Evaluation of Cynomolgus Macaque (Macaca fascicularis) Endogenous Retrovirus Expression Following Simian Immunodeficiency Virus Infection

Human endogenous retrovirus type K (HERV-K) transcripts are upregulated in the plasma of HIV-infected individuals and have been considered as targets for an HIV vaccine. We evaluated cynomolgus macaque endogenous retrovirus (CyERV) mRNA expression by RT-qPCR in PBMCs isolated from a cohort of animals previously utilized in a live attenuated SIV vaccine trial. CyERV env transcript levels decreased following vaccination (control and vaccine groups) and CyERV env and gag mRNA expression was decreased following acute SIV-infection, whereas during chronic SIV infection, CyERV transcript levels were indistinguishable from baseline. Reduced susceptibility to initial SIV infection, as measured by the number of SIV challenges required for infection, was associated with increased CyERV transcript levels in PBMCs. In vitro analysis revealed that SIV infection of purified CD4+ T-cells did not alter CyERV gene expression. This study represents the first evaluation of ERV expression in cynomolgus macaques following SIV infection, in an effort to assess the utility of cynomolgus macaques as an animal model to evaluate ERVs as a target for an HIV/SIV vaccine. This non-human primate model system does not recapitulate what has been observed to date in the plasma of HIV-infected humans suggesting that further investigation at the cellular level is required to elucidate the impact of HIV/SIV infection on endogenous retrovirus expression

<b>RT-qPCR Primers.</b>

<p>The slope and correlation represent the mean value.</p

CyERV gene expression levels in CD4⁺ T-cells following <i>in vitro</i> SIV infection.

<p>CyERV gene expression was quantitated by RT-qPCR using RNA isolated from mock- and SIV-infected CD4⁺ T-cells. CyERV envelope (A) and gag (B) gene expression levels relative to GAPDH are compared between <i>in vitro</i> mock- and SIV-infected CD4⁺ T-cells. Paired-samples t-tests (2-tailed) were performed to determine statistical significance.</p

Vaccination schedule.

<p>Baseline PBMC samples were obtained prior to any vaccinations (week 0). The animals were primed at weeks 0 and 9 with viral constructs (Δ5-CMV, Δ6-CCI) or medium only (control group). Animals were boosted with a DNA plasmid (Δ5-CMV, Δ6-CCI or control) at weeks 79 and 87. A second boost comprised of both virus and plasmid accompanied by a CpG adjuvant was given at week 114. Post-vaccination PBMCs were isolated at week 116 and intrarectal SIVmac239 challenge was initiated at week 118. PBMCs were isolated during acute SIV infection (mean 6 weeks post-infection (wpi); range: 4–10 wpi) and during chronic SIV infection (mean 79 wpi; range 25–98 wpi) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040158#pone.0040158-Willer1" target="_blank">[20]</a>.</p

CyERV gene expression in peripheral blood mononuclear cells.

<p>CyERV envelope and gag RNA was isolated from PBMCs and quantified using RT-qPCR. Log transformed CyERV envelope (A) and gag (B) gene expression levels relative to GAPDH were compared across all timepoints (baseline, post-vaccination, acute and chronic SIV infection). Post-vaccination PBMCs are comprised of samples from both week 114 and 116. Vaccine groups include controls, Δ5-CMV group, and Δ6-CCI group. Paired-samples t-tests (2-tailed) were performed to determine statistical significance, bars represent the mean.</p

CyERV gene expression negatively associated with CD4⁺ T-cell counts and SIV viral load.

<p>During acute SIV infection, CyERV envelope (n = 11) and gag (n = 10) gene expression levels negatively correlated with CD4⁺ T-cell counts (CD4⁺ T-cells/ul of PBMCs) (A-B) and negatively associated with acute SIV viral load for CyERV envelope (n = 9) (C). CyERV gag (n = 8) expression showed a slight trend towards a positive association with acute SIV viral load (D). Bivariate correlations were performed using Pearson’s correlation coefficient (r), statistical significance values (p) are shown.</p

Amino acid alignment of CyERV gag with HERV-K102 gag.

<p>The protein sequence of CyERV gag (GenBank: JN985533) was aligned with HERV-K102 (GenBank: P63130) using ClustalW and shaded using Geneious Pro 5.4.6 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040158#pone.0040158-Drummond1" target="_blank">[31]</a>. HERV-K102 is annotated with the matrix protein (Gag_p10) and the nucleocapsid protein (Gag_p24). Black shading indicates identical residues found at both sites amongst the aligned proteins and grey shading indicates similar amino acid residues.</p