HPV polyepitope constructs and uses thereof

FIELD OF THE INVENTION

The present invention is directed to Human Papillomavirus (HPV) polyepitope constructs and the use thereof for the prevention and/or treatment of HPV infection.

BACKGROUND OF THE INVENTION

Cancer of the cervix uteri is the second most common cancer among women worldwide, with an estimated 493,000 new cases and 274,000 deaths in 2002. The field of cervical cancer prevention is rapidly evolving as a consequence of the identification of the cause of the disease: a limited number of viral types from the family of the Human Papillomaviruses (HPV). Indeed, HPV has been recognized as a cause of cervical cancer, and 2 of the oncogenic types, 16 and 18, are together responsible for 70% of the world's cervical cancer cases. Recently the use of 2 prophylactic vaccines was licensed. Nevertheless, it is likely to be decades before the impact of HPV vaccination on the incidence of cervical cancer can be evaluated. Optimally, these vaccines should be administered before sexual debut and HPV infection. As such, they are of no benefit for women with already existing HPV infection. Treatment (surgery) for HPV infection is often unsatisfactory because of persistence of virus after treatment and recurrence of clinically apparent disease is common. The treatment may require frequent visits to clinics and is not directed at elimination of the virus but at clearing warts. Moreover, it is expected that less prevalent, oncogenic HPV genotypes will take over - at least in part - the place from the currently targeted HPV 16 and 18 genotypes. Women with pre-carcinogenic lesions resulting from the widespread HPV infections today represent a highly unmet need.

Thus, a need exists for an efficacious vaccine to prevent and/or treat persistent HPV infection and to prevent cancer that is associated with HPV infection. Effective HPV vaccines would be a significant advance in the control of sexually transmissible infections and could also

protect against clinical disease, particularly cancers such as cervical cancer, (see, e. g., Rowen, P. and Lacey, C, Dermatologic Clinics 16 (4): 835-838,1998).

In the majority of individuals, HPV infections presumably induce strong, local, cell-mediated immunity that results in clearance of the virus and protection against subsequent infection. Virus-specific, human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocytes (CTL) and HLA class Il-restricted helper T lymphocytes (HTL) are known to play a major role in the prevention of chronic infection and in viral clearance in vivo (Houssaint et al, 2001; Gruters et al., 2002; Tsai et al., 1997; Murray et al., 1992; Tigges et al., 1992; Bowen and Walker, 2005).

A therapeutic vaccine candidate targeting HPV should elicit strong and multi-specific cellular immune responses. The induction of a strong HPV-specific cellular response - comprising activity of cytotoxic T-cells (CTL) and helper T-cells (HTL) - may be achieved using an epitope-based vaccine approach.

The polyepitope approach to vaccine development is to rationally create a multi-specific cellular response, causing the immune system to be specifically stimulated against multiple selected epitopes that meet stringent criteria. These include CTL epitopes that are presented by MHC-I and are recognized by cytotoxic T-cells, and HTL epitopes that bind MHC II and are recognized by helper T-cells. The epitopes are selected in view of their capacity to elicit responses in humans, thereby aiming for a large population coverage (by targeting major HLA class-I alleles as well as major HLA class-II alleles).

The technology relevant to polyepitope vaccines is developing and a number of different approaches are available which allow simultaneous delivery of multiple epitopes. Several independent studies have established that induction of simultaneous immune responses against multiple and individual peptides can be achieved (Doolan et al (1997), Bertoni and colleagues (1997)). In terms of immunization with polyepitope nucleic acid vaccines, several examples have been reported where multiple T cell responses were induced (Thomson et al., 1995; Woodberry et al., 1999; Mateo et al., 1999; Ishioka et al., 1999; WO04/031210, Innogenetics N.V. et al.).

The efforts to develop an effective treatment for HPV-related disease are narrowly focused. Most studies are concentrating on the HPV type 16 E6 and/or E7 protein. Also WO05/089164 (Pharmexa et al.) discloses HPV polyepitope constructs focussing on E6 and E7,

and additionally El and E2 proteins. During the papillomavirus life cycle, the HPV proteins (El, E2, E4, E5, E6, E7, Ll and L2) are differentially expressed. Moreover, during progression from CINl to CIN3, the extent of expression of the different HPV proteins is changing (Doorbar, 2005). Targeting all 6 early proteins (El, E2, E4, E5, E6 and E7) thus provides a way to induce efficient immune responses directed to all stages of the virus life cycle, irrespective of the CIN grade.

Although E4 and E5 were screened for immunogenic epitopes, WO05/089164 was unsuccessful in obtaining reactive peptides. It is indeed known that most of the HPV proteins are comparatively small and might therefore not comprise many reactive epitopes. The present inventors however have now determined several immunogenic epitopes in the E4 and E5 proteins of the high risk HPV genotypes HPV16, 18, 31 and 45. Moreover, the present inventors were successful in creating a potent, multi-specific and full-spectrum vaccine addressing the different stages of HPV infection, and thereby broadening the treatment window. Where others, focusing on E6 and/or E7 are mainly targeting CIN2 and and CIN3, this vaccine allows to treat earlier stages of disease as well as persistent infection, thereby further reducing the chance of developing cervical cancer.

The polyepitope constructs are designed to induce an immune response to at least 4 distinct CTL and 1-3 HTL epitopes per HPV genotype in the majority of subjects infected with one of the four most prevalent, high risk HPV genotypes (HPV16, 18, 31 and 45) irrespective of their ethnic origin.

SUMMARY OF THE INVENTION

The present invention encompasses epitopes derived from the El, E2, E4, E5, E6 and/or E7 protein of the Human Papillomavirus (HPV). Each of the epitopes given in Tables 1 and 2, or any combination of two, more or all of these epitopes, are part of the invention, as well as their application in the treatment and/or prevention of HPV infection or HPV-related disease. The epitopes are those which elicit a HLA class I- and/or class II-restricted T lymphocyte response in an immunized host.

In a particular embodiment, the present invention relates to an isolated CTL inducing peptide derived from a Human Papillomavirus protein, consisting of 8 to 13 amino acids and

comprising the sequence represented by SEQ ID NO 1-88. More specific, the invention encompasses an isolated CTL inducing peptide derived from the Human Papillomavirus protein E4 or E5, consisting of 8 to 13 amino acids and comprising the sequence represented by SEQ ID NO 8, 17, 29, 42, 43, 51, 64, 74, 75, 81 and 86. The invention also covers an isolated poly epitope construct comprising one or more of the herein described CTL inducing peptides.

The present invention is furthermore directed to a polynucleotide, a polypeptide, a vector or a composition comprising a polyepitope construct encoding or comprising specifically selected epitopes derived of HPV.

In one embodiment, the polyepitope construct encodes or comprises at least the following HPV derived CTL epitopes: SEQ ID NO 1 to 44, and/or the polyepitope construct encodes or comprises at least the following HPV CTL epitopes: SEQ ID NO 1, 23, 39 and 45 to 88. In another embodiment, the current invention relates to a polynucleotide comprising a polyepitope construct comprising nucleic acids encoding all the epitopes given in Table 1 (SEQ ID NO 1- 88). Specifically, the construct does not encode a full-length protein from HPV.

In a further embodiment, the polyepitope construct of the invention further encodes or comprises at least one CTL and/or HTL epitope. Preferably, the epitopes are isolated. In a specific embodiment, the at least one CTL and/or HTL epitope is derived from HPV. More specific, the at least one HTL epitope is selected from the group consisting of SEQ ID NO 89 to 121. Preferably, the polyepitope construct furthermore comprises a PADRE® epitope. Specifically, the PADRE® epitope is characterized by SEQ ID NO 122.

Optionally, the epitopes in the polyepitope construct are linked to each other by one or more, preferably 1 to 8, spacer amino acids. In a specific embodiment, the one or more spacer amino acids are selected from the group consisting of: K, R, N, Q, G, A, S, C, G, P, and T. More specifically, the spacer between one or more CTL epitopes is selected from the group consisting of G, K, A or N, and the spacer between one or more HTL epitopes is selected from the group consisting of G, N and P.

In a further embodiment, the CTL and/or HTL epitopes comprised in the polyepitope construct are sorted to minimize the number of CTL and/or HTL junctional epitopes.

Specifically, the HPV CTL epitopes are directly or indirectly linked in the order as shown in Figure IA, 2 A, 3 or 4. The HPV HTL epitopes can be directly or indirectly linked in the order as shown in Figure IB or 2B.

Optionally, the polynucleotide of the present invention further comprises one or more regulatory sequences. Preferably, said regulatory sequence is an internal ribosome binding site (IRES).

In a specific embodiment, the polynucleotide of the present invention further comprises one or more promoters. Preferably, the promoter is a CMV promoter.

In a further embodiment, the polynucleotide of the present invention further comprises one or more signal sequences. Preferably, the signal sequence is a Igkappa signal sequence.

In another embodiment, the polynucleotide of the invention comprises one or more MHC class I and/or MHC class Il-targeting sequences. Preferably, the targeting sequence is selected from the group consisting of: tissue plasminogen activator signal sequence, insulin signal sequence, endoplasmic reticulum signal sequence, LAMP-I lysosomal targeting sequence, LAMP-2 lysosomal targeting sequence, HLA-DM lysosomal targeting sequence, HLA-DM-association sequences of HLA-DO, Ig-alpha cytoplasmic domain, Ig-beta cytoplasmic domain, Ii protein, influenza matrix protein, HBV surface antigen, HBV core antigen, and yeast Ty protein.

In a specific embodiment, the polynucleotide of the present invention comprises a polyepitope construct encoding the amino acid sequence consisting of, comprised in or comprising the sequence represented by SEQ ID NO 123, SEQ ID NO 125, SEQ ID NO 127 or SEQ ID NO 129. In another embodiment the polyepitope construct is characterized by or comprised in the nucleic acid sequence represented by SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 128 or SEQ ID NO 130.

In a further embodiment, the polyepitope construct encodes the amino acid sequence consisting of or comprising the sequence represented by SEQ ID NO 156, SEQ ID NO 158, SEQ ID NO 160, or SEQ ID NO 162. In another embodiment, polyepitope construct consists of or comprises the nucleic acid sequence represented by SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 161, or SEQ ID NO 163.

Furthermore, the invention encompasses a vector comprising the polynucleotide as described herein. Preferably, the vector is an expression vector. More preferably, the vector is a plasmid (pDNA), a viral, a bacterial or a yeast vector. In a further embodiment, the viral vector is a pox virus. Preferably, the pox virus is a vaccinia virus. More preferably, the vaccinia virus is MVA.

Moreover, the current invention also relates to an isolated polypeptide encoded by the polynucleotide as described herein.

The current invention also relates to a composition comprising the polynucleotide, the polypeptide, or the vector as described herein, or any combination thereof.

Preferably, the composition further comprises a pharmaceutical acceptable excipient or carrier. In a specific embodiment, the composition is a vaccine.

In another embodiment, the present invention relates to the composition, the polynucleotide, the vector or the polypeptide as described herein, for use as a medicament.

Specifically, the invention includes the use of the composition, the polynucleotide, the vector or the polypeptide for the manufacture of a medicament for treating and/or preventing persistent HPV infection. The invention also encompasses the composition, the polynucleotide, the vector or the polypeptide for use in the treatment and/or prevention of persistent HPV infection. Specifically, the invention is directed to the treatment and/or prevention of HPV-related disease.

Moreover, the present invention includes a cell comprising the polynucleotide, the polypeptide, or the vector as described herein.

In a further embodiment, the invention relates to a method of inducing an immune response against HPV in an individual, comprising administering the polynucleotide, the polypeptide, the vector, the composition, or the cell as described herein, or a combination thereof, to said individual. Specifically, the method is directed to the treatment and/or prevention of HPV infection and/or HPV-related disease.

Furthermore, the invention covers a method of making the polynucleotide, the polypeptide, the vector, the composition, or the cell as described herein.

