JP6795468B2 - Human papillomavirus vaccine and how to use it - Google Patents

Description

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

Papillomavirus is a small DNA virus containing up to 7 early genes and 2 late genes. Generally, the early genes of papillomavirus are named E1 to E7, and the late genes of papillomavirus are named L1 and L2. Some animal species can be infected by members of the papillomavirus family.

Human papillomavirus (HPV) infection is routine and can be transmitted by sexual contact. HPV was classified into more than 56 types based on DNA sequence homology. HPV types 16 and 18 that cause epithelial dysplasia and other injuries often increase the risk of cancer, especially carcinoma in situ and wet cancer of the cervix, vagina, vulva and anal canal. Related. Globally, 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 proportion in cervical cancer, but in 2.6-5.2% in all mild cervical lesions. .. In addition, HPV6 and HPV11 are today of grade 2 and grade 3 cervical intraepithelial neoplasia and mild squamous intraepithelial lesions now considered precursors to cervical cancer, including mild cervical dysplasia. It is associated with about 20%. As research progresses, these two serotypes become genital warts, recurrent respiratory papillomatosis, lung cancer, tonsillar cancer, laryngeal cancer, and other malignant transformations of otherwise benign tumors as well as head and neck abnormalities. It has been shown to be associated with various forms of otolaryngological disorders, including dysplasia. The findings of this study reveal the use of consensus-sequence DNA vaccines for HPV6 and HPV11, promising their future.

DNA vaccines have many conceptual advantages over more traditional vaccination methods, such as live attenuated virus vaccines and recombinant protein-based vaccines. DNA vaccines are safe, stable, easily produced, and well tolerated in humans, and preclinical studies show no evidence of plasmid integration (Martin, T., et al., Plasmad DNA). maria vaccine: the potential for genomic integration after plasmid immune Ther, 1999.10 (5): p.759-68: Nichols, W. W.W. NY Acad Sci, 1995.772: p.30-9). Moreover, the fact that the efficacy of the DNA vaccine is not affected by the pre-existing antibody titer against the vector makes the DNA vaccine well suited for repeated doses (Chattergon, M., J. Boyer, and D.B. Winer, DNAimmunization: a newera in vaccines and antibodies. FASEB J, 1997.11 (10): p.753-63). However, when transitioning to large animals, one major obstacle to the clinical introduction of DNA vaccines was a decrease in platform immunogenicity (Liu, MA and JB Ulmer, Human clinical trials). of plasmid DNA vaccines. Adv Genet, 2005.55: p.25-40). Recent technological advances in the genetic engineering of DNA vaccine immunogens, such as codon optimization, RNA optimization and addition of immunoglobulin leader sequences, have improved the expression and immunogenicity of DNA vaccines (Andre, S.,. et al., Increased immune response elicited by DNA vaccine with a synthetic gp120 sequence with optimized codon sage. J Virol, 1998. usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein.J Virol, 2001.75 (22):. p.10991-1001; Laddy, D.J., et al, Immunogenicity of novel consensus-based DNA vaccines against avian influenza.Vaccine, 2007.25 (16):. p.2984-9; Frelin, L., et al, Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3 / 4A gene. Gene Ther, 2004.11 (6): p.522-33), and in recent years, techniques for vaccine delivery systems such as electric perforation have been developed (Hirao, LA, et al., Intradermal / subcutaneous immunization by electroporation immunization vaccines vaccine vaccine and potenc y in pigs and rhesus macaques. Vaccine, 2008.26 (3): p. 440-8; Lucky, A. et al. , Et al. , Effect of plasmid DNA vaccine design and in vivo electroporation on the returning vaccine-special immune responses in rhesus macaques. J Virol, 2007.81 (10): p. 5257-69; Ahlen, G. et al. , Et al. , In vivo electroporation enhancement the immunogenicity of hepatitis C virus non-structural 3/4A DNA by increased local DNA enhancement, protein, protein, protein. J Immunol, 2007.179 (7): p. 4741-53). In addition, studies have suggested that the use of consensus immunogens may be able to broaden the range of cell-mediated immune responses compared to native antigens alone (Yan., J. et al. , Et al., Enhanced cell-mediated immune response by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ter, et. Reconstruction and function of envelope center-of-tree human immunification virus type 1 antigens.J Virol, 2007.81 (16): p.8507-14).

There is still a need for improved vaccines and methods to prevent and treat HPV infections, especially those that cause cervical and / or lung, tonsillar, and pharyngeal cancers.

Aspects of the invention encode HPV antigens 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. A nucleic acid molecule containing a nucleotide sequence is included. In some embodiments, these nucleic acid molecules are nucleotide sequences 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 and at least. A nucleotide sequence encoding an amino acid fragment having 90% homology; a fragment of a 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 Alternatively, it comprises a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of fragments of the nucleotide sequence encoding an amino acid fragment having at least 90% homology with SEQ ID NO: 8.

In some embodiments, pharmaceutical compositions comprising the nucleic acid molecules described herein are provided.

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

The present invention also includes recombinant vaccines containing the nucleic acid molecules described herein. These recombinant vaccines include recombinant vaccinia vaccines, live attenuated vaccines, and DNA plasmids.

