SIV-infected Monkeys Research Articles

Translating the brain transcriptome in neuroAIDS: from non-human primates to humans.

In the post-human genome project era, high throughput techniques to detect and computational algorithms to analyze differentially expressed genes have proven to be powerful tools for studying pathogenesis of neuroAIDS. Concurrently, discovery of non-coding RNAs and their role in development and disease has underscored the importance of examining the entire transcriptome instead of protein coding genes alone. Herein, we review the documented changes in brain RNA expression profiles in the non-human primate model of neuroAIDS (SIV infected monkeys) and compare the findings to those resulting from studies in post-mortem human samples of neuroAIDS. Differential expression of mRNAs involved in inflammation and immune response are a common finding in both monkey and human samples - even in HIV infected people on combination antiretroviral therapy, a shared set of genes is upregulated in the brains of both infected monkeys and humans: B2M, IFI44, IFIT3, MX1, STAT1. Additionally, alterations in ion channel encoding genes have been observed in the human studies. Brain miRNA profiling has also been performed, and up-regulation of two miRNAs originating from the same transcript, miR-142-3p and miR-142-5p, is common to human and monkey neuroAIDS studies. With increases in knowledge about the genome and advances in technology, unraveling alterations in the transcriptome in the SIV/monkey model will continue to enrich our knowledge about the effects of HIV on the brain.

Monocyte Mobilization, Activation Markers, and Unique Macrophage Populations in the Brain: Observations from SIV Infected Monkeys Are Informative with Regard to Pathogenic Mechanisms of HIV Infection in Humans

Non-human primate models of AIDS and neuroAIDS have been useful to study AIDS in humans in general and neuroAIDS in particular. Important information concerning target cells of infection, mechanisms of immune activation and pathology and cell traffic has been made in non-human primate models. To date observations in SIV infected monkey models have predicted or paralleled monocyte/macrophage biology with HIV infection and neuroAIDS. In this brief review we discuss a CD8+ T lymphocyte depletion model of rapid AIDS which results in a high incidence of SIV encephalitis. Specifically we review recent observations we have made using this model concerning monocyte turnover, monocyte/macrophage activation, macrophage derived biomarkers of disease and novel therapeutic approaches to AIDS and CNS pathology. Importantly, all observations made in the rapid model of AIDS discussed here are important and relevant to HIV infection of humans, even in the current era of anti-retroviral therapy that maintains HIV in plasma below the limit of detection.

Association of TLR7 variants with AIDS-like disease and AIDS vaccine efficacy in rhesus macaques.

In HIV infection, TLR7-triggered IFN-α production exerts a direct antiviral effect through the inhibition of viral replication, but may also be involved in immune pathogenesis leading to AIDS. TLR7 could also be an important mediator of vaccine efficacy. In this study, we analyzed polymorphisms in the X-linked TLR7 gene in the rhesus macaque model of AIDS. Upon resequencing of the TLR7 gene in 36 rhesus macaques of Indian origin, 12 polymorphic sites were detected. Next, we identified three tightly linked single nucleotide polymorphisms (SNP) as being associated with survival time. Genotyping of 119 untreated, simian immunodeficiency virus (SIV)-infected male rhesus macaques, including an ‘MHC adjusted’ subset, revealed that the three TLR7 SNPs are also significantly associated with set-point viral load. Surprisingly, this effect was not observed in 72 immunized SIV-infected male monkeys. We hypothesize (i) that SNP c.13G>A in the leader peptide is causative for the observed genotype-phenotype association and that (ii) the underlying mechanism is related to RNA secondary structure formation. Therefore, we investigated a fourth SNP (c.-17C>T), located 17 bp upstream of the ATG translation initiation codon, that is also potentially capable of influencing RNA structure. In c.13A carriers, neither set-point viral load nor survival time were related to the c.-17C>T genotype. In c.13G carriers, by contrast, the c.-17C allele was significantly associated with prolonged survival. Again, no such association was detected among immunized SIV-infected macaques. Our results highlight the dual role of TLR7 in immunodeficiency virus infection and vaccination and imply that it may be important to control human AIDS vaccine trials, not only for MHC genotype, but also for TLR7 genotype.

