Analysis of genetic diversity and VRC01 pressure on HIV-1 breakthrough viruses from the AMP trial (HVTN 703/HPTN 081 and HVTN 704/085)


BACKGROUND: The Antibody Mediated Prevention (AMP) trial evaluated if VRC01, a CD4 binding site broadly neutralizing antibody, could prevent HIV-1 acquisition. To inform our understanding of how prevention efficacy depended on viral env gene charateristics, viral quasispecies and neutralization sensitivity of HIV-1 breakthrough infections were analyzed.
METHODS: The trial evaluated VRC01 in women from sub-Saharan Africa (703/081); and men and transgender persons who have sex with men (704/085), from the Americas. Control samples from Thailand, Kenya and South Africa are being used for calibrating infection timing using sequence data. Rev-env-nef (REN) sequences were generated using Sanger and PacBio Single Molecule Real-Time (SMRT) sequencing platforms. To improve PacBio accuracy and enable quantitation, each viral RNA molecule was labelled with a unique molecular identifier (SMRT-UMI). Rev-env genes from imputed founder viruses were synthesized for pseudovirus production and in vitro sensitivity to VRC01 determined using the TZM-bl assay.
RESULTS: Approximately 84,000 unique REN sequences were generated from the first HIV-1 positive visit from 218 infected individuals in both trials, and 2-4 weeks later from a subset of individuals, providing a median of 174 unique env sequences per participant per time point. In approximately 2/3 of individuals, viral diversity was low, consistent with infection with a single founder virus. Of the 1/3 of individuals with higher viral diversity, consistent with infection with multiple founders, a third had low frequency variants (<5% of sequences). We synthesized 1-4 Env-pseudoviruses per person, and identified some individuals infected with variants with different neutralization phenotype, including infection with both sensitive (IC80<1ug/ml) and resistant (IC80>3ug/ml) viruses. In some individuals, the unique VRC01 resistance mutations were associated with distinct viral lineages, suggestive of infection with resistant viruses. However, we also found evidence of low frequency resistant mutations, that were likely derived from resistance acquired after infection.
CONCLUSIONS: SMRT-UMI sequencing allowed a very detailed evaluation of viral populations following infection, including identification of minor founders not detected using conventional approaches. We found evidence of infection with viruses of mixed neutralization phenotypes, and in some cases, low frequency resistance mutations that were likely to be due VRC01 pressure. The study is not yet unblinded.