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HIV-1-specific CD4(+) T cells (T(CD4+)) play a critical role in controlling HIV-1 infection. Canonically, T(CD4+) are activated by peptides derived from extracellular (“exogenous”) antigens displayed in complex with major histocompatibility complex class II (MHCII) molecules on the surfaces of “professional” antigen presenting cells (APC) such as dendritic cells (DCs). In contrast, activated human T(CD4+), which express MHCII, are not typically considered for their APC potential due to their low endocytic capacity and the exogenous antigen systems historically used for assessment. Using primary T(CD4+) and monocyte-derived DCs from healthy donors, we show that activated human T(CD4+) are highly effective at MHCII-restricted presentation of an immunodominant HIV-1-derived epitope following infection and subsequent noncanonical processing and presentation of endogenously produced antigen. Our results indicate that, in addition to marshalling HIV-1-specific immune responses during infection, T(CD4+) also act as APCs, leading to the activation of HIV-1-specific T(CD4+)

The novel coronavirus SARS-CoV-2, the causative agent of COVID-19 disease, has killed over five million people worldwide as of December 2021 with infections rising again due to the emergence of highly transmissible variants. Animal models that faithfully recapitulate human disease are critical for assessing SARS-CoV-2 viral and immune dynamics, for understanding mechanisms of disease, and for testing vaccines and therapeutics. Pigtail macaques (PTM, Macaca nemestrina) demonstrate a rapid and severe disease course when infected with simian immunodeficiency virus (SIV), including the development of severe cardiovascular symptoms that are pertinent to COVID-19 manifestations in humans. We thus proposed this species may likewise exhibit severe COVID-19 disease upon infection with SARS-CoV-2. Here, we extensively studied a cohort of SARS-CoV-2-infected PTM euthanized either 6- or 21-days after respiratory viral challenge. We show that PTM demonstrate largely mild-to-moderate COVID-19 disease. Pulmonary infiltrates were dominated by T cells, including CD4+ T cells that upregulate CD8 and express cytotoxic molecules, as well as virus-targeting T cells that were predominantly CD4+. We also noted increases in inflammatory and coagulation markers in blood, pulmonary pathologic lesions, and the development of neutralizing antibodies. Together, our data demonstrate that SARS-CoV-2 infection of PTM recapitulates important features of COVID-19 and reveals new immune and viral dynamics and thus may serve as a useful animal model for studying pathogenesis and testing vaccines and therapeutics., Author summary The COVID-19 pandemic has claimed the lives of millions in a span of less than two years. Despite the development of several highly effective vaccines, many millions remain unvaccinated, and several highly transmissible variants have emerged, clearly suggesting the need for new approaches to treat those that become severely ill. The development of new drugs will rely on having animal models that reproduce the most severe disease seen in humans. To date, nonhuman primate models have not exhibited this severe disease. In this study we tested whether pigtail macaques (PTM) might exhibit such severe disease, based on previous work showing this species is prone to more rapid and severe disease when infected with other viruses.

Graft-versus-host disease (GVHD) remains the most common treatment-related complication after allogeneic hematopoietic cell transplantation (allo-HCT). Lymphocyte migration plays a critical role in the pathogenesis of GVHD. A previous phase I/II trial demonstrated that CCR5 blockade with maraviroc in the first 30days after allo-HCT resulted in a low incidence of early acute GVHD, primarily in visceral organs, but with no impact on late acute or chronic GVHD. We conducted a phase II trial to examine the efficacy of an extended course of maraviroc, administered through post-transplantation day +90 in addition to standard prophylaxis in 37 recipients of reduced-intensity-conditioned unrelated donor allo-HCT performed to treat hematologic malignancies. Extended maraviroc treatment was safe and feasible. The primary study endpoint, day +180 rate of grade II-IV acute GVHD, was 22 ± 7%, liver GVHD was not observed, and gut GVHD was uncommon. The day +180 rate of grade III-IV acute GVHD was 5 ± 4%. The 1-year rate of moderate to severe chronic GVHD was 8 ± 5% and that of disease relapse was 30 ± 8%. Overall survival at 1 year was 70 ± 8%. Compared with the previously studied short course of maraviroc, the extended course resulted in a significantly higher GVHD-free, relapse-free survival (adjusted hazard ratio [HR], .45; 95% confidence interval [CI], .25 to .82; P = .009) and overall survival (adjusted HR, .48; 95% CI, .24 to .96; P = .037). A combined analysis of both trials showed that high maraviroc trough concentrations on the day of hematopoietic cell infusion were associated with lower rates of acute GVHD. An extended course of maraviroc after reduced-intensity-conditioned unrelated donor allo-HCT is safe and effective in preventing acute and chronic GVHD and is associated with favorable survival.

SUMMARYThe HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-dependent trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting of Env. Despite extensive characterization, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which the Tyr-based signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY) replicates to high levels acutely in pigtail macaques (PTM) but is rapidly controlled. We previously reported that rhesus macaques and PTM can progress to AIDS following SIVmac239ΔGY infection in association with novel amino acid changes in the Env cytoplasmic domain. These included an R722G flanking the ΔGY deletion and a nine nucleotide deletion that encodes amino acids 734-736 (ΔQTH) and overlaps with the rev and tat open reading frames. We show that molecular clones containing these mutations reconstitute signals for both endocytosis and polarized sorting. In one PTM, a novel genotype was selected, which generated a new signal for polarized sorting but not endocytosis. This mutation by itself was sufficient to maintain high viral loads for several months when introduced into naïve PTMs. These findings reveal, for the first time, strong selection pressure for Env endocytosis and, in particular, for polarized sorting during pathogenic SIV infection in vivo.

An effective strategy to cure HIV will likely require a potent and sustained antiviral T cell response. Here we explored the utility of chimeric antigen receptor (CAR) T cells, expressing the CD4 ectodomain to confer specificity for the HIV envelope, to mitigate HIV-induced pathogenesis in bone marrow, liver, thymus (BLT) humanized mice. CAR T cells expressing the 4–1BB/CD3-ζ endodomain were insufficient to prevent viral rebound and CD4(+) T cell loss after the discontinuation of antiretroviral therapy. Through iterative improvements to the CAR T cell product, we developed Dual-CAR T cells that simultaneously expressed both 4–1BB/CD3-ζ and CD28/CD3-ζ endodomains. Dual-CAR T cells exhibited expansion kinetics that exceeded 4–1BB-, CD28- and third-generation costimulated CAR T cells, elicited effector functions equivalent to CD28-costimulated CAR T cells and prevented HIV-induced CD4(+) T cell loss despite persistent viremia. Moreover, when Dual-CAR T cells were protected from HIV infection through expression of the C34-CXCR4 fusion inhibitor, these cells significantly reduced acute-phase viremia, as well as accelerated HIV suppression in the presence of antiretroviral therapy and reduced tissue viral burden. Collectively, these studies demonstrate the enhanced therapeutic potency of a novel Dual-CAR T cell product with the potential to effectively treat HIV infection.

