A Possible Sterilizing Cure of HIV-1 Infection Without Stem Cell Transplantation.

BACKGROUND: A sterilizing cure of HIV-1 infection has been reported in 2 persons living with HIV-1 who underwent allogeneic hematopoietic stem cell transplantations from donors who were homozygous for the CCR5Δ32 gene polymorphism. However, this has been considered elusive during natural infection. OBJECTIVE: To evaluate persistent HIV-1 reservoir cells in an elite controller with undetectable HIV-1 viremia for more than 8 years in the absence of antiretroviral therapy. DESIGN: Detailed investigation of virologic and immunologic characteristics. SETTING: Tertiary care centers in Buenos Aires, Argentina, and Boston, Massachusetts. PATIENT: A patient with HIV-1 infection and durable drug-free suppression of HIV-1 replication. MEASUREMENTS: Analysis of genome-intact and replication-competent HIV-1 using near-full-length individual proviral sequencing and viral outgrowth assays, respectively; analysis of HIV-1 plasma RNA by ultrasensitive HIV-1 viral load testing. RESULTS: No genome-intact HIV-1 proviruses were detected in analysis of a total of 1.188 billion peripheral blood mononuclear cells and 503 million mononuclear cells from placental tissues. Seven defective proviruses, some of them derived from clonally expanded cells, were detected. A viral outgrowth assay failed to retrieve replication-competent HIV-1 from 150 million resting CD4+ T cells. No HIV-1 RNA was detected in 4.5 mL of plasma. LIMITATIONS: Absence of evidence for intact HIV-1 proviruses in large numbers of cells is not evidence of absence of intact HIV-1 proviruses. A sterilizing cure of HIV-1 can never be empirically proved. CONCLUSION: Genome-intact and replication-competent HIV-1 were not detected in an elite controller despite analysis of massive numbers of cells from blood and tissues, suggesting that this patient may have naturally achieved a sterilizing cure of HIV-1 infection. These observations raise the possibility that a sterilizing cure may be an extremely rare but possible outcome of HIV-1 infection. PRIMARY FUNDING SOURCE: National Institutes of Health and Bill & Melinda Gates Foundation.

Persistence of an intact HIV reservoir in phenotypically naive T cells.

Despite the efficacy of antiretroviral therapy (ART), HIV persists in a latent form and remains a hurdle to eradication. CD4+ T lymphocytes harbor the majority of the HIV reservoir, but the role of individual subsets remains unclear. CD4+ T cells were sorted into central, transitional, effector memory, and naive T cells. We measured HIV DNA and performed proviral sequencing of more than 1900 proviruses in 2 subjects at 2 and 9 years after ART initiation to estimate the contribution of each subset to the reservoir. Although our study was limited to 2 subjects, we obtained comparable findings with publicly available sequences. While the HIV integration levels were lower in naive compared with memory T cells, naive cells were a major contributor to the intact proviral reservoir. Notably, proviral sequences isolated from naive cells appeared to be unique, while those retrieved from effector memory cells were mainly clonal. The number of clones increased as cells differentiated from a naive to an effector memory phenotype, suggesting naive cells repopulate the effector memory reservoir as previously shown for central memory cells. Naive T cells contribute substantially to the intact HIV reservoir and represent a significant hurdle for HIV eradication.

Peripheral blood lymphocyte proviral DNA predicts neurocognitive impairment in clade C HIV.

