Early Simian Immunodeficiency Virus Infection Research Articles

Identification of macaque dendritic cell precursors in blood and tissue reveals their dysregulation in early SIV infection

Distinct dendritic cell (DC) subsets play important roles in shaping immune responses. Circulating DC precursors (pre-DCs) are more susceptible to HIV infection invitro, which may explain the inefficiency of immune responses against HIV. However, the interplay between HIV and pre-DC is not defined invivo. We identify human pre-DC equivalents in the cynomolgus macaque and then analyze their dynamics during simian immunodeficiency virus (SIV) infection to illustrate a sharp decrease of blood pre-DCs in early SIV infection and accumulation in lymph nodes (LNs), where they neglect to upregulate CD83/CD86 or MHC-II. Additionally, SIV infection attenuates the capacity of stimulated LN pre-DCs to produce IL-12p40. Analysis of HIV cohorts provides correlation between costimulatory molecule expression on pre-DCs and Tcell activation in spontaneous HIV controllers. These findings pinpoint certain dynamics and functional changes of pre-DCs during SIV infection, providing a deeper understanding of immune dysregulation mechanisms elicited in people living with HIV.

CD8+ lymphocytes do not impact SIV reservoir establishment under ART

Persistence of the human immunodeficiency virus type-1 (HIV-1) latent reservoir in infected individuals remains a problem despite fully suppressive antiretroviral therapy (ART). While reservoir formation begins during acute infection, the mechanisms responsible for its establishment remain unclear. CD8+ T cells are important during the initial control of viral replication. Here we examined the effect of CD8+ T cells on formation of the latent reservoir in simian immunodeficiency virus (SIV)-infected macaques by performing experimental CD8+ depletion either before infection or before early (that is, day 14 post-infection) ART initiation. We found that CD8+ depletion resulted in slower decline of viremia, indicating that CD8+ lymphocytes reduce the average lifespan of productively infected cells during acute infection and early ART, presumably through SIV-specific cytotoxic T lymphocyte (CTL) activity. However, CD8+ depletion did not change the frequency of infected CD4+ T cells in the blood or lymph node as measured by the total cell-associated viral DNA or intact provirus DNA assay. In addition, the size of the persistent reservoir remained the same when measuring the kinetics of virus rebound after ART interruption. These data indicate that during early SIV infection, the viral reservoir that persists under ART is established largely independent of CTL control.

Rapid Loss of CD4 T Cells by Pyroptosis during Acute SIV Infection in Rhesus Macaques.

ABSTRACTThe mechanisms underlying depletion of CD4 T cells during acute HIV-1 infection are not well understood. Here we show that caspase-1-induced pyroptosis, a highly inflammatory programmed cell death pathway, is the dominant mechanism responsible for the rapid depletion of CD4 T cells in gut-associated lymphatic tissue (GALT), spleen, and lymph nodes during acute simian immunodeficiency virus (SIV) infection in rhesus macaques. Upregulation of interferon-gamma inducible factor 16, a host DNA sensor that triggers pyroptosis, was also observed in tissue-resident CD4 T cells and correlated with viral loads and CD4 T cell loss. In contrast, caspase-3-mediated apoptosis and viral cytotoxicity only accounted for a small fraction of CD4 T cell death. Other programmed cell death mechanisms, including mitochondria-induced caspase-independent cell death, necroptosis, and autophagy, did not significantly contribute to CD4 T cell depletion. These data support a model in which caspase-1-mediated pyroptosis is the principal mechanism that results in CD4 T cell loss in the GALT and lymphoid organs and release of proinflammatory cytokines. These findings contribute to our understanding of the pathogenesis of acute SIV infection and have important implications for the development of therapeutic strategies.IMPORTANCE Different mechanisms for CD4 T cell depletion during acute HIV-1 infection have been proposed. In this study, we demonstrate that in early simian immunodeficiency virus infection, depletion of CD4 T cells is primarily due to pyroptosis. Other mechanisms may also contribute in a minor way to CD4 T cell depletion.

CD4 Tcells are rapidly depleted from tuberculosis granulomas following acute SIV co-infection.

HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals have an increased risk of tuberculosis prior to loss of peripheral CD4 Tcells, raising the possibility that HIV co-infection leads to CD4 Tcell depletion in lung tissue before it is evident in blood. Here, we use rhesus macaques to study the early effects of simian immunodeficiency virus (SIV) co-infection on pulmonary granulomas. Two weeks after SIV inoculation of Mtb-infected macaques, Mtb-specific CD4 Tcells are dramatically depleted from granulomas, before CD4 Tcell loss in blood, airways, and lymph nodes, or increases in bacterial loads or radiographic evidence of disease. Spatially, CD4 Tcells are preferentially depleted from the granuloma core and cuff relative to B cell-rich regions. Moreover, live imaging of granuloma explants show that intralesional CD4 Tcell motility is reduced after SIV co-infection. Thus, granuloma CD4 Tcells may be decimated before many co-infected individuals experience the first symptoms of acute HIV infection.

Jejunal epithelial barrier disruption triggered by reactive oxygen species in early SIV infected rhesus macaques

Intestinal epithelial barrier destruction occurs earlier than mucosal immune dysfunction in the acute stage of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections. At present, however, the cause of compromised gastrointestinal integrity in early SIV infection remains unknown. In the current study, we investigated the effects of SIV infection on epithelial barrier integrity and explored oxidative stress-mediated DNA damage and apoptosis in epithelial cells from early acute SIVmac239-infected Chinese rhesus macaques (Macaca mulatta). Results showed that the sensitive molecular marker of small intestinal barrier dysfunction, i.e., intestinal fatty acid-binding protein (IFABP), was significantly increased in plasma at 14 days post-SIV infection. SIV infection induced a profound decrease in the expression of tight junction proteins, including claudin-1, claudin-3, and zonula occludens (ZO)-1, as well as a significant increase in the active form of caspase-3 level in epithelial cells. RNA sequencing (RNA-seq) analysis suggested that differentially expressed genes between pre- and post-SIV-infected jejuna were enriched in pathways involved in cell redox homeostasis, oxidoreductase activity, and mitochondria. Indeed, a SIV-mediated increase in reactive oxygen species (ROS) in the epithelium and macrophages, as well as an increase in hydrogen peroxide (H2O2) and decrease in glutathione (GSH)/glutathione disulfide (GSSG) antioxidant defense, were observed in SIV-infected jejuna. In addition, the accumulation of mitochondrial dysfunction and DNA oxidative damage led to an increase in senescence-associated β-galactosidase (SA-β-gal) and early apoptosis in intestinal epithelial cells. Furthermore, HIV-1 Tat protein-induced epithelial monolayer disruption in HT-29 cells was rescued by antioxidant N-acetylcysteine (NAC). These results indicate that mitochondrial dysfunction and oxidative stress in jejunal epithelial cells are primary contributors to gut epithelial barrier disruption in early SIV-infected rhesus macaques.

HIV-1 and SIV Infection Are Associated with Early Loss of Lung Interstitial CD4+ T Cells and Dissemination of Pulmonary Tuberculosis

SUMMARYLung interstitial CD4+ T cells are critical for protection against pulmonary infections, but the fate of this population during HIV-1 infection is not well described. We studied CD4+ T cells in the setting of HIV-1 infection in human lung tissue, humanized mice, and a Mycobacterium tuberculosis (Mtb)/simian immunodeficiency virus (SIV) nonhuman primate co-infection model. Infection with a CCR5-tropic strain of HIV-1 or SIV results in severe and rapid loss of lung interstitial CD4+ T cells but not blood or lung alveolar CD4+ T cells. This is accompanied by high HIV-1 production in these cells in vitro and in vivo. Importantly, during early SIV infection, loss of lung interstitial CD4+ T cells is associated with increased dissemination of pulmonary Mtb infection. We show that lung interstitial CD4+ T cells serve as an efficient target for HIV-1 and SIV infection that leads to their early depletion and an increased risk of disseminated tuberculosis.

High-Resolution Sequencing of Viral Populations during Early Simian Immunodeficiency Virus Infection Reveals Evolutionary Strategies for Rapid Escape from Emerging Env-Specific Antibody Responses.

