Pegivirus avoids immune recognition but does not attenuate acute-phase disease in a macaque model of HIV infection

<div><p>Human pegivirus (HPgV) protects HIV+ people from HIV-associated disease, but the mechanism of this protective effect remains poorly understood. We sequentially infected cynomolgus macaques with simian pegivirus (SPgV) and simian immunodeficiency virus (SIV) to model HIV+HPgV co-infection. SPgV had no effect on acute-phase SIV pathogenesis–as measured by SIV viral load, CD4+ T cell destruction, immune activation, or adaptive immune responses–suggesting that HPgV’s protective effect is exerted primarily during the chronic phase of HIV infection. We also examined the immune response to SPgV in unprecedented detail, and found that this virus elicits virtually no activation of the immune system despite persistently high titers in the blood over long periods of time. Overall, this study expands our understanding of the pegiviruses–an understudied group of viruses with a high prevalence in the global human population–and suggests that the protective effect observed in HIV+HPgV co-infected people occurs primarily during the chronic phase of HIV infection.</p></div

SIV pathogenesis in SIV-only vs. SIV+SPgV infected macaques.

<p>(<b>A</b>) Peripheral CD4+ T cell counts were obtained by multiplying absolute lymphocyte counts by the percentage of lymphocytes that were CD3+ CD4+ CD20- CD8- (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006692#ppat.1006692.g003" target="_blank">Fig 3</a> for gating strategy details). (<b>B</b>) Gut CD4+ T cells were stained within sections of colonic tissues via IHC with an anti-CD4 antibody and manually quantified. Significant differences between the SIV-only and SPgV+SIV groups were analyzed using a two-tailed unpaired t-test (solid line) with error bars representing SEM. Significant changes in all animals over the course of acute SIV infection were quantified using a two-tailed paired t-test (dashed line). (<b>C</b>) A representative set of colonic tissue from Cy0883 (SIV+SPgV) and Cy0887 (SIV-only) are shown pre and post SIV infection at 400x for comparison. Arrows highlight representative cells with membranous CD4 staining.</p

Peripheral immune activation in SIV-only vs. SIV+SPgV infected macaques.

<p>(<b>A</b>) Flow cytometry gating strategy used for defining immune cell subsets. Fresh whole blood was used for staining and flow cytometry at each time point. (<b>B-D</b>) Activation of immune cell subsets. <i>P</i> values represent a two-tailed unpaired t-test with error bars reflecting SEM. Note: Cy0886 did not exhibit a distinct peak or nadir of CD69+ Ki67+ expression in the CD3+ CD8+ T cell population, and so is not included in these analyses.</p

Activation of immune tissues in SIV-only vs. SIV+SPgV infected macaques.

<p>Proliferating cells were stained within sections of lymph nodes (<b>A</b>) and colon (<b>B</b>) via IHC with an anti-Ki67 antibody and manually quantified. Significant changes over time were quantified using a two-tailed paired t-test (dashed line). A representative set of lymph nodes from Cy0883 (SIV+SPgV) and Cy0881 (SIV-only) is shown at 400X pre and post SIV infection for comparison in (<b>A</b>). A representative set of colon tissues from Cy0886 (SIV+SPgV) and Cy0887 (SIV-only) is shown pre and post SIV infection at 400x for comparison in (<b>B</b>).</p

Immune activation following SPgV vs. SIV infection.

<p>(<b>A-C</b>) Fresh whole blood was used for analysis by flow cytometry at each time point. <i>P</i> values are from a two-tailed paired t-test comparing the average immune activation pre-any-virus-infection to the average of all post-SPgV or post-SIV data points within the first 26 days of infection for each virus for which flow cytometry data was available. (<b>D</b>) Proliferating cells were stained within sections of lymph nodes via IHC with an anti-Ki67 antibody and manually quantified (SPgV: day -8 vs day 24; SIV: day -34 vs day 25). Significant changes over time were quantified using a two-tailed paired t-test. (<b>E</b>) Representative set of lymph node tissue from Cy0885 is shown at 400x.</p

SPgV and SIV viral loads in infected macaques.

