WO2013093629A2

WO2013093629A2 - Modular vaccines, methods and compositions related thereto

Info

Publication number: WO2013093629A2
Application number: PCT/IB2012/003006
Authority: WO
Inventors: Koen OOSTERHUIS; Jan Batist Anna Gerardus HAANEN; Antonius Nicolaas Maria Schumacher
Original assignee: Netherlands Cancer Institute
Priority date: 2011-12-20
Filing date: 2012-12-19
Publication date: 2013-06-27
Also published as: WO2013093629A3

Abstract

The invention relates to the field of methods and related compositions for the preparation and administration of vaccines, such as DNA vaccines, for the treatment of one or more diseases. In other embodiments, the invention relates to the field of methods for the prevention or treatment of disease comprising administration of one or more vaccines, such as DNA vaccines, for the treatment of a disease to a patient in need thereof. In a particular embodiment, the invention provides a DNA vaccine for the prevention and treatment of HPV and/or an HPV -induced cancer.

Description

TITLE OF THE INVENTION

MODULAR VACCINES, METHODS AND COMPOSITIONS RELATED THERETO

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority of U.S. Provisional Application Serial Number 61/577,811, filed December 20, 2011. The foregoing application is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of methods and related compositions for the preparation and administration of vaccines, such as DNA vaccines, for the treatment of one or more diseases. In other embodiments, the invention relates to the field of methods for the prevention or treatment of disease comprising administration of one or more vaccines, such as DNA vaccines, for the treatment of a disease to a patient in need thereof.

BACKGROUND OF THE INVENTION

High risk HPV E6 and E7 are ideal tumor-associated antigens. Their expression is necessary and sufficient for cellular transformation (1). As a consequence, any HPV- induced (pre)malignancy expresses these proteins (2). As E6 and E7 are of non-self origin it should in theory be feasible to elicit strong and specific immune responses against these proteins, resulting in eradication of (pre)malignant cells without affecting healthy cells (3). Importantly E6 and E7 specific T cell responses are correlated with the clearance of HPV induced lesions (4-6). Different therapeutic vaccination strategies have been developed that aim to induce HPV 16 E6 and E7 specific T cell responses (7,8). This includes DNA vaccination. DNA vaccination benefits from its relative simplicity, excellent safety record, and its ability to elicit strong cellular immunity (9-11).

Most of these vaccine candidates consist of a genetic fusion of the antigen and a so called "carrier protein." The nature of the carrier proteins used for this purpose widely varies, examples include: heat shock proteins (HSPs) (13,14), calreticulin (CRT) (15), E. Coli Beta glucoronidase (GUS) (16), interferon gamma inducible protein 10 (IP10) (17), herpes simplex virus viral protein 22 (HSV VP22) (18), Herpes simplex virus glycoprotein D (HSV gD) (19) and Tetanus toxin fragment C (TTFC) (20). The mechanisms proposed to explain the enhanced immunogenicity resulting from the fusion with these carriers also vary widely. In many cases, the authors hypothesize that the fusion to a carrier leads to enhanced antigen presentation as a result of the specific function of the carrier protein. ER

chaperones such as calreticulin are believed to deliver the antigen directly into the antigen presentation pathway thereby increasing the likelihood of antigen presentation (15,21,22). DC binding molecules such as HSPs or Flt3 ligand are thought to result in an enhanced uptake of the coupled antigen by antigen presenting cells (13,23). HSV VP22 fusion is believed to result in spreading of the coupled antigen to neighboring cells thereby increasing antigen cross presentation (18). IP- 10 is thought to attract T cells to the vaccination site by its chemo attractive function (17). Most of these carrier function- dependent mechanisms are speculative.

In case the carrier is a foreign molecule, it is well accepted that provision of CD4+ T cell help in the form of carrier encoded CD4+ epitopes, at least partly, explains the carrier effect (13,22,24,25). This could for example be the case for mycobacterial HSP-70, TTFC, HSV VP22 or Pseudomonas Aaeruginose exototoxin A, and HSV glycoprotein D. In case the carrier is of self origin, provision of CD4+ T cell help is highly unlikely (25).

HPV DNA vaccines that are not genetically coupled to a carrier are very poorly immunogenic. Furthermore, current used carriers suffer from limitations, such as when the carrier protein is of foreign origin, CD8+ T cell responses against epitopes within the carrier may prevent or limit the induction of CD8+ T cell responses against the antigen of interest. Furthermore, where the carrier contains substantial amounts of genetic material encoding an endogenous protein, the risk of inducing T cell responses against the endogenous protein also exists.

SUMMARY OF THE INVENTION

The instant invention relates to compositions and related methods for enhancing the immunogenicity of an antigen. The inventors have surprisingly discovered that the combination of a minimal subcellular localization element and a minimal CD4 help element fused to an antigen synergistically enhances the immunogenicity of the antigen over use of the localization element or CD4 help element fused to the antigen alone.

Typically, the fusion protein is transcribed from a genetically-encoded vaccine, such as a DNA vaccine or viral vector. In certain embodiments, the genetically-encoded vaccine comprises an expression cassette, wherein the expression cassette comprises a subcellular localization element (e.g., a signal peptide), a CD4 help element (e.g., a CD4+ T cell epitope such as PADRE), and an antigen (e.g., HPV 16 E7) that is transcribed as a fusion protein in an animal after administration to the animal. Accordingly, in certain embodiments, the invention relates to enhancing the immunogenicity of an antigen by adding one or more short signal peptide nucleic acid sequences and minimal CD4 helper epitope nucleic acid sequences to the nucleic acid sequence encoding an antigen, wherein the fusion protein that is transcribed comprises an antigen with enhanced immunogenicity after administration to an animal.

In certain embodiments, the instant application relates to a genetically-encoded vaccine comprising an expression cassette transcribing (i) at least one antigen, (ii) at least one subcellular localization element, and (iii) at least one CD4 help element.

In certain embodiments, the at least one antigen is selected from the group consisting of: a tumor-associated antigen, a viral antigen, a bacterial antigen, a parasite- derived antigen, and disease-associated self antigen. Examples of tumor-associated antigens include HPV antigens, such as HPV 16 E6 and/or E7.

In some embodiments, expression of the at least one subcellular localization element results in export of the antigen out of the cytosol. In some embodiments, the at least one subcellular localization element is a signal peptide or protein sequence that results in export of the antigen from the cytosol. In certain embodiments, expression of the at least one subcellular localization element results in localization of the antigen to an organelle selected from the group consisting of: the endoplasmic reticulum (ER), the Golgi apparatus, an endosome, a lysosome, and a mitochondrion. In a particular embodiment, expression of the at least one subcellular localization element localizes the antigen to the ER.

In some embodiments, the at least one subcellular localization element is or comprises a portion of a self carrier protein. In other embodiments, the at least one subcellular localization element enhances antigen stability.

In certain embodiments, the at least one CD4 help element is selected from the group consisting of: P30 (FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 17)), PADRE (AKFVAAWTLKAAA (SEQ ID NO: 16)), NEF, P23TT (VSIDKFRIFCKANPK (SEQ ID NO: 7)), P32TT (LKFIIKRYTPNNEIDS (SEQ ID NO: 8)), P21TT

(IREDNNITLKLDRCNN (SEQ ID NO: 9)), PfT3 (EKKIAAKMEKAS S VFNVVN (SEQ ID NO: 10)), P2TT (QYIKANSKFIGITE (SEQ ID NO: 11)), HBVnc

(PHHTALRQAILCWGELMTLA (SEQ ID NO: 12)), HA (PKYVKQNTLKLAT (SEQ ID NO: 13)), HBsAg (FFLLTRILTIPQSLD (SEQ ID NO: 14)), and MT

(YSGPLKAEIAQRLEDV (SEQ ID NO: 15)). In certain embodiments, the at least one subcellular localization element comprises a signal peptide. In other embodiments, the at least one subcellular localization element comprises an ER retention signal.

In some embodiments, the at least one CD4 help element comprises at least one CD4+ epitope. In other embodiments, the at least one CD4 help element comprises at least two CD4+ epitopes. In further embodiments, the at least two CD4+ epitopes are encoded by nucleic acid sequences separated by a glycine proline repeat or other peptide sequence that prevents the formation of one or more neo-epitopes.

In some embodiments, a genetically-encoded vaccine of the invention comprises a pharmaceutically acceptable carrier. In certain embodiments, the vaccine is a DNA vaccine or a viral vaccine. In further embodiments, the viral vaccine is an adenoviral vaccine or a vaccinia viral vaccine.

In some embodiments, administration of the vaccine to an animal elicits an immunogenic response against the at least one antigen that is encoded by and expressed from the genetically-encoded vaccine by the animal after administration. In certain embodiments, the immunogenic response elicited is an increase in CD8+ T cells specific for the at least one antigen.

In yet other embodiments, the instant application relates to a method of treating a tumor or cancer or a method of inhibiting tumor cell growth or cancer cell growth in a mammal comprising administering to the mammal an effective amount of the genetically- encoded vaccine, wherein the genetically-encoded vaccine comprises an expression cassette transcribing (i) at least one antigen, (ii) at least one subcellular localization element, and (iii) at least one CD4 help element. In a particular embodiment, the mammal is a human.

In certain embodiments, the tumor or cancer is induced by HPV. In further embodiments, the HPV is HPV 16. In certain embodiments, the genetically-encoded vaccine is a DNA vaccine comprising an HPV E6 and/or E7 antigen.

In some embodiments, the at least one subcellular localization element of the genetically-encoded vaccine is selected from the group consisting of: a signal peptide or protein sequence that results in export of the antigen from the cytosol.

In some embodiments, expression of the at least one subcellular localization element results in export of the antigen out of the cytosol. In some embodiments, expression of the at least one subcellular localization element in the genetically-encoded vaccine localizes the antigen to an organelle selected from the group consisting of: the endoplasmic reticulum (ER), the Golgi apparatus, an endosome, a lysosome, and a mitochondrion. In a particular embodiment, expression of the at least one subcellular localization element localizes the antigen to the ER.

In certain embodiments, the at least one subcellular localization element of the genetically-encoded vaccine is a self carrier protein. In some embodiments, the at least one subcellular localization element of the genetically-encoded vaccine enhances antigen stability.

In certain embodiments, the at least one CD4 help element of the genetically- encoded vaccine is selected from the group consisting of: P30

(FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 17)), PADRE (AKFVAAWTLKAAA (SEQ ID NO: 16)), NEF, P23TT (VSIDKFRIFCKANPK (SEQ ID NO: 7)), P32TT

(LKFIIKRYTPNNEIDS (SEQ ID NO: 8)), P21TT (IREDNNITLKLDRCNN (SEQ ID NO: 9)), PfT3 (EKKIAAKMEKAS S VFNVVN (SEQ ID NO: 10)), P2TT (QYIKANSKFIGITE (SEQ ID NO: 11)), HBVnc (PHHTALRQAILCWGELMTLA (SEQ ID NO: 12)), HA (PKYVKQNTLKLAT (SEQ ID NO: 13)), HBsAg (FFLLTRILTIPQSLD (SEQ ID NO: 14)), and MT (YSGPLKAEIAQRLEDV (SEQ ID NO: 15)).

In some embodiments, administration of the vaccine elicits an immunogenic response against the at least one antigen that is encoded by and expressed from the genetically-encoded vaccine by the mammal after administration. In certain embodiments, the immunogenic response elicited is an increase in CD8+ T cells specific for the at least one antigen.

In yet other embodiments, the instant application relates to a vaccine comprising a nucleic acid expression cassette transcribing (i) at least one antigen, (ii) at least one subcellular localization element, and (iii) at least one CD4 help element.

In certain embodiments, the nucleic acid expression cassette is expressed from a viral expression vector. In some embodiments, the viral expression vector is selected from the group consisting of: an adenovirus, an adeno-associated virus (AAV), an alphavirus, a retrovirus, and a poxvirus. In a particular embodiment, the alphavirus is Semliki Forest virus. In certain embodiments, the adenovirus is selected from the group consisting of Ad5, Adl 1, Ad26, Ad35, Ad48, and Ad49. In a particular embodiment, the poxvirus is a vaccinia virus. In a further embodiment, the vaccinia virus is MVA.

In some embodiments, the nucleic acid expression cassette is expressed from a DNA vector. In other embodiments, a vaccine of the invention further comprises at least one additional therapeutically active agent. In some embodiments, the at least one additional therapeutically active agent is a second vaccine.

In some embodiments, the instant application relates to a kit comprising (a) a vaccine of of the invention and (b) instructions for use. In certain embodiments, the kit further comprises at least one additional therapeutically active agent. In certain

embodiments, the additional therapeutically active agent is a second vaccine.

In some embodiments, the instant application provides a genetically-encoded vaccine for use in a method for treating a tumor or cancer in a mammal, the method comprising administering to the mammal an effective amount of a genetically-encoded vaccine, wherein the genetically-encoded vaccine comprises an expression cassette transcribing (i) at least one antigen, (ii) at least one subcellular localization element, and (iii) at least one CD4 help element. In a particular embodiment, the mammal is a human.

In yet other embodiments, the instant application provides a genetically-encoded vaccine for use in a method for inhibiting tumor cell growth or cancer cell growth in a mammal, the method comprising administering to the mammal an effective amount of a genetically-encoded vaccine, wherein the genetically-encoded vaccine comprises an expression cassette transcribing (i) at least one antigen, (ii) at least one subcellular localization element, and (iii) at least one CD4 help element. In a particular embodiment, the mammal is a human.