In some embodiments of the invention, at least 90% homology with 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 SEQ ID NO: 8 Included are proteins containing amino acid sequences selected from the group consisting of fragments of the sequences having.

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

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

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

FIG. 1 is a phylogenetic tree based on neighbor-joining 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 SDS-PAGE gels and detected using autoradiography. Both the E6 / E7 proteins of HPV6 and HPV11 are about 32 kDa, respectively (A). Human rhabdomyosarcoma (RD) cells were also transfected with p6E6E7 and p11E6E7, followed by immunofluorescence staining and then fixed. FITC fluorescence confirms the expression of p6E6E7 and p11E6E7 (B). DAPI fluorescence supports nuclear localization by Hoechst staining. FIG. 3 shows that the IFN-γ ELISpot assay induces a strong cellular response by p6E6E7 (A) and p11E6E7 (B) in C57BL / 6 mice. Assays were performed with splenocytes isolated from each group of mice (5 per group) after 3 immunizations every other week. Each immunization consisted of 20 μg per construct. Mice in the combination group received both p6E6E7 and p11E6E7 at 20 μg per construct for a total of 40 μg of DNA per vaccination. DNA was administered by IM injection, followed by electroporation ( ^* indicates P <0.0001, † indicates P = 0.0001). FIG. 4 shows an additional IFN-γ ELISpot assay with individual peptides to characterize the dominant epitope. Spleen cells and negative controls isolated from immunized mice were stimulated with overlapping peptides across the complete HPV6 E6 / E7 fusion protein (A) or HPV11 E6 / E7 fusion protein (B). FIG. 5A represents 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 dot spots above show the difference in the proportions of IFN-γ, IL-2, and TNF-α produced by either total CD4 + cells or CD8 + cells minus background. The P value for plots with an asterisk could not be determined because the mean of the negative control group was 0. FIG. 5B represents 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 dot spots above show the difference in the proportions of IFN-γ, IL-2, and TNF-α produced by either total CD4 + cells or CD8 + cells minus background. The P value for plots with an asterisk could not be determined because the mean of the negative control group was 0.

As used herein, the phrase "stringent hybridization condition" or "stringent condition" is a condition in which one nucleic acid molecule hybridizes with another nucleic acid molecule but not with another sequence. Represents. Stringent conditions are sequence-dependent and vary in different environments. Longer sequences specifically hybridize at higher temperatures. In general, stringent conditions are selected about 5 ° C. below the thermal melting point (Tm) of the specific sequence at the specified ionic strength and pH. Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probe complementary to the target sequence hybridizes to this target sequence in equilibrium. In general, these target sequences are in excess at Tm, so 50% of these probes occupy in equilibrium. Typically, stringent conditions are sodium ions with a salt concentration of less than about 1.0 M, typically about 0.01-1.0 M sodium ions (or other) at pH 7.0-8.3. Salt) and the temperature is 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 can also be achieved by the addition of stabilizers such as formamide.

Sequence homology of nucleotides and amino acids, 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. ^* Measurement may be performed using 4.0b10 software (DL Software, Sinauer Associates, Massachusetts). The "percentage of similarity" is calculated using PAUP ^* 4.0b10 software (DL Software, Sinauer Associates, Massachusetts). The mean similarity of the consensus sequence is calculated by comparing it with the entire sequence of the phylogenetic tree.

Briefly, the BLAST algorithm representing the Basic Local Sequence Search Tool is suitable for determining sequence similarity (Altschul et al., J. Mol. Biol., 1990, 215, 403-410, which is a reference. Incorporates this entire specification herein). Software for performing BLAST analysis is published through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm is a length in a query sequence that matches or satisfies a positive-valued threshold score T when aligned with words of the same length in the database array. Includes first identification of high-score sequence pairs (HSPs) by identifying short words of W. T is referred to as the neighborhood word score area value (neighborhood word score threshold) (Altschul et al., Supra). These first neighborhood word hits serve as seeds to initiate a search 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 by X from its maximum achievement; 2) one or more negative score residue alignments (negative) -When the cumulative score becomes 0 or less due to the accumulation of -scoring word alignments); or 3) when the end of any sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of alignment. In this Blast program, by default, the word length (W) is 11, see BLOSUM62 scoring matrix (Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is a reference. Alignment (B) is 50, expected value (E) is 10, M = 5, N = 4, and a comparison of both chains is used. This 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 are two sequences. Perform a statistical analysis of the similarity between them. One measure of similarity provided by this BLAST algorithm is the minimum sum probability (P (N)), which provides an indicator of the probability that a match between two nucleotide sequences will occur by chance, eg, a test. A nucleic acid is another if the minimum total probability in comparison of nucleic acid to other nucleic acids 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. It is considered to be similar to nucleic acid.

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

As used herein, the term "expressible form" refers to an essential regulatory sequence that is operably linked to a protein-encoding coding sequence so that the coding sequence is expressed when present in an individual cell. Represents a gene construct containing.