The H196 residue in SIVmac239 Nef is conserved across the majority of HIV and SIV isolates, lies immediately adjacent to the AP-2 (adaptor protein 2) binding di-leucine domain (ExxxLM195), and is critical for several described AP-2 dependent Nef functions, including the downregulation of tetherin (BST-2/CD317), CD4, and others. Surprisingly, many stocks of the closely related SIVmac251 swarm virus harbor a nef allele encoding a Q196. In SIVmac239, this variant is associated with loss of multiple AP-2 dependent functions. Publicly available sequences for SIVmac251 stocks were mined for variants linked to Q196 that might compensate for functional defects associated with this residue. Variants were engineered into the SIVmac239 backbone and in Nef expression plasmids and flow cytometry was used to examine surface tetherin expression in primary CD4 T cells and surface CD4 expression in SupT1 cells engineered to express rhesus CD4. We found that SIVmac251 stocks that encode a Q196 residue in Nef uniformly also encode an upstream R191 residue. We show that R191 restores the ability of Nef to downregulate tetherin in the presence of Q196 and has a similar but less pronounced impact on CD4 expression. However, a published report showed Q196 commonly evolves to H196 in vivo, suggesting a fitness cost. R191 may represent compensatory evolution to restore the ability to downregulate tetherin lost in viruses harboring Q196.

The functional properties of circulating CD8+ T cells have been associated with immune control of HIV. However, viral replication occurs predominantly in secondary lymphoid tissues, such as lymph nodes (LNs). We used an integrated single-cell approach to characterize effective HIV-specific CD8+ T cell responses in the LNs of elite controllers (ECs), defined as individuals who suppress viral replication in the absence of antiretroviral therapy (ART). Higher frequencies of total memory and follicle-homing HIV-specific CD8+ T cells were detected in the LNs of ECs compared with the LNs of chronic progressors (CPs) who were not receiving ART. Moreover, HIV-specific CD8+ T cells potently suppressed viral replication without demonstrable cytolytic activity in the LNs of ECs, which harbored substantially lower amounts of CD4+ T cell–associated HIV DNA and RNA compared with the LNs of CPs. Single-cell RNA sequencing analyses further revealed a distinct transcriptional signature among HIV-specific CD8+ T cells from the LNs of ECs, typified by the down-regulation of inhibitory receptors and cytolytic molecules and the up-regulation of multiple cytokines, predicted secreted factors, and components of the protein translation machinery. Collectively, these results provide a mechanistic framework to expedite the identification of novel antiviral factors, highlighting a potential role for the localized deployment of noncytolytic functions as a determinant of immune efficacy against HIV.

BACKGROUND. The effect of a brief analytical treatment interruption (ATI) on the HIV-1 latent reservoir of individuals who initiate antiretroviral therapy (ART) during chronic infection is unknown. METHODS. We evaluated the impact of transient viremia on the latent reservoir in participants who underwent an ATI and at least 6 months of subsequent viral suppression in a clinical trial testing the effect of passive infusion of the broadly neutralizing Ab VRC01 during ATI. RESULTS. Measures of total HIV-1 DNA, cell-associated RNA, and infectious units per million cells (IUPM) (measured by quantitative viral outgrowth assay [QVOA]) were not statistically different before or after ATI. Phylogenetic analyses of HIV-1 env sequences from QVOA and proviral DNA demonstrated little change in the composition of the virus populations comprising the pre- and post-ATI reservoir. Expanded clones were common in both QVOA and proviral DNA sequences. The frequency of clonal populations differed significantly between QVOA viruses, proviral DNA sequences, and the viruses that reactivated in vivo. CONCLUSIONS. The results indicate that transient viremia from ATI does not substantially alter measures of the latent reservoir, that clonal expansion is prevalent within the latent reservoir, and that characterization of latent viruses that can reactivate in vivo remains challenging. TRIAL REGISTRATION. ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02463227","term_id":"NCT02463227"}}NCT02463227 FUNDING. Funding was provided by the NIH.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Lymphocyte trafficking via chemokine receptors such as CCR5 plays a critical role in alloreactive responses, and previous data suggest that CCR5 blockade with maraviroc results in a low incidence of visceral GVHD. However, the full scope of clinical and immunologic effects of CCR5 blockade in HSCT has not been described. We compared a cohort of patients enrolled on a trial of reduced-intensity allo-HSCT with standard GVHD prophylaxis plus maraviroc to a contemporary control cohort receiving standard GVHD prophylaxis alone. Maraviroc treatment was associated with a lower incidence of acute GVHD without increased risk of disease relapse, as well as reduced levels of gut-specific markers. At day 30, maraviroc treatment increased CCR5 expression on T cells and dampened T-cell activation in peripheral blood without impairing early immune reconstitution or increasing risk for infections. Patients who developed acute GVHD despite maraviroc prophylaxis showed increased T-cell activation, naive T-cell skewing, and elevated serum CXCL9 and CXCL10 levels. Collectively, these data suggest that maraviroc effectively protects against GVHD by modulating alloreactive donor T-cell responses, and that CXCR3 signaling may be an important resistance mechanism to CCR5 blockade in GVHD.

HIV is a major driver of tuberculosis (TB) reactivation. Depletion of CD4(+) T cells is assumed to be the basis behind TB reactivation in individuals with latent tuberculosis infection (LTBI) coinfected with HIV. Nonhuman primates (NHPs) coinfected with a mutant simian immunodeficiency virus (SIVΔGY) that does not cause depletion of tissue CD4(+) T cells during infection failed to reactivate TB. To investigate the contribution of CD4(+) T cell depletion relative to other mechanisms of SIV-induced reactivation of LTBI, we used CD4R1 antibody to deplete CD4(+) T cells in animals with LTBI without lentiviral infection. The mere depletion of CD4(+) T cells during LTBI was insufficient in generating reactivation of LTBI. Instead, direct cytopathic effects of SIV resulting in chronic immune activation, along with the altered effector T cell phenotypes and dysregulated T cell homeostasis, were likely mediators of reactivation of LTBI. These results revealed important implications for TB control in HIV-coinfected individuals.

