Expansion and diversification of virus-specific T cells following immunization of human immunodeficiency virus type 1 (HIV-1)-infected individuals with a recombinant modified vaccinia virus Ankara/HIV-1 Gag vaccine

Abstract

Affordable therapeutic strategies that induce sustained control of human immunodeficiency virus type 1 (HIV-1) replication and are tailored to the developing world are urgently needed. Since CD8(+) and CD4(+) T cells are crucial to HIV-1 control, stimulation of potent cellular responses by therapeutic vaccination might be exploited to reduce antiretroviral drug exposure. However, therapeutic vaccines tested to date have shown modest immunogenicity. In this study, we performed a comprehensive analysis of the changes in virus-specific CD8(+) and CD4(+) T-cell responses occurring after vaccination of 16 HIV-1-infected individuals with a recombinant modified vaccinia virus Ankara-vectored vaccine expressing the consensus HIV-1 clade A Gag p24/p17 sequences and multiple CD8(+) T-cell epitopes during highly active antiretroviral therapy. We observed significant amplification and broadening of CD8(+) and CD4(+) gamma interferon responses to vaccine-derived epitopes in the vaccinees, without rebound viremia, but not in two unvaccinated controls followed simultaneously. Vaccine-driven CD8(+) T-cell expansions were also detected by tetramer reactivity, predominantly in the CD45RA(-) CCR7(+) or CD45RA(-) CCR7(-) compartments, and persisted for at least 1 year. Expansion was associated with a marked but transient up-regulation of CD38 and perforin within days of vaccination. Gag-specific CD8(+) and CD4(+) T-cell proliferation also increased postvaccination. These data suggest that immunization with MVA.HIVA is a feasible strategy to enhance potentially protective T-cell responses in individuals with chronic HIV-1 infection.

Figures

FIG. 1.

Responses to overlapping HIVA Gag 24/p17 15-mer and non-Gag epitope peptides obtained in ex vivo IFN-γ ELISPOT assays. (A) Total Gag-specific response (sum of peptide matrix pool responses divided by 2) obtained in assays with fresh undepleted (top panels) or CD8+-cell-depleted (bottom panels) PBMC from 16 vaccinees and 2 unvaccinated control subjects (014 and 019) (solid black symbols in left panels) at the time points indicated. Immunizations were given at days 0 and 28. Negative control values have been subtracted. Broken lines indicate that no sample was available for the intervening time point. (B) Box plots (interquartile range with median indicated by horizontal line) of total Gag responses of the 16 vaccinees in assays with undepleted (top) or CD8-depleted (bottom) PBMC at the time points indicated. (C) Breadth of Gag-specific responses in IFN-γ ELISPOT assays with undepleted PBMC before (white bars) and at the peak of the response (black bars) after one or two MVA.HIVA immunizations or, for the control subjects, the highest response seen at any time after day zero. (D) Preimmunization (white bars) and peak postimmunization (black bars) responses in IFN-γ ELISPOT assays with optimal CTL epitope peptides derived from Pol, Nef, and Env expressed in the epitope string (14).

FIG. 2.

Postimmunization expansion of CD8+ T cells specific for HIV-1 epitopes in MVA.HIVA, identified by tetramer staining. (A) Representative profile showing evolution of the HLA-B*2705/p24-specific response of subject 015 at the times indicated. Ex vivo PBMC were stained with an HLA-B*2705 tetramer refolded with the KRWIILGLNK peptide. Dot plots show gated lymphocytes identified by forward and side scatter. (B) Top panel: frequencies of HIV-1-specific CD8+ T cells in seven vaccinees determined by staining with various tetramers at the times indicated. Tetramers were refolded with the following peptides: A2 p17, SLYNTVATL; A2 Pol, ILKEPVHGV; A3 Pol, AIFQSSMTK; A3 Nef, QVPLRPMTYK; B7 Nef, TPGPGVRYPL; B8 p24, GEIYKRWII; B8 Nef, FLKEKGGL; B27 p24, KRWIILGLNK. Bottom panel: frequencies of CD8+ T cells specific for non-vaccine-derived epitopes from EBV proteins (subjects 012, 013, and 015) or HLA-B35 Nef (GPGVRYPLTF) (subject 015) at corresponding time points. (C) Representative plots (subject 015) showing expression in tetramer-reactive cells (top panels) of CD45RA and CCR7 before and 6 and 27 weeks after the first MVA.HIVA immunization. Bottom panels show profiles obtained by gating on total CD8bright lymphocytes. The fold change in each compartment within tetramer-positive populations detected in seven donors is shown in Table 3.