Targeting the glycans of the HIV envelope as a therapeutic tool

Carbohydrate-binding agents (CBA) represent a broad family of agents that recognize specific glycan conformations. Several members of this compound family including peptidicprokaryotic, plant, invertebrate and vertebrate lectins, but also the well-defined non-peptidicpradimicin and benanomicin antibiotics have shown to be endowed with anti-HIV activity. These compounds inhibit entry of virus particles in susceptible T-lymphocytes and macrophages, block syncytia formation between HIV-infected and non-infected T-lymphocytes, prevent capture of HIV by DC-SIGN-expressing cells and by the macrophage mannose receptor and prevent subsequent transmission of captured HIV to susceptible T-lymphocytes. No other antiviral class of compounds have been demonstrated to block these four important ways of HIV infection/transmission, which make the CBAs an interesting new family of potential anti-HIV drugs that may be particularly useful for both systemic and topical (i.e. microbicidal) applications. CBAs bind to the glycans of the HIV envelope gpl 20 and block viral entry, most likely by freezing the envelope conformation during viral entry. Escalating drug pressure in HIV-infected cell cultures result in the selection of mutant virus strains predominantly having one or several N-glycan deletions in their envelope gp120.ln this respect, many CBAs have a high genetic barrier since several N-glycan deletions seem to be required for significant phenotypic drug resistance. Interestingly, mutant virus strains containing an increasing amount of N-glycan deletions in gp120 tend to show a compromised infectivity potential. Moreover, we have found one specific, highly conserved N-glycan in HIV-1 gp120, whose deletion results in the production of noninfectious virus particles lacking gp41gp120 in their envelope. This N-glycan may be regarded as a hot-spot for a targeted therapeutic intervention. Also, it is expected that if mutant virus particles lacking N-glycans in gp120 are selected under CBA pressure in vivo, as shown to occur in drug-exposed HIV-and SIV-infected cell cultures, previously hidden conserved immunogenic epitopes become exposed, allowing the immune system to specifically trigger an efficient immune response against these viruses. An in vivo study using SIV-infected monkeys has been planned to explore this new therapeutic concept.

Changes in the plasma proteome follows chronic opiate administration in simian immunodeficiency virus infected rhesus macaques

Background Substantive plasma proteomic changes follow lentiviral infection and disease pathobiology. We posit that such protein alterations are modified during drug abuse, further serving to affect the disease. To this end, we investigated the effect of opiate administration on the plasma proteome of Indian-strain rhesus monkeys infected with simian immunodeficiency virus (SIV) strain smm9. Methods Whole blood was collected at 7 weeks prior to and 1.4 and 49 weeks after viral infection. Viral load, CD4 + T cell subsets, and plasma protein content were measured from monkeys that did or did not receive continuous opiate administrations. The plasma proteome was identified and quantified by isobaric tags for relative and absolute quantitation labeling (iTRAQ) and mass spectrometry. Results While substantive changes in plasma proteins were seen during SIV infection, the addition of opiates led to suppression of these changes as well as increased variance of the proteome. These changes demonstrate that opiates induce broad but variant immune suppression in SIV-infected monkeys. Conclusion The broad suppressive changes seen in plasma of SIV-infected monkeys likely reflect reduced multisystem immune homeostatic responses induced by opiates. Such occur as a consequence of complex cell-to-cell interactions operative between the virus and the host. We conclude that such changes in plasma proteomic profiling may be underappreciated and as such supports the need for improved clinical definitions.

Cutting Edge: T Cells Monitor N-Myristoylation of the Nef Protein in Simian Immunodeficiency Virus-Infected Monkeys

The use of the host cellular machinery is essential for pathogenic viruses to replicate in host cells. HIV and SIV borrow the host-derived N-myristoyl-transferase and its substrate, myristoyl-CoA, for coupling a saturated C(14) fatty acid (myristic acid) to the N-terminal glycine residue of the Nef protein. This biochemical reaction, referred to as N-myristoylation, assists its targeting to the plasma membrane, thereby supporting the immunosuppressive activity proposed for the Nef protein. In this study, we show that the host immunity is equipped with CTLs capable of sensing N-myristoylation of the Nef protein. A rhesus macaque CD8(+) T cell line was established that specifically recognized N-myristoylated, but not unmodified, peptides of the Nef protein. Furthermore, the population size of N-myristoylated Nef peptide-specific T cells was found to increase significantly in the circulation of SIV-infected monkeys. Thus, these results identify N-myristoylated viral peptides as a novel class of CTL target Ag.