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIVmac239 and 47 animals infected with SIVmac239Δnef Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVΔnef In particular, polymorphisms at positions 43 and 111 (P43 and H111) were associated with lower acute-phase viral loads for SIVΔnef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef.IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin.

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIV(mac)239 and 47 animals infected with SIV(mac)239Δnef. Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVΔnef. In particular, polymorphisms at positions 43 and 111 (P(43) and H(111)) were associated with lower acute-phase viral loads for SIVΔnef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef. IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef-deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin.

Background: Cellular tropism of human immunodeficiency virus (HIV-1) is closely linked to interactions between the viral envelope glycoprotein (Env) with CD4 and chemokine receptor family members, CCR5 and CXCR4. This interaction plays a key role in determining anatomic sites that are infected in vivo and the cascade of early and late events that result in chronic immune activation, immunosuppression and ultimately, AIDS. CD4+ T cells are critical to adaptive immune responses, and their early and rapid infection in gut lamina propria and secondary lymphoid tissues in susceptible hosts likely contributes to viral persistence and progression to disease. CD4+ macrophages are also infected, although their role in HIV-1 pathogenesis is more controversial.Methods: Pathogenic infection by simian immunodeficiency viruses (SIV) in Asian macaques as models of HIV-1 infection has enabled the impact of cellular tropism on pathogenesis to be directly probed. This review will highlight examples in which experimental interventions during SIV infection or the introduction of viral mutations have altered cellular tropism and, subsequently, pathogenesis.Results: Alterations to the interaction of Env and its cellular receptors has been shown to result in changes to CD4 dependence, coreceptor specificity, and viral tropism for gut CD4+ T cells and macrophages.Conclusion: Collectively, these findings have yielded novel insights into the critical role of the viral Env and tropism as a driver of pathogenesis and host control and have helped to identify new areas for targeted interventions in therapy and prevention of HIV-1 infection.

Defining a macrophage-tropic phenotype for HIV-1 to assess a role in pathogenesis is complicated by the fact that HIV-1 isolates vary continuously in their ability to enter monocyte-derived macrophages (MDMs) in vitro , and MDMs vary in their ability to support HIV-1 entry. To overcome these limitations, we identified consistent differences in entry phenotypes between five paired blood-derived, T cell-tropic HIV-1 env genes, four of which are CCR5-using (R5) and one of which is CXCR4-using (X4), and cerebrospinal fluid (CSF)-derived, R5 macrophage-tropic env genes. We performed entry assays using the CD4- and CCR5-inducible Affinofile cell line, expressing a range of CD4 levels that approximates the range from MDMs to CD4 + T cells. The macrophage-tropic viruses were significantly better at infecting cells expressing low levels of CD4 than the T cell-tropic viruses from the same subjects, with the titration of CD4 providing a distinctive and quantitative phenotype. This difference in CD4 utilization was not due to macrophage-tropic viruses being CD4 independent. Furthermore, macrophage-tropic viruses did not differ from paired T cell-tropic viruses in their ability to use low levels of CCR5 ( t paired = −1.39; P = 0.24) or their use of an alternative conformation of CCR5. We also infected MDMs with a panel of viruses and observed that infectivity of each virus differed across four donors and between three preparations from a single donor. We concluded that the evolutionary transition from replication in T cells to that in macrophages involves a phenotypic transition to acquire the ability to infect cells expressing low levels of CD4 and that this phenotype is more reliably measured in Affinofile cells than in macrophages. IMPORTANCE HIV-1 typically infects memory T cells by using CD4 and CCR5 to enter cells. The virus evolves to infect new cell types by changing the coreceptor from CCR5 to CXCR4 to infect naive T cells or adapting to the use of low levels of CD4 to infect macrophages. However, defining the phenotype of macrophage tropism has been difficult due to inherent variability in the use of macrophages generated in culture to support entry of HIV-1. We describe the use of Affinofile cells with inducible and variable levels of CD4 to identify a signature phenotype for macrophage-tropic HIV-1. The ability to define HIV-1 variants that have evolved an entry phenotype that allows more efficient entry into cells with low levels of CD4 sets the stage for a clearer placement of these variants in HIV-associated pathogenesis.

The discovery of potent and broadly neutralizing antibodies (bNAbs) against human immunodeficiency virus (HIV) has made passive immunization a potential strategy for the prevention and treatment of HIV infection. We sought to determine whether passive administration of VRC01, a bNAb targeting the HIV CD4-binding site, can safely prevent or delay plasma viral rebound after the discontinuation of antiretroviral therapy (ART).We conducted two open-label trials (AIDS Clinical Trials Group [ACTG] A5340 and National Institutes of Health [NIH] 15-I-0140) of the safety, side-effect profile, pharmacokinetic properties, and antiviral activity of VRC01 in persons with HIV infection who were undergoing interruption of ART.A total of 24 participants were enrolled, and one serious alcohol-related adverse event occurred. Viral rebound occurred despite plasma VRC01 concentrations greater than 50 μg per milliliter. The median time to rebound was 4 weeks in the A5340 trial and 5.6 weeks in the NIH trial. Study participants were more likely than historical controls to have viral suppression at week 4 (38% vs. 13%, P=0.04 by a two-sided Fisher's exact test in the A5340 trial; and 80% vs. 13%, P0.001 by a two-sided Fisher's exact test in the NIH trial) but the difference was not significant at week 8. Analyses of virus populations before ART as well as before and after ART interruption showed that VRC01 exerted pressure on rebounding virus, resulting in restriction of recrudescent viruses and selection for preexisting and emerging antibody neutralization-resistant virus.VRC01 slightly delayed plasma viral rebound in the trial participants, as compared with historical controls, but it did not maintain viral suppression by week 8. In the small number of participants enrolled in these trials, no safety concerns were identified with passive immunization with a single bNAb (VRC01). (Funded by the National Institute of Allergy and Infectious Diseases and others; ACTG A5340 and NIH 15-I-0140 ClinicalTrials.gov numbers, NCT02463227 and NCT02471326 .).

