, E. Appella, and A. Sette, 2002, "A decaepitope polypeptide primes for multiple CD8+ IFN-gamma and Th lymphocyte responses: evaluation of multiepitope polypeptides as a mode for vaccine delivery", J. Immunol. 755:6189-6198.

80. Wall, K.A., J.Y. Hu, P. Currier, S. Southwood, A. Sette, and AJ. Infante, 1994, "A disease-related epitope of Torpedo acetylcholine receptor. Residues involved in I- Ab binding, self-nonself discrimination, and TCR antagonism", J. Immunol. 752:4526- 4536.

81. Townsend, A. and H. Bodmer, 1989, "Antigen recognition by class I- restricted T lymphocytes", Annu. Rev. Immunol. 7:601-624. 82. Germain, R.N. and D.H. Margulies, 1993, "The biochemistry and cell biology of antigen processing and presentation", Annu. Rev. Immunol. 77:403-450.

83. Sette, A. and H.M. Grey, 1992, "Chemistry of peptide interactions with MHC proteins", Current Opinion in Imunology 4:79-86.

84. Sinigaglia, F. and J. Hammer, 1994, "Defining rules for the peptide-MHC class II interaction", Current Opinion in Imunology. 5:52-56.

85. Engelhard,V.H., 1994, "Structure of peptides associated with MHC class I molecules", Current Opinion in Imunology. 5:13-23.

86. Brown, K., T.S. Jardetzky, J.C. Gorga, LJ. Stern, J.L. Strominger, and D. C. Wiley, 1993, "Three-dimensional structure of the human class II histocompatibility antigen HLA-DRl", Nature 364:33-39.

87. Guo, H.C., D.R. Madden, M.L. Silver, T.S. Jardetzky, J.C. Gorga, J.L. Strominger, and D. C. Wiley, 1993, "Comparison of the P2 specificity pocket in three human histocompatibility antigens: HLA-A*6801, HLA-A*0201, and HLA-B*2705", Proc. Nat. Acad. Sci. USA 90:8053-8057.

88. Guo, H.C., T.S. Jardetzky, T.P. Garrett, W.S. Lane, J.L. Strominger, and D.C. Wiley, 1992, "Different length peptides bind to HLA- Aw68 similarly at their ends but bulge out in the middle", Nature 550:364-366.

89. Silver, M.L., H.C. Guo, J.L. Strominger, and D.C. Wiley, 1992, "Atomic structure of a human MHC molecule presenting an influenza virus peptide", Nature 550:367-369.

90. Matsumura, M., D.H. Fremont, P.A. Peterson, and LA. Wilson, 1992, "Emerging principles for the recognition of peptide antigens by MHC class I molecules". Science 257:927-934.

91. Madden, D.R., J.C. Gorga, J.L. Strominger, and D.C. Wiley, 1995, "The three-dimensional structure of HLA-B27 at 2.1 A resolution suggests a general mechanism for tight peptide binding to MHC" Cell 70:1035-1048.

92. Fremont, D.H., M. Matsumura, E.A. Stura, P.A. Peterson, and LA. Wilson, 1992, "Crystal structures of two viral peptides in complex with murine MHC class I H-2K^b", Science 257:919-927.

93. Sapp, M., PJ. Bjorkman, and D.C. Wiley, 1991, "Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 A resolution", J. MoI. Biol. 279:277- 319. 94. Ruppert, J., J. Sidney, E. Celis, R.T. Kubo, H.M. Grey, and A. Sette, 1993, "Prominent role of secondary anchor residues in peptide binding to HLA-A2.1 molecules", Cell 74:929-37.

95. Parker, K.C., M.A. Bednarek, and J.E. Coligan, 1994, "Scheme for ranking potential HLA- A2 binding peptides based on independent binding of individual peptide side-chains", J. Immunol. 752:163-175.

96. De Groot, A.S., A. Bosma, N. Chinai, J. Frost, B.M. Jesdale, M.A. Gonzalez, W. Martin, and C. Saint- Aubin, 2001, Vaccine 79:4385-4395.

97. Schueler-Furman, O., Y. Altuvia, A. Sette, and H. Margalit, 2000, "Structure-based prediction of binding peptices to MHC class I molecules: Application to a broad range of MHC alleles", Protein Science P: 1838-1846.

98. Meister, G.E., C.G.P. Roberts, J.A. Berzofsky, and A.S. DeGroot, 1995, "Two novel T cell epitope prediction algorithms based on MHC-binding motifs; comparison of predicted and published epitopes from Mycobacterium tuberculosis and HIV protein sequences", Vaccine 75:581-591.

99. Bhasin, M., and G.P.S. Raghava, 2004, "Prediction of CTL epitopes using QM, SVM and ANN techniques", Vaccine 22:3195-3204.

100. Altuvia, Y., O. Schueler, and H. Margalit, 1995, "Ranking potential binding peptides to MHC molecules by a computational threading approach", J. MoI. Biol. 249:244-250.

101. Methods for prediction of peptide binding to MHCmolecules: a comparative study, Molecular Medicine 5:137-148.

102. Gulukota, K., J. Sidney, and A. Sette, 1997, "Two complementary methods for predicting peptide binding major histocompatibility complex molecules", J. MoI. Biol. 2(57:1258-1267.

103. Kubo, R.T., A. Sette, H.M. Grey, E. Appella, K. Sakaguchi, N.Z. Zhu, D. Arnott, N. Sherman, J. Shabanowitz, and H. Michel, 1994, "Definition of specific peptide motifs for four major HLA-A alleles", J. Immun. 752:3913-24.

104. Threlkeld, S.C., P.A. Wentworth, S.A. Kalams, B.M. Wilkes, DJ. Ruhl, E. Keogh, J. Sidney, S. Southwood, B.D. Walker, and A. Sette, 1997, "Degenerate and promiscuous recognition by CTL of peptides presented by the MHC class I A3 -like superfamily - Implications for vaccine development", J. Immun. 759:1648-1657.

105. Sidney, J., H.M. Grey, S. Southwood, E. Celis, P.A. Wentworth, M.F. Del Guercio, R.T. Kubo, R.C. Chesnut, and A. Sette, 1996, "Definition of an HLA-A3-like supermotif demonstrates the overlapping peptide-binding repertoires of common HLA molecules", Hum. Immunol. 45:79-93.

