Preexisting infection with human T-cell lymphotropic virus type 2 neither exacerbates nor attenuates simian immunodeficiency virus SIVmac251 infection in macaques

Abstract

Coinfection with human T-cell lymphotropic virus type 2 (HTLV-2) and human immunodeficiency virus type 1 (HIV-1) has been reported to have either a slowed disease course or to have no effect on progression to AIDS. In this study, we generated a coinfection animal model and investigated whether HTLV-2 could persistently infect macaques, induce a T-cell response, and impact simian immunodeficiency virus SIV(mac251)-induced disease. We found that inoculation of irradiated HTLV-2-infected T cells into Indian rhesus macaques elicited humoral and T-cell responses to HTLV-2 antigens at both systemic and mucosal sites. Low levels of HTLV-2 provirus DNA were detected in the blood, lymphoid tissues, and gastrointestinal tracts of infected animals. Exposure of HTLV-2-infected or naïve macaques to SIV(mac251) demonstrated comparable levels of SIV(mac251) viral replication, similar rates of mucosal and peripheral CD4(+) T-cell loss, and increased T-cell proliferation. Additionally, neither the magnitude nor the functional capacity of the SIV-specific T-cell-mediated immune response was different in HTLV-2/SIV(mac251) coinfected animals versus SIV(mac251) singly infected controls. Thus, HTLV-2 targets mucosal sites, persists, and importantly does not exacerbate SIV(mac251) infection. These data provide the impetus for the development of an attenuated HTLV-2-based vectored vaccine for HIV-1; this approach could elicit persistent mucosal immunity that may prevent HIV-1/SIV(mac251) infection.

FIG. 1.

HTLV-2 establishes an infection in rhesus macaques, replicating in lymphoid and mucosal tissues. (A) Intracellular HTLV-2 Gag p19 expression in the Mo-T and RhM304 cell lines measured by flow cytometry. The isotype control (ctrl) is shown in blue, and p19 Gag expression is shown in black. (B) Number of HTLV-2 genome copies per 10⁶ cells in Mo-T and RhM304 cells. (C) Serum antibodies to p24 Gag measured by ELISA 1 to 3 months after HTLV-2 infection in animals L900, M214, and M304. OD, optical density. (D) HTLV-2 proviral DNA load in the spleen, ileum, and jejunum from animals M214 and M304. (E) Tax protein expression in plasmacytoid dendritic cells infected in vitro with cell-free HTLV-2 (black) or uninfected plasmacytoid dendritic cells (blue).

FIG. 2.

HTLV-2 infection stimulates humoral and cell-mediated responses. (A) Serum antibodies specific to several HTLV-2 proteins measured by Western blotting in animals M893, M897, M905, and M906. The months postinfection (p.i.) are shown below the blot. (B) Nested semiquantitative PCR to amplify either HTLV-2 tax or β-actin. DNA was extracted from the lymph nodes of macaques 3 months after HTLV-2 infection, and the mononuclear cells were CD8 enriched (+) or CD8 depleted (−). (C) CD4⁺ and CD8⁺ T-cell counts in blood from HTLV-2-infected macaques. Repeated-measure analysis of variance with square root-transformed counts over time demonstrated that CD8⁺ T cells significantly increase over time with a P value of 0.0017, while the CD4⁺ cells show a trend toward significance with a P value of 0.049. (D and E) Representative flow cytometric pseudocolor plots showing the frequency of IFN-γ and/or TNF-α production by CD8⁺ T cells in blood (D) or the gastrointestinal (GI) tract (E) after stimulation with HTLV-2 Tax and Gag overlapping peptides or in unstimulated cells. (F and G) Mean HTLV-2-specific CD4⁺ and CD8⁺ T-cell responses, after background subtraction, 10 weeks after HTLV-2 infection in blood (F) and the gastrointestinal tract (G). The frequency of Gag- or Tax-specific cells was measured by intracellular cytokine staining for IFN-γ/TNF-α or IL-2.

FIG. 3.

