Anti-PD-1 chimeric antigen receptor T cells efficiently target SIV-infected CD4+ T cells in germinal centers.

Programmed cell death protein 1 (PD-1) is an immune checkpoint marker commonly expressed on memory T cells and enriched in latently HIV-infected CD4+ T cells. We engineered an anti-PD-1 chimeric antigen receptor (CAR) to assess the impact of PD-1 depletion on viral reservoirs and rebound dynamics in SIVmac239-infected rhesus macaques (RMs). Adoptive transfer of anti-PD-1 CAR T cells was done in 2 SIV-naive and 4 SIV-infected RMs on antiretroviral therapy (ART). In 3 of 6 RMs, anti-PD-1 CAR T cells expanded and persisted for up to 100 days concomitant with the depletion of PD-1+ memory T cells in blood and tissues, including lymph node CD4+ follicular helper T (TFH) cells. Loss of TFH cells was associated with depletion of detectable SIV RNA from the germinal center (GC). However, following CAR T infusion and ART interruption, there was a marked increase in SIV replication in extrafollicular portions of lymph nodes, a 2-log higher plasma viremia relative to controls, and accelerated disease progression associated with the depletion of CD8+ memory T cells. These data indicate anti-PD-1 CAR T cells depleted PD-1+ T cells, including GC TFH cells, and eradicated SIV from this immunological sanctuary.

Optimization and use of near infrared imaging to guide lymph node collection in rhesus macaques (Macaca mulatta).

BACKGROUND: Identification of lymph nodes (LNs) draining a specific site or in obese macaques can be challenging. METHODS: Indocyanine Green (ICG) was administered intradermal (ID), intramuscular, in the oral mucosa, or subserosal in the colon followed by Near Infrared (NIR) imaging. RESULTS: After optimization to maximize LN identification, intradermal ICG was successful in identifying 50-100% of the axillary/inguinal LN at a site. Using NIR, collection of peripheral and mesenteric LNs in obese macaques was 100% successful after traditional methods failed. Additionally, guided collection of LNs draining the site of intraepithelial or intramuscular immunization demonstrated significantly increased numbers of T follicular helper (Tfh) cells in germinal centers of draining compared to nondraining LNs. CONCLUSION: These imaging techniques optimize our ability to evaluate immune changes within LNs over time, even in obese macaques. This approach allows for targeted serial biopsies that permit confidence that draining LNs are being harvested throughout the study.

Rapamycin limits CD4+ T cell proliferation in simian immunodeficiency virus-infected rhesus macaques on antiretroviral therapy.

Proliferation of latently infected CD4+ T cells with replication-competent proviruses is an important mechanism contributing to HIV persistence during antiretroviral therapy (ART). One approach to targeting this latent cell expansion is to inhibit mTOR, a regulatory kinase involved with cell growth, metabolism, and proliferation. Here, we determined the effects of chronic mTOR inhibition with rapamycin with or without T cell activation in SIV-infected rhesus macaques (RMs) on ART. Rapamycin perturbed the expression of multiple genes and signaling pathways important for cellular proliferation and substantially decreased the frequency of proliferating CD4+ memory T cells (TM cells) in blood and tissues. However, levels of cell-associated SIV DNA and SIV RNA were not markedly different between rapamycin-treated RMs and controls during ART. T cell activation with an anti-CD3LALA antibody induced increases in SIV RNA in plasma of RMs on rapamycin, consistent with SIV production. However, upon ART cessation, both rapamycin and CD3LALA-treated and control-treated RMs rebounded in less than 12 days, with no difference in the time to viral rebound or post-ART viral load set points. These results indicate that, while rapamycin can decrease the proliferation of CD4+ TM cells, chronic mTOR inhibition alone or in combination with T cell activation was not sufficient to disrupt the stability of the SIV reservoir.

The ingenol-based protein kinase C agonist GSK445A is a potent inducer of HIV and SIV RNA transcription.