106. Sette, A. and Sidney, J., "Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism", Immunogenetics 50(3-4), 201-212, 1999.

107. Sidney, J., S. Southwood, M.F. Del Guercio, H.M. Grey, R.C. Chesnut, R.T. Kubo, and A. Sette, 1996, "Specificity and degeneracy in peptide binding to HLA- B7-like class I molecules", J. Immunol. 757:3480-3490.

108. Del Guercio, M.F., J. Sidney, G. Hermanson, C. Perez, H.M. Grey, R.T. Kubo, and A. Sette, 1995, "Binding of a peptide antigen to multiple HLA alleles allows definition of an A2-like supertype" J. Immunol. 54:685-693.

109. Fruci, D., P. Rovero, G. Falasca, A. Chersi, R. Sorrentino, R. Butler, N. Tanigaki, and R. Tosi, 1993, "Anchor residue motifs of HLA class-I-binding peptides analyzed by the direct binding of synthetic peptides to HLA class I alpha chains", Hum. Immunol. 55:187-192.

110. Bertoni, R., J. Sidney, P. Fowler, R.W. Chesnut, F.V. Chisari, and A. Sette, 1997, "Human histocompatibility leukocyte antigen-binding supermotifs predict broadly cross-reactive cytotoxic T lymphocyte responses in patients with acute hepatitis", J. Clin. Invest. 700:503-513.

111. Khanna, R., S.R. Burrows, A. Neisig, J. Neefjes, DJ. Moss, and S. L. Silins, 1997, "Hierarchy of Epstein-Barr virus-specific cytotoxic T-cell responses in individuals carrying different subtypes of an HLA allele: Implications for epitope-based antiviral vaccines", J. Virol. 77:7429-7435.

112. Bertoletti, A., S. Southwood, R. Chesnut, A. Sette, M. Falco, G.B. Ferrara, A. Penna, C. Boni, F. Fiaccadori, and C. Ferrari, 1997, "Molecular features of the hepatitis B virus nucleocapsid T-cell epitope 18-27: interaction with HLA and T-cell receptor", Hepatology 26:1027-1034.

113. Fleischhauer, K., S. Tanzarella, HJ. Wallny, C. Bordignon, and C. Traversari, 1996, "Multiple HLA-A alleles can present an immunodominant peptide of the human melanoma antigen Melan- A/MART- 1 to a peptide-specific HLA-A*0201+ cytotoxic T cell line", J. Immunol. 757:787-297.

114. Kawashima, L, SJ. Hudson, V. Tsai, S. Southwood, K. Takesako, E. Appella, A. Sette, and E. Celis, 1998, "The multi-epitope approach for immunotherapy for cancer: Identification of several CTL epitopes from various tumor- associated antigens expressed on solid epithelial tumors", Hum. Immunol. 59: 1-14.

115. Wang, R.F., S. L. Johnston, S. Southwood, A. Sette, and S. A. Rosenberg, 1998, "Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by CTL in the context of HLA-A31 and -A33", J. Immunol. 760:890-897.

116. Southwood, S., J. Sidney, A. Kondo, M.F. Del Guercio, E. Appella, S. Hoffman, R.T. Kubo, R.W. Chesnut, H.M. Grey, and A. Sette, 1998, "Several Common HLA-DR Types Share Largely Overlapping Peptide Binding Repertoires", J. Immunol. 7(50:3363-3373.

117. Schaeffer, E.B., A. Sette, D.L. Johnson, M.C. Bekoff, J.A. Smith, H.M. Grey, and S. Buus, 2000, "Relative contribution of "determinant selection" and "holes in the T-cellrepertoire" to T-cell responses", Proc. Nat. Acad. Sci. USA 5(5:4649-4653.

118. Currier, J.R., E.G. Kuta, E. Turk, B. L.B. Earhart, L. Loomis-Price, S. Janetzki, G. Ferrari, D.L. Birx, and J.H. Cox, 2002, "A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays", J. Immunological Meth. 260:157-172.

119. Bartlett, J.A., S.S. Wasserman, CB. Hicks, R.T. Dodge, KJ. Weinhold, CO. Tacket, N. Ketter, A.E. Wittek, T.J. Palker, and B.F. Haynes, 1998, "Safety and immunogenicity of an HLA-based HIV envelope polyvalent synthetic peptide immunogen DATRI 010 study group. Division of AIDS Treatment Research Inititive", AIDS 72:1291-1300.

120. Pinto, L.A., J.A. Berzofsky, K.R. Fowke, R.F. Little, F. Merced-Galindez, R. Humphrey, J. Ahlers, N. Dunlop, R.B. Cohen, S.M. Steinberg, P. Nara, G.M. Shearer, and R. Yarchoan, 1999, "HIV-specific immunity following immunization with HIV synthetic envelope peptides in asymptomatic HIV-infected patients", AIDS 73:2003- 2012.

121. Gahery-Segard, H., G. Pialoux, B. Charmeteau, S. Sermet, H. Poncelet, M. Raux, A. Tartar, J.P. Levy, H. Gras-Masse, and J.G. Guillet, 2000, "Multiepitopic B- and T-cell responses induced in humans by a human immunodeficiency virus type 1 lipopeptide vaccine", J. Virol. 74:1694-1703.

122. Serwold, T. and N. Shastri, 1999, "Specific Proteolytic Cleavages Limit the Diversity of the Pool of Peptides Available to MHC Class I Molecules in Living Cells", J. Immunol. 7(52:4712-4719. 123. Shastri, N., T. Serwold, and F. Gonzalez, 1995, "Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C- terminal flanking residues", J. Immunol. 755:4339-4346.

124. Chou, K.C., 2000, "Prediction of tight turns and their types in proteins", Anal. Biochem. 255:1-16.

125. Davis, N.L., A. West, E. Reap, G. MacDonald, M. Collier, S. Dryga, MM Maughan, M. Connell, C. Walker, K. McGrath, C. Cecil, L.H. Ping, J. Frelinger, R. Olmstged, P. Keith, R. Swanstrom, C. Williamson, P. Johnson, D. Montefiori, and R.E. Johnston, 2002, "Alphavirus replicon particles as candidate HIV Vaccines", IUBMB Life 55:209-211.