FIG. 2.

Postimmunization expansion of CD8+ T cells specific for HIV-1 epitopes in MVA.HIVA, identified by tetramer staining. (A) Representative profile showing evolution of the HLA-B*2705/p24-specific response of subject 015 at the times indicated. Ex vivo PBMC were stained with an HLA-B*2705 tetramer refolded with the KRWIILGLNK peptide. Dot plots show gated lymphocytes identified by forward and side scatter. (B) Top panel: frequencies of HIV-1-specific CD8+ T cells in seven vaccinees determined by staining with various tetramers at the times indicated. Tetramers were refolded with the following peptides: A2 p17, SLYNTVATL; A2 Pol, ILKEPVHGV; A3 Pol, AIFQSSMTK; A3 Nef, QVPLRPMTYK; B7 Nef, TPGPGVRYPL; B8 p24, GEIYKRWII; B8 Nef, FLKEKGGL; B27 p24, KRWIILGLNK. Bottom panel: frequencies of CD8+ T cells specific for non-vaccine-derived epitopes from EBV proteins (subjects 012, 013, and 015) or HLA-B35 Nef (GPGVRYPLTF) (subject 015) at corresponding time points. (C) Representative plots (subject 015) showing expression in tetramer-reactive cells (top panels) of CD45RA and CCR7 before and 6 and 27 weeks after the first MVA.HIVA immunization. Bottom panels show profiles obtained by gating on total CD8bright lymphocytes. The fold change in each compartment within tetramer-positive populations detected in seven donors is shown in Table 3.

FIG. 3.

Up-regulation of CD38 on CD8+ T cells specific for vaccine-encoded epitopes. (A) Top panels: CD38 expression in KRWIILGLNK-specific T cells (gated on tetramer-positive cells as shown in Fig. 2C) from subject 015. Bottom panels: Nef-specific (epitope not in the vaccine) tetramer-positive cells from this donor showed stable levels of CD38 expression over the same period. Triangles indicate immunization times. (B) Kinetics of CD38 expression in tetramer-positive cells with different specificities (solid lines) (see Fig. 2B for specificities) and total CD8+ T cells (broken lines) obtained from seven donors before and after MVA.HIVA immunization. (C) Kinetics of perforin expression on tetramer-positive cells is similar to that of CD38. The percentage of HIV-1-specific tetramer-positive cells expressing perforin pre- and post-MVA.HIVA immunization are shown for subject 015 (representative of four vaccinees). The percentage of total CD8+ cells expressing perforin remained stable over this time (6% [not shown]).

FIG. 4.

Proliferative capacity of Gag-specific CD8+ and CD4+ T cells postimmunization. Proliferation was determined by flow cytometric analysis of CFSE dilution after 6-day in vitro culture with HIVA peptide pools 1 to 4 (A) or pool 90 (B and C). (A) Percentage of CFSElo CD3+ CD8+ cells (top panel) or CFSElo CD3+ CD4+ cells (bottom panel) from six subjects at the time the peak response was detected by ELISPOT, i.e., after one (subject 022) or two (subjects 015, 016, 018, 019, and 021) MVA.HIVA immunizations (weeks 2 and 8, respectively). Values from unstimulated samples have been subtracted. Responses shown were at least three times the unstimulated control sample value. No responses to pool 4 were seen. (B) Representative plots showing total Gag-specific (pool 90 peptides) CD8+ T-cell proliferation, after gating on CD3+ lymphocytes, of subject 006 at the time points indicated. Representative positive (staphylococcal enterotoxin B [SEB]) and negative (medium alone) control responses at day zero are also shown for comparison. (C) Total Gag-specific proliferative responses before and after MVA.HIVA immunization of eight vaccinees who had received pTHr.HIVA vaccine 2 years earlier. Cell division index = [% CD8+ (top panels) or CD4+ (bottom panels) CFSElo cells in CD3+ lymphocyte gates obtained after peptide stimulation]/(% obtained from unstimulated samples).