CD4 + T cells rather than macrophages are the principal cells infected by human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) in vivo . Macrophage tropism has been linked to the ability to enter cells through CCR5 in conjunction with limiting CD4 levels, which are much lower on macrophages than on T cells. We recently reported that rhesus macaques (RM) experimentally depleted of CD4 + T cells before SIV infection exhibit extensive macrophage infection as well as high chronic viral loads and rapid progression to AIDS. Here we show that early-time-point and control Envs were strictly CD4 dependent but that, by day 42 postinfection, plasma virus of CD4 + T cell-depleted RM was dominated by Envs that mediate efficient infection using RM CCR5 independently of CD4. Early-time-point and control RM Envs were resistant to neutralization by SIV-positive (SIV + ) plasma but became sensitive if preincubated with sCD4. In contrast, CD4-independent Envs were highly sensitive to SIV + plasma neutralization. However, plasma from SIV-infected CD4 + T cell-depleted animals lacked this CD4-inducible neutralizing activity and failed to neutralize any Envs regardless of sCD4 pre-exposure status. Enhanced sensitivity of CD4-independent Envs from day 42 CD4 + T cell-depleted RM was also seen with monoclonal antibodies that target both known CD4-inducible and other Env epitopes. CD4 independence and neutralization sensitivity were both conferred by Env amino acid changes E84K and D470N that arose independently in multiple animals, with the latter introducing a potential N-linked glycosylation site within a predicted CD4-binding pocket of gp120. Thus, the absence of CD4 T cells results in failure to produce antibodies that neutralize CD4-independent Envs and CD4-pretriggered control Envs. In the absence of this constraint and with a relative paucity of CD4 + target cells, widespread macrophage infection occurs in vivo accompanied by emergence of variants carrying structural changes that enable entry independently of CD4.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans., Author Summary HIV-1 infection persists and requires life-long therapy. Approaches to prevent viral replication in the absence of treatment will likely require effective antiviral immune responses, but this goal has been confounded by HIV-1’s ability to target CD4 T cells that coordinate adaptive immunity. We describe a novel approach to confer HIV-resistance to CD4 T cells using peptides from the HIV-1 gp41 heptad repeat-2 (HR2) domain to inhibit infection. By linking a 34 amino acid HR2 peptide to the amino terminus of CCR5 or CXCR4 we were able to use the physiologic trafficking of these coreceptors to deliver the inhibitory peptide to sites of viral fusion where they exerted potent, specific and broad resistance irrespective of viral clade or tropism. This effect was highly dependent on the positioning of the peptide and most effective when conjugated to CXCR4. In vitro and in humanized mice, primary CD4 T cells were protected and expanded following HIV-1 infection. This work represents a proof of concept that T cells can be genetically engineered to resist infection in vivo and provides a rationale to explore this approach as a novel cell-based therapeutic in strategies to augment antiviral immune responses that target viral reservoirs and for long-term control of HIV-1.

CD4 tropism is conserved among all primate lentiviruses and likely contributes to viral pathogenesis by targeting cells that are critical for adaptive antiviral immune responses. Although CD4-independent variants of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) have been described that can utilize the coreceptor CCR5 or CXCR4 in the absence of CD4, these viruses typically retain their CD4 binding sites and still can interact with CD4. We describe the derivation of a novel CD4-independent variant of pathogenic SIVmac239, termed iMac239, that was used to derive an infectious R5-tropic SIV lacking a CD4 binding site. Of the seven mutations that differentiate iMac239 from wild-type SIVmac239, a single change (D178G) in the V1/V2 region was sufficient to confer CD4 independence in cell-cell fusion assays, although other mutations were required for replication competence. Like other CD4-independent viruses, iMac239 was highly neutralization sensitive, although mutations were identified that could confer CD4-independent infection without increasing its neutralization sensitivity. Strikingly, iMac239 retained the ability to replicate in cell lines and primary cells even when its CD4 binding site had been ablated by deletion of a highly conserved aspartic acid at position 385, which, for HIV-1, plays a critical role in CD4 binding. iMac239, with and without the D385 deletion, exhibited an expanded host range in primary rhesus peripheral blood mononuclear cells that included CCR5 + CD8 + T cells. As the first non-CD4-tropic SIV, iMac239-ΔD385 will afford the opportunity to directly assess the in vivo role of CD4 targeting on pathogenesis and host immune responses. IMPORTANCE CD4 tropism is an invariant feature of primate lentiviruses and likely plays a key role in pathogenesis by focusing viral infection onto cells that mediate adaptive immune responses and in protecting virions attached to cells from neutralizing antibodies. Although CD4-independent viruses are well described for HIV and SIV, these viruses characteristically retain their CD4 binding site and can engage CD4 if available. We derived a novel CD4-independent, CCR5-tropic variant of the pathogenic molecular clone SIVmac239, termed iMac239. The genetic determinants of iMac239's CD4 independence provide new insights into mechanisms that underlie this phenotype. This virus remained replication competent even after its CD4 binding site had been ablated by mutagenesis. As the first truly non-CD4-tropic SIV, lacking the capacity to interact with CD4, iMac239 will provide the unique opportunity to evaluate SIV pathogenesis and host immune responses in the absence of the immunomodulatory effects of CD4 + T cell targeting and infection.

Background CD4 T cell depletion during HIV-1 infection is associated with AIDS disease progression, and the HIV-1 Env protein plays an important role in the process. Together with CXCR4, CCR5 is one of the two co-receptors that interact with Env during virus entry, but the role of CCR5 in Env-induced pathogenesis is not clearly defined. We have investigated CD4 T cell depletion mechanisms caused by the Env of a highly pathogenic CXCR4/CCR5 dual-tropic HIV-1 isolate R3A. Results We report here that R3A infection induced depletion of both infected and uninfected “bystander” CD4 T cells, and treatment with CCR5 antagonist TAK-779 inhibited R3A-induced bystander CD4 T cell depletion without affecting virus replication. To further define the role of Env-CCR5 interaction, we utilized an Env-mutant of R3A, termed R3A-5/6AA, which has lost CCR5 binding capability. Importantly, R3A-5/6AA replicated to the same level as wild type R3A by using CXCR4 for viral infection. We found the loss of CCR5 interaction resulted in a significant reduction of bystander CD4 T cells death during R3A-5/6AA infection, whereas stimulation of CCR5 with MIP1-β increased bystander pathogenesis induced by R3A-5/6AA. We confirmed our findings using a humanized mouse model, where we observed similarly reduced pathogenicity of the mutant R3A-5/6AA in various lymphoid organs in vivo. Conclusion We provide the first evidence that shows CCR5 interaction with a dual-tropic HIV-1 Env played a significant role in Env-induced depletion of CD4 T cells. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0255-z) contains supplementary material, which is available to authorized users.

