Evolution of HIV-1 envelope towards reduced neutralization sensitivity, as demonstrated by contemporary HIV-1 subtype B from the United States.

Subtype B HIV-1 has been the primary driver of the HIV-1 epidemic in the United States (U.S.) for over forty years and is also a prominent subtype in the Americas, Europe, Australia, the Middle East and North Africa. In this study, the neutralization profiles of contemporary subtype B Envs from the U.S. were assessed to characterize changes in neutralization sensitivities over time. We generated a panel of 30 contemporary pseudoviruses (PSVs) and demonstrated continued diversification of subtype B Env from the 1980s up to 2018. Neutralization sensitivities of the contemporary subtype B PSVs were characterized using 31 neutralizing antibodies (NAbs) and were compared with strains from earlier in the HIV-1 pandemic. A significant reduction in Env neutralization sensitivity was observed for 27 out of 31 NAbs for the contemporary as compared to earlier-decade subtype B PSVs. A decline in neutralization sensitivity was observed across all Env domains; the NAbs that were most potent early in the pandemic suffered the greatest decline in potency over time. A meta-analysis demonstrated this trend across multiple subtypes. As HIV-1 Env diversification continues, changes in Env antigenicity and neutralization sensitivity should continue to be evaluated to inform the development of improved vaccine and antibody products to prevent and treat HIV-1.

Tracking coreceptor switch of the transmitted/founder HIV-1 identifies co-evolution of HIV-1 antigenicity, coreceptor usage and CD4 subset targeting: the RV217 acute infection cohort study.

BACKGROUND: The CCR5 (R5) to CXCR4 (X4) coreceptor switch in natural HIV-1 infection is associated with faster progression to AIDS, but the mechanisms remain unclear. The difficulty in elucidating the evolutionary origin of the earliest X4 viruses limits our understanding of this phenomenon. METHODS: We tracked the evolution of the transmitted/founder (T/F) HIV-1 in RV217 participants identified in acute infection. The origin of the X4 viruses was elucidated by single genome amplification, deep sequencing and coreceptor assay. Mutations responsible for coreceptor switch were confirmed by mutagenesis. Viral susceptibility to neutralization was determined by neutralization assay. Virus CD4 subset preference was demonstrated by sequencing HIV-1 RNA in sorted CD4 subsets. FINDINGS: We demonstrated that the earliest X4 viruses evolved de novo from the T/F strains. Strong X4 usage can be conferred by a single mutation. The mutations responsible for coreceptor switch can confer escape to neutralization and drive the X4 variants to replicate mainly in the central memory (CM) and naïve CD4 subsets. Likely due to the smaller viral burst size of the CM and naïve subsets, the X4 variants existed at low frequency in plasma. The origin of the X4 viruses preceded accelerated CD4 decline. All except one X4 virus identified in the current study lost the conserved V3 N301 glycan site. INTERPRETATIONS: The findings demonstrate co-evolution of HIV-1 antigenicity, coreceptor usage and CD4 subset targeting which have implications for HIV-1 therapeutics and functional cure. The observations provide evidence that coreceptor switch can function as an evolutionary mechanism of immune evasion. FUNDING: Institute of Human Virology, National Institutes of Health, Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc, Thai Red Cross AIDS Research Centre, Gilead Sciences, Merck, and ViiV Healthcare.

Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype.

Nearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, detection was not at the earliest stages of acute infection. Here, we identified an X4-tropic T/F HIV-1 in a participant in acute infection cohort. Coreceptor assays demonstrated that this T/F virus is strictly CXCR4 tropic. The participant experienced significantly faster CD4 depletion compared with R5 virus infected participants in the same cohort. Naïve and central memory CD4 subsets declined faster than effector and transitional memory subsets. All CD4 subsets, including naïve, were productively infected. Increased CD4+ T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions. These findings demonstrate that X4-tropic HIV-1 is transmissible through the mucosal route in people with the wild-type CCR5 genotype and have implications for understanding the transmissibility and immunopathogenesis of X4-tropic HIV-1.

Transmission of highly virulent CXCR4 tropic HIV-1 through the mucosal route in an individual with a wild-type CCR5 genotype.