FIGURE LEGENDS

Figure 1: A. Specific order of CTL epitopes in construct ICCG6150

B. Specific order of HTL epitopes in construct ICCG6150 or ICCG6149

Figure 2: A. Specific order of CTL epitopes in construct ICCG6138

B. Specific order of HTL epitopes in construct ICCG6138 or ICCG6137

Figure 3: Specific order of CTL epitopes in construct ICCG6137

Figure 4: Specific order of CTL epitopes in construct ICCG6149

Figure 5: Construct ICCG6137

A. Amino acid sequence of the signal sequence (in italics) and the poly epitope; B. DNA sequence: The start and stop codons are underlined. The signal sequence is shown in italics, and the epitope-coding sequence is bolded.

C. Amino acid sequence of the polyepitope;

D. DNA sequence of the polyepitope.

Figure 6: Construct ICCG6138

A. Amino acid sequence of the signal sequence (in italics) and the polyepitope;

B. DNA sequence: The start and stop codons are underlined. The signal sequence is shown in italics, and the epitope-coding sequence is bolded.

C. Amino acid sequence of the polyepitope; D. DNA sequence of the polyepitope.

Figure 7: Construct ICCG6149

A. Amino acid sequence of the signal sequence (in italics) and the polyepitope;

B. DNA sequence: The start and stop codons are underlined. The signal sequence is shown in italics, and the epitope-coding sequence is bolded.

C. Amino acid sequence of the polyepitope;

D. DNA sequence of the polyepitope.

Figure 8: Construct ICCG6150

A. Amino acid sequence of the signal sequence (in italics) and the poly epitope;

B. DNA sequence: The start and stop codons are underlined. The signal sequence is shown in italics, and the epitope-coding sequence is bolded.

C. Amino acid sequence of the polyepitope; D. DNA sequence of the polyepitope.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a polynucleotide or polypeptide comprising a polyepitope construct encoding or comprising epitopes derived from the El, E2, E4, E5, E6 and/or E7 protein of the Human Papillomavirus (HPV). The epitopes are those which elicit a HLA class I- and/or class II- restricted T-lymphocyte response in an immunized host. More specifically, the present invention describes highly optimized and effective polyepitope constructs characterized by efficient processing and comprising highly immunogenic epitopes allowing efficient treatment of patients at different stages of HPV-related disease.

Identification of the epitopes

CTL binding epitopes were evaluated for their immunogenicity in different HLA transgenic mice. To this, a single immunization with CTL peptide pools together with a common HTL epitope emulsified in IFA was performed and up to 14 days later, CD8+ spleen cells were isolated and evaluated for epitope specificity using a direct ex vivo IFNγ ELISPOT assay. The majority of high affinity binding CTL epitopes proved to be immunogenic. HTL binding epitopes were evaluated for their induction of (ex vivo) recall T cell responses using PBMC from HPV patients. To this, PBMC from subjects were cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen-presenting cells (APC to allow activation of "memory" T cells, as compared to "naive" T cells). At the end of the culture period, T cell activity was detected using assays such as ⁵¹ Cr release involving peptide-loaded target cells, T cell proliferation, or cytokine release.

The CTL epitopes of the present invention are given in Table 1. Each individual epitope is part of the invention as well as combinations of two, more, or all of said epitopes. In a specific aspect of the invention, epitopes have been identified in the E4 and E5 proteins of the high risk HPV genotypes HPV16, 18, 31 and 45, whereby said epitopes are being

characterized by SEQ ID NO 8, 17, 29, 42, 43, 51, 52, 64, 74, 75, 81 and 86. As such, the present invention also relates to a combination comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or all of the epitopes characterized by SEQ ID NO 8, 17, 29, 42, 43, 51, 52, 64, 74, 75, 81 and 86, optionally linked to each other in a polyepitope construct.

Starting from said pool of CTL epitopes, the present inventors were successful in creating a potent, multi- specific and full-spectrum vaccine addressing the different stages of HPV infection, and broadening the treatment window. The polyepitope construct of the present invention is particularly useful to prevent and/or treat HPV infection, more specific HPV-related disease, and even more specific the precancerous stages of HPV infection, i.e. CIN 1-3. HPV-related disease includes neoplasia and HPV related cancers such as but not limited to cervical cancer and head and neck carcinoma. The neoplasia to be treated with the methods and medicaments according to the current invention may be any HPV induced neoplasia, preferably in an epithelial tissue, in the ano-genital area and/or ano-genital tract, comprising the vulva, vagina, cervix, penis, scrotum, anus and rectum. The neoplastic disorders to be treated comprise Cervial Intraepithelial Neoplasia of various grades (CIN 1, 2 and 3), Vulvar intraepithelial neoplasias of various grades (VIN 1, 2 and 3) and Vaginal intraepithelial neoplasias (VAIN) and anal intraepithelial neoplasia (AIN). Also male subjects suffering from virally induced neoplasias in the ano-genital area and/or tract, such as but not limited to, Penile intraepithelial neoplasia (PIN) and Anal intraepithelial neoplasia (AIN), may be treated according to this invention.

Although most HPV infections do not progress to cervical cancer, infections that persist for many years are more likely to do so. Most cervical cancers develop slowly through a series of abnormal changes in the cells of the cervix. Regular Pap tests can detect these changes and the abnormal tissue can be removed, preventing it from ever developing into cancer. Various terms have been used to describe the abnormal cells that may be seen in Pap tests. Samples with cell abnormalities may be divided into different categories.

Cervical intraepithelial neoplasia (CIN) is a term that is often used to describe abnormal tissue findings. Neoplasia means an abnormal growth of cells. The term CIN along with a number (1, 2, or 3) describes how much of the thickness of the lining of the cervix contains abnormal cells. CIN-3 is considered to be a precancerous condition that includes carcinoma in situ.

Another classification is:

• ASC — Atypical Squamous Cells. Squamous cells are the thin, flat cells that form the surface of the cervix.

• AGC — Atypical Glandular Cells. Glandular cells are mucus-producing cells found in the endocervical canal (opening in the center of the cervix) or in the lining of the uterus. The glandular cells do not appear normal, but doctors are uncertain what the cell changes mean.

• AIS — endocervical Adenocarcinoma In Situ. Precancerous cells are found in the glandular tissue.

• LSIL — Low-grade Squamous Intraepithelial Lesion. Low-grade means there are early changes in the size and shape of the cells. The word lesion refers to an area of abnormal tissue. LSILs are considered mild abnormalities caused by HPV infection and are a common condition, especially among young women. The majority of LSILs return to normal over months to a few years.

• HSIL — High-grade Squamous Intraepithelial Lesion. High-grade means that the cells look very different in size and shape from normal cells. HSILs are more severe abnormalities and may eventually lead to cancer if left untreated.

Pap test results may also be described using an older set of categories called the "dysplasia scale." Dysplasia is a term used to describe abnormal cells. Although dysplasia is not cancer, it may develop into very early cancer of the cervix. The cells look abnormal under the microscope, but they do not invade nearby healthy tissue. There are four degrees of dysplasia: mild, moderate, severe, and carcinoma in situ. Carcinoma in situ is a precancerous condition that involves only the layer of cells on the surface of the cervix, and has not spread to nearby tissues. Currently, mild dysplasia is classified as LSIL; moderate or severe dysplasia and carcinoma in situ are combined into HSIL.

Overview:

The polyepitope constructs are designed to induce an immune response to at least 4 distinct CTL and 1-3 HTL epitopes per HPV genotype in the majority of subjects infected with one of the four most prevalent, high risk HPV genotypes (HPV16, 18, 31 and 45) irrespective of their ethnic origin. The epitopes in the constructs were sorted and optimized using the method as described in WO04/031210 (Pharmexa Inc. et al; incorporated herein by reference). Epitopes were included in one or more constructs. The constructs were subsequently tested in HLA transgenic mice and immunogenicity was measured for the encoded epitopes.

Evidently, the T cell epitopes as given in Tables 1 and/or 2 can be combined into one or more constructs in any manner appropriate for a specific therapeutic application or patient group. For example, only the HPV 16 CTL and/or HTL epitopes of Tables 1 and/or 2 can be combined into 1 construct. Alternatively, the epitopes in the construct can be limited to HPVl 8, HPV31 or HPV45. A different approach is to combine the epitopes for the different HPV genotypes, but to limit to certain proteins, e.g. El, E2, E4, E5, E6 or E7, or combinations of proteins, e.g. El and E2, or E4 and E5, or E6 and E7, or other combinations of T cell epitopes derived from 2 or more HPV proteins, this for one genotype, or alternatively for two, three or four of the herein described genotypes.

In one embodiment, the present invention relates to an isolated polynucleotide comprising a polyepitope construct encoding at least the following HPV derived cytotoxic T lymphocyte (CTL) epitopes: SEQ ID NO 1-44 and/or encoding at least the following HPV derived cytotoxic T lymphocyte (CTL) epitopes: SEQ ID NO 1, 23, 39, and 45-88. Furthermore, the present invention also encompasses a polypeptide encoded by the polynucleotide as described herein. Preferably, the epitopes of the polyepitope construct are directly or indirectly linked to one another in the same reading frame.

The term "construct" as used herein generally denotes a composition that does not occur in nature. As such, the polynucleotide construct of the present invention does not encode a wild-

type full-length protein from HPV but encodes a chimeric protein containing isolated epitopes from at least one HPV protein not necessarily in the same sequential order as in nature. A construct may be a "polynucleotide construct" or a "polypeptide construct". The polynucleotides or (polypeptides as described herein are "isolated" or "biologically pure". The term "isolated" refers to material that is substantially free from components that normally accompany it as found in its naturally occurring environment. However, it should be clear that the isolated polynucleotide or (polypeptide of the present invention might comprise heterologous cell components or a label and the like. An "isolated" epitope refers to an epitope that does not include the neighboring amino acids of the whole sequence of the antigen or polynucleotide from which the epitope was derived. As such, the present invention relates to a polynucleotide comprising a polyepitope construct comprising the following isolated HPV CTL epitopes: SEQ ID NO 1-44 and/or SEQ ID NO 1, 23, 39, and 45-88. It is thus to be understood that the construct of the present invention comprises isolated epitopes that are not embedded in the naturally occurring full length protein from HPV. The specific epitopes in the construct can be directly or indirectly linked in any order or in the order as given in Figures IA, 2 A, 3 and 4. A construct can be produced by synthetic technologies, e.g. recombinant DNA preparation and expression or chemical synthetic techniques for nucleic acids and amino acids. A construct can also be produced by the addition or affiliation of one material with another such that the result is not found in nature in that form.

With regard to a particular amino acid sequence, an "epitope" is a set of amino acid residues which is involved in recognition by a particular immunoglobulin, or in the context of T cells, those residues necessary for recognition by T cell receptor proteins and/or Major Histocompatibility Complex (MHC) molecules. With regard to a particular nucleic acid sequence, a "nucleic acid epitope" is a set of nucleic acids that encode for a particular amino acid sequence that forms an epitope. In an immune system setting, in vivo or in vitro, an epitope is the collective features of a molecule, such as primary, secondary and tertiary peptide structure, and charge, that together form a site recognized by a T cell receptor and MHC molecule, or an immunoglobulin. The term "polypeptide" is used interchangeably with "oligopeptide" and designates a series of amino acids, connected one to the other, typically by peptide bonds between the amino and carboxyl groups of adjacent amino acids.

The term "polyepitope construct" when referring to nucleic acids and polynucleotides can be used interchangeably with the terms "minigene" and "polyepitope nucleic acid" and other equivalent phrases, and comprises multiple nucleic acid epitopes that encode peptides of any length that can bind to a molecule functioning in the immune system, preferably a HLA class I or a HLA class II and a T-cell receptor. All disclosures herein with regard to nucleic acid epitopes comprised in a polynucleotide construct apply mutatis mutandis to epitopes comprised in an amino acid construct. The epitopes in a polyepitope construct can be HLA class I epitopes and/or HLA class II epitopes. HLA class I epitopes are referred to as CTL epitopes and HLA class II epitopes are referred to as HTL epitopes. Some polyepitope constructs can have a subset of HLA class I epitopes and another subset of HLA class II epitopes. A CTL epitope usually consists of 13 or less amino acid residues in length, 12 or less amino acids in length, or 11 or less amino acids in length, preferably from 8 to 13 amino acids in length, more preferably from 8 to 11 amino acids in length (i.e. 8, 9, 10, or 11), and most preferably 9 or 10 amino acids in length. A HTL epitope consists of 50 or less amino acid residues in length, and usually from 6 to 30 residues, more usually from 12 to 25, and preferably consists of 15 to 20 (i.e. 15, 16, 17, 18, 19, or 20) amino acids in length.