Tyrosine sulfate-mediated interactions play an important role in HIV-1 entry. After engaging the CD4 receptor at the cell surface, the HIV-1 gp120 glycoprotein binds to the CCR5 co-receptor via an interaction that requires two tyrosine sulfates, at positions 10 and 14 in the CCR5-N terminus. Building on previous structure determinations of this interaction, here we report the targeting of these tyrosine sulfate binding sites for drug design through in silico screening of small molecule libraries, identification of lead compounds, and characterization of biological activity. A class of tyrosine sulfate-mimicking small molecules containing a "phenyl sulfonate-linker-aromatic" motif was identified that specifically inhibited binding of gp120 to the CCR5-N terminus as well as to sulfated antibodies that recognize the co-receptor binding region on gp120. The most potent of these compounds bound gp120 with low micromolar affinity and its CD4-induced conformation with K(D)'s as tight as ∼50 nM. Neutralization experiments suggested the targeted site to be conformationally inaccessible prior to CD4 engagement. Primary HIV-1 isolates were weakly neutralized, preincubation with soluble CD4 enhanced neutralization, and engineered isolates with increased dependence on the N terminus of CCR5 or with reduced conformational barriers were neutralized with IC(50) values as low as ∼1 μM. These results reveal the potential of targeting the tyrosine sulfate interactions of HIV-1 and provide insight into how mechanistic barriers, evolved by HIV-1 to evade antibody recognition, also restrict small-molecule-mediated neutralization.

CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.

During the course of infection, transmitted HIV-1 isolates that initially use CCR5 can acquire the ability to use CXCR4, which is associated with an accelerated progression to AIDS. Although this coreceptor switch is often associated with mutations in the stem of the viral envelope (Env) V3 loop, domains outside V3 can also play a role, and the underlying mechanisms and structural basis for how X4 tropism is acquired remain unknown. In this study we used a V3 truncated R5-tropic Env as a starting point to derive two X4-tropic Envs, termed ΔV3-X4A.c5 and ΔV3-X4B.c7, which took distinct molecular pathways for this change. The ΔV3-X4A.c5 Env clone acquired a 7-amino-acid insertion in V3 that included three positively charged residues, reestablishing an interaction with the CXCR4 extracellular loops (ECLs) and rendering it highly susceptible to the CXCR4 antagonist AMD3100. In contrast, the ΔV3-X4B.c7 Env maintained the V3 truncation but acquired mutations outside V3 that were critical for X4 tropism. In contrast to ΔV3-X4A.c5, ΔV3-X4B.c7 showed increased dependence on the CXCR4 N terminus (NT) and was completely resistant to AMD3100. These results indicate that HIV-1 X4 coreceptor switching can involve (i) V3 loop mutations that establish interactions with the CXCR4 ECLs, and/or (ii) mutations outside V3 that enhance interactions with the CXCR4 NT. The cooperative contributions of CXCR4 NT and ECL interactions with gp120 in acquiring X4 tropism likely impart flexibility on pathways for viral evolution and suggest novel approaches to isolate these interactions for drug discovery.

Developing an HIV-1 vaccine that can elicit antibodies to prevent infection has been a formidable challenge. Although no single immunogen has generated antibodies that can neutralize diverse isolates, progress has been made in understanding (a) the structure of the HIV-1 envelope glycoprotein, which is targeted by neutralizing antibodies, (b) how HIV-1 evades antibodies made by an infected host, and (c) how rare monoclonal antibodies can exhibit broadly neutralizing activity. Advances in structural and molecular biology coupled with new approaches to isolate neutralizing antibodies from HIV-1-infected individuals are enhancing our understanding of what humoral immune responses will be required for a vaccine. This review summarizes progress in understanding the host antibody response to HIV-1 and current strategies for applying this information to develop an effective vaccine.

The HIV-1 envelope glycoprotein (Env) functional spike has evolved multiple immune evasion strategies, and only a few broadly neutralizing determinants on the assembled spike are accessible to Abs. Serological studies, based upon Ab binding and neutralization activity in vitro, suggest that vaccination with current Env-based immunogens predominantly elicits Abs that bind nonneutralizing or strain-restricted neutralizing epitopes. However, the fractional specificities of the polyclonal mixture of Abs present in serum, especially those directed to conformational Env epitopes, are often difficult to determine. Furthermore, serological analyses do not provide information regarding how repeated Ag inoculation impacts the expansion and maintenance of Env-specific B cell subpopulations. Therefore, we developed a highly sensitive Env-specific B cell ELISPOT system, which allows the enumeration of Ab-secreting cells (ASC) from diverse anatomical compartments directed against different structural determinants of Env. In this study, we use this system to examine the evolution of B cell responses in mice immunized with engineered Env trimers in adjuvant. We demonstrate that the relative proportion of ASC specific for defined structural elements of Env is altered significantly by homologous booster immunizations. This results in the selective expansion of ASC directed against the variable regions of Env. We suggest that the B cell specificity and compartment analysis described in this study are important complements to serological mapping studies for the examination of B cell responses against subspecificities of a variety of immunogens.

Deciphering antibody specificities that constrain human immunodeficiency virus type 1 (HIV-1) envelope (Env) diversity, limit virus replication, and contribute to neutralization breadth and potency is an important goal of current HIV/AIDS vaccine research. Transplantation of discrete HIV-1 neutralizing epitopes into HIV-2 scaffolds may provide a sensitive, biologically functional context by which to quantify specific antibody reactivities even in complex sera. Here, we describe a novel HIV-2 proviral scaffold (pHIV-2KR.X7) into which we substituted the complete variable region 3 (V3) of theenvgene of HIV-1YU2or HIV-1Cconto yield the chimeric proviruses pHIV-2KR.X7YU2 V3 and pHIV-2KR.X7Ccon V3. These HIV-2/HIV-1 chimeras were replication competent and sensitive to selective pharmacological inhibitors of virus entry. V3 chimeric viruses were resistant to neutralization by HIV-1 monoclonal antibodies directed against the CD4 binding site, coreceptor binding site, and gp41 membrane proximal external region but exhibited striking sensitivity to HIV-1 V3-specific monoclonal antibodies, 447-52D and F425 B4e8 (50% inhibitory concentration of [IC50] 50measurements ranging from 1:50 to greater than 1:40,000. Neutralization titers of B clade plasmas were as much as 1,000-fold lower when tested against the primary HIV-1YU2virus than with the HIV-2KR.X7YU2 V3 chimera, demonstrating highly effective shielding of V3 epitopes in the native Env trimer. This finding was replicated using a second primary HIV-1 strain (HIV-1BORI) and the corresponding HIV-2KR.X7BORI V3 chimera. We conclude that V3 is highly immunogenic in vivo, eliciting antibodies with substantial breadth of reactivity and neutralizing potential. These antibodies constrain HIV-1 Env to a structure(s) in which V3 epitopes are concealed prior to CD4 engagement but do not otherwise contribute to neutralization breadth and potency against most primary virus strains. Triggering of the viral spike to reveal V3 epitopes may be required if V3 immunogens are to be components of an effective HIV-1 vaccine.