126. Lee, J.S., B.K. Dyas, S.S. Nystrom, CM. Lind, J.F. Smith, and R.G. Ulrich, 2002, "Immune protection against Staphylococcal enterotoxin-induced toxic shock by vaccination with a Venezuelan Equine Encephalitis virus replicon", J. Infec. Dis. 755:1192-1196.

127. Hevey, M., D. Negley, L. VanderZanden, R.F. Tammariello, J. Geisbert, C. Schmaljohn, J.F. Smith, P.B. Jahrling, and A.L. Schmaljohn, 2002, "Marburg virus vacciness: comparing classical and new approaches", Vaccine 20:586-593.

128. Pratt, W.D., N.L. Davis, R.E. Johnston, J.F. Smith, 2003, "Genetically engineered, live attenuated vaccines for Venezuelan equine encephalitis: testing in animal models. 2003", Vaccine 27:3854-3862.

129. Gipson, C.L., N.L. Davis, R.E. Johnston, and A.M. deSilva, 2003, "Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease", Vaccine 27:3875-3884.

130. Nelson, E.L., D. Prieto, T.G. Alexander, P. Pushko, L. A. Lofts, J.O. Rayner, K.I. Kamrud, B. Fralish, and J.F. Smith, 2003, "Venezuelan equine encephalitis replicon immunization overcomes intrinsic tolerance and elicits effective anti-tumor immujnity to the 'self tumor-associated antigen, neu in a rat mammary tumor model", Breast Cancer Research and Treatment 52:169-183.

131. Leitner, W. W., L.N. Hwang, MJ. DeVeer, A. Zhou, R.H. Silverman, B.R.G. Williams, T.W. Dubensky, H. Ying, and N.P. Restifo, 2003, "Alphavirus-based DNA vaccine breaks immunological tolerance by activating innate antiviral pathways", Nat. Med. 9:33-39. 132. Leitner, W. W., L.N. Hwang, E.S. Bergmann-Leitner, S.E. Finkelstein, S. Frank, and N.P. Restifo, 2004, "Apoptosis is essential for the increased efficacy of alphaviral replicase-based DNA vaccines", Vaccine 22:1537-1544.

133. Garland, S.M., 2003, "Imiquimod", Curr. Opin. Infect. Dis. 76:85-89.

134. Jurk, M., F. Heil, J. Vollmer, C. Schetter, A.M. Krieg, H. Wagner, F. Lipford, and S. Bauer, 2002, "Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848", Nat. Immunol. 3:499.

135. Tyring, S.T., IArany, L, M. A. Stanley, M.A. Tomai, R.L. Miller, M.H. Smith, DJ. McDermott and H.B. Slade, 1998, "A randomized, controlled, molecular study of condlomata acuminate clearance during treatment with imiquimod", J. Infect. Dis. 775:551-555.

136. Fritz, J.H., S. Brunner, M.L. Birnstiel, M. Buschle, A. V. Gabain, F. Mattner, and W. Zauner, 2004, "The artificial antimicrobial peptide KLKLLLLLKLK induces predominantly a TH2-type immune response to co-injected antigens", Vaccine 22:3274-3284.

137. Alexander, J., J. Sidney, S. Southwood, J. Ruppert, C. Oseroff, A. Maewal, K. Snoke, H.M. Serra, R.T. Kubo, A. Sette and 1994, "Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR- blocking peptides", Immunity. 7:751-761.

138. del Guercio, M.F., J. Alexander, R.T. Kubo, T. Arrhenius, A. Maewal, E. Appella, S. L. Hoffman, T. Jones, D. Valmori, K. Sakaguchi, H.M. Grey, and A. Sette, 1997, "Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper epitopes (PADRE^®) for antibody responses in vivo", Vaccine 75:441-448.

139. Alexander, J., M.F. delGuercio, B. Frame, A. Maewal, A. Sette, M.H. Nahm, and MJ. Newman, 2004, "Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lyphocyte epitope (PADRE^®)", Vaccine 22:2362-2367.

140. Rosa, D. S., F. Tzelepis, M. G. Cunha, LS. Soares, and M.M. Redriques, 2004, "The pan HLA DR-binding epitope improves adjuvant-assisted immunization with a recombinant protein containing a malaria vaccine candidate", Immunol. Lett. 92:259- 268.

141. Vichier-Guerre, S., R. Lo-Man, L. BenMohamed, E. Deriaud, S. Kovats, C. Leclerc, and S. Bay, 2003, "Induction of carbohydrate-specific antibodies in HLA-DR transgenic mice by a synthetic glycopeptide: a potential anti cancer vaccine for human use", J. Peptide Res. 62:117-124.

142. Pamonsinlapatham, P., N. Decroix, L. Mihaila-Amrouche, A. Bouvet, and J.P. Bouvet, 2004, "Idnuction of a mucosal immune response to the Streptococcal M protein by intramuscular administration of a PADRE- ASREAK peptide.

143. Ressing, M.E., WJ. vanDriel, R.M.P. Brandt, G.G. Renter, J.H. deJong, T. Bauknecht, GJ. Fleuren, P. Hoogerhout, R. Offringa, A. Sette, E. Celis, H. Grey, BJ. Trimbos, W.M. Kast, and C.J.M. Melief, 2000, "Detection of T helper respoonses, but not a f human papillomavirus-specific cytotoxic T lymphocyte responses, after peptide vaccination of patients with cervical carcinoma", J. Immunother. 25:255-266.

144. Crotty, S., R.D. Aubert, J. Glidewell, and R. Ahmed, 2004, "Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system", J. Immunological Meth. 256:111-122.

145. Wilson, C, D.M. McKinney, M. Anders, S. MaWhinney, J. Forster, C. Crimi, S. Southwood, A. Sette, R. Chesnut, M. Newman, and B. Livingston, 2003, "Development of a DNA Vaccine Designed to Induce Cytotoxic T Lymphocyte Responses to Multiple Conserved Epitopes in HIV-I", J. Immunol. 777:5611-5623.

146. Claas, E.C., A.D. Osterhaus, R. Van Beek, J.C. De Jong, G.F. Rimmelzwaan, D. A. Senne, S. Krauss, K.F. Shortridge, and R. G. Webster, 1998, "Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus" Lancet 357:472-477.

