Development Of HPV16 Recombinant Epitope Vaccine

Human papillomavirus (HPV) is a papillomavirus that infects the epidermis and mucous membranes of humans. In 1976, Zur Hausen first linked HPV to cervical cancer which had been the second leading cause of cancer-related death in women worldwide. In present, there are approximate 500,000 women developing cervical cancer each year, and 274,000 die of the disease, 80% of those being in the developing country. In addition, HPV infection also leads to genital warts.More than 120 different HPV types have been identified based on DNA homology, and approximately 40 of them are known to infect the genital tract. HPV genotypes can be classified as the high risk types and the low risk types. The high risk types (HPV 16, 18, 31, 33, 35, 45, 58, 59) are associated with cevical, vulvar, vaginal, and anal cancers. Among them, type 16 is the most cancerigenic, causes about 50% of all cervical cancers.The ideal strategy to block HPV infection is to construct prophylactic vaccines. Studies on the structure of HPV show that HPV capsid can induce the product of neutralizing antibody which can block HPV infection. However, there are some problems to restrict the application of HPV inactivated vaccines and attenuated vaccines. First, HPVs are difficult to be cultured in vitro. Second, gene E6 and E7 are cancerigenic. Further studies show that the major capsid protein L1 can self-assemble into Virus-Like Particles (VLPs) in vitro. VLPs, similar with natrual virion, can induce high level of neutralizing antibody. So VLPs vaccine has been an available choice of HPV prophylactic vaccine. However, there are some disadvantages of VLPs vaccine, such as complicated procedures of manufacturing, high price, and limited range of protection.Recombinant epitope vaccine is a vaccine based on the identified epitopes and constructed by genetic engineering. Recombinant epitope vaccine containing specific epitopes is able to induce specific neutralizing antibody expressed in prokaryotic system and it is quite safe.In previous studies in our lab, three plasmids carrying the encoding genes for B cell epitope and Th cell epitope of L1, and B cell epitope of L2, respectively have been constructed. To isolate the encoding genes for the recombinant epitopes, the plasmids were digested with EcoRⅠa nd HindⅢt o release the recombinant-epitope- encoding genes, designated as R5, R6 and R8 respectively. The R5, R6 and R8 were subsequently cloned into pET-28a vectors. The vectors were transformed into E.coli BL21 (DE3) and were induced to express three recombinant proteins, designated as rR5, rR6 and rR8, respectively. In order to detect the expression way of the recombinat proteins, sonication method was implied to lyse the E.coli. The result showed that rR5, rR6 and rR8 weren't released by sonication, suggesting that rR5, rR6 and rR8 were expressed in inclusion bodies and that they must be released from E.coli by 8mol/L Urea that could denature the recombinant proteins. To harvest active proteins with the high purity, rR5, rR6 and rR8 were renatured by affinity chromatography (AFC) and gel filtration chromatography (GFC). Briefly, bacterial lysate was loaded into AFC column packed with the resin that could catch recombinant proteins with N-terminal His-tag. With successively decreased concentration of Urea in washing buffers from 3mol/L to 1mol/L, rR5, rR6 and rR8 were gradually refolded. Then rR5, rR6 and rR8 eluted from AFC were loaded into GFC column to remove the Urea from the proteins by molecular sieve. So recombinant proteins were renatured completely.To prepare poly-clonal antibodies for detecting rR5, rR6 and rR8, female Balb/c mice were immunized with 50μg of rR5, rR6 or rR8 for 3 times, respectively. One week after the last immunization, the sera were collected and tested by Elisa coated with rR5, rR6 and rR8. The results showed that high titers of antibodies were induced in Balb/c mice and the antibodies could be caught by rR5, rR6 and rR8 in Elisa.To detect whether the sera induced by rR5, rR6 and rR8 contain neutralizing antibodies that block HPV infection, we next to produce VLPs composing of L1 proteins of HPV 16 using Bac to Bac System in insect cells. In previous studies in our lab, from the lesioned tissues of condyloma acuminatum patients, HPV genomic DNAs have been isolated, from which, the encoding genes of L1 protein of HPV 16 were amplified by PCR. The genes were subcloned into the pFastBac1, the transfer vector with the mini-Tn7 element for site-specific transposition, by using multiple cloning site (MCS) in the vector. The resultant recombinant pFastBac1-L1 was transformed into E.coli DH10Bac containing the helper plasmid and baculovirus shuttle vector (Bacmid) with LacZαgene and the mini-Tn7 element for site-specific transposition with pFastBac1 vector. Under the effect of the transposase expressed by helper plasmid, L1 gene was transposed into the Bacmid to substitute the LacZαgene, generating recombinant Bacmid-L1 by homologous recombination between pFastBac1-L1 and Bacmid. Because of the disruption of LacZαgene, E.coli containing recombinant Bacmid-L1 failed to resolve the X-gal, presented a white phenotype in LB plate containing X-gal, and therefore could be selected by White Screen. From the selected DH10Bac clones, Bacmid-L1 was isolated to generate recombinant baculovirus for expressing recombinant L1 protein. Briefly, Bacmid-L1 DNA was isolated from DH10Bac in 5ml of culture and dissolved by 80μl of 1×TE Buffer (pH 8.0). The resultant Bacmid-L1 was transfected into insect cells (sf9) to generate the recombinant baculovirus. Beginning at 7 days after transfection, viral-infections were observed using an inverted phase microscope, characterized by increased cell diameter, cessation of cell growth and cell lysis, indicating that recombinant baculovirus had been generated and multiplied. To detect the expression of L1 protein in sf9, the infected cells were lysed in lysis buffer and the lysate were analyzed by SDS-PAGE. The result showed that L1 protein was expressed in sf9, with molecular weight (Mw) about 55kD, as expected.To confirm whether rR5, rR6 and rR8 are the candidate vaccines against HPV 16, Western Blot was applied to detect the recognition between L1 protein and the antibodies in sera induced by rR5, rR6 and rR8. The Blot revealed that the L1 protein could be recognized by the antibodies in the sera from the mice immunized with either rR5 or rR6, suggesting that the rR5 and rR6 might be used as the candidate peptides for developing vaccines to induce specific neutralizing antibodies against HPV 16.