MTD: a unique pipeline for host and meta-transcriptome joint and integrative analyses of RNA-seq data.

Ribonucleic acid (RNA)-seq data contain not only host transcriptomes but also nonhost information that comprises transcripts from active microbiota in the host cells. Therefore, joint and integrative analyses of both host and meta-transcriptome can reveal gene expression of the microbial community in a given sample as well as the correlative and interactive dynamics of the host response to the microbiome. However, there are no convenient tools that can systemically analyze host-microbiota interactions through simultaneously quantifying the host and meta-transcriptome in the same sample at the tissue and the single-cell level. This poses a challenge for interested researchers with limited expertise in bioinformatics. Here, we developed a software pipeline that can comprehensively and synergistically analyze and correlate the host and meta-transcriptome in a single sample using bulk and single-cell RNA-seq data. This pipeline, named meta-transcriptome detector (MTD), can extensively identify and quantify microbiome, including viruses, bacteria, protozoa, fungi, plasmids and vectors, in the host cells and correlate the microbiome with the host transcriptome. MTD is easy to install and run, involving only a few lines of simple commands. It offers researchers with unique genomics insights into host responses to microorganisms.

Duration of antiretroviral therapy impacts the degree of residual SIV infection in the gut in long-term non-progressing Chinese rhesus macaques.

The gut is a major reservoir in HIV-infected individuals on antiretroviral therapy (ART) and in long-term non-progressors (LTNPs). Whether ART reduces gut infection and reservoirs in LTNPs is unknown. Herein, SIV-infected LTNP Rhesus macaques were treated with short- or long-term ART, and SIV envelope gp120 sequences obtained from single genome amplification were analyzed before and after ART in peripheral blood and the intestine. Although ART does not eliminate SIV in these LTNPs, a longer ART period dramatically reduces SIV infection in the gut. This study highlights the importance of long-term ART in LTNPs to minimize gut infection and prolong remission.

Discovery of Novel Small-Molecule Inhibitors of LIM Domain Kinase for Inhibiting HIV-1.

A dynamic actin cytoskeleton is necessary for viral entry, intracellular migration, and virion release. For HIV-1 infection, during entry, the virus triggers early actin activity by hijacking chemokine coreceptor signaling, which activates a host dependency factor, cofilin, and its kinase, the LIM domain kinase (LIMK). Although knockdown of human LIM domain kinase 1 (LIMK1) with short hairpin RNA (shRNA) inhibits HIV infection, no specific small-molecule inhibitor of LIMK has been available. Here, we describe the design and discovery of novel classes of small-molecule inhibitors of LIMK for inhibiting HIV infection. We identified R10015 as a lead compound that blocks LIMK activity by binding to the ATP-binding pocket. R10015 specifically blocks viral DNA synthesis, nuclear migration, and virion release. In addition, R10015 inhibits multiple viruses, including Zaire ebolavirus (EBOV), Rift Valley fever virus (RVFV), Venezuelan equine encephalitis virus (VEEV), and herpes simplex virus 1 (HSV-1), suggesting that LIMK inhibitors could be developed as a new class of broad-spectrum antiviral drugs.IMPORTANCE The actin cytoskeleton is a structure that gives the cell shape and the ability to migrate. Viruses frequently rely on actin dynamics for entry and intracellular migration. In cells, actin dynamics are regulated by kinases, such as the LIM domain kinase (LIMK), which regulates actin activity through phosphorylation of cofilin, an actin-depolymerizing factor. Recent studies have found that LIMK/cofilin are targeted by viruses such as HIV-1 for propelling viral intracellular migration. Although inhibiting LIMK1 expression blocks HIV-1 infection, no highly specific LIMK inhibitor is available. This study describes the design, medicinal synthesis, and discovery of small-molecule LIMK inhibitors for blocking HIV-1 and several other viruses and emphasizes the feasibility of developing LIMK inhibitors as broad-spectrum antiviral drugs.

Persistence of SIV in the brain of SIV-infected Chinese rhesus macaques with or without antiretroviral therapy.