Nearly all transmitted/founder (T/F) HIV-1 are CCR5 (R5)-tropic. While previous evidence suggested that CXCR4 (X4)-tropic HIV-1 are transmissible, detection was not at the earliest stages of acute infection. Here, we identified an X4-tropic T/F HIV-1 in a participant in acute infection cohort. Coreceptor assays demonstrated that this T/F virus is strictly CXCR4 tropic. The participant experienced significantly faster CD4 depletion compared with R5 virus infected participants in the same cohort. Naïve and central memory CD4 subsets declined faster than effector and transitional memory subsets. All CD4 subsets, including naïve, were productively infected. Increased CD4 + T cell activation was observed over time. This X4-tropic T/F virus is resistant to broadly neutralizing antibodies (bNAbs) targeting V1/V2 and V3 regions. These findings demonstrate that X4-tropic HIV-1 is transmissible through the mucosal route in people with the wild-type CCR5 genotype and have implications for understanding the transmissibility and immunopathogenesis of X4-tropic HIV-1.

Single-cell transcriptomics identifies prothymosin α restriction of HIV-1 in vivo.

Host restriction factors play key roles in innate antiviral defense, but it remains poorly understood which of them restricts HIV-1 in vivo. Here, we used single-cell transcriptomic analysis to identify host factors associated with HIV-1 control during acute infection by correlating host gene expression with viral RNA abundance within individual cells. Wide sequencing of cells from one participant with the highest plasma viral load revealed that intracellular viral RNA transcription correlates inversely with expression of the gene PTMA, which encodes prothymosin α. This association was genome-wide significant (Padjusted < 0.05) and was validated in 28 additional participants from Thailand and the Americas with HIV-1 CRF01_AE and subtype B infections, respectively. Overexpression of prothymosin α in vitro confirmed that this cellular factor inhibits HIV-1 transcription and infectious virus production. Our results identify prothymosin α as a host factor that restricts HIV-1 infection in vivo, which has implications for viral transmission and cure strategies.

Evolution of Antibody Responses in HIV-1 CRF01_AE Acute Infection: Founder Envelope V1V2 Impacts the Timing and Magnitude of Autologous Neutralizing Antibodies.

Understanding the dynamics of early immune responses to HIV-1 infection, including the evolution of initial neutralizing and antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, will inform HIV vaccine design. In this study, we assess the development of autologous neutralizing antibodies (ANAbs) against founder envelopes (Envs) from 18 participants with HIV-1 CRF01_AE acute infection. The timing of ANAb development directly associated with the magnitude of the longitudinal ANAb response. Participants that developed ANAbs within 6 months of infection had significantly higher ANAb responses at 1 year (50% inhibitory concentration [IC50] geometric mean titer [GMT] = 2,010 versus 184; P = 0.001) and 2 years (GMT = 3,479 versus 340; P = 0.015), compared to participants that developed ANAb responses after 6 months. Participants with later development of ANAb tended to develop an earlier, potent heterologous tier 1 (92TH023) neutralizing antibody (NAb) response (P = 0.049). CRF01_AE founder Env V1V2 loop lengths correlated indirectly with the timing (P = 0.002, r = -0.675) and directly with magnitude (P = 0.005, r = 0.635) of ANAb responses; Envs with longer V1V2 loop lengths elicited earlier and more potent ANAb responses. While ANAb responses did not associate with viral load, the viral load set point correlated directly with neutralization of the heterologous 92TH023 strain (P = 0.007, r = 0.638). In contrast, a striking inverse correlation was observed between viral load set point and peak ADCC against heterologous 92TH023 Env strain (P = 0.0005, r = -0.738). These data indicate that specific antibody functions can be differentially related to viral load set point and may affect HIV-1 pathogenesis. Exploiting Env properties, such as V1V2 length, could facilitate development of subtype-specific vaccines that elicit more effective immune responses and improved protection. IMPORTANCE Development of an effective HIV-1 vaccine will be facilitated by better understanding the dynamics between the founder virus and the early humoral responses. Variations between subtypes may influence the evolution of immune responses and should be considered as we strive to understand these dynamics. In this study, autologous founder envelope neutralization and heterologous functional humoral responses were evaluated after acute infection by HIV-1 CRF01_AE, a subtype that has not been thoroughly characterized. The evolution of these humoral responses was assessed in relation to envelope characteristics, magnitude of elicited immune responses, and viral load. Understanding immune parameters in natural infection will improve our understanding of protective responses and aid in the development of immunogens that elicit protective functional antibodies. Advancing our knowledge of correlates of positive clinical outcomes should lead to the design of more efficacious vaccines.

