Neutralization sensitivity of genital tract HIV-1: shift in selective milieu shapes the population available to transmit.

OBJECTIVE: Previous studies indicate that transmitted/founder HIV-1 isolates are sensitive to neutralization by the transmitting donor's antibodies. This is true in at least a subset of sexual transmissions. We investigated whether this selection for neutralization-sensitive variants begins in the genital tract of the donor, prior to transmission. DESIGN: Laboratory study. METHODS: HIV-1 viruses from semen and blood of two male donors living with HIV-1 were tested for neutralization sensitivity to contemporaneous autologous antibodies. RESULTS: In one donor, semen-derived clones (n = 10, geometric mean ID50 = 176) were 1.75-fold [95% confidence interval (CI) 1.11-2.76, P = 0.018] more sensitive than blood-derived clones (n = 12, geometric mean ID50 = 111) to the individual's own contemporaneous neutralizing antibodies. Enhanced overall neutralization sensitivity of the semen-derived clones could not explain the difference because these semen-derived isolates showed a trend of being less sensitive to neutralization by a pool of heterologous clade-matched sera. This relative sensitivity of semen-derived clones was not observed in a second donor who did not exhibit obvious independent HIV-1 replication in the genital tract. A Bayesian analysis suggested that the set of semen sequences that we analysed originated from a blood sequence. CONCLUSION: In some instances, selection for neutralization-sensitive variants during HIV-1 transmission begins in the genital tract of the donor and this may be driven by independent HIV-1 replication in this compartment. Thus, a shift in the selective milieu in the male genital tract allows outgrowth of neutralization-sensitive HIV-1 variants, shaping the population of isolates available for transmission to a new host.

Compartmentalization and Clonal Amplification of HIV-1 in the Male Genital Tract Characterized Using Next-Generation Sequencing.

Compartmentalization of HIV-1 between the systemic circulation and the male genital tract may have a substantial impact on which viruses are available for sexual transmission to new hosts. We studied compartmentalization and clonal amplification of HIV-1 populations between the blood and the genital tract from 10 antiretroviral-naive men using Illumina MiSeq with a PrimerID approach. We found evidence of some degree of compartmentalization in every study participant, unlike previous studies, which collectively showed that only ∼50% of analyzed individuals exhibited compartmentalization of HIV-1 lineages between the male genital tract (MGT) and blood. Using down-sampling simulations, we determined that this disparity can be explained by differences in sampling depth in that had we sequenced to a lower depth, we would also have found compartmentalization in only ∼50% of the study participants. For most study participants, phylogenetic trees were rooted in blood, suggesting that the male genital tract reservoir is seeded by incoming variants from the blood. Clonal amplification was observed in all study participants and was a characteristic of both blood and semen viral populations. We also show evidence for independent viral replication in the genital tract in the individual with the most severely compartmentalized HIV-1 populations. The degree of clonal amplification was not obviously associated with the extent of compartmentalization. We were also unable to detect any association between history of sexually transmitted infections and level of HIV-1 compartmentalization. Overall, our findings contribute to a better understanding of the dynamics that affect the composition of virus populations that are available for transmission.IMPORTANCE Within an individual living with HIV-1, factors that restrict the movement of HIV-1 between different compartments-such as between the blood and the male genital tract-could strongly influence which viruses reach sites in the body from which they can be transmitted. Using deep sequencing, we found strong evidence of restricted HIV-1 movements between the blood and genital tract in all 10 men that we studied. We additionally found that neither the degree to which particular genetic variants of HIV-1 proliferate (in blood or genital tract) nor an individual's history of sexually transmitted infections detectably influenced the degree to which virus movements were restricted between the blood and genital tract. Last, we show evidence that viral replication gave rise to a large clonal amplification in semen in a donor with highly compartmentalized HIV-1 populations, raising the possibility that differential selection of HIV-1 variants in the genital tract may occur.

Detectable HIV-1 in semen in individuals with very low blood viral loads.