Activation of the NF-κB signaling pathway by Protein Kinase C (PKC) agonists is a potent mechanism for human immunodeficiency virus (HIV) latency disruption in vitro. However, significant toxicity risks and the lack of evidence supporting their activity in vivo have limited further evaluation of PKC agonists as HIV latency-reversing agents (LRA) in cure strategies. Here we evaluated whether GSK445A, a stabilized ingenol-B derivative, can induce HIV/simian immunodeficiency virus (SIV) transcription and virus production in vitro and demonstrate pharmacological activity in nonhuman primates (NHP). CD4+ T cells from people living with HIV and from SIV+ rhesus macaques (RM) on antiretroviral therapy (ART) exposed in vitro to 25 nM of GSK445A produced cell-associated viral transcripts as well as viral particles at levels similar to those induced by PMA/Ionomycin, indicating that GSK445A can potently reverse HIV/SIV latency. Importantly, these concentrations of GSK445A did not impair the proliferation or survival of HIV-specific CD8+ T cells, but instead, increased their numbers and enhanced IFN-γ production in response to HIV peptides. In vivo, GSK445A tolerability was established in SIV-naïve RM at 15 µg/kg although tolerability was reduced in SIV-infected RM on ART. Increases in plasma viremia following GSK445A administration were suggestive of increased SIV transcription in vivo. Collectively, these results indicate that GSK445A is a potent HIV/SIV LRA in vitro and has a tolerable safety profile amenable for further evaluation in vivo in NHP models of HIV cure/remission.

Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques.

Chimeric antigen receptor (CAR) T cell therapies are being investigated as potential HIV cures and designed to target HIV reservoirs. Monoclonal antibodies (mAbs) targeting the simian immunodeficiency virus (SIV) envelope allowed us to investigate the potency of single-chain variable fragment (scFv)-based anti-SIV CAR T cells. In vitro, CAR T cells expressing the scFv to both the variable loop 1 (V1) or V3 of the SIV envelope were highly potent at eliminating SIV-infected T cells. However, in preclinical studies, in vivo infusion of these CAR T cells in rhesus macaques (RMs) resulted in lack of expansion and no detectable in vivo antiviral activity. Injection of envelope-expressing antigen-presenting cells (APCs) 1 week post-CAR T cell infusion also failed to stimulate CAR T cell expansion in vivo. To investigate this in vitro versus in vivo discrepancy, we examined host immune responses directed at CAR T cells. A humoral immune response against the CAR scFv was detected post-infusion of the anti-SIV CAR T cells; anti-SIV IgG antibodies present in plasma of SIV-infected animals were associated with inhibited CAR T cell effector functions. These data indicate that lack of in vivo expansion and efficacy of CAR T cells might be due to antibodies blocking the interaction between the CAR scFv and its epitope.

Interleukin-15 response signature predicts RhCMV/SIV vaccine efficacy.

Simian immunodeficiency virus (SIV) challenge of rhesus macaques (RMs) vaccinated with strain 68-1 Rhesus Cytomegalovirus (RhCMV) vectors expressing SIV proteins (RhCMV/SIV) results in a binary outcome: stringent control and subsequent clearance of highly pathogenic SIV in ~55% of vaccinated RMs with no protection in the remaining 45%. Although previous work indicates that unconventionally restricted, SIV-specific, effector-memory (EM)-biased CD8+ T cell responses are necessary for efficacy, the magnitude of these responses does not predict efficacy, and the basis of protection vs. non-protection in 68-1 RhCMV/SIV vector-vaccinated RMs has not been elucidated. Here, we report that 68-1 RhCMV/SIV vector administration strikingly alters the whole blood transcriptome of vaccinated RMs, with the sustained induction of specific immune-related pathways, including immune cell, toll-like receptor (TLR), inflammasome/cell death, and interleukin-15 (IL-15) signaling, significantly correlating with subsequent vaccine efficacy. Treatment of a separate RM cohort with IL-15 confirmed the central involvement of this cytokine in the protection signature, linking the major innate and adaptive immune gene expression networks that correlate with RhCMV/SIV vaccine efficacy. This change-from-baseline IL-15 response signature was also demonstrated to significantly correlate with vaccine efficacy in an independent validation cohort of vaccinated and challenged RMs. The differential IL-15 gene set response to vaccination strongly correlated with the pre-vaccination activity of this pathway, with reduced baseline expression of IL-15 response genes significantly correlating with higher vaccine-induced induction of IL-15 signaling and subsequent vaccine protection, suggesting that a robust de novo vaccine-induced IL-15 signaling response is needed to program vaccine efficacy. Thus, the RhCMV/SIV vaccine imparts a coordinated and persistent induction of innate and adaptive immune pathways featuring IL-15, a known regulator of CD8+ T cell function, that support the ability of vaccine-elicited unconventionally restricted CD8+ T cells to mediate protection against SIV challenge.

CD8+ T cells fail to limit SIV reactivation following ART withdrawal until after viral amplification.

