The Impact of SIV-Induced Immunodeficiency on Clinical Manifestation, Immune Response, and Viral Dynamics in SARS-CoV-2 Coinfection.

Persistent and uncontrolled SARS-CoV-2 replication in immunocompromised individuals has been observed and may be a contributing source of novel viral variants that continue to drive the pandemic. Importantly, the effects of immunodeficiency associated with chronic HIV infection on COVID-19 disease and viral persistence have not been directly addressed in a controlled setting. Here we conducted a pilot study wherein two pigtail macaques (PTM) chronically infected with SIVmac239 were exposed to SARS-CoV-2 and monitored for six weeks for clinical disease, viral replication, and viral evolution, and compared to our previously published cohort of SIV-naïve PTM infected with SARS-CoV-2. At the time of SARS-CoV-2 infection, one PTM had minimal to no detectable CD4+ T cells in gut, blood, or bronchoalveolar lavage (BAL), while the other PTM harbored a small population of CD4+ T cells in all compartments. Clinical signs were not observed in either PTM; however, the more immunocompromised PTM exhibited a progressive increase in pulmonary infiltrating monocytes throughout SARS-CoV-2 infection. Single-cell RNA sequencing (scRNAseq) of the infiltrating monocytes revealed a less activated/inert phenotype. Neither SIV-infected PTM mounted detectable anti-SARS-CoV-2 T cell responses in blood or BAL, nor anti-SARS-CoV-2 neutralizing antibodies. Interestingly, despite the diminished cellular and humoral immune responses, SARS-CoV-2 viral kinetics and evolution were indistinguishable from SIV-naïve PTM in all sampled mucosal sites (nasal, oral, and rectal), with clearance of virus by 3-4 weeks post infection. SIV-induced immunodeficiency significantly impacted immune responses to SARS-CoV-2 but did not alter disease progression, viral kinetics or evolution in the PTM model. SIV-induced immunodeficiency alone may not be sufficient to drive the emergence of novel viral variants.

HIV-1-Infected CD4+ T Cells Present MHC Class II-Restricted Epitope via Endogenous Processing.

HIV-1-specific CD4+ T cells (TCD4+s) play a critical role in controlling HIV-1 infection. Canonically, TCD4+s are activated by peptides derived from extracellular ("exogenous") Ags displayed in complex with MHC class II (MHC II) molecules on the surfaces of "professional" APCs such as dendritic cells (DCs). In contrast, activated human TCD4+s, which express MHC II, are not typically considered for their APC potential because of their low endocytic capacity and the exogenous Ag systems historically used for assessment. Using primary TCD4+s and monocyte-derived DCs from healthy donors, we show that activated human TCD4+s are highly effective at MHC II-restricted presentation of an immunodominant HIV-1-derived epitope postinfection and subsequent noncanonical processing and presentation of endogenously produced Ag. Our results indicate that, in addition to marshalling HIV-1-specific immune responses during infection, TCD4+s also act as APCs, leading to the activation of HIV-1-specific TCD4+s.

Broad coverage of neutralization-resistant SIV strains by second-generation SIV-specific antibodies targeting the region involved in binding CD4.

Both SIV and SHIV are powerful tools for evaluating antibody-mediated prevention and treatment of HIV-1. However, owing to a lack of rhesus-derived SIV broadly neutralizing antibodies (bnAbs), testing of bnAbs for HIV-1 prevention or treatment has thus far been performed exclusively in the SHIV NHP model using bnAbs from HIV-1-infected individuals. Here we describe the isolation and characterization of multiple rhesus-derived SIV bnAbs capable of neutralizing most isolates of SIV. Eight antibodies belonging to two clonal families, ITS102 and ITS103, which target unique epitopes in the CD4 binding site (CD4bs) region, were found to be broadly neutralizing and together neutralized all SIV strains tested. A rare feature of these bnAbs and two additional antibody families, ITS92 and ITS101, which mediate strain-specific neutralizing activity against SIV from sooty mangabeys (SIVsm), was their ability to achieve near complete (i.e. 100%) neutralization of moderately and highly neutralization-resistant SIV. Overall, these newly identified SIV bnAbs highlight the potential for evaluating HIV-1 prophylactic and therapeutic interventions using fully simian, rhesus-derived bnAbs in the SIV NHP model, thereby circumventing issues related to rapid antibody clearance of human-derived antibodies, Fc mismatch and limited genetic diversity of SHIV compared to SIV.