Persistent HTLV-2 infection despite SIV coinfection. (A) Study design. Four macaques were infected with HTLV-2. Ten months postinfection, these 4 macaques along with 4 naïve animals were challenged with SIV_mac251. Blood, lymphoid, and mucosal tissues were sampled before infection and for up to 90 days after SIV_mac251 infection. (B) Semiquantitative nested PCR for HTLV-2 tax DNA or β-actin amplified from the peripheral blood of HTLV-2-infected animals (M893, M897, M905, and M906) and uninfected control (P182) before and after SIV_mac251 infection. The number of weeks after HTLV-2 infection is shown below the blots. (C) Serum antibodies to HTLV-2 measured by Western blotting before and after SIV_mac251 infection.

FIG. 4.

Similar viral and cellular dynamics in HTLV-2/SIV_mac251 coinfected and SIV_mac251 monoinfected macaques. (A) SIV_mac251 viral load in HTLV-2 coinfected macaques (shown in black) and controls (shown in red) measured by the number of SIV_mac251 RNA copies/ml of plasma. (B) Average CD4⁺ T-cell count in blood following SIV_mac251 infection in HTLV-2/SIV_mac251-infected macaques (black) and SIVmac251-infected macaques (red). (C) Percentage of baseline CD4⁺ CCR5⁺ T-cell count following SIV_mac251 infection in HTLV-2/SIV_mac251-infected macaques (shown in black) and SIV_mac251-infected animals (shown in red). (D) Percentage of baseline CD4⁺ memory T-cell count in blood following SIV_mac251 infection. Memory cells are defined as cells that express both CD28 and CD95. (E) Average CD8⁺ T-cell count following SIV_mac251 infection. (F) Percentage of baseline CD8⁺ effector/effector memory T-cell count in blood after SIV_mac251 infection. CD8⁺ effector/effector memory T cells are CD8⁺ CD28⁻ CD95⁺.

FIG. 5.

HTLV-2 preinfection did not significantly affect either the viral load or the rate of CD4⁺ T-cell loss after SIV_mac251 infection. (A) Viral load in the lymph nodes, jejunum, and rectum in HTLV-2/SIV_mac251 coinfected macaques (black) and SIV_mac251 monoinfected macaques (white) quantified as the number of SIV DNA copies/10⁶ cells 10 and 30 days after SIV_mac251 infection. (B) The frequency of CD3⁺ CD4⁺ T cells in the bone marrow and lymph nodes of HTLV-2/SIV_mac251 coinfected and SIV_mac251 singly infected animals before infection (baseline) and 10 and 30 days after SIV_mac251 infection. (C) The frequency of CD3⁺ CD4⁺ T cells in the jejunum and rectum in HTLV-2/SIV_mac251 coinfected and SIV_mac251 singly infected animals before infection (baseline) and 10 and 30 days after SIV_mac251 infection. (D) Number of CD4⁺ expressing cells, enumerated by immunohistochemical staining of paraformaldehyde-fixed tissue sections from the rectum. A repeated-measure analysis of variance demonstrated that the difference between the baseline (before infection) values for HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques is significantly different (P < 0.05). (E) Number of CD8⁺ expressing cells, enumerated by immunohistochemical staining of paraformaldehyde-fixed rectum tissue sections.

FIG. 6.

No difference in spontaneous MIPα expression by T cells in blood from HTLV-2/SIV_mac251 coinfected and SIV_mac251 monoinfected animals. (A) Mean fluorescence intensity (MFI) of MIP1α in CD3⁺ CD4⁺ T cells in blood from HTLV-2/SIV_mac251 coinfected animals (black) and SIV_mac251 singly infected animals (red) before or after SIV_mac251 infection. (B) Mean fluorescence intensity of MIP1α in CD3⁺ CD8⁺ T cells in blood from HTLV-2/SIV_mac251 coinfected animals and SIV_mac251 singly infected animals SIV_mac251 before or after SIV_mac251 infection.

FIG. 7.

