Pathogenic SIVmac239 Research Articles

Viral escape mutations do not account for non-protection from SIVmac239 challenge in RhCMV/SIV vaccinated rhesus macaques.

Simian immunodeficiency virus (SIV) vaccines based upon 68-1 Rhesus Cytomegalovirus (RhCMV) vectors show remarkable protection against pathogenic SIVmac239 challenge. Across multiple independent rhesus macaque (RM) challenge studies, nearly 60% of vaccinated RM show early, complete arrest of SIVmac239 replication after effective challenge, whereas the remainder show progressive infection similar to controls. Here, we performed viral sequencing to determine whether the failure to control viral replication in non-protected RMs is associated with the acquisition of viral escape mutations. While low level viral mutations accumulated in all animals by 28 days-post-challenge, which is after the establishment of viral control in protected animals, the dominant circulating virus in virtually all unprotected RMs was nearly identical to the challenge stock, and there was no difference in mutation patterns between this cohort and unvaccinated controls. These data definitively demonstrate that viral mutation does not explain lack of viral control in RMs not protected by RhCMV/SIV vaccination. We further demonstrate that during chronic infection RhCMV/SIV vaccinated RMs do not acquire escape mutation in epitopes targeted by RhCMV/SIV, but instead display mutation in canonical MHC-Ia epitopes similar to unvaccinated RMs. This suggests that after the initial failure of viral control, unconventional T cell responses induced by 68-1 RhCMV/SIV vaccination do not exert strong selective pressure on systemically replicating SIV.

Vaccine plus microbicide effective in preventing vaginal SIV transmission in macaques

The human immunodeficiency virus epidemic continues in sub-Saharan Africa, and particularly affects adolescent girls and women who have limited access to antiretroviral therapy. Here we report that the risk of vaginal simian immunodeficiency virus (SIV)mac251 acquisition is reduced by more than 90% using a combination of a vaccine comprising V1-deleted (V2 enhanced) SIV envelope immunogens with topical treatment of the zinc-finger inhibitor SAMT-247. Following 14 weekly intravaginal exposures to the highly pathogenic SIVmac251, 80% of a cohort of 20 macaques vaccinated and treated with SAMT-247 remained uninfected. In an arm of 18 vaccinated-only animals without microbicide, 40% of macaques remained uninfected. The combined SAMT-247/vaccine regimen was significantly more effective than vaccination alone. By analysing immune correlates of protection, we show that, by increasing zinc availability, SAMT-247 increases natural killer cytotoxicity and monocyte efferocytosis, and decreases T-cell activation to augment vaccine-induced protection.

Direct intranodal tonsil vaccination with modified vaccinia Ankara vaccine protects macaques from highly pathogenic SIVmac251

Human immunodeficiency virus (HIV) is a mucosally transmitted virus that causes immunodeficiency and AIDS. Developing efficacious vaccines to prevent infection is essential to control the epidemic. Protecting the vaginal and rectal mucosa, the primary routes of HIV entry has been a challenge given the significant compartmentalization between the mucosal and peripheral immune systems. We hypothesized that direct intranodal vaccination of mucosa associated lymphoid tissue (MALT) such as the readily accessible palatine tonsils could overcome this compartmentalization. Here we show that rhesus macaques primed with plasmid DNA encoding SIVmac251-env and gag genes followed by an intranodal tonsil MALT boost with MVA encoding the same genes protects from a repeated low dose intrarectal challenge with highly pathogenic SIVmac251; 43% (3/7) of vaccinated macaques remained uninfected after 9 challenges as compared to the unvaccinated control (0/6) animals. One vaccinated animal remained free of infection even after 22 challenges. Vaccination was associated with a ~2 log decrease in acute viremia that inversely correlated with anamnestic immune responses. Our results suggest that a combination of systemic and intranodal tonsil MALT vaccination could induce robust adaptive and innate immune responses leading to protection from mucosal infection with highly pathogenic HIV and rapidly control viral breakthroughs.

Pharmacological Validation of Long-Term Treatment with Antiretroviral Drugs in a Model of SIV-Infected Non-Human Primates.

