Construction Of In Vitro Infection Model Of Human Papillomavirus Pseudovirion By Optimized Baculovirus Transduction Method

Human papillomavirus can cause cervical cancer and other related cancers. It is very important to research and develop HPV vaccines. The recombinant HPV vaccine is underdeveloping in our lab. And the simple and efficient method for estimation of the protective ability of the candidate vaccine was needed. However currently available in vitro infection models are technically demanding or relatively low-yield. Recently reports have described that recombinant baculovirus could serve as a new gene transfer vehicle for mammalian cells with many unique advantages compared with other gene transfer methods. In this study, the feasibility of constructing HPV pseudovirion in mammalian cells by recombinant baculoviruses was investigated. The transduction protocol of delivering exogenous genes into mammalian cells by recombinant baculoviruses was investigated and optimized. And a new convenient way for rapid and efficient expression of target genes in mammalian cells was founded. With help of the new transduction protocol, the HPV16 pseudovirion and a rapid method for estimation of baculovirus titer were obtained in this research.Based on the Bac-to-Bac system recombinant baculoviruses were constructed, which contain the enhanced green fluorescent protein (EGFP) gene driven by CMV-T7 promoter, to investigate the feasibility of delivering exogenous genes into mammalian cells with the culture supernatant of insect cells infected by recombinant baculoviruses. Directly incubating HepG2 cells with the culture supernatant of infected Sf-9 cells (≥1.0×10⁷pfu/ml) for 12 hours in 37℃could achieve hyper 95% efficiency of gene transfer. The optimization of the incubation times and dilution ratios showed that the culture supernatant of infected insect cells l:l(vol:vol) diluted by the mammalian cell complete culture medium could achieve the high efficiency of gene transfer and expression together with the least impairment to cell viability.We first developed a transduction protocol based on the recombinant baculovirus culture supernatant transduction. Comparing with lipofectAMINE, recombinant retrovirus and vaccinia virus expression systems, the baculovirus vector could achieve higher transduction rate with no obvious cytopathic effect. Twenty-seven different mammalian cell lines were used to investigate the feasibility of delivering reporter gene into various mammalian cells with the culture supernatant transduction protocol, including sixteen human cell lines, nine rodent cell lines and two monkey cell lines. Results showed that most mammalian cell lines could be transduced with recombinant baculoviruses by this way. And we found that the gene transfer efficiencies in primate cell lines by baculovirus vector were markedly higher than those from rodent, and the efficiencies in adherent culture cells were markedly higher than suspended culture cells.Refer to the recent research reports, the protocol of culture supernatant transduction was further modified and optimized, and new ways were attempted to enhance gene delivery and expression. Different surrounding solutions and incubation temperatures were investigated, and the result showed that transduction occurred at 27℃using PBS as the surrounding solution could achieve the highest efficiency. Different incubation times and dilution ratios and different concentrations of FBS in PBS solutions were investigated, and results showed that transduction efficiencies were consistent to the virus dosages and incubation times. And we first found that the presence of FBS in PBS surrounding solution could influence transduction efficiency. Different concentrations of sodium butyrate were used in the culture medium to enhance gene expression. Results showed that 0.5～1 mM sodium butyrate was comparatively fit for the CV1 cells. In this study, centrifugal method was first used in the transduction of the mammalian cells by baculoviruses. And we found that centrifugal transduction at 600g for 1h could achieve more higher gene delivery and expression efficiencies than transduction in PBS at 27℃for 8h. And different centrifugal times, incubation times post centrifugal transduction and surrounding solutions were further investigated and optimized.In this study, we first developed a new and efficient transduction protocol based on the recombinant baculovirus culture supernatant transduction and centrifugal transduction. Mammalian cells transduced with the virus supernatant at 600g for 1h in PBS surrounding solution could achieve higher transduction efficiency with more fewer time. The centrifugal transduction protocol have notable advantages: simple, rapid and efficient, and could be easily used in daily common experiments. The effect of co-infection of recombinant vaccinia virus VV-T7RP, which could efficiently express the T7 RNA polymerase, on the gene expression in cells transduced with recombinant baculovinis containing the CMV-T7 promoter was investigated, results showed that VV-T7RP coinfection could markedly enhance the gene expression level in the transduced cells. It suggested that the VV-T7RP and recombinant baculovinis vectors could combine to an high efficient transient expression protocol.In this study, the recombinant baculovinis BacCS16L1L2 was constructed and capable of expressing HPV16 L1 and L2 protein in mammalian cells simultaneously. Utilizing the recombinant baculovinis culture supernatant centrifugal transduction protocol developed in this study, the HPV16 pseudovirion was successfully obtained in the 293FT cells. The infection assays and electron microscopy showed that the HPV16 pseudovirion have the infectivity. Four anti-HPV16 Mab, which have been fully characterized, were used to identify the HPV16 pseudovirion. Results showed that the HPV16 pseudovirion obtained in this study could be used for neutralization assay. And this model was used for testing neutralizing antibodies. Two neutralizing antibodies, 3D10 and PD1, was selected from eighteen anti-HPV16 Mab lines obtained in our lab. Utilizing the HPV16 pseudovirion, we proved that the protect humoral response could be elicited in Balb/c mice immunized with the HPV16 VLP constructed in our lab. In this study, HPV16 pseudovirion was first tried to infect the insect cells, and eGFP expression was found in some infected insect cells.Numerous research and application works on the recombinant baculovinis expression system have showed that it was very important to know the exact titer of a recombinant baculovinis stock in time for the stability of experiments and protein productions. In this study, utilizing the centrifugal transduction method and VV-T7RP coinfection, a new strategy of determining the recombinant baculovinis titer in mammalian cells was developed. Recombinant baculoviruses BacDGCG and BacT7G were constructed and capable of expressing EGFP in mammalian cells and insect cells. Firstly the virus titers were determined in insect cells. Then the same virus samples were used to transduce the CV1 cells by centrifugal method, followed by coinfected with VV-T7RP. 12～24h post transduction, the numbers of EGFP-positive wells were counted by fluorescence microscopy, and the transducing titer was calculated using TCID₅₀ method. Results showed that the CMV-T7-EGFP expression cassette in the baculovirus genome has the highest detection sensitivity than single CMV or T7 promoter with presence of T7 RNA polymerase and the obtained virus titer was similar to that determined in insect cells. In this study, we first suggested that the CMV-T7-EGFP expression cassette could be used as an excellent element for virus titer detection in mammalian cells, with no interfere with the protein or virus productions in insect cells. It was a new way could be chosen by researchers for the detection of recombinant baculovirus titers.