We disclose an improved vaccine that results from a multifaceted strategy for enhancing the cellular immune response induced by immunogenicity. A modified consensus sequence was prepared. Genetic modifications including codon optimization, RNA optimization, and the addition of highly efficient immunoglobulin leader sequences are also disclosed. This novel construct was designed to elicit a broader cell-mediated immune response that is more potent than the corresponding codon-optimized immunogen.

The modified HPV vaccine is based on a protein having an epitope that makes the modified vaccine particularly effective as an immunogen capable of inducing anti-HPV, and a gene construct encoding a protein having such an epitope. Therefore, vaccines can induce an immune response therapeutically or prophylactically. In some embodiments, means of delivering this immunogen include 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 is 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. Includes 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, which regulates the activity of the individual's immune system, thereby enhancing the immune response to HPV. When the nucleic acid molecule encoding this protein is incorporated into the cells of this individual, this nucleotide sequence is expressed in these cells, thereby delivering this protein to this individual. Provided is a method of delivering the coding sequence of this protein on a nucleic acid molecule, such as a plasmid, as part of an isolated protein or vector as part of a recombinant vaccine and as part of an attenuated vaccine.

To provide compositions and methods for prophylactically and / or therapeutically immunizing an individual against HPV.

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

SEQ ID NO: 1 comprises a nucleotide sequence encoding a consensus immunogen for the E6 and E7 proteins of HPV6. 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 immunogenicity of the E6 and E7 proteins of HPV6. SEQ ID NO: 2 contains the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogenic sequence. This IgE reader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

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

In some embodiments, the fragment of SEQ ID NO: 1 contains 786 or more nucleotides, in some embodiments 830 or more nucleotides, and in some embodiments 856 or more nucleotides, in some embodiments. In form, it may contain 865 or more nucleotides. In some embodiments, the fragment of SEQ ID NO: 1 such as the fragment described herein may further comprise the coding sequence of the IgE reader sequence. In some embodiments, the fragment of SEQ ID NO: 1 does not contain the coding sequence of the IgE reader sequence.

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

SEQ ID NO: 3 comprises a nucleotide sequence encoding a consensus immunogen for the E6 and E7 proteins of HPV11. 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 the consensus immunogenicity of the E6 and E7 proteins of HPV11. SEQ ID NO: 4 contains the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogenic sequence. This IgE reader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

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

In some embodiments, the fragment of SEQ ID NO: 3 contains 786 or more nucleotides, in some embodiments 830 or more nucleotides, and in some embodiments 856 or more nucleotides, in some embodiments. In form, it may contain 865 or more nucleotides. In some embodiments, the fragment of SEQ ID NO: 3, such as the fragment described herein, may further comprise the coding sequence of the IgE reader sequence. In some embodiments, the fragment of SEQ ID NO: 3 does not contain the coding sequence of the IgE reader sequence.

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

SEQ ID NO: 5 comprises a nucleotide sequence encoding a consensus immunogen for the E6 and E7 proteins of HPV33. SEQ ID NO: 5 comprises 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 immunogenicity of the E6 and E7 proteins of HPV33. SEQ ID NO: 6 comprises the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogenic sequence. This IgE reader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

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

In some embodiments, the fragment of SEQ ID NO: 5 contains 746 or more nucleotides, in some embodiments 787 or more nucleotides, and in some embodiments 812 or more nucleotides, in some embodiments. In form, it may contain 820 or more nucleotides. In some embodiments, the fragment of SEQ ID NO: 5, such as the fragment described herein, may further comprise the coding sequence of the IgE reader sequence. In some embodiments, the fragment of SEQ ID NO: 5 does not contain the coding sequence of the IgE reader sequence.

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

SEQ ID NO: 7 comprises a nucleotide sequence encoding a consensus immunogen for the E6 and E7 proteins of HPV58. 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 immunogenicity of the E6 and E7 proteins of HPV58. SEQ ID NO: 8 comprises the IgE leader sequence of SEQ ID NO: 10 at the N-terminus of the consensus immunogenic sequence. This IgE reader sequence is SEQ ID NO: 10 and can be encoded by SEQ ID NO: 9.

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

In some embodiments, the fragment of SEQ ID NO: 7 contains 752 or more nucleotides, in some embodiments 794 or more nucleotides, and in some embodiments 819 or more nucleotides, in some embodiments. In form, it may contain 827 or more nucleotides. In some embodiments, the fragment of SEQ ID NO: 7, such as the fragment described herein, may further comprise the coding sequence of the IgE reader sequence. In some embodiments, the fragment of SEQ ID NO: 7 does not contain the coding sequence of the IgE reader sequence.

In some embodiments, the fragment of SEQ ID NO: 8 contains 235 or more amino acids, in some embodiments 249 or more amino acids, and in some embodiments 257 or more amino acids, in some embodiments. In form, it may contain 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. Consensus The amino acid sequences of immunogenics, their functional fragments, and their expressible coding sequences include administration to this individual. Some embodiments encode amino acid sequences of consensus immunogenics 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. Contains isolated nucleic acid molecules. Some embodiments encode amino acid sequences of consensus immunogens 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 recombinant vaccines. Some embodiments include amino acid sequences of consensus immunogens 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 include amino acid sequences of consensus immunogens 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 vaccines and / or inactivated vaccines.

