CA2949851A1 - Htert sequences and methods for using the same - Google Patents

Htert sequences and methods for using the same Download PDF

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CA2949851A1
CA2949851A1 CA2949851A CA2949851A CA2949851A1 CA 2949851 A1 CA2949851 A1 CA 2949851A1 CA 2949851 A CA2949851 A CA 2949851A CA 2949851 A CA2949851 A CA 2949851A CA 2949851 A1 CA2949851 A1 CA 2949851A1
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seq
fragments
sequences
nucleotides
homology
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CA2949851C (en
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David B. Weiner
Jian Yan
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University of Pennsylvania Penn
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Abstract

Proteins and nucleic acid molecules for inducing an immune response against human telomerase reverse transcriptase ("hTERT") are provided. Pharmaceutical compositions, recombinant vaccines, and live attenuated pathogens for inducing an immune response against hTERT are also provided.

Description

= CA 02949851 2016-11-28 IMPROVED HPV VACCINES
AND METHODS FOR USING THE SAME
= FIELD OF THE INVENTION
The present invention relates to improved HIV, HPV, HCV, Influenza and cancer vaccines, improved methods for inducing immune responses, and for prophylactically and/or therapeutically immunizing individuals against HIV, HPV, HCV, Influenza and cancer.
BACKGROUND OF THE INVENTION
The HIV genome is highly plastic due to a high mutation rate and functional compensation. This high mutation rate is driven by at least two mechanisms:
the low fidelity of the viral reverse transcriptase (RT) resulting in at least one mutation per replication cycle, and the dual effects of the anti-retroviral cellular factor APOBEC3G gene and viral infectivity factor Vif accessory gene. Genomes with every possible mutation and many double mutations are generated during every replication cycle, resulting in tremendous antigenic diversity.
Accordingly, it has been argued that a candidate vaccine derived from an individual isolate may not elicit sufficient cross reactivity to protect against diverse circulating HIV viruses. Recent studies have suggested that consensus immunogens (Gao, F., et al. 2005.
Antigenicity and inirnunogenicity of a synthetic human immunodeficiency virus type I group m consensus envelope glycoprotein. J Virol 79:1154-63.; Scriba, T. J., et al. 2005.
Functionally-inactive and immunogenic Tat, Rev and Nef DNA vaccines derived from sub-Saharan subtype C
human immunodeficiency virus type 1 consensus sequences. Vaccine 23:1158-69) or ancestral irnmunogens (Doria-Rose, N. A., et al. 2005. Human Immunodeficiency Virus Type I subtype B
Ancestral Envelope Protein Is Functional and Elicits Neutralizing Antibodies in Rabbits Similar to Those Elicited by a Circulating Subtype B Envelope. J. Virol. 79:11214-11224; Gao, F., et al.

2004. Centralized immunogens as a vaccine strategy to overcome HIV-1 diversity. Expert Rev.
Vaccines 3:S161-S168; Mullins, 1 1.. et al. 2004. Immunagen sequence: the fourth tier of AIDS
vaccine design.. Expert Rev. Vaccines 3:S151-S159; Nickle, D. C., et al. 2003.
Consensus and ancestral state HIV vaccines. Science 299:1515-1517) may be aselid in this regard. However, the initiai studies of these approaches showed relatively modest cellular immune enhancement induced by these immunogcns.
R.ccently Derdeyn et al. analyzed HIV-1 subtype C envelope glyeoprotein sequences in eight African heterosexual transmission pairs and found that shorter V1, V2 and V4 length and fewer glyeans are the common features shared by the sequences obtained from early transmitters (Derdeyn, C. A., et al. 2004. Envelope-constrained neutralization-sensitive 1-IIV-1 after heterosexual transmission. Science 303:2019-2022.). This data suggests that antigens that mimic such viruses might have relevance for the early-transmitted viruses. However, such early =
transmitter structures have not been observed for all subtypes (Cholla!), B., et al. 2005. Selection for Human Immunodeficiency Virus Type 1 envelope glycosylation variants with shorter V1-V2 loop sequences occurs during transmission of certain genetic subtypes and may impact viral RNA levels. J. Virol, 79:6528-6531). However, incorporation of shorter V loops itt an envelope immunogen may have other benefits, such as enhancement of sensitivity to soluble CD4 (Pickora, C., et al. 2005. Identification of two N-linked glycosylation sites within the core of the Simian Immunodificicncy virus glyeoprotcin whose removal enhances sensitivity to soluble CD4. J. Virol. 79:12575-12583), and should be considered.
Studies have shown the importance of H1V-1 specific CIL responses in controlling viral load during acute and asymptomatic infection and the development of AIDS.
However, it is unclear if current envelope based DNA vaccines are as potent as needed.
Several methods have been used to increase the expression levels of HIV-1 immunogens, such as cotton optimization (Andre, S., et al. 1998. Increased immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage. J Virol 72:1497-503; Deml, L., et al. A. 2001.
Multiple effects of eodon usage optimization on expression and immunogenicity of DNA
candidate vaccines encoding the human immunodeficiency virus type 1 gag protein. J. Virol.
75: I 0991-11001), RNA optimization (Muthumani, K., et al. 2003. Novel engineered East African Clade-A gp160 pl.asmid construct induces strong Immoral and cell-mediated immune responses in vivo. Virology 314:134-46; Schneider, R., M. et al. 1997.
Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gagfprotease and particle formation. S. Virol. 71:4892-4903) and the addition of immunoglobin leader sequences that have weak RNA secondary structure (Yang, 3.
S., et al..
2001. Induction of potent Th.1-Type immune responses from a novel DNA vaccine for West Nile Virus New York Isolate (WNV-NY1999). J. Infect Diseases 184:809-816).
Human Papillomavirus (HPV) has a circular dsDNA genome (7,0-00-8,000 base pairs).
There are up to 200 different genotypes. Phylogenetically, HPV is highly conserved. Mucosa]
HPV are Classified as "High Risk" or "Low Risk". The Low Risk group includes types 6, 11, 42, and others. Associated Diseases include: Genital Warts; Low grade cervical, anal, vulvar, vaginal dysplasia; and Recurrent Respiratory Papillomatosis, The High Risk group includes types 16, 18, 31, 33, 45, 52, 58, and others. Associated Diseases include:
Essential cause of Cervical cancer, pre-cancerous dysplasia; major cause of Anal, vulvar, vaginal, tonsillar cancer;
and co-factor for other aerodigcstive cancer. Every Day, SOO women die of cervical cancer.
HPV E6 and E7 proteins are tumor-specific antigens, required for tumorigenesis and maintenance of the tumor state. 137-specific immune responses are deleted in cervical cancer patients. Both E6 and E7 proteins interact specifically with the products of cellular human tumor suppressor genes, E6 with p53 and E7 with Rti (retinoblastoma tumor suppressor gene). E6 and E7 are ideal immunotherapeutic targets.
hTERT is a human telomerase reverse transcriptase that synthesizes a TTAGGG
tag on the end of telomercs to prevent cell death due to chromosomal shortening.
Embryonic cells and SOTTIC germ line cells normally express hTERT to regulate homeostasis of cell populations.
Cancer cells, however, exploit this mechanism of regulation to disrupt homeostasis of cell populations. For instance, hTERT over-expression occurs in .more than 85% of human cancer cells. Therefore, hTERT is an ideal immunotherapeutic target.
hTERT may also enhance immunotherapeutics against hyperproliferating cells expressing hTERT due to HCV or HPV infection. The E6 oncoprotein from high-risk 'HPV
types activates human telomerase reverse transcriptase (hTERT) transcription in human keratinocytes.

=

=Dysplastic legions and early neoplastic legions within the liver also express hTERT at abnormally high levels. Thus, immunotherapy against HPV and HCV may be enhanced by targeting cells that express hTERT at abnormal levels. Combination immunotherapy using both IITERT and EIPV or }{CV proteins or nucleic acids encoding such proteins is an attractive immunotherapy.
Influenza Hemagglutinin (HA.) is expressed on the surface of influenza viral particles and is responsible for initial contact between the virus and its host cell. HA is a well-known immunogen. Influenza A strain Ii1N5, an avian influenza strain, particularly threatens the human population because of its HA protein which, if slightly genetically reassorted by natural mutation, has greatly increased infectivity of human cells as compared to other strains of the virus, Infection ofinfants and older or immunocompromised adults humans with the viral II1N5 strain is often correlated to poor clinical outcome. Therefore, HA and other influenza molecules = of the t-IIN5 strain of Influenza are ideal inununotherapeutic targets.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid constructs and proteins encoded thereby which provide improved immunogenic targets against which an anti-HIV immune response can be generated.
The present. invention provides consensus sequences for HIV Subtype A Envelope protein, consensus sequences for HIV Subtype B Envelope protein, consensus sequences for HIV
Subtype C Envelope protein, consensus sequences for HIV Subtype D Envelope protein, consensus sequences for HIV Subtype B consensus Nef-Rev protein, and consensus sequences form HIV Gag protein subtypes A, 13, C and D.
The present invention provides constructs which encode such proteins sequences, vaccines which comprise such proteins and/or nucleic acid molecules that encode such proteins, = an.d methods of indu ii cin anti-HIV immune responses.
The present invention relates to nucleic acid molecules comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO: l; fragments of SEQ ID NO:1;
sequences having at least 90% homology to SEQ ID NO:1; fragments of sequences having at least 90%

homology to SEQ ID NO: 1; SEQ ID NO:3; fragments of SEQ ID NO:3; seq-uences having at least 90% homology to SEQ ID NO:3; fragments of sequences having at least 90%
homology to SEQ ID NO:3; SEQ ID NO:5; fragments of SEQ ID NO:5; sequences haying at least 90%
homology to SEQ ID -N0:5; fragments of sequences having at least 90% homology to SEQ ID
NO:5; SEQ ID NO:7; fragments of SEQ 1.D NO:7; sequences having at least 90%
homology to SEQ ID NO:7; fragments of sequences having at least 90% homology to SEQ ID
NO:7; SEQ ID
NO:9; fragments of SEQ ID NO:9:, sequences having at least 90% homology to SEQ
NO:9;
fragments of sequences having at least 90% homology to SEQ ID NO:9; SEQ ID
NO:1 I;
fragments of SEQ ID NO:11; sequences having at least 90% homology to SEQ ID
NO: I. I;
fragments of sequences having at least 90% homology to SEQ ID NO:11.
The present invention relates to nucleic acid molecule that encode a protein selected from the group consisting of: SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID
NO:19; SEQ
ID NO:20 and SEQ ID NO:21.
Th.e present invention relates to nucleic- acid molecules comprising a nucleotide sequence selected from the group consisting of: nucleotide sequences that encode SEQ ID
NO:2;
nucleotide sequences that encode an amino acid sequences having at least 90%
homology to SEQ .ID NO:2; fragments of nucleotide sequences that encode SEQ ID NO:2;
fragments of a nucleotide sequence that encode an amino acid sequence having at least 90%
homology to SEQ
ID NO:2; nucleotide sequences that encode SEQ ID NO:4; nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID NO:4; fragments of nucleotide sequences that encodes SEQ ID NO:4; fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:4; nucleotide sequences that encode SEQ ID NO:6; nucleotide sequences that encode an amino acid sequences haying at least 90%
homology to SEQ ID NO:6; fragments of nucleotide sequences that encode SEQ ID
NO:6;
fragments of a nucleotide sequence that encode an amino acid sequence having at least 90%
homology to SEQ ID NO:6; nucleotide sequences that encode SEQ ID NO:8;
nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID
NO:8; fragments c f nucleotide sequences that encodes SEQ .ID NO:8; fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:8;
-5-.

nucleotide sequences that encode SEQ 1D NO:10; nucleotide sequences that encode an amino acid sequences having at least 90% homology to SEQ ID NO:10; fragments of nucleotide sequences that encode SEQ ID NO:10; fragments of a nucleotide sequence that encode an amino acid sequence having at least 90%, homology to SEQ ID NO:10; nucleotide sequences that encode SEQ ID NO:12; nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ NO:12; fragments of nucleotide sequences that encodes SEQ ID
.N0:12; fragments of nucleotide sequences that encodes an amino acid sequence braving at least 90% homology to SEQ NO:12.
Thc present invention further provides pharmaceutical compositions comprising such nucleic acid molecules and their use in methods of inducing an im.mune response in an individual against HIV that comprise administering to an individual a composition comprising such nucleic acid molecules.
The present invention further provides recombinant vaccine comprising such nucleic acid = molecules and their use in methods of inducing an immune response in an individual against .H1V that comprise administering to an individual such a recombinant vaccine.
The present invention further provides live attenuated pathogens comprising such nucleic acid molecules and their use in methods of inducing an immune response in an individual against ITIV that comprise administering to an individual such live attenuated pathogens.
live attenuated pathogen The present invention further provi.dcs proteins comprising amino acid sequences selected from the group consisting of: SEQ ID NO:2, sequences having at least 90%, homology to SEQ ID NO:2; fragments of SEQ NO:2; fragments of sequences having at least 90%
homology to SEQ NO:2; SEQ ID NO:4, sequences having at least 90% homology to SEQ ID
NO:4; fragments of SEQ ID NO:; fragments of sequences having at least 90%
homology to SEQ
ID NO:4; SEQ ID NO:6, sequences having at least 90% homology to SEQ ID NO:6;
fragments of SEQ ID NO:6; fragments of sequences having at least 90% homology to SEQ ID
NO:6; SEQ
ID NO:8, sequences having at least 90% homology to SEQ 1D NO:8; fragments of SEQ ID
NO:8; .fragments of sequences having at least 90% homology to SEQ ID NO:; SEQ
ID NO:10, sequences having at least 90% homology to SEQ ID NO:10; fragments of SEQ ID
NO:10;

fragments of sequences having at least 90% homology to SEQ NO:10; SEQ. ID
NO:12, sequences having at least 90% homology to SEQ ID NO:12; fragments of SEQ ID
NO: and fragments of sequences having at least 90% homology to SEQ ID NO:12.
The present invention further provides proteins comprising amino acid sequ.ences selected from the group consisting of SEQ ID NO:16; SEQ ID NO:17; SEQ ID
NO:18; SEQ ID
NO:19; SEQ ID NO:20 and SEQ ID NO:21.
The present invention further provides pharmaceutical compositions comprising such proteins and their use in methods of inducing an immune response in an individual against HIV
that comprise administering to an individual a composition comprising such proteins.
The present invention further provides recombinant vaccine comprising such proteins and their use in methods of inducing an immune response in an individual against HIV that comprise administering to an individual such a recombinant vaccine.
The present invention further provides live attenuated pathogens comprising such proteins and their use. in methods of inducing an immune response in an individual against HIV
that comprise administering to an individual such live attenuated pathogens.
Proteins comprising consensus HPV genotype 16 E6-E7 amino acid sequences and nucleic acid molecules that comprising a. nucleotide sequence encoding such proteins are provided.
The present invention relates to nucleic acid molecules that comprising a nucleotide sequence selected =from the group consisting of: SEQ ID NO:22; fragments thereof nucleotide sequences having at least 90% homology to SEQ NO:22; and fragments thereof The present 'invention also relates to nucleic acid molecules that comprising a nucleotide sequence selected from the group consisting of: a nucleic acid sequence that encodes SEQ ID
NO:23; a nucleic acid sequence that encodes SEQ ID NO:24; a nucleic acid sequence that encodes SEQ ID NO:25; a nucleic acid sequence that encodes SEQ ID NO:26; and a nucleic acid sequence that encodes SEQ ID NO:27.
The present invention also relates to pharmaceutical composition such nucleic acid molecules and to methods of inducing an immune response in an individual against HPV

comprising administering to said individual a composition comprising such nucleic acid molecules.
The present invention further relates to recombinant vaccines comprising such nucleic acid molecules and methods of inducing an immune response in an individual against HPV
comprising administering to said individual such a recombinant vaccine.
The present invention finther relates to live attenuated pathogen comprising such nucleic acid molecules and methods of inducing an immune response in an individual against HPV
comprising administering to said individual such live attenuated pathogens.
The present invention also relates to nucleic acid molecules that comprising a nucleotide sequence selected from the group consisting of: proteins comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:23, fragments thereof;
nucleotide sequences having at least 90% homology to SEQ ID NO:23; and fragments thereof.
The present invention also relates to= proteins comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID
NO:26;
and SEQ ID NO:27.
The present invention also relates to pharmaceutical compositions comprising such proteins and to methods of inducing an immune response in an individual against HPV
comprising administering to said individual a composition comprising such proteins.
The present invention also relates to recombinant vaccines comprising such proteins and to method of inducing an immune response in an individual against HPV
comprising administering to said individual such recombinant vaccines.
The present invention. also relates to live attenuated- pathogens comprising such protein and to methods of inducing an immune response in an individual against HPV
comprising administering to said individual such live attenuated pathogens.
Proteins comprising consensus HCV genotype la and lb El -E2 amino acid sequences and nucleic acid molecules that comprising a nucleotide sequence encoding such proteins are provided.
-8..

The present invention relates to nucleic acid molecules that comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:30; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID NO:30; and fragments thereof.
The present invention also relates to nucleic acid molecules that comprising a nucleotide sequence selected from the group consisting of: a nucleic acid sequence that encodes SEQ ID
NO:3l.
The present invention also relates to pharmaceutical composition such nucleic acid molecules and to methods of inducing an immune response in an individual against HCV
comprising administering to said individual a composition comprising such nucleic acid.
molecules.
The present invention further relates to recombinant vaccines comprising such nucleic acid molecules and methods of inducing an immune response in an individual against tICV
comprising administering to said individual such a recombinant vaccine.
The present invention further relates to live attenuated pathogen comprising such nucleic acid molecules and methods of inducing an immune response in an individual against FICV
comprising administering to said individual such live attenu.ated pathogens.
The present invention also relates to nucleic acid molecules that comprising a nucleotide sequence selected from. the group consisting of: proteins comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:31; fragments thereof;
nucleotide sequences having at least 90% homology to SEQ ID NO:31; and fragments thereof.
The present invention also relates to pharmaceutical compositions comprising such proteins and to methods of inducing an immune response in an individual against HCV
comprising administering to said individual a. composition comprising such proteins.
The present invention also relates to recombinant vaccines comprising such proteins and to method of inducing an immune response- in an individual against HCV
comprising administering to said individual such recombinant vaccines.
The present invention also relates to live attenuated pathogens comprising such protein and to methods of inducing an immune response in an individual against HCV
comprising, administering to said individual such live attenuated pathogens.

Proteins comprising consensus hTERTamino acid sequences and nucleic acid molecules that comprising a nucleotide sequence encoding such proteins are provided.
The present invention further relates to nucleic acid molecules comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO: 34; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID NO: 34; and fragments thereof.
The present invention also relates to pharmaceutical composition. such nucleic acid molecules and to methods of inducing an immune response in an individual against hyperproliferative cells expressing hTERT comprising administering to said individual a composition comprising such nucleic acid molecules.
The present in.vention further relates to recombinant vaccines comprising such nucleic acid molecules and methods of inducing an immune response in art individual against hyperproliferative cells expressing hTERT comprising administering to said individual such a recombinant vaccine.
The present invention further relates to live attenuated pathogen comprising such nucleic acid molecules and methods of inducing an immune response in an individual against hyperprolifcrative cells expressing hTERT comprising administering to said individual such live attenuated pathogens.
The present invention also relates to nucleic acid molecules that comprising a nucleotide sequence selected from the group consisting of: proteins comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:35; fragments thereof;
nucleotide sequences having at least 90% homology to SEQ ID NO:35; and fragments thereof.
The present invention also relates to pharmaceutical compositions comprising such proteins and to methods of inducing an immune response in an individual against hyperproliferative cells expressing hTERT co.mprising administering to said individual a composition comprising such proteins.
The present invention also relates to recombinant vaccines comprising such proteins and to method of inducing an immune response in an individual against hyperproliferative cells expressing, hTERT comprising administering to said individual such recombinant vaccines.

The present invention also relates to live attenuated pathogens comprising such protein and to methods of inducing an immune response in an individual against hyperproliferative cells expressing hTERT comprising, administering to said individual such live attenuated pathogens.
Proteins comprising Influenza 1.15N I. consensus tiA amino acid sequences, Influenza 111N1 and H5N1. consensus NA amino acid sequences, Influenza IlIN1 and H5N1 consensus MI amino acid sequences, and Influenza H5N1 consensus 1\42E-NP amino acid sequences and nucleic acid molecules that comprising a nucleotide sequence encoding such proteins are provided.
The present invention further relates to nucleic acid molecules comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:36; fraDnents thereof; nucleotide sequences having at least 90% homology to SEQ ID NO:36; and fragments thereof.
The. present invention further relates to nucleic acid molecules comprising a nucleotide sequence selected from the group consisting of: SEQ
NO:38; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID NO:38; and fragments thereof.
The present invention further relates to nucleic acid molecules comprising: -a nucleotide sequence selected from the group consisting of: SEQ ID NO:40; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID NO:40; and fragments thereof.
The present invention further relates to nucleic acid molecules comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:42; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID NO:42; and fragments thereof.
The present invention also relates to pharmaceutical compositions comprising such nucleic acid molecules and to inethods of inducing an immune response in an individual against HPV, IHCV, and influenza virus comprising administering to said individuai a composition comprising such nucleic acid molecules.
The present invention further relates to recombinant vaccines comprising such nucleic = acid molecules and methods of inducing an immune response in an individual against. FIPV, HCV, and Influenza virus comprising administering to said individual such a recombinant vaccine.

.

The present invention further relates to live attenuated pathogens comprising such nucleic acid molecules and methods of inducing, an immune response in an individual against HPV, HCV, and Influenza virus comprising adininistering to said individual such live attenuated pathogens.
The present invention also relates to pharmaceutical compositions comprising such nucleic acid molecules and to methods of inducing an immune response in an individual against HPV. HCV, and Influenza virus comprising administering, to said individual a composition comprising such nucleic acid molecules.
The present invention further relates to recombinant vaccines comprising such nucleic acid molecules and methods of inducing: an immune response in an individual against HPV, HCV, and Influenza virus comprising administering to said individual such a recombinant vaccine.
The present invention further relates to live attenuated pathogens comprising such nucleic acid molecules and methods of inducing an immune response in an individual against HPV, HCV, and Influenza virus comprising administering to said individual such live attenuated pathogens.
The present invention further relates to protein molecules comprising an amino acid sequence selected from the group consisting of: SEQ 1D NO:37; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID NO:37; and fragments thereof.
The present invention further relates to protein molecules comprising an amino acid sequence selected .from the group consisting of: SEQ ID NO:39; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID .N0:39; and fragments thereof.
The present invention further relates to protein molecules comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:41; fragments thereof; nucleotide sequences having at least 90% homology to SEQ ID NO:41; and fragments thereof.
The present invention further relates to protein molecules comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:43; fragments thereof; nucleotide sequences havi.ng at least 90% homology to SEQ ID NO:43; and fragments thereof.