Tracking coreceptor switch of the transmitted/founder HIV-1 identifies co-evolution of HIV-1 antigenicity, coreceptor usage and CD4 subset targeting.

The CCR5 (R5) to CXCR4 (X4) coreceptor switch in natural HIV-1 infection is associated with faster progression to AIDS, but the underlying mechanisms remain unclear. The difficulty in capturing the earliest moment of coreceptor switch in vivo limits our understanding of this phenomenon. Here, by tracking the evolution of the transmitted/founder (T/F) HIV-1 in a prospective cohort of individuals at risk for HIV-1 infection identified very early in acute infection, we investigated this process with high resolution. The earliest X4 variants evolved from the R5 tropic T/F strains. Strong X4 usage can be conferred by a single mutation. The mutations responsible for coreceptor switch can confer escape to neutralization and drive X4 variants to replicate mainly in the central memory and naïve CD4+ T cells. We propose a novel concept to explain the co-evolution of virus antigenicity and entry tropism termed "escape by shifting". This concept posits that for viruses with receptor or coreceptor flexibility, entry tropism alteration represents a mechanism of immune evasion in vivo .

Fc receptor engagement of HIV-1 Env-specific antibodies in mothers and infants predicts reduced vertical transmission.

Introduction: Infants acquire maternal antibodies by Fc receptor transcytosis across the placenta during pregnancy. Fc receptors are expressed on immune cells and are important for activation of effector cell functions. Methods: In this study, we evaluated Fc receptor engagement and ADCC activity of plasma binding antibodies from human immunodeficiency virus-1 (HIV) -infected mothers and to identify factors that may contribute to protection from HIV vertical transmission. Results: HIV-specific binding and Fc receptor engagement of plasma antibodies varied between mothers by transmission status and infants by infection status. Non-transmitting (NT) mothers and HIV-uninfected infants had antibodies with higher neonatal Fc receptor (FcRn) and FcγR engagement, as compared to transmitting (T) mothers and HIV+ infants, respectively. A significant inverse correlation between plasma antibody FcRn and FcγR engagement was observed for T mothers, but not NT mothers. Conversely, a significant direct correlation was observed between plasma antibody FcRn and FcγR engagement for HIV- infants, but not for HIV+ infants. Consequently, we observed significantly higher plasma antibody ADCC potency and breadth in HIV- infants, as compared to HIV+ infants. However, no differences in overall ADCC potency and breadth were observed between mothers. FcRn-engagement of HIV-specific antibodies in both mothers and infants predicted a lack of vertical transmission of HIV. Discussion: This study indicates that HIV-uninfected infants acquire HIV-specific antibodies with greater Fc receptor engagement and thus, greater ADCC capacity.

HIV-1 infections with multiple founders associate with the development of neutralization breadth.

Eliciting broadly neutralizing antibodies (bnAbs) is a cornerstone of HIV-1 vaccine strategies. Comparing HIV-1 envelope (env) sequences from the first weeks of infection to the breadth of antibody responses observed several years after infection can help define viral features critical to vaccine design. We investigated the relationship between HIV-1 env genetics and the development of neutralization breadth in 70 individuals enrolled in a prospective acute HIV-1 cohort. Half of the individuals who developed bnAbs were infected with multiple HIV-1 founder variants, whereas all individuals with limited neutralization breadth had been infected with single HIV-1 founders. Accordingly, at HIV-1 diagnosis, env diversity was significantly higher in participants who later developed bnAbs compared to those with limited breadth (p = 0.012). This association between founder multiplicity and the subsequent development of neutralization breadth was also observed in 56 placebo recipients in the RV144 vaccine efficacy trial. In addition, we found no evidence that neutralization breath was heritable when analyzing env sequences from the 126 participants. These results demonstrate that the presence of slightly different HIV-1 variants in acute infection could promote the induction of bnAbs, suggesting a novel vaccine strategy, whereby an initial immunization with a cocktail of minimally distant antigens would be able to initiate bnAb development towards breadth.

RV144 vaccine imprinting constrained HIV-1 evolution following breakthrough infection.