Primate lentiviruses, including the human and simian immunodeficiency viruses (HIV and SIV), produce infections marked by persistent, ongoing viral replication. This occurs despite the presence of virus-specific adaptive immune responses, including antibodies targeting the viral envelope glycoprotein (Env), and evolution of antibody-escape variants is a well-documented feature of lentiviral infection. Here, we examined the evolutionary dynamics of the SIV env gene during early infection (≤29 weeks postinfection) in a cohort of four SIVmac251-infected rhesus macaques. We tracked env evolution during acute and early infection using frequent sampling and ultradeep sequencing of viral populations, capturing a transmission bottleneck and the subsequent reestablishment of Env diversity. A majority of changes in the gp120 subunit mapped to two short clusters, one in the first variable region (V1) and one in V4, while most changes in the gp41 subunit appeared in the cytoplasmic domain. Variation in V1 was dominated by short duplications and deletions of repetitive sequence, while variation in V4 was marked by short in-frame deletions and closely overlapping substitutions. The most common substitutions in both patches did not alter viral replicative fitness when tested using a highly sensitive, deep-sequencing-based competition assay. Our results, together with the observation that very similar or identical patterns of sequence evolution also occur in different macaque species infected with related but divergent strains of SIV, suggest that resistance to early, strain-specific anti-Env antibodies is the result of temporally and mutationally predictable pathways of escape that occur during the early stages of infection.IMPORTANCE The envelope glycoprotein (Env) of primate lentiviruses mediates entry by binding to host cell receptors followed by fusion of the viral membrane with the cell membrane. The exposure of Env complexes on the surface of the virion results in targeting by antibodies, leading to selection for virus escape mutations. We used the SIV/rhesus macaque model to track in vivo evolution of variation in Env during acute/early infection in animals with and without antibody responses to Env, uncovering remarkable variation in animals with antibody responses within weeks of infection. Using a deep-sequencing-based fitness assay, we found substitutions associated with antibody escape had little to no effect on inherent replicative capacity. The ability to readily propagate advantageous changes that incur little to no replicative fitness costs may be a mechanism to maintain continuous replication under constant immune selection, allowing the virus to persist for months to years in the infected host.

Is a Human CD8 T-Cell Vaccine Possible, and if So, What Would It Take? Could a CD8+ T-Cell Vaccine Prevent Persistent HIV Infection?

Vaccines that stimulate CD8+ T cells could clear early virus infection or control ongoing infection and prevent disease. This could be valuable to combat human immunodeficiency virus type 1 (HIV-1) where it has not yet been possible to generate broadly reacting neutralizing antibodies with a vaccine. However, HIV-1 vaccines aimed at stimulating CD8+ T cells have had no success. In contrast, a cytomegalovirus vectored simian immunodeficiency virus (SIV) vaccine enabled clearance of early SIV infection. This may open the door to the design of an effective HIV vaccine.

Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization.

Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8(+) cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8(+) cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)(hi)/phosphorylated AMP-activated protein kinase (AMPK)(lo) subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control. In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.

Next-Generation mRNA Sequencing Reveals Pyroptosis-Induced CD4+ T Cell Death in Early Simian Immunodeficiency Virus-Infected Lymphoid Tissues.

Lymphoid tissues (LTs) are the principal sites where human immunodeficiency virus type 1 (HIV-1) replicates and virus-host interactions take place, resulting in immunopathology in the form of inflammation, immune activation, and CD4(+) T cell death. The HIV-1 pathogenesis in LTs has been extensively studied; however, our understanding of the virus-host interactions in the very early stages of infection remains incomplete. We investigated virus-host interactions in the rectal draining lymph nodes (dLNs) of rhesus macaques at different times after intrarectal inoculation (days postinoculation [dpi]) with simian immunodeficiency virus (SIV). At 3 dpi, 103 differentially expressed genes (DEGs) were detected using next-generation mRNA sequencing (RNA-seq). At 6 and 10 dpi, concomitant with increased SIV replication, 366 and 1,350 DEGs were detected, respectively, including upregulation of genes encoding proteins that play a role in innate antiviral immune responses, inflammation, and immune activation. Notably, genes (IFI16, caspase-1, and interleukin 1β [IL-1β]) in the canonical pyroptosis pathway were significantly upregulated in expression. We further validated increased pyroptosis using flow cytometry and found that the number of CD4(+) T cells expressing activated caspase-1 protein, the hallmark of ongoing pyroptosis, were significantly increased, which is correlated with decreased CD4(+) T cells in dLNs. Our results demonstrated that pyroptosis contributes to the CD4(+) T cell death in vivo in early SIV infection, which suggests that pyroptosis may play a pivotal role in the pathogenesis of SIV, and by extension, that of HIV-1, since pyroptosis not only induces CD4(+) T cell death but also amplifies inflammation and immune activation. Thus, blocking CD4(+) T cell pyroptosis could be a complementary treatment to antiretroviral therapy. Although secondary lymphoid tissues (LTs) are principal sites of human immunodeficiency virus type 1 (HIV-1) replication, inflammation, immune activation, and CD4(+) T cell death, immunopathogenesis in LTs during early infection remains largely unknown. Using the simian immunodeficiency virus (SIV)/rhesus monkey model of HIV rectal infection, we investigated early virus-host interactions. Our results revealed elevated potent host responses in early infection in LTs, including upregulation of genes involved in antiviral immune response, inflammation, and immune activation. Importantly, genes involved in the canonical pyroptosis pathway were significantly upregulated, and there was a strong correlation between CD4(+) T cell decrease and increased number of CD4(+) T cells expressing activated caspase-1 protein, demonstrating that pyroptosis contributes to CD4(+) T cell death in vivo in very early SIV infection. Our finding suggests that blocking pyroptosis may be able to decrease CD4(+) T cell loss during early SIV infection.