These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 depicts the design and validation of a modular DNA vaccine construct according to the invention that allows separation of the CD4 help effect from the carrier effect. This approach limits the use of foreign sequences, thereby reducing the possibility for antigenic competition between carrier- and antigen-derived epitopes. A) Schematic representation of the vaccine design. In this example, the carrier and the helper cassette are both placed between identical restriction sites to allow their removal from the construct. The antigen is placed between two different restriction enzymes to enable easy cloning. B) Detail of the HELPER cassette. The GPGPGPG spacers avoid formation of neo epitopes at the junctions. C) Schematic representation of the constructs used for proof of principle and their expected molecular weight. D) Western blot analysis of HEK 293 cells transfected with the constructs shown in C. HEK 293 cells were transfected with a mixture of 3 μg GFP encoding DNA and 7 μg of the indicated DNA vaccine. Cells were harvested 24 hours after trans fection and equal trans fection efficiency was demonstrated by FACS staining (data not shown). Subsequently both HPV 16 E7 and actin (to demonstrate equal loading) were detected by western blot analysis, using a mouse monoclonal antibody against E7 and a mouse anti-human actin antibody, respectively. In all cases a dominant band of the expected size (see C for the expected molecular weights) could be detected demonstrating the correct expression of the DNA vaccine-encoded antigens.

FIGURE 2 shows that the combined addition of a self-carrier and a helper-cassette leads to superior CD8+ T cell immunogenicity. C57BL/6 mice (n=5 per group) were immunized by DNA tattoo vaccination on day 0, 3 and 6, and peripheral blood was analyzed for antigen-specific CD8+ T cells by MHC tetramer staining. A) Representative dot plots of H-2D^b E7₄ _57 MHC tetramer stainings at the peak of the response for the different constructs. B) Bar diagram depicting the mean percentage +/- S.D. of H-2D^b E7₄9_ 57-specific CD8+ T cells for the indicated groups at the peak of the response. C) Plot depicting the mean percentage +/- S.D. of H-2D^b E7₄9_57-specific CD8+ T cells for the indicated groups over time. D) Bar diagram depicting the total area under the curve (AUC) +/- S.D. for each construct as a measure for the total construct potency. E) Bar diagram showing the mean percentage +/- S.D. of interferon-γ positive CD4+ T cells in peripheral blood samples, collected on day 11, after 16h stimulation with either the P30 or the PADRE peptide. These data demonstrate that both the presence of helper T cell epitopes and of a carrier enhance immunogenicity of the fused antigen individually and that the combination of these two entities has a synergistic effect.

FIGURE 3 shows that ER-localized self-carriers provide an advantage over the addition of the helper-cassette alone. A) Schematic representation and molecular weights of DNA vaccines with different carrier proteins. B) Western blot analysis of HEK 293 cells transfected with the constructs shown in A, and performed as described before. In all cases a dominant band of the expected size could be detected demonstrating the correct expression of the DNA vaccine encoded antigens. C) Time curve depicting the mean percentage +/- S.D. of H-2D^b E7₄9_57-specific CD8+ T cells over time of C57BL6/J mice (n=5) DNA tattoo vaccinated with the indicated constructs. D) Bar diagram depicting the mean percentage +/- S.D. of H-2D^b E7₄9_57-specific CD8+ T cells for the indicated groups at the peak of the response. E) Bar diagram depicting the total area under the curve (AUC) +/-

S.D. for each construct as measure for the total construct potency. These data demonstrate that in this embodiment, compared to the foregoing localization carriers used, use of an ER- localized carrier leads to superior immunogenicity.

FIGURE 4 shows that the 'carrier effect' can be explained by ER targeting of the antigen. A) Schematic representation and molecular weights of constructs used to determine if the carrier effect can be explained by ER targeting of the antigen. B) Western blot analysis of HEK 293 cells transfected with the constructs shown in A, and performed as described before. In all cases, a dominant band of the expected size could be detected demonstrating the correct expression of the DNA vaccine-encoded antigens. C,D,E. Results demonstrating that removal of the signal peptide renders the carrier ineffective. C) Curve depicting the mean percentage +/- S.D. of H-2D^b E7₄₉_57-specific CD8+ T cells over time of C57BL6/J mice (n=5) DNA tattoo vaccinated with the indicated constructs. D) Bar diagram depicting the mean percentage +/- S.D. of H-2D^b E7₄ _57-specific CD8+ T cells for the indicated groups at the peak of the response. E) Bar diagram depicting the total area under the curve (AUC) +/- S.D. for each construct as measure for the total construct potency. F,G,H. Results demonstrating that the use of only a signal peptide and KDEL sequence is sufficient to increase the immunogenicity of HELP-E7SH. F) Time curve depicting the mean percentage +/- S.D. of H-2D^b E7₄ _57-specific CD8+ T cells over time of C57BL6/J mice (n=5) DNA tattoo vaccinated with the indicated constructs. G) Bar diagram depicting the mean percentage +/- S.D. of H-2D^b E7₄₉_57-specific CD8+ T cells for the indicated groups at the peak of the response. H) Bar diagram depicting the total area under the curve (AUC) +/- S.D. for each construct as measure for the total construct potency. These data demonstrate that a minimal immunogenic combination can be designed according to the instant invention that provides CD4 T cell help and that incorporates the 'carrier effect.' In this combination, both the likelihood of the induction of unintended CD8+ T cell responses against foreign MHC class I associated epitopes, as well as the likelihood of induction of an unintended autoimmune response against endogenous sequences in the carrier protein are markedly reduced.

FIGURE 5 shows that the combined addition of the helper-cassette, signal peptide and ER retention signal also improves the immunogenicity of E6SH. A) Schematic representation and molecular weights of E6SH encoding DNA vaccines. B) Representative dot plots at the peak of the response for the indicated constructs. C) Curve depicting the mean percentage +/- S.D. of H-2K^b E6₄₈-57-specific CD8+ T cells over time, of C57BL6/J mice (n=5) DNA tattoo vaccinated with the indicated constructs. D) Bar diagram depicting the mean percentage +/- S.D. of H-2K^b E6₄₈_57-specific CD8+ T cells for the indicated groups at the peak of the response. E) Bar diagram depicting the total area under the curve (AUC) +/- S.D. for each construct as measure for the total construct potency. These data demonstrate that the design that has been developed and described herein has a broader applicability for the induction of T cell responses against antigens of interest.

FIGURE 6 shows that the novel design allows for dose sparing and demonstrates superior functionality. A) Curve depicting the mean percentage +/- S.D. of H-2D^b E7₄9_₅₇- specific CD8+ T cells over time of C57BL6/J mice (n=5) vaccinated with a single DNA tattoo vaccination with the indicated constructs. B) Curve depicting the mean percentage +/- S.D. of H-2D^b E7₄9-57-specific CD8+ T cells over time of C57BL6/J mice (n=5) DNA tattoo vaccinated with the indicated constructs using a 5 times lower DNA concentration (0.4 mg/ml). C, D,E. Tumor regression after single DNA tattoo vaccination with the sig-HELP- E7SH-KDEL vaccine. C57BL/6 mice (n=10per group) were injected with 1 x 10⁵ TC-1 tumor cells on day 0. Subsequently, mice were immunized by DNA tattoo vaccination on day 4 (lx) after tumor challenge or on day 4, 7,10 (3x) as indicated. Tumor sizes were determined by caliper measurements 2-3 times weekly. Peripheral blood was analyzed for antigen-specific CD8+ T cells by MHC tetramer staining. C) Plot depicting the mean tumor diameter (mm) +/- S.D. for the indicated groups over time. D) Plot depicting the percentage survival for the indicated groups over time. E) Plot depicting the mean percentage +/- S.D. of H-2K^b E6₄8_₅₇-specific CD8+ T cells for the indicated groups over time. F.) Plot depicting the mean percentage +/- S.D. of H-2D^b E7₄9_5₇-specific CD8+ T cells for the indicated groups over time. These data demonstrate that in addition to the above described advantages with respect to the induction of unintended immune responses, the design that has been developed and described herein also strongly outperforms a previously developed carrier vaccine design (20) with respect to immunogenicity and tumor control.

FIGURE 7 shows that sig-HELP-E6SH-KDEL strongly outperforms TTFC-E6SH in HLA-A2 transgenic mice. HLA-A2 transgenic mouse class I knock-out (HHD) mice (n=3 per group) were immunized by DNA tattoo vaccination on day 0, 3 and 6, and peripheral blood was analyzed for antigen-specific CD8⁺ T cells by MHC tetramer staining. A) Representative dot plots of A2-K^b E6₂ -38 MHC tetramer stainings at the peak of the response for the different vaccines B) Plot depicting the mean percentage +/- S.D. of A2-K^b E6₂₉-38-specific CD8⁺ T cells for the indicated groups over time. C) Bar diagram depicting the mean percentage +/- S.D. of A2-K^b E6₂₉-38-specific CD8⁺ T cells for the indicated groups at the peak of the response. D) Bar diagram depicting the total area under the curve (AUC) +/- S.D. for each construct as measure for the total vaccine potency. These data demonstrate that the combined addition of a subcellular localization element and a CD4 help element is also superior in the context of human HLA molecules.

FIGURE 8 shows that ER localization of the antigen and not the ER-stress provoked is causing the increased immunogenicity. A) Schematic representation and estimated molecular weights of the DNA vaccines used to test if ER-stress or ER localization is causing the increase in immunogenicity. B) Westernblot analysis of HEK 293 cells transfected with the vaccines shown in A. The FM4-2A-HELP-E7SH encoding constructs shows a dominant band of the size of HELP-E7SH and only a minor band of the size of FM4-HELP-E7SH demonstrating the functionality of the 2A linker. C) Curve depicting the mean percentage +/- S.D. of H-2D^b E7₄ _57-specific CD8+ T cells over time in C57BL/6J mice (n=5) tattoo vaccinated on day 0, 3 and 6 with the indicated vaccines D) Bar diagram depicting the mean percentage +/- S.D. of H-2D^b E7₄₉_57-specific CD8+ T cells for the indicated groups at the peak of the response. E) Bar diagram depicting the total area under the curve (AUC) +/- S.D. for each vaccine as measure for the total vaccine potency. These data demonstrate that ER localization of the antigen as such is important and that ER stress as such does not explain the increase in immunogenicity.

FIGURE 9 shows the nucleic acid sequences corresponding to Sig-HELP-E6SH- KDEL (SEQ ID NO: 1) and Sig-HELP-E7SH-KDEL (SEQ ID NO: 2).

FIGURE 10 shows the amino acid sequences corresponding to Sig-HELP-E6SH- KDEL (SEQ ID NO: 3) and Sig-HELP-E7SH-KDEL (SEQ ID NO: 4).

FIGURE 11 shows the nucleic acid sequence corresponding to pVAX sig-HELP- E6SH-KDEL (SEQ ID NO: 5).

FIGURE 12 shows the nucleic acid sequence corresponding to pVAX sig-HELP- E7SH-KDEL (SEQ ID NO: 6). DETAILED DESCRIPTION

The instant invention relates to modular vaccine designs that enhance the immunogenicity of an antigen administered to a subject. In certain embodiments, the instance invention provides a nucleic acid expression cassette comprising nucleic acid sequence encoding an antigen (such as an HPV 16 E6 or E7 protein), a subcellular localization element, and a CD4 help element.

A "subcellular localization element" refers to an element that effects the subcellular localization of the antigen, and a "CD4 help element" refers to an element that elicits a CD4+ T cell response in an animal. By "element" is meant a nucleic acid sequence that encodes a particular polypeptide (e.g., native, modified, full length, fragment, fusion, etc.) and/or the polypeptide encoded thereby.

A subcellular localization element may be any suitable length to effect the subcellular localization of an antigen. Subcellular localization elements such as signal peptides typically have a length of 15 to 40 amino acids (see, e.g., Nielsen H, Krogh A. Prediction of signal peptides and signal anchors by a hidden Markov model. Proc Int Conf Intell Syst Mol Biol. 1998;6: 122-30). For example, a subcellular localization element that is a signal peptide will generally comprise a minimum length of 5 amino acids and a maximum length of 120 amino acids.

A CD4 help element may be any suitable length to elicit a CD4+ T cell response in an animal. Individual CD4 help epitopes in CD4 help elements typically have a length of 13 to 25 amino acids (see, e.g., Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol. 2008 Apr 4;4(4):el000048). For example, a CD4 help epitope in a CD4 help element according to the invention will generally comprise a minimum length of 4 amino acids and a maximum length of 75 amino acids per epitope.

A "genetically-encoded vaccine" refers to any vaccine comprising nucleic acid that encodes an antigen. Examples of genetically-encoded vaccines include DNA vaccines and viral vaccines, such as adenoviral vaccines.

Typically, the antigen, subcellular localization element, and CD4 help element are expressed as a fusion protein from an expression cassette in a vector suitable for administration to an animal, such as a human. In particular embodiments, the vector is suitable for use as a genetically-encoded vaccine, such as a DNA vaccine or an adenovirus or alphavirus (e.g., Semliki Forest virus) or other virus-based vector vaccine. In other embodiments, the fusion protein of the invention is provided as a recombinant or synthetic protein or peptide.

The antigen employed in the vaccines of the instant invention can be any suitable substance that will elicit an immunogenic response to a target pathogenic agent, such as a human papillomavirus (HPV). As used herein, the terms "antigen" or "immunogen" are used interchangeably to refer to a substance, typically a protein or peptide, which is capable of inducing an immune response in a subject. The term also refers to proteins that are immunologically active in the sense that once administered to a subject (e.g., by

administering to the subject a nucleotide sequence or vector that encodes the protein) is able to evoke an immune response of the humoral and/or cellular type directed against that protein. Examples of immunogens include glycoproteins, polypeptides, peptides, epitopes or derivatives, e.g., fusion protein, that induce an immune response, preferably of a protective nature.

In certain embodiments, the antigen is a tumor-associated antigen. In other embodiments, the antigen is a viral antigen. In certain embodiments, the antigen employed in a vaccine of the invention is an antigen derived from an HPV, such as an HPV 16 protein, for example, HPV 16 E6 or E7. In other embodiments, the antigen is a bacterial antigen, a parasitic antigen, an disease-associated self-antigen, etc.

Subcellular localization elements of the invention can be any suitable substance that impacts and/or alters the subcellular localization of the antigen. In certain embodiments, the subcellular localization element exports the antigen out of the cytosol. For example, a subcellular localization element can be an import/retention signal that leads to the accumulation of the antigen in an organelle, such as the Golgi apparatus, an endosome, or the endoplasmic reticulum (ER). An example of a suitable localization element is a signal peptide, alone or combined with a retention signal, such as an ER retention signal such as KDEL. In other embodiments, the subcellular localization element is a carrier protein or part thereof, or is a fragment of a carrier protein (e.g., protein domain) or part thereof, that localizes the antigen to a particular organelle, such as the ER.