147. Katz, J.M., X. Lu, A.M. Frace, T. Morken, S.R. Zaki, and T.M. Tumpey, 2000, "Pathogenesis and immunity to avian influenza A H5 viruses", Biomed. Pharmacother. 54:178-187.

148. Lu, X., T.M. Tumpey, T. Morken, S.R. Zaki, NJ. Cox, and J.M. Katz, 1999, "A mouse model for the evaluation of pathogenesis and immunity to influenza A (H5N1) viruses isolated from humans", J. Virol. 75:5903-5911.

149. Nicholson, K.G., A.E. Colegate, A. Podda, I. Stephenson, J. Wood, E. Ypma, and M.C. Zambon, 2001, "Safety and antigenicity of non-adjuvanted and MF59-adjuvanted influenza A/Duck/Singapore/97 (H5N3) vaccine: a randomised trial of two potential vaccines against H5N1 influenza", Lancet 357:1937.

150. Hehme, N., H. Engelmann, W. Kuenzel, E. Neumeier, and R. Saenger, 2004, "Immunogenicity of a monovalent, aluminum-adjuvanted influenza whole virus vaccine for pandemic use", Virus Res. 703:163-171. 151. Hehme, N., H. Engelmann, W. Kunzel, E. Neumeier, and R. Sanger, 2002, "Pandemic preparedness: lessons learnt from H2N2 and H9N2 candidate vaccines", Med. Microbiol. Immunol. 797:203-208.

152. Stephenson, I., K.G. Nicholson R. Gluck, R. Mischler, R.W. Newman, A.M. Palache, N.Q. Verlander, F. Warburton, J.M. Wood, and M.C. Zambon, 2003, "Safety and antigenicity of whole virus and subunit influenza A/Hong Kong/1073/99 (H9N2) vaccine in healthy adults: phase I randomised trial", Lancet 562:1959-1966.

153. Stephenson, L, K.G. Nicholson, J.M. Wood, M.C. Zambon, and J.M .Katz, 2004, "Confronting the avian influenza threat: vaccine development for a potential pandemic", The Lancet Infectious Diseases 4:499-509.

154. Wood, J.M.,and J. S. Robertson, 2004, "From lethal virus to life-saving vaccine: developing inactivated vaccines for pandemic influenza", Nat. Rev. Microbiol. 2:842- 847.

TABLE 1

-- indicates binding affinity >20,000nM

TABLE 1

-- indicates binding affinity >20,000nM

TABLE 1

-- indicates binding affinity >20,000nM

TABLE 1

-- indicates binding affinity >20,000nM

TABLE 1

-- indicates binding affinity >20,000nM

TABLE 2

indicates binding affinity >20, 00OnM

TABLE 2

-- indicates binding affinity >20,000nM

TABLE 2

-- indicates binding affinity >20,000nM

TABLE 2

(2) HLA-A03 supertype binding data of Influenza-derived 9&10mers (Minimally, peptides bind A*0301 or A*1101 ≤500nM and conserved ^30%)

-- indicates binding affinity >20,000nM

TABLE 3

-- indicates binding affinity >20,000nM

TABLE 3

( 3 ) HLA-A03 supertype binding data of Influenza-derived 9&10mers (Minimally, peptides bind A* 0301 or A*1101 ≤500nM and conserved ≥38%)

Limited to 3 peptides per protein

-- indicates binding affinity >20,000nM

TABLE 4

-- indicates binding affinity >20,000nM

TABLE 4

-- indicates binding affinity >20,000nM

TABLE 5

-- indicates binding affinity >20,000nM

TABLE 5

-- indicates binding affinity >20,000nM

TABLE 6

-- indicates binding affinity >20,000nM

TABLE 7

TABLE 7

-- indicates binding affinity >20,000nM.

TABLE 8

-- indicates binding affinity >20,000nM.

TABLE 9

-- indicates binding affinity >20,000nM

TABLE 9

-- indicates binding affinity >20,000nM

TABLE 9

-- indicates binding affinity >20,000nM

TABLE 9

-- indicates binding affinity >20,000nM

TABLE 9

-- indicates binding affinity >20,000nM

TABLE 9

-- indicates binding affinity >20,000nM

TABLE 10

-- indicates binding affinity >20,000nM

TABLE 10

-- indicates binding affinity >20,000nM

TABLE 11

-- indicates binding affinity >20,000nM

TABLE 11

(3) HLA-B44 supertype binding data of Influenza-derived 9&10mers Limited to 3 peptides per protein

-- indicates binding affinity >20,000nM

TABLE 12

-- indicates binding affinity >20,000nM.

TABLE 12

-- indicates binding affinity >20,O0OnM.

TABLE 13

-- indicates binding affinity >20, 000nM.

TABLE 14

- indicates binding affinity >20,000nM.

TABLE 15

-- indicates binding affinity >20,OO0nM.

TABLE 15

-- indicates binding affinity >20,000nM.

TABLE 16

-- indicates binding affinity >20,000nM.

TABLE 16

-- indicates binding affinity >20,000nM.

TABLE 17

-- indicates binding affinity >20,000nM.

TABLE 21

TABLE 21

TABLE 22

TABLE 23

TABLE 24

TABLE 25

TABLE 25

TABLE 25

TABLE 25

TABLE 25

TABLE 26

TABLE 26

TABLE 27

TABLE 27

TABLE 27

TABLE 27

TABLE 27

TABLE 28

TABLE 28

TABLE 28

TABLE 29

TABLE 29

TABLE 29

TABLE 29

TABLE 29

TABLE 29

TABLE 29

TABLE 30

TABLE 30

TABLE 30

TABLE 31

TABLE 31

TABLE 31

TABLE 31

TABLE 33

TABLE 34

TABLE 34

TABLE 34

A

(2) NA DRl Supertype

Minimally, peptides predicted to bind DRl ≤lOOnM and anchor conservancy ≥35%

TABLE 41

TABLE 42

TABLE 43

-- indicates binding affinity >20,000nM

TABLE 43

-- indicates binding affinity >20,000nM

TABLE 43

-- indicates binding affinity >20,000nM

TABLE 43

-- indicates binding affinity >20,000nM

TABLE 43

-- indicates binding affinity >20,000nM

TABLE 43

-- indicates binding affinity >20,000nM

TABLE 43

-- indicates binding affinity >20,000nM

TABLE 44

-- indicates binding affinity >20,0OOnM

TABLE 44

-- indicates binding affinity >20,000nM

TABLE 44

-- indicates binding affinity >20,000nM

TABLE 47

TABLE 48

— indicates binding affinity >20,OOOnM

TABLE 48

- indicates binding affinity >20,000nM

TABLE 48

- indicates binding affinity >20,000nM

TABLE 48

- indicates binding affinity >20,000nM

TABLE 48

- indicates binding affinity >20,000nM

TABLE 49

TABLE 49

TABLE 50

CTL CLASS I FLU EPITOPES

- indicates binding affinity >20,000nM

TABLE 50

TABLE SO

FEDLRVSSF NP 338 12 86 7439 9.3 9.5 25 67 35 5

GEISPLPSL NSl 158 12 86 140 4.3 5.5 40 45 82 6

- indicates binding affinity >20.000nM.