The present invention also relates to pharmaceutical compositions comprising such protein molecules and to methods of inducing an immune response in an individual- against Influenza virus comprising administering to said individual a composition comprising such protein molecules.
Thc present invention further relates to recombinant vaccines comprising such protein molecules and methods of inducing an immune response in an individual against influenza virus = comprising administering to said individual such a recombinant vaccine.
The present invention further relates to live attenuated pathogens comprising such protein molecules and method.s of inducing an immune response in an individual against Influenza virus comprising administering to said individual such live attenuated pathogens.
The present invention also relates to pharmaceutical compositions comprising such protein molecules and to methods of inducing an immune response in an individual against Influenza virus comprising administering to said individual a composition comprising such protein molecules.
The present invention further relates to recombinant vaccines comprising such protein molecules and methods of inducing an immune response in an individual against Influenza virus comprising administering to said individual such a recombinant vaccine.
The present invention further relates to live attenuated pathogens comprising such protein molecules and methods of inducing an immune response in an individual against Influenza virus comprising administering to said individual such live attenuated pathogens.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a comparison of the amino acid sequences of EY2E1-B and EK2P-B.
The IgE leader sequence is underlined. The boxed regions show variable regions.
The denotes six important residues involved in CCR5 utilization. The cleavage site is indicated by a.n arrow. The transmembrane domain is shown by the dotted line.
Figure 2 shows phylogenetic relationships of two HIV-1 subtype B envelope sequences.
Forty-two 111V-I subtype B envelope sequences, EY2EI-Bõ EK2P-B, two subtype D
and two subtype C sequences (outgroup) were included in the phylogenetic analysis. The subtype B

envelope sequences representing a broad sample of diversity were from the following 11 countries: Argentinia (1); Australia (6); China (1); France (4); Germany (1);
Great Britain (2);
Italy (1); Japan (1); The Netherlands (4); Spain (1); United States (20). The EY2E1-B and EK2P-B sequences are shown in black boxes.
Figure 3 shows expression of envelope immunogens. Panel A shows results from Western blotting analysis of EY2E1-B and EK2P-B genes. RD cells were transfected with different plasmids. 48 hours later, cell lysates were collected. Samples were analyzed by Western blotting and probed with HIV-1 gp120 monoclonal (2G12). As for loading control, the blot was stripped and reprobed with a monoclonal anti-actin antibody. Panel B shows results from imm.unotluorescence ass-ay of EY2E1-B and EK2P-B genes. The transfected RD
cells expressing envelope proteins showed typical red fluorescence. HIV-1 envelope-specific monoclonal antibody- F105 se-flied as the source of primary antibody.
Figure 4. shows total 1gG antibody titers in the sera of the immunized mice.
Panel A
shows the measurement of subtype B envelope-specific antibody responses. Panel B shows the measurement of subtype AIE envelope-specific antibody responses. Panel C shows the measurement of subtype C envelope-specific antibody responses. Humeral immune responses after immunization with DNA constructs pEY2E1-B and pEK2P-B were detected by enzyme-linked immimosorbent assay (EL1SA). Each mouse was immunized intramuscularly with three times, each of 100 ug of DNA at bi-weekly intervals. Mice from each group (n=3) were bled one week after the third immunization and equally pooled sera were diluted in blocking buffer and analyzed as described in Materials and Methods, Pooled sera collected from mice immunized with pVAX were used as a control. Absorbance (OD) was measured at 450 nm. Each data point represents averaged three OD values from three mice sera per group and values represent the = mean of ELISA obtained in three separate assays.
Figure 5 shows induction of cell-mcdiated immune responses by pEY2E1-B in both BalB/C mice and HLA-A2 transgenic rnice. Frequencies of subtype B consensus envelope-specific IFN-7 spot forming cells (SFC) per million splenoeytes after DNA
vaccination with pEY2E1-B and PEK2P-B were determined by ELISpot assay in both BalB/C mice (Panel A) and transgenic mice (Panel C). Frequencies of CD8 depleted, subtype B consensus envelope-specific IFN--.7 spot forming cells per million splenocytes after DNA vaccination with pEY2EI-B and pEK2P-I3 were also determined in both BalB/C mice (Panel B) and transgenic mice (Panel D).
The slcnocytes were isolated from individual immunized mice (three mice per group) and stimulated in vitro with overlapping consensus subtype, B envelope peptides pools. Backbone pVAX immunized mice were included as a negative control. The values are the means +
standard deviations of thc means of IFN- 7 SECs. (Panel E) Characterization of subtype 13 consensus envelope-specific dominant epitopes. The splenocytes collected from pEY2E1.-B and pEK2P-B vaccinated BalBIC mice, respectively, were cultured with 29 HIV-I
subtype B
consensus envelope peptide pools for 24 hours. 1FN- 7 secreting cells were determined by ELISpot assay as described above..
Figure 6 shows cross reactivity induced by pEY2E1-B in both 13a1B/C mice and transgenic mice. The additive T-cell immune responses in BalB/C mice induced by vaccination with pEY2E1-B and pEK2P-B against four individual peptide pools of HIV-1 MN
envelope peptides (Panel A), HIV-1 group M (Panel B), subtype C consensus envelope peptides (Panel C) and two subtype C isol.ate envelope peptides (Panels D and E) were measured by WN-7ELISpot assay. The additive T-cell imm.une responses in [-ILA.-A2 transgenic mice induced by vaccination with. pEY2E1-B and pEK2P-B against four individual peptide pools of H1V-1 MN
envelope peptides (Panel F), HIV-1 group M (Panel G), subtype C consensus envelope peptides (Panel H) and two subtype C isolate envelope peptides (Panels 1 and 3) were also measured, Backbone pVAX immunized mice were included as a negative control.
= Figure 7 show characterization of subtype 13 MN envelope-specific dominant epitopes in both 13a13/C mice (Panel A) and HI..õA.-A2 transgenic mice (Panel B) immunized with pEY2E1-B
and pEK2P-B. The splenocytes collected from pEY2EI-B and pEK2P-B vaccinated BalB/C
rniee and transgenie mice, respectively, were cultured with 29 1-i1V-1 subtype 13 MN envelope peptide pools for 24 hours. IFN-7 secreting cel.ls were determined by ELISpot assay as described above.
Figure 8 shows a schematic representation of functional domains of E72E1-B
(about 700+ amino acids).
Figure 9 shows a map of E72E1.-B construct.

Figure 10 Panels A and B, show that a strong cellular immune response is induced E72E1-B.
Figure 11 Panels A and B. show that strong and broad cross-reactive cellular immune responses are induce 72E1-B.
Figure 12 Panels A-D show that strong cross-elade cellular immune responses are induced .E72E1-B.
Figu.re 13 depicts the immunogen designed for study in Example 2.
Figure 14 shows phylogenetic relationships: Thirty-Six HIV-1 subtype C
envelope sequences, EY3E1-C, EK3P-C, two subtype B, one subtype A and one subtype D
sequences (outuoup) were included in the phylogenetic analysis. The subtype C envelope sequences representing a broad sample of diversity were from 12 countries.
Figure 15 Panels A and 13 show data from studies of cellular response elicited by pEY3E I -C.
Figure 16 shows data from studies of cellular responses elicited by pEY3E1-C.
Figure 17 Panels A-D show data from studies across-reactive cellular responses elicited by pEY3E1-C within the SaMC clade.
Figure 18 Panels A and B show data from studies of cross-reactive cellular responses elicited by pEY3E1-C. Panel. A shows data from. subtype C (Uruguay) env-Specific IFN-y ELISpot. Panel B shows data from Subtype C (S. Africa) env-Specifie IFN-yELISpot.
Figure 19 Panels A-F show data from studies of cross-reactive cellular responses elicited by pEY3E 1-C between clacks.
Figure 20 Panels A-X show data from studies ofimmune responses elicited by HIV-1 gag consensus constructs.
Figure 21 illustrates the HPV life cycle in the genital tract epithelium.
= Figure 22 shows a map of HPV-16 genome organization.
Figure 23 illustrates immunogen design: * refers to deletions or mutations important for p53 binding and degradation; A refers to mutations in Rh binding site.

.

Figure 24 includes an illustration of the genetic construct p1667 which includes coding sequences for HPV E6 and E7 proteins, and pVAX, the backpbone plasmid µvhich lacks the WV
insert and is used a negative control.
Figure 25 Panels A-I) show cellular immune responses induced by the DNA
immunogen p1667.
Figure 26 shows results of irrummodominant epitope mapping.
Figure 27 shows results from the prophylactic experim.ents using E6/E7 DNA
Vaccine to study protection in C57/11L6 Mice.
Figure 28 shows results from the tumor regression experiments using E6/E7 DNA
Vaccine to study protection in C57/13E6 .Mice.
Figure 29 shows the data from experiments detecting E7 Tetramer positive lymphocytes in spleens.
Figure 30 shows the data from experiments detecting', E7 Tetramer positive lymphocytes in tumors.
Figure 31 shows data from a. DNA Vaccine protection study in transgenic mice.
Figure 32 shows enhanced cellular immune responses to HIV-1 consensus immunogens with 1M co-injection of plasmid encoded IL-12 followed by electroporation (EP). IFNy ELISpots were performed two weeks after the (a) first immunization, (b) second immunization, and (c) third immunization (as seen in comparison to the other three). Responses to env are depicted as black bars and gag are depicted as white bars with the data shown as stacked group mean responses SEM.
Figure 33 shows enhanced cross-reactive cellular immune responses with intramuscular eleetroporation. After three immunizations, the total T-cell immune response in pEY2F1-13 immunized mac.aqucs against four peptide pools of the HIV-1 group M peptides were determined by IFNy ELISpot. The data are shown as stacked group means S.EM.
Figure 34 shows Enhanced memory responses to H1V-1 immunogens with TM
electroporation and plasmid 1-12. Five months after the last immunization, ELISpot assays were performed to determine antigen-specific memory responses to gag and env in the IM and .

EP immunized groups with and without co-immunization with the IL-12 plasmid.
The data are shown as group mean responses SEM.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions As used herein, the phrase "stringent hybridization conditions" or "stringent conditions"
refers to conditions under which a nucleic acid molecule will hybridize another a nucleic acid molecule, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 C.
lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium.
Since the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 C. for short probes, primers or oligonueleotides (e.g. 10 to 50 nucleotides) and at least about 60 C. for longer probes, primers or oligonucleotides. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.
Sequence homology for nucleotides and amino acids may be determined using FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389) and PAUP* 4.0b10 software (D. L. Swofford, Sinauer Associates, Massachusetts). "Percentage of similarity" is calculated using AAUP*
4.0b10 software (D. L. Swofford, Sinauer Associates, Massachusetts). The average similarity of the consensus sequence is calculated compared to all sequences in the phylogenic tree (see Figures 2 and 14).
Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410).

Software for performing BLAST
analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.niEgov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X
from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue aligtunents; or 3) the end of either sequence is reached. The Blast algorithm pararneters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919) alignments (B) of 50, expe,ctation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Nat/.
Acad. Sci. USA, 1993, 90, 5873-5787) and Gapped BLAST perform a statistical analysis of the similarity between two sequences.
One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
As used herein, the term "genetic construct" refers to the DNA or RNA
molecules that comprise a nucleotide sequence which encodes protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polya.denylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered.
As used herein, the term "expressible Ibrm" refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.
OverView The present invention provides improved vaccines by utilizing a multi-phase strategy to enhance cellular immune responses induced by immunogens. Modified consensus sequences for immunogcns were generated. Genetic modifications including codon optimization, RNA
optimization, and the addition of a high efficient immunoglobin leader sequence to increase the immunogenicity of constructs are also disclosed. The novel immunogens have been designed to elicit stronger and broader cellular immune responses than a corresponding codon optimized immunogens.
The invention provides improved HIV, HPV, HCV, Influenza and cancer vaccines by providing proteins and genetic constructs that encode proteins with epitopes that make them particularly effective as immunogens against which anti-ITIV, anti-HPV, anti-HCV, antri-influenze and anti-hTert immune responses, respectively, can be induced.
Accordingly, vaccines can be provided to induce a therapeutic or prophylactic immune response. In some embodiments, the means to deliver the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, an attenuated vaccine or a killed vaccine. In some -embodiments, thc vaccine comprises a com.bination selected from the groups consisting of: one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit -vaccines, one or more compositions comprising the immunogen, one or more attenuated vaccines and one or more killed vaccines.
According to some embodiments of the invention, a vaccine according to the invention is delivered to an individual to modulate the activity of the individual's immune system and thereby enhance thc immune response against HIV, HPV, HCV, Influenza or hTERT. When a nucleic acid molecules that encodes the protein is taken up by cells of the individual the nucleotide sequence is expressed in the cells and thc protein are thereby delivered to the individual.

Aspects of the invention provide, methods of delivering the coding sequences of the protein on nucleic acid molecule such as plasmid, as part of recombinant vaccines and as part of attenuated vaccines, as isolated proteins or proteins part of a vector.
According to some aspects of the present invention, compositions and methods are provided which prophylactically and/or therapeutically immunize an individual against HIV, HPV, HCV, Influenza and cancer.
The present invention relates to compositions for delivering nucleic acid molecules that comprise a nucleotide sequence that encodes a protein of the invention operably linked to regulatory elements. Aspects of the present invention relate to compositions a recombinant vaccine comprising a nucleotide sequence that encodes that encodes a protein of the invention; a live attenuated pathogen that encodes a protein of the invention and/or includes a protein of the invention; a killed pathogen includes a protein of the invention; or a composition such as a Liposome or subunit vaccine that comprises a protein of the invention. The present invention further relates to injectable pharmaceutical compositions that comprise compositions.
HIV
The present invention. provides improved anti-HIV vaccines by utilizing a multi-phase strategy to enhance cellular immune responses induced by HIV iminunogens.
Modified consensus sequences for immunogens were generated Genetic modifications including codon optimization, RNA optimization, and the addition of a high efficient immunoglobin leader sequence to increase the immunogenicity of constructs are also disclosed. The novel immunogens have been designed to elicit stronger and broader cellular immune responses than a corresponding codon optimized immunogens.
SEQ ID NO:1 is a subtype A consensus envelope DNA sequence construct. SEQ ID
NO:1 comprises coding sequence for HIV vaccine sequence that comprises an IgE
leader sequence linked to a consensus sequence for Subtype A envelope protenn SEQ ID
NO:2 comprises the amino acid sequence for HIV vaccine sequence construct that comprises an IgE
leader sequence linked to a consensus sequence for Subtype A envelope protein.
The IgE leader sequence is SEQ NO:15. SEQ ID NO:16 is the Subtype A consensus Envelope protein sequence.
In some embodiments, vaccines of the invention preferably include SEQ ID
NO:16, fragment thereof, a nucleic acid molecule that encodes SEQ ID NO:16, or fragments thereof. In some embodiments, vaccines of the invention preferably include SEQ ID NO:2 or a nucleic acid molecule that encodes it. In some embodiments, vaccines of the invention preferably include SEQ ID NO: l. Vaccines of the present invention preferably include the IgE
leader sequence SEQ ID NO:15 or nucleic acid sequence which encodes the same.
Fragments of SEQ NO:1 may comprise 90 or more nucleotides. In some embodiments, fragments of SEQ ID NO:1 may comprise 180 or more nucleotides; in some embodiments, 270 or more nucleotides; in some embodiments 360 or more nucleotides; in some embodiments, 450 or more nucleotides; in some embodiments 540 or more nucleotides; in some embodiments, 630 or more nucleotides; in some embodiments, 720 or more nucleotides; in some embodiments, 810 or more nucleotides; in some embodiments, 900 or more nucleotides; in some embodiments, 990 or more nucleotides; in some embodiments, 1.080 or more nucleotides; in some embodiments, 1170 or more nucleotides; in some embodiments, 1260 or more nucleotides;
in sonic embodiments, 1350 or more nucleotides in some embodiments, 1440 or more nucleotides; in some embodiments, 1530 Or more nucleotides; in some embodiments, 1620 or = more nucleotides; in some embodiments, 1710 or more nucleotides; in some embodiments, 1800 or more nucleotides; in some embodiments, 1890 or more nucleotides; in some embodiments, 1980 or more nucleotides; and in some embodiments, 2070 or more nucleotides.
In some embodiments, frag,ments of SEQ ID NO:1 may comprise coding sequences for the 1gE leader sequences. In some embodiments, fragments of SEQ 11) NO:1 do not comprise coding sequences for the IgE leader sequences. Fragments may comprise fewer than 180 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 450 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 630 nucleotides, in some embodiments fewer than 720 nucleotides, in some embodiments fewer than 810 nucleotides, in some embodiments fewer than 900 nucleotides, in some embodiments fewer than 990 nucleotides, in sonic embodiments fewer than 1080 nucleotides, in some embodiments fewer than 1170 nucleotides, in some embodiments fewer than 12.60 nucleotides, in some embodiments fewer than 1350 nucleotides, in some embodiments fewer than 1440 nucleotides, in some embodiments fewer than 1530 nucleotides, in .some embodiments fewer than. 1620 nucleotides, in some embodiments fewer than 1710 nucleotides, in some embodiments fewer than 1800 nucleotides, in some embodiments fewer. than 1890 nucleotides, in some embodiments fewer than 1980 nucleotides, in some embodiments fewer than 1020 nucleotides, and in some embodiments fewer than 2070 nucleotides.
Fragments of SEQ ID NO:2 may comprise 30 or more amino acids. in some embodiments, fragments of SEI) ID NO:2 may comprise 60 or more amino acids; in some embodiments, 90 or more amino acids; in some embodiments, 1.20 or more amino acids; in some embodiments; 150 or more amino acids; in some embodiments 180 or more amino acids; in some embodiments, 210 or inore amino acids; in some embodiments, 240 or more amino acids;
in some embodiments, 270 or more amino acids; in some embodiments, 300 or more amino acids; in some embodiments, 330 or more amino acids; in some embodiments, 360 or more amino acids; in some embodiments, 390 or niore amino acids; in some embodiments, 420 or more amino acids; in some embodiments, 450 or more amino acids; in some embodiments, 480 or more amino acids; in some embodiments, 510 or more amino acids; in some embodiments, 540 or more amino acids; in some embodiments, 570 or more amino acids; in some embodiments, 600 or .more a.mino acids; in some embodiments, 630 or more amino acids; in some embodiments, 660 or more amino acid; and fl some embodiments, 690 or more amino acids. Fragments may comprise fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 270 amino acids, in some embodiments fewer than 300 amino acids, in some embodiments fewer than 330 amino acids, in some embodiments fewer than 360 amino acids, in some embodiments fewer than 390 amino acids, in some embodiments fewer than 420 amino acids, in some embodiments fewer than 450 amino acids, in some embodiments fewer than 480 amino acids, in some embodiments fewer than 540 amino acids, in some embodiments fewer than 600 amino acids, in some embodiments fewer than 660 amino acids, and in some embodiments fewer than 690 amino acids.
SEQ ID NO:3 is a subtype B consensus envelope DNA sequence construct. SEQ ID
NO:3 comprises coding sequence for HIV vaccine sequence that comprises an IgE
leader sequence linked to a consensus sequence for Subtype B envelope protein. SEQ 1D
NO:4 comprises the amino acid sequence tbr HIV vaccine sequence construct that comprises an IgE
leader sequence linked to a consensus sequence for Subtype B env-elope protein. The IgE leader sequence is SEQ ID N0:15. SEQ NO:17 is the Subtype B consensus Envelope protein sequence.
In sorne embodiments, vaccines of the invention preferably include SEQ ID
NO:17, fragment thereof, a nucleic acid molecule that encodes SEQ ID NO: or fragments thereof. In some embodiments, vaccines of the invention preferably include SEQ ID NO:4 or a nucleic acid molecule that encodes it. In SOMC embodiments, vaccines of the invention preferably include SEQ ID NO:3. Vaccines of the present invention preferably include the IgE
leader sequence SEQ I.D NO:15 or nucleic acid sequence 1,vhich encodes the same.
Fragments of SEQ ID NO:3 may comprise 90 or more nucleotides. In some embodiments, fragments of SEQ ID NO:3 may comprise 180 or more nucleotides; in some embodiments, 270 or more nucleotides; in some embodiments 360 or more nucleotides: in some embodiments, 450 or more nucleotides; in somc embodiments 540 or more nucleotides; in some embodiments, 630 or more nucleotides; in some embodiments, 720 or more nucleotides; in some embodiments, 810 or more nucleotides; in some embodiments, 900 or more nucleotides; in some embodiments, 990 or more nucleotides; in some embodiments, 1080 or more nucleotides; in some embodiments, 1170 or more nucleotides; in some embodiments, 1260 or more nucleotides;
in some embodiments. 1350 or more nucleotides in some embodiments, 1440 or more nucleotides; in some embodiments, 1530 or more nucleotides; in some embodiments, 1620 or more nucleotides; in some embodiments, 1710 or more nucleotides; in some embodiments, 1800 or more nucleotides, in. some embodiments, 1890 or more nucleotides; in some embodiments, 1980 or more nucleotides; in some embodiments, 2070 or more nucleotides; in some embodiments, 2160 or more nucleotides; in some embodiments, 2250 or more nucleotides; in some embodiments, 2340 or more nucleotides; in SOIlle embodiments, 2430 or more nucleotides;
in some embodiments, 2520 or more nucleotides; in some embodiments, 2620 or rnore nucleotides; and in some embodiments, 2700 or more nucleotides. 1.11 some embodiments, fragments of SEQ ID ìO:3 may comprise coding sequences for the I,gE leader sequences. In some embodiments, fragments of SEQ 1 ì'O:3 do not comprise coding sequences for the IgE
leader sequences. Fragments may comprise fewer than 180 nucleotides, in some emhodi.ments fewer than 270 nucleotides, in some embodiments fewer than 360 nucleotides, in sonic embodiments fewer than 450 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 630 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 810 nucleotides, in some embodiments fewer than 900 nucleotides, in some embodiments fewer than 990 nucleotides, in some embodiments fewer than 1080 nucleotides, in some embodiments fewer than 1170 nucleotides, in some embodiments=
fewer than 1260 nucleotides, in sonic embodiments fewer than 1350 nucleotides, in some embodiments fewer than 1440 nucleotides, in some embodiments fewer than 1530 nucleotides, in some embodiments fewer than 1620 nucleotides, in some embodiments fewer than 1'710 nucleotides, in some embodiments fewer than 1800 nucleotides, in some embodiments fewer than 1890 nucleotides, in some embodiments fewer than 1.980 nucleotides, in some embodiments fewer than 1020 nucleotides, in some embodiments fewer than 2070 nucleotides, in some embodiments fewer than 2160 nucleotides, in some embodiments fewer than 2250 nucleotides, in some embodiments fewer than 2340 nucleotides, in some embodiments fewer than 2430 nucleotides. in some embodiments fewer than 2520 nucleotides, in some embodiments fewer .than 2610 nucleotides, and in some embodiments fewer than 2700 nucleotides.
Fragme.nts of SEQ ID N0:4 may comprise 30 or more amino acids. In some embodiments, fragments of SEQ 113 NO:4 may comprise 60 or more amino acids; in some embodiments, 90 or more amino acids; in some embodiments, 120 or more amino acids; in some embodiments; 150 or more amino acids; in some embodiments 180 or more amino acids; in some embodiments, 210 or more amino acids; in some em.bodiments, 240 or more amino acids:
in some embodiments, 270 or more amino acids; in some embodiments, 300 or more amino acids; in sonic embodiments, 330 or more amino acids; in some embodiments, 360 or more amino acids; in som.e embodiments, 390 or more amino acids; in sonic embodiments, 420 or more amino acids; in some embodiments, 450 or more amino acids; in some embodiments, 480 or more amino acids; in some embodiments, 510 or more amino acids; in some embodiments, 540 or more amino acids; in some embodiments, 570 or more amino acids; in some embodiments, 600 or more amino acids; in some embodiments, 630 or more amino acids; in some embodiments, 660 or more amino acid; and in some ernbodiments, 690 or more amino acids. Fragments may comprise fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 am.ino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 270 amino acids, in some embodiments fewer than 300 amino acids, in some embodiments fewer than. 330 amino acids, in some embodiments fewer than 360 amino acids, in some embodiments fewer than 390 amino acids, in some embodiments fewer than 420 amino acids, in some embodiments fewer than 450 amino acids, in sonic embodiments fewer than 480 amino acids, in some embodiments fewer than 540 amino acids, in sonic embodiments fewer than 600 amino acids, in some embodiments fewer than 660 amino acids, and in some embodiments fewer than 690 amino acids.
SEQ ID NO:5 is a subtype C consensus envelope DNA sequence construct. SEQ ID
NO:5 comprises coding sequence for HIV vaccine sequence that comprises an IgE
leader sequence linked to a consensus sequence for Subtype C envelope protein.
Sf...:Q ID NO:6 comprises the amino acid sequence for HIV vaccine sequence construct that comprises an IgE
leader sequence linked to a consensus sequence for Subtype C envelope protein.
The IgE leader sequence is SEQ ID NO:15. SEQ ID NO:IS is the Subtype C consensus Envelope protein sequence.
In some embodiments, vaccines of the invention preferably include SEQ ID
NO:18, fragment thereof, a nucleic acid molecule that encodes SEQ ID NO:18, or fragments thereof, in some embodiments, vaccines of the invention preferably include SEQ ID NO:6 or a11.11CiCiC acid molecule that encodes it. In some embodiments, vaccines of the invention preferably include SEQ ID NO:5. Vaccines of the present inventio preferably include the IgE
leader sequence SEQ ID NO!15 or nucleic acid sequence which encodes the same.