It is not known if proviral DNA in the periphery corresponds to cognitive status in clade C as it does in clade B and recombinant forms. A cross-sectional study was conducted on participants investigated for HIV-associated neurocognitive impairment in South Africa. HIV-1 proviral DNA was quantified using a PCR assay targeting a highly conserved HIV-1 LTR-gag region. Fifty-four (36.7%) participants were cognitively impaired and 93 (63.3%) were not impaired. Forty-three (79.6%) of the cognitively impaired participants were female and 11 (20.4%) were male. There was no significant age difference between cognitively impaired and unimpaired participants (p = 0.42). HIV-1 DNA in cognitively impaired PLWH was significantly higher than in cognitively normal individuals (p = .016). Considering impaired participants, lymphocyte HIV-1 DNA was significantly higher in males than females (p = 0.02). There was a modest positive correlation between lymphocyte HIV-1 DNA and global deficit scores (GDS) r = 0.176; p = 0.03). The two measures of viral load, lymphocyte HIV-1 DNA copies/million and plasma RNA copies/ml, were positively correlated (r = 0.39; p < .001). After adjusting for other covariates, age, sex, treatment status, and the interactions between impairment and treatment, the multivariate regression showed association between proviral load and neurocognitive impairment; omega effect size was 0.04, p value = 0.010. The burden of HIV-1 peripheral blood lymphocyte proviral DNA corresponds to neurocognitive impairment among individuals infected with clade C disease. Therefore, therapeutic strategies to reduce the HIV-1 proviral DNA reservoir in lymphocytes may improve neurocognitive outcomes in PLWH.

Neurologic, clinical, and immunologic features in a cohort of HTLV-1 carriers with high proviral loads.

A high proviral load (PVL) is recognized as a risk factor for human T cell leukemia virus-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), but there is a lack of prospective studies evaluating whether or not HTLV-1 carriers with high PVL are at risk of developing HAM/TSP or other HTLV-1-related diseases. Here, we compare the incidence of clinical manifestations and the cytokine levels in 30 HTLV-1 carriers with high (> 50,000 copies/106 PBMC) and an equal number of subjects with low proviral load. Participants were followed for 3 to 16 years (median of 11 years). The PVL, IFN-γ, TNF, and IL-10 levels were quantified at entry and at the end of the follow-up. Among the self-reported symptoms in the initial evaluation, only the presence of paresthesia on the hands was more frequent in the group with high PVL (p < 0.04). The production of IFN-γ was higher in the group with high PVL group (median of 1308 versus 686 pg/ml, p < 0.011) when compared with the control group in the first assessment. There was no difference in the occurrence of urinary symptoms or erectile dysfunction, periodontal disease, Sicca syndrome, and neurologic signs between the two groups during the follow-up. The observation that none of the HTLV-1 carriers with high PVL and with exaggerated inflammatory response progressed to HAM/TSP indicates that other factors in addition to the PVL and an exaggerated immune response are involved in the pathogenesis of HAM/TSP.

Longitudinal study reveals HIV-1-infected CD4+ T cell dynamics during long-term antiretroviral therapy.

Proliferation of CD4+ T cells harboring HIV-1 proviruses is a major contributor to viral persistence in people on antiretroviral therapy (ART). To determine whether differential rates of clonal proliferation or HIV-1-specific cytotoxic T lymphocyte (CTL) pressure shape the provirus landscape, we performed an intact proviral DNA assay (IPDA) and obtained 661 near-full-length provirus sequences from 8 individuals with suppressed viral loads on ART at time points 7 years apart. We observed slow decay of intact proviruses but no changes in the proportions of various types of defective proviruses. The proportion of intact proviruses in expanded clones was similar to that of defective proviruses in clones. Intact proviruses observed in clones did not have more escaped CTL epitopes than intact proviruses observed as singlets. Concordantly, total proviruses at later time points or observed in clones were not enriched in escaped or unrecognized epitopes. Three individuals with natural control of HIV-1 infection (controllers) on ART, included because controllers have strong HIV-1-specific CTL responses, had a smaller proportion of intact proviruses but a distribution of defective provirus types and escaped or unrecognized epitopes similar to that of the other individuals. This work suggests that CTL selection does not significantly check clonal proliferation of infected cells or greatly alter the provirus landscape in people on ART.

Different human resting memory CD4+ T cell subsets show similar low inducibility of latent HIV-1 proviruses.

