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The HIV-1 reservoir consists of latently infected cells that persist despite antiretroviral therapy (ART). Elucidating the proviral genetic composition of the reservoir, particularly in the context of pre-therapy viral diversity, is therefore important to understanding reservoir formation and the persistence of latently infected cells. Here we investigate reservoir proviral variants from 13 Zambian acutely-infected individuals with additional pre-therapy sampling for a unique comparison to the ART-naïve quasispecies. We identified complete transmitted/founder (TF) viruses from seroconversion plasma samples, and additionally amplified and sequenced HIV-1 from plasma obtained one year post-infection and just prior to ART initiation. While the majority of proviral variants in the reservoir were most closely related to viral variants from the latest pre-therapy time point, we also identified reservoir proviral variants dating to or near the time of infection, and to intermediate time points between infection and treatment initiation. Reservoir proviral variants differing by five or fewer nucleotide changes from the TF virus persisted during treatment in five individuals, including proviral variants that exactly matched the TF in two individuals, one of whom had remained ART-naïve for more than six years. Proviral variants during treatment were significantly less divergent from the TF virus than plasma variants present at the last ART-naïve time point. These findings indicate that reservoir proviral variants are archived throughout infection, recapitulating much of the viral diversity that arises throughout untreated HIV-1 infection, and strategies to target and reduce the reservoir must therefore permit for the clearance of proviruses encompassing this extensive diversity., Author summary Despite reducing viremia to levels below the limit of detection in standard assays, effective antiretroviral therapy (ART) does not eradicate cells latently infected with HIV-1. These cells serve as a reservoir for viral rebound if therapy is interrupted; thus, understanding the composition of the reservoir may yield further targets for HIV-1 cure strategies. We have taken a genetic approach to elucidating the reservoir in 13 Zambian subtype C seroconvertors who were followed longitudinally through ART initiation and virologic suppression. In five of the 13 individuals, provirus sequences identical to or differing by five or fewer nucleotides from the transmitted/founder virus were detected, indicating archiving and persistence of early infection variants for more than six years following infection. While the majority of proviral variants in latently infected cells were most closely related to plasma virus circulating immediately prior to treatment initiation, additional variants dating to intermediate time points in the infection were also observed. These findings demonstrate that virus is archived during all stages of ART-naïve infection, and these variants persist throughout ART. HIV-1 cure strategies to eliminate the reservoir must address the broad genetic diversity of a within-host proviral quasispecies including variants archived from acute through chronic infection.

HIV-1 frequently escapes from CD8 T cell responses via HLA-I restricted adaptation, leading to the accumulation of adapted epitopes (AE). We previously demonstrated that AE compromise CD8 T cell responses during acute infection and are associated with poor clinical outcomes. Here, we examined the impact of AE on CD8 T cell responses and their biological relevance in chronic HIV infection (CHI). In contrast to acute infection, the majority of AE are immunogenic in CHI. Longitudinal analyses from acute to CHI showed an increased frequency and magnitude of AE-specific IFNγ responses compared to NAE-specific ones. These AE-specific CD8 T cells also were more cytotoxic to CD4 T cells. In addition, AE-specific CD8 T cells expressed lower levels of PD1 and CD57, as well as higher levels of CD28, suggesting a more activated and less exhausted phenotype. During CHI, viral sequencing identified AE-encoding strains as the dominant quasispecies. Despite increased CD4 T cell cytotoxicity, CD8 T cells responding to AE promoted dendritic cell (DC) maturation and CD4 T cell trans-infection perhaps explaining why AE are predominant in CHI. Taken together, our data suggests that the emergence of AE-specific CD8 T cell responses in CHI confers a selective advantage to the virus by promoting DC-mediated CD4 T cell trans-infection., Author summary HIV-1 infection remains a critical public health threat across the world. Over the past two decades, CD8 T cells have been clearly shown to exert immune pressure on HIV and drive viral adaptation. Previously, our group reported that such HLA-I associated adaptations can predict clinical outcomes and are beneficial to HIV-1 as CD8 T cells are unable to recognize epitopes with adaptation in acute HIV infection. However, it is still unclear how HIV-1 adaptation impacts CD8 T cells during chronic HIV infection. In this study, we observed an enhancement of CD8 T cell responses targeting adapted epitopes in chronic infection. Although these responses were cytotoxic, they also exhibited a “helper” effect by promoting viral infection of CD4 T cells via interaction with dendritic cells. This phenomenon may contribute to the persistence of adapted viruses. In summary, these findings present a novel mechanism of CD8 T cell driven HIV-1 adaptation.