During human immunodeficiency virus type 1 (HIV-1) infection, patients develop various levels of neutralizing antibody (NAb) responses. In some cases, patient sera can potently neutralize diverse strains of HIV-1, but the antibody specificities that mediate this broad neutralization are not known, and their elucidation remains a formidable challenge. Due to variable and nonneutralizing determinants on the exterior envelope glycoprotein (Env), nonnative Env protein released from cells, and the glycan shielding that assembles in the context of the quaternary structure of the functional spike, HIV-1 Env elicits a myriad of binding antibodies. However, few of these antibodies can neutralize circulating viruses. We present a systematic analysis of the NAb specificities of a panel of HIV-1-positive sera, using methodologies that identify both conformational and continuous neutralization determinants on the HIV-1 Env protein. Characterization of sera included selective adsorption with native gp120 and specific point mutant variants, chimeric virus analysis, and peptide inhibition of viral neutralization. The gp120 protein was the major neutralizing determinant for most sera, although not all neutralization activity against all viruses could be identified. In some broadly neutralizing sera, the gp120-directed neutralization mapped to the CD4 binding region of gp120. In addition, we found evidence that regions of the gp120 coreceptor binding site may also be a target of neutralizing activity. Sera displaying limited neutralization breadth were mapped to the immunogenic V3 region of gp120. In a subset of sera, we also identified NAbs directed against the conserved, membrane-proximal external region of gp41. These data allow a more detailed understanding of the humoral responses to the HIV-1 Env protein and provide insights regarding the most relevant targets for HIV-1 vaccine design.

Insights into the process of HIV-1 neutralization may assist rational vaccine design. Here, we compared antibody neutralization against the JR-FL primary isolate and trimer binding affinities judged by native PAGE. Monovalent Fab-trimer binding and neutralization showed a direct quantitative relationship, implying that neutralization begins as each trimer is occupied by one antibody. At saturation, three Fab or soluble CD4 molecules engaged each trimer. In contrast, a maximum of one soluble CD4 molecule bound to functional SIV trimers with a truncated a gp41 tail. Remarkably, soluble CD4 was found to trigger dramatic enhancement of this virus. Unlike Fabs, a quantitative correlation between JR-FL trimer binding and neutralization was unclear for some, but not all IgGs, as neutralization was markedly increased, but trimer affinity was largely unchanged. In addition, only one molecule of certain gp41-specific IgGs appeared to be able to bind each trimer. We discuss the implications of these findings in weighing the relative contributions of size, multivalent binding and other possible effects of IgGs to explain their increased potency.

The human immunodeficiency virus type 1 (HIV-1) V3 loop is critical for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. The recent crystallographic resolution of V3 shows that its base is closely associated with the conserved coreceptor binding site on the gp120 core, whereas more distal regions protrude toward the cell surface, likely mediating interactions with coreceptor extracellular loops. However, these V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity are poorly understood. Using the dual-tropic virus HIV-1 R3A , which uses both CCR5 and CXCR4, we sought to identify subdomains within V3 that selectively mediate R5 or X4 tropism. An extensive panel of V3 mutants was evaluated for effects on tropism and sensitivity to coreceptor antagonists. Mutations on either side of the V3 base (residues 3 to 8 and 26 to 33) ablated R5 tropism and made the resulting X4-tropic Envs more sensitive to the CXCR4 inhibitor AMD3100. When mutations were introduced within the V3 stem, only a deletion of residues 9 to 12 on the N-terminal side ablated X4 tropism. Remarkably, this R5-tropic Δ9-12 mutant was completely resistant to several small-molecule inhibitors of CCR5. Envs with mutations in the V3 crown (residues 13 to 20) remained dual tropic. Similar observations were made for a second dual-tropic isolate, HIV-1 89.6 . These findings suggest that V3 subdomains can be identified that differentially affect R5 and X4 tropism and modulate sensitivity to CCR5 and CXCR4 inhibitors. These studies provide a novel approach for probing V3-coreceptor interactions and mechanisms by which these interactions can be inhibited.

Deletion of Gly-720 and Tyr-721 from a highly conserved GYxxØ trafficking signal in the SIVmac239 envelope glycoprotein cytoplasmic domain, producing a virus termed ΔGY, leads to a striking perturbation in pathogenesis in rhesus macaques ( Macaca mulatta ). Infected macaques develop immune activation and progress to AIDS, but with only limited and transient infection of intestinal CD4 + T cells and an absence of microbial translocation. Here we evaluated ΔGY in pig-tailed macaques ( Macaca nemestrina ), a species in which SIVmac239 infection typically leads to increased immune activation and more rapid progression to AIDS than in rhesus macaques. In pig-tailed macaques, ΔGY also replicated acutely to high peak plasma RNA levels identical to those for SIVmac239 and caused only transient infection of CD4 + T cells in the gut lamina propria and no microbial translocation. However, in marked contrast to rhesus macaques, 19 of 21 pig-tailed macaques controlled ΔGY replication with plasma viral loads of + T cells were preserved in blood and gut for up to 100 weeks with no immune activation or disease progression. Robust antiviral CD4 + T cell responses were seen, particularly in the gut. Anti-CD8 antibody depletion demonstrated CD8 + cellular control of viral replication. Two pig-tailed macaques progressed to disease with persisting viremia and possible compensatory mutations in the cytoplasmic tail. These studies demonstrate a marked perturbation in pathogenesis caused by ΔGY's ablation of the GYxxØ trafficking motif and reveal, paradoxically, that viral control is enhanced in a macaque species typically predisposed to more pathogenic manifestations of simian immunodeficiency virus (SIV) infection. IMPORTANCE The pathogenesis of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) reflects a balance between viral replication, host innate and adaptive antiviral immune responses, and sustained immune activation that in humans and Asian macaques is associated with persistent viremia, immune escape, and AIDS. Among nonhuman primates, pig-tailed macaques following SIV infection are predisposed to more rapid disease progression than are rhesus macaques. Here, we show that disruption of a conserved tyrosine-based cellular trafficking motif in the viral transmembrane envelope glycoprotein cytoplasmic tail leads in pig-tailed macaques to a unique phenotype in which high levels of acute viral replication are followed by elite control, robust cellular responses in mucosal tissues, and no disease. Paradoxically, control of this virus in rhesus macaques is only partial, and progression to AIDS occurs. This novel model should provide a powerful tool to help identify host-specific determinants for viral control with potential relevance for vaccine development.