The development of animal models undergoing long-term antiretroviral treatment (ART) makes it possible to understand a number of immunological, virological, and pharmacological issues, key factors in the management of HIV infection. We aimed to pharmacologically validate a non-human primate (NHP) model treated in the long term with antiretroviral drugs after infection with the pathogenic SIVmac251 strain. A single-dose pharmacokinetic study of tenofovir disoproxil fumarate, emtricitabine, and dolutegravir was first conducted on 13 non-infected macaques to compare three different routes of administration. Then, 12 simian immunodeficiency virus (SIV)-infected (SIV+) macaques were treated with the same regimen for two years. Drug monitoring, virological efficacy, and safety were evaluated throughout the study. For the single-dose pharmacokinetic study, 24-h post-dose plasma concentrations for all macaques were above or close to 90% inhibitory concentrations and consistent with human data. During the two-year follow-up, the pharmacological data were consistent with those observed in humans, with low inter- and intra-individual variability. Rapid and sustained virological efficacy was observed for all macaques, with a good safety profile. Overall, our SIV+ NHP model treated with the ART combination over a two-year period is suitable for investigating the question of pharmacological sanctuaries in HIV infection and exploring strategies for an HIV cure.

Cytokine Adjuvants IL-7 and IL-15 Improve Humoral Responses of a SHIV LentiDNA Vaccine in Animal Models.

HIV-1 remains a major public health issue worldwide in spite of efficacious antiviral therapies, but with no cure or preventive vaccine. The latter has been very challenging, as virus infection is associated with numerous escape mechanisms from host specific immunity and the correlates of protection remain incompletely understood. We have developed an innovative vaccine strategy, inspired by the efficacy of live-attenuated virus, but with the safety of a DNA vaccine, to confer both cellular and humoral responses. The CAL-SHIV-IN− lentiDNA vaccine comprises the backbone of the pathogenic SHIVKU2 genome, able to mimic the early phase of viral infection, but with a deleted integrase gene to ensure safety precluding integration within the host genome. This vaccine prototype, constitutively expressing viral antigen under the CAEV LTR promoter, elicited a variety of vaccine-specific, persistent CD4 and CD8 T cells against SIV-Gag and Nef up to 80 weeks post-immunization in cynomolgus macaques. Furthermore, these specific responses led to antiviral control of the pathogenic SIVmac251. To further improve the efficacy of this vaccine, we incorporated the IL-7 or IL-15 genes into the CAL-SHIV-IN− plasmid DNA in efforts to increase the pool of vaccine-specific memory T cells. In this study, we examined the immunogenicity of the two co-injected lentiDNA vaccines CAL-SHIV-IN− IRES IL-7 and CAL-SHIV-IN− IRES IL-15 in BALB/cJ mice and rhesus macaques and compared the immune responses with those generated by the parental vaccine CAL-SHIV-IN−. This co-immunization elicited potent vaccine-specific CD4 and CD8 T cells both in mice and rhesus macaques. Antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies were detected up to 40 weeks post-immunization in both plasma and mucosal compartments of rhesus macaques and were enhanced by the cytokines.

Expansion of Immature Neutrophils During SIV Infection Is Associated With Their Capacity to Modulate T-Cell Function.

In spite of the efficacy of combinational antiretroviral treatment (cART), HIV-1 persists in the host and infection is associated with chronic inflammation, leading to an increased risk of comorbidities, such as cardiovascular diseases, neurocognitive disorders, and cancer. Myeloid cells, mainly monocytes and macrophages, have been shown to be involved in the immune activation observed in HIV-1 infection. However, less attention has been paid to neutrophils, the most abundant circulating myeloid cell, even though neutrophils are strongly involved in tissue damage and inflammation in several chronic diseases, in particular, autoimmune diseases. Herein, we performed a longitudinal characterization of neutrophil phenotype and we evaluated the interplay between neutrophils and T cells in the model of pathogenic SIVmac251 experimental infection of cynomolgus macaques. We report that circulating granulocytes consists mainly of immature CD10- neutrophils exhibiting a prime phenotype during primary and chronic infection. We found that neutrophil priming correlates with CD8+ T cell activation. Moreover, we provide the evidence that neutrophils are capable of modulating CD4+ and CD8+ T-cell proliferation and IFN-γ production in different ways depending on the time of infection. Thus, our study emphasizes the role of primed immature neutrophils in the modulation of T-cell responses in SIV infection.

