JP2020039341A - Vaccines for human papilloma virus and methods for using the same - Google Patents

Abstract

To provide improved anti-HPV immunogens and nucleic acid molecules that encode the improved anti-HPV immunogens.SOLUTION: A nucleic acid molecule comprises a nucleotide sequence encoding an HPV antigen selected from the group consisting of fragments having specific sequences that are 90% homologous to specific sequences and combinations thereof.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an improved vaccine of human papillomavirus (HPV), an improved method for inducing an immune response to HPV, and an improved method for immunizing an individual prophylactically and / or therapeutically against HPV.

  Papillomaviruses are small DNA viruses that contain up to seven early genes and two late genes. Generally, the early genes of papillomavirus are named E1-E7, and the late genes of papillomavirus are named L1 and L2. Several animal species can be infected by members of the papillomavirus family.

  Human papillomavirus (HPV) infection is routine and can be transmitted by sexual contact. HPVs were classified into 56 or more types based on DNA sequence homology. HPV types 16 and 18 that cause epithelial dysplasia and other damage often increase the risk of cancer, especially intraepithelial and invasive cancers of the cervix, vagina, vulva and anal canal. Related. Worldwide, nearly 88% of cervical cancers are due to HPV subtypes 16, 18, 45, 31, 33, 52 and 58. In addition, various studies have revealed the presence of HPV6 and HPV11 in most outbreaks of recurrent respiratory papillomatosis. HPV6 and HPV11 are known to be associated with genital warts and are found in only a small percentage of cases of cervical cancer, but in 2.6-5.2% of all cases of mild cervical lesions . In addition, HPV6 and HPV11 are now considered for the treatment of mild squamous intraepithelial lesions, now considered precursors to cervical cancer, including grade 2 and grade 3 cervical intraepithelial neoplasia and mild cervical dysplasia. It is associated with about 20%. As the study progresses, these two serotypes will become genital warts, recurrent respiratory papillomatosis, lung cancer, tonsil cancer, laryngeal cancer, and other malignant transformations of otherwise benign tumors and head and neck differences It has been shown to be associated with various forms of otologic disease, including formation. The findings of this study reveal the use of a consensus sequence DNA vaccine against HPV6 and HPV11, and promise its future promise.

  DNA vaccines have many conceptual advantages over more traditional methods of vaccination, such as live attenuated virus vaccines and vaccines based on recombinant proteins. DNA vaccines are safe, stable, readily produced, and well-tolerated in humans, and preclinical studies show no evidence of plasmid integration (Martin, T., et al., Plasmid DNA). malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther, 1999.10 (5): p.759-integration, W.e.n., lt. NY Acad Sci, 1995.772: 30-30). Furthermore, due to the fact that the efficacy of DNA vaccines is not affected by pre-existing antibody titers against the vector, DNA vaccines are well suited for repeated administration (Cattergoon, M., J. Boyer, and D.B. Weiner, Genetic immunization: a new era in vaccines and immunotherapeutics. FASEB J, 1997.11 (10): p.753-63). However, when transferred to large animals, one significant obstacle to the clinical introduction of DNA vaccines was the reduction in platform immunogenicity (Liu, MA and JB Ulmer, Human clinical trials). of plasmid DNA vaccines. Adv Genet, 2005.55: 25-40). Recent technological advances in genetic engineering of DNA vaccine immunogens, such as codon optimization, RNA optimization and the addition of immunoglobulin leader sequences, have improved the expression and immunogenicity of DNA vaccines (Andre, S., et al. et al., Included immune response elicited by DNA vaccination with a synthetic gp120 sequence with optimized codon usage, co., et al., 1998.e.l, et. usage optimization on expression and immunogenici Yof DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol, 2001.75 (22): p.en, oid, ov, kon, et. against avian influenza.Vaccine, 2007.25 (16): p.2984-9; Frelin, L., et al., Codon optimization and mRNA amplification enhancement activities. of the hepatitis C virus nonstructural 3 / 4A gene.Gene Ther, 2004.11 (6): p.522-33), and in recent years, techniques for plasmid delivery systems such as electroporation have been developed (Hirao, L. , A., et al., Intradermal / subcutaneous immu nization by electroporation improv- es plasm vaccine delivery and potency in pigues and gasses. Vaccine, 2008.26 (3): p. 440-8; Luckay, A .; , Et al. , Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immunoresponse in massage. J Virol, 2007.81 (10): p. 5257-69; Ahlen, G .; , Et al. , In vivo electroporation enhancements the immunogenicity of hepatitis C virus nonstructural 3 / 4A DNA by incorporation of related DNA uptake and proofing in tandem. J Immunol, 2007.179 (7): p. 4741-53). In addition, several studies have suggested that the use of consensus immunogens may be able to extend the breadth of cellular immune responses compared to natural antigens alone (Yan., J. et al. , Et al., Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope, DNA DNA et al., 21: Mol. Reconstruction and function of ancestor center-of-tree human immunodeficiency virus type proteins.J Virol, 2007.81 (16): p.8507-14).

  There remains a need for improved vaccines and methods for preventing and treating HPV infection, particularly infections that cause cervical and / or lung, tonsil, and pharyngeal cancer.

  An embodiment of the present invention encodes an HPV antigen selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7; fragments thereof having 90% homology; and combinations thereof. Nucleic acid molecules that include a nucleotide sequence are included. In some embodiments, these nucleic acid molecules comprise at least a nucleotide sequence encoding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8. Nucleotide sequence encoding an amino acid fragment having 90% homology; fragments of the nucleotide sequence encoding SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 Or a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of fragments of a nucleotide sequence encoding an amino acid fragment having at least 90% homology to SEQ ID NO: 8.

  In some aspects, there is provided a pharmaceutical composition comprising a nucleic acid molecule described herein.

  In another aspect, there is provided a method of inducing an immune response to HPV in an individual, the method comprising administering to the individual a composition comprising a nucleic acid molecule described herein.

  The invention also includes a recombinant vaccine comprising a nucleic acid molecule described herein. These recombinant vaccines include recombinant vaccinia vaccines, live attenuated vaccines, and DNA plasmids.

  In some aspects of the invention, SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; and at least 90% homology to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 A protein comprising an amino acid sequence selected from the group consisting of fragments of the sequence having the amino acid sequence.

  Also provided is a method of inducing an immune response to HPV in an individual, the method comprising administering to the individual a recombinant vaccine provided herein.

  In some embodiments, there is provided a pharmaceutical composition comprising a nucleotide sequence encoding the HPV antigen of SEQ ID NO: 1 and SEQ ID NO: 3; or a fragment thereof; or a fragment having 90% homology thereto. Also provided is a method of inducing an immune response to HPV subtype 6 or 11 in an individual, the method comprising administering to the individual a pharmaceutical composition as described above.

  In some embodiments, there is provided a pharmaceutical composition comprising a nucleotide sequence encoding the HPV antigen of SEQ ID NO: 5 and SEQ ID NO: 7; or a fragment thereof; or a fragment having 90% homology thereto. Also provided is a method of inducing an immune response to HPV subtype 33 or 58 in an individual, the method comprising administering to the individual a pharmaceutical composition as described above.