Dysregulation of myelopoiesis by chronic alcohol administration during early SIV infection of rhesus macaques.

Chronic alcohol intoxication suppresses immune function and increases osteoporosis risk suggesting bone-tissue cytotoxicity. Human immunodeficiency virus infection leads to similar impairments. This study investigated the effects of chronic alcohol administration during the early stage of simian immunodeficiency virus (SIV) infection on hematopoietic stem and progenitor cells (HSPCs) and their differentiated progeny in the bone marrow and peripheral blood of rhesus macaques. Rhesus macaques were administered alcohol or sucrose daily for a period of 3 months prior to intrarectal inoculation with 250 TCID50 of SIVmac251 . Bone marrow aspirates and blood samples were taken prior to and 2 weeks after SIV infection. Bone marrow cells (BMCs) were assessed using flow cytometric phenotyping for upstream HSPCs and for differentiated cells of the monocyte-granulocyte lineages. Likewise, cells were quantitated in peripheral blood. Of the bone marrow HSPCs, only the common lymphoid progenitor (CLP) was altered by alcohol administration pre-SIV (38 ± 9.4/10(6) BMCs vs. 226 ± 64.1/10(6) BMCs, sucrose vs. alcohol). Post-SIV, the frequency of CLPs in the bone marrow of alcohol-administered macaques decreased compared with the sucrose-administered macaques (107 ± 47.6/10(6) BMCs vs. 43 ± 16.3/10(6) BMCs). However, marrow mature cells of the monocyte lineage, specifically macrophages and osteoclast progenitors, were increased by both chronic alcohol administration and SIV infection (287% and 662%, respectively). As expected, mature cells such as granulocytes (polymorphonuclear cells), B cells, and CD4+ T cells in the peripheral blood were decreased by SIV infection (37 to 62% decline from preinfection), but not affected after 3 months of chronic alcohol administration. Chronic alcohol administration disrupts myelomonocytic development in the bone marrow during the early period of SIV infection promoting macrophage and osteoclast lineages. We predict this shift in CLP:macrophage/osteoclast balance creates an environment that favors bone resorption and immunosuppression.

Blocking TLR7- and TLR9-mediated IFN-α Production by Plasmacytoid Dendritic Cells Does Not Diminish Immune Activation in Early SIV Infection

Persistent production of type I interferon (IFN) by activated plasmacytoid dendritic cells (pDC) is a leading model to explain chronic immune activation in human immunodeficiency virus (HIV) infection but direct evidence for this is lacking. We used a dual antagonist of Toll-like receptor (TLR) 7 and TLR9 to selectively inhibit responses of pDC but not other mononuclear phagocytes to viral RNA prior to and for 8 weeks following pathogenic simian immunodeficiency virus (SIV) infection of rhesus macaques. We show that pDC are major but not exclusive producers of IFN-α that rapidly become unresponsive to virus stimulation following SIV infection, whereas myeloid DC gain the capacity to produce IFN-α, albeit at low levels. pDC mediate a marked but transient IFN-α response in lymph nodes during the acute phase that is blocked by administration of TLR7 and TLR9 antagonist without impacting pDC recruitment. TLR7 and TLR9 blockade did not impact virus load or the acute IFN-α response in plasma and had minimal effect on expression of IFN-stimulated genes in both blood and lymph node. TLR7 and TLR9 blockade did not prevent activation of memory CD4+ and CD8+ T cells in blood or lymph node but led to significant increases in proliferation of both subsets in blood following SIV infection. Our findings reveal that virus-mediated activation of pDC through TLR7 and TLR9 contributes to substantial but transient IFN-α production following pathogenic SIV infection. However, the data indicate that pDC activation and IFN-α production are unlikely to be major factors in driving immune activation in early infection. Based on these findings therapeutic strategies aimed at blocking pDC function and IFN-α production may not reduce HIV-associated immunopathology.