Fragments of SEQ ID NO:5 may comprise 90 or more nucleotides. In some embodiments, fra.gments of SEQ ID NO:5 may comprise 180 or more nucleotides;
in some ernbodirnen.ts, 270 or more nucleotides; in some embodiments 360 or more nucleotides; in some embodiments, 450 or more nucleotides; in some embodiments 540 or more nucleotides; in some embodiments, 630 or more nucleotides; in some embodiments, 720 or more nucleotides; in some embodiments, 810 or more nucleotides; in some embodiments, 900 or more nucleotides; in some embodiments, 990 or more nucleotides; in soine embodiments, 1080 or more nucleotides; in some embodiments, 1170 or more nucleotides; in soine embodiments, 1260 or more nucleotides;
in some embodiments, 1350 or more nucleotides in some embodiments, 1440 or more nucleotides; in some embodiments, 1530 or more .nucleotides; in some embodiments, 1620 or rnore nucleotides; in some embodiments, 1710 or .more nucleotides; in some embodiments, 1800 or more nucleotides; in some embodiments, 1890 or more nucleotides; in some embodiments, 1980 or more nucleotides; and in some embodiments, 2070 or more nucleotides.
In some embodiments, fragments of SEQ NO:5 may comprise coding sequences for the IgE
leader sequences. In some embodiments, fragments of SEQ ID NO:5 do not cornprise coding sequences for the fgE leader sequences. Fragments may comprise fewer than 180 nucleotides, in some embodiments fewer than 270 nucleotides, in sonic embodiments fewer than nucleotides, in. sonic embodiments fel,ver than 450 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 630 nucleotides, in sonie embodiments fewer than 720 nucleotides, in some embodiments fewer than 810 nucleotides, in some embodiments fewer than 900 nucleotides, in some embodiments fewer than 990 nucleotides, in some embodiments fewer than-1080 nucleotides, in sotne embodiments fewer than 1170 nucleotides, in some embodiments fewer Man 1260 nucleotides, in some embodim.ents fewer than 1350 nucleotides, in some entbodiments fewer than. 1440 nucleotides, in some embodiments fewer than 1 530 nucleotides, in some embodiments fewer than 1620 nucleotides, in sonic embodiments fewer than 1710 nucleotides, in some embodiments fewer than 1800 nucleotides, in some embodiments fewer than 1890 nucleotides, in some embodiments fewer than 1980 nucleotides, in some embodiments fewer than 1020 nucleotides, and in sonic embodiments fewer than 2070 nucleotides.

Fragments of SEQ UD NO:6 may comprise 30 or more amino acids. In some = embodiments, fragments of SEQ ID 1'O:6 may comprise 60 or more amino acids; in some embodiments, 90 or more amino acids; in some embodiments, 120 or more amino acids; in some embodiments; 150 or more amino acids; in some embodiments 180 or more amino acids; in some embodiments, 210 or more amino acids; in some embodiments, 240 or more amino acids;
in some embodiments, 2'70 or more amino acids; in some embodiments, 300 or more amino acids; in some embodiments, 330 or more amino acids; in some embodiments, 360 or more amino acids; in some embodiments, 390 or more amino acids; in sonic embodiments, 420 or more amino acids; in some embodiments, 450 or more amino acids; in some embodiments, 480 or more amino acids; in sonic embodiments, 510 or more amino acids; in some embodiments, 540 or more amino acids; in some embodiments, 570 or more amino acids; in some embodiments, 600 or more amino acids; in some embodiments, 630 or more amino acids; in some embodiments, 660 or more amino acid; and in some embodiments, 690 or more amino acids. Fragments may comprise fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids. in some embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 270 amino acids, in some embodiments fewer than 300 amino acids, in some embodiments fewer than 330 amino acids, in some embodiments fewer than 360 amino acids, in some embodiments fewer tha.n 390 arnino acids, in some embodiments fewer than 420 amino acids, in some embodiments fci,ver than 450 amino acids, in some embodiments fewer than 480 amino acids, in some embodiments fewer than 540 amino acids, in some embodiments fewer than 600 amino acids, in some embodiments fewer than 660 amino acids, and in some embodiments fewer than 690 amino acids.
SEQ NO:7 is a subtype D consensus envelope DNA sequence construct. SEQ ID
NO:7 cornprises coding s.equen.ce for HIV vaccine sequence that comprises an IgE leader sequence linked to a consensus sequence for Subtype D envelope protein. SEQ
NO:8 comprises the amino acid sequence for HIV vaccine sequence construct that comprises an TgE
leader sequence linked to a consensus sequence for Subtype D envelope protein.
The IgE leader sequence is SEQ ID NO:15. SEQ ID NO: is the Subtype D consensus Envelope .protein sequence_ In some embodiments, vaccines of the invention preferably include SEQ ID
NO:19, fragment thereof, a nucleic acid molecule that encodes SEQ ID NO:19, or fragments thereof. In some embodiments, vaccines of the invention preferably include SEQ 10 NO:8 or a nucleic acid molecule that encodes it. In some embodiments, vaccines of the invention preferably include SFQ ID NO:7. Vaccines of the present invention preferably include the IgE
leader sequence SEQ ID NO:15 or nucleic acid sequence which encodes the same.
Fragments of SEQ 1D NO:7 may comprise 90 or more nucleotides. In some embodiments, fragments- of SEQ NO:7 may comprise 180 or more nucleotides;
in some embodiments, 270 or more nucleotides; in some embodiments 360 or more nucleotides; in some embodiments, 450 or more nucleotides; in some embodiments 540 or more nucleotides; in some embodiments, 630 or niore nucleotides; in some etnbodiments, 720 or more nucleotides; in some embodiments, 810 or more nucleotides; in some embodiments, 900 or more nucleotides; in some embodiments, 990 or more nucleotides; in some embodiments, 1080 or more nucleotides; in some embodiments, 1170 or niore nucleotides; in some embodiments, 1260 or more nucleotides;
in some embodiments, 1350 OT more nucleotides in some embodiments, 1440 or more nucleotides; in some embodiments, 1530 or more nucleotides; in some embodiments, 1620 or more nucleotides; in some embodiments, 1710 or more nucleotides; in some embodiments, 1800 or more nucleotides; in some embodiments, 1890 or more nucleotides; in some embodiments, 1980 or more nucleotides; and in some embodiments, 2070 or more nucleotides;
and in some embodiments, 2140 Or more nucleotides. In some embodiments, fra.gments of SEQ
ID NO:7 may comprise coding sequences for the I.gE leader sequences. In some embodiments, fragments of SEQ ID NO:7 do not comprise coding sequences for the IgE leader sequences.
Fragments may comprise fewer than 180 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 630 nucleotides, in sonic embodiments fewer than 720 nucleotides, in some embodiments fewer than 810 nucleotides, in some einbodiments fewer than 900 nucleotides, in some embodiments fewer than 990 nucleotides, in some embodiments fewer than 1080 nucleotides, in some embodiments fewer than 1170 nucleotides, in sorne embodiments fewer than 1260 nucleotides, in some embodiments fewer than 1350 nucleotides, in some embodiments fewer than 1440 nucleotides, in some embodiments fewer than 1530 nucleotides, in some embodiments fewer than 1620 nucleotides, in some embodiments fewer than 1710 nucleotides, in some embodiments fewer than 1800 nucleotides, in some embodiments fewer than 1890 nucleotides, in some embodiments fewer than 1980 nucleotides, in some embodiments fewer than 1020 nucleotides, in sonic embodiments fewer than 2070 nucleotides and in some embodiments fewer than 2140 nucleotid.es.
Fragments of SEQ ID NO:8 may comprise 30 or more amino acids. In some embodiments, fragments of SEQ ID NO:8 inay comprise 60 or more arnino acids;
in some embodiments,. 90 or more amino acids; in some embodiments, 120 or more amino acids; in some embodiments; 150 or more amino acids; in some embodiments 180 or more amino acids; in some embodiments, 210 or more amino acids; in some embodiments, 240 or more amino acids;
in some embodiments, 270 or more amino acids; in some embodiments, 300 or more amino acids: in some embodiments, 330 or more amino acids; in sonie embodiments, 360 or more amino acids; in some embodiments, 390 or more amino acids; in some embodiments, 420 or more amino acids; in some embodiments, 450 or more amino acids; in some embodiments, 480 or more amino acids; in some embodiments, 510 or more amino acids; in some embodiments, 540 or more amino acids; in some embodiments, 570 or more amino acids; in some = embodiments, 600 or more amino acids; in some embodiments, 630 or more amino acids; in some embodiments, 660 or more amino acid; and in SOMC embodiments, 690 or more amino acids. Fragments may comprise fewer than 90 amino acids, in sonic entbodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in sonie embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 270 amino acids, in some embodiments fewer than 300 amino acids, in some embodiments fewer than 330 amino acids, in some embodiments fewer than 360 amino acids, in some embodiments fewer than 390 amino acids, in some embodiments fewer than 420 amino acids, in some embodiments fewer than 450 amino acids, in some embodiments fewer than 480 amino acids, in some embodiments fewer than 540 amino acids, in some embodiments fewer than 600 amino acids, in some embodiments fewer than 660 amino acids, and in some embodiments fewer than 690 amino acids.
SEQ ID NO:9 is a subtype B Nef-Rev consensus envelope DNA sequence construct.
SEQ ID NO:9 comprises coding sequence for HIV vaccine sequence that comprises an IgE
leader sequence linked to a consensus sequence for Subtype B Nef-Rev protein.
SEQ JD NO:10 comprises the amino acid sequence for HIV vaccine sequence construct that comprises an IgE
leader sequence linked to a consensus sequence for Subtype B Nef-Rev protein.
The IgE leader sequence is SEQ ID NO:15. SEQ ID NO:20 is the Subtype B NeliRev consensus protein sequence-.
In some embodiments, vaccines of the invention preferably include SEQ ED NO:20 fragment- thereof, a nucleic acid molecule that encodes SEQ ID NO:20, or fragments thereof. In some embodiments, vaccines of the, invention preferably include SEQ ID N-0:10 or a nucleic acid molecule that encodes it. In some embodiments, vaccines of the invention preferably incli.ide SEQ ID NO:9. Vaccines of the present invention preferably include the IgE leader sequence SEQ ID -N0:15 or nucleic acid sequence which encodes the same.
Fragments of SEQ ID NO:9 may comprise 90 or more nucleotides. In some embodiments, fragments of SEQ 1D NO:9 may comprise 180 or more nucleotides; in some embodiments, 270 or more nucleotides; in some embodiments 360 or more nucleotides; in some embodiments, 450 or more nucleotides; in sonie embodiments 540 or more nucleotides; in some embodiments, 630 or more nucleotides; in some embodiments, 720 or more nucleotides; in some embodiments, 810 or more nucleotides; in some embodiments, 900 or more nucleotides; and in some embodiments, 990 or 111017e nucleotides; in some embodiments. In some embodiments, fragments of SEQ ID NO:9 may comprise coding sequences for the IgE leader sequences. In some embodiments, framents of SEQ ID NO:9 do not comprise coding sequences for the IgE
leader sequences. Fragments may comprise fewer than 180 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 450 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 630 nucleotides, in= some embodiments fewer than .

nucleotides, in sonic embodiments fewer than 810 nucleotides, in some embodiments fewer than 900 nucleotides, and in sonic embodiments fewer than 990 nucleotides.
Fragments of SEQ ID NO:2 may comprise 30 or more amino acids. In sonie embodiments, fragments of SEQ ID NO:2 may comprise 60 or more amino acids; in some embodiments, 90 or more amino acids; in some. embodiments, 120 or more amino acids; in some embodiments; 150 or m.ore amino acids; in sonic embodiments 180 or more amino acids; in some embodiments, 210 or more atnino acids; in some embodiments, 240 or more amino acids;
in some embodiments, 270 or more amino acids; in some embodiments, 300 or more amino acids; and in som.e embodiments, 330 or more amino acids.
SEQ ID NO:11 is a Gag consensus DNA sequence of subtype A, B, C and D DNA
sequence construct. SEQ ID NO:11 comprises coding sequence for HIV vaccine sequence that comprises an IgE leader sequence linked to a consensus sequence for Gag consensus subtype A, B, C and D protein. SEQ ID NO:12 comprises the amino acid sequence for HIV
vaccine sequence construct that comprises an IgE leader sequence linked to a consensus sequence for Gag subtype A, B, C and D protein. The IgE leader sequence is SEQ ID NO:15.
SEQ ID NO:21 is the consensus Gag subtype A, B, C and D protein sequence.
In some embodiments, vaccines of the invention preferably include SEQ ID
NO:21, fragment thereof, a nucleic acid molecule that encodes SEQ ID NO:21., or fragments thereof. In sonic embodiments, vaccines of the invention preferably include .SEQ ID NO:12 or a nucleic acid molecule that encodes it. In some embodiments, vaccines of the invention preferably include SEQ ID NO:11. Vaccines of the present invention preferably include the IgE leader sequence SEQ ID NO:15 or nucleic acid sequence which encodes the same.
Fragments of SEQ ID NO;11 may comprise 90 or more nucleotides. In some embodiments, fragments of SEQ LD NO:11 may comprise 180 or more nucleotides;
in some embodiments, 270 or more nucleotides; in some embodiments 360 or more nucleotides; in some embodiments, 450 or more nucleotides; in some embodiments 540 or more nucleotides; in some embodiments, 630 or more nucleotides; in some embodiments, 720 or more nucleotides; in some embodiments, 81.0 or mon: nucleotides; in some embodiments, 900 or more nucleotides; in some embodiments, 990 or more nucic.otides: in some embodiments, 1 080 or more nu.cleotides; in some embodiments, 1170 or more nucleotides; in some embodiments, 1260 or more nucleotides;
in some embodiments, 1350 or more nucleotides in some embodiments, 1440 or more nucleotides; in some embodiments, 1530 or more nucleotides; in some embodiments, 1620 or more nucleotides; in some embodiments, 1710 or more nucleotides; and in sorne embodiments, 1800 or more nucleotides. In some embodiments', fragments of SEQ ID NO:11 m-ay comprise = coding sequences for the IgE leader sequences. In some embodiments, fragments of SEQ ID
NO:II do not comprise coding sequences for the le leader sequences. Fragments may co.mprise fewer than 180 nucleotides, in some. embodiments .fewer than 270 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 450 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 720 nucleotides, in some embodiments fewer than 810 nucleotides., in some embodiments fewer than 900 nucleotides., in some embodiments fewer than 990 nucleotides, in SOMC embodiments- fewer than: 1080 nucleotides, in some embodiments fewer than 1170 nucleotides, in sonic embodiments fewer than 1260 nucleotides, in some embodiments fewer than 1350 nucleotides, in some embodiments fewer than 1440 nucleotides, in some embodiments fewer than 1530 nucleotides, in sonic embodiments fewer than 1620 nucleotides, in some embodiments fewer than 1710 nucleotides, and in some embodiments fewer than 1800 nucleotides.
= Fragments of SEQ ID NO:12 may comprise 30 or more amino acids. in some embodiments, fragments of SEQ ID NO:12 may comprise 60 or more= amino acids;
in some embodiments, 90 or more amino acids; in some embodiments, 120 or more amino acids; in some embodiments; 150 or more amino acids; in some embodiments 180 or more amino acids; in sonic embo-dinients, 210 or more amino acids; in some embodiments, 240 or more amino acids;
in some embodiments, 270 or more amino acids; in some embodiments, 300 or more amino acids; in some embodiments, 330 or more amino acids; in SOITIC embodiments, 360 or .more amino acids; in some embodiments, 390 or more amino acids; in some embodiments, 420 or more amino acids; in sonic embodiments, 450 or more amino acids; in some embodiments, 480 or more amino acids; and in some embodiments, 5.10 or more amino acids.
Fragments may comprise fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 arnino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 270 amino acids, in some embodiments fewer than 300 amino acids, in some embodiments fewer than 330- amino acids, in some embodiments fewer than 360 amino acids, in some embodiments fewer than 390 amino acids, in some embodiments fewer than 420 amino acids, in some embodiments fewer than 450 amino acids, in some embodiments fewer than 480 amino acids, and in some embodiments fewer than 510 amino acids.
HPV
SEQ ID NO:22 comprises coding sequence for HPV vaccine sequence that comprises and IgE leader sequence, a consensus sequence for HPV E6, linked to a consensus sequence for HPV
E7 by a proteolytic cleavage sequence. The consensus sequence for HPV E6 includes the immunodominant epitopc set forth in SEQ ID NO:24. The consensus sequence for includes the immunodorninant cpitope set forth in SEQ ID NO:25. The consensus sequence for HPV E6 is SEQ ID NO:26. The consensus sequence for HPV E6 is SEQ ID NO:27. The IgE
leader sequence is SEQ ID NO:28. A proteolytic cleavage sequence useful to link the two consensus sequences is SEQ ID NO:29.
In some embodiments, vaccines of the invention preferably include SEQ ID NO:24 and/or SEQ ID NO:25, or nucleic acid sequence which encode one of both of them. Vaccines of the invention preferably include SEQ ID NO:27 and/or the SEQ ID NO:28, or nucleic acid sequences which encode one or both of them. Vaccines of the invention preferably include SEQ
ID NO:27 linked to SEQ ID NO:28 by a proteolytic cleavage sequence such as SEQ
ID NO:29, or nucleic acid sequence which encodes the fusion protein. Vaccines of the present invention preferably include the IgE leader sequence SEQ ID NO:28 or nucleic acid sequence which = encodes the same. Vaccines of the invention preferably include SEQ ID
NO:23 or the nucleic acid sequence in SEQ ID NO:22.
hi some embodiments. proteins comprises fragments of SEQ ID NO:23. In some embodiments, proteins consist of fragments of SEQ ID NO:23. In some embodiments, nucleic acids comprises .fragment of SEQ ID NO:22, In some embodiments, nucleic acids consist of a fragment of SEQ. ID NO:22.
Fragments of SEQ ID NO:22 may comprise 30 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ
ID NO:22 may comprise 45 or more nucleotides, including, preferably sequences that encode an immunodominant cpitope. In some embodiments, fragments of SEQ ID NO:22 may comprise = 60 or more nucleotides, including preferably- sequences that encode an immun.odominant epitope.
In some embodiments, fragments of SEQ ID NO:22 may comprise 75 or more nucleotides, including preferably sequences that encode an immunodominant epitopc. In some embodiments, fragments of SEQ ID NO:22 may comprise 90 or more nucleotides, including preferably sequences that encode an itnmunodominant epitope. ln some embodiments, fragments of SEQ ID
NO:22 may comprise 120 or more nucleotides, including, preferably sequences that encode an immunodominant epitope. Iu some embodiments, fragments of SEQ ID NO:22 may.
comprise 150 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may comprise 180 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may comprise 210 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may comprise 240 or more nucleotides, including preferably sequences that encode an immtmodominant epitope. in some embodiments, fragments of SEQ H) NO:22 may comprise 270 or more nucleotides, including preferably sequences that encode an inummodominant cpitopc. In some embodiments, fragments of SEQ ID NO:22 may comprise 300 or more nucleotides, including preferably sequences that encode an immunodominant epitope, in some embodiments, fragments of SEQ 1D NO:22 may comprise 360 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In sonic -embodiments, fragments of SEQ ID NO:22 may comprise 420 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fra.gmcnts of SEQ ID NO:22 may comprise 480 or more nucleotides, including preferably sequences that encode an imintmodominant epitope. In some embodiments, fragments of SEQ ID
-35..

NO:22 may comprise 540 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may comprise 600 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may comprise 300 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may comprise 660 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may comprise 720 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:22 may comprise 780 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:22 may coinprise coding sequences for the IgE leader sequences. In some embodiments, fragments of SEQ ID
NO:22 do not comprise coding sequences for the le-E leader sequences.
Fragments may comprise fewer than 60 nucleotides, in some embodiments fewer than 75 nucleotides, in some embodiments fewer than 90 nucleotides, in some embodiments fewer than 120 nucleotides, in some embodiments fewer than 150 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 210 nucleotides, in some embodiments fewer than 240 nucleotides, in some embodiments fewer than 270 nucleotides, in SOMe embodiments fewer than 300 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 420 nucleotides, in some embodiments fewer than 480 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 600 nucleotides, in some embodiments fewer than 660 nucleotides, in some embodiments fewer than nucleotides, and in some embodiments fewer than 780 nucleotides.
Fragments of SEQ ID NO:23 may comprise 15 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ
ID NO:23 may comprise 18 or more amino acids, including preferably sequences that encode an immunodominant cpitope. In some embodiments, fragments of SEQ ID NO:23 may comprise 21 or m.ore amino acids. including preferably sequences that encode an immunodominant epitopc. In some embodiments, fragments of SEQ ID NO:23 may comprise 24 or more amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ ID NO:23 may comprise 30 or more amino acids, includina preferably sequences that encode an immunodominant epitope. In some embodiments, frapnents of SEQ ID NO:23 may comprise 36 or more amino acids, including preferably sequences that encode an irrummodorninant epitope. In some embodiments, fragments of SEQ
ID NO:23 may comprise 42 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:23 may comprise 48 or more amino acids, including preferably sequences that encode an immunodoininant epitopc. In some embodiments, fragments of SEQ Ill NO:23 may comprise 54 or more ainino acids, including preferably sequences that encode an im.munodominant epitope.
In some embodiments, fragments of SEQ ID NO:23 may comprise 60 or more amino acids, including preferably sequences that encode an inummodominant epitope. In some embodiments, framents of SEQ ID NO:23 may comprise 18 or more amino acids, including preferably sequences that encode an immunodominant epitope. In sonic embodiments, fragments of SEQ
ID NO:23 may comprise 72 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodin-icnts, fragments of SEQ TD NO:23 may comprise 90 or more amino acids, including preferably sequences that encode an immunodominant cpitope. In some embodiments, fragments of SEQ ID NO:23 may comprise 120 or more amino acids, including preferably sequences that encode an imm-unodominant epitopc.
In some embodiments, fragments of SEQ ID NO:23 may comprise 150 or more amino acids, including preferably sequences that encode, an immunodominant epitope. In some embodim.crits, fragments of SEQ ID NO:23 may comprise 180 or more amino acids, including preferably sequences that encode an iinmunodominant epitope. In some embodiments, fragments of .SEQ
11) NO:23 may comprise 210 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:23 may comprise 240 or more amino acids, including preferably sequ.ences that encode an immunodominant epitope. In some embodiments, fragments of SEQ NO:23 may comprise 260 or more amino acids, including preferably sequences that encode an immun.odorninant epitope.
In some embodiments, fragments of SEQ ID NO:23 may comprise coding sequences for the IgE leader sequences. In some embodiments, fragments of SEQ ID NO:23 do not comprise coding sequences for the igE leader sequences. Fragments may comprise fewer than 24 amino acids, in some embodiments fewer than 30 amino acids, in some embodiments fewer than 36 amino acids, in some embodiments fewer than 42 amino acids, in some embodiments fewer than 48 amino = acids, in some embodiments fewer than 54 amino acids, in some embodiments fewer than 60 amino acids, in some embodiments fewer than 72 amino acids, in some embodiments fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than. 150 amino acids, in sonic embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids in some embodiments fewer than 240 amino acids, and in some embodiments fewer than. 260 amino acids.
SEQ ID NO:30 comprises coding sequence for HCV .vaccine sequence that comprises and IgE leader sequence, a consensus sequence for FICV El, linked to a consensus sequence for HCV E2 by a proteolytic cleavage sequence. The consensus sequence for HCV El is SEQ ID
NO:32. The consensus sequence for HCV E2 is SEQ ID NO:33.
In some embodiments, vaccines of the invention preferably include SEQ ID NO:32 and/or SEQ 1D NO:33, or nucleic acid sequence which encode one of both of them. Vaccines of = the invention preferably include SEQ ID NO:32 linked to SEQ ID NO:33 by a proteolytic cleavage sequence, such as SEQ ID NO:29, or nucleic acid sequence which.
encodes the fusion protein. Vaccines o the present invention preferably include the IgE leader sequence SEQ ID
NO:28.or nucleic acid sequence which encodes the same. Vaccines of the invention preferably' include SEQ ID NO:31 or the nucleic acid sequence in SEQ fD NO:30.
In some embodiments of the invention, the vaccines of the invention include the SEQ ID
NO:30 and a nucleic acid sequence or amino acid sequence encoded by the nucleic acid sequences thereof selected from the following group: SEQ ID NO:34, SEQ ID
NO:35, and any combination thereof.
Fragments of SEQ ID NO:30 may comprise 30 or more nucleotides. In some embodiments, fragments of SEQ 1D NO:30 may comprise 45 or more nucleotides. In some embodirrients, fragments of SEQ ID NO:30 may comprise 60 or more nucleotides.
hi some .