Longitudinal diffusion tensor imaging and perfusion MRI investigation in a macaque model of neuro-AIDS: A preliminary study

The Simian immunodeficiency virus (SIV) infected macaque model exhibits neuropathological symptoms similar to those of HIV(+) patients, and is ideal for studying cognitive impairment and neuropathological sequelae of disease in repeated measurements. The aim of this study is to use Diffusion Tensor Imaging (DTI) and perfusion MRI to longitudinally access the disease development in SIV-infected monkeys under controlled conditions and to cross-validate our finding with MRI studies in HIV(+) patients. Three adult male pig-tailed macaques (Macaca nemestrina) were inoculated with the SIVsmmFGb virus. Blood was collected for enumeration of CD4+ and CD8+ T-cells. Serial time-sensitive high-resolution T(2)- weighted structural images, Cerebral Blood Flow (CBF) maps measured with the Continuous Arterial Spin Labeling (CASL) technique, and DTI images were obtained. Animals were sacrificed after 24 weeks. Cognitive behavioral tests were also carried out at each time point. Longitudinal changes in brain volume, CBF, and DTI in selected regions were analyzed statistically. In this study, CD4+ T-cell counts were found declined significantly after SIV infection in all macaques. No significant neurological behavior and brain volume changes were observed following virus inoculation. The CBF was found reduced in the caudate, inferior parietal cortex, and the prefrontal cortex. Fractional Anisotropy (FA) values in the whole brain and several Regions of Interest (ROIs) decreased significantly. These longitudinal changes in CBF and FA are correlated with CD4+ T-cell depletion and/or CD4:CD8 ratio. The MRI findings from this pilot study agree with previous results in HIV(+) patients.

Correlation between CD4+ T-cell loss and Gag-specific T cells in different intestinal sites of chronically SIV-infected rhesus monkeys

To determine the loss of CD4+ T cells and virus-specific cytotoxic T cells (CTL) in different mucosal sites of rhesus monkeys infected with simian immunodeficiency virus (SIV). A cross-sectional comparative investigation of seven different mucosal sites from chronically SIV-infected rhesus monkeys was performed by analyzing blood and mucosal lymphocytes. Mucosal lymphocytes were isolated from duodenum, jejunum, ileum and colon as well as from vagina, cervix and uterus of SIV-infected rhesus monkeys at necropsy. CD4+ T cells and SIV-Gag-specific CTL were determined in blood and mucosal samples by flow cytometry. A significant depletion of CD4+ T cells was observed in blood and all mucosal sites of SIV-infected rhesus monkeys compared to uninfected animals. But the mean percentage loss of CD4+ T cells varied between 66 and 95% between the different mucosal tissues. The frequency of CTL ranged between 0.4 and 2.4% with the highest proportions in vagina and cervix. Among the intestinal sites the mean levels of CTL correlated with mean percentage loss of CD4+ T cells. A discriminative pronounced loss of CD4+ T cells among the mucosal tissues confirmed that viral replication affects different mucosal sites in a distinct way. Despite high levels of CTL, especially in vagina and cervix, the severe loss of mucosal CD4+ T cells could not be prevented during chronic SIV infection. However, within the four sites of the intestine a high virus-specific cellular immune response correlated with better preservation of CD4+ T cells.

COX1 and COX2 expression in non-neuronal cellular compartments of the rhesus macaque brain during lentiviral infection