<p>Titers for each virus were measured from plasma using highly sensitive virus-specific quantitative RT-PCR assays. (<b>A</b>) SPgV titers in the four macaques infected with SPgV+SIV. (<b>B</b>,<b>C</b>,<b>D</b>) SIV titers in four macaques infected with SPgV+SIV (green) and four macaques infected with SIV-only (black). <i>P</i> values reflect a two-tailed unpaired t-test and error bars represent SEM. The symbols used for each animal in this figure are consistent throughout the manuscript.</p

SPgV co-infection does not alter recognition of MHC class I restricted SIV epitopes by CD8+ T cells.

<p>Lymph nodes were collected from macaques at 125/126 days post SIV infection and cells were stained for analysis with MHC class I tetramers folded with SIV peptides that are immunodominant on the M3/M4 MHC background. (<b>A</b>) Flow cytometry gating strategy used for defining tetramer-positive CD8+ T cells. (<b>B</b>) Percentage of CD8+ T cells that were positive for each tetramer. <i>P</i> values represent a two-tailed unpaired t-test with error bars reflecting SEM.</p

Microbial Translocation and Inflammation Occur in Hyperacute Immunodeficiency Virus Infection and Compromise Host Control of Virus Replication

<div><p>Within the first three weeks of human immunodeficiency virus (HIV) infection, virus replication peaks in peripheral blood. Despite the critical, causal role of virus replication in determining transmissibility and kinetics of progression to acquired immune deficiency syndrome (AIDS), there is limited understanding of the conditions required to transform the small localized transmitted founder virus population into a large and heterogeneous systemic infection. Here we show that during the hyperacute “pre-peak” phase of simian immunodeficiency virus (SIV) infection in macaques, high levels of microbial DNA transiently translocate into peripheral blood. This, heretofore unappreciated, hyperacute-phase microbial translocation was accompanied by sustained reduction of lipopolysaccharide (LPS)-specific antibody titer, intestinal permeability, increased abundance of CD4+CCR5+ T cell targets of virus replication, and T cell activation. To test whether increasing gastrointestinal permeability to cause microbial translocation would amplify viremia, we treated two SIV-infected macaque ‘elite controllers’ with a short-course of dextran sulfate sodium (DSS)–stimulating a transient increase in microbial translocation and a prolonged recrudescent viremia. Altogether, our data implicates translocating microbes as amplifiers of immunodeficiency virus replication that effectively undermine the host’s capacity to contain infection.</p></div

Longitudinal levels of SIV RNA and bacterial rDNA in plasma.

<p>Eight MHC-identical cynomolgus macaques became infected following intrarectal inoculation with SIVmac239. (<b>A</b>) The number of SIV RNA copies/ml of plasma was enumerated using qRT-PCR. Values are Log₁₀-transformed and plotted longitudinally. (<b>B</b>) 16S sequencing data was used to correct raw 16S rDNA qPCR data by removing the proportion of 16S rDNA copies that corresponded to taxa detected in matched water controls. Corrected 16S rDNA copy data was Log₁₀-transformed and plotted longitudinally. By Bonferroni-corrected one-way ANOVA, plasma levels of 16S rDNA did not change significantly between -42 and 0 DPI. Plasma levels of 16S rDNA increased significantly (P<0.0005) from both -42 to 8 DPI and 0 to 8 DPI. In both plots, the vertical checkered box positioned between 14 and 18 DPI corresponds to the acute-phase peak of SIV replication as detected by our sampling resolution.</p

Chemically inducing microbial translocation stimulates multiple host inflammatory processes and increases plasma viremia and levels of bacterial rDNA.

<p>(<b>A</b>) Log₁₀-transformed plasma SIV load. (<b>B</b>) Linear plasma 16S rDNA load. The bacteria-specific host response was assessed by monitoring plasma levels of (<b>C</b>) EndoCAb and (<b>D</b>) sCD14. Plasma IFABP levels (<b>E</b>) were used to monitor changes to the integrity of the gastrointestinal epithelium. Generalized inflammation was monitored using plama levels of (<b>F</b>) MCP-1. In all panels, 5 black vertical lines indicate the 5-day period of once-daily treatment with dextran sulfate sodium (DSS).</p