We quantified HERV RNA in plasma from 16 untreated HIV-1-positive primary infection individuals and four HIV-1-negative volunteers. We detected significantly greater levels of HERV transcripts in the plasma of most HIV-1-positive individuals compared to controls (, Mann-Whitney, p = 0.0160).
Plasma RNA Levels of HERV-K in HIV-1-Positive and -Negative Individuals' Plasma
Although HIV-1 and endogenous retroviruses are phylogenetically distant [23
], we identified several regions of clustered and distributed amino acid identity in RT and Gag elements. Sequence comparison within a well-conserved protein like RT showed amino acid identities that were both distributed and concentrated in short, contiguous regions (A). Although alignments of the entire amino acid sequence were not possible with less well-conserved proteins, short, contiguous regions of amino acid sequence identity were still present (B).
HERV/HIV-1 Amino Acid Alignments
Because of the possibility of both a cross-reactive and independent T cell response to HERV in HIV-1 infection, we sought to measure the CD8+ T cell response to a number of HERV epitopes. We manufactured peptides to test for T cell reactivity, based upon the analysis of HERV sequence with epitope prediction programs [20
] and based on similarity to known CD8+ T cell epitopes in HIV-1 [19
]. A subset of the peptides shared more amino acids in common with HIV-1 (≥4 amino acids), and others were unique to HERV (defined as ≤ 3 amino acids in common) (B; Table S1
). We tested PBMCs from HIV-1-positive and -negative individuals for HERV- and HIV-1-specific T cell responses in 29 HIV-1-positive study participants from the OPTIONS cohort of primary HIV-1 infection at UCSF [24
] and in 13 low-risk HIV-1-negative controls. Specific interferon-γ responses were detected to HERV peptides in HIV-1-infected individuals but not in HIV-1-negative controls (, Mann-Whitney, p
< 0.001). As expected, PBMCs from HIV-1-positive individuals also recognized HIV-1-specific peptides. There was no statistical difference in the mean frequency of responding cells specific for HERV peptides with similarity to HIV-1 sequences and those unique to HERVs (Mann-Whitney, p
= 0.1025). In addition to HIV-1-negative controls, we also tested three HCV+, HIV-1 negative controls. T cell responses were not detected in response to HERV peptides in HCV+ controls (). Individual T cell responses to each peptide tested for each study participant summarized in this figure are shown in detail in Figure S3
T Cell Responses to HERV and HIV-1 Antigens in HIV-1-Positive and -Negative Individuals Measured by Interferon-γ ELISPOT
In a cross-sectional analysis of the cohort of HIV-1-positive study participants, five individuals recognized the unique HERV peptide HERV-L IQ10 with variable magnitudes of response (A). In the responder with the highest T cell response magnitude (OP562), a peptide titration assay was performed (B).
Cross-Sectional and Longitudinal ELISPOT Responses to HIV-1- and HERV-Derived Peptides and Sequence Variants in HIV-1-Positive Study Participants
We measured HERV and HIV-1 T cell responses in three study participants in longitudinal series, including OP562, who naturally contained HIV-1 viremia without antiretroviral therapy over the duration of our longitudinal analysis. The unique HERV peptide HERV-L IQ10 stimulated T cell responses in all three individuals, demonstrating persistent, independent HERV-specific T cell responses at high magnitude (C). In two of the individuals tested (OP747 and OP841), highly active antiretroviral therapy (HAART) was initiated, with subsequent declines in HIV-1 plasma viral load and the level of T cell responses to the HERV-L IQ10 peptide.
We also compared responses to HIV-1 and HERV peptides in longitudinal series with a similar pair of peptides. As the peptides HIV Nef LG13 and HERV-H LI13 shared five amino acids, these responses could reflect a level of cross-reactivity. For OP562, responses to both HIV-1 and HERV were not detectable by week 18, but emerged by week 63 of HIV-1 infection (D, upper panel). Responses to the HIV Nef LG13 and HERV-H LI13 were detected in another study participant, OP747 (D, lower panel).