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines., Author summary Despite much effort, an effective vaccine against HIV/AIDS has not been developed. There are many obstacles that continue to undermine this effort, which is focused largely on inducing neutralizing antibodies that can protect against infection. We conducted a pre-clinical vaccine study in nonhuman primates that utilized a trimeric HIV-1 envelope vaccine to induce neutralizing antibodies. We found that all animals that developed very high levels of neutralizing antibodies were protected against infection. When we mapped the neutralizing antibody responses using viral mutants, we found that they mainly targeted one exposed region of the HIV-1 envelope protein that is unique to the strain we used in the vaccine. By isolating individual antibodies from a protected animal with a very high level of neutralizing antibodies, we found that an expanded antibody family was responsible for the neutralization. Our findings demonstrate that neutralizing antibodies targeting mainly one exposed region on the envelope trimer vaccine were associated with protection, and that this could be achieved by a single antibody family that develops and expands during immunization.

HIV-1 transmission is associated with a severe bottleneck in which a limited number of variants from a pool of genetically diverse quasispecies establishes infection. The IAVI protocol C cohort of discordant couples, female sex workers, other heterosexuals and men who have sex with men (MSM) present varying risks of HIV infection, diverse HIV-1 subtypes and represent a unique opportunity to characterize transmitted/founder viruses (TF) where disease outcome is known. To identify the TF, the HIV-1 repertoire of 38 MSM participants’ samples was sequenced close to transmission (median 21 days post infection, IQR 18–41) and assessment of multivariant infection done. Patient derived gag genes were cloned into an NL4.3 provirus to generate chimeric viruses which were characterized for replicative capacity (RC). Finally, an evaluation of how the TF virus predicted disease progression and modified the immune response at both acute and chronic HIV-1 infection was done. There was higher prevalence of multivariant infection compared with previously described heterosexual cohorts. A link was identified between multivariant infection and replicative capacity conferred by gag, whereby TF gag tended to be of lower replicative capacity in multivariant infection (p = 0.02) suggesting an overall lowering of fitness requirements during infection with multiple variants. Notwithstanding, multivariant infection was associated with rapid CD4+ T cell decline and perturbances in the CD4+ T cell and B cell compartments compared to single variant infection, which were reversible upon control of viremia. Strategies aimed at identifying and mitigating multivariant infection could contribute toward improving HIV-1 prognosis and this may involve strategies that tighten the stringency of the transmission bottleneck such as treatment of STI. Furthermore, the sequences and chimeric viruses help with TF based experimental vaccine immunogen design and can be used in functional assays to probe effective immune responses against TF., Author summary The development of a safe and effective HIV-1 vaccine alongside cure strategies is a major public health concern and requires in-depth knowledge of the HIV-1 populations that establish infection. Here we sequenced the HIV genetic populations present during the acute stage of HIV-1 infection in 38 Men who have sex with men (MSM) and identified the transmitted/founder variant’s sequence. We then cloned the gag gene from each patient’s transmitted/founder gag sequences in both single and multiple variant infection into a common, lab-adapted virus and measured the replicative capacity it conferred on this virus. Finally, cellular immune responses were compared between single variant and multiple variant infection at 0–3, 6–9 and 24–30 months after infection. We observed a higher frequency of multivariant infection in MSM than has been previously described in heterosexually infected participants, and this was associated with faster decline of CD4+ T cells and perturbances in the CD4+ T cell and the B cell compartment. Moreover, the replicative capacity conferred by gag was lower in multivariant infection, suggesting a less stringent transmission bottleneck that allowed for less fit variants to establish infection. These results suggest that strategies to tighten the stringency of the transmission bottleneck may be of benefit to patients by reducing the likelihood of multivariant transmission and potentially slowing down disease progression.