The polyepitope construct described herein preferably includes 2 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more, 100 or more and up till 150 epitopes, preferably up till 130 and more preferably up till 80 epitopes. More specific, the polyepitope construct consists of or comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, or more epitopes.

In a preferred embodiment, the polyepitope construct of the present invention further comprises at least one CTL and/or HTL epitope. Said "further" CTL and/or HTL epitope to be used in combination with the epitopes of the present invention can be derived from HPV or from a foreign antigen or organism (non-HP V). Accordingly, the present invention encompasses a polynucleotide comprising a polyepitope construct encoding at least the following HPV CTL epitopes characterized by: SEQ ID NO 1-44 and/or SEQ ID NO 1, 23, 39, and 45-88, and at least one additional CTL and/or HTL epitope, and wherein the construct does not encode a full- length protein from HPV. Preferably, the at least one CTL and/or HTL epitope is derived from HPV, and more specifically from the El, E2, E4, E5, E6, E7, Ll and/or L2 protein. Preferably,

the at least one HTL epitope is selected from Table 2. Any combination of HTL epitopes and even all of the HTL epitopes as given in Table 2 can be included into the construct. In a further embodiment, the present invention relates to a polynucleotide comprising nucleic acids encoding a polyepitope construct comprising respectively the following isolated HPV CTL and HTL epitopes: SEQ ID NO 1-44 and SEQ ID 89-105, or alternatively SEQ ID NO 1, 23, 39, and 45-88 and SEQ ID NO 106-121. The HTL epitopes in the construct can be directly or indirectly linked in any order or in the order as given in Figures IB and 2B. In a further embodiment, the invention encompasses a polypeptide encoded by said nucleotide.

The further epitopes can be derived from any desired antigen of interest, e.g. a viral antigen, a tumor antigen or any pathogen. Multiple HLA class I or class II epitopes present in a polyepitope construct can be derived from the same antigen, or from different antigens. For example, a polyepitope construct can contain one or more HLA binding epitopes than can be derived from two different antigens of the same virus, or from two different antigens of different viruses. In a preferred embodiment, the epitopes of the present invention are derived from HPV and more specifically from the El, E2, E4, E5, E6, E7, Ll and/or L2 protein. There is no limitation on the length of said further epitopes, these can have a length of e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more amino acids. The "at least one" can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130 or even more epitopes.

In a preferred embodiment, the polyepitope construct of the present invention further comprises the universal T cell epitope called PADRE ^® (Pharmexa, San Diego; described for example in US 5 736 142 or US 6 413 935 or International Application WO95/07707 or WO97/26784, which are enclosed herein by reference). A "PanDR binding epitope or PADRE ^® epitope" is a member of a family of molecules that binds more than one HLA class II DR molecule. The pattern that defines the PADRE ^® family of molecules can be thought of as an HLA Class II supermotif. PADRE ^® binds to most HLA-DR molecules and stimulates in vitro and in vivo human helper T lymphocyte (HTL) responses. In a preferred embodiment, the PADRE ^® epitope is characterized by SEQ ID NO 122.

Alternatively, HTL epitopes from universally used vaccines such as tetanos toxoid can be included. In an alternative embodiment, the further epitopes are B cell epitopes.

The aim of the present invention is to provide strategies to optimize antigenicity and immunogenicity of polyepitope vaccines encompassing a large number of relevant epitopes, and to provide optimized polyepitope vaccines, particularly HPV polyepitope constructs. Examples of such constructs are depicted in Figures 5-8. Said constructs comprise a plurality of HPV- specifϊc CTL and HTL epitopes that are efficiently processed, and thus highly immunogenic. Hence, the present invention is directed to a polynucleotide comprising or consisting of a polyepitope construct encoding CTL and HTL epitopes, whereby the polyepitope construct is represented by or comprised in SEQ ID NO 124, SEQ ID NO 126, SEQ ID NO 128 or SEQ ID NO 130.

In a further embodiment, the polyepitope construct encodes a polypeptide comprising or consisting of an amino acid sequence represented by or comprised in SEQ ID NO 123, SEQ ID NO 125, SEQ ID NO 127 or SEQ ID NO 129. More specific, the polyepitope construct encodes the amino acid sequence consisting of or comprising the sequence represented by SEQ ID NO 156, SEQ ID NO 158, SEQ ID NO 160, or SEQ ID NO 162. More particular, the polyepitope construct consists of or comprises the nucleic acid sequence represented by SEQ ID NO 157, SEQ ID NO 159, SEQ ID NO 161, or SEQ ID NO 163.

The term "immunogenic" or "immunogenicity" as used herein is the ability to evoke an immune response.

Immunogenicity can be manifested in several different ways. Immunogenicity corresponds to whether an immune response is elicited at all, and to the vigor of any particular response, as well as to the extent of a population in which a response is elicited. For example, a peptide might elicit an immune response in a diverse array of the population, yet in no instance produce a vigorous response. In accordance with these principles, close to 90% of high affinity binding peptides have been found to be immunogenic, as contrasted with about 50% of the peptides that bind with intermediate affinity (Sette et al., 1994; Alexander et al., 2003). Moreover, higher binding affinity peptides lead to more vigorous immunogenic responses. As a result, less peptide is required to elicit a similar biological effect if a high affinity binding peptide is used.

Various strategies can be utilized to evaluate immunogenicity, including but not limited to: 1) Evaluation of primary T cell cultures from normal individuals (see, e. g., Wentworth et al., 1995; Celis et al., 1994; Tsai et al., 1997; Kawashima et al., 1998). This procedure involves

the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen-presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a ⁵¹ Cr-release assay involving peptide sensitized target cells. 2) Immunization of HLA transgenic mice (see, e.g., Wentworth et al., 1996; Alexander et al., 1997) or surrogate mice. In this method, peptides (e.g. formulated in incomplete Freund's adjuvant) are administered subcutaneously to HLA transgenic mice or surrogate mice. Eleven to 14 days following immunization, splenocytes are removed. Cells are either cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a ⁵¹ Cr-release assay involving peptide-sensitized target cells and/or target cells expressing endogenously generated antigen. Alternatively, cells are incubated overnight together with peptide-loaded APC in the IFNg ELISPOT assay for the quantitation of peptide-specific single T cells releasing mouse interferon gamma upon stimulation. 3) Demonstration of recall T cell responses from immune individuals who have effectively been vaccinated, recovered from infection, and/or from chronically infected patients (see, e.g., Rehermann et al., 1995; Doolan et al., 1997; Bertoni et al., 1997; Threlkeld et al., 1997; Diepolder et al., 1997). In applying this strategy, recall responses are detected by culturing PBL from subjects that have been naturally exposed to the HPV antigen, for instance through infection, and thus have generated an immune response "naturally", or from patients who were vaccinated with a vaccine comprising the epitope of interest. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to "naive" T cells. At the end of the culture period, T cell activity is detected using assays including ⁵¹ Cr release involving peptide-sensitized target cells, T cell proliferation, or cytokine release.

A given epitope is stated to be immunogenic if T cell reactivity can be shown to target cells sensitized with that peptide. Immunogenicity for a given epitope can further be described by the number of individuals in a group of HLA matched infected or vaccinated subjects (e.g. humans, primates, transgenic mice, surrogate mice) that show T cell reactivity to that particular epitope, or e.g. by the number of spots detected in an ELISPOT assay. Immunogenicity for the epitopes of the invention is indicated in Tables 8 and 9.

Epitope analogs derived from naturally occurring HPV sequences exhibit increased binding to HLA molecules and immunogenicity due to the modification of specific amino acid

residues with respect to the naturally occurring HPV sequence. Accordingly, in a specific embodiment, the epitopes of the present invention may be analoged to modify binding affinity and/or the ability to bind to multiple alleles within an HLA supertype. Analog epitopes can be created by altering the presence or absence of particular residues in the primary anchor positions. Such analogs are used to modulate the binding affinity of a peptide comprising a particular motif or supermotif. Accordingly, the analoged epitopes as given in Table 1 are also part of the invention.

A "preferred primary anchor residue" is an anchor residue of a motif or supermotif that is associated with optimal binding. HLA binding motifs, supermotifs and preferred primary anchor residues are given in e.g. WO05/089164 (Pharmexa Inc. et al; incorporated herein by reference). "Heteroclitic analogs" are defined herein as epitopes with increased potency for a specific T cell, as measured by increased responses to a given dose, or by a requirement of lesser amounts to achieve the same response. Advantages of heteroclitic analogs include that the epitopes can be more potent, or more economical (since a lower amount is required to achieve the same effect). In addition, modified epitopes might overcome antigen-specific T cell unresponsiveness (T cell tolerance). (See e.g. WOO 1/36452, which is hereby incorporated by reference)

The epitopes of the polyepitope construct are directly or indirectly linked to one another in the same reading frame. More specific, the epitopes are either contiguous (directly linked) or are separated by a linker or a spacer nucleic acid encoding a spacer amino acid or spacer peptide

(indirectly linked).

"Link" or "join" refers to any method known in the art for functionally connecting peptides

(direct of via a linker), including, without limitation, recombinant fusion, covalent binding, non- covalent binding, disulfide binding, ionic binding, hydrogen binding, polymerization, cyclization, electrostatic binding and connecting through a central linker or carrier.

Polymerization can be accomplished for example by reaction between glutaraldehyde and the -

NH2 groups of the lysine residues using routine methodology.

In a specific embodiment, the polyepitope construct of the present invention further comprises three or a plurality of spacer nucleic acids, linked in the same reading frame to the CTL and/or

HTL epitope nucleic acids. A reading frame is a contiguous and non-overlapping set of three- nucleotide codons in DNA or RNA. There are 3 possible reading frames in a strand and six in a double stranded DNA molecule. "In the same reading frame" means that there is no shift from one frame to another that could lead to different genes/proteins.

To develop polyepitope constructs using the epitopes of the present invention, said epitopes can be sorted and optimized using a computer program or, for fewer epitopes, not using a computer program. "Sorting epitopes" refers to determining or designing an order of the epitopes in a polyepitope construct.

"Optimizing" refers to increasing the antigenicity of a polyepitope construct having at least one epitope pair by sorting epitopes to minimize the occurrence of junctional epitopes, and inserting a spacer residue (as described herein) to further prevent the occurrence of junctional epitopes or to provide a flanking residue. As described herein, a "flanking residue" is a residue that is positioned next to an epitope. A flanking residue can be introduced or inserted at a position adjacent to the N-terminus (N+ 1) or the C-terminus (C+ 1) of an epitope. An increase in immunogenicity or antigenicity of an optimized polyepitope construct is measured relative to a polyepitope construct that has not been constructed based on the optimization parameters by using assays known to those skilled in the art, e.g. assessment of immunogenicity in HLA transgenic mice, ELISPOT, tetramer staining, ⁵¹ Cr release assays, and presentation on antigen presenting cells in the context of MHC molecules.