KIR2DL4 copy number variation is associated with CD4+ T-cell depletion and function of cytokine-producing NK cell subsets in SIV-infected Mamu-A*01-negative rhesus macaques.

Here, we demonstrate that KIR2DL4 copy number variation (CNV) is associated with CD4(+) T-cell decline and functionality of cytokine-producing NK cells during primary simian immunodeficiency virus (SIV) infection in Mamu-A*01(-) Indian-origin rhesus macaques, with higher KIR2DL4 copy numbers being associated with a better preservation of CD4(+) T cells and an increased gamma interferon (IFN-γ) production from stimulated cytokine-producing NK cell subsets during acute SIVmac251 infection. These findings underscore the crucial role of activating killer-cell immunoglobulin-like receptors (KIRs) in NK cell-mediated SIV responses during early SIV infection.

Trivalent Live Attenuated Influenza-Simian Immunodeficiency Virus Vaccines: Efficacy and Evolution of Cytotoxic T Lymphocyte Escape in Macaques

There is an urgent need for a human immunodeficiency virus (HIV) vaccine that induces robust mucosal immunity. CD8(+) cytotoxic T lymphocytes (CTLs) apply substantial antiviral pressure, but CTLs to individual epitopes select for immune escape variants in both HIV in humans and SIV in macaques. Inducing multiple simian immunodeficiency virus (SIV)-specific CTLs may assist in controlling viremia. We vaccinated 10 Mane-A1*08401(+) female pigtail macaques with recombinant influenza viruses expressing three Mane-A1*08401-restricted SIV-specific CTL epitopes and subsequently challenged the animals, along with five controls, intravaginally with SIV(mac251). Seroconversion to the influenza virus vector resulted and small, but detectable, SIV-specific CTL responses were induced. There was a boost in CTL responses after challenge but no protection from high-level viremia or CD4 depletion was observed. All three CTL epitopes underwent a coordinated pattern of immune escape during early SIV infection. CTL escape was more rapid in the vaccinees than in the controls at the more dominant CTL epitopes. Although CTL escape can incur a "fitness" cost to the virus, a putative compensatory mutation 20 amino acids upstream from an immunodominant Gag CTL epitope also evolved soon after the primary CTL escape mutation. We conclude that vaccines based only on CTL epitopes will likely be undermined by rapid evolution of both CTL escape and compensatory mutations. More potent and possibly broader immune responses may be required to protect pigtail macaques from SIV.

Distinct Expression Patterns of CD69 in Mucosal and Systemic Lymphoid Tissues in Primary SIV Infection of Rhesus Macaques

Although the intestinal tract plays a major role in early human immunodeficiency virus (HIV) infection, the role of immune activation and viral replication in intestinal tissues is not completely understood. Further, increasing evidence suggests the early leukocyte activation antigen CD69 may be involved in the development or regulation of important T cell subsets, as well as a major regulatory molecule of immune responses. Using the simian immunodeficiency virus (SIV) rhesus macaque model, we compared expression of CD69 on T cells from the intestine, spleen, lymph nodes, and blood of normal and SIV-infected macaques throughout infection. In uninfected macaques, the majority of intestinal lamina propria CD4+ T cells had a memory (CD95+) phenotype and co-expressed CD69, and essentially all intestinal CCR5+ cells co-expressed CD69. In contrast, systemic lymphoid tissues had far fewer CD69+ T cells, and many had a naïve phenotype. Further, marked, selective depletion of intestinal CD4+CD69+ T cells occurred in early SIV infection, and this depletion persisted throughout infection. Markedly increased levels of CD8+CD69+ T cells were detected after SIV infection in virtually all tissues, including the intestine. Further, confocal microscopy demonstrated selective, productive infection of CD3+CD69+ T cells in the intestine in early infection. Combined, these results indicate CD69+CD4+ T cells are a major early target for viral infection, and their rapid loss by direct infection may have profound effects on intestinal immune regulation in HIV infected patients.