Platelets can engulf human immunodeficiency virus type 1 (HIV-1), and a significant amount of HIV-1 in the blood of infected individuals is associated with these cells. However, it is unclear how platelets capture HIV-1 and whether platelet-associated virus remains infectious. DC-SIGN and other lectins contribute to capture of HIV-1 by dendritic cells (DCs) and facilitate HIV-1 spread in DC/T-cell cocultures. Here, we show that platelets express both the C-type lectin-like receptor 2 (CLEC-2) and low levels of DC-SIGN. CLEC-2 bound to HIV-1, irrespective of the presence of the viral envelope protein, and facilitated HIV-1 capture by platelets. However, a substantial fraction of the HIV-1 binding activity of platelets was dependent on DC-SIGN. A combination of DC-SIGN and CLEC-2 inhibitors strongly reduced HIV-1 association with platelets, indicating that these lectins are required for efficient HIV-1 binding to platelets. Captured HIV-1 was maintained in an infectious state over several days, suggesting that HIV-1 can escape degradation by platelets and might use these cells to promote its spread. Our results identify CLEC-2 as a novel HIV-1 attachment factor and provide evidence that platelets capture and transfer infectious HIV-1 via DC-SIGN and CLEC-2, thereby possibly facilitating HIV-1 dissemination in infected patients.

Many viruses enter cells via an interaction of the viral envelope glycoprotein (Env) with receptor inducing fusion of viral and cellular membranes. These interactions are often evaluated in cell-cell fusion, gene-reporting systems with effector cells expressing Env and target cells expressing receptors. A common system utilizes vaccinia virus encoding T7 RNA polymerase (RNAP) in effector cells and a T7 promoted reporter plasmid in target cells. Fusion is quantified with expression of the reporter plasmid. However, direct activation of reporter plasmid from vaccinia virus can occur increasing background activity. We report here a modification of this assay in which T7 RNAP is expressed from a plasmid rather than vaccinia. This modification increased sensitivity with a ten-fold reduction in background. A novel dual T7/SP6 RNAP fusion assay was also developed to allow rapid screening for functional Env clones. Using these assays, we show that Envs from two CD4-independent HIV-2 isolates (VCP and ROD/B), which are able to fuse with chemokine receptor CXCR4 in a CD4-independent manner, are also able to fuse with alternative coreceptors GPR1 and GPR15 in the absence of CD4. The assay could also detect fusion of murine leukemia virus on target cells expressing the ecotropic MCAT-1 receptor showing its broad utility in other viral systems.

Immunogenic, broadly reactive epitopes of the HIV-1 envelope glycoprotein could serve as important targets of the adaptive humoral immune response in natural infection and, potentially, as components of an acquired immune deficiency syndrome vaccine. However, variability in exposed epitopes and a combination of highly effective envelope-cloaking strategies have made the identification of such epitopes problematic. Here, we show that the chemokine coreceptor binding site of HIV-1 from clade A, B, C, D, F, G, and H and circulating recombinant form (CRF)01, CRF02, and CRF11, elicits high titers of CD4-induced (CD4i) antibody during natural human infection and that these antibodies bind and neutralize viruses as divergent as HIV-2 in the presence of soluble CD4 (sCD4). 178 out of 189 (94%) HIV-1–infected patients had CD4i antibodies that neutralized sCD4-pretreated HIV-2 in titers (50% inhibitory concentration) as high as 1:143,000. CD4i monoclonal antibodies elicited by HIV-1 infection also neutralized HIV-2 pretreated with sCD4, and polyclonal antibodies from HIV-1–infected humans competed specifically with such monoclonal antibodies for binding. In vivo, variants of HIV-1 with spontaneously exposed coreceptor binding surfaces were detected in human plasma; these viruses were neutralized directly by CD4i antibodies. Despite remarkable evolutionary diversity among primate lentiviruses, functional constraints on receptor binding create opportunities for broad humoral immune recognition, which in turn serves to constrain the viral quasispecies.

The C-type lectins DC-SIGN and DC-SIGNR [collectively referred to as DC-SIGN(R)] bind and transmit human immunodeficiency virus (HIV) and simian immunodeficiency virus to T cells via the viral envelope glycoprotein (Env). Other viruses containing heavily glycosylated glycoproteins (GPs) fail to interact with DC-SIGN(R), suggesting some degree of specificity in this interaction. We show here that DC-SIGN(R) selectively interact with HIV Env and Ebola virus GPs containing more high-mannose than complex carbohydrate structures. Modulation of N -glycans on Env or GP through production of viruses in different primary cells or in the presence of the mannosidase I inhibitor deoxymannojirimycin dramatically affected DC-SIGN(R) infectivity enhancement. Further, murine leukemia virus, which typically does not interact efficiently with DC-SIGN(R), could do so when produced in the presence of deoxymannojirimycin. We predict that other viruses containing GPs with a large proportion of high-mannose N -glycans will efficiently interact with DC-SIGN(R), whereas those with solely complex N -glycans will not. Thus, the virus-producing cell type is an important factor in dictating both N -glycan status and virus interactions with DC-SIGN(R), which may impact virus tropism and transmissibility in vivo.