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

In another aspect, the vaccines herein are for HPV subtypes that have been found to be predominantly associated with the morphology of cervical cancer in, for example, non-US patients, more specifically Asian patients. Vaccines that provoke an immune response, in particular, these vaccines include HPV33 E6 and E7 and HPV58 E6 and E7, preferably both.

Modified vaccines include proteins having epitopes that make the improved vaccine particularly effective as immunogens capable of inducing an anti-HPV immune response, and gene constructs encoding proteins having such epitopes. Therefore, vaccines can be provided to induce an immune response therapeutically or prophylactically. In some embodiments, means of delivering this immunogen include 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, this immunogen. One or more compositions comprising, one or more attenuated vaccines, one or more inactivated vaccines.

Aspects of the invention provide a method of 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, or as a protein portion of an isolated protein or vector.

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

DNA vaccines are described in US Pat. Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, U.S. Pat. It is described in No. 5,981,505, No. 5,580,859, No. 5,703,055, No. 5,676,594, and the priority application 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 US Pat. Nos. 4,945,050 and 5,036,006, both of which are referenced. Is incorporated herein by.

The present invention relates to an improved live attenuated vaccine, an improved inactivated vaccine, an improved vaccine that uses a recombinant vector to deliver a foreign gene encoding an antigen, and a subunit vaccine and a glycoprotein vaccine. Examples of live attenuated vaccines, vaccines that deliver foreign antigens using recombinant vectors, subunit vaccines and glycoprotein vaccines are US Pat. Nos. 4,510,245, 4,797,368, No. 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 , 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. 5,451,499, No. 5,453,364, No. 5,462,734, No. 5,470,734, No. 5, No. 474,935, No. 5,482,713, No. 5,591,439, No. 5,643,579, No. 5,650,309, No. 5,698,202, It is described in No. 5,955,088, No. 6,034,298, No. 6,042,836, No. 6,156,319 and No. 6,589,529. Each of these is incorporated herein by reference.

Once incorporated into a cell, the gene construct (s) may remain present within 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 the chromosome may be introduced into this cell. When introducing DNA into these cells, a drug that promotes DNA integration into the chromosome may be added. A DNA sequence useful for facilitating integration may be included in this DNA molecule. Alternatively, RNA may be administered to this cell. It is also conceivable 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 an attenuated raw microbial or recombinant microbial vector that survives intracellularly. The genetic construct may be part of the genome of a recombinant viral vaccine, where the genetic material is either integrated into the chromosome of this cell or remains extrachromosomal. The gene construct contains regulatory elements required for gene expression of nucleic acid molecules. These elements include promoters, start codons, stop codons, and polyadenylation signals. In addition, 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 which they are administered.

The start and stop codons are generally considered to be part of the nucleotide sequence encoding the desired protein. However, these factors need to be functional in the individual to whom this gene construct is administered. The start and stop codons must be in the frame along with the coding sequence.

The promoter and polyadenylation signal used must be functional within the cell of the individual.

Examples of promoters useful for carrying out the present invention, particularly useful in the production of human gene vaccines, include the Cytomegalovirus 40 (SV40) promoter, the mouse milk cancer virus (MMTV) promoter, the BIV terminal repeat sequence (LTR) promoter, and the like. Human immunodeficiency virus (HIV) promoter, Moloney virus promoter, ALV promoter, Cytomegalovirus (CMV) promoter such as CMV earliest promoter, Epstein-Barvirus (EBV) promoter, Laus sarcoma virus (RSV) promoter and human actin , But not limited to, promoters derived from human genes such as, but not limited to, human myosin, human hemoglobin, human muscle creatin and human metallothioneine.

Examples of polyadenylation signals useful in carrying out the present invention, especially in the production of human gene vaccines, include, but are not limited to, SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal, which is present in the plasmid pCEP4 (Invitrogen, San Diego CA) and is referred to as the SV40 polyadenylation signal, is used.

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

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

In some preferred embodiments relating to the application of immunization, a nucleic acid molecule (s) comprising a nucleotide sequence encoding a protein of the invention and, in addition, a gene for a protein that further enhances the immune response to such target protein. Deliver. Examples of such genes are α-interferon, γ-interferon, platelet-derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MHC, CD80, CD86 and IL-15 containing IL-15 with signal sequences removed and optionally with IgE signal peptide, etc. Examples include genes encoding other cytokines as well as phosphorus hokine. Other genes that may be useful include MCP-1, MIP-lα, 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, SAP K, 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, TAP2 and genes encoding functional fragments thereof.

If for some reason it is desirable to eliminate cells that receive this gene 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 this gene construct. The drug ganciclovir can be administered to this individual, and the drug selectively kills all tk-producing cells, thus providing a means for the selective destruction of cells carrying this gene construct.

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

In some embodiments, a genetic construct in which the coding sequences of the proteins described herein are linked to an IgE signal peptide may be provided. In some embodiments, the proteins described herein are linked to an IgE signal peptide.