As used herein, a "self carrier protein" is produced by or is substantially similar to that produced by the target species for vaccination, such as a mammal (e.g., a human being). In certain embodiments, the subcellular localization element enhances the stability of the antigen.

CD4 help elements can be any suitable substance that elicits a CD4+ T cell response in an animal. Typically, a CD4 help element will comprise one or more epitopes that are recognized by CD4+ T cells. Examples of suitable epitopes include P30, PADRE, and NEF, as described herein. In certain embodiments, the CD4 help element comprises one or more promiscuous epitopes that are recognized by CD4+ T cells. In some embodiments, for example, where the CD4 help element comprises two or more CD4+ epitopes, to prevent the formation of neo-epitopes at the junctions, the epitopes can be separated by a non- immunogenic spacer sequence, such as glycine proline repeats. Spacer sequences that are suitable for preventing the formation of one or more neo-epitopes typically comprise a minimum of five amino acids and lack amino acid residues that fit best in the F pocket of MHC class I molecules. For example, suitable peptide sequences for preventing the formation of one or more neo-epitopes include peptide linkers containing a minimum of five amino acids such as G, P, A, and/or S residues. Such sequences are generally chosen to limit the likelihood of formation of novel CD8 epitopes at the junction of fused peptide domains. In other embodiments, the CD4 help element is a carrier protein or present in a larger protein or fragment thereof (e.g., peptide fragment comprising a CD4+ T cell epitope), such as a CD4+ T cell epitope from mycobacterial HSP 70, TTFC, HSV VP22, Pseudomonas aeruginose exototoxin A, and HSV glycoprotein D. Examples of suitable CD4 help elements include any minimal CD4 helper epitope, such as P23TT

(VSIDKFRIFCKANPK (SEQ ID NO: 7)), P32TT (LKFIIKRYTPNNEIDS (SEQ ID NO: 8)), P21TT (IREDNNITLKLDPvCNN (SEQ ID NO: 9)),

PfT3 (EK IAAKMEKAS S VFNVVN (SEQ ID NO: 10)), P2TT (QYIKANSKFIGITE (SEQ ID NO: 11)), HBVnc (PHHTALRQAILCWGELMTLA (SEQ ID NO: 12)), HA

(PKYVKQNTLKLAT (SEQ ID NO: 13)), HBsAg (FFLLTRILTIPQSLD (SEQ ID NO: 14)), and MT (YSGPLKAEIAQRLEDV (SEQ ID NO: 15)) (see, e.g., Baraldo K, et al. Infect. Immun. (2004) 72(8):4884-7 and Falugi F. Eur. J. Immunol_(2001)

Dec;31(12):3816-24, incorporated by reference herein).

Any of the modular components of a vaccine of the instant invention, e.g., the antigen, subcellular localization element, and/or the CD4 help element, can be arranged in an order that is most suitable for enhancing the expression and/or immunogenicity of the antigen in the subject to which the vaccine is administered. For example, in one embodiment, a DNA vaccine of the invention comprises an expression cassette wherein the subcellular localization element is fused N-terminal to the CD4 help element, and the CD4 help element is fused N-terminal to the antigen. In certain embodiments, the CD4 help element is fused N-terminal to the subcellular localization element, and the subcellular localization element is fused N-terminal to the antigen. In other embodiments, the antigen is fused C-terminal to the subcellular localization element and N-terminal to the CD-4 help element. In still other embodiments, the antigen is fused C-terminal to the subcellular localization element, and the subcellular localization elememt is fused C-terminal to the CD4 help element. In certain embodiments, the antigen is fused C-terminal to the CD4 element, and the CD4 element is fused C-terminal to the subcellular localization element. In other embodiments, the CD4 element is incorporated within the antigen or the localization element, or the localization element is split into two parts, such as a signal peptide and KDEL sequence.

In further embodiments, at least two subcellular localization elements are used in a vaccine of the invention. In some embodiments, at least two CD4 help elements are used in a vaccine of the invention. In other embodiments, a combination of at least one subcellular localization element and at least two CD4 help elements is used in a vaccine of the invention. In other embodiments, a combination of at least two subcellular localization elements and at least one CD4 help element is used in a vaccine of the invention. The modular nature of the vaccines of the instant invention allows for the arrangement of the one or more localization elements and one or more CD4 help elements in any suitable order.

The term "vaccine " as used herein refers to any composition containing an antigen, which composition can be used to prevent or treat a disease or condition in a subject.

As used herein, the terms "drug," "agent," and "compound" encompass any composition of matter or mixture which provides some pharmacologic effect that can be demonstrated in vivo or in vitro. This includes small molecules, antibodies,

microbiologicals, vaccines, vitamins, and other beneficial agents. As used herein, the terms further include any physiologically or pharmacologically active substance that produces a localized or systemic effect in a patient.

It has been noted that fusion of some carrier proteins results in increased, steady state levels of the antigen (16,18,21). Importantly, it has been shown that in vivo

accumulation of DNA vaccine-encoded antigens is required for the efficient induction of CD8+ T cell responses (26). The increased steady state levels of antigen are likely to lead to enhanced cross presentation, as proteasomal substrates rather than proteasomal products are subject of cross presentation (27). This mechanism might be especially relevant in case of HPV 16 E6 and E7 as both proteins are known to have a short half life (28,29). Finally, it has been noted that many of the fusions result in an altered sub cellular localization of the antigen (15,17,19,23,30,31). An altered sub cellular localization as such could also form an explanation for the improved immunogenicity as in some of these reports addition of only domains of proteins, or even only signal sequences has been shown to result in improved immunogenicity (21,32,33). The inventors hypothesize that it might be possible and sufficient to optimize HPV 16 E7- and E6-directed DNA vaccines, by using a combination of these more common mechanisms encoded by modular elements. Moreover, to determine the relative importance of these mechanisms, the inventors aimed to separate the different mechanisms in distinct building blocks. For this purpose, the inventors developed a modular DNA vaccine design comprising an antigen localization element and a separate element to provide CD4 help.

In short, as described herein, the inventors demonstrate that, the addition of an element providing CD4 help and an antigen localization element that affects subcellular localization and can, in some embodiments, also affect antigen stability, can independently improve the immunogenicity of a shuffled version of HPV 16 E7 (E7SH), albeit the effect of the addition of CD4 help was most pronounced. Interestingly the combination of the element providing CD4 help and the carrier protein had a synergistic effect. Furthermore, as described herein, the inventors demonstrate that the effect of the carrier protein can be explained in certain embodiments by ER re-localization of the antigen, suggesting an important role for ER localization in DNA vaccine immunogenicity. The resulting design rules also applied to HPV 16 E6SH. These novel vaccine candidates only contain minimal additional sequences besides the antigen, thereby minimizing the risk of antigenic competition and/or autoimmunity. Importantly the thus developed vaccine candidates strongly outperformed TTFC-E6SH and TTFC-E7SH that were developed in a previous study.

As described herein, the inventors rationally design DNA vaccines encoding HPV 16 E6 and E7. The resulting vaccine designs: sig-HELP-E6SH-KDEL and sig-HELP- E7SH-KDEL contain only minimal additional sequences apart from the antigen of interest, and induce extremely potent E6 and E7 specific CD8+ T cell responses. These results underscore the importance of the addition of CD4+ T cell help in DNA vaccination and suggest that the enhanced immunogenicity that is often observed after addition of a carrier molecule might be explained by ER localization of the fused antigen. The importance of CD4+ T cell help for the generation of effective CD8+ T cell responses has been well established (49). In the field of DNA vaccination, multiple strategies have been developed to provide CD4+ T cell help. The most commonly used method is to fuse a foreign protein towards the antigen (24,25). An extensively studied example is Tetanus Toxin fragment C that contains several promiscuous CD4+ helper epitopes (20,24). As mentioned, the provision of CD4+ T-cell help thus forms a likely explanation for the increased immunogenicity of HPV 16 E6 and E7 fusion vaccines in those cases where foreign proteins are used as carrier molecules. A drawback of using complete foreign proteins as carrier molecules is that they are likely to also contain competing CD8+ T cell epitopes. These might skew the CD8+ T cell response towards the carrier molecule by the principle of immunodominance (50). A more elegant strategy is to fuse the antigen to a version of invariant chain in which the CLIP peptide is replaced by a promiscuous CD4+ T cell epitope such as P30 or PADRE (24,51). A drawback of this approach, potentially limiting its clinical application, is that only a single CD4+ T cell epitope can be included. Furthermore there is a potential risk for inducing autoimmunity towards invariant chain. As described herein, the inventors are the first to show that CD4+ T cell help can be provided by fusing minimal CD4+ helper epitopes towards the antigen, using E6SH and E7SH as model antigens (see fig 2 and 5). The potential risk of the formation of neo-epitopes at the junctions can be avoided by the inclusion of spacers, such as the glycine proline repeats (40). The advantage of this method is that only minimal foreign sequences are added, thereby strongly decreasing the risk of antigenic competition. Without being bound to theory, Applicants hypothesize that this might explain why the instant approach also results in strong E6SH specific responses, whereas TTFC fusion that effectively enhanced the immunogenicity of E7SH, only moderately improved the immunogenicity of E6SH (20).

The finding that also genetic fusion of antigens to carrier molecules that are of self origin can improve DNA vaccine immunogenicity indicates that other mechanisms than the provision of CD4+ T cell help must play a role. Examples of such carriers that are shown to improve the immunogenicity of HPV 16 E6 and or E7 include calreticulin, HSP 60 and IP- 10. As mentioned above, the enhanced immunogenicity of these self carriers is often explained by the specific function of the carrier molecule. However, to the best of our knowledge, convincing proof that such carrier function-specific mechanisms indeed play a role is lacking. In the past we have demonstrated antigen stability can strongly affect its immunogenicity (26). We hypothesized that the improvement of the antigen stability could form a more general explanation for the so called "carrier-effect." On the other hand the self carrier molecules might influence other antigen properties, for example subcellular localization that could form an explanation for the "carrier effect." To our surprise, the experiment, where we compared the impact of 5 different self carrier molecules (FM4, H2B, ERP29, KRT14 and CD8a), on the immunogenicity of E7SH, showed no clear correlation between the extent of antigen accumulation (as a measure for antigen stability) and antigen immunogenicity (see fig 3). Remarkably, the only carrier molecules that provided an advantage over the addition of HELP alone were ER localized. Subsequently, we showed that the addition of an ER localization element sufficed to improve the immunogenicity of E7SH, HELP-E7SH and HELPE6SH. These data suggest that ER re- localization of the antigen could form a more general mechanism explaining the "carrier effect." As the accumulation of sig-E7SH-KDEL is strongly increased compared to that of the cytosolic E7SH (fig 4), without being bound to theory, Applicants hypothesize that in certain embodiments, ER localization protects the antigen from cytosolic degradation, thereby increasing protein half life and thus the opportunity to be cross presented; this would be in line with the previously reported importance of antigen stability (26). Freigang et al (52) made the observation that modifications affecting, among other things, ER localization of LCMV glycoprotein strongly enhanced its immunogenicity in a cross- presentation dependent setting, and speculated on several mechanisms that could lead to enhanced cross presentation. Firstly, it might be that upon cell death, ER localized antigens might be enclosed within ER-derived membranes/vesicles that protect it from degradation in the extracellular milieu. Secondly, slight folding defects might lead to re-association with ER chaperones that could lead to enhanced delivery to APC's (53). Thirdly, the destruction of (misfolded) proteins in the ER is known to be mediated via ERAD, possibly yielding higher numbers or qualitatively different (54) antigenic peptides. Finally, the accumulation of misfolded proteins in the ER might induce the ER-stress response, thereby triggering the transfected cells to undergo apoptosis leading to the release of the antigen in a way that it can be used by the immune system as a source of antigen for cross-presentation (47,55). This latter hypothesis is unlikely in view of our experiment where we separated the carrier (FM4) and the antigen (HELP-E7SH) by use of the 2A linker (Figure 7). This construct, named FM4-2A-HELP-E7SH, can be expected to trigger ER stress, but was not more immunogenic than HELP-E7SH, indicating the need for ER localization of the antigen per se. Taken together, our data clearly demonstrate the impact of ER localization on the immunogenicity of DNA vaccine-encoded antigens. An important observation in our current study is that the combination of the addition of CD4+ T cell help and ER targeting has a strong synergistic effect: sig-E7SH-KDEL yields a peak response of 3.10 ± 1.63 % and HELP E7SH yields a peak response of 7.62 ± 3.84, the combination (sig-HELP-E7SH-KDEL), however, yielded a peak response of 19.38 ± 8.32% Notably, the immunogenicity of sig-HELP-E7SHKDEL was hardly affected by reducing the number of vaccinations or by a 5 fold reduction in DNA dose, whereas the immunogenicity of both HELP-E7SH and TTFC-E7SH was strongly reduced under these conditions. The finding that the vaccine dose can be lowered without a significant effect on potency may be relevant for clinical translation: the inability to scale DNA doses used in mice to humans is considered as one of the main explanations for the lower efficacy of DNA vaccination in humans compared to small animals (56). Furthermore, we

demonstrated functional superiority of sig-HELP-E6SH-KDEL and sig-HELP-E7SH-KDEL compared to TTFC-E6SH and TTFC-E7SH, respectively, in a tumor treatment experiment using the TC-1 model. These data clearly demonstrate the enormous potential of optimizing DNA vaccine immunogenicity by alterations in transgene design.

In certain embodiments, a DNA vaccine of the invention is administered in combination with DNA encoding for adjuvant molecules (57), or in combination with delivery methods such as electroporation (58).

In a particular embodiment, the instant invention provides highly effective DNA vaccines for the treatment of HPV 16 positive malignancies. The resulting candidate vaccines, sig-HELP-E6SH-KDEL and sigHELP-E7SHKDEL, contain only minimal additional sequences apart from the antigen, thereby limiting the risk of induction of autoimmunity and/or antigenic competition. These results form a powerful illustration of the enormous impact of antigen design on DNA vaccine immunogenicity.

