Troy J. Kemp (United States of America)
Frederick National Laboratory for Cancer Research HPV Serology LaboratoryPresenter of 2 Presentations
HPV-16 MEMORY B-CELL RESPONSES POST-DOSE THREE OF THE QUADRIVALENT HPV VACCINE AMONG MID-ADULT AGED MEN (ID 929)
- Troy J. Kemp (United States of America)
- Martha Abrahamsen (United States of America)
- Kimberly Isaacs-Soriano (United States of America)
- Bradley Sirak (United States of America)
- Yuanji Pan (United States of America)
- Ligia A. Pinto (United States of America)
- Anna R. Giuliano (United States of America)
Abstract
Introduction
Strong quantitative and functional antibody responses to the quadrivalent HPV vaccine were previously reported in mid-adult aged men, but there are very limited data on the direct cellular response of B-cells following vaccination, especially in males. Here, we assessed HPV-16 B-cell responses via ELISPOT at Day 1 (prior to vaccination) and 1- month post-dose three of the vaccine in mid-adult aged men.
Methods
Sera and peripheral blood mononuclear cells (PBMC) at Day 1 and month 7 were obtained from 32 men, ages 27-45, from Tampa, FL, USA, who received Gardasil at 0, 2, and 6 months. Following stimulation, PBMCs were plated in triplicate at 300,000 or 100,000 cells/well in ELISPOT plates pre-coated with HPV-16 L1 VLPs. IgG spots were enumerated on the Cellular Technology Limited (CTL) Immunospot® instrument. Data were expressed as # of spots/106 cells and % of HPV16- specific IgG memory B cells.
Results
The memory B-cell response was induced following vaccination and followed a similar pattern for both types of analysis, # of spots/106 cells (2.8 spots/106 cells at Day 1 versus 150.0 spots/106 cells at Month 7) and % of IgG specific cells (0.001% of HPV-16 IgG specific cells at Day 1 versus 0.357% of HPV-16 IgG specific cells at Month 7). No significant correlations were observed between the HPV-16 ELISPOT results and anti-HPV-16 IgG ELISA or avidity responses.
Conclusions
Gardasil induces an increase in HPV-16-specific memory B-cell responses following three doses of vaccine in mid-adult aged men, along with increases in antibody levels and avidity. However, there appears to be a lack of correlation between quantity and strength of antibody responses and memory B-cell responses. Future studies are needed to better understand the role of memory B cells and antibody responses in HPV efficacy.
EVALUATION OF POLYETHYLENIMINE (PEI)-BASED TRANSFECTION METHODS FOR HPV L1L2 VLP PRODUCTION IN MAMMALIAN CELLS FOR USE IN SEROLOGY ASSAYS (ID 1032)
Abstract
Introduction
Currently licensed HPV vaccines are based on HPV L1 virus-like particles. HPV VLPs are used extensively in HPV research and as critical reagents in the assessment of HPV-induced immune responses. There are a variety of available methods to produce HPV VLPs for use in immunoassay testing; however, transfection methods are expensive and time-consuming. With the aim of reducing costs and saving time in VLP production while maintaining robust HPV particle assembly and similar performance in assays, we have investigated different transfection approaches in a mammalian-based HPV L1L2 VLP production system.
Methods
HEK293TT transfection procedures were optimized for two transfection reagents, polyethylenimine (PEI) and TransporterTM 5 (T5), in the context of final HPV-6 and HPV-18 L1L2 VLP output as compared to Lipofectamine 2000 (Lipo). XTT cell viability assay, GFP transfection efficiency, HPV-6 and HPV-18 ELISA, and electron microscopy were performed to evaluate and to optimize the HPV-6 and HPV-18 L1L2 VLPs produced.
Results
Transfection efficiency and cell viability were used to define optimal transfection reagent ratios (Lipo, 3:1; PEI and T5, 4:1). The average concentration from three transfection experiments for HPV-6 and HPV-18 L1L2 VLPs were as follows: HPV-6 (Lipo, 1.7 mg; PEI, 1.6 mg; T5, 1.8 mg) and HPV-18 (Lipo, 0.72 mg; PEI, 0.6 mg; T5, 1.3 mg). Furthermore, the ELISA antibody levels obtained using HPV-6 and HPV-18 VLPs produced by each transfection method were similar when evaluating a set of samples with a wide range range of HPV VLP antibody responses.
Conclusions
Results demonstrate PEI and TransporterTM 5 are as efficient as Lipofectamine 2000 in producing assembled particles. The use of these reagents will add alternative methods for VLP production and help reduce overall production cost.