To define the contribution of CD8+ T cell responses to control of SIV reactivation during and following antiretroviral therapy (ART), we determined the effect of long-term CD8+ T cell depletion using a rhesusized anti-CD8ß monoclonal antibody on barcoded SIVmac239 dynamics on stable ART and after ART cessation in rhesus macaques (RMs). Among the RMs with full CD8+ T cell depletion in both blood and tissue, there were no significant differences in the frequency of viral blips in plasma, the number of SIV RNA+ cells and the average number of RNA copies/infected cell in tissue, and levels of cell-associated SIV RNA and DNA in blood and tissue relative to control-treated RMs during ART. Upon ART cessation, both CD8+ T cell-depleted and control RMs rebounded in fewer than 12 days, with no difference in the time to viral rebound or in either the number or growth rate of rebounding SIVmac239M barcode clonotypes. However, effectively CD8+ T cell-depleted RMs showed a stable, approximately 2-log increase in post-ART plasma viremia relative to controls. These results indicate that while potent antiviral CD8+ T cell responses can develop during ART-suppressed SIV infection, these responses effectively intercept post-ART SIV rebound only after systemic viral replication, too late to limit reactivation frequency or the early spread of reactivating SIV reservoirs.

Role of IL-15 Signaling in the Pathogenesis of Simian Immunodeficiency Virus Infection in Rhesus Macaques.

Although IL-15 has been implicated in the pathogenic hyperimmune activation that drives progressive HIV and SIV infection, as well as in the generation of HIV/SIV target cells, it also supports NK and T cell homeostasis and effector activity, potentially benefiting the host. To understand the role of IL-15 in SIV infection and pathogenesis, we treated two cohorts of SIVmac239-infected rhesus macaques (RM; Macaca mulatta), one with chronic infection, the other with primary infection, with a rhesusized, IL-15-neutralizing mAb (versus an IgG isotype control) for up to 10 wk (n = 7-9 RM per group). In both cohorts, anti-IL-15 was highly efficient at blocking IL-15 signaling in vivo, causing 1) profound depletion of NK cells in blood and tissues throughout the treatment period; 2) substantial, albeit transient, depletion of CD8+ effector memory T cells (TEM) (but not the naive and central memory subsets); and 3) CD4+ and CD8+ TEM hyperproliferation. In primary infection, reduced frequencies of SIV-specific effector T cells in an extralymphoid tissue site were also observed. Despite these effects, the kinetics and extent of SIV replication, CD4+ T cell depletion, and the onset of AIDS were comparable between anti-IL-15- and control-treated groups in both cohorts. However, RM treated with anti-IL-15 during primary infection manifested accelerated reactivation of RM rhadinovirus. Thus, IL-15 support of NK cell and TEM homeostasis does not play a demonstrable, nonredundant role in SIV replication or CD4+ T cell deletion dynamics but may contribute to immune control of oncogenic γ-herpesviruses.

Early antiretroviral therapy limits SIV reservoir establishment to delay or prevent post-treatment viral rebound.

Prophylactic vaccination of rhesus macaques with rhesus cytomegalovirus (RhCMV) vectors expressing simian immunodeficiency virus (SIV) antigens (RhCMV/SIV) elicits immune responses that stringently control highly pathogenic SIV infection, with subsequent apparent clearance of the infection, in ~50% of vaccinees. In contrast, here, we show that therapeutic RhCMV/SIV vaccination of rhesus macaques previously infected with SIV and given continuous combination antiretroviral therapy (cART) beginning 4-9 d post-SIV infection does not mediate measurable SIV reservoir clearance during over 600 d of follow-up on cART relative to RhCMV/control vaccination. However, none of the six animals started on cART on day four or five, across both RhCMV/SIV- and RhCMV/control-vaccinated groups, those rhesus macaques with SIV reservoirs most closely resembling those of prophylactically RhCMV/SIV-vaccinated and protected animals early in their course, showed post-cART viral rebound with up to nine months of follow-up. Moreover, at necropsy, these rhesus macaques showed little to no evidence of replication-competent SIV. These results suggest that the early SIV reservoir is limited in durability and that effective blockade of viral replication and spread in this critical time window by either pharmacologic or immunologic suppression may result in reduction, and potentially loss, of rebound-competent virus over a period of ~two years.

B cell follicle sanctuary permits persistent productive simian immunodeficiency virus infection in elite controllers.