Persistence of HIV-1 reservoirs in the central nervous system (CNS) is an obstacle to cure strategies. However, little is known about residual viral distribution, viral replication levels, and genetic diversity in different brain regions of HIV-infected individuals on combination antiretroviral therapy (cART). Because myeloid cells particularly microglia are likely major reservoirs in the brain, and more microglia exist in white matter than gray matter in a human brain, we hypothesized the major viral reservoirs in the brain are the white matter reflected by higher levels of viral DNA. To address the issue, we used the Chinese rhesus macaque (ChRM) model of SIV infection, and treated 11 SIVmac251-infected animals including long-term nonprogressors with cART for up to 24 weeks. SIV reservoirs were assessed by SIV DNA levels in 16 specific regions of the brain and 4 regions of spinal cord. We found relatively high frequencies of SIV in basal ganglia and brain stem compared to other regions. cART-receiving animals had significantly lower SIV DNA levels in the gray matter than white matter. Moreover, a shortened envelope gp120 with 21 nucleotide deletions and guanine-to-adenine hypermutations were observed. These results demonstrate that SIV enters the CNS in SIV-infected ChRM with a major reservoir in the white matter after cART; the SIV/ChRM/cART is an appropriate model for studying HIV CNS reservoirs and testing new eradication strategies. Further, examining multiple regions of the CNS may be needed when assessing whether an agent is successful in reducing the size of SIV reservoirs in the CNS.

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections.

BACKGROUND: New simian-human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques. RESULTS: Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env's in the pool, a feature also observed in the HIV establishing new infections in humans. CONCLUSION: Despite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.

Genistein interferes with SDF-1- and HIV-mediated actin dynamics and inhibits HIV infection of resting CD4 T cells.

BACKGROUND: Binding of HIV to the chemokine coreceptor CXCR4 mediates viral fusion and signal transduction that promotes actin dynamics critical for HIV infection of blood resting CD4 T cells. It has been suggested that this gp120-mediated actin activity resembles the chemotactic actin dynamics mediated by chemokines such as SDF-1. To determine whether inhibiting SDF-1-mediated chemotactic activity can also inhibit HIV infection, we screened several inhibitors known to reduce SDF-1-mediated chemotaxis of T cells. RESULTS: We found that a tyrosine kinase inhibitor, genistein, inhibited both SDF-1-mediated chemotaxis and HIV infection of resting CD4 T cells. Genistein was also found to interfere with SDF-1- and HIV-mediated actin dynamics in CD4 T cells. This reduction in actin activity correlates with genistein-mediated inhibition of viral DNA accumulation in resting CD4 T cells. In addition, we also tested two other tyrosine kinase inhibitors, sunitinib and AG1478. Sunitinib, but not AG1478, inhibited HIV infection of resting CD4 T cells. We further tested the safety of genistein in 3 Chinese rhesus macaques (Macaca mulatta), and each animal was given a monotherapy of genistein at 10 mg/kg orally for 12 weeks. No adverse drug effects were observed in these animals. CONCLUSIONS: Our results suggest that novel therapeutic strategies can be developed based on targeting cellular proteins involved in HIV-dependent signaling. This approach can interfere with HIV-mediated actin dynamics and inhibit HIV infection.

Cannabinoid enhancement of lncRNA MMP25-AS1/MMP25 interaction reduces neutrophil infiltration and intestinal epithelial injury in HIV/SIV infection.

Intestinal epithelial barrier dysfunction, a hallmark of HIV/SIV infection, persists despite viral suppression by combination antiretroviral therapy (cART). Emerging evidence suggests a critical role for long noncoding RNAs (lncRNAs) in maintaining epithelial homeostasis. We simultaneously profiled lncRNA/mRNA expression exclusively in colonic epithelium (CE) of SIV-infected rhesus macaques (RMs) administered vehicle (VEH) or Δ-9-tetrahydrocannabinol (THC). Relative to controls, fewer lncRNAs were up- or downregulated in CE of THC/SIV compared with VEH/SIV RMs. Importantly, reciprocal expression of the natural antisense lncRNA MMP25-AS1 (up 2.3-fold) and its associated protein-coding gene MMP25 (attracts neutrophils by inactivating alpha-1 anti-trypsin/SERPINA1) (down 2.2-fold) was detected in CE of THC/SIV RMs. Computational analysis verified 2 perfectly matched complementary regions and an energetically stable (normalized binding free energy = -0.2626) MMP25-AS1/MMP25 duplex structure. MMP25-AS1 overexpression blocked IFN-γ-induced MMP25 mRNA and protein expression in vitro. Elevated MMP25 protein expression in CE of VEH/SIV but not THC/SIV RMs was associated with increased infiltration by myeloperoxidase/CD11b++ neutrophils (transendothelial migration) and epithelial CD47 (transepithelial migration) expression. Interestingly, THC administered in combination with cART increased MMP25-AS1 and reduced MMP25 mRNA/protein expression in jejunal epithelium of SIV-infected RMs. Our findings demonstrate that MMP25-AS1 is a potentially unique epigenetic regulator of MMP25 and that low-dose THC can reduce neutrophil infiltration and intestinal epithelial injury potentially by downregulating MMP25 expression through modulation of MMP25-AS1.