CD8 T Cells Show Protection against Highly Pathogenic Simian Immunodeficiency Virus (SIV) after Vaccination with SIV Gene-Expressing BCG Prime and Vaccinia Virus/Sendai Virus Vector Boosts.

Toward development of a dual vaccine for human immunodeficiency virus type 1 (HIV-1) and tuberculosis infections, we developed a urease-deficient bacillus Calmette-Guérin (BCG) strain Tokyo172 (BCGΔurease) to enhance its immunogenicity. BCGΔurease expressing a simian immunodeficiency virus (SIV) Gag induced BCG antigen-specific CD4+ and CD8+ T cells more efficiently and more Gag-specific CD8+ T cells. We evaluated its protective efficacy against SIV infection in cynomolgus monkeys of Asian origin, shown to be as susceptible to infection with SIVmac251 as Indian rhesus macaques. Priming with recombinant BCG (rBCG) expressing SIV genes was followed by a boost with SIV gene-expressing LC16m8Δ vaccinia virus and a second boost with SIV Env-expressing Sendai virus. Eight weeks after the second boost, monkeys were repeatedly challenged with a low dose of SIVmac251 intrarectally. Two animals out of 6 vaccinees were protected, whereas all 7 control animals were infected without any early viral controls. In one vaccinated animal, which had the most potent CD8+ T cells in an in vitro suppression activity (ISA) assay of SIVmac239 replication, plasma viremia was undetectable throughout the follow-up period. Protection was confirmed by the lack of anamnestic antibody responses and detectable cell-associated provirus in various organs. Another monkey with a high ISA acquired a small amount of SIV, but it later became suppressed below the detection limit. Moreover, the ISA score correlated with SIV acquisition. On the other hand, any parameter relating anti-Env antibody was not correlated with the protection.IMPORTANCE Because both AIDS and tuberculosis are serious health threats in middle/low-income countries, development of a dual vaccine against them would be highly beneficial. To approach the goal, here we first assessed a urease-deficient bacillus Calmette-Guérin (BCG) for improvement of immunogenicity against both Mycobacterium tuberculosis and SIV. Second, we demonstrated the usefulness of Asian-origin cynomolgus monkeys for development of a preclinical AIDS vaccine by direct comparison with Indian rhesus macaques as the only validated hosts that identically mirror the outcomes of clinical trials, since the availability of Indian rhesus macaques is limited in countries other than the United States. Finally, we report the protective effect of a vaccination regimen comprising BCG, the highly attenuated vaccinia virus LC16m8Δ strain, and nontransmissible Sendai virus as safe vectors expressing SIV genes using repeated mucosal challenge with highly pathogenic SIVmac251. Identification of CD8+ T cells as a protective immunity suggests a future direction of AIDS vaccine development.

An Automated Fluorescence-Based Method to Isolate Bone Marrow-Derived Plasma Cells from Rhesus Macaques Using SIVmac239 SOSIP.664

Simian immunodeficiency virus (SIV) infection of Indian rhesus macaques (RMs) is one of the best-characterized animal models for human immunodeficiency virus (HIV) infection. Monoclonal antibodies (mAbs) have shown promise for prevention and treatment of HIV infection. However, it has been difficult to isolate mAbs that potently neutralize the highly pathogenic SIVmac239 strain. This has been largely due to the low frequency of circulating B cells encoding neutralizing Abs. Here we describe a novel technique to isolate mAbs directly from bone marrow-derived, Ab-secreting plasma cells. We employed an automated micromanipulator to isolate single SIVmac239 SOSIP.664-specific plasma cells from the bone marrow of a SIVmac239-infected RM with serum neutralization titers against SIVmac239. After picking plasma cells, we obtained 44 paired Ab sequences. Ten of these mAbs were SIV specific. Although none of these mAbs neutralized SIVmac239, three mAbs completely neutralized the related SIVmac316 strain. The majority of these mAbs bound to primary rhesus CD4+ T cells infected with SIVmac239 and induced Ab-dependent cellular cytotoxicity. This method is a first step in successful isolation of antigen-specific bone marrow-derived plasma cells from RMs.

Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance.IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

A single lentivector DNA based immunization contains a late heterologous SIVmac251 mucosal challenge infection

Variety of conventional vaccine strategies tested against HIV-1 have failed to induce protection against HIV acquisition or durable control of viremia. Therefore, innovative strategies that can induce long lasting protective immunity against HIV chronic infection are needed. Recently, we developed an integration-defective HIV lentiDNA vaccine that undergoes a single cycle of replication in target cells in which most viral antigens are produced. A single immunization with such lentiDNA induced long-lasting T-cell and modest antibody responses in cynomolgus macaques. Here eighteen months after this single immunization, all animals were subjected to repeated low dose intra-rectal challenges with a heterologous pathogenic SIVmac251 isolate. Although the viral set point in SIVmac-infected cynomolgus is commonly lower than that seen in Indian rhesus macaques, the vaccinated group of macaques displayed a two log reduction of peak of viremia followed by a progressive and sustained control of virus replication relative to control animals. This antiviral control correlated with antigen-specific CD4+ and CD8+ T cells with high capacity of recall responses comprising effector and central memory T cells but also memory T cell precursors. This is the first description of SIV control in NHP model infected at 18 months following a single immunization with a non-integrative single cycle lentiDNA HIV vaccine. While not delivering sterilizing immunity, our single immunization strategy with a single-cycle lentivector DNA vaccine appears to provide an interesting and safe vaccine platform that warrants further exploration.

Long-Term Delivery of an Anti-SIV Monoclonal Antibody With AAV.

Long-term delivery of anti-HIV monoclonal antibodies using adeno-associated virus (AAV) holds promise for the prevention and treatment of HIV infection. We previously reported that after receiving a single administration of AAV vector coding for anti-SIV antibody 5L7, monkey 84-05 achieved high levels of AAV-delivered 5L7 IgG1 in vivo which conferred sterile protection against six successive, escalating dose, intravenous challenges with highly infectious, highly pathogenic SIVmac239, including a final challenge with 10 animal infectious doses (1). Here we report that monkey 84-05 has successfully maintained 240–350 μg/ml of anti-SIV antibody 5L7 for over 6 years. Approximately 2% of the circulating IgG in this monkey is this one monoclonal antibody. This monkey generated little or no anti-drug antibodies (ADA) to the AAV-delivered antibody for the duration of the study. Due to the nature of the high-dose challenge used and in order to rule out a potential low-level infection not detected by regular viral loads, we have used ultrasensitive techniques to detect cell-associated viral DNA and RNA in PBMCs from this animal. In addition, we have tested serum from 84-05 by ELISA against overlapping peptides spanning the whole envelope sequence for SIVmac239 (PepScan) and against recombinant p27 and gp41 proteins. No reactivity has been detected in the ELISAs indicating the absence of naturally arising anti-SIV antibodies; moreover, the ultrasensitive cell-associated viral tests yielded no positive reaction. We conclude that macaque 84-05 was effectively protected and remained uninfected. Our data show that durable, continuous antibody expression can be achieved after one single administration of AAV and support the potential for lifelong protection against HIV from a single vector administration.

Lack of therapeutic efficacy of an antibody to α4β7 in SIVmac251-infected rhesus macaques.

Sustained virologic control of human immunodeficiency virus type 1 (HIV-1) infection after discontinuation of antiretroviral therapy (ART) is a major goal of the HIV-1 cure field. A recent study reported that administration of an antibody against α4β7 induced durable virologic control after ART discontinuation in 100% of rhesus macaques infected with an attenuated strain of simian immunodeficiency virus (SIV) containing a stop codon in nef We performed similar studies in 50 rhesus macaques infected with wild-type, pathogenic SIVmac251. In animals that initiated ART during either acute or chronic infection, anti-α4β7 antibody infusion had no detectable effect on the viral reservoir or viral rebound after ART discontinuation. These data demonstrate that anti-α4β7 antibody administration did not provide therapeutic efficacy in the model of pathogenic SIVmac251 infection of rhesus macaques.

CD8β Depletion Does Not Prevent Control of Viral Replication or Protection from Challenge in Macaques Chronically Infected with a Live Attenuated Simian Immunodeficiency Virus.