Proliferation of T cells in blood and tissues from HTLV-2/SIV_mac251 coinfected animals and SIV_mac251 monoinfected animals. (A and B) Frequency of CD3⁺ CD4⁺ T cells (A) and CD3⁺ CD8⁺ T cells (B) expressing Ki67 in blood from HTLV-2/SIV_mac251 coinfected animals (black) and SIV_mac251 singly infected animals (red) after SIV_mac251 infection. A Wilcoxon signed-rank test demonstrated that the level of CD8⁺ Ki67 expression continued to be significantly greater in the chronic phase compared to preinfection levels (B). (C and D) Frequency of CD3⁺ CD4⁺ T cells (C) and CD3⁺ CD8⁺ T cells (D) expressing Ki67 in the lymphoid tissues (bone marrow and lymph nodes) of HTLV-2/SIV_mac251 coinfected animals and SIV_mac251 singly infected animals before infection (baseline) and 10 and 30 days after SIV_mac251 infection. (E) Number of cells expressing both CD4 and Ki67 in the rectum in HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques before infection (baseline) and 10 and 30 days after SIV_mac251 infection. Repeated-measure analysis of variance demonstrated that the difference between HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques at baseline is significantly different (P = 0.0007). (F) Number of cells expressing both CD8 and Ki67 in the rectum in HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques before infection (baseline) and 10 and 30 days after SIV_mac251 infection.

FIG. 8.

HTLV-2 preinfection does not affect the quality or quantity of the SIV_mac251-specific CD8⁺ T-cell response. Flow cytometric analysis of CD3⁺ CD8⁺ T cells expressing IFN-γ and/or TNF-α, IL-2, and IL-17 in blood was performed after stimulation with overlapping pools of SIV Gag and SIV Env peptides or in unstimulated cells. (A) Polyfunctional cytokine response to overlapping SIV_mac251 Env peptides produced by CD3⁺ CD8⁺ T cells in blood from HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques 7, 30, 60, and 90 days after SIV_mac251 infection. The cytokines measured after stimulation were as follows: IL-2, IL-17, and IFN-γ stained along with TNF-α. Within the pie charts, the green slice represents the proportion of cells producing 1 cytokine considered to have 1 function (i.e., either IL-2 or IL-17 or IFN-γ/TNF-α), the blue slice represents the proportion of cells concurrently producing 2 cytokines considered to have 2 functions, and the red slice represents the proportion of cells producing 3 cytokines or 3 functions (i.e., cells concurrently producing IFN-γ and/or TNF-α, IL-2, and IL-17). (B) Average total cytokine (IFN-γ and/or TNF-α, IL-2, IL-17) production, after background subtraction, in CD3⁺ CD8⁺ T cells from blood in response to stimulation with SIV Env overlapping peptide pool after SIV_mac251 infection. Cytokine production in HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques is shown. (C) Average total cytokine (IFN-γ and/or TNF-α, IL-2, IL-17) production, after background subtraction, in CD3⁺ CD8⁺ T cells from blood in response to stimulation with SIV Gag overlapping peptide pool after SIV_mac251 infection. Cytokine production in HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques is shown.

FIG. 9.

SIV_mac251-specific CD4⁺ T-cell responses are similar in HTLV-2/SIV_mac251 coinfected animals and SIV_mac251 monoinfected animals. (A) Polyfunctional cytokine response to overlapping SIV_mac251 Env peptides produced by CD3⁺ CD4⁺ T cells in blood from HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques 7, 30, 60, and 90 days after SIV_mac251 infection. The cytokines measured after stimulation were as follows: IL-2, IL-17, and IFN-γ stained along with TNF-α. Within the pie charts, the green slice represents the proportion of cells producing 1 cytokine considered to have 1 function (i.e., either IL-2 or IL-17 or IFN-γ/TNF-α), the blue slice represents the proportion of cells concurrently producing 2 cytokines considered to have 2 functions, and the red slice represents the proportion of cells producing 3 cytokines or 3 functions (i.e., cells concurrently producing IFN-γ and/or TNF-α and IL-2 and IL-17). (C) Average total cytokine (IFN-γ and/or TNF-α, IL-2, IL-17) production, after background subtraction, in CD3⁺ CD4⁺ T cells from blood in response to stimulation with SIV Env overlapping peptide pool after SIV_mac251 infection. Cytokine production in HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques is shown.(D) Average total cytokine (IFN-γ and/or TNF-α, IL-2, IL-17) production, after background subtraction, in CD3⁺ CD4⁺ T cells from blood in response to stimulation with SIV Gag overlapping peptide pool after SIV_mac251 infection. Cytokine production in HTLV-2/SIV_mac251 coinfected macaques and SIV_mac251 singly infected macaques is shown.