Nucleic Acids, Proteins, and Recombinant Technology

The present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A

Practical Approach, Irl Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is herein incorporated by reference.

The term "nucleic acid" encompasses DNA, RNA (e.g., mRNA, tRNA),

heteroduplexes, and synthetic molecules capable of encoding a polypeptide and includes all analogs and backbone substitutes such as PNA that one of ordinary skill in the art would recognize as capable of substituting for naturally occurring nucleotides and backbones thereof. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences which encode a particular amino acid sequence.

Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide," "protein," and "peptide," and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an "enzyme." The conventional one-letter or three-letter code for amino acid residues are used herein.

As used herein, a "synthetic" molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.

As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

A "gene" refers to the DNA segment encoding a polypeptide or RNA.

An "isolated" polynucleotide or polypeptide is one that is substantially free of the materials with which it is associated in its native environment. By substantially free, is meant at least 50%, advantageously at least 70%, more advantageously at least 80%>, and even more advantageously at least 90% free of these materials.

An "isolated" nucleic acid molecule is a nucleic acid molecule separate and discrete from the whole organism with which the molecule is found in nature; or a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith.

"Native" proteins or polypeptides refer to proteins or polypeptides isolated from the source in which the proteins naturally occur. "Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; e.g., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. "Synthetic" polypeptides include those prepared by chemical synthesis as well as the synthetic antigens described above.

By "homolog" is meant an entity having a certain degree of identity with the subject amino acid sequences and the subject nucleotide sequences. As used herein, the term "homolog" covers identity with respect to structure and/or function, for example, the expression product of the resultant nucleotide sequence has the enzymatic activity of a subject amino acid sequence. With respect to sequence identity, preferably there is at least 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%), 96%o, 97%), 98%o, or even 99% sequence identity. These terms also encompass allelic variations of the sequences. The term, homolog, may apply to the relationship between genes separated by the event of speciation or to the relationship between genes separated by the event of genetic duplication.

Relative sequence identity can be determined by commercially available computer programs that can calculate % identity between two or more sequences using any suitable algorithm for determining identity, using, for example, default parameters. A typical example of such a computer program is CLUSTAL. Advantageously, the BLAST algorithm is employed, with parameters set to default values. The BLAST algorithm is described in detail on the National Center for Biotechnology Information (NCBI) website.

The homologs of the peptides as provided herein typically have structural similarity with such peptides. A homolog of a polypeptide includes one or more conservative amino acid substitutions, which may be selected from the same or different members of the class to which the amino acid belongs.

In one embodiment, the sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the secondary binding activity of the substance is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

The present invention also encompasses conservative substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue with an alternative residue) that may occur e.g., like-for-like substitution such as basic for basic, acidic for acidic, polar for polar, etc. Non-conservative substitution may also occur e.g., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine and phenylglycine. Conservative substitutions that may be made are, for example, within the groups of basic amino acids (Arginine, Lysine and

Histidine), acidic amino acids (glutamic acid and aspartic acid), aliphatic amino acids

(Alanine, Valine, Leucine, Isoleucine), polar amino acids (Glutamine, Asparagine, Serine, Threonine), aromatic amino acids (Phenylalanine, Tryptophan and Tyrosine), hydroxyl amino acids (Serine, Threonine), large amino acids (Phenylalanine and Tryptophan) and small amino acids (Glycine, Alanine).

Many methods of amplifying DNA are known in the art, and any such method can be used, see for example Sambrook et al., Molecular Cloning; A Laboratory Manual 2d ed. (1989). For example, a DNA fragment of interest can be amplified using the polymerase chain reaction or some other cyclic polymerase mediated amplification reaction.

The amplified region of DNA can then be sequenced using any method known in the art. Advantageously, the nucleic acid sequencing is by automated methods (reviewed by Meldrum, Genome Res. September 2000;10(9): 1288-303, the disclosure of which is incorporated by reference in its entirety), for example using a Beckman CEQ 8000 Genetic Analysis System (Beckman Coulter Instruments, Inc.). Methods for sequencing nucleic acids include, but are not limited to, automated fluorescent DNA sequencing (see, e.g., Watts & MacBeath, Methods Mol Biol. 2001;167: 153-70 and MacBeath et al, Methods Mol Biol. 2001;167: 119-52), capillary electrophoresis (see, e.g., Bosserhoff et al, Comb Chem High Throughput Screen. December 2000;3(6):455-66), DNA sequencing chips (see, e.g., Jain, Pharmacogenomics. August 2000;l(3):289-307), mass spectrometry (see, e.g., Yates, Trends Genet. January 2000;16(l):5-8), pyrosequencing (see, e.g., Ronaghi, Genome Res. January 2001;11(1):3-11), and ultrathin-layer gel electrophoresis (see, e.g., Guttman &

Ronai, Electrophoresis. December 2000; 21 (18):3952-64), the disclosures of which are hereby incorporated by reference in their entireties. The sequencing can also be done by any commercial company. Examples of such companies include, but are not limited to, the University of Georgia Molecular Genetics Instrumentation Facility (Athens, Ga.) or SeqWright DNA Technologies Services (Houston, Tex.).

Any one of the methods known in the art for amplification of DNA may be used, such as for example, the polymerase chain reaction (PCR), the ligase chain reaction (LCR) (Barany, F., Proc. Natl. Acad. Sci. (U.S.A.) 88: 189-193 (1991)), the strand displacement assay (SDA), or the oligonucleotide ligation assay ("OLA") (Landegren, U. et al, Science 241 : 1077-1080 (1988)). Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990)). Other known nucleic acid amplification procedures, such as transcription-based amplification systems (Malek, L. T. et al., U.S. Pat. No.

5,130,238; Davey, C. et al, European Patent Application 329,822; Schuster et al, U.S. Pat. No. 5,169,766; Miller, H. I. et al, PCT Application W089/06700; Kwoh, D. et al, Proc. Natl. Acad. Sci. (U.S.A.) 86: 1173 (1989); Gingeras, T. R. et al, PCT Application

W088/10315)), or isothermal amplification methods (Walker, G. T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)) may also be used.

To perform a cyclic polymerase mediated amplification reaction according to the present invention, the primers are hybridized or annealed to opposite strands of the target DNA, the temperature is then raised to permit the thermostable DNA polymerase to extend the primers and thus replicate the specific segment of DNA spanning the region between the two primers. Then the reaction is thermocycled so that at each cycle the amount of DNA representing the sequences between the two primers is doubled, and specific amplification of gene DNA sequences, if present, results.

Any of a variety of polymerases can be used in the present invention. For thermocyclic reactions, the polymerases are thermostable polymerases such as Taq, KlenTaq, Stoffel Fragment, Deep Vent, Tth, Pfu, Vent, and UlTma, each of which are readily available from commercial sources. For non-thermocyclic reactions, and in certain thermocyclic reactions, the polymerase will often be one of many polymerases commonly used in the field, and commercially available, such as DNA pol 1 , Klenow fragment, T7 DNA polymerase, and T4 DNA polymerase. Guidance for the use of such polymerases can readily be found in product literature and in general molecular biology guides. Typically, the annealing of the primers to the target DNA sequence is carried out for about 2 minutes at about 37-55° C, extension of the primer sequence by the polymerase enzyme (such as Taq polymerase) in the presence of nucleoside triphosphates is carried out for about 3 minutes at about 70-75° C, and the denaturing step to release the extended primer is carried out for about 1 minute at about 90-95° C. However, these parameters can be varied, and one of skill in the art would readily know how to adjust the temperature and time parameters of the reaction to achieve the desired results. For example, cycles may be as short as 10, 8, 6, 5, 4.5, 4, 2, 1, 0.5 minutes or less.

Also, "two temperature" techniques can be used where the annealing and extension steps may both be carried out at the same temperature, typically between about 60-65° C, thus reducing the length of each amplification cycle and resulting in a shorter assay time.

Typically, the reactions described herein are repeated until a detectable amount of product is generated. Often, such detectable amounts of product are between about 10 ng and about 100 ng, although larger quantities, e.g. 200 ng, 500 ng, 1 mg or more can also, of course, be detected. In terms of concentration, the amount of detectable product can be from about 0.01 pmol, 0.1 pmol, 1 pmol, 10 pmol, or more. Thus, the number of cycles of the reaction that are performed can be varied, the more cycles are performed, the more amplified product is produced. In certain embodiments, the reaction comprises 2, 5, 10, 15, 20, 30, 40, 50, or more cycles.

For example, the PCR reaction may be carried out using about 25-50 μΐ samples containing about 0.01 to 1.0 ng of template amplification sequence, about 10 to 100 pmol of each generic primer, about 1.5 units of Taq DNA polymerase (Promega Corp.), about 0.2 mM dDATP, about 0.2 mM dCTP, about 0.2 mM dGTP, about 0.2 mM dTTP, about 15 mM MgCl.sub.2, about 10 mM Tris-HCl (pH 9.0), about 50 mM KC1, about 1 μg/ml gelatin, and about 10 μΐ/ml Triton X-100 (Saiki, 1988).

Those of ordinary skill in the art are aware of the variety of nucleotides available for use in the cyclic polymerase mediated reactions. Typically, the nucleotides will consist at least in part of deoxynucleotide triphosphates (dNTPs), which are readily commercially available. Parameters for optimal use of dNTPs are also known to those of skill, and are described in the literature. In addition, a large number of nucleotide derivatives are known to those of skill and can be used in the present reaction. Such derivatives include fluorescently labeled nucleotides, allowing the detection of the product including such labeled nucleotides, as described below. Also included in this group are nucleotides that allow the sequencing of nucleic acids including such nucleotides, such as chain-terminating nucleotides, dideoxynucleotides and boronated nuclease-resistant nucleotides. Commercial kits containing the reagents most typically used for these methods of DNA sequencing are available and widely used. Other nucleotide analogs include nucleotides with bromo-, iodo- , or other modifying groups, which affect numerous properties of resulting nucleic acids including their antigenicity, their replicatability, their melting temperatures, their binding properties, etc. In addition, certain nucleotides include reactive side groups, such as sulfhydryl groups, amino groups, N-hydroxysuccinimidyl groups, that allow the further modification of nucleic acids comprising them.

The term "oligonucleotide" is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use for the method. In certain embodiments, oligonucleotides that can be used as primers to amplify specific nucleic acid sequences of a gene in cyclic polymerase-mediated amplification reactions, such as PCR reactions, consist of oligonucleotide fragments. Such fragments should be of sufficient length to enable specific annealing or hybridization to the nucleic acid sample. The sequences typically will be about 8 to about 44 nucleotides in length, but may be longer. Longer sequences, e.g., from about 14 to about 50, are advantageous for certain embodiments.

In embodiments where it is desired to amplify a fragment of DNA, primers having contiguous stretches of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides from a gene sequence are contemplated.

As used herein, "hybridization" refers to the process by which one strand of nucleic acid base pairs with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Whichever probe sequences and hybridization methods are used, one ordinarily skilled in the art can readily determine suitable hybridization conditions, such as temperature and chemical conditions. Such hybridization methods are well known in the art. For example, for applications requiring high selectivity, one will typically desire to employ relatively stringent conditions for the hybridization reactions, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by about 0.02 M to about 0.10 M NaCl at temperatures of about 50° C to about 70° C. Such high stringency conditions tolerate little, if any, mismatch between the probe and the template or target strand. It is generally appreciated that conditions can be rendered more stringent by the addition of increasing amounts of formamide. Other variations in hybridization reaction conditions are well known in the art (see for example, Sambrook et al., Molecular Cloning; A Laboratory Manual 2d ed. (1989)).

Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught, e.g., in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol 152, Academic Press, San Diego CA), and confer a defined "stringency" as explained below.

Maximum stringency typically occurs at about Tm-5 °C (5 °C below the Tm of the probe); high stringency at about 5 °C to 10 °C below Tm; intermediate stringency at about 10 °C to 20 °C below Tm; and low stringency at about 20 °C to 25 °C below Tm. As will be understood by those of ordinary skill in the art, a maximum stringency hybridization can be used to identify or detect identical nucleotide sequences while an intermediate (or low) stringency hybridization can be used to identify or detect similar or related polynucleotide sequences.

In one aspect, the present invention employs nucleotide sequences that can hybridize to another nucleotide sequence under stringent conditions (e.g., 65 °C and O.lxSSC {IxSSC = 0.15 M NaCl, 0.015 M Na3 Citrate pH 7.0). Where the nucleotide sequence is double- stranded, both strands of the duplex, either individually or in combination, may be employed by the present invention. Where the nucleotide sequence is single-stranded, it is to be understood that the complementary sequence of that nucleotide sequence is also included within the scope of the present invention.

Stringency of hybridization refers to conditions under which polynucleic acid hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of ordinary skill in the art, the stability of hybrids is reflected in the melting temperature (Tm) of the hybrid which decreases approximately 1 to 1.5 °C with every 1 % decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is performed under conditions of higher stringency, followed by washes of varying stringency.

As used herein, high stringency includes conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 1 M Na+ at 65-68 °C. High stringency conditions can be provided, for example, by hybridization in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na+

pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non-specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 minutes) at the hybridization temperature in 0.2 - O.lx SSC, 0.1 % SDS.

It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g., formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of ordinary skill in the art as are other suitable hybridization buffers (see, e.g., Sambrook, et al, eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al, eds. (1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridization conditions are typically determined empirically, as the length and the GC content of the hybridizing pair also play a role.

Nucleic acid molecules that differ from the sequences of the primers and probes disclosed herein, are intended to be within the scope of the invention. Nucleic acid sequences that are complementary to these sequences, or that are hybridizable to the sequences described herein under conditions of standard or stringent hybridization, and also analogs and derivatives are also intended to be within the scope of the invention.

Advantageously, such variations will differ from the sequences described herein by only a small number of nucleotides, for example by 1, 2, or 3 nucleotides.