FIG. 1 is a phylogenetic tree based on neighborhood binding evaluation of E6 and E7 alignments (A and B, respectively). The asterisk indicates the position of the consensus sequence in each tree. FIG. 2 is data showing in vivo expression of p6E6E7 and p11E6E7. Gene products were isolated from lysed transfected 293-T cells, passed through an SDS-PAGE gel, and detected using autoradiography. Both the HPV6 and HPV11 E6 / E7 proteins are each approximately 32 kDa (A). Human rhabdomyosarcoma (RD) cells were also transfected with p6E6E7 and p11E6E7, and then fixed after immunofluorescent staining. FITC fluorescence confirms expression of p6E6E7 and p11E6E7 (B). DAPI fluorescence confirms nuclear localization by Hoechst staining. FIG. 3 shows that IFN-γ ELISpot assay induces strong cellular responses by p6E6E7 (A) and p11E6E7 (B) in C57BL / 6 mice. After three immunizations biweekly, assays were performed using splenocytes isolated from each group of mice (5 per group). Each immunization consisted of 20 μg per construct. The mice in the combination group received both p6E6E7 and p11E6E7 at 20 μg per construct for a total of 40 μg DNA per vaccination. DNA was administered by IM injection, followed by electroporation ( ^* indicates P <0.0001, Δ indicates P = 0.0001). FIG. 4 performed an additional IFN-γ ELISpot assay using individual peptides to characterize the dominant epitope. Splenocytes isolated from immunized mice and a negative control were stimulated with overlapping peptides spanning the complete HPV6 E6 / E7 fusion protein (A) or HPV11 E6 / E7 fusion protein (B). FIG. 5A depicts cytokine production by antigen-specific T cells characterized by intracellular cytokine staining. Splenocytes isolated from mice immunized with p6E6E7 were stimulated with R10 growth medium, PMA, or peptides of the consensus gene for 4 hours, and then stained with surface and intracellular markers. The upper dot spot shows the difference in the percentage of IFN-γ, IL-2, and TNF-α produced by either total CD4 + cells or CD8 + cells minus background. The P value of the plot with an asterisk could not be determined because the mean value of the negative control group was 0. FIG. 5B depicts cytokine production by antigen-specific T cells characterized by intracellular cytokine staining. Splenocytes isolated from mice immunized with p11E6E7 were stimulated with R10 growth medium, PMA, or peptides of the consensus gene for 4 hours, and then stained with surface and intracellular markers. The upper dot spot shows the difference in the percentage of IFN-γ, IL-2, and TNF-α produced by either total CD4 + cells or CD8 + cells minus background. The P value of the plot with an asterisk could not be determined because the mean value of the negative control group was 0.

  As used herein, the phrase "stringent hybridization conditions" or "stringent conditions" refers to conditions under which one nucleic acid molecule will hybridize to another nucleic acid molecule but will not hybridize to another sequence. Represents Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected to be about 5 ° C. below the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to a target sequence hybridize to this target sequence at equilibrium. Generally, 50% of these probes occupy at equilibrium since these target sequences are present in excess at the Tm. Typically, stringent conditions are at pH 7.0-8.3, wherein the salt concentration is less than about 1.0 M sodium ion, typically about 0.01-1.0 M sodium ion (or other And a temperature of at least about 30 ° C. for short probes, primers or oligonucleotides (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes, primers or oligonucleotides. Will. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

Nucleotide and amino acid sequence homology was determined by FASTA, BLAST and Gapped BLAST (Altschul et al., Nuc. Acids Res., 1997, 25, 3389, which is incorporated herein by reference in its entirety) and PAUP. ^* Measurements may be performed using 4.0b10 software (DL Swofford, Sinauer Associates, Massachusetts). "Percentage of similarity" is calculated using PAUP ^* 4.0b10 software (DL Swofford, Sinauer Associates, Massachusetts). The average similarity of the consensus sequence is calculated relative to the entire sequence of the phylogenetic tree.

  Briefly, the BLAST algorithm, which represents the Basic Local Alignment Search Tool, is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410, which is hereby incorporated by reference. Which is hereby incorporated by reference in its entirety). Software for performing BLAST analysis is published through the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). The algorithm is such that when aligned with words of the same length in the database sequence, the length of the query sequence that matches or satisfies some positive-valued threshold score T Identifying short words of length W involves first identifying a high-scoring sequence pair (HSP). T is referred to as the neighborword score score threshold (Altschul et al., Supra). These first neighbor word hits serve as seeds for initiating searches to find HSPs containing them. This word hit is expanded in both directions along each sequence as long as the cumulative alignment score can be increased. Expansion of word hits in each direction is stopped if: 1) the cumulative alignment score drops X from its maximum attainment; 2) one or more negative score residue alignments (negative). -Accumulation of scoring resin alignments results in a cumulative score of 0 or less; or 3) reaching the end of any sequence. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. In this Blast program, as an initial setting, the word length (W) is 11, and the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is referred to. The alignment (B) is 50, the expected value (E) is 10, M = 5, N = 4, and a comparison of both strands is used. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873-5787, which is incorporated herein by reference in its entirety) and Gapped BLAST have two sequences. A statistical analysis of the similarity between them is performed. One measure of similarity provided by this BLAST algorithm is the smallest sum probability (P (N)) that gives an indication of the probability that a match between two nucleotide sequences occurs by chance. If the minimum total probability of comparison of a nucleic acid to another nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the nucleic acid is another Considered to be similar to nucleic acids.

  The term "gene construct" as used herein refers to a DNA or RNA molecule that contains a nucleotide sequence that encodes a protein. A coding sequence includes start and stop signals operably linked to regulatory elements, including promoters and polyadenylation signals, that can direct expression in the cells of the individual to which the nucleic acid molecule is administered.

  The term "expressible form" as used herein refers to an essential regulatory sequence operably linked to a coding sequence encoding a protein such that the coding sequence is expressed when present in the cells of the individual. Represents the gene construct containing.

  Disclosed are improved vaccines resulting from a pleiotropic strategy to enhance the cellular immune response induced by the immunogen. A modified consensus sequence was created. Also disclosed are genetic modifications including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences. This novel construct was designed to elicit a more potent and broader cellular immune response than the corresponding codon-optimized immunogen.

  Improved HPV vaccines are based on proteins having epitopes that make the improved vaccines particularly effective as immunogens capable of inducing anti-HPV, and gene constructs encoding proteins having such epitopes. Thus, a vaccine can induce a therapeutic or prophylactic immune response. In some embodiments, the means for delivering the immunogen includes a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, an attenuated vaccine or an inactivated vaccine. In some embodiments, the vaccine comprises one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, one or more compositions comprising the immunogen, one or more And a combination selected from the group consisting of an attenuated vaccine and one or more inactivated vaccines.

  According to some embodiments, the vaccine is delivered to an individual and modulates the activity of the individual's immune system, thereby increasing the immune response to HPV. When the nucleic acid molecule encoding the protein is taken up by the cells of the individual, the nucleotide sequence is expressed in the cells, thereby delivering the protein to the individual. Methods are provided for delivering the coding sequence for this protein on a nucleic acid molecule, such as a plasmid, as part of a recombinant vaccine and as part of an attenuated vaccine, as an isolated protein or protein portion of a vector.

  Compositions and methods for prophylactically and / or therapeutically immunizing an individual against HPV are provided.

  A composition for delivering a nucleic acid molecule comprising a nucleotide sequence encoding the immunogen is operably linked to a regulatory element. The composition includes a plasmid encoding the immunogen, a recombinant vaccine comprising a nucleotide sequence encoding the immunogen, a live attenuated pathogen encoding a protein of the invention and / or comprising a protein of the invention; Inactivated pathogens comprising the protein; or compositions such as liposomes or subunit vaccines comprising the protein of the invention may be included. The invention further relates to an injectable pharmaceutical composition comprising the composition.

  SEQ ID NO: 1 contains the nucleotide sequence encoding the consensus immunogen of the HPV6 E6 and E7 proteins. SEQ ID NO: 1 contains the IgE leader sequence of SEQ ID NO: 9 linked to this nucleotide sequence at the 5 'end of SEQ ID NO: 1. SEQ ID NO: 2 comprises the amino acid sequence of the consensus immunogen of HPV6 E6 and E7 proteins. SEQ ID NO: 2 contains the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogen sequence. The IgE leader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

  In some embodiments, the vaccine comprises SEQ ID NO: 2 or a nucleic acid molecule encoding SEQ ID NO: 2.

