Baricitinib treatment resolves lower-airway macrophage inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques.

SARS-CoV-2-induced hypercytokinemia and inflammation are critically associated with COVID-19 severity. Baricitinib, a clinically approved JAK1/JAK2 inhibitor, is currently being investigated in COVID-19 clinical trials. Here, we investigated the immunologic and virologic efficacy of baricitinib in a rhesus macaque model of SARS-CoV-2 infection. Viral shedding measured from nasal and throat swabs, bronchoalveolar lavages, and tissues was not reduced with baricitinib. Type I interferon (IFN) antiviral responses and SARS-CoV-2-specific T cell responses remained similar between the two groups. Animals treated with baricitinib showed reduced inflammation, decreased lung infiltration of inflammatory cells, reduced NETosis activity, and more limited lung pathology. Importantly, baricitinib-treated animals had a rapid and remarkably potent suppression of lung macrophage production of cytokines and chemokines responsible for inflammation and neutrophil recruitment. These data support a beneficial role for, and elucidate the immunological mechanisms underlying, the use of baricitinib as a frontline treatment for inflammation induced by SARS-CoV-2 infection.

A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs.

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.

Author Correction: Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Robust and persistent reactivation of SIV and HIV by N-803 and depletion of CD8+ cells.

Human immunodeficiency virus (HIV) persists indefinitely in individuals with HIV who receive antiretroviral therapy (ART) owing to a reservoir of latently infected cells that contain replication-competent virus1-4. Here, to better understand the mechanisms responsible for latency persistence and reversal, we used the interleukin-15 superagonist N-803 in conjunction with the depletion of CD8+ lymphocytes in ART-treated macaques infected with simian immunodeficiency virus (SIV). Although N-803 alone did not reactivate virus production, its administration after the depletion of CD8+ lymphocytes in conjunction with ART treatment induced robust and persistent reactivation of the virus in vivo. We found viraemia of more than 60 copies per ml in all macaques (n = 14; 100%) and in 41 out of a total of 56 samples (73.2%) that were collected each week after N-803 administration. Notably, concordant results were obtained in ART-treated HIV-infected humanized mice. In addition, we observed that co-culture with CD8+ T cells blocked the in vitro latency-reversing effect of N-803 on primary human CD4+ T cells that were latently infected with HIV. These results advance our understanding of the mechanisms responsible for latency reversal and lentivirus reactivation during ART-suppressed infection.

Systemic HIV and SIV latency reversal via non-canonical NF-κB signalling in vivo.

Long-lasting, latently infected resting CD4+ T cells are the greatest obstacle to obtaining a cure for HIV infection, as these cells can persist despite decades of treatment with antiretroviral therapy (ART). Estimates indicate that more than 70 years of continuous, fully suppressive ART are needed to eliminate the HIV reservoir1. Alternatively, induction of HIV from its latent state could accelerate the decrease in the reservoir, thus reducing the time to eradication. Previous attempts to reactivate latent HIV in preclinical animal models and in clinical trials have measured HIV induction in the peripheral blood with minimal focus on tissue reservoirs and have had limited effect2-9. Here we show that activation of the non-canonical NF-κB signalling pathway by AZD5582 results in the induction of HIV and SIV RNA expression in the blood and tissues of ART-suppressed bone-marrow-liver-thymus (BLT) humanized mice and rhesus macaques infected with HIV and SIV, respectively. Analysis of resting CD4+ T cells from tissues after AZD5582 treatment revealed increased SIV RNA expression in the lymph nodes of macaques and robust induction of HIV in almost all tissues analysed in humanized mice, including the lymph nodes, thymus, bone marrow, liver and lung. This promising approach to latency reversal-in combination with appropriate tools for systemic clearance of persistent HIV infection-greatly increases opportunities for HIV eradication.

CD8(+) Lymphocytes Are Required for Maintaining Viral Suppression in SIV-Infected Macaques Treated with Short-Term Antiretroviral Therapy.

Infection with HIV persists despite suppressive antiretroviral therapy (ART), and treatment interruption results in rapid viral rebound. Antibody-mediated CD8(+) lymphocyte depletion in simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) shows that these cells contribute to viral control in untreated animals. However, the contribution of CD8(+) lymphocytes to maintaining viral suppression under ART remains unknown. Here, we have shown that in SIV-infected RMs treated with short-term (i.e., 8-32 week) ART, depletion of CD8(+) lymphocytes resulted in increased plasma viremia in all animals and that repopulation of CD8(+) T cells was associated with prompt reestablishment of virus control. Although the number of SIV-DNA-positive cells remained unchanged after CD8 depletion and reconstitution, the frequency of SIV-infected CD4(+) T cells before depletion positively correlated with both the peak and area under the curve of viremia after depletion. These results suggest a role for CD8(+) T cells in controlling viral production during ART, thus providing a rationale for exploring immunotherapeutic approaches in ART-treated HIV-infected individuals.