The process of optimizing polyepitope constructs is given e.g. in WO01/47541 and WO04/031210 (Pharmexa Inc. et al.; incorporated herein by reference). According to a specific embodiment, the polyepitope construct of the present invention is optimized for CTL and/or HTL epitope processing. More particular, the optimization comprises the introduction of one or more spacers. More preferred, the polyepitope construct as described herein comprises 0, 3, 6, 9, 12, 15, 18, or more spacer nucleic acids or 0, 1, 2, 3, 4, 5, 6, or more spacer amino acids between two epitopes. A "spacer" refers to a sequence that is inserted between two epitopes in a polyepitope construct to prevent the occurrence of junctional epitopes, or to facilitate cleavage between epitopes and thereby enhance epitope presentation. "Junctional epitopes" refer to epitopes recognized by the immune system, not present in the target antigen, and only created by the man-made juxtaposition of epitopes. A spacer nucleic acid may encode one or more amino acids. A spacer nucleic acid flanking a HLA class I epitope in a polyepitope construct encodes preferably 1 to 8, and more preferably 1 to 5 amino acids, i.e. 1, 2, 3, 4 or 5 amino acids. A spacer nucleic acid flanking a HLA class II epitope in a polyepitope construct encodes 1 to 8 amino acids, preferably 5, 6, 7, or more amino acids, and more preferably 5 or 6 amino acids. A spacer nucleic acid separating a HLA class I epitope and a class II epitope in a polyepitope construct encodes preferably 1 to 8, and more preferably 1 to 5 amino acids, i.e. 1, 2, 3, 4 or 5 amino acids. The number of

spacers in a construct, the number of amino acids in a spacer, and the amino acid composition of a spacer can be selected to optimize epitope processing and/or minimize junctional epitopes. It is preferred that spacers are selected by concomitantly optimizing epitope processing and preventing junctional motifs. The "spacer amino acid" or "spacer peptide" is typically comprised of one or more relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. For example, spacers flanking HLA class II epitopes preferably include G (GIy), P (Pro), and/or N (Asn) residues. A particularly preferred spacer for flanking a HLA class II epitope includes alternating G and P residues, for example, (GP)n, (PG)n, (GP)nG, (PG)nP, and so forth, where n is an integer between 1 and 10, preferably 2 or 3, and where a specific example of such a spacer is GPGPG (SEQ ID NO 155). For separating class I epitopes, or separating a class I and a class II epitope, the spacers are typically selected from, e.g., A (Ala), N (Asn), K (Lys), G (GIy), L (Leu), I (He), R (Arg), Q (GIn), S (Ser), C (Cys), P (Pro), T (Thr), or other neutral spacers of nonpolar amino acids or neutral polar amino acids, though polar residues could also be present. A preferred spacer, particularly for HLA class I epitopes, comprises or consists of 1, 2, 3 or more consecutive alanine (A), Lysine (K), Asparagine (N) or Glycine (G) residues, or a combination of K (Lys) and A (Ala) residues, e.g. KA, KAA or KAAA, a combination of N (Asn) and A (Ala) residues, e.g. NA, NAA or NAAA or a combination of G (GIy) and A (Ala) residues, e.g. GA, GAA or GAAA. The present invention is thus directed to a polynucleotide comprising a polyepitope construct as described herein, and wherein the epitopes in the construct are separated by one or more spacer amino acids. In a preferred embodiment, the one or more spacer amino acids are selected from the group consisting of: K, R, N, Q, G, A, S, C, G, P and T. In some polyepitope constructs, it is sufficient that each spacer nucleic acid encodes the same amino acid sequence. In other polyepitope constructs, one or more of the spacer nucleic acids may encode different amino acid sequences.

The only outer limit on the total length and nature of each spacer sequence derives from considerations of ease of synthesis, proteolytic processing, and manipulation of the polynucleotide.

The (polypeptides of the present invention can be in their natural (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications. Also included in the definition are peptides modified by additional substituents attached to the amino acids side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical

conversions of the chains, such as oxidation of sulfhydryl groups. Thus, "polypeptide" or its equivalent terms is intended to include the appropriate amino acid sequence referenced, and may be subject to those of the foregoing modifications as long as its functionality is not destroyed.

Moreover, the present invention also contemplates a polyepitope construct comprising or consisting of multiple repeats or combinations of any of the epitopes of the present invention, as given in Tables 1 and 2. The polyepitope construct can exist as a homopolymer comprising multiple copies of the same (combination of) peptide(s), or as a heteropolymer of various peptides. Polymers have the advantage of increased immunological reaction and, where different peptide epitopes are used to make up the polymer, the additional ability to induce HTL's and/or CTLs that react with different antigenic determinants of the pathogenic organism targeted for an immune response. As an alternative, the individual epitopes of the present invention are not linked into a construct and can be combined separately e.g. in a composition.

The present invention also encompasses a method of making a polyepitope construct. Polynucleotides or nucleic acids that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage & Caruthers, 1981, using an automated synthesizer, as described in Van Devanter et. al., 1984. Purification of polynucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson & Reanier, 1983. Other purification methods are reversed phase separation and hydroxyapatite and are well known to the skilled person. Chemically synthesized and purified polynucleotides can be assembled into longer polynucleotides by PCR-based methods (Stemmer et al., 1995; Kriegler et al., 1991). The epitopes of the polyepitope constructs are typically subcloned into an expression vector that contains a promoter to direct transcription, as well as other regulatory sequences such as enhancers and polyadenylation sites. Additional elements of the vector are e.g. signal or target sequences, translational initiation and termination sequences, 5' and 3' untranslated regions and introns, required for expression of the polyepitope construct in host cells. Polyepitope constructs can for example be prepared according to the methods set forth in Ishioka et al. , 1999; Velders et al. , 2001 ; or as described in WO04/031210 - Pharmexa Inc. (all incorporated herein by reference).

A polyepitopic polypeptide or the polypeptide comprising the polyepitope construct can be generated synthetically or recombinantly. The polyepitopic polypeptide can be expressed as one

protein. In order to carry out the expression of the polyepitopic polypeptide in bacteria, in eukaryotic cells (including yeast) or in cultured vertebrate hosts such as Chinese Hamster Ovary (CHO), Vero cells, RKl 3, COSl, BHK, and MDCK cells, or invertebrate hosts such as insect cells, the following steps are carried out: - transformation of an appropriate cellular host with a recombinant vector, or by means of adenoviruses, influenza viruses, BCG, and any other live carrier systems, in which a nucleotide sequence coding for one of the polypeptides of the invention has been inserted under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host or of the live carrier system and in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleotide sequence, culture of said transformed cellular host under conditions enabling the expression of said insert.

As such, the present invention also relates to a cell or host cell comprising a polynucleotide, a polypeptide or a vector containing a polyepitope construct. In a further embodiment, the "cell" of the present invention is an antigen presenting cell (APC) comprising the polynucleotide as described herein.

The polyepitopic polypeptide can be purified by methods well known to the person skilled in the art (see e.g. Lichty JJ et al, 2005; Gaberc-Porekar V et al, 2001).

For therapeutic or prophylactic immunization purposes, the polyepitope construct of the invention can be expressed by vectors. The present invention thus also relates to a vector comprising the polynucleotide of the present invention. The term "vector" may comprise a plasmid, a cosmid, a prokaryotic organism, a phage, a virus or an eukaryotic organism such as an animal or human cell or a yeast cell. The expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the polyepitope construct in host cells. A typical expression cassette thus contains a promoter operably linked to the polyepitope construct and signals required for efficient polyadenylation of the transcript. Additional elements of the cassette may include enhancers and introns with functional splice donor and acceptor sites.

Suitable promoters are well known in the art and described, e.g., in Sambrook et al., Molecular cloning, A Laboratory Manual (2 ^nd ed. 1989) and in Ausubel et al, Current Protocols in Molecular Biology (1994). Eukaryotic expression systems for mammalian cells are well known in the art and are commercially available. Such promoter elements include, for example,

cytomegalovirus (CMV), Rous sarcoma virus long terminal repeats (RSV LTR) and Simian Virus 40 (SV40). See, e.g. US 5 580 859 and US 5 589 466 (Vical Inc.; incorporated by reference) for other suitable promoter sequences.

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

In a particular embodiment, the polynucleotide of the present invention further comprises one or more regulatory sequences. By "regulatory sequence" is meant a polynucleotide sequence that contributes to or is necessary for the expression of an operably associated nucleic acid or nucleic acid construct in a particular host organism. The regulatory sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and an internal ribosome binding site (IRES). Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers. Promoter may be a CMV promoter or other promoter described herein or known in the art. Regulatory sequences include IRESs. Other specific examples of regulatory sequences are described herein and otherwise known in the art.

In a further embodiment, the polynucleotide of the present invention further comprises one or more MHC class I and/or MHC class II "targeting nucleic acids" or "targeting sequences".

The use of a MHC targeting sequence enhances the immune response to an antigen, relative to delivery of antigen alone, by directing the peptides to the site of MHC molecule assembly and transport to the cell surface, thereby providing an increased number of MHC molecule- peptides complexes available for binding to and activation of T cells. Examples of possible targeting sequences are well known to the skilled person and are described e.g. in

WO04/031210 (Pharmexa Inc. et al). In a specific embodiment, the epitopes of polyepitope construct of the present invention are operably linked to a nucleic acid encoding a targeting sequence selected from the group consisting of: tissue plasminogen activator signal sequence, insulin signal sequence, endoplasmic reticulum signal sequence, LAMP-I lysosomal targeting sequence, LAMP-2 lysosomal targeting sequence, HLA-DM lysosomal targeting sequence, HLA-DM-association sequences of HLA-DO, Ig-alpha cytoplasmic domain, Ig-beta cytoplasmic domain, Ii protein, influenza matrix protein, HBV surface antigen, HBV core antigen, and yeast Ty protein. In a further embodiment, the polynucleotide of the present invention further comprises at least

one signal sequence. A "signal sequence" is or encodes a 16-30 amino acid sequence, in a secreted polypeptide, that directs the protein to its target compartment or membrane. A preferred signal sequence is the Igkappa signal sequence.

The phrase "operably linked" or "operatively linked" refers to a linkage in which a nucleotide sequence is connected to another nucleotide sequence (or sequences) in such a way as to be capable of altering the functioning of the sequence (or sequences). For example, a nucleic acid or polyepitope nucleic acid construct that is operably linked to a regulatory sequence, such as a promoter/operator, places expression of the nucleic acid or construct under the influence or control of the regulatory sequence. Two nucleotide sequences (such as a protein encoding sequence and a promoter region sequence linked to the 5' end of the encoding sequence) are said to be operably linked if induction of promoter function results in the transcription of the protein encoding sequence mRNA and if the nature of the linkage between the two nucleotide sequences does not (1) result in the introduction of a frame- shift mutation nor (2) prevent the expression regulatory sequences to direct the expression of the mRNA or protein. Thus, a promoter region would be operably linked to a nucleotide sequence if the promoter were capable of effecting transcription of that nucleotide sequence.

One or more cysteine residues comprised in epitopes of the polyepitope construct may be "reversibly or irreversibly blocked". An "irreversibly blocked cysteine" is a cysteine of which the cysteine thiol-group is irreversibly protected by chemical means. In particular,

"irreversible protection" or "irreversible blocking" by chemical means refers to alkylation, preferably alkylation of a cysteine in a protein by means of alkylating agents, such as, for example, active halogens, ethylenimine or N-(iodoethyl)trifluoro-acetamide. In this respect, it is to be understood that alkylation of cysteine thiol-groups refers to the replacement of the thiol-hydrogen by (CH ₂ ) _n R, in which n is 0, 1, 2, 3 or 4 and R= H, COOH, NH ₂ , CONH ₂ , phenyl, or any derivative thereof. Alkylation can be performed by any method known in the art, such as, for example, active halogens X(CH ₂ ) _n R in which X is a halogen such as I, Br, Cl or F. Examples of active halogens are methyliodide, iodoacetic acid, iodoacetamide, and 2- bromoethylamine.

A "reversibly blocked cysteine" is a cysteine of which the cysteine thiol-groups is reversibly protected. In particular, the term "reversible protection" or "reversible blocking" as used herein contemplates covalently binding of modification agents to the cysteine thiol-groups, as

well as manipulating the environment of the protein such, that the redox state of the cysteine thiol-groups remains (shielding). Reversible protection of the cysteine thiol-groups can be carried out chemically or enzymatically. The term "reversible protection by enzymatical means" as used herein contemplates reversible protection mediated by enzymes, such as for example acyl-transferases, e.g. acyl-transferases that are involved in catalysing thio- esterifϊcation, such as palmitoyl acyltransferase. The term "reversible protection by chemical means" as used herein contemplates reversible protection, using conditions or agents well known to the person skilled in the art.

The removal of the reversibly protection state of the cysteine residues can chemically or enzymatically accomplished by e.g.: a reductant, in particular DTT, DTE, 2-mercaptoethanol, dithionite, SnCl ₂ , sodium boro hydride, hydroxylamine, TCEP, in particular in a concentration of 1-200 mM, more preferentially in a concentration of 50-200 mM; removal of the thiol stabilising conditions or agents by e.g. pH increase; - enzymes, in particular thioesterases, glutaredoxine, thioredoxine, in particular in a concentration of 0,01-5 μM, even more particular in a concentration range of 0,1-5 μM.; combinations of the above described chemical and/or enzymatical conditions. The removal of the reversibly protection state of the cysteine residues can be carried out in vitro or in vivo, e.g. in a cell or in an individual.

