David R. Morcock

Compromised gastrointestinal integrity in pigtail macaques is associated with increased microbial translocation, immune activation, and IL-17 production in the absence of SIV infection

Pigtail macaques (PTMs) rapidly progress to AIDS after simian immunodeficiency virus (SIV) infection. Given the strong association between human immunodeficiency virus (HIV) and SIV disease progression and microbial translocation and immune activation, we assessed whether high basal levels of immune activation and microbial translocation exist in PTMs. We found that before SIV infection, PTMs had high levels of microbial translocation that correlated with significant damage to the structural barrier of the gastrointestinal tract. Moreover, this increased microbial translocation correlated with high levels of immune activation and was associated with high frequencies of interleukin-17-producing T cells. These data highlight the relationship among mucosal damage, microbial translocation and systemic immune activation in the absence of SIV replication, and underscore the importance of microbial translocation in the rapid course of disease progression in SIV-infected PTMs. Furthermore, these data suggest that PTM may be an ideal model to study therapeutic interventions aimed at decreasing microbial translocation-induced immune activation.

Probiotic/prebiotic supplementation of antiretrovirals improves gastrointestinal immunity in SIV-infected macaques

HIV infection results in gastrointestinal (GI) tract damage, microbial translocation, and immune activation, which are not completely ameliorated with suppression of viremia by antiretroviral (ARV) therapy. Furthermore, increased morbidity and mortality of ARV-treated HIV-infected individuals is associated with these dysfunctions. Thus, to enhance GI tract physiology, we treated SIV-infected pigtail macaques with ARVs, probiotics, and prebiotics or with ARVs alone. This synbiotic treatment resulted in increased frequency and functionality of GI tract APCs, enhanced reconstitution and functionality of CD4+ T cells, and reduced fibrosis of lymphoid follicles in the colon. Thus, ARV synbiotic supplementation in HIV-infected individuals may improve GI tract immunity and thereby mitigate inflammatory sequelae, ultimately improving prognosis.

Simian Immunodeficiency Virus Integration Preference Is Similar to That of Human Immunodeficiency Virus Type 1

ABSTRACT Simian immunodeficiency virus (SIV) is a useful model for studying human immunodeficiency virus (HIV) pathogenesis and vaccine efficacy. As with all other retroviruses, integration is a necessary step in the replication cycle of SIV. The location of the retrovirus integration site is known to impact on viral gene expression, establishment of viral latency, and other aspects of the replication cycle of a retrovirus. In this study, 148 SIV provirus integration sites were sequenced and mapped in the human genome. Our analysis showed that SIV integration, like that of HIV type 1 (HIV-1), exhibited a strong preference for actively transcribed regions in the genome (A. R. Schroder et al., Cell 110:521-529, 2002) and no preference for the CpG islands or transcription start sites, in contrast to observations for murine leukemia virus (X. Wu et al., Science 300:1749-1751, 2003). The parallel integration target site preferences of SIV and HIV-1 suggest that these lentiviruses may share similar mechanisms for target site selection and that SIV serves as an accurate model of HIV-1 with respect to integration.

Defining HIV and SIV Reservoirs in Lymphoid Tissues

A primary obstacle to an HIV-1 cure is long-lived viral reservoirs, which must be eliminated or greatly reduced. Cure strategies have largely focused on monitoring changes in T cell reservoirs in peripheral blood (PB), even though the lymphoid tissues (LT) are primary sites for viral persistence. To track and discriminate viral reservoirs within tissue compartments we developed a specific and sensitive next-generation in situ hybridization approach to detect vRNA, including vRNA+ cells and viral particles (“RNAscope”), vDNA+ cells (“DNAscope”) and combined vRNA and vDNA with immunohistochemistry to detect and phenotype active and latently infected cells in the same tissue section. RNAscope is highly sensitive with greater speed of analysis compared to traditional in situ hybridization. The highly sensitive and specific DNAscope detected SIV/HIV vDNA+ cells, including duplexed detection of vDNA and vRNA or immunophenotypic markers in the same section. Analysis of LT samples from macaques prior to and during combination antiretroviral therapy demonstrated that B cell follicles are an important anatomical compartment for both latent and active viral persistence during treatment. These new tools should allow new insights into viral reservoir biology and evaluation of cure strategies.