Nucleic acid molecules corresponding to natural allelic variants, homologues (i.e., nucleic acids derived from other species), or other related sequences (e.g., paralogs) of the sequences described herein can be isolated based on their homology to the nucleic acids disclosed herein, for example by performing standard or stringent hybridization reactions using all or a portion of the known sequences as probes. Such methods for nucleic acid hybridization and cloning are well known in the art.

Similarly, a nucleic acid molecule detected in the methods of the invention may include only a fragment of the specific sequences described. Fragments provided herein are defined as sequences of at least 6 (contiguous) nucleic acids, a length sufficient to allow for specific hybridization of nucleic acid primers or probes, and are at most some portion less than a full-length sequence. Fragments may be derived from any contiguous portion of a nucleic acid sequence of choice. Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.

Derivatives, analogs, homologues, and variants of the nucleic acids of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids of the invention, in various embodiments, by at least about 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or even 99% identity over a nucleic acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art.

For the purposes of the present invention, sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990;87: 2264-2268, modified as in Karlin &

Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.

Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988;85: 2444-2448.

Advantageous for use according to the present invention is the WU-BLAST

(Washington University BLAST) version 2.0 software. WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from

ftp://blast.wustl.edu/blast/executables. This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al, Journal of Molecular Biology 1990;215: 403-410; Gish & States, 1993;Nature Genetics 3: 266-272; Karlin & Altschul, 1993;Proc. Natl. Acad. Sci. USA 90:

5873-5877; all of which are incorporated by reference herein).

In all search programs in the suite the gapped alignment routines are integral to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.

Alternatively or additionally, the term "homology" or "identity", for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (NrefNdif)* 100/- N_ref, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein N_ref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (N N_ref =8; N N_dif =2). "Homology" or "identity" can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur & Lipman, Proc Natl Acad Sci USA 1983;80:726, incorporated herein by reference), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics.TM. Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences. Without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.

As regards codon optimization, the nucleic acid molecules of the invention have a nucleotide sequence that encodes the antigens of the invention and can be designed to employ codons that are used in the genes of the subject in which the antigen is to be produced. Such methods, and the selection of such methods, are well known to those of skill in the art. In addition, there are several companies that will optimize codons of sequences, such as Geneart (geneart(dot)com). Thus, the nucleotide sequences of the invention can readily be codon optimized.

The nucleotide sequences of the present invention may be inserted into vectors. The term "vector" is widely used and understood by those of ordinary skill in the art, and as used herein the term "vector" is used consistent with its meaning to those of ordinary skill in the art. For example, the term "vector" is commonly used by those ordinarily skilled in the art to refer to a vehicle that allows or facilitates the transfer of nucleic acid molecules from one environment to another or that allows or facilitates the manipulation of a nucleic acid molecule.

For example, a vector is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. An "origin of replication" refers to those DNA sequences that participate in DNA synthesis. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "operably linked" and "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. When the polynucleotide encodes a polyprotein fragment,

advantageously, in the vector, an initiation codon (ATG) is placed at 5 ' of the reading frame and a stop codon is placed at 3'. Other elements for controlling expression may be present, such as enhancer sequences, stabilizing sequences and signal sequences permitting the secretion of the protein. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

Any vector that allows expression of the immunogens of the present invention may be used in accordance with the present invention. In certain embodiments, the immunogens of the present invention may be used in vitro (such as using cell-free expression systems) and/or in cultured cells grown in vitro. For such applications, any vector that allows expression of the immunogens in vitro and/or in cultured cells may be used.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A "cDNA" is defined as copy- DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, ribosome binding sites, upstream regulatory domains, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A "cis-element" is a nucleotide sequence, also termed a "consensus sequence" or "motif, that interacts with other proteins which can upregulate or downregulate expression of a specific gene locus. A "signal sequence" can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes. Not all of these control sequences need always be present in a recombinant vector so long as the desired gene is capable of being transcribed and translated.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. The promoter sequence is typically bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to enzymes which cut double-stranded DNA at or near a specific nucleotide sequence.

"Recombinant DNA technology" refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include "gene splicing", "molecular cloning" and "genetic engineering". The product of these manipulations results in a "recombinant" or

"recombinant molecule".

A cell has been "transformed" or "transfected" with exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed "transgenic".

As used herein, the term "host" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells and plant cells, such as

Arabidopsis thaliana and Tobaccum nicotiana. A number of mammalian cell lines are known in the art and include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Mandin-Darby bovine kidney ("MDBK") cells, as well as others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs. Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans,

Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica. Insert hosts useful in the present invention include, but are not limited to, Spodoptera frugiperda cells.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, the coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. For example, a polynucleotide, may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.

As used herein, "fragment" or "portion" as applied to a gene or a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments of these genes can be generated by methods known to those skilled in the art, e.g., by restriction digestion of naturally occurring or recombinant fiber or fibritin genes, by recombinant DNA techniques using a vector that encodes a defined fragment of the fiber or fibritin gene, or by chemical synthesis.

Methods for making and/or administering a vector or recombinants or plasmid for expression of gene products of genes of the invention either in vivo or in vitro can be any desired method, e.g., a method which is by or analogous to the methods disclosed in, or disclosed in documents cited in: U.S. Patent Nos. 4,603,112; 4,769,330; 4,394,448;

4,722,848; 4,745,051; 4,769,331; 4,945,050; 5,494,807; 5,514,375; 5,744,140; 5,744,141; 5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 5,591,639; 5,589,466; 5,677,178; 5,591,439; 5,552,143; 5,580,859; 6,130,066; 6,004,777; 6,130,066; 6,497,883; 6,464,984; 6,451,770; 6,391,314; 6,387,376; 6,376,473; 6,368,603; 6,348,196; 6,306,400; 6,228,846; 6,221,362; 6,217,883; 6,207,166; 6,207,165; 6,159,477; 6,153,199; 6,090,393; 6,074,649; 6,045,803; 6,033,670; 6,485,729; 6,103,526; 6,224,882; 6,312,682; 6,348,450 and 6; 312,683; U.S. patent application Serial No. 920,197, filed October 16,1986; WO 90/01543; W091/11525; WO 94/16716; WO 96/39491; WO

98/33510; EP 265785; EP 0 370 573; Andreansky et al, Proc. Natl. Acad. Sci. USA

1996;93: 11313-11318; Ballay et al, EMBO J. 1993;4:3861-65; Feigner et al, J. Biol.

Chem. 1994;269:2550-2561; Frolov et al, Proc. Natl. Acad. Sci. USA 1996;93: 11371- 11377; Graham, Tibtech 1990;8:85-87; Grunhaus et al, Sem. Virol. 1992;3:237-52; Ju et al, Diabetologia 1998;41 :736-739; Kitson et al, J. Virol. 1991;65:3068-3075; McClements et al, Proc. Natl. Acad. Sci. USA 1996;93: 11414-11420; Moss, Proc. Natl. Acad. Sci. USA 1996;93: 11341-11348; Paoletti, Proc. Natl. Acad. Sci. USA 1996;93: 11349-11353;

Pennock et al, Mol. Cell. Biol. 1984;4:399-406; Richardson (Ed), Methods in Molecular Biology 1995;39, "Baculovirus Expression Protocols," Humana Press Inc.; Smith et al. (1983) Mol. Cell. Biol. 1983;3:2156-2165; Robertson et al, Proc. Natl. Acad. Sci. USA 1996;93: 11334-11340; Robinson et al. Sem. Immunol. 1997;9:271; and Roizman, Proc. Natl. Acad. Sci. USA 1996;93: 11307-11312.

The invention also provides for transformed host cells comprising a vector of the invention. In one embodiment, the vector is introduced into the cell by transfection, electroporation or infection. The invention also provides for a method for preparing a transformed cell expressing an immunogen of the present invention comprising transfecting, electroporating or infecting a cell with an expression vector (e.g., a DNA vaccine) to produce an infected producing cell and maintaining the host cell under biological conditions sufficient for expression of the immunogen in the host cell.

According to another embodiment of the invention, the expression vectors are expression vectors used for the in vitro expression of proteins in an appropriate cell system. The expressed proteins can be harvested in or from the culture supernatant after, or not after secretion (if there is no secretion a cell lysis typically occurs or is performed), optionally concentrated by concentration methods such as ultrafiltration and/or purified by purification means, such as affinity, ion exchange or gel filtration-type chromatography methods.

It is understood to one of skill in the art that conditions for culturing a host cell varies according to the particular gene and that routine experimentation is necessary at times to determine the optimal conditions for culturing the vector depending on the host cell. A "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof.

Polynucleotides comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Polynucleotides can be introduced into host cells by any means known in the art. The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means as described above, including direct uptake, endocytosis, transfection, f-mating, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is infectious, for instance, a retroviral vector). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

For applications where it is desired that the immunogens be expressed in vivo, for example when the immunogens of the invention are used in DNA or DNA-containing vaccines, any vector that allows for the expression of the immunogens of the present invention and is safe for use in vivo may be used. In preferred embodiments the vectors used are safe for use in humans, mammals and/or laboratory animals.

The vectors used in accordance with the present invention should typically be chosen such that they contain a suitable gene regulatory region, such as a promoter or enhancer, such that the immunogens of the invention can be expressed.

For example, when the aim is to express the immunogens of the invention in vitro, or in cultured cells, or in any prokaryotic or eukaryotic system for the purpose of producing the protein(s) encoded by that immunogen, then any suitable vector can be used depending on the application. For example, plasmids, viral vectors, bacterial vectors, protozoal vectors, insect vectors, baculovirus expression vectors, yeast vectors, mammalian cell vectors, and the like, can be used. Suitable vectors can be selected by the skilled artisan taking into consideration the characteristics of the vector and the requirements for expressing the immunogens under the identified circumstances.

When the aim is to express the immunogens of the invention in vivo in a subject, for example in order to generate an immune response against an HPV antigen and/or protective or therapeutic immunity against an HPV-induced cancer, expression vectors that are suitable for expression on that subject, and that are safe for use in vivo, should be chosen. For example, in some embodiments it may be desired to express the immunogens of the invention in a laboratory animal, such as for pre-clinical testing of HPV immunogenic compositions and vaccines of the invention. In other embodiments, it will be desirable to express the immunogens of the invention in human subjects, such as in clinical trials and for actual clinical use of the immunogenic compositions and vaccine of the invention. Any vectors that are suitable for such uses can be employed, and it is well within the capabilities of the skilled artisan to select a suitable vector. In some embodiments it may be preferred that the vectors used for these in vivo applications be attenuated to prevent vector from amplifying in the subject. For example, if plasmid vectors are used, preferably they will lack an origin of replication that functions in the subject so as to enhance safety for in vivo use in the subject. If viral vectors are used, preferably they are attenuated or replication- defective in the subject, again, so as to enhance safety for in vivo use in the subject.

Any vector suitable for administration as a vaccine may be employed in the instant invention. In certain embodiments of the instant invention, vectors suitable for use as DNA vaccines are used, such as pVAX and pcDNA vectors (Invitrogen).

In other embodiments of the present invention, viral vectors are used. Viral expression vectors are well known to those skilled in the art and include, for example, viruses such as adenoviruses (e.g., adenovirus subtypes Ad5, Adl 1, Ad26, Ad35, Ad48 and Ad49), adeno-associated viruses (AAV), alphaviruses, retroviruses and poxviruses, including avipox viruses, attenuated poxviruses, and vaccinia viruses, such as the modified vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566). In certain embodiments, a vaccine of the invention comprises an adenovirus selected from Ad5, Adl 1 , Ad26, Ad35, Ad48 and Ad49. Such viruses, when used as expression vectors are innately non-pathogenic in the selected subjects such as humans or have been modified to render them nonpathogenic in the selected subjects. For example, replication-defective adenoviruses and alphaviruses are well known and can be used as gene delivery vectors.

Following expression, the antigens of the invention can be isolated and/or purified or concentrated using any suitable technique known in the art. For example, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immuno-affinity chromatography,

hydroxyapatite chromatography, lectin chromatography, molecular sieve chromatography, isoelectric focusing, gel electrophoresis, or any other suitable method or combination of methods can be used.

In certain embodiments, the nucleotide sequences and/or antigens of the invention are administered in vivo, for example where the aim is to produce an immunogenic response in a subject. A "subject" in the context of the present invention may be any animal. For example, in some embodiments it may be desired to express the immunogens of the invention in a laboratory animal, such as for pre-clinical testing of HPV immunogenic compositions and vaccines of the invention. In other embodiments, it will be desirable to express the immunogens of the invention in human subjects, such as in clinical trials and for actual clinical use of the immunogenic compositions and vaccine of the invention. In certain embodiments the subject is a human, for example a human that is infected with, or is at risk of infection with, an HPV.

For such in vivo applications the nucleotide sequences and/or antigens of the invention are preferably administered as a component of an immunogenic composition comprising the nucleotide sequences and/or antigens of the invention in admixture with a pharmaceutically acceptable carrier. The immunogenic compositions of the invention are useful to stimulate an immune response against HPV and HPV-induced cancers and may be used as one or more components of a prophylactic or therapeutic vaccine against HPV for the prevention, amelioration or treatment of HPV and/or an HPV-induced cancer. The nucleic acids and vectors of the invention are particularly useful for providing genetic vaccines, i.e., vaccines for delivering the nucleic acids encoding the antigens of the invention to a subject, such as a human, such that the antigens are then expressed in the subject to elicit an immune response.

Immunogenic Compositions

The term "immunogenic protein or peptide" as used herein also includes peptides and polypeptides that are immunologically active in the sense that once administered to the host, it is able to evoke an immune response of the humoral and/or cellular type directed against the protein. Preferably the protein fragment is such that it has substantially the same immunological activity as the total protein. Thus, a protein fragment according to the invention comprises at least one epitope or antigenic determinant. The term epitope relates to a protein site able to induce an immune reaction of the humoral type (B cells) and/or cellular type (T cells).

The term "immunogenic protein or peptide" further contemplates deletions, additions and substitutions to the sequence, so long as the polypeptide functions to produce an immunological response as defined herein.

