T cell responses to human endogenous retroviruses in HIV-1 infection

Abstract

Human endogenous retroviruses (HERVs) are remnants of ancient infectious agents that have integrated into the human genome. Under normal circumstances, HERVs are functionally defective or controlled by host factors. In HIV-1-infected individuals, intracellular defense mechanisms are compromised. We hypothesized that HIV-1 infection would remove or alter controls on HERV activity. Expression of HERV could potentially stimulate a T cell response to HERV antigens, and in regions of HIV-1/HERV similarity, these T cells could be cross-reactive. We determined that the levels of HERV production in HIV-1-positive individuals exceed those of HIV-1-negative controls. To investigate the impact of HERV activity on specific immunity, we examined T cell responses to HERV peptides in 29 HIV-1-positive and 13 HIV-1-negative study participants. We report T cell responses to peptides derived from regions of HERV detected by ELISPOT analysis in the HIV-1-positive study participants. We show an inverse correlation between anti-HERV T cell responses and HIV-1 plasma viral load. In HIV-1-positive individuals, we demonstrate that HERV-specific T cells are capable of killing cells presenting their cognate peptide. These data indicate that HIV-1 infection leads to HERV expression and stimulation of a HERV-specific CD8+ T cell response. HERV-specific CD8+ T cells have characteristics consistent with an important role in the response to HIV-1 infection: a phenotype similar to that of T cells responding to an effectively controlled virus (cytomegalovirus), an inverse correlation with HIV-1 plasma viral load, and the ability to lyse cells presenting their target peptide. These characteristics suggest that elicitation of anti-HERV-specific immune responses is a novel approach to immunotherapeutic vaccination. As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.

Figure 1. Plasma RNA Levels of HERV-K in HIV-1-Positive and -Negative Individuals' Plasma

Levels of a HERV-K transcript derived from the envelope region were measured as a percentage relative to a standard derived from peripheral blood cells. The p-value was determined with the Mann-Whitney test.

Figure 2. HERV/HIV-1 Amino Acid Alignments

Alignments were anchored based on short regions of similarity identified with BLAST [38] short nearly exact match search settings, which included both amino acid similarity estimated with evolutionary matrices (not shown in this figure) and identity. (A) HIV-1 HXB-2 and HERV-K (gi|5802821) showing a segment of the RT protein. Sequence numbering shown is based on position within individual accessions. Identical amino acids are shown in bold. (B) Short regions of similarity between reading frames within different HERV insertions [17,18] in the human genome and HIV-1 HXB-2 and their correspondence to regions of HIV-1. NT is shown when the region in HIV-1 was not a known CD8+ T cell epitope and the HIV-1 peptide was not included in the study.

Figure 3. T Cell Responses to HERV and HIV-1 Antigens in HIV-1-Positive and -Negative Individuals Measured by Interferon-γ ELISPOT

HERV peptides were grouped according to their similarity to HIV-1 peptide sequence, with “Unique HERV Peptides” having three or fewer amino acids in common with an HIV-1 peptide, and “HERV Peptides Similar to HIV-1” having four or more peptides in common with HIV-1. Subsets of peptides were tested in each patient, with the number tested (n = 6–23) varying depending on HLA type. Values shown for responses are normalized per peptide within each grouping (i.e., the sum of the response values to all peptides tested divided by the number of peptides tested for each patient). The individual peptide responses that are summarized in this figure are detailed in Figure S3. Responses in HIV-1-positive individuals are shown as closed circles and in HIV-1-negative individuals as open circles. Responses to all HERV peptides were measured for HCV+ individuals and are shown as filled triangles. p-Values are derived from the Mann-Whitney test.

Figure 4. Cross-Sectional and Longitudinal ELISPOT Responses to HIV-1- and HERV-Derived Peptides and Sequence Variants in HIV-1-Positive Study Participants

(A) Responses in five HIV-1 positive study participants to the unique HERV peptide HERV-L IQ10. (B) A titration of peptide concentrations for HERV-L IQ10 (open green triangles) peptides for an HIV-1-positive study participant. (C) Responses in three individuals to a unique HERV peptide are shown as inverted green triangles. HIV-1 plasma viral loads are represented as filled red diamonds and are determined with clinical assays for viral load (bDNA and PCR assays). (D) Responses in two representative HIV+ study participants to an HIV-1 and HERV peptide pair with a high level of amino acid identity are shown in blue as open and filled circles, respectively. (E) Responses in three study participants to an HLA-A2-restricted HIV-1 epitope peptide (HIV RT VL9) and a HERV-L peptide (HERV-L II9) are shown, along with responses to three intermediate sequence variant peptides in which selected amino acids in the HIV-1 epitope were replaced with the equivalent amino acid found in the HERV peptide.

Figure 5. Phenotypic Profile Comparison of HIV-1-, HERV-, and CMV-Specific T Cells Measured by Multicolor Cytokine Flow Cytometry

(A) Phenotypic characterization of cytokine producing cells (see Text S1) from two HIV-1-positive study participants for the markers CCR7, CD27, CD28, and CD45RA are shown as pie charts. The colors in the pie charts correspond to the phenotypic categories delineated in the table below them. (B) A subset of the data from (A) is shown, highlighting comparisons of specific phenotypes. The percentages of CD45RA+ (unfilled bars) and CD45RA− (filled bars) T cells are shown in the panels in the left column. The panels in the right column detail the percentages of CD28− T cells from the two study participants.

Figure 6. Inverse Correlation between Anti-HERV T Cell Responses and HIV-1 Plasma Viral Load

PBMCs from 20 HIV-1-positive individuals not on treatment were analyzed by ELISPOT for HERV responses. The mean response (>50 SFU/million PBMCs) values for all HERV peptides tested had a significant inverse correlation to HIV-1 plasma viral load (Spearman, two-tailed, r = −0.49, p = 0.03) and by linear regression (r ² = 0.39, p = 0.003) as shown in the figure.

Figure 7. HERV-Specific T Cells Lyse HERV Peptide-Pulsed Targets

HERV-L IQ10-specific T cells were tested against autologous B cells pulsed with HERV-L IQ10 peptide (open inverted green triangles), control peptide (filled squares), or no peptide (open squares) in independent peptide-stimulated expansions from two HIV-positive study participants.