The human and simian immunodeficiency viruses (HIV and SIV) envelope glycoprotein consists of a trimer of two noncovalently and weakly associated subunits, gp120 and gp41. Upon binding of gp120 to cellular receptors, this labile native envelope complex undergoes conformational changes, resulting in a stable trimer-of-hairpins structure in gp41. Formation of the hairpin structure is thought to mediate membrane fusion by placing the viral and cellular membranes in close proximity. An in vitro-derived variant of SIVmac251, denoted CPmac, has acquired an unusually stable virion-associated gp120-gp41 complex. This unique phenotype is conferred by five amino acid substitutions in the gp41 ectodomain. Here we characterize the structural and physicochemical properties of the N40(L6)C38 model of the CPmac gp41 core. The 1.7-A resolution crystal structure of N40(L6)C38 is very similar to the six-helix bundle structure present in the parent SIVmac251 gp41. In both structures, three N40 peptides form a central three-stranded coiled coil, and three C38 peptides pack in an antiparallel orientation into hydrophobic grooves on the coiled-coil surface. Thermal unfolding studies show that the CPmac mutations destabilize the SIVmac251 six-helix bundle by 15 kJ/mol. Our results suggest that the formation of the gp41 trimer-of-hairpins structure is thermodynamically coupled to the conformational stability of the native envelope glycoprotein and raise the intriguing possibility that introduction of mutations to destabilize the six-helix bundle may lead to the stabilization of the trimeric gp120-gp41 complex. This study suggests a potential strategy for the production of stably folded envelope protein immunogens for HIV vaccine development.

We have described a CD4-independent variant of HXBc2, termed 8x, that binds directly to CXCR4 and mediates CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4-C4 domains together with a single nucleotide deletion in the transmembrane domain which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of 27 amino acids. We demonstrate here that while introduction of the 8x FS mutation into heterologous R5, X4, or R5X4 Env proteins did not impart CD4 independence, it did affect the conformation of the gp120 surface subunit, exposing highly conserved domains involved in both coreceptor and CD4 binding. In addition, antigenic changes in the gp41 ectodomain were also observed, consistent with the idea that the effects of cytoplasmic domain truncation must in some way be transmitted to the external gp120 subunit. Truncation of gp41 also resulted in the marked neutralization sensitivity of all Env proteins tested to human immunodeficiency virus-positive human sera and monoclonal antibodies directed against the CD4 or coreceptor-binding sites. These results demonstrate a structural interdependence between the cytoplasmic domain of gp41 and the ectodomain of the Env protein. They also may help explain why the length of the gp41 cytoplasmic domain is retained in vivo and may provide a way to genetically trigger the exposure of neutralization determinants in heterologous Env proteins that may prove useful for vaccine development.

Evidence for HIV-1 infection of trophoblasts is discordant. Utilizing highly purified full-term trophoblasts, we demonstrate that full-term trophoblasts express CXCR4 but are negative for CCR5 and ...

Chimeric simian immunodeficiency virus (SIV)/human immunodeficiency virus (HIV) (SHIV) infection of macaques is commonly used to model HIV type 1 (HIV-1) transmission and pathogenesis in humans. Despite the fact that SHIVs encode SIV antagonists of the known macaque host restriction factors, these viruses require additional adaptation for replication in macaques to establish a persistent infection. Additional adaptation may be required in part because macaque CD4 (mCD4) is a suboptimal receptor for most HIV-1 envelope glycoprotein (Env) variants. This requirement raises the possibility that adaptation of HIV-1 Env to the macaque host leads to selection of variants that lack important biological and antigenic properties of the viruses responsible for the HIV-1 pandemic in humans. Here, we investigated whether this adaptation process leads to changes in the antigenicity and structure of HIV-1 Env. For this purpose, we examined how two independent mutations that enhance mCD4-mediated entry, A204E and G312V, impact antibody recognition in the context of seven different parental HIV-1 Env proteins from diverse subtypes. We also examined HIV-1 Env variants from three SHIVs that had been adapted for increased replication in macaques. Our results indicate that these different macaque-adapted variants had features in common, including resistance to antibodies directed to quaternary epitopes and sensitivity to antibodies directed to epitopes in the variable domains (V2 and V3) that are buried in the parental, unadapted Env proteins. Collectively, these findings suggest that adaptation to mCD4 results in conformational changes that expose epitopes in the variable domains and disrupt quaternary epitopes in the native Env trimer. IMPORTANCE These findings indicate the antigenic consequences of adapting HIV-1 Env to mCD4. They also suggest that to best mimic HIV-1 infection in humans when using the SHIV/macaque model, HIV-1 Env proteins should be identified that use mCD4 as a functional receptor and preserve quaternary epitopes characteristic of HIV-1 Env.

HIV-1 infection depends on effective viral entry mediated by the interaction of its envelope (Env) glycoprotein with specific cell surface receptors. Protective antiviral antibodies generated by passive or active immunization must prevent these interactions. Because the HIV-1 Env is highly variable, attention has also focused on blocking the HIV-1 primary cell receptor CD4. We therefore analyzed the in vivo protective efficacy of three potent neutralizing monoclonal antibodies (mAbs) to HIV-1 Env compared to an antibody against the CD4 receptor. Protection was assessed after mucosal challenge of rhesus macaques with simian/HIV (SHIV). Despite its comparable or greater neutralization potency in vitro, the anti-CD4 antibody did not provide effective protection in vivo, whereas the HIV-1–specific mAbs VRC01, 10E8, and PG9, targeting the CD4 binding site, membrane-proximal, and V1V2 glycan Env regions, respectively, conferred complete protection, albeit at different relative potencies. These findings demonstrate the protective efficacy of broadly neutralizing antibodies directed to the HIV-1 Env and suggest that targeting the HIV-1 Env is preferable to the cell surface receptor CD4 for the prevention of HIV-1 transmission.

The detection of viral dynamics and localization in the context of controlled HIV infection remains a challenge and is limited to blood and biopsies. We developed a method to capture total-body simian immunodeficiency virus (SIV) replication using immunoPET (antibody-targeted positron emission tomography). The administration of a poly(ethylene glycol)-modified, (64)Cu-labeled SIV Gp120-specific antibody led to readily detectable signals in the gastrointestinal and respiratory tract, lymphoid tissues and reproductive organs of viremic monkeys. Viral signals were reduced in aviremic antiretroviral-treated monkeys but detectable in colon, select lymph nodes, small bowel, nasal turbinates, the genital tract and lung. In elite controllers, virus was detected primarily in foci in the small bowel, select lymphoid areas and the male reproductive tract, as confirmed by quantitative reverse-transcription PCR (qRT-PCR) and immunohistochemistry. This real-time, in vivo viral imaging method has broad applications to the study of immunodeficiency virus pathogenesis, drug and vaccine development, and the potential for clinical translation.