In some embodiments in which the protein is used, for example, one of ordinary skill in the art can use well-known techniques to produce and isolate the protein of the invention. In some embodiments in which the protein is used, for example, one of ordinary skill in the art will use well-known techniques to insert the DNA molecule encoding the protein of the invention into a commercially available expression vector for use in a well-known expression system. 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 Saccharomyces cerevisiae strain. For example, a 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, Vector 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 lines, resulting in a series of processing forms of this protein.

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

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

In addition to protein production by recombinant techniques, automated peptide synthesizers may also be used to produce essentially pure isolated proteins. Such techniques are well known to those of skill in the art and are useful when derivatives with substitutions are not supplied in the production of DNA-encoded proteins.

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

Routes of administration include intramuscular, intranasal, intraperitoneal, intradermal, subcutaneous, intravenous, intraarterial, intraocular, and oral routes, as well as inhalants or suppositories into mucosal tissue. For example, local and percutaneous routes by washing to the vagina, rectum, urethra, cheeks and sublingual tissues are included, but not limited to. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injections. The gene construct may be administered by means including, but not limited to, methods and devices of electroperforation, conventional syringes, needleless injection devices, or "fine particle impact gene guns".

Examples of preferred electroporation devices and electroporation methods for facilitating the delivery of these DNA vaccines are US Pat. No. 7,245,963 by Draghia-Akli et al., U.S. Patent Publication No. 2005 filed by Smith et al. Included are examples described in / 0052630, the contents of which are incorporated herein by reference in their entirety. Under Article 35USC119 (e), U.S. Patent Provisional Application No. 60 / 852,149 filed on October 17, 2006 and U.S. Patent Provisional Application No. 60 / 978,982 filed on October 10, 2007. Provided in co-pending and co-owned U.S. Patent Application No. 11/847072, all of which is incorporated herein by reference in its entirety, which claims the interests of the issue, filed October 17, 2007. Electric punching devices and methods that facilitate the delivery of DNA vaccines are also preferred.

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

In some embodiments, the plurality of electrodes can deliver energy pulses in a distributed pattern. In some embodiments, the plurality of electrodes can deliver energy pulses in a distributed pattern via electrode control under a programmed order, allowing the user to electroporate the programmed order. Enter 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 at least two active electrodes with one neutral pole measuring impedance.

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

In some embodiments, the nucleic acid molecule is delivered to these cells in combination with administration of a polynucleotide function enhancer or gene vaccine promoter. The polynucleotide function enhancer is described in US Pat. Nos. 5,593,972, 5,962,428 and International Application No. PCT / US94 / 0899 filed on January 26, 1994. Each of these is incorporated herein by reference. Genetic vaccine promoters are described in US Pat. No. 021,579 filed April 1, 1994, which is incorporated herein by reference. Auxiliary agents administered in combination with the nucleic acid molecule may be administered as a mixture with the nucleic acid molecule before and after administration of the nucleic acid molecule, or may be administered separately or simultaneously. In addition, other agents that can function as transfectants and / or replicators and / or inflammatory agents and can be administered with GVF include α-interferon, γ-interferon, GM-CSF, and platelet-derived growth. Growth factors such as factors (PDGF), TNF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 and IL-15, cytokines and lymphocains. , And surface active agents such as fibroblast growth factor, immunostimulatory complex (ISCOMS), Freund's incomplete adjuvant, LPS analogs including monophosphoryl lipid A (WL), muramilpeptides, quinone analogs and squalensqualenes. Hyaluronic acid may also be used for administration in combination with this gene construct. In some embodiments, the immunomodulatory protein may be used as the GVF. In some embodiments, this nucleic acid molecule is provided in association with PLG to enhance delivery and uptake.

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

The pharmaceutical composition according to the invention is formulated according to the administration method used. When the pharmaceutical composition is an injectable pharmaceutical composition, the injectable pharmaceutical composition is sterile and contains no pyrogens or particles. Preferably, an isotonic formulation is used. In general, the isotonic additives may include sodium chloride, D-glucose, mannitol, sorbitol and lactose. In some cases, an isotonic solution such as phosphate buffered saline is 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 protein-based live attenuated vaccine, cell vaccine, recombinant vaccine or nucleic acid or DNA vaccine. In some embodiments, methods of inducing an immune response against an immunogen in an individual, including methods of inducing a mucosal immune response, are CTACK protein, TECK protein, MEC protein and functional fragments thereof or codes capable of expressing them. One or more of the sequences are isolated nucleic acid molecules encoding the proteins of the invention and / or recombinant vaccines encoding the proteins of the invention, and / or subunit vaccines and / or attenuated proteins of the invention. Includes administration to this individual in combination with a live vaccine and / or an inactivated vaccine. Includes an isolated nucleic acid molecule that encodes an immunogen; and / or a recombinant vaccine and / or an immunogen that encodes an immunogen, one or more of the CTACK protein, TECK protein, MEC protein and functional fragments thereof. The subunit vaccine and / or live attenuated vaccine and / or inactivated vaccine may be administered before, at the same time as, or after their administration. In some embodiments, the individual is administered with an isolated nucleic acid molecule encoding one or more proteins selected from the group consisting of CTACK, TECK, MEC and functional fragments thereof.