  In some embodiments, the fragment of SEQ ID NO: 1 has 786 or more nucleotides, in some embodiments 830 or more nucleotides, and in some embodiments 856 or more nucleotides, In embodiments, it may comprise 865 or more nucleotides. In some embodiments, a fragment of SEQ ID NO: 1, such as the fragments described herein, may further include a coding sequence for an IgE leader sequence. In some embodiments, the fragment of SEQ ID NO: 1 does not include a coding sequence for an IgE leader sequence.

  In some embodiments, the fragment of SEQ ID NO: 2 has 252 or more amino acids, in some embodiments, 266 or more amino acids, in some embodiments, 275 or more amino acids, In embodiments, it may include 278 or more amino acids.

  SEQ ID NO: 3 comprises the nucleotide sequence encoding a consensus immunogen for HPV11 E6 and E7 proteins. SEQ ID NO: 3 comprises the IgE leader sequence of SEQ ID NO: 9 linked to this nucleotide sequence at the 5 'end of SEQ ID NO: 3. SEQ ID NO: 4 comprises the amino acid sequence of a consensus immunogen for HPV11 E6 and E7 proteins. SEQ ID NO: 4 contains the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogen sequence. The IgE leader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

  In some embodiments, the vaccine comprises SEQ ID NO: 4 or a nucleic acid molecule encoding SEQ ID NO: 4.

  In some embodiments, the fragment of SEQ ID NO: 3 has 786 or more nucleotides, in some embodiments 830 or more nucleotides, and in some embodiments 856 or more nucleotides, In embodiments, it may comprise 865 or more nucleotides. In some embodiments, a fragment of SEQ ID NO: 3, such as the fragments described herein, may further include a coding sequence for an IgE leader sequence. In some embodiments, the fragment of SEQ ID NO: 3 does not include the IgE leader sequence coding sequence.

  In some embodiments, the fragment of SEQ ID NO: 4 has 252 or more amino acids, in some embodiments, 266 or more amino acids, in some embodiments, 275 or more amino acids, In embodiments, it may include 278 or more amino acids.

  SEQ ID NO: 5 comprises the nucleotide sequence encoding a consensus immunogen for the HPV33 E6 and E7 proteins. SEQ ID NO: 5 contains the IgE leader sequence of SEQ ID NO: 9 linked to this nucleotide sequence at the 5 'end of SEQ ID NO: 5. SEQ ID NO: 6 comprises the amino acid sequence of the consensus immunogen of HPV33 E6 and E7 proteins. SEQ ID NO: 6 contains the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogen sequence. The IgE leader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

  In some embodiments, the vaccine comprises SEQ ID NO: 6 or a nucleic acid molecule encoding SEQ ID NO: 6.

  In some embodiments, the fragment of SEQ ID NO: 5 has 746 or more nucleotides, in some embodiments 787 or more nucleotides, and in some embodiments 812 or more nucleotides, In embodiments, it may include 820 or more nucleotides. In some embodiments, a fragment of SEQ ID NO: 5, such as the fragments described herein, may further include a coding sequence for an IgE leader sequence. In some embodiments, the fragment of SEQ ID NO: 5 does not include the coding sequence for an IgE leader sequence.

  In some embodiments, the fragment of SEQ ID NO: 6 has 235 or more amino acids, in some embodiments, 248 or more amino acids, in some embodiments, 256 or more amino acids, In embodiments, it may comprise 259 or more amino acids.

  SEQ ID NO: 7 comprises the nucleotide sequence encoding a consensus immunogen for HPV58 E6 and E7 proteins. SEQ ID NO: 7 comprises the IgE leader sequence of SEQ ID NO: 9 linked to this nucleotide sequence at the 5 'end of SEQ ID NO: 7. SEQ ID NO: 8 comprises the amino acid sequence of the consensus immunogen of HPV58 E6 and E7 proteins. SEQ ID NO: 8 contains the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogen sequence. The IgE leader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

  In some embodiments, the vaccine comprises SEQ ID NO: 8 or a nucleic acid molecule encoding SEQ ID NO: 8.

  In some embodiments, the fragment of SEQ ID NO: 7 has 752 or more nucleotides, in some embodiments, 794 or more nucleotides, in some embodiments, 819 or more nucleotides, In embodiments, it may comprise 827 or more nucleotides. In some embodiments, a fragment of SEQ ID NO: 7, such as the fragments described herein, may further include a coding sequence for an IgE leader sequence. In some embodiments, the fragment of SEQ ID NO: 7 does not include the IgE leader sequence coding sequence.

  In some embodiments, the fragment of SEQ ID NO: 8 has 235 or more amino acids, in some embodiments, 249 or more amino acids, in some embodiments, 257 or more amino acids, In embodiments, it may comprise 260 or more amino acids.

  According to some embodiments, the method of inducing an immune response against an immunogen in an individual is selected from the group consisting of HPV6 E6 and E7, HPV11 E6 and E7, HPV33 E6 and E7, HPV58 E6 and E7. Administering to this individual the amino acid sequences of the consensus immunogens, their functional fragments, and their expressible coding sequences. Some embodiments encode the amino acid sequence of a consensus immunogen selected from the group consisting of E6 and E7 of HPV6, E6 and E7 of HPV11, E6 and E7 of HPV33, E6 and E7 of HPV58, and fragments thereof. Isolated nucleic acid molecule. Some embodiments encode the amino acid sequence of a consensus immunogen selected from the group consisting of E6 and E7 of HPV6, E6 and E7 of HPV11, E6 and E7 of HPV33, E6 and E7 of HPV58, and fragments thereof. Including recombinant vaccines. Some embodiments comprise an amino acid sequence of a consensus immunogen selected from the group consisting of E6 and E7 of HPV6, E6 and E7 of HPV11, E6 and E7 of HPV33, E6 and E7 of HPV58, and fragments thereof. Includes subunit vaccine. Some embodiments comprise an amino acid sequence of a consensus immunogen selected from the group consisting of E6 and E7 of HPV6, E6 and E7 of HPV11, E6 and E7 of HPV33, E6 and E7 of HPV58, and fragments thereof. Includes live attenuated and / or inactivated vaccines.

  In one aspect, the vaccine herein is a vaccine that elicits an immune response against the HPV subtype, which has been found to be primarily associated with forms of head and neck cancer, and other forms of ENT diseases, In particular, these vaccines include HPV6 E6 and E7 and HPV11 E6 and E7, preferably both.

  In another aspect, the vaccines herein are directed against HPV subtypes, which have been found to be primarily associated with forms of cervical cancer in patients outside the United States, more specifically, in Asian patients. Vaccines that provoke an immune response, in particular, these vaccines include E6 and E7 of HPV33 and E6 and E7 of HPV58, preferably both.

  Improved vaccines include proteins having epitopes that make the improved vaccines particularly effective as immunogens capable of inducing an anti-HPV immune response, and genetic constructs encoding proteins having such epitopes. Thus, vaccines can be provided to induce a therapeutic or prophylactic immune response. In some embodiments, the means for delivering the immunogen includes a DNA vaccine, a recombinant vaccine, a protein subunit vaccine, a composition comprising the immunogen, an attenuated vaccine or an inactivated vaccine. In some embodiments, the vaccine comprises a combination selected from the group consisting of: one or more DNA vaccines, one or more recombinant vaccines, one or more protein subunit vaccines, One or more compositions comprising, one or more attenuated vaccines, one or more inactivated vaccines.

  Aspects of the invention provide methods for delivering a protein coding sequence on a nucleic acid molecule, such as a plasmid, as part of a recombinant vaccine, as part of an attenuated vaccine, as an isolated protein or protein part of a vector.

  According to some aspects of the present invention, there are provided compositions and methods for prophylactically and / or therapeutically immunizing an individual.

  DNA vaccines are described in U.S. Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, Nos. 5,981,505, 5,580,859, 5,703,055, 5,676,594 and the priority applications cited herein. , Each of which is incorporated herein by reference. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in U.S. Patent Nos. 4,945,050 and 5,036,006, both of which are incorporated by reference. Hereby incorporated by reference.