Lymphoid tissues contribute to viral clonotypes present in plasma at early post-ATI in SIV-infected rhesus macaques.

The rebound-competent viral reservoir (RCVR), comprised of virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after antiretroviral therapy interruption (ATI), remains the biggest obstacle to the eradication of HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop targeted therapeutic strategies for reducing the RCVR. In this study, barcoded SIVmac239M was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood, lymphoid tissues (LTs, spleen, mesenteric and inguinal lymph nodes), and non-lymphoid tissues (NLTs, colon, ileum, lung, liver, and brain) were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX/RNAscope/ in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy although plasma viral RNA remained < 22 copies/mL. Among the tissues studied, mesenteric and inguinal lymph nodes, and spleen contained viral barcodes detected in plasma, and trended to have higher cell-associated viral loads, higher intact provirus levels, and greater diversity of viral barcodes. CD4+ T cells were the main cell type harboring viral RNA (vRNA) after ATI. Further, T cell zones in LTs showed higher vRNA levels than B cell zones for most animals. These findings are consistent with LTs contributing to virus present in plasma early after ATI. One Sentence Summary: The reemerging of SIV clonotypes at early post-ATI are likely from the secondary lymphoid tissues.

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques.

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

Copy number variation of CCL3-like genes affects rate of progression to simian-AIDS in Rhesus Macaques (Macaca mulatta).

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10(-6)) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.

Neuroinflammatory Profiling in SIV-Infected Chinese-Origin Rhesus Macaques on Antiretroviral Therapy.

The central nervous system (CNS) HIV reservoir is an obstacle to achieving an HIV cure. The basal ganglia harbor a higher frequency of SIV than other brain regions in the SIV-infected rhesus macaques of Chinese-origin (chRMs) even on suppressive combination antiretroviral therapy (ART). Since residual HIV/SIV reservoir is associated with inflammation, we characterized the neuroinflammation by gene expression and systemic levels of inflammatory molecules in healthy controls and SIV-infected chRMs with or without ART. CCL2, IL-6, and IFN-γ were significantly reduced in the cerebrospinal fluid (CSF) of animals receiving ART. Moreover, there was a correlation between levels of CCL2 in plasma and CSF, suggesting the potential use of plasma CCL2 as a neuroinflammation biomarker. With higher SIV frequency, the basal ganglia of untreated SIV-infected chRMs showed an upregulation of secreted phosphoprotein 1 (SPP1), which could be an indicator of ongoing neuroinflammation. While ART greatly reduced neuroinflammation in general, proinflammatory genes, such as IL-9, were still significantly upregulated. These results expand our understanding of neuroinflammation and signaling in SIV-infected chRMs on ART, an excellent model to study HIV/SIV persistence in the CNS.

Cannabinoid control of gingival immune activation in chronically SIV-infected rhesus macaques involves modulation of the indoleamine-2,3-dioxygenase-1 pathway and salivary microbiome.