embodiments, fragments of SEQ ID NO:30 may comprise 75 or more nucleotides. In some embodiments, fragments of SEQ ID NO:30 may comprise 90 or more nucleotides. In some embodiments, fragments of SEQ ID NO:30 may comprise 120 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 150 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 180 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 210 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 240 or more nucleotides.
In some embodiments,, fragments of SEQ ID NO:30 may comprise 270 or more nucleotides.
In some embodiments, fragments of SEQ tD NO:30 may comprise 300 or more nucleotides.
In some embodiments, fraunents of SEQ ID NO:30 may comprise 360 or more nucleotides.
In some embodiments, .fragments of SEQ ID NO:30 may comprise 420 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 480 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 540 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 600 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 660 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 720 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 780 or more nucleotides.
In some embodiments, fragments of SEQ ID .N0:30 may comprise 840 or more nucleotides.
In sorne embodiments, fragments of SEQ ID NO:30 may comprise 900 or more nucleotides.
in some embodiments, fragments of SEQ ID NO:30 may comprise 960 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 1020 or more- nucleotides.
In some = embodiments, fragments of SEQ ID NO:30 ma.y comprise 1080 or more nucleotides. In some embodiments, fragments of SEQ ID NO:30 may comprise 1140 or more nucleotides.
In some embodiments, fragments of SEQ_ ID NO:30 may comprise 1200 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 1260 or more nucleotides.
In some embodiments, fragments of SEQ ID .N0:30 may comprise 1320 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 1380 or more nucleotides.
In some embodiments, fragment's of SEQ. 1-1.) NO:30 may comprise 1440 or more nucleotides. In some embodiments, fragments of SEQ ID NO:30 may comprise 1500 or more nucleotides.
In some -39..

embodiments, fragments of SEQ ID NO:30 may comprise 1560 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:30 may comprise 1620 or more nucleotides.
In some einbodiments, fragments of SEQ. ID NO:30 may comprise 1680 or more nucleotides. In some embodiments, fragments of SEQ NO:30 may comprise 1740 or more nucleotides. In some embodiments, fragments of SEQ NO:30 may comprise coding sequences for the IgE leader = sequences. In some embodiments, fragments of SEQ ID NO:30 do not comprise coding_ sequences for the IgE leader sequences. Fragments may comprise fewer than 60 nucleotides, in some embodiments fewer than 75 nucleotides, in some embodiments fewer than 90 nucl.eotides, in some embodiments fewer than 1.20 nucleotides, in some embodiments fewer than 150 nucleotides, in sonic emboditnents fewer than 180 nucleotides, in some embodiments fewer than 210 nucleotides, in some embodiments fewer than 240 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than 300 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 420 nucleotides, in some embodiments fewer than 480 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 600 nucleotides, in some embodiments fewer than nucleotides, in sonic embodiments fewer than 720 nucleotides, in sonic embodiments fewer than 780 nucleotides, in some embodiments fewer than 840 nucleotides, in. some embodiments fewer than 900 nucleotides, in some embodiments fewer than 960 nucleotides, in some embodiments fewer than 1020 nucleotides, in some embodiments fewer than 1080 nucleotides, in some embodiments fewer than 1140 nucleotides, in some embodiments fewer than 1200 nucleotides, in some embodiments fewer than 1260 nucleotides, in some embodiments fewer than 1320 nucleotides, in some embodiments fewer than 1380 nucleotides, in some embodiments fewer than 1440 nucleotides, in some embodiments fewer than 1500 nucleotides, in some embodiments fewer than 1560 nucleotides, in some embodiments fewer than 1620 nucleotides, in some embodiments fewer than 1680 nucleotides, and in some embodiments fewer than nucleotides.
Fragments ISE() LD NO:31 may comprise 15 or more amino acids, In some embodiments, fragments of SEQ ID NO:31 may comprise 30 or more amino acids. In some em.bodiments, fragments of SEQ ID NO:3.1 may comprise 45 or more amino acids.
lri some embodiments. fragments of SEQ ID NO:31 may comprise 60 or more amino acids. In some embodiments, fragments of SEQ ID NO:31 may comprise 75 or more amino acids. In some embodiments, fragments of SEQ ID NO:31 may comprise 90 or more amino acids. In some embodiments, fraginents of SEQ 11) NO:31 may comprise 105 or more amino acids.
In some embodiments, fragments of SEQ JD NO:31 may comprise 120 or more amino acids.
In some embodiments, fragments of SEQ ID NO:31 may comprise 150 or more amino acids.
In some embodiments, fragments of SEQ ED NO:31 may comprise 1.80 or more amino acids.
In some embodiments, fragments of SEQ ID NO:31 may comprise 210 or more amino acids.
In some embodiments, fragments of SEQ ID NO:31 may comprise 240 or more amino acids.
In some embodiments, fragments of SEQ ID NO:31 may comprise 270 or more amino acids.
In some embodiments, fragments of SEQ ID NO:31 may comprise 300 or more amino acids.
In some embodiments, fragments of SEQ ID NO:31 may comprise 360 or more amino acids.
In some embodiments, fragments of SEQ ID .NO:31 may comprise 420 or more amino acids.
In some embodiments, fragments of SEQ ID NO:31 may comprise 480 or more amino acids.
In some = embodiments, fragments of SEQ ID NO:31 may comprise 540 or more amino acids. In some embodiments, fragments of SEQ ID NO:3 l may comprise 575 or more amino acids.
Fragments may comprise fewer than 30 amino acids, in some embodiments fewer than 45 amino acids, in some embodiments fewer than 60 amino acids, in some embodiments fewer than 75 amino acids, in some embodiments l'ewer than 90 amino acids, in some embodiments fewer than 120 amino acids, i.n some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 270 amino acids, in sorne embodinients fewer thart 300 amino acids, in some cmbodhnents fewer than 360 amino acids, in some embodiments fewer than 420 amino acids, in some embodiments fewer than 480 amino acids, in some embodiments fewer than 540 amino acids, and in some embodiments fewer than 575 amino acids.
hTERT
hIERT is a human telomerase reverse transeriptase that synthesizes a TTAGGG
tag on the end of telomercs to prevent cell death due to chromosomal shortening.
Ilyperproliferative .

cells with abnormally high expression of hIERT may be targeted by immunotherapy. Recent studies also support the abnormal expression oI hTERT on hyperproliferative cells infected with HCV and }VV. Thus, immunotherapy for both HPV and HCV may be enhanced by targeting cells that express hTERT at abnormal levels.
Recent studies demonstrate that hTERT expression in dendritic cells transfected with hTERT genes can induce CDS+ cytotoxie T cells and elicit a CD44- T cells in an antigen-specific fashion. Therefore, use of liTERT expression within antigen presenting cells (APCs) to delay senescence and sustain their capacity to present the antigen of choice is attractive in developing new m.ethods (3f immunotherapy.
According to some embodiments of the invention, methods of inducing an immune response in individuals against an immunogen comprise administering to the individual the hTERT protein and functional fragments thereof or expressible coding sequences thereof in combination with an isolated nucleic acid molecule that encodes protein of the invention and/or a recombinant vaccine that encodes protein of the invention and/or a subunit vaccine that protein of the invention and/or a live attenuated vaccine and/or a killed vaccine.
In some embodiments of the invention, the vaccines of the invention include the SEQ ID
NO:30 and a nucleic acid sequence or amino acid sequence encoded by the nucleic acid sequences thereof selected from the following group: SEQ ID NO:34, SEQ m NO:35, and any combination thereof. In some embodiments of the invention, the vaccines of the invention comprise SEQ ID NO:34 or SEQ ID NO:35. SEQ ID NO:34 comprises the nucleic acid sequence that encodes hTERT. SEQ ID NO:35 comprises the amino acid sequence for hTERT.
In some embodiments of the invention, the vaccines of the invention comprise SEQ JD
NO:22. and SEQ ID NO:34 or S.EQ ID NO: 35. Using nucleic acid sequences that encode hTERT
and/or protein of hTERT in combination with the HPV immunogens enhance the cell-mediated immune response against .HPV-infected cells.
Fragments of SEQ ID NO:34 may comprise 30 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ
ID NO:34 may comprise 45 or more nucleotides, including preferably sequences that encode an immunodaminant cpitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 60 or m.orc nucleotides, including preferably sequences that encode an immuriodominant epitope.
In some embodiments, frag,inents of SEQ ID NC):1 may comprise 75 or more nucleotides, including preferably sequences that encode an immunodorninant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 90 or more nucleotides, including preferably sequences that encode an inimunodominant epitope. In some embodiments, fragments of SEQ ID
-N0:34 may comprise 120 or more nucleotides, including preferably sequences that encode an immunodorninant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 150 or more nucleotides, including preferably sequences that encode an imrnunodominant epitope. .In some embodiments, fragments of SEQ ID NO:34 may comprise 180 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 ina.y comprise 210 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may cornprise 240 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:34 may comprise 270 or more nucleotides, including preferably sequences that encode an immunodominant epitopc. in some embodiments, fragments of SEQ ID NO:34 may comprise 300 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fr4..nnents of SEQ ID NO:34 may comprise 360 or more nucleotides, including preferably sequences that encode an iminunod.omin.ant epitope. In some embodime.nts, fragments of SEQ ID NO:34 may comprise 420 or more nucleotides, including preferably sequences that encode an immunodominant cpi.tope. In some embodiments, fragments of SEQ fE) NO:34 ina.y comprise 480 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:34 may comprise 540 or more nucleotides, including preferably sequences that encode an immunodominant epitope,. In some embodiments, fragments of SEQ ID NO:34 may comprise 600 or more nucleotides, including preferably sequences that encode an immunodorninant = epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 300 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 660 or more nucleotides, including preferably sequences that encode an immunedominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 720 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ 1D
NO:34 may comprise 780 or more nucleotides, including preferably seqtrences that encode an =
immunod.ominant epitope, In some em.bodiments, fragments of SEQ ID NO:34 may comprise 840 or 1110re n.ucleotidcs, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 900 or more nucleotides, including preferably sequences that encode an immunodominant epitope. . In some embodiments, fragments of SEQ ID NO:34 may comprise 960 or more nucleotides, including preferably sequences that encode an immunodominant epitope... In some embodiments, fragments of SEQ ID NO:34 may comprise 1020 or more nucleotides, including preferably sequences that encode aiì immunodominant epitope. . In some embodiments, fragments of SEQ
ID NO:34 may comprise 1080 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may coinprisc 1140 or more nucleotides, including preferably sequences that encode an immun.odominant epitope. . In some embodiments, fragments of SEQ ID NO:34 may comprise 1200 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 1260 or more nucleotides, including preferably sequences that encode an immunodominant cpitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 1320 or more nucleotides, including preferably sequences that encode an im.munodominant epitope. In some embodiments, fragments of SEQ
ID NO:34 may comprise 1380 or more nucleotides, including preferably sequences that encode an immunedominant cpitope. . In some embodirnents, fragments of SEQ ID NO:34 may comprise 1440 or more nucleotides, including preferably sequences that encode an immunodominant epitope. . In some embodiments, fragments of SEQ ID NO:34 may comprise 1500 or more nucleotides, including preferably sequences that encode an immunodominant epitope. . In some embodinients, fragments of SEQ ID NO:34 may comprise 1560 or more nucleotides, including preferably sequences that encode an immunodominant cpitope. In some embodim.ents, fragments of SEQ ID NO:34 may comprise 1620 or more nucleotides, including preferably sequences that encode an immtmodominant epitope, hi some embodiments, fragments of SEQ ID NO:34 may comprise 1680 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:34 may comprise 1740 or triorc nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 1800 or more nucleotides, including preferably sequences that encode an immunodominant epitopc. In some embodiments, fragments of SEQ NO:34 may comprise 1860 or more nucleotides, including preferably sequences that encode an irnmunodominant epitope. In some embodiments, 'fragments of SEQ ID NO:34 may comprise 1920 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 1980 or more nucleotides, including preferably sequences that encode an irnmunodominant epitope. In some embodiments, fragments of SEQ ID
NO:34 may comprise 2040 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise = 2100 or .more nucleotides, including preferably sequences that encode an immunodominant cpitope. In some embodiments, fragments of SEQ ID N0:34 may comprise 2160 or more nucleotides, including preferably sequences that encode a.n immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 2220 or more nucleotides, including preferably sequences that encode an inummodominant epitope. In some em.bodiments, fragnients of SEQ ID NO:34 may comprise 2280 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:34 may comprise 2340 or more nucleotides, including preferably sequences that encode an immunodominant epitope. ín sonic, embodiments, fragments of SEQ ID NO:34 may comprise 2400 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 2460 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodimeitts, fragments of SEQ ID NO:34 may comprise 2520 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 2580 or more nucleotides, including preferably sequences that encode an imimmodominant epitopc. In some embodiments, fragments of SEQ ID
NO:34 may comprise 2640 or more nucleotides, including preferably sequences that encode an iinnumodominant cpitope. In SOrile embodiments, fragments of SEQ ID NO:34 may comprise 2700 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 2760 or more nucleotides, including preferably sequences that encode an irnmunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 2820 or more nucleotides, including preferabl.y sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ I.D NO:34 may comprise 2880 or more nucleotides, including preferably sequences that encode an immunodominant cpitope. In sonic embodiments, fragments of SEQ ID
NO:34 may comprise 2940 or more nucleotides, including preferably sequences that encode an immunodorninant epitope. In sonic embodiments, fragments of SEQ ID NO:34 may comprise 3000 or more nucleotides, including preferably sequences that encode an iminunodominant epitope. fn some embodiments, fragments of SEQ ID NO:34 may comprise 3060 or more nucleotides, including preferably sequences that encode an immunodominant cpitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 3120 or more nucleotides, including preferably sequences that encode an imrnunoclorninant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 3180 or more nucleotides, including preferably sequences- that encode an nrimunodominant cpitope. In some embodiments, fragments of SEQ ID
NO:34 may comprise 3240 or more nucleotides, including preferably sequences that encode an immuriodorninant epitope. In some embodiments, fragments of SEQ 1D NO:34 may comprise 3300 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of' SEQ ID NO:34 may comprise 3360 or more nucleotides, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 3420 or more nucleotides, including preferably sequences that encode an inummodominant epitope. In some embodiments, fragments of SEQ ID NO:34 may comprise 3480 or more nucleotides, including preferably sequences that.
encode an immunodominant epitope. Jn S0111C embodiments. fragments of SEQ ID
NO:34 may comprise coding sequences for the IgE leader sequences. In some embodiments..
fragments of SEQ ID NO:34 do not comprise coding sequences for the íg E leader sequences.
Fragments may comprise fewer than 60 nucleotides, in some embodiments fewer than 75 nucleotides, in some embodiments fewer than 90 nucleotides, in some embodiments fewer than 120 nucleotides, in some embodiments fewer than 150 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 210 nucleotides, in some embodiments fewer than 240 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than 300 nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 420 nucleotides, in some embodiments fewer than 480 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 600 nucleotides, in some embodiments fewer than 660 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 780 nucleotides, in some embodiments fewer than 840 nucleotides, in some embodiments fewer than 900 nucleotides, in some embodiments fewer than 960 nucleotides, in some embodiments fewer than 1020 nucleotides, in some embodiments fewer than 1080 nucleotides, in some embodiments fewer than 1140 nucleotides, in some embodiments :fewer than 1200 nucleotides, in some embodiments fewer than 1260 nucleotides, in sorne embodiments fewer than 1320 nucleotides, in some embodiments fewer than 1380 nucleotides, in some embodiments fewer than 1440 nucleotides, in some embodiments fewer than 1500 nucleotides, in some embodiments fewer than 1560 nucleotides, in some embodiments fewer than. 1620 nucleotides, in some embodiments fewer than 1680 nucleotides, in some embodiments fewer than 1740 nucleotides, in some embodiments fewer than 1800 nucleotides, in some embodiments fewer than 1860 nucleotides, in sonic embodiments fewer than 1920 nucleotides, in some embodiments fewer than 1980 nucleotides, in some embodiments fewer than 2040 nucleotides, in some embodiments fewer than 2100 nucleotides, in some embodiments fewer than 2160 nucleotides, in sonic embodiments fewer than 2220 nucleotides, in SOITIC
embodiments fewer than 2280 nucleotides, in some embodiments fewer than 2340 nucleotides, in some embodiments fewer than 2400 nucleotides, in some embodiments fewer than 2460 nucleotides, in some embodiments fewer than 2520 nucleotides, in some embodiments fewer than 2580 nucleotides, in some embodiments fewer than 2640 nucleotides, in some embodiments = fewer than 2700 nucleotides, in SOMC embodiments fewer than 2760 nucleotides, in some embodiments fewer than 2820 nucleotides, in some embodiments fewer than 2860 nucleotides, in some embodiments fewer than 2940 nucleotides, in some embodiments fewer than 3000 nucleotides, in some embodiments fewer than 3060 nucleotides, in some embodiments fewer than 3120 nucleotides, in some- embodiments fewer than 3180 nucleotides, in some embodiments Fewer than 3240 nucleotides, in some embodiments fewer than 3300 nucleotides, in some embodiments fewer than 33610 nucleotides, in some embodiments fewer than 3420 nucleotides, in sonic embodiments fewer than 3480 nucleotides, and in some embodiments fewer than 3510 nucleotides.
Fragments of SEQ ID NO:35 may comprise 15 or more amino acids, including preferably sequences that encode an immunodominant epitope. In sonic embodiments, fragments of SEQ
ID NO:35 may comprise 18 or more amino acids, including preferably sequences that encode an inummodominant epitope. In sonie embodiments, fragments of SEQ ID NO:35 may comprise 21 or more a.mitio acids, including preferably sequences that encode an immtmodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 24 or more amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ ID NO:35 may comprise 30 or more amino acids, including preferably sequences that encode an immunodorninant epitope. In some embodiments, .fragments of SEQ ID NO:35 may comprise 36 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ
ID NO:35 may comprise 42 or more amino acids, including preferably sequences that encode an immunodomina-nt epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 48 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 54 or more amino including- preferably sequences that encode an immunodominant epitopc. In some embodiments, frag.ments of .SEQ ID NO:35 may comprise 60 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 66 or more amino acids, including preferably sequences that encode an immunodominant cpitope. In. some embodiments, fragments of SEQ
ID NO:35 may comprise 72 or more amino acids, including preferably sequences that encode an imniunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 90 or more amino acids, including preferably sequences that encode an immunodominant epitope. In SOITIC= embodiments, fragments of SEQ ID NO:35 may- comprise 120 or more amino acids, including preferably sequences that encode an immunodominant epitope.
In soine embodiments, fragments of SEQ ID NO:35 may comprise 150 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ fD NO:35 may comprise 180 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ
ID NO:35 may comprise 210 or more amino acids, including preferably sequences that encode an immunodominant epitope. In sonic embodiments, fragments of SEQ ID N0:35 may comprise 240 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 270 or _more amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ ID NO:35 may comprise 300 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ fD NO:35 may comprise 330 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:35 may comprise 360 or more amino acids, including preferably.sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 390 or more amino acids, including preferably sequences that encode an immunodominant cpitope. hi some embodiments, fragments of SEQ ID NO:35 may comprise 420 or more amino acids, including preferably sequences that encode an immunodorninant epitope.
In some embodiments, fragments of SEQ ID NO:35 may comprise 450 or more amino acids, including preferably sequences that encode ail immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 niay comprise 480 or more amino acids, including preferably sequences that encode an immunodorninant epitope. In some embodiments, fragments of SEQ ID
NO:35 may comprise 510 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 540 or niore amino acids, including preferably sequences that encode an immunodorninant epitope. In some embodiments, fragments of SEQ NO:35 may comprise 570 or more amino acids, including preferably sequences that encode an immunodominant epitopc.
In some embodiments, fragments of SEQ ID NO:35 may comprise 600 or more amino acids, including preferably sequences that encode an immunodominant epitope, in sotne embodiments, fragments of SEQ ID NO:35 may comprise 630 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:35 may comprise 660 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise = 690 or more amino acids, including preferably sequences that encode an immunodominant epitope. In SOITIC embodiments, fragments of SEQ
NO:35 may comprise 720 or more amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ ID NO:35 may comprise 750 or more amino acids, including preferably sequences that encode an immtmodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 780 or more amino acids, including preferably sequences that encode an immunodominant epitopc. In some embodiments, fragments of SEQ ID
NO:35 may comprise 810 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 840 or more amino acids, including preferably sequences that encode an immunodominant epi(ope. In some embodiments, fragments of SEQ ID NO:35 may comprise 870 or more amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ .1.1) NO:35 may comprise 900 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 930 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:35 may comprise 960 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 990 or more amino acids, including preferably sequences that encode an immunodorninant epitope. hi some embodiments, fragments of SEQ ID NO:35 may comprise 1020 or more amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ ID NO:3-5 may comprise 1050 or more amino acids, including preferably sequences that encode an immunodominant epitopc. In some embodiments, fragments of SEQ ID NO:35 may comprise 1080 or more amino acids, including preferably sequences that encode_ an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:35 may comprise 11 10 or more amino acids; including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 1140 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 1170 or more amino acids, including preferably sequences that encode an immunodominant epitope, 1.n some embodiments, fragnicnts of SEQ ID NO:35 may comprise 1200 or more amino acids, including preferably sequences. that encode an immunodominant epitope. In some embodiments, fragments = of SEQ ID NO:35 may comprise 1230 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID
NO:35 may comprise 1260 or more amino acids, including preferably sequences that encode an immunodominant epitope. ln some embodiments, fragments of SEQ ID NO:35 may comprise 1290 or more amino acids, including ffeferably sequences that encode an immunodominam epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 1320 or more amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ lD NO:35 may comprise 1350 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ 1.D NO:35 may comprise 1380 or more amino acids, including preferably sequences that encode an immunodominant epitope. In, some embodiments, fragments of SEQ ID
NO:35 may comprise 1410 or inore amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may com.prise 1440 or more amino acids, including preferably sequences that encode an immunodominant epitope. In some embodiments, fragments of SEQ ID NO:35 may comprise 1470 or more. amino acids, including preferably sequences that encode an immunodominant epitope.
In some embodiments, fragments of SEQ ID NO:35 may comprise 1500 or more amino acids, including preferably sequences that encode an immtmodominant epitope. In some embodiments, fragments of SEQ ID NO:3.5 may comprise coding sequences for the lgE leader sequences. In some embodiments, fragments of S.EQ ID NO:35 do not comprise coding sequences for the IgE
leader sequences. Fragments may comprise fewer than 24 amino acids, in sonic embodiments fewer than 30 amino acids, in some embodiments fewer than 36 amino acids, in some embodiments fewer than. 42 amino acids, in some embodiments fewer than 48 amino acids, in some embodiments fewer than 54 amino acids, in some enabodiments fewer than 60 amino acids, in some embodiments fewer than 72 amino acids, in some embodiments fewer than 90 amino acids, in some embodiments fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids in some embodiments fewer titan 240 amino acids, in some embodiments fewer than 260 amino acids, in some embodiments fewer than 290 amino acids, in some embodiments fewer than 320 amino acids, in some embodiments fewer than 350 amino acids, in some embodiments fewer than 380 amino acids, in some embodiments fewer than 410 amino acids in some embodiments fewer than 440 amino acids, in some embodiments fewer than 470 amino acids in some embodiments fewer titan 500 amino acids, in some embodiments fewer than 530 amino acids in some embodiments fewer than 560 amino acids, in some embodiments fewer than 590 amino acids, in some embodiments fewer than 620 amino acids, in some embodiments fewer than 650 amino acids, in some embodiments fewer than 680 amino acids, in some embodiments fewer than 710 amino acids, in some embodiments fewer than 740 amino acids, in some embodiments fewer than 770 amino acids, in some embodiments fewer than 800 amino acids, in some embodiments fewer than 830 amino acids, in some embodiments fewer than 860 amino acids, in sorne embodiments fewer than 890 amino acids, in some embodiments fewer than 920 amino acids, in some embodiments fewer than 950 amino acids, in some embodiments fewer than 980 amino acids, in some embodiments fewer than 1010 amino acids, in some embodiments fewer than 1040 amino acids, in some embodiments fewer than 1070 amino acids, in some embodiments fewer than 1200 amino acids, in some embodiments fewer than 1230 amino acids, in some embodiments fewer than 1260 amino acids, in some embodiments fewer than 1290 amino acids, in some embodiments fewer than 1320 amino acids. in sonic embodiments fewer than 1350 amino acids, in SOT11C embodiments fewer than 1380 amino acids.