Recent evidence suggests that cyclooxygenases COX1 and COX2 differentially affect brain immunity. Limited data exist about their expressional changes in neurodegenerative diseases such as neuro-AIDS. Here, we analyzed the regulation of non-neuronal COX1/2 expression in rhesus macaque brain during infection with SIV δ670 and antiretroviral treatment. COX1 was constitutively expressed in microglia and endothelial cells and was not changed in early SIV infection. Late stage of disease was characterized by increased COX1 expression in globally activated microglia, macrophage nodules, infiltrates, and multinucleated giant cells. Endothelial COX1 expression was unaltered. In contrast, COX2 was not expressed in non-neuronal cells in the brain of uninfected and asymptomatically SIV-infected monkeys but was induced in nodule- and syncytium-forming macrophages and in endothelial cells in areas with infiltrates and SIV in monkeys with AIDS. Antiretroviral treatment of AIDS-diseased monkeys with 6-chloro-2′,3′-dideoxyguanosine markedly reduced SIV burden, appearance of COX1-positive macrophage nodules, giant cells, and infiltrates, and COX2 induction in the brain. However, the number of COX1-positive diffuse microglia was still increased in antiretrovirally treated animals as compared to uninfected or asymptomatic SIV-infected monkeys. Our data imply that both COX isoforms are differentially regulated and may distinctly modulate local immune responses in the brain during lentiviral disease.

Clonal Repertoires of Virus-Specific CD8+ T Lymphocytes Are Shared in Mucosal and Systemic Compartments during Chronic Simian Immunodeficiency Virus Infection in Rhesus Monkeys

Because it is thought that mucosal tissues play a fundamental role in early HIV/SIV infection, it is crucial to understand the virus-specific responses in mucosal tissues to facilitate devising strategies to prevent and control these infections. We have employed TCR repertoire analyses to define the clonal composition of a dominant SIV epitope-specific CD8(+) T cell population in mucosal and systemic compartments of SIV-infected rhesus monkeys during both acute and chronic infection. We show that the CD8(+) T cell repertoire in mucosal tissues of uninfected rhesus monkeys is oligoclonal, whereas the CD8(+) T cell repertoire in blood is polyclonal. Early postinfection, the SIV-specific CD8(+) T cell clonal repertoire is distinct in mucosal compartments and peripheral blood. However, we observed a narrowing of the virus-specific CD8(+) T cell clonal repertoire in all sampled anatomic compartments as infection progressed from acute to chronic, and there was comparable clonal diversity in all anatomic compartments. We showed during chronic infection that the same clonal populations of virus-specific CD8(+) T cells are present in all compartments. These data indicate that the SIV-specific CD8(+) T cells in systemic and mucosal sites have a shared clonal origin and are, therefore, capable of both responding to infection in the systemic circulation and trafficking to mucosal tissues.

Killer T cells not so deadly in HIV

T cellshavean essential role in controlling HIV infectionand the corresponding SIV infection in non-human primates. The most direct evidencefor their central role in virus control isobserved when SIV-infected monkeys areinfused with anti-CD8 monoclonal antibo-dies—a virtually complete depletion of CD8

AIDS Progression Is Associated with the Emergence of IL-17–Producing Cells Early After Simian Immunodeficiency Virus Infection

IL-17 is a potent effector cytokine involved in inflammatory response and antimicrobial defense. We report that SIV infection of rhesus macaques (RMs) results in the emergence of IL-17-expressing cells during the acute phase. This subpopulation appears at day 14 postinfection concomitantly with an increase in TGF-beta and IL-18 expression. This subset, which exhibits phenotypic markers of NK T cells (NKT), rather than Th17 CD4 cells, persists during the chronic phase and is higher in noncontrollers SIV-infected RMs compared with controllers SIV-infected RMs. In contrast, in the nonpathogenic model of SIVagm infection of African green monkeys, no change in the level of IL-17-expressing cells is observed in lymphoid organs. Consistent with the emergence of TGF-beta and IL-18 during the acute phase in SIV-infected RMs, but not in SIV-infected African green monkeys, we demonstrate that in vitro TGF-beta and IL-18 induce the differentiation and expansion of IL-17+NKT+. Altogether, these results demonstrate that IL-17-producing NKT are associated with the pathogenesis of SIV in RMs and suggest that TGF-beta and IL-18 play a role in their development.

Distribution, persistence, and efficacy of adoptively transferred central and effector memory-derived autologous simian immunodeficiency virus-specific CD8+ T cell clones in rhesus macaques during acute infection.

