Establishment Of Neutralizing Antibody Detection Method Of Human Papillomavirus

Human papillomavirus (HPV) infection causes virtually all cases of cervical cancer, the second most common cause of death from cancer among women worldwide. Of the 10 million cases of cancer that develop annually throughout the world, more than 15% are estimated to be attributable to infectious agents . Infection by human papillomaviruses (HPVs) accounts for approximately 30% of these cancers (~5% of all cancers).HPV are small(55-60nm),non-enveloped icosahedral DNA viruses which infect cutaneous and mucosal epithelia。In the HPV genome, the DNA (approximately 8 kb) is maintained in a supercoiled closed circular form, and a total of eight open reading frames (ORFs), encoding two main groups of proteins, namely early (E) and late (L) groups, are observed.Despite overall similarity in genomic organization, HPVs have rather diverse tissue speci-ficities and malignant potentials . Therefore, analyses of antigenic relationships among HPVs are needed to study viral pathogenicity and to develop a prophylactic vaccine strategy. Studies on neutralizing antibodies against HPVs have been hampered by the lack of cell culture systems for producing HPV particles and for monitoring infection with HPVs.The L-genes encoding L1 and L2 proteins, are the major and minor capsid proteins constructing the viral capsids in the ratio of 30:1 . Each capsid consists of an ordered array of 72 L1 capsomers. L2 proteins are co-assembled with L1 proteins, but the exact location is still unknown . In addition, it is found that L1 capsomers alone can also form virus-like particles (VLPs) that are morphologically identical to the real viral capsids .For all HPV types tested, L1 pseudovirion infection of cells was less efficient than infection with L1+L2 pseudovirions.PsV has been useful for studying papillomavirus assembly, cellular entry and neutralization and may have future utility as laboratory gene transfer tools or vaccine vehicles. We introduce a simple method for PsV production. The production method is based on transfection of a 293 cell line, 293FT, engineered to express high levels of SV40 large T antigen. The cells are co-transfected with codon-modified papillomavirus capsid genes, L1 and L2, together with a reporter plasmid containing the SV40 origin of replication. Reporter plasmid encapsidation within L1/L2 capsids is largely sequence independent and plasmids entirely lacking PV sequences can be packaged efficiently. Efficient purification of the PsV is achieved by ultracentrifugation. Then the PsV encapsidating a reporter plasmid are used for a high throughput in vitro neutralization assay in a multi well plate format. Infection of 293FT cells is monitored by reporter plasmid activity in the culture supernatant using a highly sensitive reporter system. Antibody-mediated PsV neutralization is detected by a reduction in reporter plasmid activity.Doing as research work, on the purpose of improving the packaging efficience, we subcloned the reporter gene of pGL3 to pcDNA3.1(+) which had SV40 ori of replication using XhoⅠand HindⅢ, and we constructed a new reporter plasmid L3-3.1 which was proved very efficient.Second, we constructed two capsid gene plamids of HPV58, 58L1-pcDNA3.1 and 58L2-pcDNA3.1,the capsid gene plamid of HPV16 we used was marked as pshell which both had L1 and L2 and was presented by Professor John T. Schiller as a gift.We used L3-3.1 with structure gene plamid of both HPV16 and HPV58 to transfect 293FT, and harvested PsV after 48 hours, we made purification of the two PsV through ultracentrifugation, from the result of Western blot ,we knowed that L1 and L2 of HPV16 and HPV58 expressed very specially in 293FT, and the L1 and L2 released from 293FT under the effect of lysate, and existed in the purified product after ultracentrifugation, and the site of the band matched very well with their actuall size.Then we used the PsV which had been purified to infect 293FT according to concentration gradient,the result indicated that we succeeded to harvest HPV16 PsV which encapidated L3-3.1 that succeeded to release and express after infection to 293FT; on the other side, unfortunately,we found a negative result of HPV58. At last, we established the proper low-grade amount of HPV16 PsV required to give a robust signal in the assay that was well above background, but within the linear range of the assay, and used this kind of amount of HPV16 PsV to incubate with heparin which could be used as standard preparation, then infected 293FT,the result indicated that heparin interfered with PsV infection with great effection.Actually,besides L3-3.1,we also tried to use some other reporter vectors includingβof which detection method was different from L3-3.1.As a attemptation to improve packing efficience of PsV, we cloned the BPV packing siganal toβ,which was marked asβB. We usedβandβB doing the same reseach work as L3-3.1 except ultracentrifugation, we got the negative result of HPV58, although we got a positive result of HPV16, which must be futher checked because this kind of detection method had some flaw when used in 293FT, and we desided to give up this kind of detection method finally.There are two things which we will do next, first, we will do something to optimize this kind of HPV16 neutralizing antibody detection method to become a kind of HPV16 neutralizing antibody evaluation system which is perfect; Second, we will make some codon modification to capsid genes of HPV58 to overcome negative regulatory features of the wild-type ORFs, these codon changes do not change the primary amino acid sequence of the proteins but do lead to a large increase in capsid protein production.What we has done is to establish a kind of HPV16 neutralizing antibody detection method initially, which can be foundation of our future work.