Optimized Preparation Method Of Pseudovirus For Human Papillomaviruses And Establishment Of A High-throughput Neutralization Assay

Infection of Human papillomavirus(HPV) is the cause of various diseases of genital tract,including cervix cancer,which is the second most common cancer leading to death in women.HPV's infection has host specificity,moreover,completed life cycle of HPV relays on differentiation of epithelial cells.Absence of applicable cell culture systems for infectious virus and limited virus particles obtained from patients' tissues restrict research on HPV and development of HPV vaccine. Convenient and efficient infection models of HPV in vitro are necessary for development of prophylactic vaccine and therapeutic agents of HPV.In recent years, people use pseudovirus to imitate HPV and construct pseudovirus-based neutralization assay for HPV,which displays a enormous applied potential in detection of neutralizing antibodys and development of prophylactic vaccine.Cotransfection of plasmids with condon-optimized genes is proved more convenient and higher-yield in producing pseudovirion than other systems,which has been constructed in our lab.To evaluate the protection providing by HPV prophylactic vaccine to animals or human,a great deal pseudovirion and a rapid, simple and convenient,high-throughput neutralization assay is required.To satisfy need of development of multivalent HPV vaccine,developing pseudovirus-based neutralization assay for other HPV types is necessary.In construction of pseudovirus by cotransfection of plasmids,appropriately lowering transfected mole ratio of L2 expression plasmid and reporter plasmid enhances expression of L1,which is positive correlative to construction efficiency of pseudovirus under certain conditions.The optimized transfected ratio may be 1:1/10:1/2 of L1,L2 expression plasmids and reporter plasmid.Modified transfected mole ratios of plasmids,optimal DNA concentration in calcium phosphate solution and appropriate volume of calcium phosphate solution contribute to a 10～20 times higher efficiency in construction of HPV16,HPV18,HPV6 and HPV11 pseudovirion.To satisfy the need of high-throughput assay for neutralizing antibodies, considerring the conditions in our lab,we constructed a new neutralization assay based on pseudovirion encapsidatingβ-gal expression plasmid.We utilized Elispot reader to scan 96 wells cell culture plates and display automatic counting result of cells transducted by pseudovirion every well,what remarkably enhance efficiency of neutralization assay for HPV.The method of x-gal stainning was improved to further enhance the efficiency.The new neutralization assay for HPV has high specificity,a wide detection range and similar sensitivity comparing to that of EGFP pseudovirus -based neutralization assay,moreover,it's low-cost in reagents and easy to operate.The neutralization efficiency of HPV16,HPV18,HPV6 and HPV11 monoclonal antibodies generated in our lab were then evaluated by the new pseudovirus-base neutralization assay.All these neutralizing mAbs can be used to thoroughly elucidate the neutralizing eptitopes of HPV and evaluate the quality of HPV vaccines.To investigate a suitable dose of candidate VLP-mixed vaccine of HPV16/18/6/11,sixty mice were vaccinated with serial diluted HPV16/18/6/11 VLP mix and the ED50 was identified to be HPV16 0.017μg/mL,HPV18 0.017μg/mL,HPV6 0.025μg/mL,HPV11 0.067μg/mL,which will guide us in the determination of an appropriate dose of mix vaccine in clinical trial studies.HPV58 and HPV52 are popular high-risk HPV in China,popularity of HPV58 is second to that of HPV18,but little has been studied about the two types since always.Construction of HPV58 and HPV52 pseudovirion help to research on HPV58,HPV52 and development of corresponding vaccine.Codon-optimized expression plasmids of capsid protein and reporter plasmid are cotransfected to 293FT cells, successfully generating high titer infectious pseudovirion of HPV58 and HPV52 respectively.Expression of 58L1 and 52L1 in cells was detected by Western blotting. Immunizing codon-optimized 58L1 and 52L1 expression plasmids induced neutralizing antibody in mice which can neutralize infection of pseudovirion respectively.We found cross neutralization phenomena exists between HPV16 and HPV58,HPV58 and HPV52,but not between HPV52 and HPV16.