Table 51. M2e sequences from representative subtype isolates

Table 52 HLA-A3 vaccine candidate epitopes

-- indicates binding affinity >20,000nM

Table 52 HLA-A3 vaccine candidate epitopes

-- indicates binding affinity >20,OOOnM

Table 53 HLA-A24 vaccine candidate epitopes

-- indicates binding affinity >20,000nM

Table 54 HLA-B07 vaccine candidate epitopes

-- indicates binding affinity >20,OOOnM.

Table 55 HLA-AOl vaccine candidate epitopes

-- indicates binding affinity >20,000nM.

Table 56 HLA-A2 vaccine candidate epitopes

-- indicates binding affinity >20,000nM.

— indicates binding affinity >10,000nM.

- indicates binding affinity >10,000nM.

Table 58

- indicates binding affinity >10,000nM.

Table 58

- indicates binding affinity >10,000nM.

Claims

WHAT IS CLAIMED IS:

1. A isolated polynucleotide selected from the group consisting of:

(a) a multi-epitope construct comprising between five and fifty nucleic acids each encoding an influenza virus cytotoxic T lymphocyte (CTL) epitope, wherein the CTL epitope is any one of the epitopes listed in Tables 1-17, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(b) a multi-epitope construct comprising between five and fifty nucleic acids each encoding an influenza virus cytotoxic T lymphocyte (CTL) epitope, wherein the CTL epitope is any one of the epitopes listed in Tables 3, 6, 8, 11, 14 and 17, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(c) multi-epitope construct comprising between five and fifty nucleic acids each encoding an influenza virus cytotoxic T lymphocyte (CTL) epitope, wherein the CTL epitope is any one of the epitopes listed in Tables 52, 53, 54, 11, 55 and 56, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(d) a multi-epitope construct comprising between five and fifty nucleic acids each encoding an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 18-49, 57 and 58, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(e) a multi-epitope construct comprising between five and fifty nucleic acids each encoding an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 20, 22, 24, 26, 28, 30, 32, 35, 37, 39, 41, 44, 46, 49, 57 and 58 and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame; (f) a multi-epitope construct comprising between five and fifty nucleic acids each encoding an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 18, 33, 42 and 47, and wherein the nucleic acids are directly or indirectly joined to one another in the same reading frame;

(g) the multi-epitope construct of (a), further comprising any of said nucleic acids of (d), (e) or (f), directly or indirectly joined in the same reading frame to said CTL epitope nucleic acids of (a);

(h) the multi-epitope construct of (b), further comprising any of said nucleic acids of

(d), (e) or (f), directly or indirectly joined in the same reading frame to said HTL epitope nucleic acids of (d), (e) or (f);

(i) the multi-epitope construct of (c), further comprising any of said nucleic acids of

(d), (e) or (f), directly or indirectly joined in the same reading frame to said HTL epitope nucleic acids of (d), (e) or (f);

(j) the multi-epitope construct of (a) or (b) or (c) or (d) or (e) or (f) or (g) or (h) or (i), further comprising one or more spacer nucleic acids, directly or indirectly joined in the same reading frame to said CTL and/or HTL epitope nucleic acids;

(k) the multi-epitope construct of (j), wherein said one or more spacer nucleic acids are positioned between the CTL epitope nucleic acids of (a) or (b) or (c), between the

HTL epitope nucleic acids of (d) or (e) or (f), or between the CTL and/or HTL epitope nucleic acids of (g) or (h);

(1) the multi-epitope construct of (j) or (k), wherein said one or more spacer nucleic acids each encode 1 to 8 amino acids;

(m) the multi-epitope construct of any one of (j) to (1), wherein one or more of said spacer amino acid residues are selected from the group consisting of : K, R, N, Q, G, A,

S, C, and T at a C+l position of one of said CTL epitopes; (n) the multi-epitope construct of any of (j) to (m), wherein two or more of said spacer nucleic acids encode different (i.e., non- identical) amino acid sequences;

(o) the multi-epitope construct of any of (j) to (n), wherein two or more of said spacer nucleic acids encode an amino acid sequence different from an amino acid sequence encoded by one or more other spacer nucleic acids;

(p) the multi-epitope construct of any of (j) to (o), wherein two or more of the spacer nucleic acids encodes the identical amino acid sequence;

(q) the multi-epitope construct of any of (j) to (p), wherein one or more of said spacer nucleic acids encode an amino acid sequence comprising or consisting of three consecutive alanine (Ala) residues;

(r) the multi-epitope construct of (j) to (q), wherein one or more of said spacer nucleic acid encodes an amino acid sequence selected from the group consisting of: an amino acid sequence comprising or consisting of GPGPG (SEQ ID NO: ), an amino acid sequence comprising or consisting of PGPGP (SEQ ID NO: ), an amino acid sequence comprising or consisting of (GP)n, an amino acid sequence comprising or consisting of (PG)n, an amino acid sequence comprising or consisting of (GP)nG, and an amino acid sequence comprising or consisting of (PG)nP, where n is an integer between zero and eleven;

(s) the multi-epitope construct of any of (a) to (r), further comprising one or more nucleic acids encoding one or more HTL epitopes, directly or indirectly joined in the same reading frame to said CTL and/or HTL epitope nucleic acids and/or said spacer nucleic acids;

(t) the multi-epitope construct of (s), wherein said one or more HTL epitopes comprises a pan-DR binding epitope;