BACKGROUND: Several reports indicate that a portion (5-10%) of men living with HIV-1 intermittently shed HIV-1 RNA into seminal plasma while on long term effective antiretroviral therapy (ART). This is highly suggestive of an HIV-1 reservoir in the male genital tract. However, the status of this reservoir in men living with HIV-1 who are not under treatment is underexplored and has implications for understanding the origins and evolution of the reservoir. FINDING: Forty-three HIV-1 positive, antiretroviral therapy naïve study participants attending a men's health clinic were studied. Semen viral loads and blood viral loads were generally correlated, with semen viral loads generally detected in individuals with blood viral loads > 10,000 cp/ml. However, we found 1 individual with undetectable viral loads (<20cp/ml) and 2 individuals with very low blood viral load (97 and 333cp/ml), but with detectable HIV-1 in semen (485-1157 copies/semen sample). Blood viral loads in the first individual were undetectable when tested three times over the prior 5 years. CONCLUSIONS: Semen HIV-1 viral loads are usually related to blood viral loads, as we confirm. Nonetheless, this was not true in a substantial minority of individuals suggesting unexpectedly high levels of replication in the male genital tract in a few individuals, despite otherwise effective immune control. This may reflect establishment of a local reservoir of HIV-1 populations.

Molecular Basis of Unusually High Neutralization Resistance in Tier 3 HIV-1 Strain 253-11.

Understanding the mechanisms used by HIV-1 to evade antibody neutralization may contribute to the design of a high-coverage vaccine. The tier 3 virus 253-11 is poorly neutralized by subtype-matched and subtype C sera, even compared to other tier 3 viruses, and is also recognized poorly by V3/glycan-targeting monoclonal antibodies (MAbs). We found that sequence polymorphisms in the V3 loop and N-linked glycosylation sites contribute only minimally to the high neutralization resistance of 253-11. Interestingly, the 253-11 membrane-proximal external region (MPER) is rarely recognized by sera in the context of the wild-type virus but is commonly recognized in the context of an HIV-2 chimera, suggesting steric or kinetic hindrance of binding to MPER in the native envelope (Env). Mutations in the 253-11 MPER, which were previously reported to increase the lifetime of the prefusion Env conformation, affected the resistance of 253-11 to antibodies targeting various epitopes on HIV-1 Env, presumably destabilizing its otherwise stable, closed trimer structure. To gain insight into the structure of 253-11, we constructed and crystallized a recombinant 253-11 SOSIP trimer. The resulting structure revealed that the heptad repeat helices in gp41 are drawn in close proximity to the trimer axis and that gp120 protomers also showed a relatively compact disposition around the trimer axis. These observations give substantial insight into the molecular features of an envelope spike from a tier 3 virus and into possible mechanisms that may contribute to its unusually high neutralization resistance.IMPORTANCE HIV-1 isolates that are highly resistant to broadly neutralizing antibodies could limit the efficacy of an antibody-based vaccine. We studied 253-11, which is highly resistant to commonly elicited neutralizing antibodies. To further understand its resistance, we made mutations that are known to delay fusion and thus increase the time that the virus spends in the open conformation following CD4 binding. Interestingly, we found that these mutations affect the 253-11 envelope (Env) spike before CD4 binding, presumably by destabilizing the trimer structure. To gain further information about the structure of the 253-11 Env trimer, we generated a recombinant 253-11 SOSIP trimer. The crystal structure of the SOSIP trimer revealed that the gp41 helices and the gp120 protomers were drawn in toward the center of the molecule compared to most solved HIV-1 Env structures. These observations provide insight into the distinct molecular features of a tier 3 envelope spike.

The HIV-1 transmission bottleneck.

It is well established that most new systemic infections of HIV-1 can be traced back to one or a limited number of founder viruses. Usually, these founders are more closely related to minor HIV-1 populations in the blood of the presumed donor than to more abundant lineages. This has led to the widely accepted idea that transmission selects for viral characteristics that facilitate crossing the mucosal barrier of the recipient's genital tract, although the specific selective forces or advantages are not completely defined. However, there are other steps along the way to becoming a founder virus at which selection may occur. These steps include the transition from the donor's general circulation to the genital tract compartment, survival within the transmission fluid, and establishment of a nascent stable local infection in the recipient's genital tract. Finally, there is the possibility that important narrowing events may also occur during establishment of systemic infection. This is suggested by the surprising observation that the number of founder viruses detected after transmission in intravenous drug users is also limited. Although some of these steps may be heavily selective, others may result mostly in a stochastic narrowing of the available founder pool. Collectively, they shape the initial infection in each recipient.