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

Altered Response Pattern following AZD5582 Treatment of SIV-Infected, ART-Suppressed Rhesus Macaque Infants.

The "shock and kill" strategy for HIV-1 cure incorporates latency-reversing agents (LRA) in combination with interventions that aid the host immune system in clearing virally reactivated cells. LRAs have not yet been investigated in pediatric clinical or preclinical studies. Here, we evaluated an inhibitor of apoptosis protein (IAP) inhibitor (IAPi), AZD5582, that activates the noncanonical NF-κB (ncNF-κB) signaling pathway to reverse latency. Ten weekly doses of AZD5582 were intravenously administered at 0.1 mg/kg to rhesus macaque (RM) infants orally infected with SIVmac251 at 4 weeks of age and treated with a triple ART regimen for over 1 year. During AZD5582 treatment, on-ART viremia above the limit of detection (LOD, 60 copies/mL) was observed in 5/8 infant RMs starting at 3 days post-dose 4 and peaking at 771 copies/mL. Of the 135 measurements during AZD5582 treatment in these 5 RM infants, only 8 were above the LOD (6%), lower than the 46% we have previously reported in adult RMs. Pharmacokinetic analysis of plasma AZD5582 levels revealed a lower Cmax in treated infants compared to adults (294 ng/mL versus 802 ng/mL). RNA-Sequencing of CD4+ T cells comparing pre- and post-AZD5582 dosing showed many genes that were similarly upregulated in infants and adults, but the expression of key ncNF-κB genes, including NFKB2 and RELB, was significantly higher in adult RMs. Our results suggest that dosing modifications for this latency reversal approach may be necessary to maximize virus reactivation in the pediatric setting for successful "shock and kill" strategies. IMPORTANCE While antiretroviral therapy (ART) has improved HIV-1 disease outcome and reduced transmission, interruption of ART results in rapid viral rebound due to the persistent latent reservoir. Interventions to reduce the viral reservoir are of critical importance, especially for children who must adhere to lifelong ART to prevent disease progression. Here, we used our previously established pediatric nonhuman primate model of oral SIV infection to evaluate AZD5582, identified as a potent latency-reversing agent in adult macaques, in the controlled setting of daily ART. We demonstrated the safety of the IAPi AZD5582 and evaluate the pharmacokinetics and pharmacodynamics of repeated dosing. The response to AZD5582 in macaque infants differed from what we previously showed in adult macaques with weaker latency reversal in infants, likely due to altered pharmacokinetics and less inducibility of infant CD4+ T cells. These data supported the contention that HIV-1 cure strategies for children are best evaluated using pediatric model systems.

Transmission and Protection against Reinfection in the Ferret Model with the SARS-CoV-2 USA-WA1/2020 Reference Isolate.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has initiated a global pandemic, and several vaccines have now received emergency use authorization. Using the reference strain SARS-CoV-2 USA-WA1/2020, we evaluated modes of transmission and the ability of prior infection or vaccine-induced immunity to protect against infection in ferrets. Ferrets were semipermissive to infection with the USA-WA1/2020 isolate. When transmission was assessed via the detection of viral RNA (vRNA) at multiple time points, direct contact transmission was efficient to 3/3 and 3/4 contact animals in 2 respective studies, while respiratory droplet transmission was poor to only 1/4 contact animals. To determine if previously infected ferrets were protected against reinfection, ferrets were rechallenged 28 or 56 days postinfection. Following viral challenge, no infectious virus was recovered in nasal wash samples. In addition, levels of vRNA in the nasal wash were several orders of magnitude lower than during primary infection, and vRNA was rapidly cleared. To determine if intramuscular vaccination protected ferrets, ferrets were vaccinated using a prime-boost strategy with the S protein receptor-binding domain formulated with an oil-in-water adjuvant. Upon viral challenge, none of the mock or vaccinated animals were protected against infection, and there were no significant differences in vRNA or infectious virus titers in the nasal wash. Combined, these studies demonstrate direct contact is the predominant mode of transmission of the USA-WA1/2020 isolate in ferrets and that immunity to SARS-CoV-2 is maintained for at least 56 days. Our studies also indicate protection of the upper respiratory tract against SARS-CoV-2 will require vaccine strategies that mimic natural infection or induce site-specific immunity. IMPORTANCE The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) USA-WA1/2020 strain is a CDC reference strain used by multiple research laboratories. Here, we show that the predominant mode of transmission of this isolate in ferrets is by direct contact. We further demonstrate ferrets are protected against reinfection for at least 56 days even when levels of neutralizing antibodies are low or undetectable. Last, we show that when ferrets were vaccinated by the intramuscular route to induce antibodies against SARS-CoV-2, ferrets remain susceptible to infection of the upper respiratory tract. Collectively, these studies suggest that protection of the upper respiratory tract will require vaccine approaches that mimic natural infection.

