HIV pseudovirus assays play a crucial role in investigating the breadth of neutralization of mAbs against viral clones and this has great value in HIV vaccine development. With the enormous genetic diversity of HIV, high throughput approaches are required to keep pace with rate of mutation in the virus. The pseudovirus assay is ideal for such investigations. This study tested the hypotheses that differing HIV-1 envelope to backbone ratios during cotransfection of 293T/17 cells in generating HIV-1 env-pseudoviruses has an influence on viral titers and also that harvesting HIV-1 env-pseudoviruses at different time points, 48 hours and 72 hours, after cotransfection of 293T/17 cells affects viral titers and infectivity. Our findings suggest that using different envelope to backbone ratios has a strain dependent effect. For BaL26c11, increasing backbone DNA while keeping envelop DNA concentration constant leads an incease in pseudovirus titers. For 92Br025 and SF162 there is an optimal amount of backbone DNA that can be added beyond which pseudovirus titers decrease. Secondly, our data show that HIV env-pseudoviruses harvested 48 hours after cotransfection of 293T/17 cells are more infectious, requiring lower titers to achieve the same level of infectivity as compared to those harvested after 72 hours. We also show that neutralization profiles of four mAbs 4E10, 2G12, 2F5, and b12, and TriMab (a combiantion of 2G12, 2F5 and b12) against ghree HIV-1 env-pseudovirus batches (BaL26c11, 92Br025 and SF162) are neither affected by the change in env-backbone ratio nor the difference in harvesting time points i.e. 48 and 72 hours after cotransfection of 293T/17 cell line. TriMab had the best neutralization as compared to the four mAbs when tested individually. Our work thus shows that it is important to perform preliminary studies of each new pseudovirus to determine the optimal env:backbone ratio for economical reasons. However, its is reassuring that, when properly tittered on target cells, the Env/Backbone nor the time of harvesting the pseudoviruses will influence the results of neturalization studies. Thus our work has contributed to standardizing HIV neutralization assays, which are a cornerstone parameter in HIV clinical and preclinical vaccine studies.
|Date of Award||3 Sep 2009|
|Supervisor||Patrick De Baetselier (Promotor), Guido Vanham (Promotor), Sunita Balla (Co-promotor) & Betty Willems (Advisor)|