  The present invention relates to improved live attenuated vaccines, improved inactivated vaccines, and improved vaccines that use recombinant vectors to deliver foreign genes encoding antigens, as well as subunit and glycoprotein vaccines. Examples of live attenuated vaccines, vaccines that deliver foreign antigens using recombinant vectors, subunit vaccines and glycoprotein vaccines are described in US Pat. Nos. 4,510,245, 4,797,368, No. 4,722,848, No. 4,790,987, No. 4,920,209, No. 5,017,487, No. 5,077,044, No. 5,110,587 No. 5,112,749, No. 5,174,993, No. 5,223,424, No. 5,225,336, No. 5,240,703, No. 5 No. 5,242,829, No. 5,294,441, No. 5,294,548, No. 5,310,668, No. 5,387,744, No. 5,389,368 No. 5,424,065, No. 5,451 Nos. 499, 5,453,364, 5,462,734, 5,470,734, 5,474,935, 5,482,713, and No. 5,591,439, No. 5,643,579, No. 5,650,309, No. 5,698,202, No. 5,955,088, No. 6,034,298 Nos. 6,042,836, 6,156,319 and 6,589,529, each of which is incorporated herein by reference.

  When taken up by a cell, the gene construct (s) may remain present in the cell as a functioning extrachromosomal molecule and / or may be integrated into the chromosomal DNA of the cell. . The DNA may be introduced into the cell, where it remains as separate genetic material in the form of one or more plasmids. Alternatively, linear DNA that can be integrated into a chromosome may be introduced into the cell. When introducing DNA into these cells, an agent that promotes DNA integration into the chromosome may be added. DNA sequences useful for facilitating integration may be included in the DNA molecule. Alternatively, RNA may be administered to the cells. It is also contemplated to provide this gene construct as a linear minichromosome containing centromeres, telomeres and origins of replication. The genetic construct may remain part of the genetic material of a live attenuated microorganism or a recombinant microbial vector that survives in the cell. The genetic construct may be part of the genome of the recombinant viral vaccine, wherein the genetic material either integrates into the chromosome of the cell or stays extrachromosomally. Gene constructs contain the regulatory elements required for gene expression of the nucleic acid molecule. These elements include a promoter, start codon, stop codon, and polyadenylation signal. Furthermore, enhancers are often required for gene expression of sequences encoding target or immunomodulatory proteins. These elements need to be operably linked to the sequence encoding the desired protein, and these regulatory elements need to be operable in the individual to whom they are administered.

  The start codon and stop codon are generally considered to be part of a nucleotide sequence that encodes the desired protein. However, these elements need to be functional in the individual to whom the genetic construct is administered. Start and stop codons must be in frame with the coding sequence.

  The promoter and polyadenylation signal used must be functional in the cells of the individual.

  Examples of promoters useful in practicing the present invention, particularly in the production of human genetic vaccines, include the Simian Virus 40 (SV40) promoter, the mouse mammary tumor virus (MMTV) promoter, the BIV terminal repeat (LTR) promoter, and the like. Human immunodeficiency virus (HIV) promoter, Moloney virus promoter, ALV promoter, CMV immediate early promoter and other cytomegalovirus (CMV) promoters, Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter and human actin Promoters from human genes such as, but not limited to, human myosin, human hemoglobin, human muscle creatine, and human metallothionein.

  Examples of polyadenylation signals useful in practicing the invention, particularly in the production of human genetic vaccines, include, but are not limited to, the SV40 polyadenylation signal and the LTR polyadenylation signal. In particular, it uses the SV40 polyadenylation signal, which is present in the plasmid pCEP4 (Invitrogen, San Diego CA) and is called the SV40 polyadenylation signal.

  In addition to the regulatory elements required for DNA expression, other elements may be included in the DNA molecule. Such additional elements include enhancers. The enhancer may be selected from the group including, but not limited to: human actin enhancer, human myosin enhancer, human hemoglobin enhancer, human muscle creatine enhancer and viral enhancers such as CMV, RSV and EBV enhancers.

  The gene construct can be provided with a mammalian origin of replication to maintain the gene construct extrachromosomally and produce multiple copies of the construct in cells. The plasmids pVAX1, pCEP4 and pREP4 of Invitrogen (San Diego, CA) contain the Epstein-Barr virus origin of replication and the nuclear antigen EBNA-1 coding region that perform high copy episomal replication without integration.

  In some preferred embodiments relating to immunization applications, a nucleic acid molecule (s) comprising a nucleotide sequence encoding a protein of the invention and, in addition, a gene for the protein that further enhances the immune response to such a target protein is provided. Deliver. Examples of such genes include α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, Such as IL-15, including IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86, and optionally IL-15, including the signal peptide of IgE. Other cytokines as well as genes encoding lymphokines are included. Other genes that may be useful include MCP-1, MIP-1α, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL- 18 mutants, CD40, CD40L, vascular growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo -3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactive NIK, SAPK, SAP-1, JNK, interferon response gene, NFkB, Bax, TRAIL TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK ligand, Ox40, Ox40 ligand, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1 Genes included.

  If for some reason it is desirable to eliminate cells that receive this genetic construct, additional elements may be added that serve as targets for cell destruction. An expressible form of the herpes thymidine kinase (tk) gene can be included in the gene construct. The drug ganciclovir can be administered to the individual, and the drug selectively kills all cells that produce tk, thus providing a means for the selective destruction of cells bearing the genetic construct.

  To maximize protein production, regulatory sequences may be chosen that are well suited for gene expression in the cells to which the construct is administered. In addition, codons that are most efficiently transcribed in the cell may be selected. One of skill in the art can generate a DNA construct that is functional in a cell.

  In some embodiments, a genetic construct may be provided wherein the coding sequence of a protein described herein is linked to an IgE signal peptide. In some embodiments, the proteins described herein are linked to an IgE signal peptide.

  In some embodiments where a protein is used, for example, one skilled in the art can produce and isolate the protein of the invention using well-known techniques. In some embodiments where the protein is used, for example, one of skill in the art can use known techniques to insert a DNA molecule encoding a protein of the invention into a commercially available expression vector for use in known expression systems. can do. For example, the commercially available plasmid pSE420 (Invitrogen, San Diego, Calif.) May be used for protein production in E. coli. For example, the commercially available plasmid pYES2 (Invitrogen, San Diego, Calif.) May be used for (protein) production in the yeast strain Saccharomyces cerevisiae. For example, the commercially available MAXBAC ™ complete baculovirus expression system (Invitrogen, San Diego, Calif.) May be used for (protein) production in insect cells. For example, the commercially available plasmid pcDNA I or pcDNA3 (Invitrogen, San Diego, Calif) may be used for (protein) production in mammalian cells such as Chinese hamster ovary cells. One of skill in the art can use these commercially available expression vectors and expression systems or others to produce proteins by conventional techniques and readily available starting materials (eg, Sambrook et al., Molecular Cloning). a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989), which is incorporated herein by reference). Thus, the desired protein can be prepared in both prokaryotic and eukaryotic cell systems, resulting in a series of processed forms of the protein.

  One of skill in the art may use other commercially available expression vectors and systems, or may produce the vector using well-known methods and readily available starting materials. Expression systems for various types of hosts, including essential regulatory sequences such as promoters and polyadenylation signals, and preferably enhancers, are readily available and known in the art. See, for example, Sambrook et al. , Molecular Cloning a Laboratory Manual, Second Ed. See Cold Spring Harbor Press (1989). The genetic construct includes a protein coding sequence operably linked to a promoter that is functional in the cell line into which the construct is transfected. Examples of constitutive promoters include the cytomegalovirus or SV40 promoter. Examples of inducible promoters include the mouse mammary leukemia virus or metallothionein promoter. One of skill in the art can readily make from a readily available starting material a genetic construct useful for transfecting cells with DNA encoding a protein of the present invention. A compatible host is transformed with an expression vector containing DNA encoding this protein, and then cultured and maintained under conditions that allow expression of the foreign DNA.