The term "epitope" refers to the site on an antigen or hapten to which specific B cells and/or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site". Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

An "immunological response" to a composition or vaccine is the development in the host of a cellular and/or antibody-mediated immune response to a composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor-T cells, and/or cytotoxic T cells and/or γδ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

Generation of an immunological response may involve antigen presenting cells (APCs). APCs may be "professional" antigen presenting cells or may be another cell that may be induced to present antigen to T cells. APCs include dendritic cells (DCs) such as interdigitating DCs or follicular DCs, Langerhans cells, PBMCs, macrophages, B- lymphocytes, or other cell types such as epithelial cells, fibroblasts or endothelial cells, activated or engineered by transfection to express a MHC molecule (Class I or II) on their surfaces. APCs also include hybridomas, lymphomas, and synthetic APCs such as lipid membranes. Precursors of APCs include CD34+ cells, monocytes, fibroblasts and endothelial cells. Cytokine genes which may promote immune potentiation include IL-2, IL-12, IFN-γ, TNF-a, IL-18,etc. Such proteins include MHC molecules (Class I or Class II), CD80, CD86, or CD40. Examples of T cells include helper T cells (CD4+) and CD8+ cells.

The terms "immunogenic" protein or polypeptide as used herein also refers to an amino acid sequence which elicits an immunological response as described above. An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the protein, analogs thereof, or immunogenic fragments thereof. By "immunogenic fragment" is meant a fragment of a protein which includes one or more epitopes and thus elicits the immunological response described above. Such fragments can be identified using any number of epitope mapping techniques, well known in the art. See, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa, N.J. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81 :3998-4002; Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated herein by reference in their entireties. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, supra.

Synthetic antigens are also included within the definition, for example, polyepitopes, flanking epitopes, and other recombinant or synthetically derived antigens. See, e.g., Bergmann et al. (1993) Eur. J. Immunol. 23:2777-2781; Bergmann et al. (1996) J. Immunol. 157:3242-3249; Suhrbier, A. (1997) Immunol, and Cell Biol. 75:402-408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, Jun. 28-Jul. 3, 1998.

Immunogenic fragments, for purposes of the present invention, will usually include at least about 3 amino acids, at least about 5 amino acids, at least about 10-15 amino acids, or at least about 25 or more amino acids, of the molecule. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising at least one epitope of the protein.

As mentioned earlier, epitope determination procedures, such as, generating overlapping peptide libraries (Hemmer B. et al, Immunology Today, 1998, 19 (4), 163- 168), Pepscan (Geysen et al, (1984) Proc. Nat. Acad. Sci. USA, 81, 3998-4002; Geysen et al, (1985) Proc. Nat. Acad. Sci. USA, 82, 178-182; Van der Zee R. et al, (1989) Eur. J. Immunol, 19, 43-47; Geysen H.M., (1990) Southeast Asian J. Trop. Med. Public Health, 21, 523-533; Multipin.RTM. Peptide Synthesis Kits de Chiron) and algorithms (De Groot A. et al, (1999) Nature Biotechnology, 17, 533-561), and in PCT Application Serial No. PCT/US2004/022605 all of which are incorporated herein by reference in their entireties, can be used in the practice of the invention, without undue experimentation. Other documents cited and incorporated herein may also be consulted for methods for determining epitopes of an immunogen or antigen and thus nucleic acid molecules that encode such epitopes.

According to the invention, in certain embodiments, administration of a vaccine of the invention can be combined with other vaccinations within the framework of vaccination programs, in the form of immunization or vaccination kits or methods, or in the form of multivalent immunogenic compositions and multivalent vaccines, e.g., comprising at least one vaccine component against a target pathogenic agent, such as HPV, and at least one vaccine component against at least one other pathogenic agent. This also includes the expression by the same expression vector of genes of at least two pathogenic agents, including, e.g., HPV 16 E6 and/or E7.

The invention thus also relates to a multivalent or "cocktail" immunogenic composition or a multivalent or "cocktail" vaccine against a target pathogenic agent, such as HPV, and against at least one other pathogen of the target species, using the same in vivo expression vector containing and expressing at least one polynucleotide of the target pathogenic agent, such as HPV 16 E6 and/or E7, according to the invention and at least one polynucleotide expressing an immunogen of another pathogen

As discussed herein, these multivalent compositions or vaccines can also comprise a pharmaceutically acceptable carrier or vehicle or excipient, and optionally an adjuvant.

The immunogenic compositions or vaccines as discussed herein can also be combined with at least one conventional vaccine (e.g., inactivated, live attenuated, or subunit) directed against the same pathogen or at least one other pathogen of the species to which the composition or vaccine is directed. The immunogenic compositions or vaccines discussed herein can be administered prior to or after the conventional vaccine, e.g., in a "prime -boost" regimen.

Formulations

The compositions of the invention can include any pharmaceutically acceptable carrier known in the art.

To facilitate the administration of a vaccine of the invention, the vaccine can be formulated into suitable pharmaceutical compositions. Generally, such compositions include the active ingredient (e.g., a DNA vaccine) and a pharmacologically acceptable carrier. Such compositions can be suitable for delivery of the active ingredient to a patient for medical application, and can be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more pharmacologically or physiologically acceptable carriers comprising excipients, as well as optional auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

Thus, for injection, the active ingredient can be formulated in aqueous solutions, preferably in physiologically compatible buffers. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For oral administration, the active ingredient can be combined with carriers suitable for inclusion into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For administration by inhalation, the active ingredient is conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant. The active ingredient can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Such compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Other pharmacological excipients are known in the art.

The compositions of the invention may be injectable suspensions, solutions, sprays, lyophilized powders, syrups, elixirs and the like. Any suitable form of composition may be used. To prepare such a composition, a nucleic acid or vector of the invention, having the desired degree of purity, is mixed with one or more pharmaceutically acceptable carriers and/or excipients. The carriers and excipients must be "acceptable" in the sense of being compatible with the other ingredients of the composition. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to, water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, or combinations thereof, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as

octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine;

monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

An immunogenic or immunological composition of the invention, e.g., a DNA vaccine, can also be formulated in the form of an oil-in- water emulsion. The oil-in-water emulsion can be based, for example, on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane, squalene, EICOSANE™ or tetratetracontane; oil resulting from the oligomerization of alkene(s), e.g., isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, such as plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, e.g., isostearic acid esters. The oil advantageously is used in combination with emulsifiers to form the emulsion. The emulsifiers can be nonionic surfactants, such as esters of sorbitan, mannide {e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, such as the PLURONIC® products, e.g., L121. The adjuvant can be a mixture of emulsifier(s), micelle-forming agent, and oil such as that which is commercially available under the name PRO VAX® (IDEC Pharmaceuticals, San Diego, CA).

The immunogenic compositions of the invention can contain additional substances, such as wetting or emulsifying agents, buffering agents, or adjuvants to enhance the effectiveness of the vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, (ed.) 1980).

Adjuvants that enhance the effectiveness of the vaccine may also be added to the formulation. Adjuvants include, but are not limited to, mineral salts (e.g., A1K(S0₄)2, AlNa(S0₄)₂, A1NH(S0₄)₂, silica, alum, Al(OH)₃, Ca₃(P0₄)₂, kaolin, or carbon),

polynucleotides with or without immune stimulating complexes (ISCOMs) (e.g., CpG oligonucleotides, such as those described in Chuang, T.H. et al, (2002) J. Leuk. Biol. 71(3): 538-44; Ahmad-Nejad, P. et al (2002) Eur. J. Immunol. 32(7): 1958-68; poly IC or poly AU acids, polyarginine with or without CpG (also known in the art as IC31; see Schellack, C. et al (2003) Proceedings of the 34^th Annual Meeting of the German Society of Immunology; Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508), JuvaVax™ (U.S. Patent No.

6,693,086), certain natural substances (e.g., wax D from Mycobacterium tuberculosis, substances found in Corny ebacterium parvum, Bordetella pertussis, or members of the genus Brucella), flagellin (Toll-like receptor 5 ligand; see McSorley, S.J. et al (2002) J. Immunol. 169(7): 3914-9), saponins such as QS21 , QS17, and QS7 (U.S. Patent Nos.

5,057,540; 5,650,398; 6,524,584; 6,645,495), monophosphoryl lipid A, in particular, 3-de-

O-acylated monophosphoryl lipid A (3D-MPL), imiquimod (also known in the art as IQM and commercially available as Aldara®; U.S. Patent Nos. 4,689,338; 5,238,944; Zuber,

A.K. et al (2004) 22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (see Veazey, R.S. et al (2003) J. Exp. Med. 198: 1551-1562).

Aluminum hydroxide or phosphate (alum) are commonly used at 0.05 to 0.1% solution in phosphate buffered saline. Other adjuvants that can be used, especially with DNA vaccines, are cholera toxin, especially CTAl-DD/ISCOMs (see Mowat, A.M. et al (2001) J. Immunol. 167(6): 3398-405), polyphosphazenes (Allcock, H.R. (1998) App. Organometallic Chem. 12(10-11): 659-666; Payne, L.G. et al (1995) Pharm. Biotechnol. 6: 473-93), cytokines such as, but not limited to, IL-2, IL-4, GM-CSF, IL-12, IGF-1, IFN-a, IFN-β, and IFN-γ (Boyer et al, (2002) J. Liposome Res. 121 : 137-142; WO01/095919), immunoregulatory proteins such as CD40L (ADX40; see, for example, WO03/063899), and the CD la ligand of natural killer cells (also known as CRONY or a-galactosyl ceramide; see Green, T.D. et al, (2003) J. Virol. 77(3): 2046-2055), immunostimulatory fusion proteins such as IL-2 fused to the Fc fragment of immunoglobulins (Barouch et al., Science 290:486-492, 2000) and co-stimulatory molecules B7.1 and B7.2 (Boyer), all of which can be administered either as proteins or in the form of DNA, on the same expression vectors as those encoding the antigens of the invention or on separate expression vectors.

The oil in water emulsion, which is especially appropriate for viral vectors, can be based on: light liquid paraffin oil (European pharmacopoeia type), isoprenoid oil such as squalane, squalene, oil resulting from the oligomerization of alkenes, e.g. isobutene or decene, esters of acids or alcohols having a straight-chain alkyl group, such as vegetable oils, ethyl oleate, propylene glycol, di(caprylate/caprate), glycerol tri(caprylate/caprate) and propylene glycol dioleate, or esters of branched, fatty alcohols or acids, especially isostearic acid esters. The oil is used in combination with emulsifiers to form an emulsion. The emulsifiers may be nonionic surfactants, such as: esters of on the one hand sorbitan, mannide (e.g., anhydromannitol oleate), glycerol, polyglycerol or propylene glycol and on the other hand oleic, isostearic, ricinoleic or hydroxystearic acids, said esters being optionally ethoxylated, polyoxypropylene-polyoxyethylene copolymer blocks, such as Pluronic, e.g., L121.

For maleic anhydride-alkenyl derivative copolymers, EMA (Monsanto) may be used, which are straight-chain or crosslinked ethylene-maleic anhydride copolymers and they are, for example, crosslinked by divinyl ether. Reference is also made to J. Fields et al, Nature 186: 778-780, Jun. 4, 1960. With regard to structure, the acrylic or methacrylic acid polymers and EMA are preferably formed by basic units having the following formula in which: Rl and R2, which can be the same or different, represent H or CH3 ,x=0 or 1, preferably x=l, y=l or 2, with x+y=2. For EMA, x=0 and y=2 and for carbomers x=y=l . These polymers are soluble in water or physiological salt solution (20 g/1 NaCl) and the pH can be adjusted to 7.3 to 7.4, e.g., by soda (NaOH), to provide the adjuvant solution in which the expression vector(s) can be incorporated.

A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative.

Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996).

Persons skilled in the art can also refer to U.S. Patent No. 2,909,462 (incorporated herein by reference) which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name CARBOPOL® (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross- linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned CARBOPOL® 974P, 934P and 971P. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA® (Monsanto) which are

copolymers of maleic anhydride and ethylene, linear or cross-linked, for example cross- linked with divinyl ether, are preferred. Reference may be made to J. Fields et al., Nature, 186 : 778-780, 4 June 1960, incorporated herein by reference.

Advantageously, the immunogenic compositions and vaccines according to the invention comprise an effective quantity to elicit an immunological response and/or a protective immunological response of one or more expression vectors and/or polypeptides as discussed herein; and, an effective quantity can be determined from this disclosure, including the documents incorporated herein, and the knowledge in the art, without undue experimentation. The immunogenic compositions can be designed to introduce the antigens, nucleic acids or expression vectors to a desired site of action and release it at an appropriate and controllable rate. Methods of preparing controlled-release formulations are known in the art. For example, controlled release preparations can be produced by the use of polymers to complex or absorb the immunogen and/or immunogenic composition. A controlled-release formulation can be prepared using appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) known to provide the desired controlled release characteristics or release profile. Another possible method to control the duration of action by a controlled-release preparation is to incorporate the active ingredients into particles of a polymeric material such as, for example, polyesters, polyamino acids, hydrogels, polylactic acid, polyglycolic acid, copolymers of these acids, or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these active ingredients into polymeric particles, it is possible to entrap these materials into

microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsule and polymethylmethacrylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, micro emulsions, nano-particles and

nanocapsules) or in macroemulsions. Such techniques are disclosed in New Trends and Developments in Vaccines, Voller et al. (eds.), University Park Press, Baltimore, Md., 1978 and Remington's Pharmaceutical Sciences, 16th edition.

Administration

Suitable dosages of the antigens, nucleic acids and expression vectors of the invention (collectively, the immunogens) in an immunogenic composition of the invention can be readily determined by those of skill in the art. For example, the dosage of the immunogens can vary depending on the route of administration and the size of the subject. Suitable doses can be determined by those of skill in the art, for example by measuring the immune response of a subject, such as a laboratory animal, using conventional

immunological techniques, and adjusting the dosages as appropriate. Such techniques for measuring the immune response of the subject include but are not limited to, chromium release assays, tetramer binding assays, IFN-γ ELISPOT assays, IL-2 ELISPOT assays, intracellular cytokine assays, and other immunological detection assays, e.g., as detailed in the text "Antibodies: A Laboratory Manual" by Ed Harlow and David Lane.