We evaluated the contribution of CD8αβ+ T cells to control of live-attenuated simian immunodeficiency virus (LASIV) replication during chronic infection and subsequent protection from pathogenic SIV challenge. Unlike previous reports with a CD8α-specific depleting monoclonal antibody (mAb), the CD8β-specific mAb CD8β255R1 selectively depleted CD8αβ+ T cells without also depleting non-CD8+ T cell populations that express CD8α, such as natural killer (NK) cells and γδ T cells. Following infusion with CD8β255R1, plasma viremia transiently increased coincident with declining peripheral CD8αβ+ T cells. Interestingly, plasma viremia returned to predepletion levels even when peripheral CD8αβ+ T cells did not. Although depletion of CD8αβ+ T cells in the lymph node (LN) was incomplete, frequencies of these cells were 3-fold lower (P = 0.006) in animals that received CD8β255R1 than in those that received control IgG. It is possible that these residual SIV-specific CD8αβ+ T cells may have contributed to suppression of viremia during chronic infection. We also determined whether infusion of CD8β255R1 in the LASIV-vaccinated animals increased their susceptibility to infection following intravenous challenge with pathogenic SIVmac239. We found that 7/8 animals infused with CD8β255R1, and 3/4 animals infused with the control IgG, were resistant to SIVmac239 infection. These results suggest that infusion with CD8β255R1 did not eliminate the protection afforded to LASIV vaccination. This provides a comprehensive description of the impact of CD8β255R1 infusion on the immunological composition in cynomolgus macaques, compared to an isotype-matched control IgG, while showing that the control of LASIV viremia and protection from challenge can occur even after CD8β255R1 administration.IMPORTANCE Studies of SIV-infected macaques that deplete CD8+ T cells in vivo with monoclonal antibodies have provided compelling evidence for their direct antiviral role. These studies utilized CD8α-specific mAbs that target both the major (CD8αβ+) and minor (CD8αα+) populations of CD8+ T cells but additionally deplete non-CD8+ T cell populations that express CD8α, such as NK cells and γδ T cells. In the current study, we administered the CD8β-specific depleting mAb CD8β255R1 to cynomolgus macaques chronically infected with a LASIV to selectively deplete CD8αβ+ T cells without removing CD8αα+ lymphocytes. We evaluated the impact on control of virus replication and protection from pathogenic SIVmac239 challenge. These results underscore the utility of CD8β255R1 for studying the direct contribution of CD8αβ+ T cells in various disease states.

Immune Protection of SIV Challenge by PD-1 Blockade During Vaccination in Rhesus Monkeys.

Though immune correlates for protection are still under investigation, potent cytotoxic T lymphocyte responses are desirable for an ideal HIV-1 vaccine. PD-1 blockade enhances SIV-specific CD8+ T cells. However, little information has been reported about how it affects the immunogenicity and protection of prophylactic SIV vaccines in nonhuman primates. Here, we show that PD-1 blockade during vaccination substantially improved protective efficacy in SIV challenged macaques. The PD-1 pathway was blocked using a monoclonal antibody specific to human PD-1. Administration of this antibody effectively augmented and sustained vaccine-induced SIV-specific T cell responses for more than 42 weeks after first immunization in rhesus monkeys, as compared with SIV vaccination only. Importantly, after intrarectally repeated low-dosage challenge with highly pathogenic SIVmac239, monkeys with PD-1 blockade during vaccination achieved full protection against incremental viral doses of up to 50,000 TICD50. These findings highlight the importance of PD-1 blockade during vaccination for the development of HIV vaccines.

CXCR4- and CCR5-Tropic HIV-1 Clones Are Both Tractable to Grow in Rhesus Macaques.

A major issue for present HIV-1 research is to establish model systems that reflect or mimic viral replication and pathogenesis actually observed in infected humans. To this end, various strategies using macaques as infection targets have long been pursued. In particular, experimental infections of rhesus macaques by HIV-1 derivatives have been believed to be best suited, if practicable, for studies on interaction of HIV-1 and humans under various circumstances. Recently, through in vitro genetic manipulations and viral cell-adaptations, we have successfully generated a series of HIV-1 derivatives with CXCR4-tropism or CCR5-tropism that grow in macaque cells to various degrees. Of these viruses, those with best replicative potentials can grow comparably with a pathogenic SIVmac in macaque cells by counteracting major restriction factors TRIM5, APOBEC3, and tetherin proteins. In this study, rhesus macaques were challenged with CXCR4-tropic (MN4/LSDQgtu) or CCR5-tropic (gtu + A4CI1) virus. The two viruses were found to productively infect rhesus macaques, being rhesus macaque-tropic HIV-1 (HIV-1rmt). However, plasma viral RNA was reduced to be an undetectable level in infected macaques at 5–6 weeks post-infection and thereafter. While replicated similarly well in rhesus peripheral blood mononuclear cells, MN4/LSDQgtu grew much better than gtu + A4CI1 in the animals. To the best of our knowledge, this is the first report demonstrating that HIV-1 derivatives (variants) grow in rhesus macaques. These viruses certainly constitute firm bases for generating HIV-1rmt clones pathogenic for rhesus monkeys, albeit they grow more poorly than pathogenic SIVmac and SHIV clones reported to date.