Is the Gut the Major Source of Virus in Early Simian Immunodeficiency Virus Infection?

The acute phases of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection are characterized by rapid and profound depletion of CD4+ T cells from the guts of infected individuals. The large number of CD4+ T cells in the gut (a large fraction of which are activated and express the HIV/SIV coreceptor CCR5), the high level of infection of these cells, and the temporal coincidence of this CD4+ T-cell depletion with the peak of virus in plasma in acute infection suggest that the intestinal mucosa may be the major source of virus driving the peak viral load. Here, we used data on CD4+ T-cell proportions in the lamina propria of the rectums of SIV-infected rhesus macaques (which progress to AIDS) and sooty mangabeys (which do not progress) to show that in both species, the depletion of CD4+ T cells from this mucosal site and its maximum loss rate are often observed several days before the peak in viral load, with few CD4+ T cells remaining in the rectum by the time of peak viral load. In contrast, the maximum loss rate of CD4+ T cells from bronchoalveolar lavage specimens and lymph nodes coincides with the peak in virus. Analysis of the kinetics of depletion suggests that, in both rhesus macaques and sooty mangabeys, CD4+ T cells in the intestinal mucosa are a highly susceptible population for infection but not a major source of plasma virus in acute SIV infection.

Chronic cannabinoid administration increases CD8+ lymphocyte apoptosis during early Simian Immunodeficiency Virus infection in rhesus macaques.

Δ9‐tetrahydrocannabinol (Δ9‐THC), the primary psychoactive component of marijuana, is used to ameliorate Acquired Immune Deficiency Syndrome associated wasting. Cannabinoid receptors are expressed in cells of the immune system, raising the possibility that chronic Δ9‐THC use may impact on immune function, potentially affecting progression of human immunodeficiency virus infection. We examined the effects of chronic Δ9‐THC administration (0.32mg/kg i.m., 2 X daily, starting 30 d pre‐infection) on the early changes in lymphocyte counts, subpopulations, apoptosis, and proliferation in young adult male rhesus macaques at day 30 post simian immunodeficiency virus (SIVmac251; 100 TCID50/ml, i.v.) infection. SIV+ animals had lower (40%) lymphocyte CD4+/CD8+ ratio, 3‐7‐fold higher CD8+Ki67 expression (proliferation marker), without significant changes in total lymphocyte count, or CD4+ and CD8+caspase 3 expression (apoptosis marker) than time‐matched SIV‐ controls. THC‐treated SIV+ animals showed similarly decreased CD4+/CD8+, increased CD8+Ki67 expression, but higher expression of caspase 3 in CD8+ lymphocytes than time‐matched THC‐treated SIV‐ controls. We speculate that though modest the Δ9‐THC‐induced increase in CD8+ apoptosis may accentuate the CD4+/CD8+ imbalance during later stages of SIV infection. Supported NIDA‐020419‐01A1

The Gastrointestinal Tract and AIDS Pathogenesis

Gastrointestinal disease has been recognized as a major manifestation of human immunodeficiency virus infection since the earliest recognition of acquired immunodeficiency syndrome (AIDS). Originally, these disease manifestations were considered to be sequelae of the immune destruction that characterizes AIDS rather than being central to the pathogenesis of AIDS. Over time, it has become clear that the mucosal immune system in general and the intestinal immune system in particular are central to the pathogenesis of AIDS, with most of the critical events (eg, transmission, viral amplification, CD4+ T-cell destruction) occurring in the gastrointestinal tract. Compared with peripheral blood, these tissues are not easily accessible for analysis and have only begun to be examined in detail recently. In addition, although the resulting disease can progress over years, many critical events happen within the first few weeks of infection, when most patients are unaware that they are infected. Moreover, breakdown of the mucosal barrier and resulting microbial translocation are believed to be major drivers of AIDS progression. In this review, we focus on the interaction between primate lentiviruses and the gastrointestinal tract and discuss how this interaction promotes the pathogenesis of AIDS and drives immune dysfunction and progression to AIDS. This article draws extensively on work done in the nonhuman primate model of AIDS to fill gaps in our understanding of AIDS in humans.