  The produced protein was recovered from the culture as needed and as known to those skilled in the art by lysing or recovering these cells from the medium. One skilled in the art can use known methods to isolate proteins produced using such expression systems. The above-described method of purifying a protein from a natural source using an antibody that specifically binds to the specific protein may be similarly applied to purification of a protein produced by a recombinant DNA method.

  In addition to protein production by recombinant techniques, an automated peptide synthesizer may also be used to produce an essentially pure isolated protein. Such techniques are well known to those of skill in the art and are useful when derivatives having substitutions are not provided in producing a DNA-encoded protein.

  These nucleic acid molecules can be delivered using any of a number of well-known techniques, including recombinant injection vectors such as DNA injection (also referred to as DNA vaccination), recombinant adenovirus, recombinant adenovirus-associated virus and recombinant vaccinia. You may.

  Administration routes include intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral routes, as well as by inhalation or suppository to mucosal tissues. For example, but not limited to, topical, transdermal routes by irrigation of tissues under the vagina, rectum, urethra, cheeks and sublingual. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injections. The genetic construct may be administered by means including, but not limited to, electroporation methods and devices, conventional syringes, needle-free injection devices, or “microparticle bombardment gene guns”.

  Examples of preferred electroporation devices and electroporation methods to facilitate the delivery of these DNA vaccines include US Pat. No. 7,245,963 to Dragia-Akli et al., And US Patent Publication 2005 filed by Smith et al. No./0052630, the contents of which are incorporated herein by reference in their entirety. 35 USC 119 (e) U.S. Provisional Application No. 60 / 852,149 filed October 17, 2006 and U.S. Provisional Application No. 60 / 978,982 filed October 10, 2007 No. 11 / 874,072, filed Oct. 17, 2007, all of which are incorporated herein in their entirety, alleging the benefit of US Pat. Electroporation devices and methods that facilitate the delivery of DNA vaccines are also preferred.

  The following is an example of an embodiment using electroporation technology and is discussed in detail in the patent references discussed above: The electroporation device is similar to a current input preset by the user. A pulse of energy that produces a constant current can be arranged to deliver to a desired tissue in a mammal. The electroporation device includes an electroporation component and an electrode or handle assembly. The electroporation component is one of an electroporation device including a control device, a current waveform generator, an impedance tester, a waveform logger, an input component, a status reporting component, a transmission port, a recording component, a power supply, and a power switch. It can include and incorporate one or more various elements. The electroporation component can function as one element of the electroporation device, and the other element is a separate element (or component) in communication with the electroporation component. In some embodiments, the electroporation component can function as two or more components of the electroporation device, which is in communication with yet another component of the electroporation device that separates from the electroporation component can do. Using these electroporation techniques to deliver an improved HPV vaccine can be a single electromechanical device or a mechanical device, as these components can function as one device or as separate components communicating with each other. Are not limited to elements of the electroporation device that are present as part of the device. The electroporation component can deliver an energy pulse that produces a constant current in the desired tissue and includes a feedback mechanism. The electrode assembly includes an electrode array having a plurality of electrodes in a spatial arrangement, wherein the electrode assembly receives energy pulses from the electroporation component and sends energy pulses through the electrodes to a desired tissue. At least one of the plurality of electrodes is neutral during the delivery of the energy pulse, measures the impedance of the desired tissue, and communicates the impedance to the electroporation component. The feedback mechanism can receive the measured impedance, adjust the energy pulses delivered by the electroporation component, and maintain a constant current.

  In some embodiments, multiple electrodes can deliver energy pulses in a dispersive pattern. In some embodiments, the plurality of electrodes can deliver energy pulses in a decentralized pattern via electrode control under a programmed order, and the user can adjust the programmed order to the electroporation configuration. Fill in the element. In some embodiments, the programmed order comprises a plurality of pulses delivered in sequence, each pulse of the plurality of pulses being delivered by at least two active electrodes with one neutral pole measuring impedance; The next pulse of the plurality of pulses is delivered by another one of the at least two active electrodes with one neutral pole measuring impedance.

  In some embodiments, this feedback mechanism is performed by either hardware or software. Preferably, this feedback mechanism is provided by an analog closed loop circuit. Preferably, this feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but preferably is real-time feedback or instantaneous (ie, virtually instantaneous as determined by the available technology to determine the reaction time). ). In some embodiments, the neutral electrode measures the impedance at the desired tissue and transfers the impedance to the feedback mechanism, which responds to the impedance and provides a constant current at a value similar to the preset current. Adjust the energy pulse to maintain In some embodiments, the feedback mechanism maintains the constant current continuously and instantaneously during energy pulse delivery.

  In some embodiments, the nucleic acid molecule is delivered to these cells in conjunction with administration of a polynucleotide function enhancer or a gene vaccine promoter. Polynucleotide function enhancers are described in U.S. Patent Nos. 5,593,972, 5,962,428 and International Application No. PCT / US94 / 00899 filed January 26, 1994; Each of which is incorporated herein by reference. Genetic vaccine enhancers are described in U.S. Patent No. 021,579, filed April 1, 1994, which is incorporated herein by reference. The auxiliaries to be administered in combination with the nucleic acid molecule may be administered as a mixture with the nucleic acid molecule before or after the administration of the nucleic acid molecule, or separately and simultaneously. In addition, other agents that can function as transfection and / or replication agents and / or inflammatory agents and that can be administered with GVF include α-interferon, γ-interferon, GM-CSF, platelet-derived proliferation Growth factors such as factor (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15, cytokines and lymphokines And surfactants such as fibroblast growth factor, immunostimulatory complex (ISCOMS), incomplete Freund's adjuvant, LPS analogs including monophosphoryl lipid A (WL), muramyl peptides, quinone analogs and squalene squalene Vesicles and hyaluronic acid are also administered in combination with this gene construct May be used. In some embodiments, an immunomodulatory protein may be used as a GVF. In some embodiments, the nucleic acid molecule is provided in association with PLG to enhance delivery and uptake.

  A pharmaceutical composition according to the present invention contains about 1 ng to about 2000 μg of DNA. In some preferred embodiments, a pharmaceutical composition according to the present invention comprises about 5 ng to about 1000 μg of DNA. In some preferred embodiments, these pharmaceutical compositions contain about 10 ng to about 800 μg of DNA. In some preferred embodiments, these pharmaceutical compositions contain about 0.1 to about 500 μg of DNA. In some preferred embodiments, these pharmaceutical compositions contain about 1 to about 350 μg of DNA. In some preferred embodiments, these pharmaceutical compositions contain about 25 to about 250 μg of DNA. In some preferred embodiments, these pharmaceutical compositions contain about 100 to about 200 μg of DNA.

  The pharmaceutical composition according to the invention is formulated according to the administration method used. Where the pharmaceutical composition is an injectable pharmaceutical composition, the injectable pharmaceutical composition is sterile and free of pyrogens and particles. Preferably, an isotonic formulation is used. In general, additives for isotonicity can include sodium chloride, D-type glucose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstrictor is added to the formulation.

  According to some embodiments of the present invention, there is provided a method of inducing an immune response. The vaccine may be a live, attenuated, cellular, recombinant or nucleic acid or DNA vaccine based on protein. In some embodiments, a method of inducing an immune response to an immunogen in an individual, including a method of inducing a mucosal immune response, comprises the steps of: An isolated nucleic acid molecule encoding a protein of the invention and / or a recombinant vaccine encoding a protein of the invention, and / or a subunit vaccine and / or attenuated protein of the invention, of one or more of the sequences. Administering to this individual in combination with a live vaccine and / or an inactivated vaccine. An isolated nucleic acid molecule encoding an immunogen comprising one or more of the CTACK protein, TECK protein, MEC protein and functional fragments thereof; and / or a recombinant vaccine and / or immunogen encoding the immunogen. The subunit vaccine and / or the live attenuated vaccine and / or the inactivated vaccine may be administered before, simultaneously with, or after their administration. In some embodiments, an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of CTACK, TECK, MEC and functional fragments thereof is administered to the individual.

