Background: Heterosexual transmission of HIV-1 is generally restricted to infection by one or only a few viruses from amongst a complex quasispecies. This bottleneck has been attributed in part to preferential selection of virus utilizing CCR5 (R5) as a coreceptor. However, since R5 utilization appears to be necessary but not sufficient for transmission, we hypothesized that other env-encoded biological properties, such as neutralization escape, could contribute to virus transmissibility.

Methods: We amplified over 200 individual envs from blood samples collected from four female to male and four male to female heterosexual transmission pairs in a large discordant couple cohort in Zambia shortly after a transmission occurred. Next, we analyzed the sequence of V1 to V4 and directly compared the newly transmitted viruses in each recipient to those found in the quasispecies of the index case (donor). We then utilized a highly sensitive and quantitative single-round, antibody neutralization assay to analyze donor plasma-mediated neutralization of donor and recipient Env pseudotyped viruses from 5 of the 8 transmission pairs.

Results: Here we show that the env sequences found in all 8 recipients were completely homogeneous in the V1 to V4 region, regardless of the direction of transmission or the level of complexity in the donors. Moreover, the recipient viruses consistently encoded a minor Env variant containing one of the most compact constellations of variable-loop structures among the donor quasispecies, suggesting that variants containing large V1V2 and V4 loops were selected against during transmission. Paradoxically, we found that recipient Env-pseudotyped viruses were more sensitive to neutralization by donor plasma than the viruses pseudotyped with the corresponding donor Envs, ruling out neutralization escape as a prerequisite for transmission.

Conclusions: Our findings demonstrate that compact Env structure and neutralization sensitivity are linked with transmissibility, arguing that transmitted Envs must expose critical structural domains to achieve transmission. Furthermore, neutralization-resistant variants circulating in donor plasma were not the source of transmitted virus, suggesting that compartmentalization or cell-mediated transfer is necessary for transmission. These studies provide novel insight into the biological properties and constraints of viruses transmitted heterosexually that should be considered in HIV-1 vaccine design.