Alternatively, one cysteine residue, or 2 or more cysteine residues comprised in the HPV epitopes as described herein may be mutated to a natural amino acid, preferentially to methionine, glutamic acid, glutamine or lysine.

Compositions and vaccines

The current invention furthermore relates to compositions comprising a polynucleotide, a polypeptide or a vector comprising the HPV polyepitope construct as described herein, or a combination thereof. In a specific embodiment, the composition furthermore comprises at least one of a pharmaceutically acceptable excipient, i.e. a carrier, adjuvant or vehicle. The terms "composition", "immunogenic composition" and "pharmaceutical composition" can be used interchangeably. More particularly, said immunogenic composition is a vaccine composition. Even more particularly, said vaccine composition is a prophylactic vaccine composition. Alternatively, said vaccine composition may also be a therapeutic vaccine composition. The prophylactic vaccine composition refers to a vaccine aimed for preventing persistent HPV

infection and to be administered to healthy persons who are not yet infected with HPV. The therapeutic vaccine composition refers to a vaccine aimed for treatment of HPV infection and to be administered to patients being infected with HPV.

A vaccine or vaccine composition is an immunogenic composition capable of eliciting an immune response sufficiently broad and vigorous to provoke at least one or both of: a stabilizing effect on the multiplication of a pathogen already present in a host and against which the vaccine composition is targeted. A vaccine composition may also induce an immune response in a host already infected with the pathogen against which the immune response leading to stabilization, regression or resolving of the disease. In case of HPV, a vaccine e.g. prevents further progression of CIN and prevents the development of cervical cancer; or promotes the regression of the lesions; and an increase of the rate at which a pathogen newly introduced in a host, after immunization with a vaccine composition targeted against said pathogen, is resolved from said host. In particular the composition of the invention is a HPV immunogenic composition or vaccine. In particular, the composition or vaccine comprises an effective amount of the polynucleotide, nucleic acids, polypeptide or peptides of the present invention. In a specific embodiment, said composition or vaccine comprises a vector, a plasmid, a recombinant virus and/or host cell comprising the polyepitope construct of the present invention. Said composition or vaccine may additionally comprise one or more further active substances and/or at least one of a pharmaceutically acceptable excipient, being a carrier, adjuvant or vehicle.

An "effective amount" of a polynucleotide or polypeptide in a vaccine or composition is referred to as an amount required and sufficient to elicit an immune response. It will be clear to the skilled artisan that the immune response sufficiently broad and vigorous to provoke the effects envisaged by the vaccine or composition may require successive (in time) immunizations with the vaccine or composition as part of an administration scheme or vaccination schedule. The "effective amount" may vary depending on the health and physical condition of the individual to be treated, the age of the individual to be treated (e.g. dosing for infants may be lower than for adults), the taxonomic group of the individual to be treated (e.g. human, non-human primate, primate, etc.), the capacity of the individual's immune system to mount an effective immune response, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment, the strain of the infecting pathogen and other relevant factors. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine or composition may be

administered in conjunction with other immunoregulatory agents. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

Carriers, adjuvants and vehicles - delivery

The present invention furthermore relates to a method of inducing an immune response against HPV in an individual comprising administering the polynucleotide, the vector, the polypeptide, the host cell or the composition of the present invention. More specific, the present invention relates to a method of treating HPV infection and more specific HPV-related disease. The present invention furthermore relates to a method for inducing an immune response at various stages, e.g. CIN 1-3, of HPV-related disease thereby aiming for higher efficacy, comprising the administration of the polynucleotide, the vector, the polypeptide, the host cell or the composition containing epitopes from differentially expressed HPV antigens. Various art-recognized delivery systems may be used to deliver a polyepitope construct into appropriate cells. The polynucleotides and polypeptides encoded thereby can be delivered in a pharmaceutically acceptable carrier or as colloidal suspensions, or as powders, with or without diluents. They can be "naked" or associated with delivery vehicles and delivered using delivery systems known in the art.

A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable adjuvant" is any suitable excipient, diluent, carrier and/or adjuvant which, by themselves, do not induce the production of antibodies harmful to the individual receiving the composition nor do they elicit protection. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response elicited by an antigen.

A "pharmaceutically acceptable vehicle" includes vehicles such as water, saline, physiological salt solutions, glycerol, ethanol, etc. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives may be included in such vehicles.

Typically, a composition or vaccine is prepared as an injectable, either as a liquid solution or suspension. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intradermal, intraepidermal, or by "gene gun". Other types of administration comprise electroporation, implantation, suppositories, oral ingestion, enteric application, inhalation, aerosolization or nasal spray or drops. Solid forms, suitable for dissolving in, or

suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or encapsulated in liposomes for enhancing adjuvant effect. A liquid formulation may include oils, polymers, vitamins, carbohydrates, amino acids, salts, buffers, albumin, surfactants, or bulking agents. Any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred.

Another drug delivery system for increasing circulatory half-life is the liposome. The peptides and nucleic acids of the invention may also be administered via liposomes, which serve to target a particular tissue, such as lymphoid tissue, or to target selectively infected cells, as well as to increase the half-life of the peptide and nucleic acids composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.

After the liquid pharmaceutical composition is prepared, it is preferably lyophilized to prevent degradation and to preserve sterility. Methods for lyophilizing liquid compositions are known to those of ordinary skill in the art. Just prior to use, the composition may be reconstituted with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon reconstitution, the composition is preferably administered to subjects using those methods that are known to those skilled in the art.

The approach known as "naked DNA" is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould- Fogerite 1988; U.S. Pat No. 5279833; WO 91/06309; and Feigner et al, 1987). In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Further examples of DNA-based delivery technologies include facilitated (bupivicaine, polymers, peptide- mediated) delivery, cationic lipid complexes, particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., US 5 922 687), DNA formulated with charged or uncharged lipids, DNA formulated in liposomes, emulsified DNA, DNA included in a viral vector, DNA formulated with a transfection- facilitating protein or polynucleotide, DNA formulated with a targeting protein or polypeptide, DNA formulated with calcium precipitating

agents, DNA coupled to an inert carrier molecule, and DNA formulated with an adjuvant. In this context it is noted that practically all considerations pertaining to the use of adjuvants in traditional vaccine formulation apply to the formulation of DNA vaccines.

Recombinant virus or live carrier vectors may also be directly used as live vaccines in humans. Accordingly the present invention also relates to a recombinant virus, a bacterial vector, a yeast vector or a plasmid, and a host cell comprising the polynucleotide as described herein. In a preferred embodiment of the invention, the polynucleotide is introduced in the form of a vector wherein expression is under control of a promoter. Therefore, further embodiments of the present invention are an expression vector which comprises a polynucleotide encoding at least the poly epitope construct as described herein, and which is capable of expressing the respective peptides, a host cell comprising the expression vector and a method of producing and purifying the herein described peptides, and a pharmaceutical composition comprising the herein described peptides and a pharmaceutically acceptable carrier and/or adjuvants.

Detailed disclosures relating to the formulation and use of nucleic acid vaccines are available, e.g. by Donnelly J.J. et al, 1997 and 1997a. Examples of expression vectors include attenuated viral hosts, such as a pox virus. As an example of this approach, vaccinia virus is used as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits a host CTL and/or HTL response. Vaccinia vectors, for example Modified Vaccinia Ankara (MVA), and methods useful in immunization protocols are described in, e.g., US 4 722 848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., 1991. Preferable yeast vectors are Sacharomyces cerevisiae, Pichia pastoris and Hansenula polymorpha. Further examples are: Alphaviruses (Semliki Forest Virus, Sindbis

Vrius, Venezuelan Equine Encephalitis Virus (VEE)), Herpes simplex Virus (HSV), replication- deficient strains of Adenovirus (human or simian), SV40 vectors, CMV vectors, papillomavirus vectors, and vectors derived from Epstein Barr virus. A wide variety of other vectors useful for therapeutic administration or immunization of the epitopes of the invention, e.g. retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Additional vector modifications may be desired to optimize polynucleotide expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or

more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the polynucleotide construct. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing polynucleotide expression. In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of nucleic acid vaccines. These sequences may be included in the vector, outside the polynucleotide coding sequence, if desired to enhance immunogenicity. In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules.

Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-P) may be beneficial in certain diseases.

The use of polyepitope constructs is described in, e.g., US 6 534 482 (Pharmexa Inc.); An and Whitton, 1997; Thomson et al, 1996; Whitton et al, 1993; Hanke et al, 1998. For example, a polyepitope DNA plasmid encoding supermotif- and/or motif-bearing HPV epitopes derived from multiple regions of the HPV polyprotein sequence, the PADRE ^® universal helper T cell epitope (or multiple HTL epitopes from HPV), and an endoplasmic reticulum-translo eating signal sequence can be engineered.

All disclosures herein which relate to use of adjuvants in the context of protein or (polypeptide based pharmaceutical compositions apply mutatis mutandis to their use in nucleic acid and vector vaccination technology. The same holds true for other considerations relating to formulation and mode and route of administration and, hence, also these considerations discussed herein in connection with a traditional pharmaceutical composition apply mutatis mutandis to their use in nucleic acid and vector vaccination technology.

Medical use

In a further embodiment, the present invention relates to the polynucleotide, the vector, the host cell, the polypeptide or the composition of the present invention for use as a medicament. Preferably, said medicament is a vaccine. More specifically, the present invention relates to the use of the polyepitope construct comprising the epitopes of the present invention, or the nucleic acid sequence encoding said epitopes, for the manufacture of a medicament for preventing and/or treating an HPV infection and/or HPV related disease. In a specific embodiment the invention also relates to a vector, a plasmid, a recombinant virus or host cell comprising the polynucleotide as described herein for the manufacture of a medicament for preventing and/or treating an HPV infection and/or HPV related disease.

The present invention also encompasses the polyepitope construct comprising the epitopes of the present invention, or the nucleic acid sequence encoding said epitopes, or a vector, a plasmid, a recombinant virus or host cell comprising the polynucleotide as described herein, for use in the prevention and/or treatment an HPV infection and/or HPV related disease.

In a further embodiment, the present invention relates to the use of the polynucleotide, the vector, the host cell, the polypeptide or the composition for inducing an immune response against HPV in an individual. Said use can be characterized in that said polynucleotide, vector, host cell, polypeptide or composition is used as part of a series of time and compounds. In this regard, it is to be understood that the term "a series of time and compounds" refers to administering with time intervals to an individual the compounds used for eliciting an immune response. The latter compounds may comprise any of the following components: polynucleotide, vector, host cell, polypeptide or composition of the present invention.

The polyepitope construct of this invention can be provided in kit form together with instructions for vaccine administration. Typically the kit would include desired polynucleotide compositions in a container, preferably in unit dosage form and instructions for administration. An alternative kit would include a polynucleotide construct with desired nucleic acids of the invention in a container, preferably in unit dosage form together with instructions for administration. Lymphokines such as IL-2 or IL- 12 may also be included in the kit. Other kit components that may also be desirable include, for example, a sterile syringe, booster dosages, and other desired excipients.

Other arrangements of the methods and tools embodying the invention will be obvious for those skilled in the art.

It is to be understood that although preferred embodiments, specific constructions and configurations, as well as materials, have been discussed herein for the methods and tools according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention.

Table 1: HPV derived CTL epitopes

Column 1 contains the analoged epitope when no WT sequence is indicated in column 4.

Table 2: HPV HTL epitopes

The present invention is illustrated by the following Examples, which should not be understood to limit the scope of the invention to the specific embodiments therein.

EXAMPLES

Example 1: HLA Class I competition binding assays using soluble HLA

The following example of peptide binding to soluble HLA molecules demonstrates quantification of binding affinities of HLA class I and class II peptides.

Epstein-Barr virus (EBV)-transformed homozygous cell lines, fibroblasts or transfectants were used as sources of HLA class I molecules. Cell lysates were prepared and HLA molecules purified in accordance with disclosed protocols (Sidney et al, 1998; Sidney et al, 1995; Sette, et al, 1994).