To address potential cross-reactivity of HERV- and HIV-1-specific T cells in other study participants, we compared responses to an HLA-A2-restricted HIV-1 peptide HIV RT VL9 with responses to a HERV-L peptide, HERV-L II9. The HERV-L II9 peptide is classified as a unique HERV peptide for this study because it shares only three amino acids with its closest corresponding peptide in HIV-1, HIV RT VL9 (see ). To test the effect of amino acid replacements in the HIV-1 peptide that increased the amino acid sequence similarity to the HERV peptide, we included in this analysis a number of intermediate sequence variant peptides, in which selected amino acids in the HIV-1 peptide were replaced with the corresponding amino acid from the HERV sequence (E). One individual (OP478) responded to the HERV peptide, but not to the HIV-1 peptide or any of the intermediate sequence variants. Two individuals who responded to the HIV-1 peptide and the sequence variant peptides (to varying degrees) did not respond to the HERV peptide.
HERV and HIV-1-Derived Peptides Tested in This Study, Divided into the Three Categories Based on Amino Acid Sequence Identity
To qualitatively compare HERV-specific CD8+ T cells with those specific for other viruses, we determined the phenotype and function of HIV-1-, HERV-, and CMV-specific T cells from OP562 and OP841, who responded to the three viruses. For this analysis, we selected HIV-1 and HERV peptides (HERV-L IQ10 and HIV RT VY10) with only two amino acids in common, minimizing potential cross-reactivity. Upon stimulation with respective HERV, HIV-1, and CMV peptides, we ascertained the phenotypes of those cells having a cytokine production profile that were associated with degranulation (A; Text S1
). The HIV-1-specific T cells of both study participants were skewed towards CD45RA−, whereas CD8+ T cells responding to the HERV peptide had a greater percentage of the terminally differentiated cells (CCR7−CD45RA+) (B, left panels). In one study participant (OP562), HERV- and CMV-specific populations shared a lower percentage of CD28−CD27+ CD8+ T cells compared to their HIV-1-specific counterparts (B, upper right panel). In contrast, in the other study participant (OP841), HERV- and CMV-specific populations shared a higher percentage of CD28−CD27+ CD8+ T cells (B, lower right panel). Overall, the phenotype of the HERV-specific CD8+ T cells more closely resembled the phenotype of CMV-specific than HIV-1-specific T cells.
Phenotypic Profile Comparison of HIV-1-, HERV-, and CMV-Specific T Cells Measured by Multicolor Cytokine Flow Cytometry
As these data suggest possible functionality of HERV specific T cells, we measured the relationship of these responses to HIV-1 viral load within the cohort. For the untreated time points available for 20 study participants, HERV-specific T cell responses were significantly inversely correlated with HIV-1 plasma viral load by Spearman non-parametric correlation analysis and linear regression (Spearman, two-tailed, r = −0.49, p = 0.03; linear regression r2 = 0.39, p = 0.003; ).
Inverse Correlation between Anti-HERV T Cell Responses and HIV-1 Plasma Viral Load
Because the ability to control viral load by eliminating infected cells depends on killing, we measured the ability of CD8+ T cells specific for the unique HERV peptide HERV-L IQ10 to kill autologous B cells presenting their target peptide. We peptide stimulated PBMCs from two individuals (OP562 and OP841) to enrich for responsive CD8+ T cells. After a 2-wk peptide stimulation, we used the 51
Cr-release assay to measure the ability of the enriched CD8+ T cells to kill EBV-transformed B cell targets presenting cognate peptide. CD8+ T cells enriched by stimulation with HERV peptide were able to kill B cell targets presenting their cognate peptide but did not lyse targets loaded with a non-cognate or no peptide (). Similar treatment of PBMCs from HIV-1-negative study participants did not produce HERV-specific effectors capable of killing peptide-pulsed targets (Figure S2
HERV-Specific T Cells Lyse HERV Peptide-Pulsed Targets