When provided prophylactically, the immunogenic compositions of the invention are ideally administered to a subject in advance of infection with a target pathogenic agent, such as HPV, or evidence of infection with the target pathogenic agent, or in advance of any symptom due to the target pathogenic agent. The prophylactic administration of the immunogenic compositions can serve to provide protective immunity of a subject against infection by the target pathogenic agent, such as an HPV (e.g., HPV 16) or to prevent or attenuate the progression of an HPV-induced tumor in a subject already infected with HPV. When provided therapeutically, the immunogenic compositions can serve to ameliorate and treat e.g., HPV-induced cancer cell progression.

The immunogenic compositions can be administered using any suitable delivery method including, but not limited to, intramuscular, intravenous, intradermal, mucosal, and topical delivery. Such techniques are well known to those of skill in the art. More specific examples of delivery methods are intramuscular injection, intradermal injection, and subcutaneous injection. However, delivery need not be limited to injection methods.

Further, delivery of DNA to animal tissue has been achieved by cationic liposomes

(Watanabe et al, (1994) Mol. Reprod. Dev. 38:268-274; and WO 96/20013), direct injection of naked DNA into animal muscle tissue (Robinson et al., (1993) Vaccine 11 :957- 960; Hoffman et al, (1994) Vaccine 12: 1529-1533; Xiang et al, (1994) Virology 199: 132- 140; Webster et al, (1994) Vaccine 12: 1495-1498; Davis et al, (1994) Vaccine 12: 1503- 1509; and Davis et al, (1993) Hum. Mol. Gen. 2: 1847-1851), or intradermal injection of DNA using "gene gun" technology (Johnston et al, (1994) Meth. Cell Biol. 43:353-365). Additional methods of delivery of DNA to animal tissue include electroporation, jet injection, sonoporation, microneedle-assisted delivery, etc. Alternatively, delivery routes can be oral, intranasal or by any other suitable route. Delivery also be accomplished via a mucosal surface such as the anal, vaginal or oral mucosa.

Immunization schedules (or regimens) are well known for animals (including humans) and can be readily determined for the particular subject and immunogenic composition. Hence, the immunogens can be administered one or more times to the subject. In certain embodiments, there is a set time interval between separate administrations of the immunogenic composition. While this interval varies for every subject, typically it ranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the interval is typically from 2 to 6 weeks and up to 6 months or more. With DNA tatooing, the interval is typically only 3 days (e.g., 0, 3, and 6 days). The immunization regimes typically have from

1 to 6 administrations of the immunogenic composition, but may have as few as one or two or four. The methods of inducing an immune response can also include administration of an adjuvant with the immunogens. In some instances, annual, biannual or other long interval (5-10 years) booster immunization can supplement the initial immunization protocol.

The present methods also include a variety of prime -boost regimens. In these methods, one or more priming immunizations are followed by one or more boosting immunizations. The actual immunogenic composition can be the same or different for each immunization and the type of immunogenic composition (e.g., containing protein or expression vector), the route, and formulation of the immunogens can also be varied. For example, if an expression vector is used for the priming and boosting steps, it can either be of the same or different type (e.g., DNA or bacterial or viral expression vector).

The immunogenic compositions of the invention can be administered alone, or can be co-administered, or sequentially administered, with other immunogens and/or

immunogenic compositions, e.g., with "other" immunological, antigenic or vaccine or therapeutic compositions thereby providing multivalent or "cocktail" or combination compositions of the invention and methods of employing them. Again, the ingredients and manner (sequential or co -administration) of administration, as well as dosages can be determined taking into consideration such factors as the age, sex, weight, species and condition of the particular subject, and the route of administration.

Those of ordinary skill in the art can easily make a determination of the proper dosage of the DNA vaccine. Generally, certain factors will impact the dosage that is administered; although the proper dosage is such that, in one context, the exogenous gene is expressed and the gene product is produced in the particular cell of the mammal. Preferably, the dosage is sufficient to have a therapeutic and/or prophylactic effect on the animal. The dosage also will vary depending upon the exogenous gene to be administered.

The invention will now be further described by way of the following non-limiting examples.

EXAMPLE 1

Materials and Methods

Mice

C57BL/6 mice (6-10 weeks) were obtained from JAX® Mice (The Jackson

Laboratory). All experiments were approved by the Experimental Animal Committee of The Netherlands Cancer Institute and in accordance with institutional and national guidelines.

DNA vaccines:

DNA vaccines based on HPV 16 E6 and E7 genes were generated by the

introduction of target genes or gene fragments into VAX 1 (Invitrogen). The generation of E7SH, TTFC-E7SH, E6SH and TTFC-E6SH was described before (20). FM4 consists of 4 moieties of mutated human protein FKBP12 (97% homology to the mouse variant) with the signal peptide of hGH fused to the N-terminus (Rivera, VM et al. (2000) Science 287:826- 830). FM4-HELP-E7SH (see figure la for a schematic representation) was ordered from GeneArt® with codon optimization for expression in human cells, and was cloned between the Hindlll and Xbal sites of pVAX. FM4E7SH and HELP-E7SH were made by removal of either the BamHI flanked helper cassette or the Spel flanked FM4 moiety. Histone 2B, endoplasmic reticulum protein 29, keratin 14 and cluster of differentiation antigen 8a, all from mouse origin, were ordered from GeneArt® with codon optimization for expression in human cells and flanked by Spel sites. FM4(minus_sig)HELP-E7SH was made by PCR using FM4HELP-E7SH as a template. sigHELPE7SHKDEL was constructed by replacing the complete FM4 with only the signal peptide. The KDEL sequence was fused to E7SH and E6SH by PCR. The different E6SH encoding DNA vaccines were constructed by simply replacing E7SH with E6SH or E7SH-KDEL by E6SH-KDEL. Correctness of all sequences was confirmed by sequence analysis. Plasmids were expressed and amplified in E. Coli DH5a and were purified using an endotoxin free DNA purification kit (Qiagen). DNA vaccines for intradermal tattoo application were dissolved at a concentration of 2mg/ml in water for injections (Aqua B. Braun).

Transfection and immunoblotting

HEK 293 T cells were transfected with 10 μg of a mixture of a GFP encoding plasmid and the different constructs at a ratio of 3:7 by use of FuGENE 6 (Roche) according to the manufacturer's instructions. Cells were harvested 24 hours after transfection and equal transfection efficiency was confirmed by analyzing the percentage of GFP positive cells by Flow cytometry. Subsequently the rest of the samples was lysed on ice in RIPA buffer (Sigma- Aldrich) supplemented with 1 :50 protease inhibitor cocktail (Roche) and 1 : 100 PMSF (Thermo Fisher Scientific). Cell lysates were subsequentely cleared by centrifugation at 40 °C. Total cellular protein was determined using a Bradford assay (Bio-

Rad Corporation) and was separated at 30 ug per lane on 4-12 % NuPage Bis-Tris gradient gels (Invitrogen) in MES buffer, according to the manufacturer's instructions. Subsequent immunoblotting was performed by using, the iBlot® system (Invitrogen) according to the manufacturer's instructions. E7 specific signal was detected using a monoclonal mouse anti- E7 HPV- 16 antibody (Invitrogen, clone 8C9) 1 : 100. Actin was detected using a mouse anti- human actin antibody at a 1 : 10,000 (Millipore, clone C4) dilution. In both cases an HRP- rabbit anti mouse antibody (DAKO, P 0161) was used as secondary antibody at a 1 :7,500 dilution. For detection, we used the enhanced chemiluminescence kit from Pierce

Biotechnology.

Tattoo vaccination

Intradermal DNA tattoo vaccination was performed at day 0, 3 and 6, as described previously (20). In short, the hair on the hind leg was removed using depilating cream (Veet®, Reckitt Benckiser) one day before the start of vaccination. On the day of vaccination, mice were anesthetized and 10 μΐ of a 2 mg/ml DNA solution in water was applied to the hairless skin of the hind leg. In cases mice were vaccinated with a single tattoo vaccination, 15 ul of the DNA solution was used. The DNA vaccine was applied with a Permanent Make Up (PMU) tattoo machine (kindly provided by MT Derm), using a sterile disposable 9-needle bar with a needle depth of 1 mm and oscillating at a frequency of 100 Hz for 30 seconds.

Detection of HPV-specific CD8+ T cells in peripheral blood

Peripheral blood cells were obtained via tail bleeding, and erythrocytes were removed by incubation in erythrocyte lysis buffer (155 mM NH4C1, 10 mM KHC03, 0.1 mM EDTA (pH 7.4)) on ice. The cells were subsequently stained in FACS buffer (l x PBS, 0.5 % BSA and 0.02 % sodium azide) with allophycocyanin (APC)-conjugated anti-CD8a mAb (BD Pharmingen) plus phycoerythrin (PE)-conjugated H-2Db E749-57 or H-2Kb E648-57 tetramers for 15 min at 20 °C. Subsequently, cells were washed two times in FACS buffer before analysis. Live cells were selected based on PI exclusion. MHC tetramers were produced by UV-induced peptide exchange, as described previously (34).

Detection of P30 and PADRE specific CD4+ T cells in peripheral blood Intracellular IFN-γ staining was performed using the BD Cytofix/Cytoperm kit (BD

Biosciences) according to the manufacturer's protocol. Peripheral blood cells were stimulated for 16 h at a 1 μg/ml concentration of either the PADRE peptide

(AKFVAAWTLKAAA (SEQ ID NO: 16)) or P30 peptide

(FNNFTVSFWLRVPKVSASHLE (SEQ ID NO: 17)), and subsequently stained using PE- conjugated anti-INF-γ mAb (BD Pharmingen), and APC-conjugated anti-CD8a mAb (BD Pharmingen). All samples were analyzed on a FACScalibur (BD Biosciences), using Flow- Jo® software for data analysis.

TC-1 tumor challenge

C57BL/6 mice were injected subcutaneously with 1 x 10⁵ TC-1 tumor cells that express both HPV 16 E6 and E7 (35). DNA tattoo vaccination was subsequently performed on day 4, 7 and 10 after tumor challenge. Tumor growth was monitored 1-3 times per week using caliper measurements in two dimensions. The volume of the tumors was calculated as follows: volume = (width x length)/2. Mice were sacrificed when the tumor diameter reached 15 mm or when the tumor volume exceeded 1000 mm .

Statistical analysis

Statistical analysis was performed using a student's t-test. A p-value < 0.05 was considered to be significant (two-tailed). For evaluation of survival data a log-rank test was used.

Results

Construction of a modular DNA vaccine

Applicants designed a DNA vaccine consisting of a self carrier protein and a separate element to provide CD4 T cell help (see fig 1 a). As a carrier protein, we selected an engineered human protein called FM4 (Rivera, VM et al. (2000) Science 287:826-830). This protein contains four repeats of a point mutated version of the human protein FKBP12, and is targeted to the endoplasmic reticulum (ER) by the use of a signal peptide. As a result of these modifications FM4 forms stable aggregates inside the ER (36). The rationale for selecting FM4 as a carrier is that the resulting fusion product was expected to be highly stable. The element, that was designed to provide CD4+ T cell help, consists of a set of promiscuous CD4+ T cell epitopes that were fused together in a so called 'helper-cassette' (fig IB). The selected epitopes are respectively a pan DP (P30) (37), pan DR (PADRE) (38) and a pan DQ (NEF) (39) epitope. In order to avoid the formation of neo-epitopes at the junctions, spacers containing GPGPGPG (SEQ ID NO: 18) motifs were used (40). The rational of using minimal CD4 + epitopes instead of a complete foreign protein, is that this minimizes the risk for antigenic competition on the level of CD8+ T cells. Finally a gene- shuffled version of E7 (E7SH) was used as antigen in order to avoid the risk of cellular transformation at the vaccination site (20). Applicants also made constructs that only contained the carrier protein or the helper-cassette in combination with the antigen (see figure 1C). To confirm that the fusion constructs were properly expressed, constructs were transfected into HEK 293 cells and western blotting was performed using an E7 specific antibody. As can be seen in figure 1C,D all constructs have the expected size and the expression levels of all fusion constructs are strongly increased compared to E7SH alone.

The combined addition of the carrier and the helper-cassette leads to superior CD8+ T cell responses.

In order to reveal possible differences in immunogenicity between the constructs described above, C57BL6/J mice (n=5) were immunized by short interval DNA tattooing (11,41) and CD8+ T cell responses were analyzed over time. As can be seen in figure 2a, HPV 16 E7 specific CD8+ T cells could be detected directly ex vivo using E749-57 specific H2 Db tetramers in all groups except for the empty vector and E7SH vaccinated mice. Looking at the peak of the response (fig 2B,C), it can be seen that both the addition of the helper cassette (HELPE7SH) and the carrier alone (FM4E7SH) resulted in a significantly improved immunogenicity of E7SH, albeit the effect of the helper cassette was most pronounced. Clearly, the combination of the carrier and the helper-cassette (FM4-HELP- E7SH) induces superior immunogenicity with a mean primary peak response of 19.05 ± 5.02 E749-57 specific CD8+ T cells. Importantly, this combination also significantly outperforms a highly potent construct consisting of a fusion of Tetanus toxin fragment C and E7SH (TTFC-E7SH) that was developed by applicants before (20). These differences were not only observed at the peak of the response but remained visible over time and were maintained after a secondary challenge, best reflected in figure 2D that shows the total area under the curve for each group.

In order to demonstrate that addition of the helper cassette indeed results in CD4+ T cell responses towards the encoded epitopes, an intracellular interferon-gamma assay was performed. For this purpose P30 and PADRE were used for stimulation as these epitopes are also recognized in the context of mouse class II (38,42). As can be seen in figure 2E, P30 and PADRE specific CD4+ T cell responses were observed in those groups that received vaccines encoding the helper cassette. In case of TTFC-E7SH, P30 directed CD4+ T cell responses could be demonstrated, this is as expected since TTFC only encodes P30 and not PADRE.