Decreases in Colonic and Systemic Inflammation in Chronic HIV Infection after IL-7 Administration

Despite antiretroviral therapy (ART), some HIV-infected persons maintain lower than normal CD4+ T-cell counts in peripheral blood and in the gut mucosa. This incomplete immune restoration is associated with higher levels of immune activation manifested by high systemic levels of biomarkers, including sCD14 and D-dimer, that are independent predictors of morbidity and mortality in HIV infection. In this 12-week, single-arm, open-label study, we tested the efficacy of IL-7 adjunctive therapy on T-cell reconstitution in peripheral blood and gut mucosa in 23 ART suppressed HIV-infected patients with incomplete CD4+ T-cell recovery, using one cycle (consisting of three subcutaneous injections) of recombinant human IL-7 (r-hIL-7) at 20 µg/kg. IL-7 administration led to increases of both CD4+ and CD8+ T-cells in peripheral blood, and importantly an expansion of T-cells expressing the gut homing integrin α4β7. Participants who underwent rectosigmoid biopsies at study baseline and after treatment had T-cell increases in the gut mucosa measured by both flow cytometry and immunohistochemistry. IL-7 therapy also resulted in apparent improvement in gut barrier integrity as measured by decreased neutrophil infiltration in the rectosigmoid lamina propria 12 weeks after IL-7 administration. This was also accompanied by decreased TNF and increased FOXP3 expression in the lamina propria. Plasma levels of sCD14 and D-dimer, indicative of systemic inflammation, decreased after r-hIL-7. Increases of colonic mucosal T-cells correlated strongly with the decreased systemic levels of sCD14, the LPS coreceptor - a marker of monocyte activation. Furthermore, the proportion of inflammatory monocytes expressing CCR2 was decreased, as was the basal IL-1β production of peripheral blood monocytes. These data suggest that administration of r-hIL-7 improves the gut mucosal abnormalities of chronic HIV infection and attenuates the systemic inflammatory and coagulation abnormalities that have been linked to it.

Elimination of Retroviral Infectivity by N-Ethylmaleimide with Preservation of Functional Envelope Glycoproteins

ABSTRACT The zinc finger motifs in retroviral nucleocapsid (NC) proteins are essential for viral replication. Disruption of these Cys-X2-Cys-X4-His-X4-Cys zinc-binding structures eliminates infectivity. To determine if N-ethylmaleimide (NEM) can inactivate human immunodeficiency virus type 1 (HIV-1) or simian immunodeficiency virus (SIV) preparations by alkylating cysteines of NC zinc fingers, we treated infectious virus with NEM and evaluated inactivation of infectivity in cell-based assays. Inactivation was rapid and proportional to the NEM concentration. NEM treatment of HIV-1 or SIV resulted in extensive covalent modification of NC and other internal virion proteins. In contrast, viral envelope glycoproteins, in which the cysteines are disulfide bonded, remained intact and functional, as assayed by high-performance liquid chromatography, fusion-from-without analyses, and dendritic cell capture. Quantitative PCR assays for reverse transcription intermediates showed that NEM and 2,2′-dipyridyl disulfide (aldrithiol-2), a reagent which inactivates retroviruses through oxidation of cysteines in internal virion proteins such as NC, blocked HIV-1 reverse transcription prior to the formation of minus-strand strong-stop products. However, the reverse transcriptase from NEM-treated virions remained active in exogenous template assays, consistent with a role for NC in reverse transcription. Since disruption of NC zinc finger structures by NEM blocks early postentry steps in the retroviral infection cycle, virus preparations with modified NC proteins may be useful as vaccine immunogens and probes of the role of NC in viral replication.

Reduced Inflammation and Lymphoid Tissue Immunopathology in Rhesus Macaques Receiving Anti–Tumor Necrosis Factor Treatment During Primary Simian Immunodeficiency Virus Infection