The scale of the HIV-1 epidemic underscores the need for a vaccine. The multitude of circulating HIV-1 strains together with HIV-1's high evolvability hints that HIV-1 could adapt to a future vaccine. Here, we wanted to investigate the effect of vaccination on the evolution of the virus post-breakthrough infection. We analyzed 2,635 HIV-1 env sequences sampled up to a year post-diagnosis from 110 vaccine and placebo participants who became infected in the RV144 vaccine efficacy trial. We showed that the Env signature sites that were previously identified to distinguish vaccine and placebo participants were maintained over time. In addition, fewer sites were under diversifying selection in the vaccine group than in the placebo group. These results indicate that HIV-1 would possibly adapt to a vaccine upon its roll-out.

Limited Evidence for a Relationship between HIV-1 Glycan Shield Features in Early Infection and the Development of Neutralization Breadth.

Identifying whether viral features present in acute HIV-1 infection predetermine the development of neutralization breadth is critical to vaccine design. Incorporating such features in vaccine antigens could initiate cross-reactive antibody responses that could sufficiently protect vaccinees from HIV-1 infection despite the uniqueness of each founder virus. To understand the relationship between Env determinants and the development of neutralization breadth, we focused on 197 individuals enrolled in two cohorts in Thailand and East Africa (RV144 and RV217) and followed since their diagnosis in acute or early HIV-1 infection. We analyzed the distribution of variable loop lengths and glycans, as well as the predicted density of the glycan shield, and compared these envelope features to the neutralization breadth data obtained 3 years after infection (n = 121). Our study revealed limited evidence for glycan shield features that associate with the development of neutralization breadth. While the glycan shield tended to be denser in participants who subsequently developed breadth, no significant relationship was found between the size of glycan holes and the development of neutralization breadth. The parallel analysis of 3,000 independent Env sequences showed no evidence of directional evolution of glycan shield features since the beginning of the epidemic. Together, our results highlight that glycan shield features in acute and early HIV-1 infection may not play a role determinant enough to dictate the development of neutralization breadth and instead suggest that the glycan shield's reactive properties that are associated with immune evasion may have a greater impact. IMPORTANCE A major goal of HIV-1 vaccine research is to design vaccine candidates that elicit potent broadly neutralizing antibodies (bNAbs). Different viral features have been associated with the development of bNAbs, including the glycan shield on the surface of the HIV-1 Envelope (Env). Here, we analyzed data from two cohorts of individuals who were followed from early infection to several years after infection spanning multiple HIV-1 subtypes. We compared Env glycan features in HIV-1 sequences obtained in early infection to the potency and breadth of neutralizing antibodies measured 1 to 3 years after infection. We found limited evidence of glycan shield properties that associate with the development of neutralization breadth in these cohorts. These results may have important implications for antigen design in future vaccine strategies and emphasize that HIV-1 vaccines will need to rely on a complex set of properties to elicit neutralization breadth.

Correction: RV144 HIV-1 vaccination impacts post-infection antibody responses.

[This corrects the article DOI: 10.1371/journal.ppat.1009101.].

Factors influencing estimates of HIV-1 infection timing using BEAST.

While large datasets of HIV-1 sequences are increasingly being generated, many studies rely on a single gene or fragment of the genome and few comparative studies across genes have been done. We performed genome-based and gene-specific Bayesian phylogenetic analyses to investigate how certain factors impact estimates of the infection dates in an acute HIV-1 infection cohort, RV217. In this cohort, HIV-1 diagnosis corresponded to the first RNA positive test and occurred a median of four days after the last negative test, allowing us to compare timing estimates using BEAST to a narrow window of infection. We analyzed HIV-1 sequences sampled one week, one month and six months after HIV-1 diagnosis in 39 individuals. We found that shared diversity and temporal signal was limited in acute infection, and insufficient to allow timing inferences in the shortest HIV-1 genes, thus dated phylogenies were primarily analyzed for env, gag, pol and near full-length genomes. There was no one best-fitting model across participants and genes, though relaxed molecular clocks (73% of best-fitting models) and the Bayesian skyline (49%) tended to be favored. For infections with single founders, the infection date was estimated to be around one week pre-diagnosis for env (IQR: 3-9 days) and gag (IQR: 5-9 days), whilst the genome placed it at a median of 10 days (IQR: 4-19). Multiply-founded infections proved problematic to date. Our ability to compare timing inferences to precise estimates of HIV-1 infection (within a week) highlights that molecular dating methods can be applied to within-host datasets from early infection. Nonetheless, our results also suggest caution when using uniform clock and population models or short genes with limited information content.

