| Cervical cancer is the second most prevalent cancer in gynecological malignancies. Persistent infections with high-risk human papillomavirus (HPVs) have been proved to be major etiological agents of cervical cancer.Globally, high-risk HPVs such as HPV-16and-18are the most prevalent types in cervical cancers, and low-risk HPVs such as HPV-6and-11are the most prevalent types in genital warts. In China, HPV58ranks as the third most prevalent in cervical cancer. So it is of great importance to develop vaccines against HPV-16,-18,-58.Currently there are two licensed HPV L1virus-like particles (VLPs) vaccines, Cervarix, a bivalent HPV-16,-18vaccine produced in Hi-5insect cells by GSK, and Gardasil, a quadrivalent HPV-16,-18,-6,-11vaccine produced in Saccharomyces cerevisiae by Merck. Results of clinical trials showed that tow vaccines both are highly immunogenic, security and protective ability. Different from most genetic engineered proteins, HPV L1VLPs are highly assembled recombinant proteins, and epitopes of L1VLPs which are able to induce neutralizing mAbs are mostly conformational ones. Therefore, in the production of recombinant vaccines, it is necessary to determine the activity of L1recombinant proteins prepared in vitro. H16.V5and H18.J4are reported type-specific neutralizing mAbs against HPV-16,-18respectively. Both of them were utilized in competitive ELISA for the quality control of HPV-16,-18L1VLPs vaccines, as well as in competitive Luminex immunoassay (cLIA) for high-quantitive evaluation of type-specific neutralizing mAbs against HPV-16,-18in immunized people. H18.J4-based cLIA showed some limitation in detection sensitivity, while H16.V5-based cLIA showed high sensitivity. Therefore, it is necessary to prepare mAbs for optimizing the method.Studies of mechanisms of HPV infection indicated that L1protein contains many key sites reacted in the HPV infection. Antibodies with different characteristics might facilitate the study of these sites. In our lab, HPV-16,-18,-58L1VLPs vaccines have been prepared. For meeting the need of developing HPV vaccines suitable for Chinese, mAbs against HPV-16,-18,-58were prepared by hybridoma fusion technology in the present study.50%inhibitory concentrations (IC50) of mAbs were determined by pseudovirus neutralization assay. Characteristics of epitopes recognized by mAbs were analyzed by ELISA. By additivity assay, we analyzed whether mAbs recognized the same epitope, and whether they were different from H16.V5or H18.J4. Then sandwich ELISA based on mAbs against HPV-16was established to determine the quality of HPV-16L1VLPs. Results of the study set the groundwork for studies of HPV-16,-18,-58L1VLPs vaccines and mechanisms of HPV infection.Ten conformational neutralizing mAbs against HPV-16were gained, IC50of which against HPV-16ranged from0.03nM to1.72nM. Their epitopes all depended on capsomers just like the epitope recognized by H16.V5. Results of additivity tests indicated that epitopes recognized by them were different from each other or from H16.V5except that XM16-1/XM16-5might recognize the same epitopes, and epitopes of XM16-3/XM16-6might be the same one. One of them, XM16-17, could cross-neutralize HPV-18. Ten linear neutralizing mAbs were also gained, IC50of which against HPV-16ranged from1.78nM to4.17nM. Five of them could cross-neutralize HPV-18and/or-58. XM16-13, XM16-16and XM16-20could cross-neutralize HPV-18, XM16-15could cross-neutralize HPV-58, and XM16-14could cross-neutralize HPV-18,-58. The sandwich ELISA based on XM16-6(capturing mAb) and XM16-12-HRP (detecting mAb) could determine concentrations of HPV-16L1VLPs from0.05μg/mL to1.5μg/mL, and it owned good linear range and stability.Seven type-specific conformational neutralizing mAbs against HPV-18were gained, IC50of which against HPV-18ranged from0.04nM to0.89nM. All of them recognized inter-capsomeric epitopes, while epitope of H18.J4depended on intact VLPs. Results of additivity tests revealed that epitopes recognized by them were different from each other or from H18.J4. Six linear neutralizing mAbs were also gained, IC50of which against HPV-18ranged from0.44nM to2.34nM. Three of them (XM18-12, XM18-13, XM18-14) could cross-neutralize HPV-6.Seven type-specific conformational neutralizing mAbs against HPV-58were gained, IC50of which against HPV-58ranged from0.04nM to1,0nM. Three of them recognized intra-capsomeric epitopes, while others recognized inter-capsomeric epitopes. Results of additivity tests revealed that epitopes recognized by them were different from each other. Nine linear neutralizing mAbs were also gained, IC50of which against HPV-58ranged from0.44nM to1.17nM. Of them, XM-21and XM58-14could cross-neutralize HPV-18, XM-24could cross-neutralize HPV-6, and XM58-13could cross-neutralize HPV-11. Then indirect ELISA based on XM-22was established, with poor stability.Results of the study showed that most of conformational mAbs against HPV-16,-18,-58L1VLPs were type-specific, and most epitopes of them depended on capsomers like H16.V5, while epitope of H18.J4depended on intact VLPs. It is suggested that mAbs recognizing inter-capsomeric epitopes could be well utilized in quality control of vaccines. We first analyzed the dependence of HPV-58mAbs to VLPs or capsomeres and found that only3of them depended on intact VLPs. The present study also showed that half of mAbs recognizing linear epitopes could cross-neutralize other HPV besides immunogens, and they showed weak neutralizing activity against immunogens than type-specific conformational mAbs. We found that5mAbs against HPV-16could cross-neutralize HPV-18and/or-58,3mAbs against HPV-18could cross-neutralize HPV-6and4mAbs against HPV-58could cross-neutralize HPV-18or-6or-11. The sandwich ELISA based on mAbs against HPV-16could provide effective quality control in HPV-16vaccines preparation. And mAbs gained in the study could provide useful materials for further researches on mechanisms of virus infection and studies of HPV L1VLPs vaccines. |
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