The latent reservoir of HIV-1 in resting CD4+ T cells is a major barrier to cure. It is unclear whether the latent reservoir resides principally in particular subsets of CD4+ T cells, a finding that would have implications for understanding its stability and developing curative therapies. Recent work has shown that proliferation of HIV-1-infected CD4+ T cells is a major factor in the generation and persistence of the latent reservoir and that latently infected T cells that have clonally expanded in vivo can proliferate in vitro without producing virions. In certain CD4+ memory T cell subsets, the provirus may be in a deeper state of latency, allowing the cell to proliferate without producing viral proteins, thus permitting escape from immune clearance. To evaluate this possibility, we used a multiple stimulation viral outgrowth assay to culture resting naïve, central memory (TCM), transitional memory (TTM), and effector memory (TEM) CD4+ T cells from 10 HIV-1-infected individuals on antiretroviral therapy. On average, only 1.7% of intact proviruses across all T cell subsets were induced to transcribe viral genes and release replication-competent virus after stimulation of the cells. We found no consistent enrichment of intact or inducible proviruses in any T cell subset. Furthermore, we observed notable plasticity among the canonical memory T cell subsets after activation in vitro and saw substantial person-to-person variability in the inducibility of infectious virus release. This finding complicates the vision for a targeted approach for HIV-1 cure based on T cell memory subsets.

Reduction of peak viremia by an integration-defective SIV proviral DNA vaccine in rhesus macaques.

An integrase-defective SIV (idSIV) vaccine delivered by a DNA prime and viral particle boost approach can suppress viral loads (VLs) during the acute infection stage after intravenous SIVmac239 challenge. This study investigated how idSIV DNA and viral particle immunization alone contributed to the suppression of VLs in Chinese rhesus macaques after SIV challenge. Two macaques were immunized with idSIV DNA five times and two macaques were immunized with idSIV viral particles three times. Cellular and humoral immune responses were measured in the vaccinated macaques after immunization. The VLs and CD4+ T cell counts were monitored for 28 weeks after the intravenous SIVmac239 challenge. The SIV-specific T cell responses were only detected in the DNA-vaccinated macaques. However, binding and neutralizing antibodies against autologous and heterologous viruses were moderately better in macaques immunized with viral particles than in macaques immunized with DNA. After the challenge, the mean peak viremia in the DNA group was 2.3 logs lower than that in the control group, while they were similar between the viral particle immunization and control groups. Similar CD4+ T cell counts were observed among all groups. These results suggest that idSIV DNA immunization alone reduces VLs during acute infection after SIV challenge in macaques and may serve as a key component in combination with other immunogens as prophylactic vaccines.

Association between bovine leukemia virus proviral load and severity of clinical mastitis.

The purpose of this study was to clarify the effect of Bovine leukemia virus (BLV) infection on natural immunity in the bovine mammary gland and on the severity of clinical mastitis. We classified milk samples from clinical mastitic cows into BLV-positive (n=76) and BLV-negative (n=12). BLV-positive cows were further divided into cows with High BLV proviral load (H-PVL) (n=23) and Low BLV proviral load (L-PVL) (n=53). Severity of clinical mastitis was classified as MILD, MODERATE, or SEVERE. Multiple logistic regression analysis was performed on the host factors and environmental factors with severity of clinical mastitis as the objective variable. BLV proviral load (PVL) and season at onset of mastitis showed significant correlation with the severity of clinical mastitis. Binary logistic regression analysis was performed on natural immunity factors lactoferrin and lingual antimicrobial peptide (LAP) concentration in milk, with PVL as the objective variable. Of these natural immunity factors, LAP concentration in milk showed significant correlation with PVL. The results of the present study suggested that PVL and season are associated with severity of clinical mastitis, and that the immune function in the mammary gland is decreased in cows with H-PVL compared to that in cows with L-PVL.

Prostaglandin E2-Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti-PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection.

Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.

Strength of T cell signaling regulates HIV-1 replication and establishment of latency.