  The present invention is further described in the following examples. While these examples illustrate preferred embodiments of the present invention, it should be understood that they are provided for illustrative purposes only. From the above description and these examples, those skilled in the art can ascertain the essential characteristics of the present invention, and apply the present invention to various uses and conditions without departing from the spirit and scope of the present invention. Various modifications and improvements of the present invention can be made. Accordingly, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

  Each of the U.S. patents, U.S. patent applications, and references cited throughout this disclosure is hereby incorporated by reference in its entirety.

Examples The present invention is further defined by the following examples. While these examples illustrate preferred embodiments of the present invention, it should be understood that they are provided for illustrative purposes only. From the above description and these examples, those skilled in the art can ascertain the essential features of the present invention without departing from the spirit and scope of the invention and to adapt it to various uses and conditions. Various changes and modifications of the invention can be made. Thus, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1
Vaccine design and expression of HPV6 and HPV11 E6 / E7 Construction of fusion immunogens based on HPV6 and HPV11 E6 / E7 consensus HPV6 or HPV11 type E6 / E7 gene sequences were collected from GeneBank and consensus E6 and E7 nucleotides Sequences were obtained after performing multiple alignments. The consensus sequence for the HPV6 E6 or E7 protein was made from 98 or 20 sequences, respectively, and the consensus sequence for the HPV11 E6 or E7 protein was made from 76 or 13 sequences, respectively. Multiple alignment procedures applied to phylogenetic studies included the application of Clustal X (version 2.0). As shown in FIGS. 1A and 1B, there was approximately 0-2% sequence variance between HPV strains and among HPV strains belonging to the same type of their E6 and E7 proteins. However, the genetic distance could be increased between HPV6 and HPV11 by 19.3% for the E6 protein and by 16.3% for the E7 protein. Based on these results of phylogenetic analysis, two type-specific E6 / E7 consensus DNA vaccines were developed.

  After making the consensus E6 / E7 fusion sequence, several changes were made (FIG. 1C). To facilitate expression, a high performance leader sequence was fused in frame upstream of the start codon. Codon and RNA optimization was also performed as previously described (J. Yan, et al., Cellular immunity induced by a novel HPV18 DNA vaccine encoding a E6 / E7 fusion konsane sice usainse konse sice usain s e s s a s s e s e s e s e s e n s s e s e s e n s e s s e n s e s e s s e n s e s e n s e s e n s s e n e s e n e n e n e n e n e n e n e n e n e n e n e e n e n s e s e s e s e s e s e n s e s e n s e s e s e n s e s e s e n s e s e s e n s e s e n s e n e n e n e n e n c e s e n s e n e n e n e s .26 (2008) 5210-5215; and J. Yan, et al., Induction of antitumor immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding anEconi ng / Eng. 440). An intracellular proteolytic cleavage site was introduced between the E6 and E7 proteins for proper protein folding and good CTL processing. Both the genetically engineered synthetic 6E6E7 and 11E6E7 genes were 840 bp in length. For further studies, the sequence-verified synthetic genes were subcloned into the BamHI and XhoI sites of the pVAX expression vector, respectively. The consensus amino acid sequence was obtained by translating the consensus nucleotide sequence.

  After obtaining the consensus E6 sequence of HPV6 and 11 and the consensus E7 sequence of HPV6 and 11, codon optimization and RNA optimization were performed as described previously (J. Yan, et al., Vaccine. 26 ( 2008) 5210-5215; and J. Yan, et al., Induction, Vaccine. 27 (2009) 431-440). A fusion gene (6E6E7 or 11E6E7) encoding either HPV type 6 or type 11 consensus E6 / E7 fusion protein was synthesized and sequence confirmed. The synthesized 6E6E7 or 11E6E7 was digested with BamHI and XhoI, cloned into the expression vector pVAX (Invitrogen) under the control of the cytomegalovirus immediate early promoter, and these constructs were named p6E6E7 or p11E6E7.

  293T cells were cultured in 6-well plates and transfected with pVAX, p6E6E7, or p11E6E using FuGENE6 transfection reagent (Roche Applied Science, Indianapolis, IN). Two days after transfection, these cells were lysed using a modified RIPA cell lysis buffer and the cell lysate was collected. Western blot analysis was performed using an anti-HA monoclonal antibody (Sigma-Aldrich, St. Louis, Mo.) and horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma) using an ECLTM Western Blot Analysis System (Amersham, Piscataway, NJ). -Aldrich, St. Louis, MO).

  To confirm the expression of p6E6E7 and p11E6E7, an indirect immunofluorescence assay was performed using human rhabdomyosarcoma cells (RD cells). RD cells cultured in chamber slides were transfected with pVAX, p6E6E7 or p11E6E7 using Turbofectin 8.0 (Origene, Rockville, MD). Thereafter, these cells were fixed with PFA and permeabilized with 0.1% Triton-X in PBS. The cells were incubated with the primary and secondary antibodies for 1-2 hours, respectively, and washed with PBS supplemented with glycine and BSA between the two incubations. The primary and secondary antibodies used were monoclonal mouse anti-HA (Sigma-Aldrich, St. Louis, Mo.) and FITC-conjugated anti-mouse IgG (Abcam, Cambridge, Mass.), Respectively. Hoechst staining was also performed to identify cell nuclei and localize RD cells. After all incubations were completed, the cells were mounted and glass slides were fixed with Fluormount-G (Southern Biotech, Birmingham, AL). These samples were observed and imaged using a confocal microscope (CDB Microscopy Core, University of Pennsylvania, Cell Developmental Biology, Philadelphia, PA).

Example 2
Immunization of Mice and Treatment Groups Female C57BL / 6 mice, 6-8 weeks of age, were used in this experiment. Mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were housed and maintained at the University of Pennsylvania's Institute of Animal Resources, according to the policies of the National Institutes of Health and the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania. The mice used in these experiments were divided into four groups for immunization. Mice were immunized with p6E6E7, p11E6E7, or both constructs, with the pVAX group as a negative control.

DNA vaccination and electroporation Each mouse received three doses of 20 μg of each DNA plasmid at 14 day intervals. The group of mice that received both p6E6E7 and p11E6E7 received 20 μg of both plasmids per vaccination, for a total of 40 μg of DNA. These DNA constructs were administered by intramuscular injection into the right quadriceps muscle, after which short waves were generated with a CELLECTRA ™ electroporator (Inovio Pharmaceuticals, Bluebell, PA). The electroporator was configured to send two electrical pulses at 1 ms delay at 52 ms / pulse, 0.2 amps. The electroporation procedure was performed as previously described (12, 13).