Despite extensive research on the mechanisms of HLA-mediated immune control of HIV-1 pathogenesis, it is clear that much remains to be discovered, as exemplified by protective HLA alleles like HLA-B*81 which are associated with profound protection from CD4+ T cell decline without robust control of early plasma viremia. Here, we report on additional HLA class I (B*1401, B*57, B*5801, as well as B*81), and HLA class II (DQB1*02 and DRB1*15) alleles that display discordant virological and immunological phenotypes in a Zambian early infection cohort. HLA class I alleles of this nature were also associated with enhanced immune responses to conserved epitopes in Gag. Furthermore, these HLA class I alleles were associated with reduced levels of lipopolysaccharide (LPS) in the plasma during acute infection. Elevated LPS levels measured early in infection predicted accelerated CD4+ T cell decline, as well as immune activation and exhaustion. Taken together, these data suggest novel mechanisms for HLA-mediated immune control of HIV-1 pathogenesis that do not necessarily involve significant control of early viremia and point to microbial translocation as a direct driver of HIV-1 pathogenesis rather than simply a consequence., Author summary During acute HIV infection, there exists a complex interplay between the host immune response and the virus, and the balance of these interactions dramatically affects disease trajectory in infected individuals. Variations in Human Leukocyte Antigen (HLA) alleles dictate the potency of the cellular immune response to HIV, and certain well-studied alleles (HLA-B*57, B*27) are associated with control of HIV viremia. However, though plasma viral load is indicative of disease progression, the number of CD4+ T cells in the blood is a better measurement of disease severity. Through analysis of a large Zambian acute infection cohort, we identified HLA alleles that were associated with protection for CD4+ T cell loss, without dramatic affect on early plasma viremia. We further link these favorable HLA alleles to reduction in a well-known contributor to HIV pathogenesis, the presence of microbial products in the blood, which is indicative of damage to the gastrointestinal tract, a process which accelerates disease progression in HIV infected individuals. Ultimately, these results suggest a new mechanism by which the cellular immune response can combat HIV-associated pathogenesis, and further highlight the contribution of gut damage and microbial translocation to accelerating disease progression, even at early stages in HIV infection.

A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity., Author Summary HIV-1 has proven difficult to vaccinate against due to its ability to generate high levels of genetic diversity, particularly in the envelope glycoproteins. An ideal prophylactic vaccine would therefore elicit immune responses capable of blocking the full range of HIV-1 variants to which a population might be exposed. An essential component of protective immunity against HIV-1 is likely to be an antibody response that is capable of neutralizing genetically diverse HIV-1 viral variants. In an effort to understand how this type of ‘broad’ antibody response develops during natural HIV-1 infection, a large cohort study recently found that certain factors, such as high viral load, HLA-A*03 genotype, and subtype C infection, were correlated with the development of greater neutralization breadth. Here we investigated the viral envelope proteins and antibody responses in early infection in a small subset of individuals from two of the African sites included in the larger cohort study. We found that a marker for the efficiency of envelope glycosylation in the infecting viral variant was strongly correlated with the development of antibodies with greater neutralization breadth. We also found that extensive viral changes in the V2, V4, and V5 regions of the envelope gp120 protein, were strongly associated with the development of antibodies with greater neutralization breadth. Based on these results, we propose that more efficient glycosylation of the envelope protein of the infecting viral variant elicits neutralizing antibodies that drive early and complex amino acid changes in gp120, which triggers the development of antibodies whose neutralization breadth can be augmented over time by additional viral and host factors. These findings suggest that a better understanding of the efficiency of envelope glycosylation in HIV-1 could inform current vaccine strategies aimed at eliciting antibodies with neutralization breadth.