A major barrier to curing HIV-1 is the long-lived latent reservoir that supports re-emergence of HIV-1 upon treatment interruption. Targeting this reservoir will require mechanistic insights into the establishment and maintenance of HIV-1 latency. Whether T cell signaling at the time of HIV-1 infection influences productive replication or latency is not fully understood. We used a panel of chimeric antigen receptors (CARs) with different ligand binding affinities to induce a range of signaling strengths to model differential T cell receptor signaling at the time of HIV-1 infection. Stimulation of T cell lines or primary CD4+ T cells expressing chimeric antigen receptors supported HIV-1 infection regardless of affinity for ligand; however, only signaling by the highest affinity receptor facilitated HIV-1 expression. Activation of chimeric antigen receptors that had intermediate and low binding affinities did not support provirus transcription, suggesting that a minimal signal is required for optimal HIV-1 expression. In addition, strong signaling at the time of infection produced a latent population that was readily inducible, whereas latent cells generated in response to weaker signals were not easily reversed. Chromatin immunoprecipitation showed HIV-1 transcription was limited by transcriptional elongation and that robust signaling decreased the presence of negative elongation factor, a pausing factor, by more than 80%. These studies demonstrate that T cell signaling influences HIV-1 infection and the establishment of different subsets of latently infected cells, which may have implications for targeting the HIV-1 reservoir.

Ancestral sequences from an elite neutralizer proximal to the development of neutralization resistance as a potential source of HIV vaccine immunogens.

A major challenge in HIV vaccine development is the identification of immunogens able to elicit broadly neutralizing antibodies (bNAbs). While remarkable progress has been made in the isolation and characterization of bNAbs, the epitopes they recognize appear to be poorly immunogenic. Thus, none of the candidate vaccines developed to date has induced satisfactory levels of neutralizing antibodies to the HIV envelope protein (Env). One approach to the problem of poor immunogenicity is to build vaccines based on envelope (env) genes retrieved from rare individuals termed elite neutralizers (ENs) who at one time possessed specific sequences that stimulated the formation of bNAbs. Env proteins selected from these individuals could possess uncommon, yet to be defined, structural features that enhance the immunogenicity of epitopes recognized by bNAbs. Here we describe the recovery of envs from an EN that developed unusually broad and potent bNAbs. As longitudinal specimens were not available, we combined plasma and provirus sequences acquired from a single time-point to infer a phylogenetic tree. Combining ancestral reconstruction data with virus neutralization data allowed us to sift through the myriad of virus quasi-species that evolved in this individual to identify envelope sequences from the nodes that appeared to define the transition from neutralization sensitive envs to the neutralization resistant envs that occur in EN plasma. Synthetic genes from these nodes were functional in infectivity assays and sensitive to neutralization by bNAbs, and may provide a novel source of immunogens for HIV vaccine development.

Extensive editing of cellular and viral double-stranded RNA structures accounts for innate immunity suppression and the proviral activity of ADAR1p150.

The interferon (IFN)-mediated innate immune response is the first line of defense against viruses. However, an IFN-stimulated gene, the adenosine deaminase acting on RNA 1 (ADAR1), favors the replication of several viruses. ADAR1 binds double-stranded RNA and converts adenosine to inosine by deamination. This form of editing makes duplex RNA unstable, thereby preventing IFN induction. To better understand how ADAR1 works at the cellular level, we generated cell lines that express exclusively either the IFN-inducible, cytoplasmic isoform ADAR1p150, the constitutively expressed nuclear isoform ADAR1p110, or no isoform. By comparing the transcriptome of these cell lines, we identified more than 150 polymerase II transcripts that are extensively edited, and we attributed most editing events to ADAR1p150. Editing is focused on inverted transposable elements, located mainly within introns and untranslated regions, and predicted to form duplex RNA structures. Editing of these elements occurs also in primary human samples, and there is evidence for cross-species evolutionary conservation of editing patterns in primates and, to a lesser extent, in rodents. Whereas ADAR1p150 rarely edits tightly encapsidated standard measles virus (MeV) genomes, it efficiently edits genomes with inverted repeats accidentally generated by a mutant MeV. We also show that immune activation occurs in fully ADAR1-deficient (ADAR1KO) cells, restricting virus growth, and that complementation of these cells with ADAR1p150 rescues virus growth and suppresses innate immunity activation. Finally, by knocking out either protein kinase R (PKR) or mitochondrial antiviral signaling protein (MAVS)-another protein controlling the response to duplex RNA-in ADAR1KO cells, we show that PKR activation elicits a stronger antiviral response. Thus, ADAR1 prevents innate immunity activation by cellular transcripts that include extensive duplex RNA structures. The trade-off is that viruses take advantage of ADAR1 to elude innate immunity control.

