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

Abstract

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.

Fig 1. SPgV and SIV viral loads in infected macaques.

Titers for each virus were measured from plasma using highly sensitive virus-specific quantitative RT-PCR assays. (A) SPgV titers in the four macaques infected with SPgV+SIV. (B,C,D) SIV titers in four macaques infected with SPgV+SIV (green) and four macaques infected with SIV-only (black). P 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.

Fig 2. SIV pathogenesis in SIV-only vs. SIV+SPgV infected macaques.

(A) Peripheral CD4+ T cell counts were obtained by multiplying absolute lymphocyte counts by the percentage of lymphocytes that were CD3+ CD4+ CD20- CD8- (see Fig 3 for gating strategy details). (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). (C) 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.

Fig 3. Peripheral immune activation in SIV-only vs. SIV+SPgV infected macaques.

(A) 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-D) Activation of immune cell subsets. P 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.

Fig 4. Activation of immune tissues in SIV-only vs. SIV+SPgV infected macaques.

Proliferating cells were stained within sections of lymph nodes (A) and colon (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 (A). 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).

Fig 5. Immune activation following SPgV vs. SIV infection.

(A-C) Fresh whole blood was used for analysis by flow cytometry at each time point. P 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. (D) 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. (E) Representative set of lymph node tissue from Cy0885 is shown at 400x.

Fig 6. Antibody responses to linear epitopes in the SPgV and SIV envelope proteins.

Amino acid sequences from the envelope proteins of the viruses used in this study (E1/E2 of SPgV and env of SIV) were represented as 12–16 amino acid length peptides. Overlapping peptides (step size of 2 amino acids with peptides overlapping by 10–14 amino acids) were then synthesized and tiled on an array. Plasma from each animal in the study, collected 125 or 126 days post SIV infection, was then incubated on the array and antibody binding to each peptide was quantified using a mouse anti-primate-IgG secondary antibody. Signal intensity for each peptide spanning SPgV E1/E2 (A) and SIV env (C) is shown for animals infected with SIV-only (black), SIV+SPgV (green), or uninfected controls. Total signal intensity, normalized to the average signal per peptide in the SPgV E1/E2 array (B) and the SIV env array (D), is shown for each animal in the study along with uninfected controls. P values represent a two-tailed unpaired t-test with error bars reflecting SEM.

Fig 7. SPgV co-infection does not alter recognition of MHC class I restricted SIV epitopes by CD8+ T cells.

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. (A) Flow cytometry gating strategy used for defining tetramer-positive CD8+ T cells. (B) Percentage of CD8+ T cells that were positive for each tetramer. P values represent a two-tailed unpaired t-test with error bars reflecting SEM.