Mucosal antibody responses to vaccines targeting SIV protease cleavage sites or full-length Gag and Env proteins in Mauritian cynomolgus macaques.

HIV mutates rapidly and infects CD4+ T cells, especially when they are activated. A vaccine targeting conserved, essential viral elements while limiting CD4+ T cell activation could be effective. Learning from natural immunity observed in a group of highly HIV-1 exposed seronegative Kenyan female sex workers, we are testing a novel candidate HIV vaccine targeting the 12 viral protease cleavage sites (PCSs) (the PCS vaccine), in comparison with a vaccine targeting full-length Gag and Env (the Gag/Env vaccine) in a Mauritian cynomolgus macaque/SIV model. In this study we evaluated these vaccines for induction of mucosal antibodies to SIV immunogens at the female genital tract. Bio-Plex and Western blot analyses of cervicovaginal lavage samples showed that both the PCS and Gag/Env vaccines can elicit mucosal IgG antibody responses to SIV immunogens. Significantly higher increase of anti-PCS antibodies was induced by the PCS vaccine than by the Gag/Env vaccine (p<0.0001). The effect of the mucosal antibody responses in protection from repeated low dose pathogenic SIVmac251 challenges is being evaluated.

Lack of T-cell mediated IL2 and TNFa production is linked with the decreased CD58 expression in intestinal tissue during acute simian immunodeficiency virus infection

Abstract Human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection causes a progressive impairment of the immune system characterized by massive CD4+ T-cell loss, CD8+ T-cell expansion and sustained immune activation and inflammation. Our data also suggest that pathogenic SIVMAC251 infection in rhesus macaques (RMs) leads to decreased production of several T-helper 1 (TH1) and TH2 cytokines and increased production of IL-17, IFNg, CCL4 and GM-CSF by intestinal CD8+ T-cells 21 days after infection. For an effective T-cell activation and response, costimulation is required in addition to the antigen-specific signal from their antigen receptors. The CD2/CD58 interaction is considered as one of the important T-cell costimulatory pathway for T-cell activation and proliferation. Intestinal lamina propria T cells were reported to be highly responsive to a stimulus delivered by CD2 pathway compared to the conventional CD3/TCR pathway. The role of CD2/CD58 in regulating intestinal T-cell function of acute and chronic SIV infected RMs is poorly documented. Here, we demonstrated a significant reduction of CD58 expression in both T- and B-cell population during acute SIV infection along with the loss of intestinal CD4+ T-cells and high plasma viral load compared to SIV-uninfected normal RMs. The reduction of CD58 expression in T-cells was positively correlated with the reduced expression of T-cell mediated IL2 and TNFa production. Together, these results indicate that the reduction in CD2/CD58 interaction pathway in mucosal lymphocytes might play a crucial role in mucosal T-cell dysfunction during acute SIV/HIV infection.

Vector Order Determines Protection against Pathogenic Simian Immunodeficiency Virus Infection in a Triple-Component Vaccine by Balancing CD4+ and CD8+ T-Cell Responses.