BACKGROUND: HIV/SIV-associated periodontal disease (gingivitis/periodontitis) (PD) represents a major comorbidity affecting people living with HIV (PLWH) on combination anti-retroviral therapy (cART). PD is characterized by chronic inflammation and dysbiosis. Nevertheless, the molecular mechanisms and use of feasible therapeutic strategies to reduce/reverse inflammation and dysbiosis remain understudied and unaddressed. METHODS: Employing a systems biology approach, we report molecular, metabolome and microbiome changes underlying PD and its modulation by phytocannabinoids [delta-9-tetrahydrocannabinol (Δ9-THC)] in uninfected and SIV-infected rhesus macaques (RMs) untreated (VEH-untreated/SIV) or treated with vehicle (VEH/SIV) or Δ9-THC (THC/SIV). FINDINGS: VEH- untreated/SIV but not THC/SIV RMs showed significant enrichment of genes linked to anti-viral defense, interferon-ß, NFκB, RIG-1, and JAK-STAT signaling. We focused on the anti-microbial DUOX1 and immune activation marker IDO1 that were reciprocally regulated in the gingiva of VEH-untreated/SIV RMs. Both proteins localized to the gingival epithelium and CD163+ macrophages, and showed differential expression in the gingiva of THC/SIV and VEH/SIV RMs. Additionally, inflammation-associated miR-21, miR-142-3p, miR-223, and miR-125a-5p showed significantly higher expression in the gingiva of VEH/SIV RMs. In human primary gingival epithelial cells, miR-125a-5p post-transcriptionally downregulated DUOX1 and THC inhibited IDO1 protein expression through a cannabinoid receptor-2 mediated mechanism. Interestingly, THC/SIV RMs showed relatively reduced plasma levels of kynurenine, kynurenate, and the neurotoxic quinolinate compared to VEH/SIV RMs at 5 months post SIV infection (MPI). Most importantly, THC blocked HIV/SIV-induced depletion of Firmicutes and Bacteroidetes, and reduced Gammaproteobacteria abundance in saliva. Reduced IDO1 protein expression was associated with significantly (p<0.05) higher abundance of Prevotella, Lactobacillus (L. salivarius, L. buchneri, L. fermentum, L. paracasei, L. rhamnosus, L. johnsonii) and Bifidobacteria and reduced abundance of the pathogenic Porphyromonas cangingivalis and Porphyromonas macacae at 5MPI. INTERPRETATION: The data provides deeper insights into the molecular mechanisms underlying HIV/SIV-induced PD and more importantly, the anti-inflammatory and anti-dysbiotic properties of THC in the oral cavity. Overall, these translational findings suggest that phytocannabinoids may help reduce gingival/systemic inflammation, salivary dysbiosis and potentially metabolic disease/syndrome in PLWH on cART and those with no access to cART or do not suppress the virus under cART. FUNDING: Research reported in this publication was supported by the National Institutes of Health Award Numbers R01DA052845 (MM and SNB), R01DA050169 (MM and CO), R01DA042524 and R56DE026930 (MM), and P51OD011104 and P51OD011133. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Microglia-Specific Promoter Activities of HEXB Gene.

Adeno-associated virus (AAV)-mediated genetic targeting of microglia remains a challenge. Overcoming this hurdle is essential for gene editing in the central nervous system (CNS). Here, we characterized the minimal/native promoter of the HEXB gene, which is known to be specifically and stably expressed in the microglia during homeostatic and pathological conditions. Dual reporter and serial deletion assays identified the critical role of the natural 5' untranslated region (-97 bp related to the first ATG) in driving transcriptional activity of the mouse Hexb gene. The native promoter region of mouse, human, and monkey HEXB are located at -135, -134, and -170 bp to the first ATG, respectively. These promoters were highly active and specific in microglia with strong cross-species transcriptional activities, but did not exhibit activity in primary astrocytes. In addition, we identified a 135 bp promoter of CD68 gene that was highly active in microglia but not in astrocytes. Considering that HEXB is specifically expressed in microglia, these data suggest that the newly characterized microglia-specific HEXB minimal/native promoter can be an ideal candidate for microglia-targeting AAV gene therapy in the CNS.

The level of monocyte turnover predicts disease progression in the macaque model of AIDS.

It is widely accepted that destruction of CD4(+) T cells and viral load are the primary markers for immunodeficiency in HIV-1-infected humans and in simian immunodeficiency virus (SIV)-infected macaques. However, monocyte/macrophages are also important targets of HIV/SIV infection and a critical link between innate and adaptive immunity. We therefore examined whether changes in cells of the monocyte/macrophage lineage could be linked to the pathogenesis of AIDS in the rhesus macaque model. Here, we show that massive turnover of peripheral monocytes associated with death of tissue macrophages correlates with AIDS progression in macaques. More importantly, the level of monocyte turnover was not linked to the CD4(+) T-cell count and was a better predictive marker for AIDS progression than was viral load or lymphocyte activation. Our results show the importance of monocyte/macrophages in the pathogenesis of AIDS and suggest the dynamic changes of the monocyte/macrophages as a new marker for AIDS progression.