CD8 lymphocyte depletion enhances the latency reversal activity of the SMAC mimetic AZD5582 in ART-suppressed SIV-infected rhesus macaques.

Inducing latency reversal to reveal infected cells to the host immune system represents a potential strategy to cure HIV infection. In separate studies, we have previously shown that CD8+ T cells may contribute to the maintenance of viral latency and identified a novel SMAC mimetic/IAP inhibitor (AZD5582) capable of reversing HIV/SIV latency in vivo by activating the non-canonical (nc) NF-κB pathway. Here, we use AZD5582 in combination with antibody-mediated depletion of CD8α+ cells to further evaluate the role of CD8+ T cells in viral latency maintenance. Six rhesus macaques (RM) were infected with SIVmac239 and treated with ART starting at week 8 post-infection. After 84-85 weeks of ART, all animals received a single dose of the anti-CD8α depleting antibody (Ab), MT807R1 (50mg/kg, s.c.), followed by 5 weekly doses of AZD5582 (0.1 mg/kg, i.v.). Following CD8α depletion + AZD5582 combined treatment, 100% of RMs experienced on-ART viremia above 60 copies per ml of plasma. In comparator groups of ART-suppressed SIV-infected RMs treated with AZD5582 only or CD8α depletion only, on-ART viremia was experienced by 56% and 57% of the animals respectively. Furthermore, the frequency of increased viremic episodes during the treatment period was greater in the CD8α depletion + AZD5582 group as compared to other groups. Mathematical modeling of virus reactivation suggested that, in addition to viral dynamics during acute infection, CD8α depletion influenced the response to AZD5582. This work suggests that the latency reversal induced by activation of the ncNF-κB signaling pathway with AZD5582 can be enhanced by CD8α+ cell depletion.

Combination of CD8ß Depletion and Interleukin-15 Superagonist N-803 Induces Virus Reactivation in Simian-Human Immunodeficiency Virus-Infected, Long-Term ART-Treated Rhesus Macaques.

The "shock and kill" strategy predicates that virus reactivation in latently infected cells is required to eliminate the human immunodeficiency virus (HIV) reservoir. In a recent study, we showed robust and persistent induction of plasma viremia in antiretroviral therapy (ART)-treated simian immunodeficiency virus-infected rhesus macaques (RMs) undergoing CD8α depletion and treated with the interleukin-15 (IL-15) superagonist N-803 (J. B. McBrien et al., Nature 578:154-159, 2020, https://doi.org/10.1038/s41586-020-1946-0). Of note, in that study we used an antibody targeting CD8α, thereby depleting NK cells, NKT cells, and γδ T cells, in addition to CD8+ T cells. In the current proof-of-concept study, we tested whether virus reactivation can be induced by administration of N-803 to simian-human chimeric immunodeficiency virus-infected, ART-treated RMs that are selectively depleted of CD8+ T cells via the CD8ß-targeting antibody CD8b255R1. CD8ß depletion was performed in five SHIVSF162P3-infected RMs treated with ART for 12 months and with plasma viremia consistently below 3 copies/ml. All animals received four weekly doses of N-803 starting at the time of CD8b255R1 administration. The induction of detectable plasma viremia was observed in three out of five RMs, with the level of virus reactivation seemingly correlated with the frequency of CD8+ T cells following CD8ß depletion as well as the level of virus reactivation observed when the same animals underwent CD8α depletion and N-803 administration after 24 weeks of ART. These data indicate that CD8ß depletion and N-803 administration can induce virus reactivation in SHIVSF162P3-infected RMs despite suboptimal depletion of CD8+ T cells and profound ART-induced suppression of virus replication, confirming a critical role for these cells in suppressing virus production and/or reactivation in vivo under ART.IMPORTANCE The "shock and kill" HIV cure strategy attempts to reverse and eliminate the latent viral infection that prevents eradication of the virus. Latency-reversing agents tested in clinical trials to date have failed to affect the HIV viral reservoir. IL-15 superagonist N-803, currently involved in a clinical trial for HIV cure, was recently shown by our laboratory to induce robust and persistent induction of plasma viremia during ART in three in vivo animal models of HIV infection. These results suggest a substantial role for CD8+ lymphocytes in suppressing the latency reversal effect of N-803 by promoting the maintenance of viral latency. In this study, we tested whether the use of a CD8ß-targeting antibody, which would specifically deplete CD8+ T cells, would yield similar levels of virus reactivation. We observed the induction of plasma viremia, which correlated with the efficacy of the CD8 depletion strategy.