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential., Author Summary Despite the available HIV-1 diversity present in a chronically infected individual, single viral variants are transmitted in 80–90% of heterosexual transmission events. These breakthrough viruses may have unique properties that confer a higher capacity to transmit. Determining these properties could help inform the rational design of vaccines and enhance our understanding of viral transmission. We isolated the transmitted variant and a set of related non-transmitted variants from the transmitting partner near the estimated date of transmission from six epidemiologically linked transmission pairs to investigate viral correlates of transmission. The simplest explanation that transmitted variants are inherently more infectious or faster replicators in vitro did not hold true. In addition, transmitted variants did not replicate more efficiently than their non-transmitted counterparts in dendritic cells or in the presence of interferon-alpha in vitro, suggesting that they are not uniquely adapted to these components of the innate immune system. More ancestral genomes that were relatively sensitive to antibody neutralization tended to transmit, supporting previous reports that mutational escape away from the adaptive immune response likely reduces the ability to transmit. Our investigation into the traits of transmitted HIV-1 variants adds to the understanding of viral determinants of transmission.

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus's ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization.

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus's ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization., Author Summary A significant obstacle to developing an HIV vaccine is the potential for the virus to escape from the immune response induced by immunization. We previously showed that subjects in a Zambian cohort developed potent neutralizing antibody responses shortly after becoming infected by subtype C HIV-1, and here we have extended those findings to demonstrate that cycles of viral escape occurred in two of these subjects despite a potent immune response. We investigated the determinants of immune escape, and found that a single common mutational pathway was not sufficient to facilitate viral escape. Instead, we demonstrate that multiple strategies, including potential changes in glycosylation pattern, direct alteration of an epitope sequence, and cooperative envelope interactions, were used independently or together to evade neutralization. We also recovered individual monoclonal antibodies from one of the subjects and found that a single mutation can confer escape from different neutralizing antibody specificities. The studies demonstrate the remarkable flexibility of subtype C HIV-1, and suggest that the envelope glycoproteins are uniquely equipped to adjust to the specific properties of the immune response in each newly infected host.

The HIV-1 epidemic in sub-Saharan Africa is driven largely by heterosexual transmission of non-subtype B viruses, of which subtypes C and A are predominant. Previous studies of subtype B and subtype C transmission pairs have suggested that a single variant from the chronically infected partner can establish infection in their newly infected partner. However, in subtype A infected individuals from a sex worker cohort and subtype B individuals from STD clinics, infection was frequently established by multiple variants. This study examined over 1750 single-genome amplified viral sequences derived from epidemiologically linked subtype C and subtype A transmission pairs very early after infection. In 90% (18/20) of the pairs, HIV-1 infection is initiated by a single viral variant that is derived from the quasispecies of the transmitting partner. In addition, the virus initiating infection in individuals who were infected by someone other than their spouse was characterized to determine if genital infections mitigated the severe genetic bottleneck observed in a majority of epidemiologically linked heterosexual HIV-1 transmission events. In nearly 50% (3/7) of individuals infected by someone other than their spouse, multiple genetic variants from a single individual established infection. A statistically significant association was observed between infection by multiple genetic variants and an inflammatory genital infection in the newly infected individual. Thus, in the vast majority of HIV-1 transmission events in cohabiting heterosexual couples, a single genetic variant establishes infection. Nevertheless, this severe genetic bottleneck can be mitigated by the presence of inflammatory genital infections in the at risk partner, suggesting that this restriction on genetic diversity is imposed in large part by the mucosal barrier., Author Summary Previous studies of HIV transmission have yielded conflicting results regarding the genetic heterogeneity of the virus establishing infection in the newly infected individual. In this study of populations from Zambia and Rwanda that are infected by two distinct viral genetic subtypes, we compared viral sequences that encode the entry-mediating envelope glycoproteins from newly infected individuals (recipients) and their spouses (donors) very early after infection, as well as newly infected individuals infected by someone other than their spouse. In spite of the genetically diverse virus population in the donor, approximately 90% of newly infected individuals were infected by a single viral variant, while the rest were infected by multiple viral variants. The homogeneity of the virus population in the newly infected recipient, as well as the presence, in some cases, of identical virus variants in the donor, allowed us to precisely identify the transmitted variant. We were able to examine the clinical history of each newly infected individual and observed that all individuals infected by multiple variants also showed evidence of inflammatory genital infections. Our results suggest that the genital mucosa provides a natural barrier to infection by multiple genetic variants of HIV-1, but that this barrier can be lowered by inflammatory genital infections.