Chronic-phase HIV and simian immunodeficiency virus (SIV) replication is reduced by as much as 10,000-fold in elite controllers (ECs) compared with typical progressors (TPs), but sufficient viral replication persists in EC tissues to allow viral sequence evolution and induce excess immune activation. Here we show that productive SIV infection in rhesus monkey ECs, but not TPs, is markedly restricted to CD4(+) follicular helper T (TFH) cells, suggesting that these EC monkeys' highly effective SIV-specific CD8(+) T cells can effectively clear productive SIV infection from extrafollicular sites, but their relative exclusion from B cell follicles prevents their elimination of productively infected TFH cells. CD8(+) lymphocyte depletion in EC monkeys resulted in a dramatic re-distribution of productive SIV infection to non-TFH cells, with restriction of productive infection to TFH cells resuming upon CD8(+) T cell recovery. Thus, B cell follicles constitute 'sanctuaries' for persistent SIV replication in the presence of potent anti-viral CD8(+) T cell responses, potentially complicating efforts to cure HIV infection with therapeutic vaccination or T cell immunotherapy.

Genetic similarity of circulating and small intestinal virus at the end stage of acute pathogenic simian-human immunodeficiency virus infection.

To understand the pathogenicity of acquired immune deficiency syndrome (AIDS), it is important to clarify where, when and how the virus replicates in the body of infected individuals. To identify the major virus replication site at the end stage of SHIV infection, we investigated the systemic tissues of SHIV-infected monkeys that developed AIDS-like disease. We quantified proviral DNA, and compared the mutation patterns of the viruses in various systemic tissues and in peripheral blood through phylogenetic analysis of the full genome sequence. We found that the amounts of proviral DNA detected in internal tissues were higher than those in peripheral blood mononuclear cells. In the sequence and phylogenetic tree analyses, the mutation patterns of the viruses in each tissue were generally different. However, the mutation pattern of the viruses in the jejunum and mesenteric lymph node were most similar to that of plasma viral RNA among the tissues examined in all three monkeys. In two of the three monkeys, which were euthanized earlier, viruses in the jejunum and mesenteric lymph node occupied the root position of the phylogenetic tree. Furthermore, in these tissues, more than 50% of SHIV-expressing cells were identified as macrophages based on co-expression of CD68. These results suggest that macrophages of the small intestine and/or mesenteric lymph node are the major virus production site at the end stage of SHIV infection of macaques.

Lymph node T cell responses predict the efficacy of live attenuated SIV vaccines.

Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.

[Protective immune responses against HIV/SIV by live attenuated vaccine in non-human primate models].

Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) are currently the most effective vaccines in nonhuman primate models for AIDS, yet the basis of their robust protection remains poorly understood. Our recent immune correlate study revealed that degree of protection against pathogenic SIV challenge strongly correlated with the SIV-specific CD4+ and CD8+ T cell responses in the lymph node but neither with the responses of such T cells in the peripheral blood and mucosal tissues nor with humoral immune responses. Interestingly, the maintenance of protective T cell responses in the lymph node was associated with the amount of persistent LAV replication in the lymph node, which localized almost exclusively in follicular helper T cells. The protected monkeys manifested greater magnitude of functional effector CD8+ T cells in the lymph node, suggesting that the induction and maintenance of antiviral effector memory T cells derived by persistent antigen production have a vital role in establishment of protection. This article reviews the mechanisms of the protection in monkeys vaccinated with LAV and their implication for development of successful AIDS vaccine.

In vivo analysis of a new R5 tropic SHIV generated from the highly pathogenic SHIV-KS661, a derivative of SHIV-89.6.

Although X4 tropic SHIVs have been studied extensively, they show distinct infection phenotypes from those of R5 tropic viruses, which play an important role in HIV-1 transmission and pathogenesis. To augment the variety of R5 tropic SHIVs, we generated a new R5 tropic SHIV from the highly pathogenic X4 tropic SHIV-KS661, a derivative of SHIV-89.6. Based on consensus amino acid alignment analyses of subtype B R5 tropic HIV-1, five amino acid substitutions in the third variable region successfully changed the secondary receptor preference from X4 to R5. Improvements in viral replication were observed in infected rhesus macaques after two passages, and reisolated virus was designated SHIV-MK38. SHIV-MK38 maintained R5 tropism through in vivo passages and showed robust replication in infected monkeys. Our study clearly demonstrates that a minimal number of amino acid substitutions in the V3 region can alter secondary receptor preference and increase the variety of R5 tropic SHIVs.

Small intestine CD4+ cell reduction and enteropathy in simian/human immunodeficiency virus KS661-infected rhesus macaques in the presence of low viral load.