SMAC Mimetic Plus Triple-Combination Bispecific HIVxCD3 Retargeting Molecules in SHIV.C.CH505-Infected, Antiretroviral Therapy-Suppressed Rhesus Macaques.

The "shock-and-kill" human immunodeficiency virus type 1 (HIV-1) cure strategy involves latency reversal followed by immune-mediated clearance of infected cells. We have previously shown that activation of the noncanonical NF-κB pathway using an inhibitor of apoptosis (IAP), AZD5582, reverses HIV/simian immunodeficiency virus (SIV) latency. Here, we combined AZD5582 with bispecific HIVxCD3 DART molecules to determine the impact of this approach on persistence. Rhesus macaques (RMs) (n = 13) were infected with simian/human immunodeficiency virus SHIV.C.CH505.375H.dCT, and triple antiretroviral therapy (ART) was initiated after 16 weeks. After 42 weeks of ART, 8 RMs received a cocktail of 3 HIVxCD3 DART molecules having human A32, 7B2, or PGT145 anti-HIV-1 envelope (Env) specificities paired with a human anti-CD3 specificity that is rhesus cross-reactive. The remaining 5 ART-suppressed RMs served as controls. For 10 weeks, a DART molecule cocktail was administered weekly (each molecule at 1 mg/kg of body weight), followed 2 days later by AZD5582 (0.1 mg/kg). DART molecule serum concentrations were well above those considered adequate for redirected killing activity against Env-expressing target cells but began to decline after 3 to 6 weekly doses, coincident with the development of antidrug antibodies (ADAs) against each of the DART molecules. The combination of AZD5582 and the DART molecule cocktail did not increase on-ART viremia or cell-associated SHIV RNA in CD4+ T cells and did not reduce the viral reservoir size in animals on ART. The lack of latency reversal in the model used in this study may be related to low pre-ART viral loads (median, <105 copies/ml) and low preintervention reservoir sizes (median, <102 SHIV DNA copies/million blood CD4+ T cells). Future studies to assess the efficacy of Env-targeting DART molecules or other clearance agents to reduce viral reservoirs after latency reversal may be more suited to models that better minimize immunogenicity and have a greater viral burden.IMPORTANCE The most significant barrier to an HIV-1 cure is the existence of the latently infected viral reservoir that gives rise to rebound viremia upon cessation of ART. Here, we tested a novel combination approach of latency reversal with AZD5582 and clearance with bispecific HIVxCD3 DART molecules in SHIV.C.CH505-infected, ART-suppressed rhesus macaques. We demonstrate that the DART molecules were not capable of clearing infected cells in vivo, attributed to the lack of quantifiable latency reversal in this model with low levels of persistent SHIV DNA prior to intervention as well as DART molecule immunogenicity.

Simian-Human Immunodeficiency Virus SHIV.C.CH505 Persistence in ART-Suppressed Infant Macaques Is Characterized by Elevated SHIV RNA in the Gut and a High Abundance of Intact SHIV DNA in Naive CD4+ T Cells.

Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) continues to cause new pediatric cases of infection through breastfeeding, a setting where it is not always possible to initiate early antiretroviral therapy (ART). Without novel interventions that do not rely on daily ART, HIV-1-infected children face lifelong medications to control infection. A detailed analysis of virus persistence following breast milk transmission of HIV-1 and ART has not been performed. Here, we used infant rhesus macaques orally infected with simian/human immunodeficiency virus (SHIV) (SHIV.C.CH505) to identify cellular and anatomical sites of virus persistence under ART. Viral DNA was detected at similar levels in blood and tissue CD4+ T cells after a year on ART, with virus in blood and lymphoid organs confirmed to be replication competent. Viral RNA/DNA ratios were elevated in rectal CD4+ T cells compared to those of other sites (P ≤ 0.0001), suggesting that the gastrointestinal tract is an active site of virus transcription during ART-mediated suppression of viremia. SHIV.C.CH505 DNA was detected in multiple CD4+ T cell subsets, including cells with a naive phenotype (CD45RA+ CCR7+ CD95-). While the frequency of naive cells harboring intact provirus was lower than in memory cells, the high abundance of naive cells in the infant CD4+ T cell pool made them a substantial source of persistent viral DNA (approximately 50% of the total CD4+ T cell reservoir), with an estimated 1:2 ratio of intact provirus to total viral DNA. This viral reservoir profile broadens our understanding of virus persistence in a relevant infant macaque model and provides insight into targets for cure-directed approaches in the pediatric population.IMPORTANCE Uncovering the sanctuaries of the long-lived HIV-1 reservoir is crucial to develop cure strategies. Pediatric immunity is distinct from that of adults, which may alter where the reservoir is established in infancy. Thus, it is important to utilize pediatric models to inform cure-directed approaches for HIV-1-infected children. We used an infant rhesus macaque model of HIV-1 infection via breastfeeding to identify key sites of viral persistence under antiretroviral therapy (ART). The gastrointestinal tract was found to be a site for low-level viral transcription during ART. We also show that naive CD4+ T cells harbored intact provirus and were a major contributor to blood and lymphoid reservoir size. This is particularly striking, as memory CD4+ T cells are generally regarded as the main source of latent HIV/simian immunodeficiency virus (SIV) infection of adult humans and rhesus macaques. Our findings highlight unique features of reservoir composition in pediatric infection that should be considered for eradication efforts.

Short-Term Pegylated Interferon α2a Treatment Does Not Significantly Reduce the Viral Reservoir of Simian Immunodeficiency Virus-Infected, Antiretroviral Therapy-Treated Rhesus Macaques.

The major obstacle to human immunodeficiency type 1 (HIV-1) eradication is a reservoir of latently infected cells that persists despite long-term antiretroviral therapy (ART) and causes rapid viral rebound if treatment is interrupted. Type I interferons are immunomodulatory cytokines that induce antiviral factors and have been evaluated for the treatment of HIV-infected individuals, resulting in moderate reduction of viremia and inconclusive data about their effect on reservoir size. Here, we assessed the potential of pegylated IFN-α2a (pIFN-α2a) to reduce the viral reservoir in simian immunodeficiency virus (SIV)-infected, ART-treated rhesus macaques (RMs). We found that pIFN-α2a treatment of animals in which virus replication is effectively suppressed with ART is safe and well tolerated, as no major clinical side effects were observed. By monitoring the cellular immune response during this intervention, we established that pIFN-α2a administration is not associated with either CD4+ T cell depletion or increased immune activation. Importantly, we found that interferon-stimulated genes (ISGs) were significantly upregulated in IFN-treated RMs compared to control animals, confirming that pIFN-α2a is bioactive in vivo To evaluate the effect of pIFN-α2a administration on the viral reservoir in CD4+ T cells, we performed cell-associated proviral SIV DNA measurements in multiple tissues and assessed levels of replication-competent virus by a quantitative viral outgrowth assay (QVOA). These analyses failed to reveal any significant difference in reservoir size between IFN-treated and control animals. In summary, our data suggest that short-term type I interferon treatment in combination with suppressive ART is not sufficient to induce a significant reduction of the viral reservoir in SIV-infected RMs.IMPORTANCE The potential of type I interferons to reduce the viral reservoir has been recently studied in clinical trials in HIV-infected humans. However, given the lack of mechanistic data and the potential for safety concerns, a more comprehensive testing of IFN treatment in vivo in SIV-infected RMs is critical to provide rationale for further development of this intervention in humans. Utilizing the SIV/RM model in which virus replication is suppressed with ART, we addressed experimental limitations of previous human studies, in particular the lack of a control group and specimen sampling limited to blood. Here, we show by rigorous testing of blood and lymphoid tissues that virus replication and reservoir size were not significantly affected by pIFN-α2a treatment in SIV-infected, ART-treated RMs. This suggests that intensified and/or prolonged IFN treatment regimens, possibly in combination with other antilatency agents, are necessary to effectively purge the HIV/SIV reservoir under ART.

Antibody-Mediated CD4 Depletion Induces Homeostatic CD4+ T Cell Proliferation without Detectable Virus Reactivation in Antiretroviral Therapy-Treated Simian Immunodeficiency Virus-Infected Macaques.