Emerging Proviral Roles of Caspases during Lytic Replication of Gammaherpesviruses.

Due to their roles in the regulation of programmed cell death and inflammation, the cellular caspase proteases are considered antiviral factors. However, recent studies have revealed examples of proviral functions for caspases. Here, we review a growing body of literature on the role of caspases in promoting the replication of human gammaherpesviruses. We propose that gammaherpesviruses have evolved ways to redirect these enzymes and to use their activation to support viral replication and immune evasion.

Analysis of drug resistance mutations in whole blood DNA from HIV-1 infected patients by single genome and ultradeep sequencing analysis.

In HIV-1 infected patients blood CD4+ T lymphocytes could be a valuable target to analyse drug resistance mutations (DRM) selected over the course of the infection. However, detection of viral resistance mutations in cellular DNA by standard genotype resistance techniques (SGRT) is suboptimal. Whole blood DNA (wbDNA) from 12 HIV-1 infected patients on ART was studied by Single Genome Sequencing (SGS) and 8 of them also by Ultradeep pyrosequencing (UDP). Results were compared with contemporary and historical DRM detected in plasma by SGRT during follow up. All the contemporary DRM detected in plasma from the viremic patients were detected by SGS and UDP (20 from 7 patients and 4 from 5 patients respectively). Out of the 67 historical DRM detected in plasma and no longer present at the time of testing, 38 (57%) were detected by SGS in 12 patients and 27 out of 46 (59%) by UDP in 8 patients. Additional DRM never reported in plasma by SGRT were detected by SGS (12 from 8 patients) and UDP (10 from 8 patients). Analysis of wbDNA from HIV-1 infected patients by SGS and UDP provides proof of concept of the value of blood DNA to investigate current and archived DRM in HIV-1 infected patients on ART.

CD32 expression is associated to T-cell activation and is not a marker of the HIV-1 reservoir.

CD32 has been shown to be preferentially expressed in latently HIV-1-infected cells in an in vitro model of quiescent CD4 T cells. Here we show that stimulation of CD4+ T cells with IL-2, IL-7, PHA, and anti-CD3/CD28 antibodies induces T-cell proliferation, co-expression of CD32 and the activation of the markers HLA-DR and CD69. HIV-1 infection increases CD32 expression. 79.2% of the CD32+/CD4+ T cells from HIV+ individuals under antiretroviral treatment were HLA-DR+. Resting CD4+ T cells infected in vitro generally results in higher integration of provirus. We observe no difference in provirus integration or replication-competent inducible latent HIV-1 in CD32+ or CD32- CD4+ T cells from HIV+ individuals. Our results demonstrate that CD32 expression is a marker of CD4+ T cell activation in HIV+ individuals and raises questions regarding the immune resting status of CD32+ cells harboring HIV-1 proviruses.

CD32-Expressing CD4 T Cells Are Phenotypically Diverse and Can Contain Proviral HIV DNA.

Efforts to both characterize and eradicate the HIV reservoir have been limited by the rarity of latently infected cells and the absence of a specific denoting biomarker. CD32a (FcγRIIa) has been proposed to be a marker for an enriched CD4 T cell HIV reservoir, but this finding remains controversial. Here, we explore the expression of CD32 on CD3+CD4+ cells in participants from two primary HIV infection studies and identify at least three distinct phenotypes (CD32low, CD32+CD14+, and CD32high). Of note, CD4 negative enrichment kits remove the majority of CD4+CD32+ T cells, potentially skewing subsequent analyses if used. CD32high CD4 T cells had higher levels of HLA-DR and HIV co-receptor expression than other subsets, compatible with their being more susceptible to infection. Surprisingly, they also expressed high levels of CD20, TCRαß, IgD, and IgM (but not IgG), markers for both T cells and naïve B cells. Compared with other populations, CD32low cells had a more differentiated memory phenotype and high levels of immune checkpoint receptors, programmed death receptor-1 (PD-1), Tim-3, and TIGIT. Within all three CD3+CD4+CD32+ phenotypes, cells could be identified in infected participants, which contained HIV DNA. CD32 expression on CD4 T cells did not correlate with HIV DNA or cell-associated HIV RNA (both surrogate measures of overall reservoir size) or predict time to rebound viremia following treatment interruption, suggesting that it is not a dominant biomarker for HIV persistence. Our data suggest that while CD32+ T cells can be infected with HIV, CD32 is not a specific marker of the reservoir although it might identify a population of HIV enriched cells in certain situations.