(u) the multi-epitope construct of any of (a) to (t), further comprising one or more

MHC Class I and/or MHC Class II targeting nucleic acids; (v) the multi-epitope construct of (u), wherein said one or more targeting nucleic acids encode one or more targeting sequences selected from the group consisting of : an Ig kappa signal sequence, a tissue plasminogen activator signal sequence, an insulin signal sequence, an endoplasmic reticulum signal sequence, a LAMP-I lysosomal targeting sequence, a LAMP-2 lysosomal targeting sequence, an HLA-DM lysosomal targeting sequence, an HLA-DM-association sequence of HLA-DO, an Ig-a cytoplasmic domain, Ig-ss cytoplasmic domain, a Ii protein, an influenza matrix protein, an HCV antigen, and a yeast Ty protein;

(w) the multi-epitope construct of any of (a) to (v), which is optimized for CTL and/or HTL epitope processing;

(x) the multi-epitope construct of any of (a) to (w), wherein said CTL and/or HTL nucleic acids are sorted to minimize the number of CTL and/or HTL junctional epitopes encoded therein;

(y) the multi-epitope construct of any of (a)-(x), wherein said multi-epitope construct consists of the epitopes listed in Table 50;

(z) the multi-epitope construct of any of (a)-(x) wherein the influenza virus CTL and/or HTL epitopes are directly or indirectly joined in the order shown in Figure 6.

2. The polynucleotide of claim 1, wherein said CTL epitope is from about 8 to about 13 amino acids in length.

3. The polynucleotide of any one of claims 1-2, wherein said HTL epitope is from about 6 to about 30 amino acids in length.

4. The polynucleotide of any one of claims 1-3, wherein said influenza virus CTL and/or HTL epitope is from a polypeptide at least 90% identical to an influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), RNA polymerase subunit PA, RNA polymerase basic protein 1 (PBl), RNA polymerase basic protein 2 (PB2), nonstructural gene 1 (NSl), nonstructural gene 2 (NS2), matrix protein 1 (Ml) or matrix protein 2 (M2) polypeptide.

5. The polynucleotide of any one of claims 1-4, wherein said influenza virus CTL and/or HTL epitope is from an influenza strain selected from the group consisting of: Human A/Viet Nam/1203/2004 (H5N1), Human A/Hong Kong/156/97 (H5N1), Human A/Hong Kong/483/97 (H5N1), Human A/Hong Kong/1073/99 (H9N2), Avian A/Chicken/HK/G9/97 (H9N2), Swine A/Swine/Hong Kong/10/98 (H9N2), Avian A/FPV/Rostock/34 (H7N1), Avian A/Turkey/Italy/4620/99 (H7N1), Avian A/FPV/Weybridge/34 (H7N7 ), Human A/New Caledonia/20/99 (HlNl), Human A/Hong Kong/1/68 (H3N2), Human A/Shiga/25/97 (H3N2), Human A/Singapore/ 1/57 (H2N2), Human A/Leningrad/ 134/57 (H2N2), Human A/Ann Arbor/6/60 (H2N2), Human A/Brevig Mission/1/18 (HlNl), Swine A/Swine/Wisconsin/464/98 (HlNl), Human A/Netherlands/219/03 (H7N7).

6. The polynucleotide of any one of claims 1-5, wherein said CTL epitope comprises a Class I HLA motif selected from the group consisting of HLA-Al, HLA- A2, HLA- A3, HLA- A24, HLA-B7 and HLA-B44.

7. The polynucleotide of any one of claims 1-6 comprising at least one HLA-Al epitope, at least one HLA- A2 epitope, at least one HL A- A3 /Al 1 epitope, at least one HLA- A24 epitope, at least one HLA-B7 epitope, or at least one HLA-B44 epitope; or any combinations thereof.

8. The polynucleotide of any one of claims 1 -7, wherein said CTL epitope is any one of the HLA- A3 epitopes listed in Tables 1-3 or 52.

9. The polynucleotide of claim 8, wherein said HLA- A3 epitope is any one of the epitopes listed in Table 3.

10. The polynucleotide of claim 8, wherein said HLA- A3 epitope is any one of the epitopes listed in Table 52.

11. The polynucleotide of any one of claims 1-10, wherein said CTL epitope is any one of the HLA- A24 epitopes listed in Tables 4-6 or 53.

12. The polynucleotide of claim 11, wherein said HLA-A24 epitope is any one of the epitopes listed in Table 6.

13. The polynucleotide of claim 11, wherein said HLA- A24 epitope is any one of the epitopes listed in Table 53.

14. The polynucleotide of any one of claims 1-13, wherein said CTL epitope is any one of the HLA-B7 epitopes listed in Tables 7-8 or 54.

15. The polynucleotide of claim 14, wherein said HLA-B7 epitope is any one of the epitopes listed in Table 8.

16. The polynucleotide of claim 14, wherein said HLA-B7 epitope is any one of the epitopes listed in Table 54.

17. The polynucleotide of any one of claims 1-16, wherein said CTL epitope is any one of the HLA-B44 epitopes listed in Tables 9-11.

18. The polynucleotide of claim 17, wherein said HLA-B44 epitope is any one of the epitopes listed in Table 11.

19. The polynucleotide of any one of claims 1-18, wherein said CTL epitope is any one of the HLA-Al epitopes listed in Tables 12-14 or 55.

20. The polynucleotide of claim 19, wherein said HLA-Al epitope is any one of the epitopes listed in Table 14.

21. The polynucleotide of claim 19, wherein said HLA-Al epitope is any one of the epitopes listed in Table 55.

22. The polynucleotide of any one of claims 1-21, wherein said CTL epitope is any one of the HLA-A2 epitopes listed in Tables 15-17 or 56.

23. The polynucleotide of claim 22, wherein said HLA- A2 epitope is any one of the epitopes listed in Table 17.

24. The polynucleotide of claim 22, wherein said HLA-A2 epitope is any one of the epitopes listed in Table 56.

25. The polynucleotide of any one of claims 1-24, wherein said HTL epitope comprises a Class π HLA motif selected from the group consisting of HLA-DRl and HLA-DR3.

26. The polynucleotide of any one of claims 1-25, wherein said HTL epitope is any of the DR epitopes listed in Tables 48-49, 57 or 58.