A major barrier to human immunodeficiency virus (HIV) eradication is the long-term persistence of latently infected CD4+ T cells harboring integrated replication-competent virus. It has been proposed that the homeostatic proliferation of these cells drives long-term reservoir persistence in the absence of virus reactivation, thus avoiding cell death due to either virus-mediated cytopathicity or immune effector mechanisms. Here, we conducted an experimental depletion of CD4+ T cells in eight antiretroviral therapy (ART)-treated, simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs) to determine whether the homeostatically driven CD4+ T-cell proliferation that follows CD4+ T-cell depletion results in reactivation of latent virus and/or expansion of the virus reservoir. After administration of the CD4R1 antibody, we observed a CD4+ T cell depletion of 65 to 89% in peripheral blood and 20 to 50% in lymph nodes, followed by a significant increase in CD4+ T cell proliferation during CD4+ T cell reconstitution. However, this CD4+ T cell proliferation was not associated with detectable increases in viremia, indicating that the homeostatic activation of CD4+ T cells is not sufficient to induce virus reactivation from latently infected cells. Interestingly, the homeostatic reconstitution of the CD4+ T cell pool was not associated with significant changes in the number of circulating cells harboring SIV DNA compared to results for the first postdepletion time point. This study indicates that, in ART-treated SIV-infected RMs, the homeostasis-driven CD4+ T-cell proliferation that follows experimental CD4+ T-cell depletion occurs in the absence of detectable reactivation of latent virus and does not increase the size of the virus reservoir as measured in circulating cells.IMPORTANCE Despite successful suppression of HIV replication with antiretroviral therapy, current treatments are unable to eradicate the latent virus reservoir, and treatment interruption almost invariably results in the reactivation of HIV even after decades of virus suppression. Homeostatic proliferation of latently infected cells is one mechanism that could maintain the latent reservoir. To understand the impact of homeostatic mechanisms on virus reactivation and reservoir size, we experimentally depleted CD4+ T cells in ART-treated SIV-infected rhesus macaques and monitored their homeostatic rebound. We find that depletion-induced proliferation of CD4+ T cells is insufficient to reactivate the viral reservoir in vivo Furthermore, the proportion of SIV DNA+ CD4+ T cells remains unchanged during reconstitution, suggesting that the reservoir is resistant to this mechanism of expansion at least in this experimental system. Understanding how T cell homeostasis impacts latent reservoir longevity could lead to the development of new treatment paradigms aimed at curing HIV infection.

Intragastric Administration of Lactobacillus plantarum and 2,2'-Dithiodipyridine-Inactivated Simian Immunodeficiency Virus (SIV) Does Not Protect Indian Rhesus Macaques from Intrarectal SIV Challenge or Reduce Virus Replication after Transmission.

A major obstacle to development of an effective AIDS vaccine is that along with the intended beneficial responses, the immunization regimen may activate CD4+ T cells that can facilitate acquisition of human immunodeficiency virus (HIV) by serving as target cells for the virus. Lu et al. (W. Lu et al., Cell Rep 2:1736-1746, 2012, https://doi.org/10.1016/j.celrep.2012.11.016) reported that intragastric administration of chemically inactivated simian immunodeficiency virus SIVmac239 and Lactobacillus plantarum (iSIV-L. plantarum) protected 15/16 Chinese-origin rhesus macaques (RMs) from high-dose intrarectal SIVmac239 challenge at 3 months postimmunization. They attributed the observed protection to induction of immune tolerance, mediated by "MHC-Ib/E-restricted CD8+ regulatory T cells that suppressed SIV-harboring CD4+ T cell activation and ex vivo SIV replication in 15/16 animals without inducing SIV-specific antibodies or cytotoxic T." J.-M. Andrieu et al. (Front Immunol 5:297, 2014, https://doi.org/10.3389/fimmu.2014.00297) subsequently reported protection from infection in 23/24 RMs immunized intragastrically or intravaginally with iSIV and Mycobacterium bovis BCG, L. plantarum, or Lactobacillus rhamnosus, which they ascribed to the same tolerogenic mechanism. Using vaccine materials obtained from our coauthors, we conducted an immunization and challenge experiment with 54 Indian RMs and included control groups receiving iSIV only or L. plantarum only as well as unvaccinated animals. Intrarectal challenge with SIVmac239 resulted in rapid infection in all groups of vaccinated RMs as well as unvaccinated controls. iSIV-L. plantarum-vaccinated animals that became SIV infected showed viral loads similar to those observed in animals receiving iSIV only or L. plantarum only or in unvaccinated controls. The protection from SIV transmission conferred by intragastric iSIV-L. plantarum administration reported previously for Chinese-origin RMs was not observed when the same experiment was conducted in a larger cohort of Indian-origin animals.IMPORTANCE Despite an increased understanding of immune responses against HIV, a safe and effective AIDS vaccine is not yet available. One obstacle is that immunization may activate CD4+ T cells that may act as target cells for acquisition of HIV. An alternative strategy may involve induction of a tolerance-inducing response that limits the availability of activated CD4+ T cells, thus limiting the ability of virus to establish infection. In this regard, exciting results were obtained for Chinese-origin rhesus macaques by using a "tolerogenic" vaccine, consisting of intragastric administration of Lactobacillus plantarum and 2,2'-dithiodipyridine-inactivated SIV, which showed highly significant protection from virus transmission. In the present study, we administered iSIV-L. plantarum to Indian-origin rhesus macaques and failed to observe any protective effect on virus acquisition in this experimental setting. This work is important because it contributes to the overall assessment of the clinical potential of a new candidate AIDS vaccine platform based on iSIV-L. plantarum.