RV144 HIV-1 vaccination impacts post-infection antibody responses.

The RV144 vaccine efficacy clinical trial showed a reduction in HIV-1 infections by 31%. Vaccine efficacy was associated with stronger binding antibody responses to the HIV Envelope (Env) V1V2 region, with decreased efficacy as responses wane. High levels of Ab-dependent cellular cytotoxicity (ADCC) together with low plasma levels of Env-specific IgA also correlated with decreased infection risk. We investigated whether B cell priming from RV144 vaccination impacted functional antibody responses to HIV-1 following infection. Antibody responses were assessed in 37 vaccine and 63 placebo recipients at 6, 12, and 36 months following HIV diagnosis. The magnitude, specificity, dynamics, subclass recognition and distribution of the binding antibody response following infection were different in RV144 vaccine recipients compared to placebo recipients. Vaccine recipients demonstrated increased IgG1 binding specifically to V1V2, as well as increased IgG2 and IgG4 but decreased IgG3 to HIV-1 Env. No difference in IgA binding to HIV-1 Env was detected between the vaccine and placebo recipients following infection. RV144 vaccination limited the development of broadly neutralizing antibodies post-infection, but enhanced Fc-mediated effector functions indicating B cell priming by RV144 vaccination impacted downstream antibody function. However, these functional responses were not associated with clinical markers of disease progression. These data reveal that RV144 vaccination primed B cells towards specific binding and functional antibody responses following HIV-1 infection.

Evaluation of HIV-1 neutralizing and binding antibodies in maternal-infant transmission in Thailand.

Despite anti-retroviral therapy (ART) interventions for HIV+ pregnant mothers, over 43,000 perinatal infections occur yearly. Understanding risk factors that lead to mother-to-child transmission (MTCT) of HIV are critical. We evaluated maternal and infant plasma binding and neutralizing antibody responses in a drug-naïve, CRF01_AE infected MTCT cohort from Thailand to determine associations with transmission risk. Env V3-specific IgG and neutralizing antibody responses were significantly higher in HIV- infants, as compared to HIV+ infants. In fact, infant plasma neutralizing antibodies significantly associated with non-transmission. Conversely, increased maternal Env V3-specific IgG and neutralizing antibody responses were significantly associated with increased transmission risk, after controlling for maternal viral load. Our results highlight the importance of evaluating both maternal and infant humoral immune responses to better understand mechanisms of protection, as selective placental antibody transport may have a role in MTCT. This study further emphasizes the complex role of Env-specific antibodies in MTCT of CRF01_AE HIV.

Neutralizing antibody VRC01 failed to select for HIV-1 mutations upon viral rebound.

Infusion of the broadly neutralizing antibody VRC01 has been evaluated in individuals chronically infected with HIV-1. Here, we studied how VRC01 infusions affected viral rebound after cessation of antiretroviral therapy (ART) in 18 acutely treated and durably suppressed individuals. Viral rebound occurred in all individuals, yet VRC01 infusions modestly delayed rebound and participants who showed a faster decay of VRC01 in serum rebounded more rapidly. Participants with strains most sensitive to VRC01 or with VRC01 epitope motifs similar to known VRC01-susceptible strains rebounded later. Upon rebound, HIV-1 sequences were indistinguishable from those sampled at diagnosis. Across the cohort, participant-derived Env showed different sensitivity to VRC01 neutralization (including 2 resistant viruses), yet neutralization sensitivity was similar at diagnosis and after rebound, indicating the lack of selection for VRC01 resistance during treatment interruption. Our results showed that viremia rebounded despite the absence of HIV-1 adaptation to VRC01 and an average VRC01 trough of 221 µg/mL. Although VRC01 levels were insufficient to prevent a resurgent infection, knowledge that they did not mediate Env mutations in acute-like viruses is relevant for antibody-based strategies in acute infection.

Molecular dating and viral load growth rates suggested that the eclipse phase lasted about a week in HIV-1 infected adults in East Africa and Thailand.