Plasma viremia decreases coincident with the appearance of virus-specific CD8(+) T cells during acute HIV or SIV infection. This finding, along with demonstrations of viral mutational escape from CD8(+) T cell responses and transient increase in plasma viremia after depletion of CD8(+) T cells in SIV-infected monkeys strongly suggest a role for CD8(+) T cells in controlling HIV/SIV. However, direct quantitative or qualitative correlates between CD8(+) T cell activity and virus control have not been established. To directly assess the impact of large numbers of virus-specific CD8(+) T cells present at time of SIV infection, we transferred in vitro expanded autologous central and effector memory-derived Gag CM9-, Nef YY9-, and Vif WY8-specific CD8(+) T cell clones to acutely infected rhesus macaques. The cells persisted in PBMCs between 4 and 9 d, but were not detected in gut-associated lymphoid tissue or lymph nodes. Interestingly, a high frequency of the infused cells localized to the lungs, where they persisted at high frequency for >6 wk. Although persisting cells in the lungs were Ag reactive, there was no measurable effect on virus load. Sequencing of virus from the animal receiving Nef YY9-specific CD8(+) T cells demonstrated an escape mutation in this epitope <3 wk postinfection, consistent with immune selection pressure by the infused cells. These studies establish methods for adoptive transfer of autologous SIV-specific CD8(+) T cells for evaluating immune control during acute infection and demonstrate that infused cells retain function and persist for at least 2 mo in specific tissues.

Towards a genetic AIDS vaccine

We discuss a recent Nature Medicine publication by Philip Johnson and co-workers (Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys. Nat. Med. 2009, 15: 901-906) in which an effective HIV-1 vaccine was designed that is based on gene therapy. The introduced gene produces an antibody-like immunoadhesin in the blood that neutralizes the virus.

P16-50. Immune reconstitution and antiviral control of SIV following adoptive transfer of anti-CD3/28 expanded CD4+ T cells: Induction of antiviral control

Results Antiviral control was characterized with the extended presence multifunctional antigen specific CD4 and CD8+ T cells detected even 1 year post transfer while viral loads remained mostly undetectable. Monitoring of trafficking and turnover of adoptively transferred anti-CD3/28 expanded CFSE labeled CD4+ T cells demonstrated extensive recirculation and dissemination of these cells with relatively slow homeostatic proliferation over time and long time engraftment in LTNP monkeys. In efforts to examine the long-term contribution of adoptively transferred anti-CD3/28 expanded CD4+ T cells, another set of monkeys was treated with transduced with a retroviral vector directing intracellular expression of the HSV-tk suicide gene and the extracellular expression of the nerve growth factor receptor (NGFR). Transduced cells enriched based on NGFR expression were expanded with anti-CD3/ 28 coated beads and transfused in SIV infected monkeys under short term ART. Two weeks following the last adoptive transfer, the transferred cells were selectively eliminated by ganciclovir administration, resulting in various levels of viral rebound after initial control. The data lends support to a long term therapeutic effect of adoptively transferred in vitro expanded CD4+ T cells even in the absence of transduction of antiviral moiety, these cells appear refractory to SIV production, for at least 2 weeks post expansion.

P04-41. Kinetics of antibody neutralization and viral evolution following envelope vaccination in SIV-infected rhesus monkeys

Background While neutralizing antibodies should limit the replication of HIV/SIV in the infected individual, autologous neutralizing antibodies are generated too late following HIV/SIV infection to contribute to early virus control. The present study addresses whether vaccine-induced immune responses accelerate the generation of autologous neutralizing antibodies following SIV challenge in rhesus monkeys, and if this antibody response shapes viral sequence evolution.

X Chromosomal Variation Is Associated with Slow Progression to AIDS in HIV-1-Infected Women

AIDS has changed from a mostly male-specific health problem to one that predominantly affects females. Although sex differences in HIV-1 susceptibility are beyond doubt, the extent to which sex affects the onset and progression of AIDS has remained elusive. Here, we provide evidence for an influence of X chromosomal variation on the course of retroviral infection, both in HIV-1-infected patients and in the rhesus macaque model of AIDS. A two-stage, microsatellite-based GWAS of SIV-infected monkeys revealed MHC class I markers and a hitherto-unknown X chromosomal locus as being associated with a nominal score measuring progression to AIDS (Fisher's exact p < 10(-6)). The X chromosomal association was subsequently confirmed in HIV-1-infected patients with published SNP genotype data. SNP rs5968255, located at human Xq21.1 in a conserved sequence element near the RPS6KA6 and CYLC1 genes, was identified as a significant genetic determinant of disease progression in females (ANOVA p = 8.8 x 10(-5)), but not in males (p = 0.19). Heterozygous female carriers of the C allele showed significantly slower CD4 cell decline and a lower viral load at set point than TT homozygous females and than males. Inspection of HapMap revealed that the CT genotype is significantly more frequent among Asians than among Europeans or Africans. Our results suggest that, in addition to the individual innate and adaptive immunity status, sex-linked genetic variation impacts upon the rate of progression to AIDS. Elucidating the mechanisms underlying this sex-specific effect will promote the development of antiretroviral therapies with high efficacy in both sexes.