in some embodiments fewer than 1410 amino acids, in some embodiments fewer than 1440 amino acids, in some embodiments fewer than 1470 amino acids, and in some embodiments fewer than 1500 amino acids.
Influenza A.ccording to some embodiments of the invention, methods of inducing an immune response in individuals against ari immunogen comprise administering to the individual the Influenza strain H5N1 hemagedutinin (HA) protein and functional fragments thereof or expressible coding sequences thereof in combination with an isolated nucleic acid molecule that encodes protein of the invention and/or a recombinant vaccine that encodes protein of the invention and/or a subunit vaccine that protein of the invention and/or a live attenuated vaccine and/or -a killed vaccine. In SOIThe embodiments, the Influenza vaccine compositions and methods comprise the use of a nucleic acid sequence that encodes HA protein from Influenza virus species. In some embodiments, the Influezna vaccine compositions and method comprise the use of nucleic acid sequences that encode HA from Influenza viral strain H1N5 and nucleic acid sequences encoding Influenza proteins selected from the group consisting of:
SEQ ID NO:38, SEQ ID NO:40, and SEQ ID NO:42. In some embodiments of the invention, the vaccines of the invention comprise SEQ TD NO:36 or SEQ ID NO:37. SEQ ID NO:36 comprises the nucleic acid sequence that encodes 111N5 HA of Influenza virus. SEQ ID NO:37 comprises the amino acid sequence for H1N5 HA of Influenza virus. In some embodiments of the invention, the vaccines of the invention comprise SEQ ID NO:38 or SEQ ID NO:39. SEQ ID NO:38 comprises the nucleic acid sequence that encodes Influenza H1N1 and H5N1 NA
consensus sequences. SEQ ID NO:39 comprises the amino acid sequence for Influenza MN]
and H5N1 NA consensus sequences. In some embodiments of the invention, the vaccines of the invention comprise SEQ ID NO:40 or SEQ ID NO:41. SEQ ID NO:40 comprises the nucleic acid sequence that encodes Influenza RINI and II5N1 M1 consensus sequences. SEQ 1.D
NO:41 comprises the amino acid sequence for Influenza H1N1 and H5N1. M1 consensus sequences. In some embodiments of the invention, the vaccines of the invention comprise SEQ
ID NO:42 or SEQ ID .N0:43. SEQ ID NO:42 comprises the nucleic acid sequence that encodes Influenza H5N1 M2E-NE consensus sequence. SEQ ID NO:43 comprises the amino acid sequence for Influenza H.5N I M2E-NP consensus sequence. In some embodiments of the invention, the vaccines of the invention include the SEQ ID NO:36 and a sequence selected from the following group: SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ
ID NO:42, SEQ ID NO:43. and any combination thereof. The consensus sequence for Influenza virus strain H5N1 HA includes the inummodominant epitope set forth in SEQ ID
NO:36. The influenza virus :145N1 HA amino acid sequence encoded by SEQ ID NO:36 is SEQ
ID NO:37.
The consensus sequence for Influenza virus HiN1/115N I NA includes the immunodominant epitope set forth in SEQ ID NO:38. The Influenza virus strains IIIN11H5N1 NA
a.mino acid sequence encoded by- SEQ. ID NO:38 is SEQ I.D NO:39. The consensus sequence for influenza virus strains RINI/115N1 MI includes the immunodorninant epitope set forth in SEQ ID NO:40.
The 'Influenza virus WIN1/11.5N1 1\41 amino -acid sequence encoded by SEQ ID
NO:40 is SEQ
ID NO:41. The consensus sequence for Influenza virus H5NI M2E-NP includes the irnmunod.ontinant cpitope set forth in SEQ ID NO:42. The Influenza virus H5N 1 amino acid sequence encoded by SEQ ID NO:42 is SE() ID NO:43. Vaccines of the present invention may include protein products encoded by the nucleic acid molecules defined above or any fragments of proteins.
Fragments of SEQ NO:36 rriaµ,' comprise 30 or more nucleotides. In some embodiments, .fragments of SEQ ID NO:36 may comprise 45 or more nucleotides.
in some embodiments, fragments of SEQ ID NO:36 may comprise 60 or more nucleotides. In SOTIle embodiments, fragments of SEQ ID NO:36 may comprise 75 or more nucleotides. In some embodiments, fragments of SEQ ID NO:36 may comprise 90 or more nucleotides. In some embodiments, fragments of SEQ ID NO:36 rnay comprise 120 or more nucleotides.
in some embodiments, fragments of SEQ ID NO:36 may comprise 1 50 or more nucleotides.
In some embodi men ts, fragments of SEQ NO:36 may comprise 180 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 210 or more nucleotides.
In SOITIC
embodiments, fragments of SEQ ID NO:36 may- comprise 240 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 270 or more nucleotides.
In SOIlle embodiments, fragments of SEQ ID NO:36 may comprise 300 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 360 or more nucleotides.
In some -54-.

embodiments, fragments of SEQ ID NO:36 may comprise 420 or more nucleotides.
In some embodim.ents, fragments of SEQ ID NO:36 may comprise 480 or more nucleotides, In some embodiments, fragments of SEQ ID NO:36 may comprise 540 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 600 or more nucleotides.
In some embodiments, fragments of SEQ 1D NO:36 may comprise 660 or more nucleotides.
In some embodiments, framents of SEQ ID NO:36 may comprise 720 or more nucleotides. In some embodiments, fragments of SEQ ID NO:36 may comprise 780 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 840 or mom nucleotides. In some embodiments, fragments of SEQ ID NO:36 may comprise 900 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 960 or more nucleotides.
In some embodiments, fragments of SEQ NO:36 may comprise 1020 or more nucleotides. In some embodiments, fragments of SEQ ID NO:36 may comprise 1080 or more nucleotides.
In some embodiments, fragments of SEQ ID .NO:36 may comprise 1140 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 1200 or more nucleotides.
In somc embodiments, fragments of SEQ ID NO:36 may comprise 1260 or more nucleotides.
Tii some embodiments, fragments of SEQ ID NO:36 may comprise 1320 or more nucleotides.
in some embodiments, fragments of SEQ ID NO:36 may comprise 1380 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 1440 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 1500 or more nucleotides.
In some embodiments, fragments of SEQ CD NO:36 may comprise 1560 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 1620 or more .nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 1680 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise 1740 or more nucleotides.
In some embodiments, fragments of SEQ ID NO:36 may comprise coding sequences for the IgE leader sequences. In some embodiments, fragments of SEQ ID NO:36 do not comprise coding sequences for the igE leader sequences. Fragments of SEQ ID NO:36 may comprise fewer than 60 nucleotides, in some embodiments fewer than 75 nucleotides, in sonic embodiments fev,Ter than 90 nucleotides, in some embodiments fewer than 120 nucleotides, in some embodiments fewer than 150 nucleotides, in some embodiments fewer than I 80 nucleotides, in some embodiments fewer than 210 nucleotides, in some embodiments fewer than 240 nucleotides, in some embodiments fewer than 270 nucleotides, in some embodiments fewer than.

nucleotides, in some embodiments fewer than 360 nucleotides, in some embodiments fewer than 420 nucleotides, in some embodiments fewer than 480 nucleotides, in some embodiments fewer than 540 nucleotides, in some embodiments fewer than 600 nucleotides, in some embodiments fewer than 660 nucleotides, in some embodiments fewer than 720 nucleotides, in some embodiments fewer than 780 nucleotides, in some embodiments fewer than 840 nucleotides, in sonic embodiments fewer than 900 nucleotides, in some embodiments fewer than nucleotides, in some embodiments fewer than 1020 nucleotides, in some embodiments fewer than 1080 nucleotides, in some embodiments fewer than 1140 nucleotides, in some embodiments fewer than 1200 nucleotides, in some embodiments fewer than 1260 nucleotides, in some embodiments fewer than 1320 nucleotides, in some embodiments fewer than. l 380 nucleotides, in sonic embodiments fewer than 1440 nucleotides, in some embodiments fewer than 1500 nucleotides, in some embodiments fewer than 1560 nucleotides, in some embodiments fewer than 1620 nucleotides, in some embodiments fewer than 1680 nucleotides, and in some embodiments fewer than 1740 nucleotides.
Fragments of SEQ NO:37 may comprise 15 or more amino acids. In some embodiments, fragments of SEQ ID NO:37 may comprise 30 or more amino acids. In some embodiments, fragments of SEQ ID NO:37 may comprise 45 or more amino acids. In some embodiments, &ailments of SEQ 1D NO:37 may comprise 60 or more amino acids. In some embodiments, fragments of SEQ ID NO:37 may comprise 75 or more amino acids. In some embodiments, fragments of SEQ ID NO:37 may comprise 90 or more amino acids. In some embodiments, fragments of SEQ ID NO:37 may comprise 105 or more amino acids.
ln some embodiments, fragments of SEQ ID NO:37 may comprise 120 or more amino acids.
In some embodiments, fragments of SEQ ID NO:37 may comprise 150 or more amino acids.
In sonic embodiments, fragments of SEQ ID NO:37 may comprise 180 or more amino acids.
In some embodiments, fragments of SEQ ID NO:37 may comprise 210 or more amino acids.
In some embodiments, fragments of SEQ NO:37 may comprise 240 or more amino acids. In sonic embodiments, fragments of SEQ ID NO:37 may comprise 270 or more amino acids.
In some embodiments, fragments of SEQ IID NO:37 may comprise 300 or more amino acids.
In some embodiments, fragments of SEQ ID NO:37 may comprise 360 or more amino acids.
In some embodiments, fragments of SEQ ID NO:37 may comprise 420 or more amino acids.
In some embodiments, fragments of SEQ ID .NO:37 may comprise 480 or more amino acids.
in some = embodiments, fragments of SEQ ID NO:37 may comprise 540 or more amino acids. In some embodiments, fragments of SEQ I.D NO:37 may comprise 565 or more amino acids.
Fragments of SEQ NO:37 mav comprise fewer than 30 amino acids, in some embodiments fewer than 45 amino acids, in some embodiments fewer than 60 amino acids, in some embodiments fewer than 75 amino acids, in some embodiments fewer than 90 amino acids, in some embodiments 'fewer than 120 amino acids, in some embodiments fewer than 150 amino acids, in some embodiments fewer than 180 amino acids, in some embodiments fewer than 210 amino acids, in some embodiments fewer than 240 amino acids, in some embodiments fewer than 270 amino acids, in some embodiments fewer than 300 amino acids, in some embodiments fewer than 360 amino acids, in some embodiments fewer than. 420 amino acids, in some embodiments fewer than 480 amino acids, in some embodiments fewer than 540 amino acids, and in some embodiments fewer than 565 amino acids.
According to some embodiments of the invention, methods of inducing an immune response in individuals against an immunogen comprise administering to the individual the Influenza strain HINI and Influenza strain H5NI NA protein and functional fragments thereof or expressible coding sequences thereof in combination with an isolated nucleic acid molecule that encodes protein of the invention andlor a recombinant vaccine that encodes protein of the invention and/or a subunit vaccine that protein of the invention and/or a live attenuated vaccine and/or a killed vaccine.
According to some embodiments of the invention, methods of inducing an immune response in individuals against an immunogen comprise administering to the individual the influenza strain H1NII and Influenza strain 115N1 M1 protein and functional fragments thereof or expressible coding sequences thereof in combination with an isolated nucleic acid molecule that encodes protein of the invention and/or a recombinant vaccine that encodes protein of the invention and/or a subunit vaccine that protein of the invention andlor a live attenuated vaccine and/or a killed vaccine.
=
According to some embodiments of the invention, methods of inducing an immune response in individuals against an immunogen comprise administering to the individual the Influenza strain H5N1 M2E-NP protein and functional fragments thereof or expressible coding sequences thereof in combination with an isolated nucleic acid molecule that encodes protein of the invention and/or a recombinant vaccine that encodes protein of the invention and/or a subunit vaccine that protein of the invention and/or a live attenuated vaccine and/or a killed vaccine.
Vaccines The invention provides improved vaccines by providing proteins and genetic constructs that encode proteins with epitopcs that make them particularly effective as immunogens against which immune responses can be induced. Accordingly, vaccines can be provided to induce a therapeutic or prophylactic immune response. In some embodiments, the means to deliver the immunogen is a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the imm.unogen, an attenuated vaccine or a killed vaccine. In some embodiments, the vaccine comprises a combination selected from the groups consisting of:
one or more DNA
vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions comprising the immunogen, one or more attenuated vaccines and one or -more killed vaccines.
According to some embodiments of the invention, a vaccine according to the invention is delivered to an individual to modulate the activity of the individual's immune system and thereby enhance the immune response. When a nucleic acid molecules that encodes the protein is taken up by cells of the individual the nucleotide sequence is expressed in the cells and the protein are thereby delivered to the individual. Aspects of the invention provide methods of delivering the coding sequences of thc protein on nucleic acid molecule such as plasmid, as part of recom.binant vaccines and. as part of attenuated vaccines, as isolated proteins or proteins part ofa vector.
According to some aspects of the present invention, compositions and methods are provided which prophylactically and/or therapeutically- immunize an individual DNA vaccines are described in US. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, 5,676,594, and the priority applications cited therein. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in US. Patent Nos. 4,945,050 and 5,036,006.
The present invention relates to improved attenuated live vaccines, improved killed vaccines and improved vaccines that use recombinant vectors to deliver foreign genes that encode antigens and well as subunit and glycoprotein vaccines. Examples of attenuated live vaccines, those using recombinant vectors to deliver foreign antigens, subunit vaccines and glycoprotein vaccines are described in U.S. Patent Nos.: 4,510,245; 4,797,368;
4,722,848;
4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993;
5,223,424;
5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744;
5,389,368;
5,424,065; 5,451,499; 5,453,3 64; 5,462,734; 5,470,734; 5,474,935; 5,482,713;
5,591,439;
5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529 When taken up by a cell, the genetic construct(s) may remain present in the cell as a.
functioning extrachromosomal molecule and/or integrate into the cell's chromosomal DNA.
DNA may be introduced into cells where it remains as separate genetic material in the form of a plasmid or plasmids. Alternatively, linear DNA that can integrate into the chromosome may be introduced into the cell. When introducing DNA into the cell, reagents that promote DNA
integration into chromosomes may be added. DNA sequences that are useful to promote integration may also be included in the DNA molecule. Alternatively, RNA may be administered to the cell. It is also contemplated to provide the genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Gene constructs may remain part of the genetic material in attenuated live microorganisms or recombinant microbial vectors which live in cells. Gene constructs may be part of genomes of recombinant viral vaccines where the genetic material either integrates into the chromosome of the cell or remains extrachromosomal. Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenvlation signal. In addition, enhancers are often required for gene expression of the sequence that encodes the target protein or the immunomodulating protein. h is necessary that = these elements be operable linked to the sequence that encodes the desired proteins and that the regulatory elements are operably in die individual to whom they are administered.
Initiation codons and stop eodon are generally considered to be part of a nucleotide sequence that encodes the desired protein. However, it is necessary that these elements are functional in the individual to whom the gene construct is administered. The initiation and termination codons must be in frame with the coding sequence.
Promoters and polyadenylation signals used must be functional within the cells of the individual.
=.Examples of promoters useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (MV) such as the BIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovinis (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metalothionein.
Examples of polyadenylation signals useful to practice the present invention, especially in the production of a genetic vaccine for humans, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the = polyadenylation signal that is in pCEP4 plasmid (Invitrogen, San Diego CA), referred to as the SV40 polyadenylation signal, is used.
In addition to the regulatory elements required for DNA expression, other elements may also be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including but not limited to: human Actin, human Myosin, human Hemoglobin, human muscle ereatine and viral enhancers such as those from CMV, RSV and EBV.
Genetic constructs can be provided with mammalian origin of replication in order to maintain the construct extrachromosomally and produce multiple copies of the construe( in the cell. Plasmids pVAX1, pCEP4 and p.REP4 from Invitrogen (San Diego, CA) contain the Epstein Barr virus origin of replication and nuclear antigen EBNA-I coding region which produces high copy episomal replication without integration.
In some preferred embodiments related to immunization applications, nucleic acid molccule(s) are delivered which include nucleotide sequences that encode protein of the invention , and, additionally, genes for proteins which further enhance the immune response = against such target proteins. Examples of such genes are those which encode other cytokines and lymphokines such as alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFa, TNFO, GM-CSF, epidemal growth factor (EGF), IL-1, 1L-4, IL-5, 1L-6, IL-10, IL-12, EL-18, MFIC, CD80,CD86 and IL- 15 including IL-I5 having the signal sequence deleted and optionally including the signal peptide from IgE. Other genes which ma.y be useful include those encoding: MCP-1, MIP-la, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, C1334, GlyCAM-I, MadCAM-1, [.FA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD4OL, vascular growth factor, 1L-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-I, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, .NGRE, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, IVIyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JI\TIC, interferon response genes, NEkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP l, TA.P2 and functional fragments thereof An additional element may be added which serves as a target for cell destruction if it is desirable to eliminate cells receiving the genetic construct for any reason. A
herpes thymidine kinase (tk) gene in an expressible form can be included in the genetic construct. The drug gangcyclovir can be administered to the individual and that drug will cause the selective killing of any cell producing tic, thus, providing the means for the selective destruction of cells with the genetic construct.
In order to maximize protein production, regulatory sequences may be selected which are well suited for gene expression in the cells the construct is administered into. Moreover, codons may be selected which are most efficiently transcribed in the cell. One having ordinary skill in the art can produce DNA constructs that are functional in the cells.
In some embodiments, gene constructs may be provided in which the coding sequences for the proteins described herein are linked to IgE signal peptide. In some embodiments, proteins described herein are linked to IgE signal peptide.
In some embodiments for which protein is used, for example, one having ordinary skill in the art can, using well known techniques, produce and isolate proteins of the invention using well known techniques. In some embodiments for which protein is used, for example, one having ordinary skill in the art can, using well known techniques, inserts DNA
molecules that encode a protein of the invention into a commercially available expression vector for use in well known expression systems. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) may be used for production of protein in E.
coli. The commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) may, for example, be used for production in S. cerevisiae strains of yeast. The commercially available MAXBACTM
complete baculovirus expression system (Invitrogen, San Diego, Calif.) may, for example, be used for production in insect cells. The commercially available plasmid pcDNA
I or pcDNA3 (Invitrogen, San Diego, Calif.) may, for example, be used for production in mammalian cells such as Chinese Hamster Ovary cells. One having ordinary skill in the art can use these commercial expression vectors and systems or others to produce protein by routine techniques and readily available starting materials. (See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989) .) Thus, the desired proteins can be prepared in both prokaryotic and eukaryotie systems, resulting in a spectrum of processed forms of the protein.
One having ordinary skill in the art may use other commercially available expression vectors and systems or produce vectors using well known methods and readily available starting materials. Expression systems containing the requisite control sequences, such as promoters and polyadenylation signals, and preferably enhancers are readily available and known in the art for a variety of hosts. See e.g., Sambrook et al., Molecular Cloning a Laboratory Manual, Second Ed.
Cold Spring Harbor Press (1989). Genetic constructs include the protein coding sequence operably linked to a promoter that is functional in the cell line into which the constructs are transfected. Examples of constitutive promoters include promoters from cytomegalovirus or SV4O. Examples of inducible promoters include mouse mamm.ary leukemia virus or metallothionein. promoters. Those haying ordinary skill in the art can readily produce genetic constructs useful for transfeeting with cells with DNA. that encodes protein of the invention from readily available starting materials. The expression vector including the DNA
that encodes the protein is used to transform the compatible host which is then cultured and maintained under conditions wherein expression of the foreign DNA takes place.
The protein produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art. One having ordinary skill in the art can, using well known techniques, isolate protein that is produced using such expression systems. The methods of purifying protein from natural sources using antibodies .which specifically bind to a specific protein as described above may be equally applied to purifying protein produced by recombinant DNA methodology.
In addition to producing proteins by recombinant techniques, automated peptide synthesizers may also be employed to produce isolated, essentially pure protein. Such techniques arc .well known to those having ordinary skill in the art and are useful if derivatives which have substitutions not provided for in DNA-encoded protein production.
The nucleic acid molecules may be delivered using any of several well known technologies including- DNA injection (also referred to as DNA vaccination), recombinant vectors such as recombinant adenovirus, recombinant adenovirus associated virus and recom.binant vaccinia.
Routes of administration include, but are not limited to, intramuscular, intransally, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterially, intraoccularly and oral as well as topically, transdermally, by inhalation or suppository or to mucosal tissue such as by = lavage to vaginal, rectal, urethral, buccal and sublingual tissue.
Preferred .routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. Genetic constructs may be administered by means including, but not limited to, traditional syringes, needleless injection devices, or "microprojectile bombardment gone guns".

= In some embodiments, the nucleic acid molecule is delivered to the cells in conjunction with administration of a polynucleotide function enhancer or a genetic vaccine facilitator agent.
Folynucleotide function enhancers are described in U.S. Serial Number 5,593,972, 5,962,428 and International Application Serial Number PCT/US94/00899 filed January 26, 1994.
Genetic vaccine facilitator agents are described in US.
Serial Number 021,579 filed April 1, 1994.
The co-agents that are administered in conjunction with nucleic acid molecules may be administered as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after administration of nucleic acid molecules. In addition, other agents which may function transfecting agents and/or replicating agents and/or inflammatory agents and which may be co-administered with a GVF include growth factors, cytokines and lymphokines such as a-interferon, gamma-interferon, GM-CSF, platelet derived growth factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, 1L-6, IL-10, IL-12 and IL-15 as well as fibroblast growth factor, surface active agents such as immune-stimulating complexes (1SCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl Lipid A (WL), muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct In some embodiments, an immunomodulating protein may be used as a GVF. In some embodiments, the nucleic acid molecule is provided in association with PLG to enhance delivery/uptake.
The pharmaceutical compositions according to the present invention comprise about 1 nanogram to about 2000 micrograms of DNA. In some preferred embodiments, pharmaceutical compositions according to the present invention comprise about 5 nanogram to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 10 nanograms to about 800 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 0.1 to about 500 micrograms of DNA.
In some preferred embodiments, the pharmaceutical compositions contain about 1 to about 350 = micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 100 to about 200 microgram DNA.

The pharmaceutical com.positions according to the present invention are formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free and particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin, In some embodiments, a vasoconstriction agent is added to the formulation.
According to some embodim.ents of the invention, methods of inducing inunune responses are. provided. The vaccine ma.y be a protein based, live attennatcd vaccine, a cell vaccine, a recombinant vaccine or a nucleic acid or DNA vaccine. In some embodiments, methods of inducing an immune response in individuals against an immtmogcn, including methods of inducing mucosal immune responses, comprise administering to the individual one Or MOTC of CTACK protein, TECK protein, MEC protein and functionai fragments thereof or expressible coding sequences thereof in combination with an isolated nucleic acid molecule that encodes protein of the invention andlor a recombinant vaccine that encodes protein of the invention and/or a subunit vaccine that protein of the invention and/or a live attenuated vaccine and/or a killed vaccine. The one or more of CTACK protein, TECK protein, MEC
protein and functional fragments thereof may be administered prior to, simultaneously with or after administration of the isolated nucleic acid molecule that encodes an immunogen; and/or recombinant vaccine that encodes an im.munogen and/or subunit vaccine that comprises an immtmogen and/or live attenuate.d vaccine and/or killed vaccine. In some embodiments, an isolated nucleic acid molecule that encodes one or more proteins of selected from the group consisting of: CTACK, TECK, 'MEC and functional fragments thereof is administered to the individual.
EXAMPLES
Example 1 MATERIALS AND METHODS

HIV-1 subtype B envelope sequences. To generate HIV-1 subtype B consensus envelope sequence, forty-two subt)pc II envelope gene sequences collected from eleven countries were selected front GenBank to avoid sampling bias. Each sequence represents a different patient. All sequences used are non-recombinant.
Multiple alignment. The alignment procedure applied in the phylogcnetic study included the application of Clustal X (version 1.81) (Thompson, J. D., et al. 1997. The Clusta.IX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
Nucleic Acids Research 25:4876-4882). Panwise alignment parameters were set to the dynamic "slow-accurate" programming, using 10 as the gap opening penalty and 0.1 as the gap extension penalty. Multiple alignment parameters included a gap extension penalty equal to 0.2.
Construction of RIV-1 subtype 13 envelope consensus sequence. The HIV-1 subtype B
envelope consensus nucleotide sequence was obtained after performing multiple alignment and minor final manual adjustment. Deduced amino acid sequences were used to guide the introduction of alignment gaps. so that they were inserted between codons. The consensus amino acid sequence was obtained by translating the consensus nucleotide sequence.
Phylogenetic tree. The neighbor-joining (NJ) method was employed for amino acid phylogenetie tree-building using the program PAUP* 4.0b10 (Swofford, D. L.
1999. PAUP* 4.0:
phylogenetic analysis -using parsimony (* and other methods), version 4.0b2a.
Sinauer Associates, Inc.õ Sunderland, Mass.). Two additional. sequences from subtype D
(K03454 and AAA44873) and two sequences from subtype C (AAD12103 and AAD12112) were used as an outgroup for rooting (Kuiken, C., B. T. Korber, and R. W. Shafer. 2003. 'HIV
sequence databases. AIDS Rev. 5:52-61).
Modifications of H1V-1 subtype B envelope consensus sequence. Several modifications were performed after obtaining HIV-1 subtype B consensus envelope sequence:
highly variable VI and V2 regions were shortened, V3 loop was designed for CCR5 utilization, the cytoplasmic tail region was removed from the C-terminal, a leader sequence and an upstream Kozak sequence were added to the N-tenninal, codon optimization and RNA optimization was performed by using GeneOptimizerTM (GENEART, Germany).