The present invention will be further described in the following examples. Although these examples show preferred embodiments of the invention, it should be understood that they are given for purposes of illustration only. From the above description and examples thereof, one of ordinary skill in the art can identify the essential features of the invention and to apply the invention to various uses and conditions without departing from the spirit and scope of the invention. , Various modifications and improvements of the present invention can be made. Therefore, in addition to the improvements shown and described herein, various improvements of the invention will be apparent to those skilled in the art from the earlier description. Such improvements are also intended to fall within the appended claims.

Each of the US patents, US patent applications, and references cited throughout this disclosure are incorporated herein by reference in their entirety.

Examples The present invention is further defined in the following examples. It should be understood that these examples represent preferred embodiments of the present invention, but are provided for purposes of illustration only. From the above description and examples thereof, one of ordinary skill in the art can confirm the essential features of the present invention without departing from the spirit and scope of the present invention, in order to adapt to various uses and conditions. Various changes and modifications of the present invention can be made. Therefore, various modifications of the present invention will be apparent to those skilled in the art from the above description in addition to those shown and described herein. It is also intended that such changes are within the scope of the appended claims.

Example 1
Vaccine design and expression of HPV6 and HPV11 E6 / E7 Construction of fusion immunogen based on HPV6 and HPV11 E6 / E7 consensus HPV6 or HPV11 E6 / E7 gene sequences were collected from GeneBank and nucleotides of consensus E6 and E7 Sequences were obtained after multiple alignments. The consensus sequence of the E6 or E7 protein of HPV6 was made from 98 or 20 sequences, respectively, and the consensus sequence of the E6 or E7 protein of HPV11 was made from 76 or 13 sequences, respectively. The multiple alignment procedure applied to phylogenetic studies included the application of Clustal X (version 2.0). As shown in FIGS. 1A and 1B, there was about 0-2% sequence dispersion between HPV strains and between HPV strains belonging to the same type of their E6 and E7 proteins. However, the genetic distance could be increased between HPV6 and HPV11 up to 19.3% for the E6 protein and 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, some changes were made (Fig. 1C). High-performance leader sequences were fused in-frame upstream of the start codon to promote expression. The optimization of codons and RNAs was also performed as previously described (J. Yan, et al., Cellular immunity integrated by a vaccine HPV18 DNA vaccine encoding an E6 / E7 fusion vaccine protein infection protein). .26 (2008) 5210-5215; and J. Yan, et al., Induction of protein immunity in vivo following delivery of a novel HPV-16 DNA vaccine encoding 9E6 / 26 (2008) 5210-5215; 440). For proper protein folding and good CTL processing, intracellular proteolytic cleavage sites were introduced between the E6 and E7 proteins. Both the genetically engineered synthetic 6E6E7 and 11E6E7 genes were 840 bp in length. For further study, sequenced 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 previously described (J. Yan, et al., Vaccine.26 (J. Yan, et al., Vaccine.26). 2008) 5210-5215; and J. Yan, et al., Optimization, Vaccine. 27 (2009) 431-440). A fusion gene (6E6E7 or 11E6E7) encoding either the HPV6 or 11 consensus E6 / E7 fusion protein was synthesized and sequenced. The synthesized 6E6E7 or 11E6E7 was digested with BamHI and XhoI and cloned into the expression vector pVAX (Invitrogen) under the control of the cytomegalovirus earliest 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, Indiana). Two days after transfection, these cells were lysed with modified RIPA cell lysis buffer and the cell solubilized solution was recovered. Western blot analysis was performed using an anti-HA monoclonal antibody (Sigma-Aldrich, St. Louis, Missouri), and Western wasabi peroxidase-conjugated goat anti-mouse IgG (Sigma) using an ECLTM Western blot analysis system (Amersham, Piscataway, NJ). -Visualized by Aldrich, St. Louis, Missouri.

Indirect immunofluorescence assays were performed using human rhabdomyosarcoma cells (RD cells) to confirm expression of p6E6E7 and p11E6E7. RD cells cultured on chamber slides were transfected with Turbofectin 8.0 (Origene, Rockville, Maryland) at pVAX, p6E6E7 or p11E6E7. These cells were then fixed with PFA and permeabilized with 0.1% Triton-X in PBS. These cells were incubated with 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-binding anti-mouse IgG (Abcam, Cambridge, Mass.), Respectively. Hoechst staining was also performed to identify cell nuclei and localize RD cells. After all incubations were complete, these cells were mounted and glass slides fixed with Fluoromount-G (Southern Biotech, Birmingham, Alabama). These samples were observed and imaged using a confocal microscope (CDB Microscope Core, University of Pennsylvania, Cell Developmental Biology, Philadelphia, PA).

Example 2
Immunization of mice and treatment group Female C57BL / 6 mice 6-8 weeks old were used in this experiment. Mice were obtained from the Jackson Laboratory (Bar Harbor, Maine). These mice were housed and maintained at the University of Pennsylvania Animal Resources Institute in accordance with the policies of the National Institutes of Health and the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). The mice used in these experiments were divided into 4 groups for immunization. Mice were immunized with p6E6E7, p11E6E7, or both constructs and the pVAX group was used as a negative control.