Simian Immunodeficiency Virus Persistence in Cellular and Anatomic Reservoirs in Antiretroviral Therapy-Suppressed Infant Rhesus Macaques.

Worldwide, nearly two million children are infected with human immunodeficiency virus (HIV), with breastfeeding accounting for the majority of contemporary HIV transmissions. Antiretroviral therapy (ART) has reduced HIV-related morbidity and mortality but is not curative. The main barrier to a cure is persistence of latent HIV in long-lived reservoirs. However, our understanding of the cellular and anatomic sources of the HIV reservoir during infancy and childhood is limited. Here, we developed a pediatric model of ART suppression in orally simian immunodeficiency virus (SIV)-infected rhesus macaque (RM) infants, with measurement of virus persistence in blood and tissues after 6 to 9 months of ART. Cross-sectional analyses were conducted to compare SIV RNA and DNA levels in adult and infant RMs naive to treatment and on ART. We demonstrate efficient viral suppression following ART initiation in SIV-infected RM infants with sustained undetectable plasma viral loads in the setting of heterogeneous penetration of ART into lymphoid and gastrointestinal tissues and low drug levels in the brain. We further show reduction in SIV RNA and DNA on ART in lymphoid tissues of both infant and adult RMs but stable (albeit low) levels of SIV RNA and DNA in the brains of viremic and ART-suppressed infants. Finally, we report a large contribution of naive CD4+ T cells to the total CD4 reservoir of SIV in blood and lymph nodes of ART-suppressed RM infants that differs from what we show in adults. These results reveal important aspects of HIV/SIV persistence in infants and provide insight into strategic targets for cure interventions in a pediatric population.IMPORTANCE While antiretroviral therapy (ART) can reduce HIV replication, the virus cannot be eradicated from an infected individual, and our incomplete understanding of HIV persistence in reservoirs greatly complicates the generation of a cure for HIV infection. Given the immaturity of the infant immune system, it is critically important to study HIV reservoirs specifically in this population. Here, we established a pediatric animal model to simulate breastfeeding transmission and study SIV reservoirs in rhesus macaque (RM) infants. Our study demonstrates that ART can be safely administered to infant RMs for prolonged periods and that it efficiently controls viral replication in this model. SIV persistence was shown in blood and tissues, with similar anatomic distributions of SIV reservoirs in infant and adult RMs. However, in the peripheral blood and lymph nodes, a greater contribution of the naive CD4+ T cells to the SIV reservoir was observed in infants than in adults.

Reduced Chronic Lymphocyte Activation following Interferon Alpha Blockade during the Acute Phase of Simian Immunodeficiency Virus Infection in Rhesus Macaques.