IFN-γ ELISpot assay Both the treatment and control mice were sacrificed one week after the third immunization. Spleens were harvested from each mouse and transferred to RPMI-1640 medium (R10) containing 10% FBS and antibiotics. The spleen was ground using a stomacher (Secard Laboratory Systems, Bohemia, NY), then transferred through a cell strainer and suspended in ACK lysis buffer. After removing erythrocytes, splenocytes were isolated and suspended in R10 medium. High protein IP 96-well Multiscreen ™ plates (Millipore, Bedford, Mass.) Were pre-coated with monoclonal mouse IFN-γ capture antibody (R & D Systems, Minneapolis, MN) and incubated at 4 ° C. Incubated overnight. After washing three times with 1 × X PBS, the plates were blocked with 1% BSA and 5% sucrose in 1 × PBS for 2 hours at ambient temperature. Isolated splenocytes in R10 medium were counted and added to triplicate wells at 2 × 10 ⁵ cells per well. Two sets of peptides spanning the consensus E6 / E7 sequences of HPV6 and HPV11 were reconstituted in DMSO (USA GenScript, Piscataway, NJ). These peptides contained a 15 amino acid sequence, of which 8 residues overlapped with each sequential peptide. The HPV6 and 11 peptides were split into two pools (one pool at E6 and another at E7) at a concentration of 2 μg / ml in DMSO, respectively. Wells for positive and negative controls received Concanavalin A (Sigma-Aldrich, St. Louis, MO) and R10 medium instead of peptide, respectively. The plates were then placed in an incubator with a 5% CO2 atmosphere. After incubation at 37 ° C. for 24 hours, the wells were washed with 1 × PBS. Biotinylated anti-mouse IFN-γ detection antibody (R & D Systems, Minneapolis, MN) was added to each well and then incubated overnight at 4 ° C. The plates were then washed and treated with streptavidin-AP and BCIP / NBT Plus (R & D Systems, Minneapolis, MN) according to the color development protocol provided by R & D Systems. The wells were air-dried overnight and the spots inside the wells were scanned and counted using an ELISpot plate reader system using ImmunoSpot® 3 and ImmunoSpot® 4 software (Cellular Technology Ltd., Shaker Heights, Ohio). The reported number of spot forming cells was converted and the arithmetic was used to show the spot forming units per 1 × 10 ⁶ splenocytes.

  Given their sensitivity and the ability to demonstrate T cell activity, antigen-specific responses to stimulation with either HPV6 or 11 E6 and HPV6 or 11 E7 peptides using the IFN-γ ELISpot assay The number of IFN-γ secreting cells was determined.

  As shown in FIGS. 3A and 3B, the average number of SFU / 106 splenocytes from mice immunized with p6E6E7 was 1442.8 and the average number of SFU / 106 splenocytes from mice immunized with p11E6E7 was 2845, All significantly exceeded the negative control group. Thus, both p6E6E7 and p11E6E7 were effective in eliciting strong type-specific E6 and E7-specific immune responses in mice.

  Interestingly, a cross-reactive cellular immune response was also induced by immunization with p6E6E7 or p11E6E7. The frequency of addition of SFU / 106 splenocytes to HPV6 E6 / E7 peptide in mice immunized with p11E6E7 was 552.8 SFU / 106 splenocytes, and HPV11 E6 / E7-specific immune response in mice immunized with p6E6E7 Was 888.3 SFU / 106 splenocytes. The HPV6 or 11 E6 proteins shared about 80% identity and the HPV6 or 11 E7 proteins shared about 84% identity. There may be several co-immune epitopes between the HPV6 or 11 E6 antigen and the HPV6 or 11 E7 antigen. Cross-reactivity observed from the IFN-γ ELISpot assay indicated that there was a covalent immune epitope between the HPV6 and HPV11 E6 antigens and the HPV6 and HPV11 E7 antigens.

  The ELISpot assay described above was also used to determine whether there was any immune interference when immunizing these two constructs together when spleen cells from mice that received both p6E6E7 and p11E6E7 (combination group). (FIGS. 3A and 3B). The mean value of the combination group for E6 and E7 peptides of HPV6 was 1670 SFU / 106 splenocytes (σ = 55.7, P <0.001). The same group of splenocytes for HPV11 E6 and E7 peptides resulted in 2010 SFU / 106 splenocytes (σ = 247.8, P = 0.002). These data indicate that co-immunization with the two constructs can elicit statistically significant E6- and E7-specific cellular responses to HPV6 and HPV11, and these responses do not interfere with each other. Suggest.

Epitope Mapping To determine immunodominant peptides within the E6 / E7 consensus antigen, epitope mapping studies were performed to determine dominant epitopes within the peptide pool (FIGS. 4A and 4B). These tests were performed similarly to the IFN-γ ELISpot assay described above. Splenocytes were stimulated with individual peptides instead of pools.

  Each peptide used in this single peptide analysis showed a partially overlapping fragment of the HPV6 or HPV11 E6 antigen and the HPV6 or HPV11 E7 antigen. Mapping data indicated that peptide 7 of SEQ ID NO: 11 (TAEIYSYAYKQLKVL) was the dominant epitope of the HPV6 E6 and E7 immunogens (FIG. 4A). TAEIYSYAYKQLKVL of SEQ ID NO: 11 contained 8mer, 9mer, and 10mer amino acid epitopes, respectively, identified as H2-Kb restricted by the HLA binding prediction software available from NIH BIMAS. In addition, an E6 and E7-specific T cell immune response of HPV11 is described (FIG. 4B). Peptide analysis was also performed using overlapping fragments of E6 and E7 of HPV11. Epitope mapping indicated that the dominant epitopes of the HPV11 E6 and E7 antigens were peptide 7 of SEQ ID NO: 12 (TAEIYAYAYKNLKVV) and peptide 27 of SEQ ID NO: 13 (HCYEQLEDSSEDEVD). Similar to peptide 7 in the HPV6 epitope mapping assay, BIMAS HLA binding prediction software confirmed that TAEIYAYAYKNLKVV of SEQ ID NO: 12 was an H2-Kb restricted epitope. Another HLA-binding peptide database, immune epitope database and analytical resources provided by NIH NIAID confirmed that HCYEQLEDS SEDEVD of SEQ ID NO: 13 is a H2-Kb restricted epitope. Three sub-significant immunopeptides, SEQ ID NO: 14, No. 6 (FCKNALTTAEIYSYA), SEQ ID NO: 15, No. 9 (LFRGGGYPYAACACCL), and SEQ ID NO: 16, No. 13 (YAGYATTVEEEETKQD) were identified through this epitope mapping study.

Intracellular cytokine staining In light of the high immune response exhibited by the IFN-γ ELISpot assay, an intracellular cytokine staining assay was performed to provide a more comprehensive overview of the cellular responses induced by p6E6E7 and p11E6E7. . Splenocytes of the immunized and untreated groups of mice were isolated and stimulated with peptides spanning the HPV6 and HPV11 E6 and HPV6 and HPV11 E7 regions in a 5% CO2 environment at 37 ° C. for 4 hours. Positive and negative controls were used in this assay by placing cells in phorbol 12-myristate 13-acetate (PMA) and R10 cell medium, respectively. After incubation, the cells were first stained with ViViD dye (Invitrogen, Carlsbad, CA) to distinguish live and dead cells, and then all cells were APC-Cy7 hamster anti-mouse CD3e, PerCP-Cy5. 5 rat anti-mouse CD4 and APC rat anti-mouse CD8a (BD Biosciences, San Diego, CA) were stained with surface marker antibodies. The cells were then fixed using a Cytofix / Cytoperm kit (BD Biosciences, San Diego, CA). After fixation according to the manufacturer's protocol, the cells were purified from Alexa Fluor700 rat anti-mouse IFN-γ, PE-Cy7 rat anti-mouse TNF clone, and PE rat anti-mouse IL-2 (BD Biosciences, San Diego, CA). The cells were stained with a cell marker antibody. After staining, these cells were fixed with a PBS solution containing 2% paraformaldehyde. The prepared cells were obtained using an LSRII flow cytometer equipped with BD FACSDiva software (BD Biosciences, San Jose, CA). The acquired data was analyzed using the latest version of FlowJo software (Tree Star, Ashland, OR). CD4 + and CD8 + events were analyzed using FSC-A vs. FSC-H singlet, FSC-A vs. SSC-A whole splenocytes, ViViD dye (Pacific Blue) vs. live cells from SSC-A, CD3 (APC -Cy7) vs. CD3 + cells from SSC-A, CD4 (PerCP-Cy5.5: Positive-CD4 +, Negative-CD8 +) vs. CD4 + or CD8 + from SSC-A using the gate sequence. The last two populations were gated on Alexa Fluor700, PE-Cy7, and PE to observe changes in IL-2, IFN-γ and TNF-α production, respectively.