DNA Vaccination and Electroporation Each mouse was dosed with 20 μg of each DNA plasmid three times at 14-day intervals. Mice in the group vaccinated with both p6E6E7 and p11E6E7 were vaccinated with 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 a square wave was generated by a CELLECTRA ™ electroporator (Inovio Pharmaceuticals, Bluebell, PA). The electroporator was configured to deliver two electrical pulses at 52 ms / pulse, 0.2 amps, with a 1 second delay interval. The electroporation procedure was performed as previously described (12, 13).

Both the IFN-γ ELISpot assay treated and control mice were sacrificed 1 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 (Sward 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 IP96-Well Multiscreen (TM) plate (Millipore, Bedford, Mass.) Is pre-coated with monoclonal mouse IFN-γ capture antibody (R & D Systems, Minneapolis, Minnesota) at 4 ° C. Incubated evening. After washing 3 times with 1 × XPS, these 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 triple 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 15 amino acid sequences, of which 8 residues overlapped with each sequential peptide. The HPV6 and 11 peptides were divided into two pools (one pool E6 and the other E7) at a concentration of 2 μg / ml in DMSO, respectively. Concanavalin A (Sigma-Aldrich, St. Louis, Missouri) and R10 medium were placed in the positive and negative control wells in place of the peptides, respectively. The plate was then placed in an incubator with a 5% CO2 atmosphere. After incubation at 37 ° C. for 24 hours, these wells were washed with 1 x PBS. A biotinylated anti-mouse IFN-γ detection antibody (R & D Systems, Minneapolis, Minnesota) was added to each well and then incubated overnight at 4 ° C. These plates were then washed and treated with streptavidin-AP and BCIP / NBT Plus (R & D Systems, Minneapolis, Minnesota) according to the color development protocol provided by R & D Systems. These wells were air dried overnight and spots inside the wells were scanned and counted by the ELISpot plate reader system using the software of ImmunoSpot® 3 and ImmunoSpot® (Cellular Technology Ltd., Cellular Technology Ltd., Shaker Heights, Ohio). The reported number of spot-forming cells was converted and an operation was used to indicate the number of spot-forming units per 1 × 10 ⁶ splenocytes.

Given their sensitivity and ability to exhibit T cell activity, the IFN-γ ELISpot assay is used to respond to stimulation with either HPV6 or 11 E6 and HPV6 or 11 E7 peptides and is antigen-specific. The number of IFN-γ secretory cells was determined.

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

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

Mouse splenocytes that received both p6E6E7 and p11E6E7 (combination group) also had the ELISpot assay described above to determine if there was any immune interference when immunizing these two constructs together. (FIGS. 3A and 3B). The mean value of the combination group of HPV6 for E6 and E7 peptides was 1670SFU / 106 splenocytes (σ = 55.7, P <0.001). The same group of splenocytes against the E6 and E7 peptides of HPV11 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 the immunodominant peptide within the E6 / E7 consensus antigen, an epitope mapping test was performed to determine the dominant epitope within the peptide pool (FIGS. 4A and 4B). These tests were performed in the same manner as the IFN-γ ELISpot assay described above. Individual peptides were used instead of pools to stimulate splenocytes.

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

Intracellular Cytokine Staining Given 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. .. Spleen cells from immunized and untreated mice were isolated and stimulated in a 5% CO2 environment at 37 ° C. for 4 hours with peptides spanning the E6 regions of HPV6 and HPV11 and the E7 regions of HPV6 and HPV11. Positive and negative controls were used in this assay by placing cells in phorbol 12-millistate 13-acetate (PMA) and R10 cell culture medium, respectively. After incubation, these cells were first stained with ViViD dye (Invitrogen, Carlsbad, CA) to distinguish between live and dead cells, and then whole cells were subjected to APC-Cy7 hamster anti-mouse CD3e, PerCP-Cy5. They were stained with surface marker antibodies of 5 rat anti-mouse CD4 and APC rat anti-mouse CD8a (BD Biosciences, San Diego, CA). These cells were then fixed using the Cytofix / Cytoperm kit (BD Biosciences, San Diego, CA). After fixation according to the manufacturer's protocol, these cells were subjected to Alexa Fluor 700 rat anti-mouse IFN-γ, PE-Cy7 rat anti-mouse TNF clone, and PE rat anti-mouse IL-2 (BD Biosciences, San Diego, CA). Stained with cell marker antibody. After staining, these cells were fixed in PBS solution containing 2% paraformaldehyde. 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, Oregon). CD4 + and CD8 + events, singlets from FSC-A vs. FSC-H, total splenocytes from FSC-A vs. SSC-A, live cells from ViViD dye (Pacific Blue) vs. SSC-A, CD3 (APC) -Cy7) CD3 + cells from SSC-A, CD4 (PerCP-Cy5.5: positive-CD4 +, negative-CD8 +) vs. CD4 + or CD8 + from SSC-A were isolated using the gate order. The last two populations were gated against Alexa Fluor 700, PE-Cy7, and PE and changes in the production of IL-2, IFN-γ, and TNF-α were observed, 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 p6E6E7 immunized mice was 0.163% (σ =), respectively. It was 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 were 3.323% (σ = 1.39) and 0.838% (σ = 0.), respectively. 32), and 1.172% (σ = 1.81). The same intracellular cytokine data was collected using mouse splenocytes immunized with p11E6E7 after incubation of HPV11 with E6 and E7 antigens (FIG. 5B). Of all the 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), the average value of cells producing IL-2 was 0.026% (σ = 0.037). Furthermore, in mice immunized with p11E6E7, the mean values of all CD8 + cells that produced IFN-γ, TNF-α, and IL-2 were 4.52% (σ = 2.53) and 2.08%, respectively. σ = 1.56), and 0.21% (σ = 0.22). With the exception of some panels, the proportion of cytokine-producing cells in the treated group versus their respective untreated group was statistically significant at a confidence level of 89% or higher. Observation of the degree of cytokine production of CD4 + T cells vs. CD8 + T cells concludes that the immune responses evoked by p6E6E7 and p11E6E7 are significantly biased towards the drive of CD8 + lymphocytes associated with their cell clearance in all models. Can be attached.