Pathogenic human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) infection of humans and rhesus macaques (RMs) induces persistently high production of type I interferon (IFN-I), which is thought to contribute to disease progression. To elucidate the specific role of interferon alpha (IFN-α) in SIV pathogenesis, 12 RMs were treated prior to intravenous (i.v.) SIVmac239 infection with a high or a low dose of an antibody (AGS-009) that neutralizes most IFN-α subtypes and were compared with six mock-infused, SIV-infected controls. Plasma viremia was measured postinfection to assess the effect of IFN-α blockade on virus replication, and peripheral blood and lymphoid tissue samples were analyzed by immunophenotypic staining. Consistent with the known antiviral effect of IFN-I, high-dose AGS-009 treatment induced a modest increase in acute-phase viral loads versus controls. Four out of 6 RMs receiving a high dose of AGS-009 also experienced an early decline in CD4+ T cell counts that was associated with progression to AIDS. Interestingly, 50% of the animals treated with AGS-009 (6/12) developed AIDS within 1 year of infection compared with 17% (1/6) of untreated controls. Finally, blockade of IFN-α decreased the levels of activated CD4+ and CD8+ T cells, as well as B cells, as measured by PD-1 and/or Ki67 expression. The lower levels of activated lymphocytes in IFN-α-blockaded animals supports the hypothesis that IFN-α signaling contributes to lymphocyte activation during SIV infection and suggests that this signaling pathway is involved in controlling virus replication during acute infection. The potential anti-inflammatory effect of IFN-α blockade should be explored as a strategy to reduce immune activation in HIV-infected individuals.IMPORTANCE Interferon alpha (IFN-α) is a member of a family of molecules (type I interferons) that prevent or limit virus infections in mammals. However, IFN-α production may contribute to the chronic immune activation that is thought to be the primary cause of immune decline and AIDS in HIV-infected patients. The study presented here attempts to understand the contribution of IFN-α to the natural history and progression of SIV infection of rhesus macaques, the primary nonhuman primate model system for testing hypotheses about HIV infection in humans. Here, we show that blockade of IFN-α action promotes lower chronic immune activation but higher early viral loads, with a trend toward faster disease progression. This study has significant implications for new treatments designed to impact the type I interferon system.

High Doses of GM-CSF Inhibit Antibody Responses in Rectal Secretions and Diminish Modified Vaccinia Ankara/Simian Immunodeficiency Virus Vaccine Protection in TRIM5α-Restrictive Macaques.

We tested, in rhesus macaques, the effects of a 500-fold range of an admixed recombinant modified vaccinia Ankara (MVA) expressing rhesus GM-CSF (MVA/GM-CSF) on the immunogenicity and protection elicited by an MVA/SIV macaque 239 vaccine. High doses of MVA/GM-CSF did not affect the levels of systemic envelope (Env)-specific Ab, but it did decrease the expression of the gut-homing receptor α4ß7 on plasmacytoid dendritic cells (p < 0.01) and the magnitudes of Env-specific IgA (p = 0.01) and IgG (p < 0.05) in rectal secretions. The protective effect of the vaccine was evaluated using 12 weekly rectal challenges in rhesus macaques subgrouped by tripartite motif-containing protein 5α (TRIM5α) genotypes that are restrictive or permissive for infection by the challenge virus SIVsmE660. Eight of nine TRIM5α-restrictive animals receiving no or the lowest dose (1 × 105 PFU) of MVA/GM-CSF resisted all 12 challenges. In the comparable TRIM5α-permissive group, only 1 of 12 animals resisted all 12 challenges. In the TRIM5α-restrictive animals, but not in the TRIM5α-permissive animals, the number of challenges to infection directly correlated with the magnitudes of Env-specific rectal IgG (r = +0.6) and IgA (r = +0.6), the avidity of Env-specific serum IgG (r = +0.5), and Ab dependent cell-mediated virus inhibition (r = +0.6). Titers of neutralizing Ab did not correlate with protection. We conclude that 1) protection elicited by MVA/SIVmac239 is strongly dependent on the presence of TRIM5α restriction, 2) nonneutralizing Ab responses contribute to protection against SIVsmE660 in TRIM5α-restrictive animals, and 3) high doses of codelivered MVA/GM-CSF inhibit mucosal Ab responses and the protection elicited by MVA expressing noninfectious SIV macaque 239 virus-like particles.

Replicative fitness of transmitted HIV-1 drives acute immune activation, proviral load in memory CD4+ T cells, and disease progression.

HIV-1 infection is characterized by varying degrees of chronic immune activation and disruption of T-cell homeostasis, which impact the rate of disease progression. A deeper understanding of the factors that influence HIV-1-induced immunopathology and subsequent CD4(+) T-cell decline is critical to strategies aimed at controlling or eliminating the virus. In an analysis of 127 acutely infected Zambians, we demonstrate a dramatic and early impact of viral replicative capacity (vRC) on HIV-1 immunopathogenesis that is independent of viral load (VL). Individuals infected with high-RC viruses exhibit a distinct inflammatory cytokine profile as well as significantly elevated T-cell activation, proliferation, and CD8(+) T-cell exhaustion, during the earliest months of infection. Moreover, the vRC of the transmitted virus is positively correlated with the magnitude of viral burden in naive and central memory CD4(+) T-cell populations, raising the possibility that transmitted viral phenotypes may influence the size of the initial latent viral reservoir. Taken together, these findings support an unprecedented role for the replicative fitness of the founder virus, independent of host protective genes and VL, in influencing multiple facets of HIV-1-related immunopathology, and that a greater focus on this parameter could provide novel approaches to clinical interventions.