  Cells were gated so that intracellular cytokine staining data could be distinguished by the response of CD4 + and CD8 + cells. When stimulated with HPV6 E6 and E7 specific peptides, the average value of all CD4 + cells producing IFN-γ, TNF-α, and IL-2 in mice immunized with p6E6E7 was 0.163% (σ = 0.09), 0.003% (σ = 0.07), and 0.188% (σ = 0.20) (FIG. 5A). The mean values of all CD8 + cells producing IFN-γ, TNF-α, and IL-2 in the same group of mice are 3.323% (σ = 1.39) and 0.838% (σ = 0.39, respectively). 32), and 1.172% (σ = 1.81). The same intracellular cytokine data was collected using splenocytes of mice immunized with p11E6E7 after incubation of HPV11 with E6 and E7 antigens (FIG. 5B). Among all CD4 + cells of p11E6E7-immunized mice, the average value of cells that produced IFN-γ was 0.051% (σ = 0.04), and the average value of cells that produced TNF-α was 0.068% ( σ = 0.09), and the average value of cells that produced IL-2 was 0.026% (σ = 0.037). Furthermore, in mice immunized with p11E6E7, the average values of all CD8 + cells that produced IFN-γ, TNF-α, and IL-2 were 4.52% (σ = 2.53) and 2.08% ( σ = 1.56) and 0.21% (σ = 0.22). With the exception of some panels, the proportion of cytokine-producing cells in the treated groups versus their respective untreated groups was statistically significant at a confidence level of 89% or higher. Observation of the extent of cytokine production of CD4 + T cells versus CD8 + T cells concludes that the immune responses elicited by p6E6E7 and p11E6E7 are highly biased in all models towards the drive of CD8 + lymphocytes associated with their cell clearance. Can be attached.

Statistical Analysis A Student's t-test was performed to analyze the statistical significance of all quantitative data generated in this study. Unless otherwise indicated, p-values were calculated and statistical significance was determined at various confidence levels.

  This study provides compelling evidence that DNA vaccines can achieve the levels of immunogenicity found in experiments with other proven and conventionally used vaccine platforms. Indicated. High levels of cellular responses measured in other similar E6 and E7-specific HPV DNA vaccine studies were associated with data suggesting prophylactic and therapeutic anti-tumor effects. Most HPV-related studies and disease burden models focus on cervical cancer. Given that HPV6 and HPV11 are not as associated with cervical cancer as other HPV serotypes, the therapeutic effects of p6E6E7 and p11E6E7 cannot be fully evaluated using conventional HPV disease burden models. Determining the protection and therapeutic potential of p6E6E7 and p11E6E7 is insightful when appropriate disease burden models are available. Thus, by examining high levels of IFN-γ and cytokine production quantified by ELISpot assay and intracellular cytokine staining, the immunogenic efficacy of p6E6E7 and p11E6E7 can be inferred. Nevertheless, such levels were found to be significantly stronger in magnitude and show promise that the two constructs would elicit substantial levels of cellular immune responses. Because HPV-related malignancies are commonly secondary to co-infection of multiple serotypes, there is a great need to further investigate the feasibility of combining vaccines directed to different serotypes of the virus. Further studies examining T-cell kinetics and vaccine competition ensure that the effects of co-immunization of these two plasmids are further characterized.

  Intracellular cytokine staining showed that immunization with p6E6E7 and p111E6E7 could elicit a significant proportion of T cells producing IFN-γ, TNF-α, and IL-2. In view of currently known functions in the immune system, IFN-γ has traditionally been used as an indicator of a cellular immune response. Some of these important roles include the ability to modulate and stimulate innate and acquired immunity. In addition, it is widely accepted that the major producers of IFN-γ are T cells that cause IFN-γ production to be in an acceptable mode to measure cellular immunogenicity after a given vaccine exposure to the antigen. ing. TNF-α is another cytokine involved in the regulation of the immune system. The known ability of TNF-α to induce apoptosis and regulate tumor growth makes TNF-α an important parameter to consider in characterizing the post-vaccination immune response. TNF-α production can be even more interesting given the potential tumor growth properties of HPV6 and HPV11. IL-2 is another signaling molecule that has been observed to play a central role in T cell proliferation and differentiation in the immune system. As such, IL-2 is often considered in combination with other cytokines to gain further insight into the magnitude and quality of a particular immune response. In view of the above, CD4 + and CD8 + cells producing a significant proportion of IFN-γ, TNF-α and IL-2 after immunization with p6E6E7 indicate that this vaccine induces a strong immune response. Indicates success. The same applies to cells isolated from mice immunized with p11E6E7, except for CD4 + cells that secrete IL-2. In addition, it is noteworthy to observe that CD8 + cells significantly enhanced the immune response in immunized mice, a property that is important in assessing the antitumor effects of these two plasmids.

Claims (26)

  SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 5 and SEQ ID NO: 7 fragments having 90% homology with the sequence of SEQ ID NO: 7, and combinations thereof. A nucleic acid molecule comprising a nucleotide sequence encoding an HPV antigen selected from the group consisting of:   The nucleic acid molecule of claim 1, comprising the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or a combination thereof.   The nucleic acid molecule of claim 1, comprising a fragment having at least 95% homology to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.   The nucleic acid molecule of claim 1, comprising a fragment having at least 98% homology to the sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.   The nucleic acid molecule of claim 1, comprising a fragment having at least 99% homology with a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7.   A nucleotide sequence encoding the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; having at least 90% homology with the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 A nucleotide sequence encoding an amino acid fragment having; a fragment of a nucleotide sequence encoding the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or sequence A nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of a fragment of a nucleotide sequence encoding an amino acid fragment having at least 90% homology to the sequence of No. 8.   7. The nucleic acid molecule of claim 6, comprising a nucleotide sequence encoding the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.   The nucleic acid molecule according to any one of claims 1 to 7, wherein the molecule is a plasmid.   A pharmaceutical composition comprising the nucleic acid molecule according to any one of claims 1 to 8.   A method of inducing an immune response to HPV in an individual, comprising administering to the individual a composition comprising the nucleic acid molecule of any one of claims 1 to 8.   The method of claim 10, further comprising introducing the nucleic acid molecule into the individual by electroporation.   A recombinant vaccine comprising the nucleic acid molecule according to any one of claims 1 to 8.   13. The recombinant vaccine according to claim 12, wherein said recombinant vaccine is a recombinant vaccinia vaccine.   A live attenuated vaccine comprising the nucleic acid molecule according to any one of claims 1 to 8.   SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; and fragments of a sequence having at least 90% homology with the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8 A protein comprising an amino acid sequence selected from the group consisting of:   16. The protein of claim 15, comprising the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.   16. The protein of claim 15, comprising a fragment having at least 95% homology to the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.   16. The protein of claim 15, comprising a fragment having at least 98% homology to the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.   16. The protein of claim 15, comprising a fragment having at least 99% homology to the sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.   A method for inducing an immune response to HPV in an individual, comprising administering to the individual a recombinant vaccine according to any one of claims 12 to 13.   15. A method for inducing an immune response to HPV in an individual, comprising administering to the individual a live attenuated vaccine according to claim 14.   12. The method of any one of claims 10 to 11, further comprising diagnosing the individual having an HPV infection.   A pharmaceutical composition comprising a nucleotide sequence encoding the HPV antigen of SEQ ID NO: 1 and SEQ ID NO: 3, or a fragment thereof having 90% homology.   24. A method of inducing an immune response to HPV subtype 6 or HPV subtype 11 in an individual, comprising administering to the individual the pharmaceutical composition of claim 23.   A pharmaceutical composition comprising a nucleotide sequence encoding the HPV antigen of SEQ ID NO: 5 and SEQ ID NO: 7, or a fragment thereof having 90% homology.   26. A method of inducing an immune response in an individual against HPV subtype 33 or HPV subtype 58, comprising administering to the individual a pharmaceutical composition according to claim 25.