Envelope Immunogens. 'The gene encoding modified HIV-1 subtype B early transmitter consensus envelope glycoprotein (EY2E1-B) was synthesized and sequence verified by GENEART. The synthesized EY2E1-11 was digested with BamH1 and Notl, cloned into the expression vector pVAX (hwitrogen) under the control of the cytomegalovirus immediate-early promoter and this construct was named as pEY2E1-B.
The primary subtype B immunogen (EK2P-B) was generated from a human codon biased, primary subtype B isolate 6101 gp140 envelope gene that was a gift of M. Sidh.m (Wyeth). Basically, the optimized 6101 envelope gene was mutated by removing the native leader sequence and cytoplasmic tail. Then the IgE-leader sequence and Kozak sequence were introduced by designing forward and reverse specific- primers: Env-F: 5'-GTCGCTCCGCTAGCTTGTGGGTCACAGTCTATTATGGGGTACC-3' (SEQ ID NO:13) Env-R: 5'-GGTCGGATCCTTACTCCACCACTCTCCTTTTTGCC-3' (SEQ ID NO:14). The purified PCR product was cloned into pVAX plasmid vector, which was also linearized with EcoR1 and Xbai. This construct was named as pEK2P-B.
In vivo Expression and Reactivity of EY2E1-B with Monoclonal Antibodies. Human rhabdom.yosareoma (RD) cells (2 x 106) were transfected in 60 mm dishes with 3 Eig of pEY2E1-B and pEK2P-B plasmids using FuGENE 6 Transfection Reagent (Roche, Germany), respectively. Forty-eight hours after transfection, cells were washed three times with 1 x PBS
and lysed in 150 pi of lysis buffer (Cell Signaling Technology). The total protein lysates (50 ti.g) were fractional on a SDS-PAGE gel, transferred to a PVDE membrane (Amersham).
Inummoblot analyses were performed with an envelope-specific monoclonal antibody 2G12 (NIH AIDS Research and Reference Reagent Program, Rockville, MD, USA) and a monoclonal anti-actin antibody (Sigma-Aldrich) and visualized with HRP-conjugated goat anti-hiunan Tg0 (Sigma- Aldrich) using an ECLTM Western blot analysis system (Amersha.m).
Actin was used as = a loading control for Western Blot.
To detect thc reactivity of EY2E1-B with monoclonal antibodies, the total protein lysates from transfection (10) ttg) were immunoprecipitated with 5 i.tg envelope-specific monoclonal antibodies including 2012, 4010 and ID6 (NIH ArDs Research and Reference Reagent Program, Rockville, MD, USA). The sa.me amount of total protein lysates from cells transfected with empty vector pVAX was used as a negative- control. The immunoprecipitated proteins were fractioned on a SDS-PAGE gel and detected by Western Blotting described as above.
Indirect Immunalluorescent Assay. An indirect immunofluoresc.entassay for confirming the expression of EY2E1-B and EK2P-B genes was performed. Haman rhabdomyosarcoma (RD) cells were plated in tissue culture chambered slides (BD Biosciences), at a density to obtain 60-70% confluency the next day in complete DMEM medium with 10% FBS (GIBCO) and allow to adhere overnight. The next day cells were transfected with pEY2E1-B, pEK2P-B
and the control plasmid pVAX (1 jig/well) using FuGENE 6 Transfection Reagent (Roche) according to the manufa.cturer's instructions. Forty-eight hours after transfeetion, the cells were washed twice with cold 1XPBS and fixed on slides using methanol for 15 min. Upon removal of the residual solvents from the sl.ides, the cells were incubated with anti-mouse HIV-1 env monoclonal F1.05 (NIH. AIDS Research and Reference Reagent Program, Rockville, MD, USA) for 90 min. The slides were then incubated with TRITC-conjugated secondary antibody (Sigma-Aldrich) for 45 min. 4', 6-.Diamido-2-phenylindole hydrochloride (Sigma-Aldrich) was added to the solution of secondary antibody to counter stain nuclei to show the nuclei of the total number of cells available in the given ticld. The slides were mounted with mounting medium containing antifading reagent (Molecular Probes). The images were analyzed using the Phase 3 Pro program for fluorescent microscopy (Media Cybernetics).
Envelope-specific Antibody determination The measurement of IgG antibodies specific for Envelope was performed by ELISA (enzyme linked immunosorbent assay) in both inimunized and control mice. Nunc-ImmunoTM Plates (Nalge Nunc International, Rochester, NY) were coated with of clade B recombinant HIV-1 IIIB glycoprotein soluble gp160 (Immuno Diagnostics, MA), clade A/E primary envelope protein HIV-1 93TH975 gp120 and clade C primary envelope protein HIV-1 962M651 gp120 (N114 AIDS Research and Reference Reagent Program, Rockville, MD, USA), respectively, and incubated overnight at room temperature. After washing, plates were blocked with 3% BSA in PBST (1 x PBS -1 0.05%
Tween-20) for 1 h at 37 C. Then plates were washed again and incubated with the specific mouse sera, diluted with 3% BSA in PBST overnight at 4 C, followed by incubation with a 1/10,000 dilution of-IMP-conjugated goat anti-mouse igG (Jackson ImmunoResearch, West Grove, PA) for 1. h at 37C. The reaction was developed with the substrate TMB
(3, 3E1, 5, 5L1 -tetramethylbenzidine) (Sigma-Aldrich). Reaction was stopped with 100 Al of
2.5M sulfuric acid per well and the plates were read on the E.L808 plate reader (Biotech instrument Inc.) at OD of 450 nrn.
Immunization of Mice Female 4-6-week-o1d BALB/c mice were purchased from The Jackson Laboratory, Bar Harbor, ME. The breeding, pairs of transgenic B6.Cg-Tg (HLA-A/H2-D)2Enge/J mice were purchased from the. Jackson Laboratory and bred by Dr.
Michelle Kutzler in our lab. These trans.genic mice express an interspecies hybrid class I MHC
gene, AAD, which contains the alpha-I and alpha-2 domains of the human HLA-A2.1 gene and the alpha-3 transmembrane and cytopla.smic domains of th.e mouse H-2Dd gene, under the direction of the human HLA-A2.1. promoter. The mouse alpha-3 domain expression enhances the immune response in this system. Compared to unmodified HLA-A2.1, the chimeric HLA-A2.1/H2-Dd MIIC Class I molecule mediated efficient positive selection of mouse T cells to provide a more complete T cell repertoire capable of recognizing peptides presented by IILA-A2.1 Class I
molecules. The peptide epitopes presented and recognized by mouse T cells in the context of the 1-ILA-A2.I Class 1 molecule are the same as those presented in FILA-A2.1+
humans. The female 4-6-week-old transgenic mice were used for further study described below.
Their care was in accordance with the guidelines of the National Institutes of Health and the University of Pennsylvania Institutional Care and Use Committee (IACUC). Each mouse was immunized intramuscularly with three times, each of 100 ptg of DNA at biweekly intervals. There are three mice in each group and the control group was vaccinated with pVAX DNA. Mice were sacrificed one week after the third immunization and the spleens were removed aseptically. The spleen cells were collected and resuspended in RBC lysis buffer to remove erythrocytes. After lysis, the spleenocytes from the same group wcrc pooled and resuspended in RPM' 1640 medium with 10% FBS. Cells were counted and prepared for analysis.
IFN-TELISpot Assay. High-Protein Binding IP 96 well Mu1tiscrcciìTM plates (Millipore, Bedford, MA, USA) were used. Plates were coated with mAb to mouse IFNI/ (R&D
Systems, Minneapolis, MN) diluted in 1XPBS. overnight at 4 C. Plates were washed three times with PBS
and then blocked for 2 h at room temperature with 1XPBS supplemented with I%
BSA and 5%

sucrose. Mice Splenocytes were added in triplicates at an input cell number of 2 x 10 cells per well resuspended in complete culture medium (RPM} 1640 supplemented with 10%
'FBS arid antibiotics). Six sets of peptides each containing 15 amino acid residues overlapping by 11 = amino acids representing the entire protein consensus sequences of H1V-1 subtype 13, subtype C, group M and the entire protein sequences of HIV-1 MN (a subtype B isolate), C.UY.01,TRA3011 and C.ZA.01...154Ma (two subtype C isolates) envelope were obtained front NIH AIDS Research and Reference Reagent Program. Each set of env peptides were pooled at a concentration of 2 Az/ml/peptide into 4 pools as antigens for specific stimulation of the IFN-y release. Coneavalin A (Sigma- Aldrich, St. Louis, MO), at 5 g/ml, and complete culture medium were used as positive and negative control, respectively. Plates were washed four times after a 24 h incubation at 37 C, in a 5% CO2 atmosphere incubator. Then, a biotinilated anti-mouselEN-y detection antibody was added, and plates were incubated. overnight at 4 C. The plates were washed, and color development was followed according to the manufacturer's instructions (EL1SPOT Blue Color Module, R&D Systems, Minneapolis, MN). Plates were air-dried and the spots were counted using an automated EL1SPOT reader system (CTL Analyzers, Cleveland, OH) with the himmunoSpott software. The average number of spot .forming cells (SFC) was adjusted to 1 x 106 splenocytes for data display. The EL1Spot assay was repeated three times in three separate experiments.
CD8+ T-cell depletion study. CD8 lymphocytes were depleted from splcnocytes by using immune-magnetic beads coated with antibody to CDS (Dynal Biotech Inc., Lake Success, NY) Following manufacturer's instructions. After depletion of CDS+ T-cells,1FN-y ELISpot assay was performed as described above.
Epitope mapping study. In order to map the reactive epitopes, two sets of peptides containing 15 amino acid residues overlapping by 11 amino acids representing the entire envelope proteins of HIV-1 consensus subtype B and HIV-1 MN were pooled into 29 pools of 14-15 peptides/per pool, respectively, and IFN-y EL1Spot assay was performed as described above. These different sets of 29 -pooled stimulators were used in a matrix assay which facilitates epitope mapping.

Statistical Analysis. Student paired t-test was used for comparison of the cellular immune response between mice immunized with pEY2E1-B and pEK2P-B. In this study, p<0.05 has been considered statistically significant.
RESULTS
Construction and design of a novel subtype B early transmitter consensus-based envelope gene. The consensus sequence ofilIV-1 subtype B was generated from 42 subtype B sequences retrieved .from GenBank. As summarized in Fig. 1, several modifications were carried out after generating the consensus sequence. Briefly, to produce a CCR5-tropic version of HIV-1 envelope that mimicked rnucosally transmitted viruses, six important amino acids in the V3 loop were designed according to the sequences of early transmitter isolates.
Further, ten amino acids in VI loop and one amino acid in V2 loop was also deleted from the consensus sequence. A
highly efficient leader sequence was fused in frame upstream of the start codon to facilitate the expression. The transmembrane domain was kept intact to facilitate surface expression and the cleavage site was kept intact to obtain proper folding and host protcinase cleavage of the envelope protein. The cytoplasmic tail was removed to prevent envelope recycling and to promote more stable and higher surface expression (Berlioz-Torrent, C., et al.
1999. Interactions of the cytoplasmic domains of human and simian retroviral tran.smembranc proteins with components of the clathrin adaptor complexes modulate intracellular and cell surface expressioii of envelope glycoproteins. J. Virol. 73:1350-1359; Bultmann, A., et al.. 2001.
Identification of two sequences in the cytoplamic tail of the human immunodeficiency virus type 1 envelope glycoprotein that inhibit cell surface expression. J. Viral, 75:5263-5276).
Furthermore, in order to have a higher level of expression, the cotton usage of this gene was adapted to the codon bias of Homo Sapiens genes (Andre, S., et al. B. 1998. Increased immune response elicited by DNA
vaccination with a synthetic gp120 sequence with optimized codon usage. .1 Virol 72:1497-503;
Demi, L., et al. 2001. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immtmodeficiency virus type 1 gag protein. J. Virol. 75:10991-11001). In addition, RNA optimization (Schneid.er, R., et al..
1997. Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independeni expression of Gail, and Gag/protease and particle formation. J.
Viral. 71:4892-4903) .

was also perfomied: regions of very high (>80%) or very low (<30%) GC content and the cis-acting sequence motifs such as internal TATA boxes, chi-sites and ribosomal entry sites were avoided. The synthetic engineered EY2E1-13 gene was constructed and was 2734 bp in length.
The EY2E1-B gene was subcioned into pVAX at the BamEE and Notl sites fOr further study.
= Phylogenetic analysis. To assess the distribution of the distance from a randomly sampled envelope subtype B sequence to the EV2E1-B sequence, a phylogenetic analysis was performed.
As shown in Fig. 2, there was an observed relative closeness of the EY2E1-B
sequence to all sampled sequences. The EV2E] -B sequence, when compared with the primary isolate EK2P-B
sequence, has comparable distributions of similarity scores (Table 1). The average percent similarity score for ENT2E1-B was 85.7%, while it was 79.4% for EK2P-B.
Table 1 Average percent similarity Range of percent simi1¨arity-1 scores scores EY2E1-1.3 85.7 92.1-79.6 EK2P-B 79.4 86.3-73.9 Table I. The average and range of percent similarity scores between potential envelope vaccine candidates and an alignment of subtype B envelope sequences.
In Vivo Expression and Antigenic Determination of EV2E1-B. In order to test the in vivo expression of pEY2E1-B and pEK2P-B. RD cells were transfected with these plasmids as described in Materials and Methods section. Total proteins were extracted from cell lysates after transfection and immunoblotted with the envelope-specific monoclonal antibody mentioned in Materials and Methods section to detect the expression of pEV2EI-B. Western blot results indicated that these two constructs expressed envelope protein (Fig.
3A), The envelope protein detected was about 120 KD. Table 2 shows a comparison of pEY2E1-B and pEK2P-11.
Table 2 Consensus) Early Con- RNA- IELS Cytoplasmic PrinarY ttasmitter optimized optimized EYZEI-B Coasensits Yes Yes Yes No EP- 3 Priniary ci Yes es I vs To determine the antigenic epitopes, the expressed envelope proteins from the RD cell = lysates were immunoprecipitated with three different gp120-specific antibodies 2G12, 4G10 and 1D6. Following the immunoprecipitation, Western Blotting wa.s performed to detect the immnoprecipitated proteins. Our results showed that the synthetic immunogen could bind to antibodies 2G12 and ID6. but not 4G10. Since antibody 2G12 neutralizes a broad variety of primary isolates and reacts with a conformational and carbohydrate-dependent ap120 epitope, and antibody 1D6 binds to gp120 and gp1.60 and is directed against the first 204 aa of gp120, our results suggested that the synthetic engineered immunogen EY2E1-B might be able to fold into a relatively native conformation and preserve some native antigenic epitopes.
Furthermore, since the antibody 4G10 is a HIV.]: LAIIBRU V3 monoclonal antibody that recognizes LAI gp160, a 'f'-cell line adapted strain, our data also suggested that this synthetic envelope would not utilize the coreeeptor CXCR4.
To further confirm the expression and determine the antigenic epitopes, an indirect immunofluoreseent assay was performed using transfected RD cells. High specific expression was observed under fluorescent microscope in the pEY2E1-B and pEK.2P-B
transfected cells.
The HIV-1 env monoclonal E105 that reacts with a discontinuous, or conformational, gp120 epitope was used in the assay. As indicated in Fig. 3B, the transfected cells expressing Env proteins showed the typical rhodamine fluorescence, again suggesting the synthetic protein expressed and had a relatively native conformation. As a control, the expression was not detected in pVAX transfected RD cells.
Induction of Immoral response. To determine whether the synthetic im.munogen could elicit higher-titer envelope-specific antibody response, sera were collected from BalB/C mice immunized pVAX, 1EY2E1-B and pEK2P-B and ELISA was performed. As shown in Fig.
4A, we observed the relatively higher level of elude B envelope-specific antibody responses with sera collected from pEY2E1.-B immunized mice compared to these in pEK2P-B immunized mice. In contract, the vector alone mice didn't develop specific antibody responses.
However, there were not any detectable antibody responses against clade AIE and clade C proteins in both pEY2E1-13 and pEK2P-B injected mice (Fig. 4B and 4C), indicating that although the synthetic consensus-.

based immunogen has a relatively native conformation and preserve native antigenic epitopes, it may not be able to induce broad cross-clade antibody immune responses.
Strong and broad cellular immune responses measured by ELISpot. The Bal.B/C
mice were immunized with pEY2E1-B and pEK2P-B and ELISpot analysis was perfortned to determine the number of antigen-specific IEN-y secreting cells in response to four pools of peptides from H1V-1 consensus subtype B protein (Fig. 5A). The magnitude of the response as measured by the number of spot thrtning units (SFU) per million cells ranged from 27.5 to 520 in pEY2E1-B vaccinated mice. In comparison, splenocytes from pEK2P-B vaccinated mice only showed the range of spots from 2 to 237.5 (p<0.05). The additive frequency of SFU/per minion splenocytes for all four pools in pEY2E1-B immunized mice was 1976.25 + 260, while the number of STU/per million cells in pEK2P-13 immunized mice was 519 + 45. Cells from mice immunized with pVAX vector were used as a negative control, showing only 60 +
5 SFUiper million splenocytes for consensus envelope 13 peptides pools (p < 0.05). We observed similar results in three separate studies. Therefore, the pEY2E1-B construct is up to four times more potent in driving cell-mediated immune responses. We also determined whether lymphocytes were responsible for the IFN--y secretion detected in BalB/C mice immunized with pEY2E1.-B. As shown in Fig. 5E, the number of SFU/per million cells was reduced to 127.5 + 11 after CD84- depletion, indicating that there was about 90% of decrease in the frequencies ofIEN-producing cells observed by CD8+ T-cell depleted ELISpot. The IEN-7 production induced by pEY2E1-B is mediated mainly by CM+ T-cells.
In addition, iiì order to model human T cell immune responses to HLA-A2 presented antigens and identify those antigens, we performed the same ELISpot assay mentioned above using transgenic HLA-A2.1/H2-Dd mice. As shown in Fig 5C, the additive frequency of SFUlper million splenocytes for all four pools in pEY2E1-B immunized transgenie mice was 2362 257, while the number of SFUlper cells in pEK2P-B immunized transgenic mice was only 493 4. 57. These results indicated that the pEY2E1-B construct is up to four times more potent in driving cell-mediated immune responses in the transgenie mice. The E.LISpot data after C08 depiction suggested that the IFN-y production induced by pEY2E1-13 is primarily mediated by C.D8+ (Fig. 5D).
-74-.

M.oreover, we were interested in further detailing the cellular immune responses that were observed in the EL1Spot assay. Accordingly, an additional set of ELISpot assay was performed against libraries of peptides spanning the consensus subtype B
envelope protein. A
complete set of 15-mer peptides overlapped by 11 amino acids, which comprise the subtype B
consensus envelope protein, was used to perform this 'napping study. The study illustrated that there was no clear dominant epitope induced by the synthetic envelope.
However, ITN-7 ELISpot analysis of splenocytes derived from the pEY2E1-B-vaccinated BalB/C
mice revealed that there were 18 pools out of 29 pools showing more than .50 spots, while there were only 6 pools in pEK2P-B vaccinated BalBIC mice. (Fig. 5E). These results illustrated that there is a significant increase in the breadth and magnitude of cellular immune responses induced.by the EY2E1-B immunogen.
Strong cross-reactive cellular immune responses induced by pEY2E1-B. To determine whether the EY2E1-B immunogen could induce broad and cross-reactive cellular immune responses, IFN-7 ELISpot was performed both in BalB/C and HLA-A2 transgenic mice using HIV-1 group M, consensus subtype C, HIV-1 MN (subtype B isolate), HIV-1 = C.UY.01.TRA3011 and C.ZA.01.154Ma (two subtype C isolates) envelope peptides. These assays will further determine if the results observed in Fig. 5A, C and E
alone are related to the peptide targets or actually due to the increase in immune breadth. As shown in Fig. 6A, the additive number of SFU/per million splenocytes against four pools of HIV-1 MN
envelope peptides in pEY2E1-B vaccinated BalB/C mice was 1855 + 215.8, which was about two times more than those in pEK2P-B immunized BalB/C mice (SFU/per million splenocytes was 700 +
168.2), indicating that pEY2E1-B had stronger cross reactivity than pEK2P-B
within subtype B.
The numbers of IF-N-y spots in response to stimulation with four HIV group M
(Fig. 6B) and subtype C (Fig. 6C) consensus envelope peptides pools in pEY2E1-B immunized BalB/C mice were 1150 + 191.3 and 715 + 116.1, respectively. Compared to the numbers of spots against group M and subtype C peptides which were 635 + 152.3 and 345 + 82.3 in pEK2P-B vaccinated BalB/C mice, these data illustrate that the cross-clade immune responses elicited by pEY2E1-B
is approximately 45% stronger than those induced by pEK2P-B in Ba.1.13/C mice.