Only ER localized carriers provide an advantage over the addition of the helper- cassette alone

Having demonstrated that the combination of a carrier molecule and the helper- cassette results in superior immunogenicity, the role of the carrier molecule was further explored. To this end, 4 new carrier molecules were selected, namely: histone 2B (H2B, nuclear localization) (43), endoplasmic reticulum protein 29 (ERP-29, ER localized) (44), keratin 14 (KRT 14, cytosolic localization) (45) and cluster of differentiation antigen 8a (CD8a, plasma membrane localized) (46). All of these molecules are abundantly expressed self proteins. Applicants selected molecules with totally different cellular functions and different subcellular localization. As can be seen in figure 3B, transfection of HEK 293 T cells with the different constructs lead to expression of proteins with the expected size (see fig 3A). Although the expression levels vary markedly, the expression level of the fusion vaccine is strongly enhanced compared to E7SH in all cases. In order to study possible differences in construct immunogenicity, mice were vaccinated and the immune responses were monitored as described above. As can be seen in figure 3 C,D,E, both FM4HELP- E7SH and ERP29-HELP-E7SH are significantly more immunogenic than HELP-E7SH, whereas the immunogenicity of H2B-HELP-E7SH and CD8a-HELP-E7SH is comparable to HELP-E7SH, finally KRT14-HELP-E7SH is significantly less immunogenic than HELP- E7SH. Thus, surprisingly, these differences in construct immunogenicity did not reflect the differences in expression levels as displayed in figure 3B. For example, the expression levels of CD8-HELP-E7SH are extremely high, much higher than the expression levels of FM4-HELP-E7SH, however FM4-HELP-E7SH is more immunogenic. Also, the expression levels of KRT 14-HELP-E7SH and FM4-HELP-E7SH are comparable, yet the

immunogenicity of KRT14-HELP-E7SH is strongly reduced. Apparently the carrier effect can not simply be explained by an effect on accumulation of the antigen/antigen stability. An interesting observation is that the carriers that show an advantage over the addition of HELP alone are both ER localized, supporting a role for ER localization of the antigen.

The carrier effect can be explained by ER targeting of the antigen In order to determine if ER localization as such could explain the role of the carrier protein, the following constructs were generated. Firstly, a construct expressing FM4 without the signal peptide called FM4(minus_sig)HELP-E7SH. Secondly, a construct expressing only the FM4 derived signal peptide to redirect the construct towards the ER and a C-terminal KDEL (to avoid secretion) called sig-HELP-E7SH-KDEL. Thirdly, as a control, a variant that also expressed the antigen in combination with the signal peptide and the ER retention signal but that lacked the helper cassette called sigE7SHKDEL (figure 4 A). As can be seen in figure 4B, removal of the signal peptide did not negatively affect the expression level of FM4-HELP-E7SH. Addition of the signal peptide and KDEL sequence moderately increased the expression level of HELP-E7SH, and strongly enhanced the expression levels of E7SH. To study the differences in construct immunogenicity, mice were vaccinated as described above. As can be seen figure 4 C, D, E, removal of the signal peptide resulted in a strong decrease in immunogenicity of FM4-HELP-E7SH. Note that the immunogenicity is now significantly lower than in the case of addition of HELP alone. As a confirmation, we also removed the signal peptide from ERP29, and observed the same pattern (data not shown). On the other hand, sig-HELP-E7SH-KDEL, that only contained the carrier derived signal peptide and an ER retention signal, is significantly more immunogenic than HELP-E7SH, suggesting that the carrier effect can indeed be explained by ER localization of the antigen. A possible explanation is that the accumulation of the antigen inside the ER leads to ER-stress, thereby inducing "immunogenic cell death" (47). To test if ER stress might contribute to the improved immunogenicity, a construct was made that contained a 2A linker in between the carrier and the antigen (FM4-2A-HELP-E7SH). In this case the FM4 molecule will still accumulate inside the ER and cause ER-stress, but the antigen will be localized in the cytosol. The immunogenicity of this construct was equal to that of HELP-E7SH and not FM4-HELP-E7SH (Figure 7), indicating that the increase in immunogenicity relies on ER-localization of the antigen and not so much on the induction of ER-stress. Note that ER localization alone is not sufficient to make the antigen highly immunogenic, as sig-E7SH-KDEL is significantly less immunogenic than sig-HELP- E7SHKDEL.

Combination of the helper-cassette and ER targeting also improves the

immunogenicity of E6SH

Applicants also made a set of constructs that encoded E6SH (fig 5A). Mice were tattoo vaccinated with these constructs as described above and E6 specific CD8+ T cell responses were monitored directly ex vivo by tetramer staining. As can be seen in figure 5B,

E648-57 specific CD8+ T cells could be detected in all groups except for the empty vector vaccinated mice. Similar to what was observed for the E7SH encoding constructs, the addition of helper cassette alone strongly improved the immunogenicity of E6SH, the combined addition of the helper cassette and the signal peptide plus ER retention signal improved the immunogenicity even more, albeit the latter difference was not significant (fig 5 C, D, E). Importantly, the CD8+ T cell responses induced by sig-HELP-E6SH-KDEL strongly outperformed those elicited by TTFC-E6SH that applicants described before. This finding was also confirmed in HLA-A2 transgenic mice (HHD), (48) demonstrating that the design rules described herein also apply in the context of human MHC class I (see Figure 8).

Combination of the helper cassette and ER targeting allows for dose sparing and shows superior anti-tumor effect after TC-1 challenge

As the CD8+ T cell responses observed after vaccination with sig-HELP-E7SH- KDEL were so potent, applicants evaluated whether it might be possible to lower the number of vaccinations or to reduce the dose. As can be seen in figure 6A, the CD8+ T cell responses against the sig-HELP-E7SH-KDEL group were hardly affected when the mice were only vaccinated with a single tattoo (compare with figure 4 F,G). Notably, the responses against HELP-E7SH were strongly reduced, and the responses against TTFC- E7SH were hardly above background after a single vaccination. The same result was obtained when the mice were vaccinated 3 times with a 5 times reduced DNA dose (0,4 mg/ml instead of 2mg/ml) (fig 6B). These results underline the superiority of the combined use of the helper-cassette and ER targeting.

In order to assess whether the CD8+ T cell responses induced by sig-HELP-E7SH- KDEL and sig-HELP-E6SH-KDEL are also functionally superior to the TTFC-fusion constructs, we performed a TC-1 tumor challenge. For this purpose mice (n=10) were challenged with HPV E6/E7-expressing TC-1 cells and vaccination was started at day 4 after tumor challenge. Notably, a single vaccination with sig-HELP-E7SH-KDEL induced initial regression of tumors in 10 out of 10 mice, whereas a single vaccination with TTFC- E7SH only induced regression in 2 out of 10 mice. This difference also translated into a significant effect on survival (P=0.0021) (Fig. 6D). A similar pattern was observed in mice vaccinated (3x on day 4, 7, and 10) with the E6SH encoding variants: sig-HELP-E6SH- KDEL outperformed TTFC-E6SH in terms of tumor control leading to significantly (p=0.0004) improved survival. These results show that both sig-HELP-E7SH-KDEL and sig-HELP-E6SH-KDEL vaccines are superior to a previously developed carrier vaccine (20) with respect to CD8+ T cell induction and with respect to in-vivo tumor clearance.

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* * *

Having thus described in detail embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Each patent, patent application, and publication cited or described in the present application is hereby incorporated by reference in its entirety as if each individual patent, patent application, or publication was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A genetically-encoded vaccine comprising an expression cassette

transcribing (i) at least one antigen, (ii) at least one subcellular localization element, and (iii) at least one CD4 help element.

2. The genetically-encoded vaccine of claim 1, wherein the at least one antigen is selected from the group consisting of: a tumor-associated antigen, a viral antigen, a bacterial antigen, a parasite-derived antigen, and disease-associated self antigen.

3. The genetically-encoded vaccine of any preceding claim, wherein the at least one subcellular localization element is a signal peptide or protein sequence that results in export of the antigen from the cytosol.

4. The genetically-encoded vaccine of any preceding claim, wherein expression of the at least one subcellular localization element results in localization of the antigen to an organelle selected from the group consisting of: the endoplasmic reticulum (ER), the Golgi apparatus, an endosome, a lysosome, and a mitochondrion.

5. The genetically-encoded vaccine of any preceding claim, wherein expression of the at least one subcellular localization element localizes the antigen to the ER.

6. The genetically-encoded vaccine of any preceding claim, wherein the at least one subcellular localization element is or comprises a portion of a self carrier protein.

7. The genetically-encoded vaccine of any preceding claim, wherein the at least one subcellular localization element enhances antigen stability.

8. The genetically-encoded vaccine of any preceding claim, wherein the at least one CD4 help element is selected from the group consisting of: P30

9. The genetically-encoded vaccine of claim 2, wherein the tumor-associated antigen is an HPV antigen.

10. The genetically-encoded vaccine of claim 9, wherein the HPV antigen is

HPV 16 E6 and/or E7.

11. The genetically-encoded vaccine of any preceding claim, wherein the at least one subcellular localization element comprises a signal peptide.

12. The genetically-encoded vaccine of claim 11, wherein the at least one subcellular localization element comprises an ER retention signal.

13. The genetically-encoded vaccine of any preceding claim, wherein the at least one CD4 help element comprises at least one CD4+ epitope.

14. The genetically-encoded vaccine of claim 13, wherein the at least one CD4 help element comprises at least two CD4+ epitopes.

15. The genetically-encoded vaccine of claim 14, wherein the at least two CD4+ epitopes are encoded by nucleic acid sequences separated by a glycine proline repeat or other peptide sequence that prevents the formation of one or more neo-epitopes.

16. A method of treating a tumor or cancer or a method of inhibiting tumor cell growth or cancer cell growth in a mammal comprising administering to the mammal an effective amount of the genetically-encoded vaccine of claim 1.

17. The method of claim 16, wherein the tumor or cancer is induced by HPV.

18. The method of claim 17, wherein the HPV is HPV 16.

19. The method of any of claims 16-18, wherein the genetically-encoded vaccine is a DNA vaccine comprising an HPV E6 and/or E7 antigen.

20. The method of any of claims 16-19, wherein the at least one subcellular localization element of the genetically-encoded vaccine is selected from the group consisting of: a signal peptide or protein sequence that results in export of the antigen from the cytosol.

21. The method of any of claims 16-20, wherein expression of the at least one subcellular localization element in the genetically-encoded vaccine localizes the antigen to an organelle selected from the group consisting of: the endoplasmic reticulum (ER), the Golgi apparatus, an endosome, a lysosome, and a mitochondrion.

22. The method of any of claims 16-19, wherein expression of the at least one subcellular localization element localizes the antigen to the ER.

23. The method of any of claims 16-22, wherein the at least one subcellular localization element of the genetically-encoded vaccine is a self carrier protein.

24. The method of any of claims 16-23, wherein the at least one subcellular localization element of the genetically-encoded vaccine enhances antigen stability.

25. The method of any of claims 16-24, wherein the at least one CD4 help element of the genetically-encoded vaccine is selected from the group consisting of: P30 (FNNFTVSFWLPvVPKVSASHLE (SEQ ID NO: 17)), PADRE (AKFVAAWTLKAAA (SEQ ID NO: 16)), NEF, P23TT (VSIDKFRIFCKANPK (SEQ ID NO: 7)), P32TT

26. The method of claim 16, wherein the mammal is a human.

27. The genetically-encoded vaccine of any of claims 1-15, further comprising a pharmaceutically acceptable carrier.

28. The genetically-encoded vaccine of claim 27, wherein administration of the vaccine to an animal elicits an immunogenic response against the at least one antigen that is encoded by and expressed from the genetically-encoded vaccine by the animal after administration.

29. The genetically-encoded vaccine of claim 28, wherein the immunogenic response elicited is an increase in CD8+ T cells specific for the at least one antigen.

30. The method of any of claims 16-25, wherein administration of the vaccine elicits an immunogenic response against the at least one antigen that is encoded by and expressed from the genetically-encoded vaccine by the mammal after administration.

31. The method of claim 30, wherein the immunogenic response elicited is an increase in CD8+ T cells specific for the at least one antigen.

32. The genetically-encoded vaccine of any of claims 1-15, wherein the vaccine is a DNA vaccine or a viral vaccine.

33. The genetically-encoded vaccine of claim 32, wherein the viral vaccine is an adenoviral vaccine or a vaccinia viral vaccine.

34. A vaccine comprising a nucleic acid expression cassette transcribing (i) at least one antigen, (ii) at least one subcellular localization element, and (iii) at least one CD4 help element.

35. The vaccine of claim 34, wherein the nucleic acid expression cassette is expressed from a viral expression vector.

36. The vaccine of claim 35, wherein the viral expression vector is selected from the group consisting of: an adenovirus, an adeno-associated virus (AAV), an alphavirus, a retrovirus, and a poxvirus.

37. The vaccine of claim 36, wherein the alphavirus is Semliki Forest virus.

38. The vaccine of any of claims 1-15, wherein expression of the at least one subcellular localization element results in export of the antigen out of the cytosol.

39. The method of any of claims 16-25, wherein expression of the at least one subcellular localization element results in export of the antigen out of the cytosol.

40. The vaccine of claim 36, wherein the adenovirus is selected from the group consisting of Ad5, Adl 1, Ad26, Ad35, Ad48, and Ad49.

41. The vaccine of claim 36, wherein the poxvirus is a vaccinia virus.

42. The vaccine of claim 41 , wherein the vaccinia virus is MVA.

43. The vaccine of claim 34, wherein the nucleic acid expression cassette is expressed from a DNA vector.

44. The vaccine of any of claims 1-15 and 34-43, further comprising at least one additional therapeutically active agent.

45. The vaccine of claim 44, wherein the at least one additional therapeutically active agent is a second vaccine.

46. A kit comprising:

(a) a vaccine of any of claims 1-15 and 34-43; and

(b) instructions for use.

47. The kit of claim 46, further comprising at least one additional therapeutically active agent.

48. The kit of claim 47, wherein the additional therapeutically active agent is a second vaccine.