27. The polynucleotide of claim 26, wherein said DR epitope is any one of the epitopes listed in Table 49.

28. The polynucleotide of claim 26, wherein said DR epitope is any one of the epitopes listed in Table 58.

29. The polynucleotide of any one of claims 1-22, wherein said HTL epitope is any one of the DRl epitopes listed in Tables 18-39.

30. The polynucleotide of any one of claims 1-29, wherein said HTL epitope is any one of the NA DRl epitopes listed in Table 18.

31. The polynucleotide of any one of claims 1-30, wherein said HTL epitope is any one of the NP DRl epitopes listed in Tables 19-20.

32. The polynucleotide of claim 31, wherein said NP DRl epitope is any one of the epitopes listed in Table 20.

33. The polynucleotide of any one of claims 1-32, wherein said HTL epitope is any one of the NSl DRl epitopes listed in Tables 21-22.

34. The polynucleotide of claim 33, wherein said NSl DRl epitope is any one of the epitopes listed in Table 22.

35. The polynucleotide of any one of claims 1-34, wherein said HTL epitope is any one of the NS2 DRl epitopes listed in Tables 23-24.

36. The polynucleotide of claim 35, wherein said NS2 DRl epitope is any one of the epitopes listed in Table 24.

37. The polynucleotide of any one of claims 1-36, wherein said HTL epitope is any one of the PA DRl epitopes listed in Tables 25-26.

38. The polynucleotide of claim 37, wherein said PA DRl epitope is any one of the epitopes listed in Table 26.

39. The polynucleotide of any one of claims 1-38, wherein said HTL epitope is any one of the PBl DRl epitopes listed in Tables 27-28.

40. The polynucleotide of claim 39, wherein said PBl DRl epitope is any one of the epitopes listed in Table 28.

41. The polynucleotide of any one of claims 1-40, wherein said HTL epitope is any one of the PB2 DRl epitopes listed in Tables 29-30.

42. The polynucleotide of claim 41, wherein said PB2 DRl epitope is any one of the epitopes listed in Table 30.

43. The polynucleotide of any one of claims 1-42, wherein said HTL epitope is any one of the HA DRl epitopes listed in Tables 31-33.

44. The polynucleotide of claim 43, wherein said HA DRl epitope is any one of the epitopes listed in Table 32.

45. The polynucleotide of claim 44, wherein said HA DRl epitope is any one of the epitopes listed in Table 33.

46. The polynucleotide of any one of claims 1-45, wherein said HTL epitope is any one of the Ml DRl epitopes listed in Tables 34-35.

47. The polynucleotide of claim 46, wherein said Ml DRl epitope is any one of the epitopes listed in Table 35.

48. The polynucleotide of any one of claims 1-47, wherein said HTL epitope is any one of the M2 DRl epitopes listed in Tables 36-37.

49. The polynucleotide of claim 48, wherein said M2 DRl epitope is any one of the epitopes listed in Table 37.

50. The polynucleotide of any one of claims 1-49, wherein said HTL epitope is any one of the NA DRl epitopes listed in Tables 38-39.

51. The polynucleotide of claim 50, wherein said NA DRl epitope is any one of the epitopes listed in Table 39.

52. The polynucleotide of claims 1-51, wherein said HTL epitope is any one of the DR3 epitopes listed in Tables 40-47.

53. The polynucleotide of any one of claims 1-52, wherein said HTL epitope is any one of the DR3 epitopes listed in Tables 43-44.

54. The polynucleotide of claim 53, wherein said NA DR3 epitope is any one of the epitopes listed in Table 44.

55. The polynucleotide of any one of claims 1-54, wherein said HTL epitope is any one of the NA DR3 epitopes listed in Tables 40-42.

56. The polynucleotide of claim 55, wherein said NA DR3 epitope is any one of the epitopes listed in Table 41.

57. The polynucleotide of claim 56, wherein said NA DR3 epitope is any one of the epitopes listed in Table 42.

58. The polynucleotide of any one of claims 1-57, wherein said HTL epitope is any one of the HA DR3 epitopes listed in Tables 45-47.

59. The polynucleotide of claim 58, wherein said HA DR3 epitope is any one of the epitopes listed in Table 46.

60. The polynucleotide of claim 59, wherein said HA DR3 epitope is any one of the epitopes listed in Table 47.

61. The polynucleotide of any one of claims 1-60, wherein said pan-DR binding epitope comprises the amino acid sequence AFKV AA WTLKAAA (SEQ ID NO:_).

62. The polynucleotide of any one of claims 1-61, further comprising a nucleic acid encoding a targeting sequence located at the N-terminus of said construct.

63. The polynucleotide of claim 62, wherein said targeting sequence is selected from the group consisting of: an Ig kappa signal sequence, a tissue plasminogen activator signal sequence, an insulin signal sequence, an endoplasmic reticulum signal sequence, a LAMP-I lysosomal targeting sequence, a LAMP-2 lysosomal targeting sequence, an HLA-DM lysosomal targeting sequence, an HLA-DM-association sequence of HLA-DO, an Ig-a cytoplasmic domain,Ig-ss cytoplasmic domain, a Ii protein, an influenza matrix protein, an HCV antigen, and a yeast Ty protein.

64. The polynucleotide of any one of claims 1-63, wherein said multi-epitope construct comprises between 10 and 70 nucleic acids encoding influenza virus CTL and/or HTL epitopes.

65. The polynucleotide of any one of claims 1-64, wherein said multi-epitope construct comprises between 10 and 60 nucleic acids encoding influenza virus CTL and/or HTL epitopes.

66. The polynucleotide of any one of claims 1-65, wherein said multi-epitope construct comprises between 10 and 50 nucleic acids encoding influenza virus CTL and/or HTL epitopes.

67. The polynucleotide of any one of claims 1-66, wherein said CTL epitope is from about 8 to about 11 amino acids in length.

68. The polynucleotide of any one of claims 1-67, wherein said CTL epitope is from about 9 to about 10 amino acids in length.

69. The polynucleotide of any one of claims 1-68, wherein said HTL epitope is from about 8 to about 20 amino acids in length.

70. The polynucleotide of any one of claims 1-69, wherein said HTL epitope is from about 12 to about 18 amino acids in length.