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

This study provides compelling evidence that DNA vaccines may be able to achieve the levels of immunogenicity found in experiments with other proven and traditionally used vaccine platforms. Indicated. High levels of cellular response measured in other similar E6 and E7 specific HPV DNA vaccine studies were associated with data suggesting prophylactic and therapeutic antitumor effects. Most HPV-related studies and disease load 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 assessed using conventional HPV disease loading models. Determining the potential for protection and treatment of p6E6E7 and p11E6E7 is insightful when appropriate disease loading models are available. Therefore, the immunogenicity efficacy of p6E6E7 and p11E6E7 can be inferred by examining the high levels of IFN-γ and cytokine production quantified by the ELISpot assay and intracellular cytokine staining. Nonetheless, it was found that such levels are significantly stronger in size and that the two constructs are likely to elicit substantial levels of cell-mediated immune response. Since HPV-related malignancies are generally secondary to co-infection with multiple serotypes, there is a great need to further investigate the feasibility of combining vaccines for different serotypes of the virus. Further studies investigating T cell kinetics and vaccine competition ensure that the co-immunization effect of these two plasmids is further characterized.

Intracellular cytokine staining showed that immunization with p6E6E7 and p111E6E7 could induce T cells producing a significant proportion of IFN-γ, TNF-α, and IL-2. Given the currently known functions of the immune system, IFN-γ has traditionally been used as an indicator of cell-mediated immune response. Some of these important roles include the ability to regulate and stimulate innate and adaptive immunity. In addition, it is widely accepted that the major producer of IFN-γ is T cells that put IFN-γ production into an acceptable mode for measuring cell-mediated 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 when characterizing the post-vaccination immune response. Given the potential tumor growth properties of HPV6 and HPV11, TNF-α production can be even more interesting. IL-2 is another signaling molecule that has been observed to play a central role in the proliferation and differentiation of T cells in the immune system. Therefore, IL-2 is often examined in combination with other cytokines to gain further insight into the extent and quality of a particular immune response. Considering the above, CD4 + and CD8 + cells that produce a significant proportion of IFN-γ, TNF-α, and IL-2 after immunization with p6E6E7 are such that this vaccine induces a potent immune response. Suggests success. The same tendency applies to cells isolated from p11E6E7-immunized mice, with the exception of CD4 + cells that secrete IL-2. Furthermore, it is noteworthy to observe that CD8 + cells significantly enhanced the immune response in immunized mice, a property important in assessing the antitumor effects of these two plasmids.

Claims (10)

Comprising a nucleotide sequence encoding an HPV antigen of SEQ ID NO: 3, a nucleic acid molecule. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a plasmid. A pharmaceutical composition or an attenuated live virus vaccine comprising the nucleic acid molecule according to claim 1 or 2. Comprising a nucleic acid molecule comprising a nucleotide sequence encoding an HPV antigen of SEQ ID NO: 3, a pharmaceutical composition. The pharmaceutical composition according to claim 4, for use in inducing an immune response against HPV subtype 6 or HPV subtype 11 in an individual. A recombinant vaccine comprising the nucleic acid molecule according to claim 1 or 2. The recombinant vaccine according to claim 6, which is a recombinant vaccinia vaccine. The composition comprising the nucleic acid molecule of claim 1 or 2, the attenuated live vaccine of claim 3, or claim 6 or 7, for use in inducing an immune response against HPV in an individual. Recombinant vaccine. The composition, live attenuated virus vaccine, or recombinant vaccine according to claim 8, wherein the nucleic acid molecule is introduced into the individual by electroporation. The composition comprising the nucleic acid molecule of claim 1 or 2, the attenuated live virus vaccine of claim 3, or claim 6 or for use in therapeutically immunizing an individual against HPV. 7. The recombinant vaccine according to 7.