Interleukin-18 predicts atherosclerosis progression in SIV-infected and uninfected rhesus monkeys (Macaca mulatta) on a high-fat/high-cholesterol diet

Interleukin (IL)-18 levels have been identified as important predictors of cardiovascular mortality and are often elevated in human immunodeficiency virus (HIV)-infected individuals. To investigate a possible function for IL-18 in atherogenesis in the context of early HIV infection, we used the simian immunodeficiency model of HIV infection. Acutely simian immunodeficiency virus-infected and uninfected rhesus monkeys (Macaca mulatta) on an atherogenic diet were evaluated prospectively for atherosclerotic lesion development relative to a panel of plasma markers including IL-18, IL-8, IL-1β, IL-6, C-reactive protein, soluble vascular cell adhesion molecule-1, soluble E-selectin, and soluble intercellular adhesion molecule-1. Although no significant differences in lesion development were identified between groups after 35 days of infection, levels of plasma IL-18 measured 1 month before virus inoculation correlated significantly with atherosclerotic plaque cross-sectional area at the carotid bifurcation (P<0.001, R=0.946), common iliac bifurcation (P<0.01, R=0.789), and cranial abdominal aorta (P<0.01, R=0.747), as well as with extent of CD3+ and CD68+ cellular infiltration in vascular lesions (both P<0.001, R≥0.835) in both groups. Atherosclerotic plaque area at the carotid and common iliac bifurcations also showed a weaker inverse correlation with baseline IL-8 levels, as did CD68+ signal area. Results implicate a strong role for IL-18 in early atherosclerosis progression and raise the possibility that the chronically elevated IL-18 levels seen in later stages of HIV infection may contribute significantly to accelerated atherogenesis in this population.

Cerebrospinal Fluid Proteomics Reveals Potential Pathogenic Changes in the Brains of SIV-Infected Monkeys

The HIV-1-associated neurocognitive disorder occurs in approximately one-third of infected individuals. It has persisted in the current era of antiretroviral therapy, and its study is complicated by the lack of biomarkers for this condition. Since the cerebrospinal fluid is the most proximal biofluid to the site of pathology, we studied the cerebrospinal fluid in a nonhuman primate model for HIV-1-associated neurocognitive disorder. Here we present a simple and efficient liquid chromatography-coupled mass spectrometry-based proteomics approach that utilizes small amounts of cerebrospinal fluid. First, we demonstrate the validity of the methodology using human cerebrospinal fluid. Next, using the simian immunodeficiency virus-infected monkey model, we show its efficacy in identifying proteins such as alpha-1-antitrypsin, complement C3, hemopexin, IgM heavy chain, and plasminogen, whose increased expression is linked to disease. Finally, we find that the increase in cerebrospinal fluid proteins is linked to increased expression of their genes in the brain parenchyma, revealing that the cerebrospinal fluid alterations identified reflect changes in the brain itself and not merely leakage of the blood-brain or blood-cerebrospinal fluid barriers. This study reveals new central nervous system alterations in lentivirus-induced neurological disease, and this technique can be applied to other systems in which limited amounts of biofluids can be obtained.

118. Pathological Characterizations of Atherosclerosis in HIV-Infected Patients and SIV-Infected Rhesus Monkeys

Clinical studies suggest that patients with human immunodeficiency virus (HIV) infection have an increased risk for cardiovascular events including coronary artery disease, while there is little evidence about direct virus involvement in the vascular lesion formation. The objective of this study was to characterize vascular tissues obtained from HIV (+) and HIV (−) patients as well as from simian immunodeficiency virus (SIV)-infected and control Rhesus monkeys.