A cellular trafficking signal in the SIV envelope protein cytoplasmic domain is strongly selected for in pathogenic infection.

The HIV/SIV envelope glycoprotein (Env) cytoplasmic domain contains a highly conserved Tyr-based trafficking signal that mediates both clathrin-dependent endocytosis and polarized sorting. Despite extensive analysis, the role of these functions in viral infection and pathogenesis is unclear. An SIV molecular clone (SIVmac239) in which this signal is inactivated by deletion of Gly-720 and Tyr-721 (SIVmac239ΔGY), replicates acutely to high levels in pigtail macaques (PTM) but is rapidly controlled. However, 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 encoding amino acids 734-736 (ΔQTH) that overlaps 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 that generated a new signal for polarized sorting but not endocytosis. This genotype, together with the ΔGY mutation, was conserved in association with high viral loads for several months when introduced into naïve PTMs. For the first time, our findings reveal strong selection pressure for Env endocytosis and particularly for polarized sorting during pathogenic SIV infection in vivo.

The pigtail macaque (Macaca nemestrina) model of COVID-19 reproduces diverse clinical outcomes and reveals new and complex signatures of disease.

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.

Dual CD4-based CAR T cells with distinct costimulatory domains mitigate HIV pathogenesis 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.

Tetherin downmodulation by SIVmac Nef lost with the H196Q escape variant is restored by an upstream variant.

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.

Elite control of HIV is associated with distinct functional and transcriptional signatures in lymphoid tissue CD8+ T cells.

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.

Mechanisms of reactivation of latent tuberculosis infection due to SIV coinfection.

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.

Extended CCR5 Blockade for Graft-versus-Host Disease Prophylaxis Improves Outcomes of Reduced-Intensity Unrelated Donor Hematopoietic Cell Transplantation: A Phase II Clinical Trial.

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.

Polymorphisms in Rhesus Macaque Tetherin Are Associated with Differences in Acute Viremia in Simian Immunodeficiency Virus Δnef-Infected Animals.

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.

Increased surface expression of HIV-1 envelope is associated with improved antibody response in vaccinia prime/protein boost immunization.

HIV-1 envelope (Env)-based vaccines have so far largely failed to induce antibodies that prevent HIV-1 infection. One factor proposed to limit the immunogenicity of cell-associated Env is its low level of expression on the cell surface, restricting accessibility to antibodies. Using a vaccinia prime/protein boost protocol in mice, we explored the immunologic effects of mutations in the Env cytoplasmic tail (CT) that increased surface expression, including partial truncation and ablation of a tyrosine-dependent endocytosis motif. After vaccinia primes, CT-modified Envs induced up to 7-fold higher gp120-specific IgG, and after gp120 protein boosts, they elicited up to 16-fold greater Tier-1 HIV-1 neutralizing antibody titers, although results were variable between isolates. These data indicate that the immunogenicity of HIV-1 Env in a prime/boost vaccine can be enhanced in a strain-dependent manner by CT mutations that increase Env surface expression, thus highlighting the importance of the prime in this vaccine format.

The SIV Envelope Glycoprotein, Viral Tropism, and Pathogenesis: Novel Insights from Nonhuman Primate Models of AIDS.

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.

Clinical and immunologic impact of CCR5 blockade in graft-versus-host disease prophylaxis.

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.

Effect of HIV Antibody VRC01 on Viral Rebound after Treatment Interruption.

BACKGROUND: 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). METHODS: 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. RESULTS: 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%, P<0.001 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. CONCLUSIONS: 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 .).

Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus.

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.

Derivation and Characterization of a CD4-Independent, Non-CD4-Tropic Simian Immunodeficiency Virus.

UNLABELLED: 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.

CCR5 interaction with HIV-1 Env contributes to Env-induced depletion of CD4 T cells in vitro and in vivo.

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-ß 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.

Elite Control, Gut CD4 T Cell Sparing, and Enhanced Mucosal T Cell Responses in Macaca nemestrina Infected by a Simian Immunodeficiency Virus Lacking a gp41 Trafficking Motif.

UNLABELLED: 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 <15 to 50 RNA copies/ml. CD4(+) 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.

Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy-treated macaques.

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.