An effective AIDS vaccine should elicit strong humoral and cellular immune responses while maintaining low levels of CD4+ T-cell activation to avoid the generation of target cells for viral infection. The present study investigated two prime-boost regimens, both starting vaccination with single-cycle immunodeficiency virus, followed by two mucosal boosts with either recombinant adenovirus (rAd) or fowlpox virus (rFWPV) expressing SIVmac239 or SIVmac251 gag/pol and env genes, respectively. Finally, vectors were switched and systemically administered to the reciprocal group of animals. Only mucosal rFWPV immunizations followed by systemic rAd boost significantly protected animals against a repeated low-dose intrarectal challenge with pathogenic SIVmac251, resulting in a vaccine efficacy (i.e., risk reduction per exposure) of 68%. Delayed viral acquisition was associated with higher levels of activated CD8+ T cells and Gag-specific gamma interferon (IFN-γ)-secreting CD8+ cells, low virus-specific CD4+ T-cell responses, and low Env antibody titers. In contrast, the systemic rFWPV boost induced strong virus-specific CD4+ T-cell activity. rAd and rFWPV also induced differential patterns of the innate immune responses, thereby possibly shaping the specific immunity. Plasma CXCL10 levels after final immunization correlated directly with virus-specific CD4+ T-cell responses and inversely with the number of exposures to infection. Also, the percentage of activated CD69+ CD8+ T cells correlated with the number of exposures to infection. Differential stimulation of the immune response likely provided the basis for the diverging levels of protection afforded by the vaccine regimen.IMPORTANCE A failed phase II AIDS vaccine trial led to the hypothesis that CD4+ T-cell activation can abrogate any potentially protective effects delivered by vaccination or promote acquisition of the virus because CD4+ T helper cells, required for an effective immune response, also represent the target cells for viral infection. We compared two vaccination protocols that elicited similar levels of Gag-specific immune responses in rhesus macaques. Only the animal group that had a low level of virus-specific CD4+ T cells in combination with high levels of activated CD8+ T cells was significantly protected from infection. Notably, protection was achieved despite the lack of appreciable Env antibody titers. Moreover, we show that both the vector and the route of immunization affected the level of CD4+ T-cell responses. Thus, mucosal immunization with FWPV-based vaccines should be considered a potent prime in prime-boost vaccination protocols.

Simian Immunodeficiency Virus Targeting of CXCR3+ CD4+ T Cells in Secondary Lymphoid Organs Is Associated with Robust CXCL10 Expression in Monocyte/Macrophage Subsets.

Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4+ T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4+ T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Here, we studied the host responses relevant to SIV targeting of CXCR3+ CCR5+ CD4+ T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3+ T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14+ CD16+ monocytes and MAC387+ macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387+ macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages.IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4+ T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3+ CCR5+ CD4+ T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3+ cells, in CD14+ CD16+ monocytes and MAC387+ macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes.

Chronic Δ(9)-Tetrahydrocannabinol Administration Reduces IgE(+)B Cells but Unlikely Enhances Pathogenic SIVmac251 Infection in Male Rhesus Macaques of Chinese Origin.

Delta9-tetrahydrocannabinol (Δ(9)-THC) is the major psychoactive component of the cannabis plant. Δ(9)-THC has been used in the active ingredient of Marinol as an appetite stimulant for AIDS patients. Its impact on progression of HIV-1 infection, however, remains debatable. Previous studies indicated that Δ(9)-THC administration enhanced HIV-1 infection in huPBL-SCID mice but seemingly decreased early mortality in simian immunodeficiency virus (SIV) infected male Indian-derived rhesus macaques. Here, we determine the chronic effect of Δ(9)-THC administration using0.32mg/kg or placebo (PBO), i.m., twice daily for 428days on SIVmac251 infected male Chinese-derived rhesus macaques. Sixteen animals were divided into four study groups: Δ(9)-THC(+)SIV(+), Δ(9)-THC(+)SIV(-), PBO/SIV(+) and PBO/SIV(-) (n=4/group). One-month after daily Δ(9)-THC or PBO administrations, macaques in groups one and three were challenged intravenously with pathogenic SIVmac251/CNS, which was isolated from the brain of a Chinese macaque with end-staged neuroAIDS. No significant differences in peak and steady state plasma viral loads were seen between Δ(9)-THC(+)SIV(+) and PBO/SIV(+) macaques. Regardless of Δ(9)-THC, all infected macaques displayed significant drop of CD4/CD8 T cell ratio, loss of CD4(+) T cells and higher persistent levels of Ki67(+)CD8(+) T cells compared with uninfected animals. Moreover, long-term Δ(9)-THC treatment reduced significantly the frequency of circulating IgE(+)B cells. Only one Δ(9)-THC(+)SIV(+) macaque died of simian AIDS with paralyzed limbs compared with two deaths in the PBO/SIV(+) group during the study period. These findings indicate that chronic Δ(9)-THC administration resulted in reduction of IgE(+)B cells, yet it unlikely enhanced pathogenic SIVmac251/CNS infection in male Rhesus macaques of Chinese origin.