Human immunodeficiency virus type 1, simian immunodeficiency virus and simian/human immunodeficiency virus (SHIV) infection generally lead to death of the host accompanied by high viraemia and profound CD4(+) T-cell depletion. SHIV clone KS661-infected rhesus macaques with a high viral load set point (HVL) ultimately experience diarrhoea and wasting at 6-12 months after infection. In contrast, infected macaques with a low viral load set point (LVL) usually live asymptomatically throughout the observation period, and are therefore referred to as asymptomatic LVL (Asym LVL) macaques. Interestingly, some LVL macaques exhibit diarrhoea and wasting similar to the symptoms of HVL macaques and are termed symptomatic LVL (Sym LVL) macaques. This study tested the hypothesis that Sym LVL macaques have the same degree of intestinal abnormalities as HVL macaques. The proviral DNA loads in lymphoid tissue and the intestines of Sym LVL and Asym LVL macaques were comparable and all infected monkeys showed villous atrophy. Notably, the CD4(+) cell frequencies of lymphoid tissues and intestines in Sym LVL macaques were remarkably lower than those in Asym LVL and uninfected macaques. Furthermore, Sym LVL and HVL macaques exhibited an increased number of activated macrophages. In conclusion, intestinal disorders including CD4(+) cell reduction and abnormal immune activation can be observed in SHIV-KS661-infected macaques independent of virus replication levels.

Small intestine CD4+ T cells are profoundly depleted during acute simian-human immunodeficiency virus infection, regardless of viral pathogenicity.

To analyze the relationship between acute virus-induced injury and the subsequent disease phenotype, we compared the virus replication and CD4(+) T-cell profiles for monkeys infected with isogenic highly pathogenic (KS661) and moderately pathogenic (#64) simian-human immunodeficiency viruses (SHIVs). Intrarectal infusion of SHIV-KS661 resulted in rapid, systemic, and massive virus replication, while SHIV-#64 replicated more slowly and reached lower titers. Whereas KS661 systemically depleted CD4(+) T cells, #64 caused significant CD4(+) T-cell depletion only in the small intestine. We conclude that SHIV, regardless of pathogenicity, can cause injury to the small intestine and leads to CD4(+) T-cell depletion in infected animals during acute infection.

Impaired T-cell differentiation in the thymus at the early stages of acute pathogenic chimeric simian-human immunodeficiency virus (SHIV) infection in contrast to less pathogenic SHIV infection.

One of the mechanisms by which HIV infection induces the depletion of CD4+ T cells has been suggested to be impairment of T-cell development in the thymus, although there is no direct evidence that this occurs. To examine this possibility, we compared T-cell maturation in the intrathymic progenitors between macaques infected with an acute pathogenic chimeric simian-human immunodeficiency virus (SHIV), which causes profound and irreversible CD4+ T-cell depletion, and macaques infected with a less pathogenic SHIV, which causes only a transient CD4+ T-cell decline. Within 27 days post-inoculation (dpi), the two virus infections caused similar increases in plasma viral loads and similar decreases in CD4+ T-cell counts. However, in the thymus, the acute pathogenic SHIV resulted in increased thymic involution, atrophy and the depletion of immature T cells including CD4(+)CD8(+) double-positive (DP) cells, whereas the less pathogenic SHIV did not have these effects. Ex vivo differentiation of CD3(-)CD4(-)CD8(-) triple-negative (TN) intrathymic progenitors to DP cells was assessed by a monkey-mouse xenogenic fetal thymus organ culture system. Differentiation was impaired in the TN intrathymic progenitors of the acute pathogenic SHIV-infected monkeys, while differentiation was not impaired in the TN intrathymic progenitors of the less pathogenic SHIV-infected monkeys. These differences suggest that dysfunction of thymic maturation makes an important contribution to the irreversible depletion of circulating CD4+ T cells in vivo.

Intrathymic effect of acute pathogenic SHIV infection on T-lineage cells in newborn macaques.

We intrarectally infected newborn macaques with a pathogenic simian/human immunodeficiency virus (SHIV) that induced rapid and profound CD4 (+) T cell depletion, and examined the early effects of this SHIV on the thymus. After intrarectal infection, viral loads were much higher in the thymus than in other lymphoid tissues in newborns. In contrast, no clear difference was seen in the viral loads of different tissues in adults. Histological and immunohistochemical observations showed severe thymic involution. Depletion of CD4 (+) thymocytes began in the medulla at 2 weeks post infection and spread over the whole thymus. After in vivo infection, the CD2 (+) subpopulation, which represents a relatively later stage of T cell progenitors, was selectively reduced and development of thymocytes from CD3 (-) CD4 (-) CD8 (-) cells to CD4 (+) CD8 (+) cells was impaired. These results suggest that profound and irreversible loss of CD4 (+) cells that are observed in the peripheral blood of SHIV-infected monkeys are due to destruction of the thymus and impaired thymopoiesis as a result of SHIV infection in the thymus.