The large intestine as a major reservoir for simian immunodeficiency virus in macaques with long-term, nonprogressing infection.

Although patients with human immunodeficiency virus type 1 infection who are receiving antiretroviral therapy and those with long-term, nonprogressive infection (LTNPs) usually have undetectable viremia, virus persists in tissue reservoirs throughout infection. However, the distribution and magnitude of viral persistence and replication in tissues has not been adequately examined. Here, we used the simian immunodeficiency virus (SIV) macaque model to quantify and compare viral RNA and DNA in the small (jejunum) and large (colon) intestine of LTNPs. In LTNPs with chronic infection, the colon had consistently higher viral levels than did the jejunum. The colon also had higher percentages of viral target cells (memory CD4(+) CCR5(+) T cells) and proliferating memory CD4(+) T cells than did the jejunum, whereas markers of cell activation were comparable in both compartments. These data indicate that the large intestine is a major viral reservoir in LTNPs, which may be the result of persistent, latently infected cells and higher turnover of naive and central memory CD4(+) T cells in this major immunologic compartment.

Oral administration of tipranavir with long-chain triglyceride results in moderate intestinal lymph targeting but no efficient delivery to HIV-1 reservoir in mesenteric lymph nodes.

The introduction of combination antiretroviral therapy (cART) led to substantial improvement in mortality and morbidity of HIV-1 infection. However, the poor penetration of antiretroviral agents to HIV-1 reservoirs limit the ability of the antiretroviral agents to eliminate the virus. Mesenteric lymph nodes (MLNs) are one of the main HIV-1 reservoirs in patients under suppressive cART. Intestinal lymphatic absorption pathway substantially increases the concentration of lipophilic drugs in mesenteric lymph and MLNs when they are co-administered with long-chain triglyceride (LCT). Chylomicrons (CM) play a crucial role in the intestinal lymphatic absorption as they transport drugs to the lymph lacteals rather than blood capillary by forming CM-drug complexes in the enterocytes. Thus, lipophilic antiretroviral drugs could potentially be delivered to HIV-1 reservoirs in MLNs by LCT-based formulation approach. In this study, protease inhibitors (PIs) were initially screened for their potential for intestinal lymphatic targeting using a computational model. The candidates were further assessed for their experimental affinity to CM. Tipranavir (TPV) was the only-candidate with substantial affinity to both artificial and natural CM in vitro and ex vivo. Pharmacokinetics and biodistribution studies were then performed to evaluate the oral bioavailability and intestinal lymphatic targeting of TPV in rats. The results showed similar oral bioavailability of TPV with and without co-administration of LCT vehicle. Although LCT-based formulation led to 3-fold higher concentrations of TPV in mesenteric lymph compared to plasma, the levels of the drug in MLNs were similar to plasma in both LCT-based and lipid-free formulation groups. Thus, LCT-based formulation approach alone was not sufficient for effective delivery of TPV to MLNs. Future efforts should be directed to a combined highly lipophilic prodrugs/lipid-based formulation approach to target TPV, other PIs and potentially other classes of antiretroviral agents to viral reservoirs within the mesenteric lymphatic system.

The most common Chinese rhesus macaque MHC class I molecule shares peptide binding repertoire with the HLA-B7 supertype.

Of the two rhesus macaque subspecies used for AIDS studies, the Simian immunodeficiency virus-infected Indian rhesus macaque (Macaca mulatta) is the most established model of HIV infection, providing both insight into pathogenesis and a system for testing novel vaccines. Despite the Chinese rhesus macaque potentially being a more relevant model for AIDS outcomes than the Indian rhesus macaque, the Chinese-origin rhesus macaques have not been well-characterized for their major histocompatibility complex (MHC) composition and function, reducing their greater utilization. In this study, we characterized a total of 50 unique Chinese rhesus macaques from several varying origins for their entire MHC class I allele composition and identified a total of 58 unique complete MHC class I sequences. Only nine of the sequences had been associated with Indian rhesus macaques, and 28/58 (48.3%) of the sequences identified were novel. From all MHC alleles detected, we prioritized Mamu-A1*02201 for functional characterization based on its higher frequency of expression. Upon the development of MHC/peptide binding assays and definition of its associated motif, we revealed that this allele shares peptide binding characteristics with the HLA-B7 supertype, the most frequent supertype in human populations. These studies provide the first functional characterization of an MHC class I molecule in the context of Chinese rhesus macaques and the first instance of HLA-B7 analogy for rhesus macaques.

