Background
Broadly neutralizing antibodies (bNAbs) provide a useful tool for HIV cure strategies because of their ability to target conserved regions on the envelope (Env) protein in the context of both virions and infected cells. Multiple clinical trials of single and combination bNAb therapies in people living with HIV demonstrated transient viral suppression after infusion. The ease of viral escape, however, is a major obstacle to durable suppression by bNAbs. We are investigating common escape pathways from bNAbs to inform future bNAb combination trials.
Methods
We designed an in vitro viral escape assay to test bNAb and Env combinations in a high throughput manner. From PBMC, we isolated single non-subtype B viruses from Nigerian, Ugandan and Kenyan participants in the African Cohort Study (AFRICOS). Each virus stock was sequenced, subtyped and characterized for sensitivity to 9 bNAbs. Virus E01-0085.V2 was a subtype AG virus sensitive to VRC01.23 and 10E8v4-5R+100cF. To test the ability of this virus to escape bNAb neutralization, ex vivo CD4+ T cells were infected in the presence of varying bNAb concentrations in duplicate. Cultures were maintained for 60 days with increasing suboptimal concentrations of bNAbs to induce escape. Replication kinetics were monitored by p24 every 3 days. Every 14 days, target cells were replenished and cultures were tested for genotypic and phenotypic measures of bNAb resistance. This was accomplished by single genome sequencing env genes and using the remaining supernatant to spinoculate new CD4+ cells to propagate the escape assay. The remaining infected cells were washed and replenished with bNAb-free media for virus production, which was then tested by neutralization assay for bNAb resistance.
Results
Nine non-subtype B viruses were tested against 8 bNAbs, and we observed CD4bs bNAbs to have the greatest breadth, but less potency than V2-apex or V3 bNAbs. In our experiments with E01-0085.V2, complete neutralization resistance to both bNAbs was observed by day 56 of the assay, but sequence signatures begin by day 28. Fixed escape mutations were observed within the epitopes of both antibodies by day 42 and possible compensatory mutations were observed by day 56.
Conclusions
Our data reveal escape from both bNAbs was achieved by single non-synonymous nucleotide changes. This in vitro bNAb escape assay will lead to a deeper understanding of viral escape to better inform the design of highly effective bNAb cocktails targeting multiple conserved sites.
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