HLA molecules were purified from lysates by affinity chromatography. The lysate was passed over a column of Sepharose CL-4B beads coupled to an appropriate antibody. The antibodies used for the extraction of HLA from cell lysates are W6/32 (for HLA-A), and LB3.1 (for HLA-DR).

The anti-HLA column was then washed with 1OmM Tris-HCL, pH8, in 1% NP-40,

PBS, and PBS containing 0,4% n-octylglucoside and HLA molecules were eluted with 5OmM diethylamine in 0,15M NaCl containing 0,4% n-octylglucoside, pH 11,5. A 1/25 volume of

2M Tris, pH6,8, was added to the eluate to reduce the pH to +/- pH8. Eluates were then concentrated by centrifugation in Centriprep 30 concentrators (Amicon, Beverly, MA). Protein content was evaluated by a BCA protein assay (Pierce Chemical Co., Rockford, IL) and confirmed by SDS-PAGE.

A detailed description of the protocol utilized to measure the binding of peptides to Class I and Class II MHC has been published (Sette et al, 1994; Sidney et al, 1998). Briefly, purified MHC molecules (5 to 50OnM) were incubated with various unlabeled peptide inhibitors and 1-1OnM 1251-radio labeled probe peptides for 48h in PBS containing 0,05% Nonidet P-40 (NP40) in the presence of a protease inhibitor cocktail. All assays were at pH7 with the exception of DRBl*0301, which was performed at pH4,5 , and DRB1*16O1 (DR2w21 1) and DRB4*0101 (DRw53), which were performed at pH5.

Following incubation, MHC-peptide complexes were separated from free peptide by gel filtration on 7,8 mm x 15 cm TSK200 columns (TosoHaas 16215, Montgomeryville, PA). The eluate from the TSK columns was passed through a Beckman 170 radioisotope detector, and radioactivity was plotted and integrated using a Hewlett-Packard 3396A integrator, and the fraction of peptide bound was determined. Alternatively, MHC-peptide complexes were separated from free peptide by capturing onto ELISA plates coated with anti-HLA antibodies. After free peptide has been washed away, remaining reactivities were measured using the same method as above.

Radiolabeled peptides were iodinated using the chloramine-T method. Typically, in preliminary experiments, each MHC preparation was titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays were performed using these HLA concentrations.

Since under these conditions [label] < [HLA] and IC50 > [HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1,2 ng/ml, and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC50 of a positive control for inhibition by the IC50 for each tested peptide (typically unlabeled versions of the

radiolabeled probe peptide). For database purposes, and inter-experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation has proven to be the most accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC. Table 3-6 contain the IC 50 values for the CTL epitopes.

Because the antibody used for HLA-DR purification (LB3.1) is alpha-chain specific, beta-1 molecules are not separated from beta-3 (and/or beta-4 and beta-5) molecules. The beta-1 specificity of the binding assay is obvious in the cases of DRBl*0101 (DRl), DRBl*0802 (DR8w2), and DRB 1*0803 (DR8w3), where no beta-3 is expressed. It has also been demonstrated for DRBl*0301 (DR3) and DRB3*0101 (DR52a), DRBl*0401 (DR4w4), DRBl*0404 (DR4wl4), DRBl*0405 (DR4wl5), DRBl*1101 (DR5), DRB1*12O1 (DR5wl2), DRB1*13O2 (DR6wl9) and DRBl*0701 (DR7). The problem of beta chain specificity for DRBl* 1501 (DR2w2beta-l), DRB5*0101 (DR2w2beta-2), DRBl* 1601 (DR2w21beta-l), DRB5*0201 (DR51Dw21), and DRB4*0101 (DRw53) assays is circumvented by the use of fibroblasts. Development and validation of assays with regard to DRbeta molecule specificity have been described previously (see, e. g., Southwood et al, 1998). Table 7 contains the IC50 values for the HTL epitopes.

Table 3: Binding of HLA AOl-restricted peptides

SEQ ID Sequence source HLA

A0101 A2601 A2902 A3002

23 GTGCNGWFY HPV18/31.E1.1 1 136 3081 1 16 9.1

24 LQDKIIDHY HPV18.E2.15 68 - - 2770

25 ATCVSHRGLY HPV18.E2.154 421 646 4212 28

26 TLEKLTNTGLY HPV18.E6.89 174 265 2378 476

2 QVDYYGLYY HPV16.E2.151 2.7 251 25 160

3 STDLRDHIDY HPV16.E2.23 1 13 758 - 17

4 KSAIVTLTY HPV16.E2.329 9 - 96 6

5 ISDYRHYCY HPV16.E6.80.D3 10 - 10 192

1 LSQMVQWAY HPV16/31. E1.357 21 6448 122 0.93

45 VMDDSEIAY HPV31.E1.349 18 - 95 2551

46 LSSALEIPY HPV31.E6.15 35 4107 261 175

47 AFTDLTIVY HPV31.E6.46 31 36 71

48 QTEPDTSNY HPV31.E7.44.T2 19 - - 2322

67 LQDKILDHY HPV45.E2.17 32 5000 - 836

68 NTGILTVTY HPV45.E2.332 337 3147 1403 8722

69 LTDVSIACVY HPV45.E6.25.T2 2.9 8201 764 72

70 NTELYNLLI HPV45.E6.95 32 17294 - -

71 ELDPVDLLCY HPV45.E7.20 26 2107 4291 -

- : IC50 > 20 μM

Empty field: no data available

Table 4: Binding of HLA A02-restricted peptides

SEQ ID Sequence Source HLA

A0201 A0202 A0203 A0206 A6802

(nM) (nM) (nM) (nM) (nM)

27 ILYAHIQCL HPV18.E1 .266 33 8,5 10,2 236 -

28 VAWDSVYYM HPV18.E2.136 172 603 1332 790 5154

29 TVYVFCFLL HPV18.E5.51 175 587 769 26 41

30 SLQDIEITCV HPV18.E6.24 153 25 38 205 -

31 KLTNTGLYNV HPV18.E6.92.V10 106 2,9 4,7 83 688

6 CLYLHIQSL HPV16.E1 .259 73 45 21 100 Mil

7 KLLSKLLCV HPV16.E1 .292 31 67,7 23 124,6 -

8 FVYIPLFLI HPV16.E5.66 206 323 63 59 251

9 TLHDIILECV HPV16.E6.29.L2 3,6 0,54 1 ,9 92 2947

10 YMLDLQPETV HPV16.E7.11 .V10 19 1 ,9 4,5 86 5446

1 1 GTLGIVCPV HPV16.E7.85.V9 20 49 68 33 32

49 KLLEKLLCI HPV31.E1 .272 19 21 2,4 51 -

50 YTNWKFIYL HPV31.E2.131 69 14 31 84 146

51 FLLCFCVLL HPV31 .E5.15 9,6 13 34 2,6 237

52 FIYIPLFVI HPV31 .E5.66 26 97 47 8,1 157

53 FLFTDLTIV HPV31 .E6.45.L2 17 1 ,3 3,5 20 1904

54 KLTNKGICDL HPV31 .E6.90 205 440 585 484 -

72 TLYAHIQCL HPV45.E1 .252 34 4,9 6,2 417 1613

73 YVVWDSIYYI HPV45.E2.137 77 27 248 20 30

74 SLVFLLCFSV HPV45.E5.3 8,5 27 49 7,9 20

75 FLLCFSVCL HPV45.E5.6 38 81 66 59 353

76 YQFAFKDLCV HPV45.E6.45.V10 15 1 ,3 4,2 10 3698

77 TLQEIVLHV HPV45.E7.7.V9 19 30 5,1 309 2457

- : IC50 > 20 μM

Table 5: Binding of HLA All-restricted peptides

SEQ lD Sequence Source HLA

A0301 A1101 A3101 A3301 A6801

(nM) (nM) (nM) (nM) (nM)

32 KQGAMLAVFK HPV18.E1.210 35 11 519 - 235

33 TVSATQLVK HPV18.E2.211 7 4 6137 - 31

34 STVSVGTAK HPV18.E2.230 34 6 1760 - 10

35 QVVPAYNISK HPV18.E2.61 308 14 6665 - 8

36 FWYRDSIPK HPV18.E6.53.K10 3437 2504 8 473 176

37 DSVYGDTLER HPV18.E6.83.R10 193 73 246 1425 44

38 ATLQDIVLH HPV18.E7.6 211 11 543 - -

12 WLLLVRYK HPV16.E1.274 6,3 4,1 0,1 6,1 76

13 STAAALYWYK HPV16.E1.314 5 2 35 128 6

14 ATMCRHYKR HPV16.E1.406 90 14 10 17 35

15 KSLFGMSLMK HPV16.E1.483 3,9 2 230 - 887

16 SVICFVNSK HPV16.E1.497 109 2,2 25 51 5,4

17 AATKYPLLK HPV16.E4.9 59 20 - - -

55 MVMLMLVRFK HPV31.E1.253 10 16 53 20 10

56 STAAALYWYR HPV31.E1.294 198 10 5,6 47 12

57 ISFAGIVTK HPV31.E2.205 17 1 ,6 115 4366 16

58 ATTPIIHLK HPV31.E2.291 10 1 ,2 34 - 2,5

59 KVSEFRWYRY HPV31.E6.72 213 25 3 338 192

60 SVYGTTLER HPV31.E6.82.R9 22 7 75 853 4

78 AVMCRHYKR HPV45.E1.399 311 38 52 24 29

79 RQMNMSQWIK HPV45.E1.411 15 11 45 - 2557

80 STWHWTGCNK HPV45.E2.322 14 2,8 54 428 18

81 VTTRYPLLR HPV45.E4.8 158 18 483 - 3147

82 RTEVYQFAFR HPV45.E6.41.R10 755 211 8 696 439

83 SVYGETLEK HPV45.E6.84 21 9,5 6506 - 7,2

-: IC50 > 20 μM

Table 6: Binding of HLA A24-restricted peptides

SEQ ID Sequence Source HLA

A2302 A2402 A2902 A3002

(nM) (nM) (nM) (nM)

39 SYFGMSFIHF HPV18/45.E1.491 4,3 17,5 62 1544

40 YYMTDAGTW HPV18.E2.142 14 3,2 341 1 2151

41 GYNTFYIEF HPV18.E2.168 13 16 835 1264

42 MYVCCHVPL HPV18.E5.14 24 15 9442 4717

43 VYVFCFLLPM HPV18.E5.52 57 178 5276 -

44 LYNLLIRCF HPV18.E6.98.F9 10 32 - 2255

18 LYGVSFSEL HPV16.E1.214 7,2 3,1 - -

19 RFHNIRGRF HPV16.E6.131.F9 2,4 29 346 0,69

20 VYDFAFRDLCI HPV16.E6.49 8,9 22 - -

21 PYAVCDKCF HPV16.E6.66.F9 6,1 7,2 641 157

22 CYSLYGTTF HPV16.E6.87.F9 1 1 28 2088 7823

61 PYLHSRLWF HPV31.E1.557 2,8 38,7 4365 2229

62 VFTFPNPFPF HPV31.E1.565 12 17 603 -

63 HYTNWKFIF HPV31 .E2.130.F9 16 8,5 433 4847

64 WFIYIPLF HPV31 .E5.64 108 276 302 268

65 FYSKVSEFRW HPV31 .E6.69 8,8 4,5 1777 1361

66 VYGTTLEKL HPV31 .E6.83 8,2 26 - 1237

84 VFTFPHAFPF HPV45.E1.578 15,9 90,4 57 -

85 YYITETGIW HPV45.E2.144 2 8,5 - -

86 VYVCAFAWLL HPV45.E5.26 8,1 8,4 2346 -

87 VYQFAFKDL HPV45.E6.44 1 ,1 4 - 165

88 FYSRIRELRF HPV45.E6.71.F10 1 3,2 83 - - : IC50 > 20 μM

Table 7: Binding data of HTL epitopes (1): binding data shown for the 15-mer (IAA less at the carboxy-terminus) (2): binding data for crude peptide batch

IC ₅₀ nM to purified HLA

No.

DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB1 DRB3 DRB4 DRB5

SEQ ID Sequence Source al l el es '0101 ^* θ3θ1 ^* θ4θ1 ^* θ4θ4 ^* θ4θ5 ^* θ7θ1 ^* θ8θ2 ^* θ9θ1 '1101 '1201 ^* 1302 '1501 ^* 0101 ^* 0101 ^* 0101 bound

89 LYWYKTGISNISEVY HPV16. E1 .319 13 12.666 6,2 3543 96 25 2965 96 2757 938 1754 19.572 588 7

96 PEWIQRQTVLQHSFN HPV16. E1 .337 2G86 11 .652 14 21 161 -- 137 3181 117 662 9450 4,3 1528 7

91 WKHMRLECAIYYKAR HPV16. E2.033 7θ1 437 245 6858 557 564 96 6335 2401 248 1393 766 280 9

95 LQAIELQLTLETIYN (2) HPV16. E2.G7G 42 534 37 282 384 3286 7612 1433 4378 268 17 688 2 - 9

93 GLYYVHEGIRTYFVQ HPV16. E2.156 67 17.766 322 -- 1876 55 3143 431 5331 -- 156 -- 1000 6

94 QRFHNIRGRWTGRCM HPV16. E6.13θ 36 -- 2897 4116 4469 277 894 -- 574 14.594 5308 17.214 -- 4553 919 5

95 LDLQPETTDLYCYEQ HPV16. E7.13 -- 596 1964 -- -- -- -- -- -- -- -- -- -- 17 -- 2

96 LCTELQTTIHDIILE HPV16. E6.22 2714 86 3662 7966 -- -- -- -- 5529 -- -- 1044 -- 1

97 IRTLEDLLMGTLGIV HPV16. E7.76 142 449 677 277 2668 4618 -- -- -- 950 1548 10.272 4236 923 -- 6

98 FKTLIQPFILYAHIQ HPV18. E1 .258 54 -- 949 111 3255 1696 6666 1226 6695 4286 7,9 317 11 .878 5

99 IEFITFLGALKSFLK HPV18. E1 .458 36 -- 168 32 3645 -- 11 .661 -- 1613 -- 49 1229 664 5

1GG FLNTVAIPDSVQILV HPV18. E2.346 718 68 269 555 616 15 -- 534 -- 90 8957 91 446 -- 10

1θ1 HKAIELQMALQGLAQ HPV18. E2.θ74 41 5648 56 27 - - 811 12.764 844 3536 573 3 126 8

1G5 FQQLFLNTLSFVCPW HPV18. E7.86 2G4 6522 139 496 376 237 8996 1325 6522 11 555 586 17.298 8

1θ6 LFWYRDSIPHAACHK(1 ) HPV18. E6.52 356 73 4,7 28 2159 2666 168 8598 786 566 64 15.242 3770 8

1G2 EVFEFAFKDLFWYR HPV18. E6.43 2865 73 347 553 1326 14.415 1415 -- 6679 624 165 1522 -- 316 -- 6

1θ7 TNTGLYNLLIRCLRCQ(I ) HPV18. E6.94 367 9439 1485 62 466 4638 41 13.626 17 4057 719 36 19.877 366 3656 8

1G6 NGWFYVEAVIDRQTG HPV31 . E1 .15 1859 2797 845 18.761 178 1459 -- 3676 -- -- -- 627 1203 3

1θ7 PEWIERQTVLQHSFN HPV31 . E1 .317 5969 3857 35 23 127 -- 954 1767 535 2855 13.492 23 6231 6

1G8 WKHIRLECVLMYKAR HPV31 . E2.G33 1444 146 263 367 71 16449 476 11831 81 54 134 102 412 10

1θ9 TTPIIHLKGDANILK HPV31 . E2.292 168 74 8823 627 19514 3714 4486 17365 7331 527 205 4755 49 -- 6

112 IRILQELLMGSFGIV HPV31 . E7.76 56 5376 361 143 3668 267 7543 141 4637 398 94 24 5383 8

111 VLDFAFTDLTIVYRD HPV31 . E6.42 7561 36 938 3369 2839 269 14.678 796 13.666 NT 753 2622 NT 307 -- 6

110 TGRCIACWRRPRTET HPV31 . E6.132 3167 1633 758 1146 733 11 .466 512 13.556 158 4949 2771 84 5961 225 6047 6

113 PRKLHELSSALEIPY HPV31 . E6.9 183 1435 1557 117 32 62 17.333 342 2126 59 299 835 13.207 8

114 DWVMAIFGVNPTVAEGF HPV45. E1 .228 487 3749 91 26 92 52 4265 17945 4957 68 10 571 387 9

115 FKTLIKPATLYAHIQ HPV45. E1 .244 269 5197 316 111 2234 148 11 8163 24 164 195 69 1004 9

116 PINISKSKAHKAIEL HPV45. E2.67 122 1 .668 255 97 1912 17 3172 176 327 309 231 -- 1724 452 9

117 TIPNSVQISVGYMTI HPV45. E2.354 52 483 515 18 196 9 6226 91 487 14 10 10775 16 8328 11

118 LCIVYRDCIAYAACH HPV45. E6.52 462 964 481 1643 762 1686 1164 2156 1867 965 262 1525 673 7

119 FHSIAGQYRGQCNTC HPV45. E6.127 167 -- 8,6 3365 358 2463 7644 1992 416 10.126 -- 574 -- 1296 43 6

12G EIVLHLEPQNELDPV HPV45. E7.1θ 967 354 12.652 947 273 3496 -- -- -- -- 5352 -- -- 713 -- 5

121 LRTLQQLFLSTLSFV HPV45. E7.84 41 3388 127 19 166 39 4161 394 1675 33 17 21 1254 9

(1): binding data shown for the 15-mer (1AA less at the carboxy-terminus) (2): binding data for crude peptide batch

Example 2: Poly epitope construct design

Each designed DNA construct contains HLA-restricted epitopes which bind to at least one HLA molecule with an affinity < 500 nM and preferably less than 100 nM (tables 3-6 in Example 1). Most of these epitopes were demonstrated to be immunogenic in the respective HLA transgenic mice when administered as a pool of peptides emulsified in IFA (tables 8-9 in Example 3).

The epitope order and amino acid spacers in the constructs were designed to avoid generation of junctional epitopes and to maximize proteosomal processing. The amino acid sequences were back-translated using a mammalian codon usage table and the on-line back-translation tool both provided by Entelechon (Germany). The DNA sequences were inserted into pMB75.6 vector using Pstl and BamHI restriction sites. A Kozak sequence, a mouse Igkappa signal sequence (MGMQVQIQSLFLLLL WVPGSRG, SEQ ID NO 131), and a stop codon were also included. The respective amino acid and DNA sequences of the constructs ICCG6137, ICCG6138, ICCG6149 and ICCG6150 are given in Figures 5-8.

Example 3: Immunogenicity of HPV-derived HLA-class I-restricted epitopes encoded in a HPV DNA polyepitope construct

The immunogenicity of HPV HLA class I-restricted epitopes encoded in the DNA constructs was tested in the relevant HLA transgenic mice. The evaluation of the immunogenicity of HPV-derived HLA- A02-, HLA-AI l- and HLA- A24 and HLA-AOl -restricted epitopes encoded in different DNA constructs was done using the following protocol. HLA transgenic mice (Fl HLA-A02/KbxBalb/c, HLA-Al I/Kb, HLA A01/Kb and HLA-A24/Kb transgenic mice) were immunised with one of the selected DNA constructs. To ensure an equal distribution of mice between different groups, a randomisation procedure based on body weight was performed. Female and male mice (age between 8 and 24 weeks) were ranked by body weight, extreme light or heavy animals were excluded. The remaining animals were grouped by sequentially assigning animals for the 2 experiments.

DNA immunization

Mice were pre-treated with cardiotoxin (Sigma, C9759) on day - 4 by bilateral intramuscular injection of lOOμl (2x50μl) of a lOμM cardiotoxin solution. Four days later, all mice were immunised with lOOμg HPV-DNA plasmid (diluted to 1 mg/ml in PBS) by bilateral injection

of 50μg in the m. tibialis anterior. Mice were euthanised between 13 and 15 days after the DNA immunisation.

Peptide immunization Peptides were dissolved in DMSO at a concentration of 10 mg/mL. For each of the 4 DNA constructs, the corresponding HLA-restricted peptides were pooled per HLA type (5 to 7 CTL peptides/pool). A known HTL peptide (TPPAYRPPNAPIL) was added and the peptide pools were diluted in PBS to the required peptide concentration (i.e. 25 μg of each CTL peptide and 120 μg HTL peptide in 50 μl). After adding an equal volume of IFA (Pierce, lot DG56079), the mixture was emulsified by forcing it through a small orifice. Mice were immunized subcutaneously at the base of the tail with 50 μl peptide/IFA mixture.

Immunization scheme

From each line, 4 groups of 12 mice (HLA- A02 and HLA- A24) or 6 mice (HLA-Al 1) were immunized intramuscularly with one of the 4 HPV DNA constructs (ICCG6149, ICCG6150, ICCG6137, ICCG6138) respectively. For HLA AOl, only 2 constructs were evaluated. To this, 2 groups of 12 mice were immunized intramuscularly with either ICCG6150 or ICCG6138. A final group of 12 mice was not DNA- immunized nor peptide-immunized and was included as a negative control (CT treatment only) For the evaluation of the peptide immunogenicity, 4 groups of 12 mice (HLA-AOl), 6 mice (HLA- A02 and HLA- A24) or 3 mice (HLA-Al 1) were injected subcutaneously with one of the HLA-restricted peptide pools in IFA. As negative control for the peptide-immunized mice, 18 mice (in total) were injected with a PBS/IFA mixture.

In vitro experimental set-up

Mice were euthanized and spleen cells (SPC) were isolated 13 days after immunization. SPC of DNA- and peptide-immunized HLA-A01/Kb, Fl HLA-A02/KbxBalb/c and HLA-A24/Kb mice were pooled per 2 mice, resulting in 6, 6 and 3 data points per condition, respectively. Spleen cells of DNA- and peptide-immunized HLA-Al I/Kb mice were analyzed individually. SPC of all negative control mice were pooled per 2 mice, resulting in 3 data points for each peptide tested. CD8+ cells were purified by positive magnetic bead selection on SPC, using CD8a MicroBeads (MACS 130-049-401) according to the manufacturer's protocol.

A direct ex vivo IFNγ ELISPOT assay was used as surrogate CTL readout. Basically, purified CD8+ cells were incubated with the individual HPV-specific HLA-restricted peptides (10 μg/mL) loaded on the appropriate antigen presenting cells (APC), in anti-mouse IFNγ antibody-coated ELISPOT plates. Because of the limited availability, purified CD8+ cells of peptide-immunized HLA-Al I/Kb mice were seeded in the coated ELISPOT plates at 10 ⁵ cells/well, whereas for all other treatment groups, 2x10 ⁵ CD8+ cells/well were used. After 20 h incubation, IFNγ-producing cells were visualized by further developing the plates with biotinylated anti-mouse IFNγ antibody, streptavidin-HRP and AEC as substrate. APC used for the different HLA-restricted peptides were spleen cells from non- immunized HLA-A01/Kb mice for HLA-AOl, JA2.1Kb cells (2xlO ⁴ cells/well) for HLA-A02, LCL 721.22 IHLA Al I/Kb (10 ⁴ cells/well) for HLA-Al 1, and LCL 721.22 IHLA A24/Kb (10 ⁴ cells/well) for HLA- A24.

Some of the CTL epitopes included in the DNA constructs are analoged sequences of the wild type viral epitopes. For these particular epitopes, only the wild type sequences were used as in vitro stimulus for CTL readout. In addition, 3 HLA-A24-restricted sequence variant epitopes were used as in vitro stimulus to check for potential cross-variant reactivity. Baseline responses towards all HLA-restricted peptides used for in vitro readout were evaluated in naϊve HLA-AO I/Kb, Fl HLA-A02/KbxBalb/c, HLA-Al I/Kb, or HLA-A24/Kb transgenic mice. No significant responses could be detected (results not shown).

Data analysis

Peptides eliciting a specific delta CTL response of > 30 specific spots/10 ⁶ CD8 cells and a response ratio > 2 in at least one pool are categorised as immunogenic. A minimum of 2 pools is to be tested.