BACKGROUND Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections induce robust, generalized inflammatory responses that begin during acute infection and lead to pathological systemic immune activation, fibrotic damage of lymphoid tissues, and CD4⁺ T-cell loss, pathogenic processes that contribute to disease progression. METHODS To better understand the contribution of tumor necrosis factor (TNF), a key regulator of acute inflammation, to lentiviral pathogenesis, rhesus macaques newly infected with SIVmac239 were treated for 12 weeks in a pilot study with adalimumab (Humira), a human anti-TNF monoclonal antibody. RESULTS Adalimumab did not affect plasma SIV RNA levels or measures of T-cell immune activation (CD38 or Ki67) in peripheral blood or lymph node T cells. However, compared with untreated rhesus macaques, adalimumab-treated rhesus macaques showed attenuated expression of proinflammatory genes, decreased infiltration of polymorphonuclear cells into the T-cell zone of lymphoid tissues, and weaker antiinflammatory regulatory responses to SIV infection (ie, fewer presumed alternatively activated [ie, CD163⁺] macrophages, interleukin 10-producing cells, and transforming growth factor β-producing cells), along with reduced lymphoid tissue fibrosis and better preservation of CD4⁺ T cells. CONCLUSIONS While HIV/SIV replication drives pathogenesis, these data emphasize the contribution of the inflammatory response to lentiviral infection to overall pathogenesis, and they suggest that early modulation of the inflammatory response may help attenuate disease progression.

Rate of AIDS Progression Is Associated with Gastrointestinal Dysfunction in Simian Immunodeficiency Virus–Infected Pigtail Macaques

During HIV/SIV infection, mucosal immune system dysfunction and systemic immune activation are associated with progression to AIDS; however, it is unclear to what extent pre-existing gastrointestinal damage relates to disease progression postinfection. Pigtail macaques (PTM) are an excellent model in which to assess mucosal dysfunction in relation to HIV/SIV pathogenesis, as the majority of these animals have high levels of gastrointestinal damage, immune activation, and microbial translocation prior to infection, and rapidly progress to AIDS upon SIV infection. In this study, we characterized the mucosal immune environment prior to and throughout SIV infection in 13 uninfected PTM and 9 SIV-infected PTM, of which 3 were slow progressors. This small subset of slow progressors had limited innate immune activation in mucosal tissues in the periphery, which was associated with a more intact colonic epithelial barrier. Furthermore, we found that preinfection levels of microbial translocation, as measured by LPS-binding protein, in PTM correlated with the rate of progression to AIDS. These data suggest that pre-existing levels of microbial translocation and gastrointestinal tract dysfunction may influence the rate of HIV disease progression.

Experimental colitis in SIV-uninfected rhesus macaques recapitulates important features of pathogenic SIV infection

Mucosal damage to the gastrointestinal (GI) tract with resulting microbial translocation is hypothesized to significantly contribute to the heightened and persistent chronic inflammation and immune activation characteristic to HIV infection. Here we employ a non-human primate model of chemically induced colitis in SIV-uninfected rhesus macaques that we developed using dextran sulfate sodium (DSS), to directly test this hypothesis. DSS treatment results in GI barrier damage with associated microbial translocation, inflammation and immune activation. The progression and severity of colitis are longitudinally monitored by a magnetic resonance imaging approach. DSS treatment of SIV-infected African green monkeys, a natural host species for SIV that does not manifest GI tract damage or chronic immune activation during infection, results in colitis with elevated levels of plasma SIV RNA, sCD14, LPS, CRP and mucosal CD4+ T-cell loss. Together these results support the hypothesis that GI tract damage leading to local and systemic microbial translocation, and associated immune activation, are important determinants of AIDS pathogenesis.

Fluorescence and nucleic acid binding properties of the human T-cell leukemia virus-type 1 nucleocapsid protein

We used intrinsic tryptophan fluorescence to study the nucleocapsid protein from human T-cell leukemia virus-type one, HTLV-1 p15, an 85-amino-acid protein with two Trp-containing zinc-finger motifs. Fluorescence spectra suggested an interaction between the two zinc fingers and another interaction involving the C-terminal tail and the zinc fingers. Titrations with nucleic acid revealed similar, sub-micromolar affinity for poly(dT) and poly(U) in 1 mM sodium phosphate, pH 7. Double-stranded DNA bound an order of magnitude weaker, suggesting helix-destabilizing activity. Base preference of p15 was T approximately U>I approximately C approximately G>A; affinity spanned about one order of magnitude. HTLV-1 p15 bound weaker and with less variation than reported values for either human or simian immunodeficiency virus homologues. The low affinity of p15 for nonspecific nucleic acids distinguishes it from other nucleocapsid proteins, and may suggest its involvement in additional steps of the virus life cycle other than RNA packaging.