Importantly, we observed much stronger cross reactive cellular immune responses induced by pEY2E.1-B in transgenic mice (Fig. 6F-1). The additive number of SFU/per million splenocytes against four pools of HIV-1 MN envelope peptides in pEY2E1-B
vaccinated transizenie mice was. 1087 + 153, which was about three times more than those in pEK2P-B
immunized HEA-A2 mice (SFU/per million splenocytes was 316 + 63) (Fi(2,-. 6F), indicating that pEY2E1-B could also elicit stronger cross reactivity than pEK2P-B within subtype B in transgenic mice. The numbers of TFN-y spots in response to stimulation with four HIV group M
(Fig. 6G) and subtype C (Fig. 6H) consensus envelope peptides pools in pEY2E1-13 immunized transizenic mice were 2116 + 216 and 893 + 154, respectively. Compared to the numbers of spots against. group M and subtype C peptides which were 473 + 50 and 266 + 55 in pEK.2P-B
vaccinated transgenic mice, these data indicated that the cross-clade immune responses elicited by pEY2E1-B is about three to four times stronger than those induced by pEK2P-B in trausgenic mice. Moreover, two subtype C isolate peptide sets that should serve as a stringent control for evaluating breadth and cross-reactivity achieved by other peptide sets were used to further determine the cross-clade C immune responses. Although there were not too many differences of cross reactivity against these two subtype C isolate sets elicited by pEY2E1-B
and pEK2P-B in BalB/C mice (Fig. 6D and E), the cross-clade reactivity against these two subtype C isolate sets induced by pEY2E1-B is about three times stronger than those induced by pEK2P-B (Fig. 61 and ..1). The numbers of spots against C.ZA.01.,154Ma and CUY.01.TRA3011 peptides were 1080 +
= 206 and 800 + 150 in pEY2E1-B vaccinated transgenic mice, while the numbers were only 305 +
38 and 310 62 in pEK2P-B vaccinated transgenic mice.
Finally, we determined 'whether there was also an increase in the breadth of cross-reactive cellular immune responses against subtype specific targets induced by the EY2E143 immunogen by detailing the cellular immune responses against HIV-1 MN observed above both in BalB/C
tind 11LA-A2 transgenic mice. An cpitopc mapping assay was performed against the library of peptides spanning the subtype B MN envelope protein. The results suggested that there was no clear dominant epitope induced by the synthetic envelope in both mouse strains. However, ITN-7 ELISpot analysis of splenocytes derived from the pEY2EI-B-vaccinated Bal.B/C
mice revealed that there were 14 pools out of 29 pools showing more than 50 spots, while there were only 9 pools in pE.K2P-B vaccinated BalB/C mice (Fig. 7A). Similarly, in transgenic mice, there were 18 pools out of 29 pools showing more than 50 spots in pEY2E.1-B immunized transgenic mice, while there were only 6 pools in pEK2P-B vaccinated transgenic tnice (Fig, 7B). These data indicated that there is a significant increase in the breadth and magnitude of cross. reactive cellular immune responses induced by the EY2E1-B immunogen both in BalBIC and transgenic mice.
DISCUSSION
Worldwide HIV-1 DNA vaccine efforts have been guided by the principle that HIV-specific T-cell responses may provide some contribution to protection from infection or control of replication post-infection. DNA vaccines can impact viral replication although in general they are not as potent in immune induction as attenuated live viral vectors (Almond, N., et al.. 1995.
Protection by attenuated simian immunodeficiency virus in macaques against challenge with virus-infected cells. Lancet 345:1342-1344; Berman, P. W., et al. 1996..
Protection of MN-rgp120-immunized chimpanzees from heterologous infection with a primary isolate of human immunodeficiency- virus type 1. J Infect .Dis 173:52-9; Boyer, J., et al.
1997. Protection of chimpanzees from high-dose heterologous 111V-1 challenge by DNA vaccination.
Nat Med = 3:526-532; Daniel, M. C., et al. 1992. Protective effects of a live attenuated SIV vaccine with a deletion in the nef gene. Science 258:1938-1941). Strategies aimed at improving the breadth and magnitude of the cellular immune responses are therefore important. The present invention provides a novel antigen using several features of immunogcns that have been reported in the literature as separate approaches, but have not been previously assembled together in one vaccine modality. As proof of concept, a synthetic engineered consensus-based envelope immunogen was developed and compared with an optimized primary sequence immunogen for induction of cell-mediated immune responses. Expression data showed that this engineered ncw envelope gene could be efficiently expressed in mammalian cell lines although the expression levels of these two immunogens were very similar (Fig. 3A). We observed in the immunogenicity studies that the cellular immune responses induced by this functional immunogen exhibited increased diversity and magnitude compared to the primary- envelope vaccine. Epitope mapping data obtained in both BalB/C and HLA-A2 transgenic mice demonstrated that this diversity and magnitude improvement was maintained across these haplotypes. To further confirm this finding, we also developed a consensus-based subtype C envelope immunogen and compared it with a primary subtype C immtmogen, again the synthetic consensus-based subtype C envelope immunogen exhibited enhanced diversity and magnitude of cellular immune responses compared to the primary C it munogen (unpublished data).
From the point oíview of vaccine design strategy, sequen.ce homology between the vaccine candidate and the infecting or challenging virus may be an important consideration. An effective approach to minimize the degree of sequence dissimilarity between a vaccine strain and contemporary circulating viruses is to create artificial sequences that are "central" to these viruses. One strategy to design such a sequence is to use a consensus sequence derived from the most common amino acid in every position in an alignment lri this study, we developed a conscnsus-based subtype B envelope vaccine and thought this synthetic immunogen would have higher cross reactivity. Our results did show that there was a diversity ofeellular immune responses induced by the 1iEY2E1.-B vaccine. Peptide mapping results in both Balb/e and transgenic mice as well. indicated that the EY2E1-B immunogen broadened the immune responses. Moreover, the results of cross-reactive cellular immune responses study indicated that pEY2E1-B could elicit significantly stronger and broader cross-reactive cellular immune responses. Therefore, the artificial consensus envelope immunogens contain more conserved epitopes than found in any individual natural isolate and they induce broader cross-clade CTL
responses.
A consensus sequence theoretically has advantages and disadvantages. Since a consensus sequence is generated based on contemporary isolates, it may be genetically closer to current circulating viral strains than any given natural virus isolate. However, since global sequencing is generally conducted with viruses sampled during chronic infections instead of viruses sampled during acute infection, developing a consensus vaccine response on epitopcs that for the .most = part have escaped may be a disadvantage. To minimize this disadvantage, one useful strategy for vaccine design would be to take early transmitter sequences into account.
Envelope proteins are among the most difficult HIV proteins to construct artificially because the hypervariable regions in HIV-1 envelope gene evolve by rapid insertion and deletion and not by point mutation. The difference of hypervariable regions in length makes it hard to generate the consensus sequences of these regions. Recently, Gao et al. (Gao, F., Eet. al. 2005. Antigenicity and im.munogenicity of a synthetic human immunodeficiency virus type 1 group in consensus envelope glycoprotein, = Virol 79:1154-63) generated a group M consensus envelope sequence, however, the nonconsensus sequences from corresponding regions of a CRFOS BC recombinant strain were used in these variable regions. Studies have indicated that subtype C viruses encoding envelope glycoproteins with shorter VI, V2 and V4 regions arc transmitted in recipients with a frequency significantly greater than µvould be expected by chance. The subtype A
envelope sequences from early infection also had significant shorter V1 and V2 loop sequences and fewer potential N-linked glycosylation sites (Chohan, B., D. et al. 2005. Selection for Human Immunodeficiency Virus Type 1 envelope glycosylation variants with shorter V1-V2 loop sequences occurs during transmission of certain genetic subtypes and may impact viral RNA levels. J.
Virol. 79:6528,-6531). In contrast, recently transmitted subtype B variants didn't have shorter V1 and V2 loops.
However, it may be important to note the subtype B infection eases were primarily the result of homosexual transmission or drug injection use. Moreover, studies have suggested that a possible functional consequence of having a compact V1. V2 region is to increase exposure of the CD4 binding, domain, and then to enhance susceptibility to neutralization (Edwards, T. G., et al. 2001.
Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a Human Immunodeficiency Virus type 1 envelope protein. J.
Virol. 75:5230-5239; Kolchinsky, P., et al. 2001. 'Increased neutralization sensitivity of CD4-independent Human Iminnuodeficiency Virus variants. J. Virol. 75:2041-2050; Piekora, C., et al. 2005.
Identification of two N-linked glycosylation sites within the core of the Simian lininunodificiency virus glycoprotein whose removal enhances sensitivity to soluble CD4. J.
Virol. 79:12575-12583; Puffer, 13. A., et al.. 2002. CD4 independent of Simian Immunodeficiency Virus Envs is associated with macrophage tropism, neutralization sensitivity, and attenuated pathogenicity. J. Virol. 76:2595-2605). We shortened the VI and V2 regions when we generated the subtype 13 consensus sequence.
The early phase of HIV-1 infection is dominated by non-syncytium-induciug (NS1) viruses, which replicate slowly and use CCR5 as their rnain coreceptor.
Syncytium-inducing (SI) viruses, which emerge in about 50% of infected individuals preceding an accelerated CD4 cell decline and progressive clinical course of infection, use CXCR4 as the main coreceptor. A
differential coreceptor usage of HIV variants has been demonstrated for all subtypes. Subtype C
viruses appear to be different from most other subtypes because an underrepresentation of CXCR4 using HIV variants in subtype C has frequently been reported. Therefore, utilization should be a very crucial consideration for a vaccine design.
Previous reports showed that the V3 region of gp120 plays an important role in coreceptor utilization.
Six residues in V3 loop has been identified to be critical for CCR5 interaction: arginine307, lysine314, isoleueine316, arginine322, phenylalanine324 and alanine337. However, based on the sequences of subtype C early transmitters, the residue at position 322 should be glutamine instead of arginine. In. summary, based on the previous studies showing residues important for CCR5 utilization and the sequences of early transmitters, we designed the subtype B
consensus envelope immunogen that could drive immune responses that may in theory target coreceptor utilization.
To maximize potential cross-reactivity, a HIV-1 group M consensus envelope sequence has been created. However, it is possible that subtype-specific envelope consensus vaccines may represent a compromise for the overall sequence similarity of the vaccine antigen relative to circulating viruses at least at the level of cellular immune responses.
Studies have shown that there were high rates of selection identified in different regions of subtype B and C envelope proteins. This may be caused by different immune pressure on different regions of the envelope protein in subtype B and C. Therefore, there may be advantages in using a subtype-specific envelope vaccine, as the immune responses to the vaccine and the circulating virus would share antigenic domains. More experiments comparing group M and subtype-specific envelope vaccines are needed to further clarify this issue.
Another important concern about using a CODSCILSUS sequence is that its sequence may associate polymorphisms in combinations not found in any natural virus, thus potentially resulting,- in improper protein conformations. Previous studies has indicated that a group M
consensus immunogen could fold into native confomation, preserve envelope antigenic epitopes and elicit weak neutralizing antibody response. Based on thc facts that the synthetic protein -80-.

=
could bind to antibodies 2G12, 106 and F 1 05, we think that the pEY2E1.-13 may have somewhat native structural confirmations. Importantly, our data also demonstrated that immunogen could induce a higher-titer subtype B envelope-specific antibody, indicating this synthetic immunogen m.ay preserve more Class Il epitopes as well. More studies in this arca will be important.
With the generation of new IIIV-1 vaccine strategies, there is also an increasing demand to predict the efficacy of these vaccines in human using preclinical models.
In our study, HLA-A2 transgcnic mice were used to study the cellular immune responses elicited by the synthetic immunogcn. Studies have shown that this transgenic strain is an important preclinical model for design and testing of vaccines for infectious diseases involving optimal stimulation of human CD8+ cytolytic T cells, In this model the results indicated that EY2E1 -B
could elicit much broader and stronger cellular immune responses compared to EK2P-B, suggesting that this new vaccine may have more potential to induce HLA-A2-restricted cellular responses. Further study or this immunogen in non-human primates are being planned.
Taken together, our results suggest that EY2E1-B could serve as an immunogen that increases both the magnitude and breadth of CTL responses as a DNA vaccine cassette. In more general terms, this construct may be useful in other platforms for induction of stronger and = broader cellular immune responses against HIV strains in non-DNA vector approaches.
Example 2 Development of a Novel Engineered ITIV-1 Clade C Envelope DNA
Vaccine that Enhances Diversity and Breadth of the Elicited Cellular Immune Response Strong HIV-1 specific CTL responses have an important role in managing viral load during acute and asymptomatic infection. However, recent studies on consensus immunogcns have not been able_ to noticeably demonstrate improved cellular immune responses. Here we test a novel engineered Clade C consensus-based envelope immunogen for improved cellular = immune response. The novel vaccine (pEY3E1-C) was created from the HIV-1 Clade C
consensus c-nvelopc sequence. Several modifications were performed including shortening the highly variable V1 and V2 regions based on early transmitter sequence, retention of the V3 loop for CCR5 utilization, removal of the cytoplasmic tail region from the C-terminus to prevent envelope recycling, and retention of the cleavage site and TMD for proper folding. Also, an IgE

leader sequence was added to the. N-terminus. This consensus DNA vaccine was also RNA
optimized and codon optimized. The cellular immune response was studied in Bal.B/C mice via ELISpot and epitope mapping assays. Whetì studied as a DNA vaccine, compared to pEK3P-C
(derived from a primary isolate of Clade C env); our construct (pEY3E1-C) was more efTective at driving a cellular immune response. pEY3El-C elicited a cellular immune response greater in magnitude than pEK3P-C when stimulated by Consensus Clade C peptides.
Additionally, the consensus immunogen elicited an increase in the magnitude of the cellular immune response when stimulated by two other sets of primary isolate peptides also from Clade C. In addition to augmented magnitude, enhanced breadth of the CTL response was supported by the pEY3EI-C's ability to induce at least 15 out of 29 strongly reactive peptide pools (having more than 50 spotsiper million splenocytes), white pEK3P-C only induced 3 out of 29 pools and 9 out of 29 pools with strong reactivity in response to two primary- isolate peptide sets, which were selected for their uniqueness and ability to serve as a stringent control for evaluating breadth, Furthermore, pEY3E1-C elicited a stronger Cross-Clade cellular immune response when stimulated with Clack B peptides. The consensus immunogen pEY3E1-C enhances both the magnitude and breadth of CTL responses as a DNA vaccine cassette, suggesting that the potential for consensus immunogcns to serve as a component antigen in a HIV
vaccine cocktail merits further examination.
With wide genetic diversity, rapid mutation, and recombination of the existing strains, the difficulty of generating an effective vaccine i.s tremendous. A candidate DNA
vaccine derived from an individual isolate may not be able to elicit the cross-reactivity necessary for protection against the diverse circulating strains of HIV-1.
Additionally, it has been reported that DNA vaccines expressing the 1I1V-1 envelope glycoprotein are not very immunogenic.
We have used a multiphase strategy to increase the potency of the CTL response elicited by the DNA vaccine to possibly provide protection against circulating strains of the virus.
Recent studies have shown that a consensus immunogen may overcome the diversity obstacle created by the rapidly evolvingRIV-1 virus.

Derdc.lyn et al.. found that a shorter V1-V4 region is characteristic of early transmitting subtype C virus and our construct has been designed to carry this feature which might be useful in producing a immune response resulting from early transmitted viruses.
Furthermore, the expression levels of OUT DNA vaccine have been enhanced by codon optimization, RNA optimization, and the addition of an immunoglobulin leader sequence.
H1V-1 specific CTL responses have been shown to be important in controlling viral load during acute and asymptomatic infection and the development of AIDS, thus the following data focuses on the CTL responses elicited by our novel immunogen.
Figure 13 depicts the immunogen design for development of a novel engineered chile C Envelope DNA Vaccine that enhances diversity and breadth of the elicited cellular immune, responses.
Figure 14 shows phylogenetic Relationships: Thirty-Six HIV-1 subtype C
envelope sequences, EY3E1.-C, EK3P-C, two subtype B, one subtype A and one subtype D
sequences (outgroup) were included in the phylogenetic analysis. The subtype C envelope sequences representing a broad sample of diversity were from 12 countries.
Table 3 shows the average and range of percent similarity scores between potential envelope vaccine candidates and an alignment of subtype C envelope sequences.
Table 3 Average % Similarity Scores Range of % Similarity Scores pEY3E1-C 85.3 82.7-93.1 pliK3P-C 87.4 83.6-90.2 Three groups of three Balb/C mice were immunized with 100 pg of DNA 3 times with two weeks between immunizations. On the seventh week, spleens were harvested for cellular studies.
As shown in Figure 15 Panels A and B, strong cellular response elicited by pEY3E1-C.
Figure 16 shows strong and broad cellular responses elicited by pEY3E1-C. When stimulated with 29 pools of Consensus C env peptides: pEY3E1-C vaccinated mice elicited more than 50 spots/million splenocytes from 23 pools; pEK3P-C vaccinated mice elicited more than 50 spots/Million splenocytes from 2 pools.
Figure 17 Panels A-D show strong cross-reactive cellular responses elicited by pEY3E1-C within the same clade.
Figure 1S Panels A and 13 show strong and broad cross-reactive cellular responses elicited by pEY3E1-C. Panel A shows data from subtype C (Uruguay) env-Specific 'FN.-7 EL1Spot. When stimulated with 29 pools of Clade C (Uruguay) env peptides:
pEY3E1-C
vaccinated mice clici.ted more than 50 spots/million splenocytes from 12 pools; pEK3P-C
vaccinated mice elicited more than 50 spots/million splenocytes from 3 pools.
Panel B shows data from Subtype C (S. Africa) env-Specific 1EN-7 ELISpot. When stimulated with 29 pools of Clade C. (S. Africa) env peptides: pEY3E1-C. vaccinated mice elicited more than 50 spotslmillion splenocytes from 13 pools; pEK3P-C vaccinated mice elicited more than 50 spots/million splenocytes from 5 pools.
Figure 19 Panels A-f show strong cross-reactive cellular responses elicited by pEY3E1-C
between clades.
There is a significant increase in the breath and magnitude of cellular immune responses induced by the EOC immunogen. Broader cross-clade reactivity appears as an additional benefit of this immunogem Example 3:
Efficacy of a novel engineered HPV-16 DNA vaccine encoding a E61E7 fusion protein=
The imniunogen has been designed to be expressed as a polyprotein whereby E6 and E7 sequences are separated by a proteolytie cleavage site. The polyprotein is also expressed with an IgE leader sequence. The polyprotein design includes deletions or mutations in the E6 sequence which. are important for p53 binding and degradation and mutations in Rb binding site on the E7 protein. Figure 23 provides an illustration of the immunogen design.
Coding sequences encoding the polyprotein were inserted into the vector pVAX
to prod.uce plasmic] p1667 Figure 24 shows maps of pVax and p1667.
TC1 tumor cells were immortalized with HPV-16 E.7 and transformed with the e-Ha-ras oncogene. These cells express low levels of E7 and are very tumorigenic.

In the im.munogenicity study in mice, 3 mice/per group of C57BL6 mice were administered 100 /Iv, DNAiper mouse. Groups included 1) control which were administered pVAX- control vector and 2) test which were administered p1667. Mice were vaccinated on days 0, 14 and 28. On day 35, mice were sacrificed and EL1SPOT was perforated (Focus on CMI).
The data for cellular immune responses induced by the DNA Immunogen p1667 is shown on Figure 25. HPV16 consensus E6 and E7 peptides (37, 15-mers overlapping by 9 aa) were used in two pools -pool 1: 18 peptides; pool 2: 19 peptides. Pan.els A and C show data from total spleenocyt.es. Panels B and D show data from samples with CD8 depletion.
Figure 26 shows results of immunodominant epitope mapping. Two sequences are noted.
in prophylactic experiments in mice, 5 mice/per group of C57BL6 mice were administered 100 ktg- DNA/per mouse. Groups included 1) naïve (JIBS injected), 2) control which were administered pVAX- control vector and 3) test which were administered p1667.
Mice were vaccinated on clays 0, 14 and 28. On day 35, mice were challenged with TC-1 cells and thereafter tumor size measurements were made. Results arc shown in Figure 27. Data from a group in which IL-15 construct was co-administered is also shown.
In tumor regression experiments in mice, 5 mice/per group of C57BL6 mice were administered 100 ktg DNA/per mouse. Groups included 1) naïve (PBS injected), 2) control which were administered p VAX- control vector and 3) test which were administered p1667.
= Mice were challenged with 5 x 104 'I'C-1 cells at Day O. Mice were administered DNA vaccine on days 3, 10 and 17. Tumors were measured starting at day 8. Results are shown in Figure 28.
Data from a group in which 1L-15 construct was co-administered is also shown.
The level of E7 Tetramcr positive lymphocytes in spleens was determined.
Fi$rure 29 shows the data as the percent E7 Tetramer positive lymphocytes. DNA vaccine p1667 induces the activation of F7-specific CD8+ T cells that are CD62LI within spleens.
The level of E7 Tctramer positive lymphocytes in tumors was determined. Figure shows the data as the percent E7 Tetramer positive lymphocytes. DNA vaccine p1667 induces the activation of ET-specific CD8* T cells that are CD62LI within tumors A E6/E7 DNA Vaccine protection study in transgenic mice was undertaken. A
comparison was made among naive, pVAX, p1667, p1667 + IL-15 and E7/HisB. Data is shown in Figure 31. p1667 and p1667 + IL-15 protected completely.
The data presented herein support the following conclusions. The p i 667 construct induces a strong cellular immune response capable of inducing E7-specific CD8+
lymphocytes that mediate the elevated IFN-g responses. We have identified both dominant and novel sub-dominant HPV-16 epitopes against which antigen-specific CTL are generated after administration of the DNA construct. The p1667 construct is capable of preventing tumor growth and causing the regression of tumors in both C57/BL6 and transgenic mice. DNA
vaccine pl 667 shows great potential for a novel therapeutic strategy to target microscopic HPV-associated cancer.
Example 4 Nucleic acid sequences encoding HIV Env consensus sequences may be administered as DNA vaccines in combination with nucleic acid sequences encoding various other HIV proteins such as Gag, Poi, Gag/Pol, Nef, Vif, and Vpr using for example electoporation technology for intramuscular or intradermal delivery. Multivalent/polyvalent HIV vaccine constructs may provide enhanced immune responsed and be particularly useful. In some embodiments, 1L-12 coding sequences are additional provided. U.S. Patent application pubicaton number 20070106062, discloses an HIV Vif DNA vaccine.
U.S. Patent application pubicaton number 20040106100, discloses HIV vaccines comprising HIV accessory proteins as well as the sequences of such proteins which may be used to prepare additional vaccine constructs. U.S.
Patent Nos.
6,468,982, 5,817,637, and 5,593,972, disclose DNA
vaccines including HIV gag, HIV pol and HIV gag/pol constructs.
Electroporation is described in U.S. Patent No. 7,245,963 PCT application PCTIUS97/19502 discloses IL-12 constructs. U.S.
Application Publication No. 20070041941 discloses constructs encoding IL-15.
Example 5 Two groups of macaques were IM immunized three times with optimized plasmid gag and env constructs with or without plasmid IL-12. The same immunization strategy was used for two additional groups but the plasmids were delivered with or without iu vivo electroporation.
Cellular responses were determined by 1FNy EL1Spot after each immunization and five months later for memory responses. 'throughout the study humoral responses were evaluated by recombinant p24 and gp160 ELISA. The proliferative capacity of antigen-specific T cells were determined by CFSE staining. Intracellular cytokine staining was done to further characterize the functional characteristics oldie induced T-cell response.
Plasmid 1L-12 enhanced cellular responses to our optimized constructs. However the use of electroporation to enhance the delivery of plasmids was able to improve both the cellular and Immoral response compared to 1M immunization with plasmid IL-12. The combination of plasmid IL-12 and electroporation resulted in the best immune responses, both primary and memory, as measured by a variety of parameters.
Optimized DNA constructs encoding HIV gag and env in rhesus macaques in the presence or absence of plasmid IL-12 as a DNA adjuvant was compared. 1L-12 could substantially increase T cell responses 5-fold in a quantitative ELISpot format resulting in substantially better memory T cell responses. However, EP delivered DNA was more efficient at generating T cell responses and memory that were 2-fold higher compared to the adjuvanted DNA vaccine. The best responses were observed in th.e combination arm of EP + IL-12 adjuvant. Memory responses in this arm were 10-fold higher than the IM DNA
alone and almost 2-fold higher than EP alone. We also Observed 4-fold better immune expansion by CFSE
in the EP + 1L-12 arm compared to EP alone. The presence of polyfunetional T
cells also suggested that the DNA eytokinc + EP arm is most. effective.
Materials and Methods Animals:
= Rhesus macaques (Maceica tail a) were housed at BIOQUAL, Inc.
(Rockville, MD), in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. Animals were allowed to acclimate for at least 30 days in quarantinc..- prior to any experimentation.
Immunization:
Five rhesus macaques were immunized at weeks 0, 4, and 11 with 1.0mg of pGag4).' and pEY2E1-B. The DNA at each immunization time point was delivered into two injection sites, one in each quadriceps muscle. Three of the macaques were eleetroporated following IM
injection. Another group of five macaques were immunized at weeks 0, 4, and 8 with 1.0mg of pGag4Y, pEY2E1-B, and WI,V104. Of the five animals, two animals received the immunization hy IM injection and three animals were electroporated following IM injection.
All electroporation procedures were performed using the constant current Cellectraml device (VGX
Immune Therapeutics Division of VGX Pharmaceuticals, The Woodlands, TX).
Electroporation conditions were 0.5 Amps, 3 pulses, 52 msec pulse length with I sec between pulses. This software-controlled device was designed to measure the tissue resistance immediately prior to plasmid delivery and generation of constant current square wave pulses, eliminating the risk of delivery outside the muscle tissue and potential plasmid loss.
Moot/ Collection:
Animals were bled every two weeks for the duration of the study. 10 mL. of blood were coll.ected in EDTA tubes. PBMCs were isolated by standard Ficoll-hypaque centrifugation and then resuspended in complete culture medium (RPM' 1640 with 2mM11_, L-glutamine supplemented with 10% heat-inactivated fetal bovine scrum, 100 1.1)/mL
penicillin, 100pgitn1..
streptomycin, and 55tiMiL P-mercaptoethanol.) RBCs were lysed with ACK lysis buffer Wambrex Bio Science, 'East Rutherford, NJ).
Plasmids and plasmid products:
Gag4Y contains an expression cassette encoding for a consensus sequence of the gag protein of HIV clades A. B, C, and D with several modifications including: the addition of a kozak sequence, a substituted IgE leader sequence, codon and RNA optimization for expression in mammalian cells (SEQ ID NO:11 discloses HIV Gag consensus sequence.). The Gag4Y gene was subcioned into the expression vector, pVax, for further study. pEY-2E1-B
contains an expression cassette encoding for a consensus sequence of the envelope of HIV
clade B. (SEQ ID