Differential CD4+ T-lymphocyte apoptosis and bystander T-cell activation in rhesus macaques and sooty mangabeys during acute simian immunodeficiency virus infection.

In contrast to pathogenic lentiviral infections, chronic simian immunodeficiency virus (SIV) infection in its natural host is characterized by a lack of increased immune activation and apoptosis. To determine whether these differences are species specific and predicted by the early host response to SIV in primary infection, we longitudinally examined T-lymphocyte apoptosis, immune activation, and the SIV-specific cellular immune response in experimentally infected rhesus macaques (RM) and sooty mangabeys (SM) with controlled or uncontrolled SIV infection. SIVsmE041, a primary SIVsm isolate, reproduced set-point viremia levels of natural SIV infection in SM but was controlled in RM, while SIVmac239 replicated to high levels in RM. Following SIV infection, increased CD8(+) T-lymphocyte apoptosis, temporally coinciding with onset of SIV-specific cellular immunity, and elevated plasma Th1 cytokine and gamma interferon-induced chemokine levels were common to both SM and RM. Different from SM, SIV-infected RM showed a significantly higher frequency of peripheral blood activated CD8(+) T lymphocytes despite comparable magnitude of the SIV-specific gamma interferon enzyme-linked immunospot response. Furthermore, an increase in CD4(+) and CD4(-)CD8(-) T-lymphocyte apoptosis and plasma tumor necrosis factor-related apoptosis-inducing ligand were observed only in RM and occurred in both controlled SIVsmE041 and uncontrolled SIVmac239 infection. These data suggest that the "excess" activated T lymphocytes in RM soon after SIV infection are predominantly of non-virus-specific bystander origin. Thus, species-specific differences in the early innate immune response appear to be an important factor contributing to differential immune activation in natural and nonnatural hosts of SIV infection.

Alterations of the gut bacterial microbiota in rhesus macaques with SIV infection and on short- or long-term antiretroviral therapy.

Gut dysbiosis and microbial translocation are associated with chronic systemic immune activation and inflammation in HIV-1 infection. However, the extent of restoration of gut microbiota in HIV-1 patients with short or long-term antiretroviral therapy (ART) is unclear. To understand the impact of ART on the gut microbiota, we used the rhesus macaque model of SIV infection to characterize and compare the gut microbial community upon SIV infection and during ART. We observed altered taxonomic compositions of gut microbiota communities upon SIV infection and at different time points of ART. SIV-infected animals showed decreased diversity of gut microbiome composition, while the ART group appeared to recover towards the diversity level of the healthy control. Animals undergoing ART for various lengths of time were observed to have differential gut bacterial abundance across different time points. In addition, increased blood lipopolysaccharide (LPS) levels during SIV infection were reduced to near normal upon ART, indicating that microbial translocation and immune activation can be improved during therapy. In conclusion, while short ART may be related to transient increase of certain pathogenic bacterial microbiome, ART may promote microbiome diversity compromised by SIV infection, improve the gut microbiota towards the healthy compositions and alleviate immune activation.

CRISPR based editing of SIV proviral DNA in ART treated non-human primates.

Elimination of HIV DNA from infected individuals remains a challenge in medicine. Here, we demonstrate that intravenous inoculation of SIV-infected macaques, a well-accepted non-human primate model of HIV infection, with adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing construct designed for eliminating proviral SIV DNA, leads to broad distribution of editing molecules and precise cleavage and removal of fragments of the integrated proviral DNA from the genome of infected blood cells and tissues known to be viral reservoirs including lymph nodes, spleen, bone marrow, and brain among others. Accordingly, AAV9-CRISPR treatment results in a reduction in the percent of proviral DNA in blood and tissues. These proof-of-concept observations offer a promising step toward the elimination of HIV reservoirs in the clinic.