Targeting the Latent Reservoir for HIV-1.

Antiretroviral therapy can effectively block HIV-1 replication and prevent or reverse immunodeficiency in HIV-1-infected individuals. However, viral replication resumes within weeks of treatment interruption. The major barrier to a cure is a small pool of resting memory CD4+ T cells that harbor latent HIV-1 proviruses. This latent reservoir is now the focus of an intense international research effort. We describe how the reservoir is established, challenges involved in eliminating it, and pharmacologic and immunologic strategies for targeting this reservoir. The development of a successful cure strategy will most likely require understanding the mechanisms that maintain HIV-1 proviruses in a latent state and pathways that drive the proliferation of infected cells, which slows reservoir decay. In addition, a cure will require the development of effective immunologic approaches to eliminating infected cells. There is renewed optimism about the prospect of a cure, and the interventions discussed here could pave the way.

Tackling HIV Persistence: Pharmacological versus CRISPR-Based Shock Strategies.

Jan Svoboda studied aspects of viral latency, in particular with respect to disease induction by avian RNA tumor viruses, which were later renamed as part of the extended retrovirus family. The course of retroviral pathogenesis is intrinsically linked to their unique property of integrating the DNA copy of the retroviral genome into that of the host cell, thus forming the provirus. Retroviral latency has recently become of major clinical interest to allow a better understanding of why we can effectively block the human immunodeficiency virus type 1 (HIV-1) in infected individuals with antiviral drugs, yet never reach a cure. We will discuss HIV-1 latency and its direct consequence-the formation of long-lasting HIV-1 reservoirs. We next focus on one of the most explored strategies in tackling HIV-1 reservoirs-the "shock and kill" strategy-which describes the broadly explored pharmacological way of kicking the latent provirus, with subsequent killing of the virus-producing cell by the immune system. We furthermore present how the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system can be harnessed to reach the same objective by reactivating HIV-1 gene expression from latency. We will review the benefits and drawbacks of these different cure strategies.

HIV persistence: clonal expansion of cells in the latent reservoir.

While antiretroviral therapy (ART) can reduce HIV-1 to undetectable levels, the virus generally reappears if treatment is stopped. Resurgence of the virus is due to the reactivation of T cells harboring latent integrated provirus, and recent studies indicate that proliferation of these latently infected cells helps maintain the HIV-1 reservoir. In this issue of the JCI, Lee et al. evaluated CD4+ T cell subsets to determine whether certain populations are more likely to harbor full-length, replication-competent provirus. The authors identified an enrichment of clonally expanded Th1 cells containing intact HIV-1 proviruses, suggesting that this polarized subset contributes to the persistence of the reservoir. Strategies to target these provirus-harboring cells need to be considered for future therapies aimed toward HIV-1 cure.

Quantitative analyses reveal distinct sensitivities of the capture of HIV-1 primary viruses and pseudoviruses to broadly neutralizing antibodies.

Development of vaccines capable of eliciting broadly neutralizing antibodies (bNAbs) is a key goal to controlling the global AIDS epidemic. To be effective, bNAbs must block the capture of HIV-1 to prevent viral acquisition and establishment of reservoirs. However, the role of bNAbs, particularly during initial exposure of primary viruses to host cells, has not been fully examined. Using a sensitive, quantitative, and high-throughput qRT-PCR assay, we found that primary viruses were captured by host cells and converted into a trypsin-resistant form in less than five minutes. We discovered, unexpectedly, that bNAbs did not block primary virus capture, although they inhibited the capture of pseudoviruses/IMCs and production of progeny viruses at 48h. Further, viruses escaped bNAb inhibition unless the bNAbs were present in the initial minutes of exposure of virus to host cells. These findings will have important implications for HIV-1 vaccine design and determination of vaccine efficacy.