Most HIV-1 infected individuals do not know their infection dates. Precise infection timing is crucial information for studies that document transmission networks or drug levels at infection. To improve infection timing, we used the prospective RV217 cohort where the window when plasma viremia becomes detectable is narrow: the last negative visit occurred a median of four days before the first detectable HIV-1 viremia with an RNA test, referred below as diagnosis. We sequenced 1,280 HIV-1 genomes from 39 participants at a median of 4, 32 and 170 days post-diagnosis. HIV-1 infections were dated by using sequence-based methods and a viral load regression method. Bayesian coalescent and viral load regression estimated that infections occurred a median of 6 days prior to diagnosis (IQR: 9-3 and 11-4 days prior, respectively). Poisson-Fitter, which analyzes the distribution of hamming distances among sequences, estimated a median of 7 days prior to diagnosis (IQR: 15-4 days) based on sequences sampled 4 days post-diagnosis, but it did not yield plausible results using sequences sampled at 32 days. Fourteen participants reported a high-risk exposure event at a median of 8 days prior to diagnosis (IQR: 12 to 6 days prior). These different methods concurred that HIV-1 infection occurred about a week before detectable viremia, corresponding to 20 days (IQR: 34-15 days) before peak viral load. Together, our methods comparison helps define a framework for future dating studies in early HIV-1 infection.

Dynamics of Human Immunodeficiency Virus-1 Genetic Diversification During Acute Infection.

We analyzed human immunodeficiency virus envelope diversity in 98 acute infections. The within-host genetic diversity, divergence from transmitted/founder (T/F) strain, and the observed frequency of multiple T/F infections increased with Fiebig stage. These data identify rapid viral dynamics during acute infection with implications for clinical trials conducted in this setting.

Activated PD-1+ CD4+ T cells represent a short-lived part of the viral reservoir and predict poor immunologic recovery upon initiation of ART.

OBJECTIVE: Activated (CD38HLA-DR) PD-1 CD4 T cells are strongly associated with virus replication and disease progression in untreated HIV-1 infection, and viral persistence in individuals on ART. Few studies have examined cell-associated viral load (CAVL) in different activated CD4 T-cell populations to measure relative contributions to viral reservoirs. DESIGN: Longitudinal assessment of HIV-1 chronically infected Ugandans initiating ART, to investigate activated CD4 T-cell populations and their contribution to viral reservoirs. METHODS: We followed 32 HIV-1 chronically infected individuals from Kampala, Uganda, and determined their CD4 T-cell counts and viral load at baseline, 6, and 12 months after the initiation of ART. T-cell populations were sorted based on activation profiles and gag DNA was measured to determine CAVL within these populations. Soluble factors associated with inflammation were measured in plasma using a multiplexed platform. RESULTS: Concomitant with viral load decline and CD4 T-cell count rebound, the activated PD-1 CD4 T-cell population contracted upon initiation of ART. Baseline levels of activated PD-1 CD4 T cells correlated with plasma levels of IP-10 and TNFRII. Interestingly, a higher baseline level of activated PD-1 CD4 T cells was associated with poorer CD4 T-cell recovery after 12 months of ART. This population contributed significantly to the cell-associated HIV DNA load at baseline, whereas their contribution declined on ART, indicating high turnover. CONCLUSION: Activated PD-1 CD4 T cells are predictors of poor immunologic recovery on ART and may represent a short-lived component of HIV-1 reservoirs.

Deep Sequencing Reveals Central Nervous System Compartmentalization in Multiple Transmitted/Founder Virus Acute HIV-1 Infection.

HIV-1 disseminates to a broad range of tissue compartments during acute HIV-1 infection (AHI). The central nervous system (CNS) can serve as an early and persistent site of viral replication, which poses a potential challenge for HIV-1 remission strategies that target the HIV reservoir. CNS compartmentalization is a key feature of HIV-1 neuropathogenesis. Thus far, the timing of how early CNS compartmentalization develops after infection is unknown. We examined whether HIV-1 transmitted/founder (T/F) viruses differ between CNS and blood during AHI using single-genome sequencing of envelope gene and further examined subregions in pol and env using next-generation sequencing in paired plasma and cerebrospinal fluid (CSF) from 18 individuals. Different proportions of mostly minor variants were found in six of the eight multiple T/F-infected individuals, indicating enrichment of some variants in CSF that may lead to significant compartmentalization in the later stages of infection. This study provides evidence for the first time that HIV-1 compartmentalization in the CNS can occur within days of HIV-1 exposure in multiple T/F infections. Further understanding of factors that determine enrichment of T/F variants in the CNS, as well as potential long-term implications of these findings for persistence of HIV-1 reservoirs and neurological impairment in HIV, is needed.