Inactivated Simian Immunodeficiency Virus-Pulsed Autologous Fresh Blood Cells as an Immunotherapy Strategy

Practical immunotherapies for human immunodeficiency virus infection are needed. We evaluated inactivated simian immunodeficiency virus (SIV) pulsed onto fresh peripheral blood mononuclear cells in 12 pigtail macaques with chronic SIV(mac251) infection for T-cell immunogenicity in a randomized cross-over design study. The immunotherapy was safe and convincingly induced high levels of SIV-specific CD4(+) T-cell responses (mean, 5.9% +/- 1.3% of all CD4(+) T cells) and to a lesser extent SIV-specific CD8(+) T-cell responses (mean, 0.7% +/- 0.4%). Responses were primarily directed toward Gag and less frequently toward Env but not Pol or regulatory/accessory SIV proteins. T-cell responses against Gag were generally broad and polyfunctional, with a mean of 2.7 CD4(+) T-cell epitopes mapped per animal and more than half of the SIV Gag-specific CD4(+) T cells expressing three or more effector molecules. The immunogenicity was comparable to that found in previous studies of peptide-pulsed blood cells. Despite the high-level immunogenicity, no reduction in viral load was observed in the chronically viremic macaques. This contrasts with our studies of immunization with peptide-pulsed blood cells during early SIV infection in macaques. Future studies of inactivated virus-pulsed blood cell immunotherapy during early infection of patients receiving antiretroviral therapy are warranted.

Expression and regulation of the two cyclooxygenase isoforms in rhesus macaque brain during lentivirus infection: differentiation of reversible inflammatory and irreversible neuronal sequelae

The regulation of cyclooxygenase type-1 and type-2 (COX-1, COX-2), key enzymes in the conversion of arachidonic acid to prostanoids and mediators of inflammation, was investigated in the brain of simian immunodeficiency virus (SIV)-infected rhesus macaques. COX-1 and COX-2 were constitutively expressed in hippocampal and cortical neurons. COX-1 was additionally found to be expressed in cholinergic basal forebrain neurons (septum, diagonal band of Broca and nucleus basalis of Meynert). This expression was highly species-specific, as COX-1 was coexpressed with specific cholinergic markers choline acetyl transeferase, vesicular acetyl choline transporter (VAChT) and high affinity choline transporter in these neurons, in both the rhesus macaque and human, but was absent from basal forebrain cholinergic neurons in rat, mouse and guinea pig brain. COX-1 immunoreactivity was decreased more than 50% in cholinergic projection neurons of the basal forebrain in AIDS-diseased monkeys. Decreased COX-1 expression was accompanied by a decrease of VAChT-positive fibers and terminals in parietal and frontal cortex, and hippocampus. Additionally, COX-1 and COX-2 were downregulated in frontal cortex neurons in AIDS. Nonneuronally, only COX-1 was expressed constitutively in microglia and endothelial cells, and was not changed in early SIV infection. Late stage of disease was characterized by upregulation of COX-1 in scattered microglia/macrophages, macrophage nodules and multinucleated giant cells. Endothelial COX-1 expression was unchanged during SIV infection. In AIDS-symptomatic monkey brains COX-2 was induced focally in nodule and syncytium forming macrophages and in endothelial cells in areas of viral burden and inflammatory cell infiltrates. Treatment with the antiretroviral brain-permeant 6-chloro-2',3'-dideoxyguanosine (6-Cl-ddG) substantially reduced focal expression of COX-1 and COX-2 in AIDS, but diffuse COX-1 microgliosis was not. 6-Cl-ddG did not decrease COX-1 immunoreactivity in cortical and cholinergic basal forebrain neurons and cortical cholinergic fiber loss. We conclude that COX-1 coexpression is a feature of the cholinergic basal forebrain telecephalic projection system specific to primates, and may be important in primate-specific functions of this system, and in the cholinergic dysregulation that accompanies primate lentiviral encephalitis. Nonneuronal COX-2 induction is associated with reversible overt inflammatory events to areas of active viral replication. Treatment with cyclooxygenase-inhibitors and central cholinomimetica might be beneficial beside highly active antiretroviral therapy in immunodeficiency virus-induced encephalopathy and encephalitis.