Antiretroviral therapy, antibody and CD8+ T cell-mediated responses targeting human immunodeficiency virus-1 (HIV-1) exert selection pressure on the virus necessitating escape; however, the ability of CD4+ T cells to exert selective pressure remains unclear. Using a computational approach on HIV gag/pol/nef sequences and HLA-II allelic data, we identified 29 HLA-II associated HIV sequence polymorphisms or adaptations (HLA-AP) in an African cohort of chronically HIV-infected individuals. Epitopes encompassing the predicted adaptation (AE) or its non-adapted (NAE) version were evaluated for immunogenicity. Using a CD8-depleted IFN-γ ELISpot assay, we determined that the magnitude of CD4+ T cell responses to the predicted epitopes in controllers was higher compared to non-controllers (p, Author Summary In HIV, CD4+ T cells are best known as the primary targets of infection. Although emerging data has suggested a more active role in viral pathogenesis, the CD4+ T cell population remains relatively understudied. Using a novel computational approach, we predicted 29 different epitopes with mutations that potentially represent escape from CD4+ T cell responses. The predicted escaped epitopes were found to be less immunogenic than the wild type forms, suggesting that the identified escapes allow HIV to reduce its visibility to the immune system. Using longitudinal samples, we were able to show CD4+ T cells driving viral escape following acute infection. Overall, these findings significantly expand our knowledge of how CD4+ T cells can exert HIV control and influence HIV evolution, providing important implications to future vaccine development strategies.

Initial studies of 88 transmission pairs in the Zambia Emory HIV Research Project cohort demonstrated that the number of transmitted HLA-B associated polymorphisms in Gag, but not Nef, was negatively correlated to set point viral load (VL) in the newly infected partners. These results suggested that accumulation of CTL escape mutations in Gag might attenuate viral replication and provide a clinical benefit during early stages of infection. Using a novel approach, we have cloned gag sequences isolated from the earliest seroconversion plasma sample from the acutely infected recipient of 149 epidemiologically linked Zambian transmission pairs into a primary isolate, subtype C proviral vector, MJ4. We determined the replicative capacity (RC) of these Gag-MJ4 chimeras by infecting the GXR25 cell line and quantifying virion production in supernatants via a radiolabeled reverse transcriptase assay. We observed a statistically significant positive correlation between RC conferred by the transmitted Gag sequence and set point VL in newly infected individuals (p = 0.02). Furthermore, the RC of Gag-MJ4 chimeras also correlated with the VL of chronically infected donors near the estimated date of infection (p = 0.01), demonstrating that virus replication contributes to VL in both acute and chronic infection. These studies also allowed for the elucidation of novel sites in Gag associated with changes in RC, where rare mutations had the greatest effect on fitness. Although we observed both advantageous and deleterious rare mutations, the latter could point to vulnerable targets in the HIV-1 genome. Importantly, RC correlated significantly (p = 0.029) with the rate of CD4+ T cell decline over the first 3 years of infection in a manner that is partially independent of VL, suggesting that the replication capacity of HIV-1 during the earliest stages of infection is a determinant of pathogenesis beyond what might be expected based on set point VL alone., Author Summary In the majority of HIV-1 cases, a single virus establishes infection. However, mutations in the viral genome accumulate over time in order to avoid recognition by the host immune response. Certain mutations in the main structural protein, Gag, driven by cytotoxic T lymphocytes are detrimental to viral replication, and we showed previously that, upon transmission, viruses with higher numbers of escape mutations in Gag were associated with lower early set point viral loads. We hypothesized that this could be attributed to attenuation of the transmitted virus. Here, we have cloned the gag gene from 149 newly infected individuals from linked transmission pairs into a clade C proviral vector and determined the replicative capacity in vitro. We found that the replicative capacity conferred by the transmitted Gag correlated with set point viral loads in newly infected individuals, as well as with the viral load of the transmitting partner, and we identified previously unrecognized residues associated with increasing and decreasing replicative capacity. Importantly, we demonstrate that transmitted viruses with high replicative capacity cause more rapid CD4+ decline over the first three years, independent of viral load. This suggests that the trajectory of pathogenesis may be affected very early in infection, before adaptive immunity can respond.