Diversity, fidelity and antibody control: broad approaches in restricting HIV

© 2016 Dr. Sarah Blayer LloydThe HIV pandemic creates an immense worldwide burden of morbidity, mortality and economic costs. Use of antiretroviral therapy is currently the best treatment for infected patients. The low fidelity of HIV replication facilitates immune and drug escape. Some reverse transcriptase (RT) inhibitor drug-resistance mutations increase RT fidelity in biochemical assays, but the link between fidelity and fitness remains unclear. We investigated the effect of the RT mutations K65R, Q151N and V148I on SIV replication in vitro and in pigtailed macaques. SIVmac239-K65R and SIV mac239-V148I viruses had reduced replication capacity compared to wild-type SIVmac239. Direct virus competition assays demonstrated a rank order of wild-type>K65R>V148I mutants in terms of viral fitness. In a novel infection-based fidelity assay, SIVmac239-K65R demonstrated significantly higher fidelity than wild-type, though its fidelity was lower than previously reported in biochemical assays employing purified protein. K65R could infect macaques, but rapidly reverted, while SIVmac239-Q151N was replication incompetent in vitro and in macaques. Understanding HIV diversity and the fitness of drug-resistant mutants may inform the development of interventions against HIV. HIV diversity necessitates interventions that are effective against numerous strains. Broadly neutralising antibodies (bnAbs) protect macaques from cell-free SHIV challenge, but their efficacy against cell-associated SHIV is unclear. Pigtail macaques were infused with the bnAb PGT121 or an isotype control and challenged intravenously with splenocytes from a SHIVSF162P3-infected donor. All six controls had high-level viremia one week post-challenge. Three of six PGT121-infused animals were completely protected from the cell-associated challenge, two of six had a one-week delay in viremia and one animal had a seven-week delay. Infused PGT121 decayed to 2.2μg/ml on average by one week post-infusion and was well below 1μg/ml by eight weeks. Transfer of 22 million P-MCs stored at weeks 1-4 from the seven-week delay animal into uninfected macaques did not initiate infection. Our results indicate HIV-1-specific neutralising antibodies have partial efficacy against high dose cell-associated iivirus exposure. The delay in SHIV infection observed in a subset of animals raises concerns for bnAb treatments. While bnAbs are a promising preventative and therapeutic tool, production is difficult and expensive. Production of antibody-like fragments in bacterial cytoplasm provides a cheaper alternative. This work explored the transplantation of the complementarity determining regions of the bnAbs PGT121 and 10E8 onto a single-chain variable fragment (scFv) scaffold, previously discovered through a novel screening platform. The 10E8 scFv was soluble in cytoplasm, while the PGT121 scFv was not. Three additional constructs employing the 10E8 scFv were produced and tested for binding and neutralisation function. The constructs were found to have lost function, however this provided a proof of concept for bnAb construct production in bacterial cytoplasm. With future refinement of these technologies it may be possible to produce cheap, effective bnAb-like tools for the control of HIV