NO:3 discloses HIV Env consensus sequence.) WI.V104M is a plasmid encoding a rhesus IL-I?
gene. Plas.mids were produced at Aldevron (Fargo, ND), and re-formulated at VGX Immune Therapeutics (The Woodlands, TX), in sterile water for injection with low molecular weight 0.1% poly-L-glutamate sodium salt CFSE of Cryo-preserved PBMCs Cryo-preserved PBMC.s were quick-thawed in a 37 C water bath and washed with complete media. Cells were incubated overnight in a 37 C incubator and cell counts were obtained the following day. Cells were pclIcted and resuspended in 1 ml CFDA
SE (Molecular Probes, Eugene, OR) in PBS (1:2000 dilution). Cells were incubated at 37 C for 10 min. Cells were washed with complete media and resuspended to a concentration of 1x106 cells/100 ul and plated in 96 well round bottom plates with 100 ul of 2 ug/m1 recombinant HIV-I
p24 or gp120 (ImmunoDiagnostics, Woburn, MA) plus peptide pools. 5 1.tgiml Concavalin A
(positive) and complete media (negative) were used as controls. Cultures were incubated for 5 days. Cells were first stained with Vivid dye violet, a live/dead cell marker, for 15 min on ice. Cells were washed once with PBS. Cells were then stained using anti-human CD3-PE (clone SP34-2) (BD
Phariningen) and anti-human CD4-PerCP (clone L200), anti-human CD8-APC (SKI) for 1 hour at 4 C. Cells were then washed twice with PBS and fixed with I%
paraformaldchyde. Data was collected using a LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ).
Flow cytometry data was analyzed using Howie, software (Tree Star, Ashland, OR), gating on C1)3-lymphocytes. Thirty to fifty thousand C13' lymphocytes were collected per sample.
Enzyme Linked hnimmosorbant Assav (ELISA):
Ninety-six well plates were coated overnight with 100nglwell of recombinant p24 or gp120 (ImmunoDiagnosties) to determine HIV gag and env responses respectively. Plates coated with 10Ong/we11 of bovine scrum albumin served as a negative control.
Plates were blocked with 3%BSA-PBST for 1 hour at 37 C. Plates were then incubated with four-fbld serial serum dilutions for 1 hour at 37 C. Goat anti-monkey IgG horseradish peroxidasc conjugated antibody was then added at a 1:10,000 dilution (MP Biomedicals, Aurora, OH) to the plates and incubated for 1 hour at 37 C, Tetramethylbenzidine (R&D systems, Minneapolis, MN) was used to develop the plates and reactions were stopped with 2N 1-12SO4. Optical densities (OD) were then measured.
IgG end-point titers were defined as the reciprocal serum dilution that resulted in OD
values that were greater than twice the average OD value of the BSA wells.
Enzyme Linked Innnunospot Assay (ELISpoi) Antigen specific responses were determined by subtracting the number of spots in the negative control wells from the wells containing peptides. Results are shown as the mean value (spots/million splenocytes) obtained for triplicate wells.
1. Intracellular Cytokine Staining Antibody Rea.gents Directly conjugated antibodies were obtained from the following: BD
Biosciences (San Jose, CA): 1L-2 (PE), CD3 (Pacific Blue), IFN-71 (PE-Cy'), and TNF-n- (Alexa Fluor 700), CD8 (APC) and CD4 (PerCP).
Cell stimulation and. staining Pf3MCs were resuspended to 1 x 106 cells/100 ul in complete RPMI and plated in 96 well plates with stimulating peptides l 00u1 of 1:200 dilutions. An unstimulated and positive control (StaploVococcus enterotoxin B, 1 us/mL; Sigma-Aldrich) was included in each assay. Cells were incubated for 5 hours at 37C. Following incubation, the cells were washed (PBS) and stained with surface antibodies. The cells were washed and fixed using the Cytofix/Cytoperm kit (BD
PharMingen, San Diego, CA) according to instructions. Following fixation, the cells were washed twice in the perm buffer and stained with antibodies against intracellular markers.
Following stainingõ the cells were washed, fixed (PBS containing 1%
paraformaldehyde), and stored at 4C until analysis.
Flow cytometry Cells were analyzed on a modified LSR 11 flow cytometer (BD Immunocytometry Systems, San Jose, CA). Fifty thousand CD3+ events were collected per sample.
Data analysis was performed using Flowio version 8.4.1 (TreeStar, San Carlos, CA). Initial gating used a forward scatter area (FSC-A) versus height (FSC-H) plot to remove doublets.
The events were subjected to a lymphocyte gate by a FSC-A versus SSC plot. Following this, events are sequentially gated on CD3+, CDS', and CD4- events versus IFN-2 to account for down-regulation. Following identification of CDS' T cells, a gate was made for each respective function using combinations that provided optimal separation. After the gates for each functioîì.
were created, we used the Boolean gate platform to create the full array of possible combinations, equating to 8 response patterns when testing 3 functions. Data are reported after background correction. Thresholds for positive responses were 10 events or 0.05%.
Statistical Analysis Data are analyzed using Prisrn Graphpad software, and is expressed as means SEM.
= Results EL1Spot Analysis the induction of the cellular immune response was evaluated after each immunization by 1FNy ELISpot. After a single immunization (Figure 1), the group receiving plasmid DNA by IM
injection alone displayed weak cellular responses (74 29 SFU/106PBMCs). Co-immunization with rhesus FL-12 plasmid resulted in a higher response (136 51.4 SFU/106PBMCs). The electroporated (EP) group had an average response that was six times higher than tile IM group (482 181 SFU/106PBMCs). The combination of IL-12 co-inununization with EP
further doubled the number of IFNy-producing cells (1030 494 SFU/106PBMCs).
After two immunizations (Figure I), thc IM and IM +IL-12 groups had a modest increase in ELISpot counts (104 67.9 SFUI1OO PBMCs and 223 1 76.6 SFUJIOb PBMCs, respectively).
EP group had responses that were almost four fold higher (1924 417 SFU/106 PBMCs) than the previous immunization and the EP+IL-12 group had again doubled the number of IFNy-producing cells (2819 872 SFU/106 PRIVICs) compared to the EP arm alone.
After the third immunization (Figure the nu.mber of antigen specific cells in the EP
group was more than a log higher than that of the 1M group (5300 3781 and 370 l I() SFL1/106 PBMCs, respectively.). The IM+11,-12 group also had a dramatic increase in cellular responses with ELISpot C01.1111S that were nearly a log higher than the previous immunization (2042 311 SRJ/106PBMCs). As with the other two immunizations, the EP+IL.-12 group was the most potent of all the vaccination groups (7228 2227 STU11.06 PBMCs).
Induction of cross-reactive envelope responses A successful HIV vaccine µvill require the induction of a cross-reactive immune responses in this regard it was interesting to see if EP IL-12 could improve the magnitude of cross-reactivity to divergent peptide libraries. We compared the cross-reactive CTL responses induced by the env antigen using a peptide library from a consensus group M.
Cross-reactivity was observed in all groups. However the results displayed the same magnitude differences observed in the subtype 13 ELI-Spot analysis (Figure 2). After 3 immunizations, the IM group had the lowest response to the group M envelope peptides (222 iL SEM SFU/106 PBMCs), The addition of IL-12 doubled the response (540 + SEM SFU/106 PBMCs). Higher group M
envelope responses were induced with EP (830 SEM SRI/106 PBMCs), which were further enhanced with IL-12 co-injection (1238 SEIM. SFU/1)6PBMCs).
I. Memory T cell Responses An important issue is to bc able to improve the generation of memory responses with the DNA platform. We performed ELISpot analysis five months after the last DNA
vaccination (Figure 3). In the IM groups, the addition of plasmid IL-12 resulted in nearly a 10-fold increase in memory cells (751 11.1 and 78,6 -L 16.9 SFU/106 PBMCs), It is clear that IL-12 can positively impact this important T cell phenotype. The number of antigen-specific 1FNy producing cells was substantial in the EP group as well, however the IL-12 adjuvant + EP
resulted in the most robust memory response (1231 + 523.5 and 3795 + 1336 SFU/106 PBMCs respectively), a response showing, that the combined technology drives very strong T cell memory responses.
Humoral immune responses to DNA vaccines A weakness of IM 'DNA vaccine technology lies in its inability to induce clear antibody responses in non-human primates and in human clinical studies. We evaluated each group's ability to induce both HIV-1 gag and env specific antibody titers to recombinant p24 and gp160 antigens in an ELLSA format. For both antigens, the 1M and 1M 1L-12 groups did not show significant 'antibody titers ('1:50 endpoint tiler). The electroporated groups exhibited dramatically higher gag antibody titers that were able to bind to recombinant p24. Although both the EP arid the EP + IL-12 groups had similar endpoint titers at week 12 (22,400 and 12,800 respectively). the EP + 1L-12 group generated a more efficient antibody response. That response = appeared earlier in the immunization scheme and rose to the maximum level quickest. The env antibody responses also reflected the results we observed with the gag antigen, albeit with lower endpoint titers.
CD44 and CD8+ T cell proliferation Having observed substantial ELISpot responses, we next examined additional parameters of cellular immunity. We examined the ability of gag specific CD4+ and CD8'- T
cells to proliferate in vitro following peptide stimulation among the different immunization arms. Cryo-preserved samples, collected two weeks after thc final immunization, were stimulated and analyzed by CFSE assay. The average CD4 response increased similar to that observed in the ELISpot assay. By comparison, the CD8 proliferation induction was much more dramatic in magnitude. We observed that 1L-12 increased CDS' T cell proliferation over 1M
alone and EP
was substantially higher. The EP + IL-12 group had the highest percentage of CD8+ cells that were able to proliferate after in vitro stimulation (2.51 SEM //0 and 4.88 SE-M
respectively). Obvious CDS T cell proliferation bands were observed in the EP
1L-12 arm, demonstrating the potent proliferative potential of this combined immunization.
Polyfunctional CD 8' T cell responses Although we have clearly observed the induction of a robust IFNy effector response following EP and IL-12 co-immunization, we wanted to further characterize the functions of the antigen specific CDS'. T cell responses in the various arms. Samples taken three months following the final immunization were stimulated with gag peptides and stained thr intracellular cytokine production of IFNy, INFa and IL-2. Out of all groups, only one animal in the 1M + IL--12 and one animal in the EP only group had a detectable IFN7 response.
However two out oldie three animals in the EP + 1L-12 immunized group had gag-specific IFNy producing CD8' T
cells. The 14 1L-12 respond.er had a small percentage of polyfunctional cells that stained for all three cytokines as well as a population that had lost its ability to produce 1L-2. The EP
responder had slightly higher polyfunctional responses that were comprised of four different populations. The rnost dramatic response was seen in the second EP f IL-12 animal. More than 2% of its CD8' T cells were able to produce all three cytokines and 2% were able to produce both ITN-y and TNFoc. Clearly the number of animals in each group is low and requires additional primate studies to confirm these results, however collectively the trends observed appear clear and encouraging.
Discussion 1L-12 as a DNA vaccine adjuvant improved ELISpot responses several fold over piasmid = alone. In addition proliferation was clearly enhanced. The EP group exhibited a higher average response than either N. group alone or the IM + IL-12 iirm exhibiting a combined ELISpot response that was 3x higher than the 1M -4" IL-12 group. The best ELISpot responses were observed in the EP + IL-12 arm, which was almost 4x over the IMAL-12 ann 19x IM alone.
After each immunization the magnitude of the antigen-specific response by IENT.7 EL1Spot was determined. After a single imimmization all of the animals in the EP and EP + IL-12 groups not only had detectable responses, they had averages that were higher than those seen in the IM group after three immunizations. After two immunizations, IFN7 responses in the EP
and EP + 1L-12 groups were comparable to responses that have been reported in studies using viral vectors. Substantial memory responses were observed in the 1M + IL-12 and both EP
uroups five months after the last immunization.
1M immunization, with or without 1L-12, did not result in a significant amount of antibody. Eleetroporation was able to enhance the humor immune response as reported previously. All of the animals in the electroporated groups seroconvertcd.
Although the EP and the EP + 1L-12 groups had similar endpoint titers after three immunizations the kinetics of antibody induction was slightly faster in the EP + IL-12 group.
The proliferative capacity of CD8 T cells appeared to be enhanced with EP and plasmic' it- I 2_ This data supports the memory expansion observed in the EL1Spot assay where expansion of antigen specific T cell is likely a result of the enhanced proliferative potential of the EP + IL-12 arm.

Claims (34)

CLAIMS:
1. A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:1; fragments of SEQ ID NO:1; sequences having at least 90%
homology to SEQ ID NO:1; fragments of sequences having at least 90% homology to SEQ ID
NO:1; SEQ ID NO:3; fragments of SEQ ID NO:3; sequences having at least 90%
homology to SEQ ID NO:3; fragments of sequences having at least 90% homology to SEQ ID
NO:3; SEQ ID
NO:5; fragments of SEQ ID NO:5; sequences having at least 90% homology to SEQ
ID NO:5;
fragments of sequences having at least 90% homology to SEQ ID NO:5; SEQ ID
NO:7;
fragments of SEQ ID NO:7; sequences having at least 90% homology to SEQ ID
NO:7;
fragments of sequences having at least 90% homology to SEQ ID NO:7; SEQ ID
NO:9;
fragments of SEQ ID NO:9; sequences having at least 90% homology to SEQ ID
NO:9;
fragments of sequences having at least 90% homology to SEQ ID NO:9; SEQ ID
NO:11;
fragments of SEQ ID NO:11; sequences having at least 90% homology to SEQ ID
NO:11;
fragments of sequences having at least 90% homology to SEQ ID NO:11; SEQ ID
NO:34;
fragments of SEQ ID NO:34; sequences having at least 90% homology to SEQ ID
NO:34; and fragments of sequences having at least 90% homology to SEQ ID NO:34.
2. The nucleic acid molecule of claim 1 comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:1; SEQ ID NO:3; SEQ ID NO:5;
SEQ ID
NO:7; SEQ ID NO:9; SEQ ID NO:11; and SEQ ID NO:34.
3. The nucleic acid molecule of claim 1 comprising a sequence having at least 95% homology to a nucleotide sequence selected from the group consisting of:
SEQ ID NO:1;
SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; and SEQ ID
NO:34.
4. The nucleic acid molecule of claim 1 comprising a sequence having at least 98% homology to a nucleotide sequence selected from the group consisting of:
SEQ ID NO:1;
SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; and SEQ ID
NO:34.
5. The nucleic acid molecule of claim 1 comprising a sequence having at least 99% homology to a nucleotide sequence selected from the group consisting of:
SEQ ID NO:1;
SEQ ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:9; SEQ ID NO:11; and SEQ ID
NO:34.
6. The nucleic acid molecule of claim 1 comprising a nucleotide sequence that encodes a protein selected from the group consisting of: SEQ ID NO:16; SEQ ID
NO:17; SEQ
ID NO:18; SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:21; and SEQ ID NO:35.
7. A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: nucleotide sequences that encode SEQ ID NO:2; nucleotide sequences that encode an amino acid sequences having at least 90% homology to SEQ ID NO:2;
fragments of nucleotide sequences that encode SEQ ID NO:2; fragments of a nucleotide sequence that encode an amino acid sequence having at least 90% homology to SEQ ID NO:2; nucleotide sequences that encode SEQ ID NO:4; nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID NO:4; fragments of nucleotide sequences that encodes SEQ ID
NO:4; fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:4; nucleotide sequences that encode SEQ ID NO:6;
nucleotide sequences that encode an amino acid sequences having at least 90% homology to SEQ ID NO:6;
fragments of nucleotide sequences that encode SEQ ID NO:6; fragments of a nucleotide sequence that encode an amino acid sequence having at least 90% homology to SEQ ID NO:6;
nucleotide sequences that encode SEQ ID NO:8; nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID NO:8; fragments of nucleotide sequences that encodes SEQ ID NO:8; fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:8; nucleotide sequences that encode SEQ ID NO:10; nucleotide sequences that encode an amino acid sequences having at least 90%
homology to SEQ ID NO:10; fragments of nucleotide sequences that encode SEQ ID
NO:10;
fragments of a nucleotide sequence that encode an amino acid sequence having at least 90%
homology to SEQ ID NO:10; nucleotide sequences that encode SEQ ID NO:12;
nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID

NO:12; fragments of nucleotide sequences that encodes SEQ ID NO:12; fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID
NO:12; nucleotide sequences that encode SEQ ID NO:35; nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID NO:35; fragments of nucleotide sequences that encodes SEQ ID NO:35; and fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:35.
8. The nucleic acid molecule of claim 7 comprising a nucleotide sequence that encodes a protein selected from the group consisting of: SEQ ID NO:16; SEQ ID
NO:17; SEQ
ID NO:18; SEQ ID NO:19; SEQ ID NO:20 and SEQ ID NO:21; and SEQ ID NO:35.
9. The nucleic acid molecule of claim 1 comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:1; fragments of SEQ ID NO:1;
sequences having at least 90% homology to SEQ ID NO:1; fragments of sequences having at least 90%
homology to SEQ ID NO:1; SEQ ID NO:3; fragments of SEQ ID NO:3; sequences having at least 90% homology to SEQ ID NO:3; fragments of sequences having at least 90%
homology to SEQ ID NO:3; SEQ ID NO:5; fragments of SEQ ID NO:5; sequences having at least 90%
homology to SEQ ID NO:5; fragments of sequences having at least 90% homology to SEQ ID
NO:5; SEQ ID NO:7; fragments of SEQ ID NO:7; sequences having at least 90%
homology to SEQ ID NO:7; fragments of sequences having at least 90% homology to SEQ ID
NO:7; SEQ ID
NO:9; fragments of SEQ ID NO:9; sequences having at least 90% homology to SEQ
ID NO:9;
fragments of sequences having at least 90% homology to SEQ ID NO:9; SEQ ID
NO:11;
fragments of SEQ ID NO:11; sequences having at least 90% homology to SEQ ID
NO:11; and fragments of sequences having at least 90% homology to SEQ ID NO:11.
10. The nucleic acid molecule of claim 7 comprising a nucleotide sequence selected from the group consisting of: nucleotide sequences that encode SEQ ID
NO:2;
nucleotide sequences that encode an amino acid sequences having at least 90%
homology to SEQ ID NO:2; fragments of nucleotide sequences that encode SEQ ID NO:2;
fragments of a nucleotide sequence that encode an amino acid sequence having at least 90%
homology to SEQ
ID NO:2; nucleotide sequences that encode SEQ ID NO:4; nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID NO:4; fragments of nucleotide sequences that encodes SEQ ID NO:4; fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:4; nucleotide sequences that encode SEQ ID NO:6; nucleotide sequences that encode an amino acid sequences having at least 90%
homology to SEQ ID NO:6; fragments of nucleotide sequences that encode SEQ ID
NO:6;
fragments of a nucleotide sequence that encode an amino acid sequence having at least 90%
homology to SEQ ID NO:6; nucleotide sequences that encode SEQ ID NO:8;
nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID
NO:8; fragments of nucleotide sequences that encodes SEQ ID NO:8; fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:8;
nucleotide sequences that encode SEQ ID NO:10; nucleotide sequences that encode an amino acid sequences having at least 90% homology to SEQ ID NO:10; fragments of nucleotide sequences that encode SEQ ID NO:10; fragments of a nucleotide sequence that encode an amino acid sequence having at least 90% homology to SEQ ID NO:10; nucleotide sequences that encode SEQ ID NO:12; nucleotide sequences that encodes an amino acid sequences having at least 90% homology to SEQ ID NO:12; fragments of nucleotide sequences that encodes SEQ ID
NO:12; and fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:12;
11. The nucleic acid molecule of claim 1 comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:34; fragments of SEQ ID
NO:34; sequences having at least 90% homology to SEQ ID NO:34; and fragments of sequences having at least 90% homology to SEQ ID NO:34.
12. The nucleic acid molecule of claim 7 comprising a nucleotide sequence selected from the group consisting of: nucleotide sequences that encode SEQ ID
NO:35;
nucleotide sequences that encodes an amino acid sequences having at least 90%
homology to SEQ ID NO:35; and fragments of nucleotide sequences that encodes SEQ ID NO:35;
fragments of nucleotide sequences that encodes an amino acid sequence having at least 90% homology to SEQ ID NO:35.
13. The nucleic acid molecule of any of claims 1-12 wherein said molecule is a plasmid.
14. A pharmaceutical composition comprising a nucleic acid molecule of any of claims 1-13.
15. An injectable pharmaceutical composition comprising a nucleic acid molecule of any of claims 1-13.
16. A recombinant vaccine comprising a nucleic acid molecule any of claims 1-13.
17. The recombinant vaccine of claim 16 wherein said recombinant vaccine is a recombinant vaccinia vaccine.
18. A live attenuated pathogen comprising a nucleic acid molecule of any of claims 1-13.
19. A protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:2, sequences having at least 90% homology to SEQ ID
NO:2;
fragments of SEQ ID NO:2; fragments of sequences having at least 90% homology to SEQ ID
NO:2; SEQ ID NO:4, sequences having at least 90% homology to SEQ ID NO:4;
fragments of SEQ ID NO:; fragments of sequences having at least 90% homology to SEQ ID
NO:4; SEQ ID
NO:6, sequences having at least 90% homology to SEQ ID NO:6; fragments of SEQ
ID NO:6;
fragments of sequences having at least 90% homology to SEQ ID NO:6; SEQ ID
NO:8, sequences having at least 90% homology to SEQ ID NO:8; fragments of SEQ ID
NO:8;
fragments of sequences having at least 90% homology to SEQ ID NO:8; SEQ ID
NO:10, sequences having at least 90% homology to SEQ ID NO:10; fragments of SEQ ID
NO:10;
fragments of sequences having at least 90% homology to SEQ ID NO:10; SEQ ID
NO:12, sequences having at least 90% homology to SEQ ID NO:12; fragments of SEQ ID
NO:12;
fragments of sequences having at least 90% homology to SEQ ID NO:12;; SEQ ID
NO:35, sequences having at least 90% homology to SEQ ID NO:35; fragments of SEQ ID
NO:35; and fragments of sequences having at least 90% homology to SEQ ID NO:35.
20. The protein of claim 19 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ
ID
NO:10; and SEQ ID NO:12; and SEQ ID NO: 35.
21. The protein of claim 19 comprising a sequences having at least 95%
homology to an amino acid sequence selected from the group consisting of: SEQ
ID NO:2; SEQ
ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; and SEQ ID NO:12; and SEQ ID
NO:
35.
22. The protein of claim 19 comprising a sequences having at least 98%
homology to an amino acid sequence selected from the group consisting of: SEQ
ID NO:2; SEQ
ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; and SEQ ID NO:12; and SEQ ID
NO:
35.
23. The protein of claim 19 comprising a sequences having at least 99%
homology to an amino acid sequence selected from the group consisting of: SEQ
ID NO:2; SEQ
ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:10; and SEQ ID NO:12; and SEQ ID
NO:
35.
24. The protein of claim 19 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:2, sequences having at least 90% homology to SEQ ID NO:2;
fragments of SEQ ID NO:2; fragments of sequences haying at least 90% homology to SEQ ID
NO:2; SEQ ID NO:4, sequences having at least 90% homology to SEQ ID NO:4;
fragments of SEQ ID NO:; fragments of sequences having at least 90% homology to SEQ ID
NO:4; SEQ ID
NO:6, sequences having at least 90% homology to SEQ ID NO:6; fragments of SEQ
ID NO:6;
fragments of sequences haying at least 90% homology to SEQ ID NO:6; SEQ ID
NO:8, sequences haying at least 90% homology to SEQ ID NO:8; fragments of SEQ ID
NO:8;
fragments of sequences having at least 90% homology to SEQ ID NO:8; SEQ ID
NO:10, sequences having at least 90% homology to SEQ ID NO:10; fragments of SEQ ID
NO:10;
fragments of sequences having at least 90% homology to SEQ ID NO:10; SEQ ID
NO:12, sequences having at least 90% homology to SEQ ID NO:12; fragments of SEQ ID
NO:12; and fragments of sequences having at least 90% homology to SEQ ID NO:12.
25. The protein of claim 19 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19;
SEQ ID
NO:20 and SEQ ID NO:21.
26. The protein of claim 19 comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:35, sequences having at least 90% homology to SEQ ID
NO:35; fragments of SEQ ID NO:35; and fragments of sequences having at least 90% homology to SEQ ID NO:35.
27. The protein of claim 26 comprising an amino acid sequence SEQ ID NO:35.
28. A pharmaceutical composition comprising a protein of any of claims 19-27.
29. An injectable pharmaceutical composition comprising a protein of any of claims 19-27.
30. A recombinant vaccine comprising a protein of any of claims 19-27.
31. The recombinant vaccine of claim 30 wherein said recombinant vaccine is a recombinant vaccinia vaccine.
32. A live attenuated pathogen comprising a protein of any of claims 19-27.
33. A method of inducing an immune response in an individual against HIV
comprising administering to said individual a composition comprising a nucleic acid molecule of claim 9 or 10 or a protein of claim 24 or 25.
34. A method of inducing an immune response in an individual against hTERT
comprising administering to said individual a composition comprising a nucleic acid molecule of claim 11 or 12 or a protein of claim 26 or 27.
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