Development And Preliminary Application, Of False Virus-based Human Papillomavirus (hpv) And Antibody Detection Methods And Animal Infection Models

The persistent infection of HPV is the main cause of cervical cancer and genital warts, while the development of HPV prophylactic vaccine is the basic method to prevent HPV infection. A successful vaccine is judged by whether effective humoral immunity or higher level of neutralizing antibody can be induced. Thus, it is imperative to establish an effective system to evaluate HPV vaccines. In this study, we established the pseudovirus-based neutralization assay successfully and performed a preliminary application.Totally, HPV16, HPV18, HPV58, HPV45, HPV6 and HPV11 EGFP pseudoviruses were generated by co-transfection of plasmid containing codon-modified HPV L1, L2 genes and plasmid containing EGFP gene into 293FT cells. The titers of the six pseudovirus stocks were 10^7.38/100μl, 10^6.57/100μl, 10^6.59/100μl, 10⁷/100μl, 10^6.33/100μl, 10^4.8/100μl TCID50, respectively. Then 200-300 TCID50/50μl pseudovirions were determined as the inoculum dose and the pseudovirus-based neutralization assays for HPV types 16, 18, 58, 45, 6 and 11 were established.The pseudovirus-based neutralization assay was applied to detect the neutralizing antibodies induced by different HPV58 L1 DNAvaccines(L1h, L1hΔc, L1S, L1SM and L1wt). All of the vaccines were constructed by cloning different HPV58 L1 genes into the pcDNA3.1 vector. DNA vaccine L1h contains the condon-modified HPV58 L1 gene; L1hΔc contains the condon-modified HPV58 L1 gene with the carboxyl terminus deleted; L1S was constructed by inserting the HPV58 L1 gene, which was from the plasmid p58LLw, into the pcDNA3.1; L1SM was the mutated L1S in which the base of G was changed to A at the site of 485bp of the L1 gene; L1wt contains the wide type HPV58 L1 gene. The results showed that L1S and L1h could induce neutralizing antibody. And the average titer of neutralizing antibody for L1S (6400) was much higher than that for L1h (48), however the other three vaccines were not able to induce neutralizing antibody. The results demonstrated that the vaccines containing different gene fragments have the different capacity of inducing the neutralizing antibody.The neutralization assay was used to detect HPV type-specific neutralizing antibody in different populations. As the results showed, the positive rate of neutralizing antibody was 28.0% in the 82 cervical cancer serum samples with the neutralization titers at the range of 160-640. In these positive samples, more than 80% were positive for HPV16. In the 50 samples with other genital disease, only one case was positive for HPV6 with the titer of 2560. The positive rate was only 2%, which was different from that of cervical cancer cases significantly(X²=16.1, P < 0.05). The HPV seropositive rate in the genital wart patients was 36.0% with the neutralization titers at the range of 160-2560. In the positive cases, 66.7% were positive for HPV6.Hybribio HPV genotyping kit was used to detect and determine the genotype of the HPV DNA. The relationship between the HPV DNA and the neutralizing antibody were analyzed. Among the 182 samples, 114 (62.6%) samples were positive for HPV DNA whilst 42 (23.1%) samples were positive for neutralizing antibody. The concordance between the detection rate of HPV DNA and that of neutralizing antibody was 49.5% with significant difference (X²=56.3, P < 0.05). 17.6% of the samples were positive for both neutralizing antibody and HPV DNA; 5.5% of the samples were positive for neutralizing antibody but negative for HPV DNA; 45.1% of the samples were negative for neutralizing antibody but positive for HPV DNA; 31.9% of the samples were negative for both neutralizing antibody and HPV DNA. Among the neutralzing antibody positive samples, the geometric means of the neutralizing titers were 281.0 and 422.2 in the DNA positive and negative samples, respectively. The concordance between HPV16 DNA and HPV16 type-specific neutralizing antibody was 65.9%, while the difference was significant (X²=31.2, P < 0.05)The establishment of the infection mouse models based on the HPV pseudovirions will lay foundation for the research of HPV infection intervention, the evaluation of HPV vaccines and the screening of the prophylactic agents. The 293FT cells were co-transfected with a plasmid containing codon-modified HPV capsids genes together with a reporter plasmid containing the luciferase gene. 48h later, the cells were collected and lysed, then the pseudoviruses were collected and titered. The titers of HPV16, HPV45, HPV58 and HPV6 pseudoviruses were 3.7×10⁸ TU/ml, 1.5×10⁸ TU/ml, 1.2×10⁸ TU/ml and 3.3×10⁷ TU/ml, respectively. The mice were subcutaneously injected with Depo-Provera. 4 days later, the mice were intravaginally instilled with nonoxynol-9 and 6 hours later pseudoviruses were inoculated intravaginally. After 7 days, the mouse was instilled luciferin substrate intravaginally, and expression level of the luciferase gene was detected by the IVIS system. The luminescent signal intensity of the 15μl pseudovirus inoculum was 1.779e+06 p/s, 1.829e+06 p/s, 5.738e+05 p/s and 5.718e+05 p/s, respectively and the luminescent signal intensity of the 5μl pseudovirus inoculum was 3.715e+05 p/s, 3.715e+05 p/s, 3.942e+05 p/s and 0 p/s for HPV16, HPV45, HPV58 and HPV6, respectively.We amplified 9 types (HPV16, HPV58, HPV11, HPV33, HPV59, HPV52, HPV53, HPV42 and HPV68) of HPV genomes from the DNA extracted and typed from the cervical swab by the PCR procedure with two steps and the annealing time increasing gradually. The PCR products were cloned into the vector of pEASY-T3 and sequenced, The sequence analysis showed that 8 of 9 genomes has about 99% identity with their reported genome respectively except for the genome of HPV68. The cloned HPV68 in this study showed 93% identity with HPV68a and 99% identity with ME180-HPV (HPV68b) at the full length level, and was 92.6% and 99.5% identical to HPV68a and ME180-HPV respectively at the L1 ORF region. Thus, the genomes of HPV16, HPV58, HPV11, HPV33, HPV59, HPV52, HPV53 and HPV42 were the variates of those genotypes published while HPV68 amplified in this study was HPV68b.