Results

The results for the different HPV constructs are shown in table 8 and 9. The majority of epitopes that bind with high affinity to the HLA (IC50 less than 500 nM and preferably less than 100 nM) are able to induce positive T cell responses in the respective HLA transgenic mice, indicating that HLA binding affinity is a proper selection criterion for identifying potential immunogenic epitopes. These immunogenic responses can be induced with the isolated peptides and/or with a plasmid DNA encoding these peptides.

Table 8: Immunogenicity data for HLA class-I- restricted HPV 16 and HPV 18 epitopes encoded in constructs ICCG 6149 and ICCG 6150

775 7,8 115 4,9 170 1,7

38 ATLQDIVLH (10-1290) (i.1-40.5) Y (1 - 1420) (i.0-36 .5) Y (1 - 440) (1.0-2.2) Y

1 1,0 15 1,4 540 22,0

12 WLLLVRYK (1-40) (1 .0-2.0) N (1-40) (1.0-2. 0) Y (420 - 630) (14.5-64.0) Y

1 1,0 40 1,6 320 17,0

16 SVICFVNSK (1 - 20) (1 .0-2.0) N (1-110) (1 .0-5. 0) Y (320 - 620) (9.0-63.0) Y

1 1,0 10 1,3 170 1,5

32 KQGAMLAVFK ((1-20) (1.0-1.4) N (1 - 150) (1.0-1. 5) N (1 - 220) (1.0-1.9) N

1 1,0 1 1,0 110 1,3

138 FWYRDSIPH (1-20) (1 .0-2.0) N (1 - 90) (1 .0-5. 5) Y (1-310) (1 .0-2.2) Y

HLA-A24

18 1200 21,1 1120 20,7 685 13,5

LYGVSFSEL (915-1510) (17 .6-25.3) Y (250-1565) (48-33 .8) Y (590-1070) (10.1-31.6) Y

40 5 1,1 3 1,1 135 2,7

J-. YYMTDAGTW (1-15) (1.0-1.2) N (1-20) (1.0-1. 3) N (1-135) (1.0-3.3) Y

41 80 2,2 113 3,0 110 2,2

GYNTFYIEF (1-230) (1.0-5.6) Y (1-435) (1.0-9. 7) Y (50-110) (1.6-2.8) Y

39 808 14,1 498 8,3 65 1,8

SYFGMSFIHF (245-1070) (5.5-19.7) Y (81-1150) (32-24 .0) Y (45-175) (1 .5-3.9) Y

139 3 1,0 1 1,0 1 1,0

CYSLYGTTL (1-5) (1.0-1.1) N (1 - 10) (1.0-1. 3) N (1-1) (1.0-1.0) N

20 435 8,0 760 14,0 1 1,0

VYDFAFRDLCI (285-1005) (5.3-21.1) Y (155-1075) (36-20 •4) Y (1-110) (1.0-4.1) N

140 3 1,1 1 1,0 150 4,2

PYAVCDKCL (1-25) (1.0-1.4) N (1-5) (1.0-1. 1) N (35-175) (1.5-5.3) Y

141 3 1,1 1 1,0 205 6,9

RFHNIRGRW (1-35) (1.0-1.5) N (1-20) (1.0-1. 4) N (75 - 325) (2.2-6.9) Y

42 1 1,0 1 1,0 1 1,0

MYVCCHVPL (1-15) (1 .0-1.2) N (1-25) (1.0-1. 4) N (1-1) (1.0-1.0) N

43 1 1,0 1 1,0 365 5,3

VYVFCFLLPM (1-1) (1.0-1.0) N (1-6) (1.0-1. 2) N (345 - 505) (5.1-9.4) Y

142 1 1,0 N 1 1,0 170 3,8

LYNLLIRCL (1 - 10) (1 .0-1.2) (1-1) (1.0-1. 0) N (100-450) (2.3-6.0) Y

3 1, 0 1 1, 0 30 1, 5

143 LYNLLIRWL (1 -10) (1.0- -1 •2) N (1 -i) (1.0- -1. 0) N (10 -40) (1.1- -1. 5) N

3 1, 0 1 1, 0 5 1, 1

144 LYGVSFTEL (1 -5) (1.0- -1 •1) N (1 -20) (1.0- -1. 4) N (1 -15) (1.0- -1. 2) N

23 1, 4 90 2, 6 55 1, 8

145 CYSVYGTTL (10 -35) (1.2- -1 .6) N O- -865) O- 0- 13 •4) Y O- -150) (1.0- -5. 3) Y

( ^b ): results expressed as: : median, minimum and maximum CTL responses from the individual mouse pools

(min-max)

Immun = Immunogenic

Table 9: Immunogenicity data for HLA class-I- restricted HPV 31 and HPV 45 epitopes encoded in constructs ICCG 6137 and ICCG 6138

46 1, 9 Y 93 1, 3 Y 715 12 ,8

64 WFIYIPLF (1 - 65) (1.0- -2.8) (5 -555) (1.0- -7 .5) (710-1055 (6.3- 21 •4) Y

712 16 ,4 Y 75 1, 4 Y 125 6, 0

86 VYVCAFAWLL (535 - 870) (H .8- - 29.2) (35 -410) (1.2- -5 .8) (105-295) (4 .5- -9 4) Y

45 2, 0 Y 33 1, 3 Y 165 7, 3

154 FYSRIRELRY (1-210) (1.0- -9.4) (1 -420) (1.0- -5 •9) (115-220) (4.8- -7 6) Y

Immun = Immunogenic

Example 4: HTL epitope recall responses in HPV infected women

The purpose of this set of experiments is to demonstrate in vitro human recall HTL responses in a panel of HPV-infected subjects towards the final selection of HTL epitopes comprised in the HPV polyepitope constructs, using an optimized human T-cell proliferation assay.

Following table 10 gives an overview of the peptides used in the T-cell proliferation assay. Peptide sequence and source are listed. All peptides are dissolved in 100% dimethyl sulfoxide (DMSO) at a concentration of 5, 10, or 20 mg/ml and stored at -20 ⁰ C. Peptides were used in the T-cell proliferation assay at a final concentration of lOμg/ml. As a positive control antigen , Tetanus Toxoid (TT) was used at a final concentration of 3 μg/ml.

Table 10

PBMC from HPV-infected subjects, currently or previously diagnosed with cervical intraepithelial neoplasia (CIN) 1, 2, or 3, and preferentially HPV-genotyped were used to determine the HPV-specific HTL responses. PBMC were thawed following standard procedures for use in the T-cell proliferation assay.

PBMC samples from HPV-infected subjects were screened for in vitro recall HTL responses towards the whole panel of HTL peptides using a T-cell proliferation assay. Briefly, 5x10 ⁴ cells/well were seeded in 5-fold replicates, in round bottom plates in RPMI total (= RPMIbic + non essential amino acids (NEAA) + sodium pyruvate + gentamycine + beta mercapto ethanol (βME)), supplemented with 5% heat-inactivated human AB serum (ihuAB), and incubated with 10 μg/ml HTL peptide or 3 μg/ml TT during 6 days in a CO2 incubator at 37°C. After this incubation period, ³ H-thymidine (1 μCi/well) was added for overnight labeling (18 hours). Then cells were harvested and the amount of incorporated ³ H-thymidine was measured. TT was included as a positive control, as most people are expected to show a response towards this antigen. Additionally, cells from a healthy control PBMC sample were seeded (5xlO ⁴ c/well, 5-fold replicates) and stimulated with TT and Varicella Zoster in order

to have an internal assay control on each plate and to evaluate inter and intra assay variability.

Data analysis - All raw data points, calculated and reported values are collected in an Excel database per experimental setup.

The magnitude of the antigen-specific response as determined in the T-cell proliferation assay is expressed as stimulation index (the median cpm of stimulated/non- stimulated cultures) and delta cpm (the median cpm value of stimulated wells subtracted from the median cpm value of non- stimulated wells).

A response is considered positive when the delta cpm values > 200 cpm and the stimulation index is > 2.

The highest, positive responses per HTL epitope are shown (SI values only) in Figure 9. These data clearly show that - although tested in only a limited set of blood samples from HPV infected patients - T cell reactivity can be picked up for the majority of the selected HTL epitopes.

REFERENCES

Alexander, J. et al, J Immunol. 159:4753, 1997

Alexander J. et al., Hum Immunol 64(2): 211-223, 2003

An, L. and Whitton, J. L., J Virol. 71 :2292,1997 Bertoni, R. et al., J Clin. Invest. 100:503, 1997

Beaucage & Caruthers, Tetrahedron Letts. 22:1859- 1862, 1981

Bowen, DG and Walker, MW, Nature, 436:946, 2005

Celis, E. et al., Proc. Natl. Acad Sci. USA 91:2105, 1994

Diepolder, H. M. et al., J Virol. 71 :6011, 1997 Donnelly JJ, et al. Annu Rev Immunol. 1997;15:617-48.

Donnelly JJ, Ulmer JB, Liu MA. DNA vaccines. Life Sci. 1997a; 60(3): 163-72.

Doolan, D. L. et al., Immunity 7:97-112, 1997

Doorbar J., J Clin Virol 32S:S7-S15, 2005

Feigner, et al., Proc. Nat'l Acad. Sci. USA 84:7413, 1987 Gaberc-Porekar V. et al., J. Biochem. Biophys. Methods 49: 335-360, 2001

Gruters RA, van Baalen CA, Osterhaus AD.Vaccine. 2002 May 6;20(15):2011-5. The advantage of early recognition of HIV-infected cells by cytotoxic T-lymphocytes.

Hanke, R. et al., Vaccine 16:426, 1998

Houssaint E, Saulquin X, Scotet E, Bonneville M Biomed Pharmacother. 2001 Sep; 55(7): 373- 80. Immunodominant CD8 T cell response to Epstein -Barr virus.

Ishioka, et al., J. Immunol. (1999) 162(7):3915-3925

Kawashima, 1. et al., Human Immunol. 59:1, 1998

Kriegler M. Gene transfer and expression: a laboratory manual. W.H. Freeman and Company,

New York, 1991 : 60-61 and 165-172. Lichty J.J. et al., Protein Expression and Protein Purification 41 : 98-105, 2005

Mannino & Gould- Fogerite, BioTechniques 6(7): 682, 1988

Mateo et al., J Immunol, 163(7): 4058-63, 1999

Murray N, McMichael A. Curr Opin Immunol. 1992 Aug;4(4):401-7. Antigen presentation in virus infection. Pearson & Reanier, J. Chrom. 255 : 137-149, 1983

Rehermann, B. et al., J Exp. Med. 181:1047, 1995

Rowen, P. and Lacey, C, Dermatologic Clinics 16 (4): 835-838,1998

Sette, et al, J Immunol 153:5586-5592, 1994

Sette, et al., MoI. Immunol. 31 : 813 (1994).

Sidney et al, Current Protocols in Immunology, Ed., John Wiley & Sons, NY, Section 18.3

(1998)

Sidney, et al., J Immunol. 154: 247 (1995)

Southwood et al. J Immunology 160:3363-3373,1998 Stemmer WP et al. Gene 164 : 49-53 , 1995

Stover et al., Nature 351:456- 460, 1991

Threlkeld, S. C. et al., J Immunol. 159:1648, 1997

Tigges MA, et al., J Virol; 66(3): 1622-34 1992

Thomson et al., Proc Natl Acad Sci USA, 92(13):5845-9, 1995 Thomson, S. A. et al., J Immunol. 157:822, 1996

Tsai SL, Huang SN. J Gastroenterol Hepatol. 1997 Oct;12(9-10):S227-35. T cell mechanisms in the immunopathogenesis of viral hepatitis B and C.

Tsai, V. et al., J Immunol. 158:1796, 1997

Van Devanter et. al., Nucleic Acids Res. 12:6159-616S, 1984 Velders MP et al. J Immunol, 166(9): 5366-73, 2001

Wentworth, P. A. et al., MoI. Immunol. 32:603, 1995

Wentworth, P. A. et al. , J Immunol. 26:97, 1996

Whitton, J. L. et al., J Prol. 67:348, 1993

Woodberry et al, J Virol, 73(7):5320-5, 1999