71. The polynucleotide of any one of claims 1-70, further comprising one or more regulatory sequences.

72. The polynucleotide of claim 71, wherein said one or more regulatory sequences comprises an IRES element.

73. The polynucleotide of claim 71, wherein said one or more regulatory sequences comprises a promoter.

74. A polypeptide encoded by the polynucleotide of any one of claims 1-73.

75. The polypeptide of claim 74, further comprising a pan-DR binding epitope.

76. The polypeptide of claim 75, wherein said pan-DR binding epitope comprises the amino acid sequence aiKXV AA WTLKAAa₂, where "X" is selected from the group consisting of cyclohexylalanine, phenylalanine, and tyrosine; and "ai" is either D-alanine or L-alanine; and "a₂" is either D-alanine or L-alanine.

77. The polypeptide of claims 74-76, wherein said polypeptide is from about 10 to about 2000 amino acids in length.

78. A vector comprising the polynucleotide of any one of claims 1-73.

79. The vector of claim 78, wherein said vector is an expression vector.

80. A composition comprising the polynucleotide of any one of claims 1-73, the polypeptide of any one of claims 74-77, or the vector of any one of claims 78-79.

81. The composition of claim 80, further comprising an influenza HA or NA polypeptide, wherein said HA polypeptide is encoded by a sequence 90% identical to a wild-type HA sequence from an influenza strain selected from the group consisting of: Human A/Viet Nam/1203/2004 (H5N1), Human A/Hong Kong/156/97 (H5N1), Human A/Hong Kong/483/97 (H5N1), Human A/Hong Kong/1073/99 (H9N2), Avian A/Chicken/HK/G9/97 (H9N2), Swine A/Swine/Hong Kong/10/98 (H9N2), Avian A/FPV/Rostock/34 (H7N1), Avian A/Turkey/Italy/4620/99 (H7N1), Avian A/FPV/Weybridge/34 (H7N7 ), Human A/New Caledonia/20/99 (HlNl), Human A/Hong Kong/1/68 (H3N2), Human A/Shiga/25/97 (H3N2), Human A/Singapore/ 1/57 (H2N2), Human A/Leningrad/ 134/57 (H2N2), Human A/Ann Arbor/6/60 (H2N2), Human A/Brevig Mission/1/18 (HlNl), Swine A/Swine/Wisconsin/464/98 (HlNl), Human A/Netherlands/219/03 (H7N7).

82. A composition comprising the polypeptide of any one of claims 74-77 and a carrier.

83. The composition comprising the polypeptide of any one of claims 74-77 and a lipid.

84. The composition comprising the polypeptide of any one of claims 74-77 and a liposome.

85. The composition comprising the polypeptide of any one of claims 74-77 and a virosome.

86. The composition of claim 85, wherein said virosome is an immunopotentiating reconstituted influenza virosome (IRTV).

87. A cell comprising the polynucleotide of any one of claims 1-73, the polypeptide of any one of claims 74-77, or the vector of any one of claims 78-79.

88. A method of making the polynucleotide of any of claims 1-73, the polypeptide of any one of claims 74-77, or the vector of any one of claims 78-79, comprising culturing the cell of claim 87, and recovering said polynucleotide, vector, or polypeptide.

89. A method of inducing an immune response against influenza virus in an individual in need thereof, comprising administering to said individual the composition of any one of claims 80-86.

90. The polynucleotide of any one of claims 1-73, comprising, consisting essentially of, or consisting of the epitopes listed in Table 50.

91. The polynucleotide of claim 90 comprising, consisting essentially of, or consisting of SEQ ID NO:_.

92. A polypeptide composition selected from the group consisting of:

(a) a composition comprising between five and fifty peptides, each peptide comprising an influenza virus cytotoxic T lymphocyte (CTL) epitope, wherein the CTL epitope is any one of the epitopes listed in Tables 1-17, or 52-56; (b) a composition comprising between five and fifty peptides, each peptide comprising an influenza virus cytotoxic T lymphocyte (CTL) epitope, wherein the CTL epitope is any one of the epitopes listed in Tables 3, 6, 8, 11, 14 ,17 52-56;

(c) a composition comprising between five and fifty peptides, each peptide comprising an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 18-49 or 57-58;

(d) a composition comprising between five and fifty peptides, each peptide comprising an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 20, 22, 24, 26, 28, 30, 32, 35, 37, 39, 41, 44, 46, 49, 57 and 58;

(e) a composition comprising between five and fifty peptides, each peptide comprising an influenza virus helper T lymphocyte (HTL) epitope wherein the HTL epitope is any one of the epitopes listed in Tables 18, 33, 42 and 47;

(f) the composition of (a), further comprising one or more of said epitopes of (c), (d), or (e); and

(g) the composition of (b), further comprising one or more of said epitopes of (c), (d) or (e).

93. The polypeptide composition of claim 92, wherein said CTL epitope is from about 8 to about 11 amino acids in length.

94. The polypeptide composition of claim 92, wherein said CTL epitope is from about 9 to about 10 amino acids in length.

95. The polypeptide composition of claims 92, wherein said HTL epitope is from about 8 to about 20 amino acids in length.

96. The polypeptide composition of claim 92, wherein said HTL epitope is from about 12 to about 18 amino acids in length.

97. The polypeptide composition of claim 92, further comprising a carrier.

98. The polypeptide composition of claim 92, further comprising a heterologous polypeptide.

99. The polypeptide composition of claim 92, further comprising a lipid.

100. The polypeptide composition of claim 92, further comprising a liposome.

101. The polypeptide composition of claim 92, further comprising a virosome.

102. The polypeptide composition of claim 92, further comprising an immunopotentiating reconstituted influenza virosome (ERTV).

103. The polypeptide composition of claim 92, further comprising a targeting sequence.

104. The polypeptide composition of claim 103, wherein said targeting sequence is selected from the group consisting of: an Ig kappa signal sequence, a tissue plasminogen activator signal sequence, an insulin signal sequence, an endoplasmic reticulum signal sequence, a LAMP-I lysosomal targeting sequence, a LAMP-2 lysosomal targeting sequence, an HLA- DM lysosomal targeting sequence, an HLA-DM-association sequence of HLA-DO, an Ig-a cytoplasmic domain, Ig-ss cytoplasmic domain, a Ii protein, an influenza matrix protein, an HCV antigen, and a yeast Ty protein.