Received 6 August 2009/Accepted 18 November 2009

Transcription

1 JOURNAL OF VIROLOGY, Mar. 2010, p Vol. 84, No X/10/$12.00 doi: /jvi Copyright 2010, American Society for Microbiology. All Rights Reserved. Preexisting Infection with Human T-Cell Lymphotropic Virus Type 2 neither Exacerbates nor Attenuates Simian Immunodeficiency Virus SIV mac251 Infection in Macaques Shari N. Gordon, 1 Anna R. Weissman, 1 Valentina Cecchinato, 1 Claudio Fenizia, 1 Zhong-Min Ma, 7 Tzong-Hae Lee, 10 Lorenzo Zaffiri, 1 Vibeke Andresen, 1 Robyn Washington Parks, 1 Kathryn S. Jones, 3 Jean Michel Heraud, 6 Maria Grazia Ferrari, 4 Hye Kyung Chung, 4 David Venzon, 2 Renaud Mahieux, 7 Edward L. Murphy, 9,10 Steven Jacobson, 5 Christopher J. Miller, 8 Francis W. Ruscetti, 3 and Genoveffa Franchini 1 * Animal Models and Retroviral Vaccines Section 1 and Biostatistics and Data Management Section, 2 National Cancer Institute, NIH, Bethesda, Maryland 20892; Basic Research Program, SAIC-Frederick, Inc., National Cancer Institute, NIH, Frederick, Maryland 3 ; Advanced BioScience Laboratories, Inc., Kensington, Maryland ; Viral Immunology Section, Neuroimmunology Branch, NINDS, Bethesda, Maryland ; World Health Organization-National Influenza Laboratory, Institut Pasteur de Madagascar, Antananarivo, Madagascar 6 ; Oncogenèse Rétrovirale, INSERM U758, Ecole Normale Supérieure, and IFR 128 BioSciences Lyon-Gerland, Lyon Cedex 07, France 7 ; Center for Comparative Medicine and California National Primate Research Center, University of California, Davis, Davis, California ; and Department of Laboratory Medicine and of Epidemiology and Biostatistics, University of California, 9 and Blood Systems Research Institute, 10 San Francisco, California Received 6 August 2009/Accepted 18 November 2009 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. Human T-cell lymphotropic virus type 2 (HTLV-2) was discovered in 1982 and recognized as the second human retrovirus found (29). HTLV-2 is closely related to the first human retrovirus discovered, HTLV-1 (49, 50), a pathogenic virus that causes adult T-cell leukemia/lymphoma (ATLL) and an inflammatory neurologic disorder called HTLV-1-associated myelopathy or tropical spastic paraparesis (HAM/TSP) (22, 45). HTLV-2 is prevalent in Amerindian populations of North and South America and in Africa (57). The prevalence of HTLV-2 is generally low; however, in the past 20 years, an epidemic of HTLV-2 infection has occurred among intravenous drug users (8, 24, 54, 57). HTLV-2 establishes a lifelong infection and replicates at low levels in most infected individuals. While anecdotal cases of TSP/HAM-like neurological manifestations (1, 44) and hematopoietic diseases, such as * Corresponding author. Mailing address: National Cancer Institute, NIH, 9000 Rockville Pike, Bldg. 41, Room D804, Bethesda, MD Phone: (301) Fax: (301) franchig@mail.nih.gov. Published ahead of print on 13 January large granular lymphoma (LGL), in HTLV-2-infected individuals have been reported (3, 37 39, 46), the extent to which HTLV-2 can induce disease in humans remains unclear. Indeed, even in the condition of immune deficiency, such as infection with human immunodeficiency virus type 1 (HIV-1), HTLV-2 coinfection has not been reported to be associated with cancer or neurological diseases. However, more studies are necessary to fully understand the role of HTLV-2 in human disease. While HTLV-1 infection has been connected with an accelerated course of disease in HIV-1 coinfected patients (2, 34), HTLV-2 has been reported to either have no effect (26) or suggested to exert a potential protective role during HIV-1 infection (12, 23). This protective role is thought to be due to a maintenance of CD4 T cells, lowering immune activation, and delayed progression to AIDS (4, 5). In addition, modulation of cytokine and chemokine networks by HTLV-2 has been suggested to contribute to the control of HIV-1 infection (12, 36, 47). Since studies on the immunological interactions between HIV-1 and HTLV-2 have been performed in patients coinfected with HIV-1 and HTLV-2 in the chronic phase of HIV-1 disease, little is known about the effects of HTLV

2 3044 GORDON ET AL. J. VIROL. infection during acute HIV-1 replication, mucosal CD4 T-cell depletion, or HIV-1-specific immune responses. Furthermore, the potential protective effect of an HTLV-2 vector that would target both CD4 and CD8 T cells and induce a low-grade persistent infection makes HTLV-2 an interesting potential vaccine platform for an HIV-1 vaccine. Current HIV-1 vaccine strategies have focused on viral vectors delivering HIV-1 antigens. These vectors stimulate strong, systemic antigen-specific responses but are unable to protect from infection, since they generate only limited mucosal responses and do not persist. The only vaccine approach that has conferred protection in the simian immunodeficiency virus SIV mac251 macaque model is a live attenuated virus (17), suggesting that persistent expression of viral antigens in mucosal and lymphoid tissues may be necessary. An HTLV-2 vector expressing HIV-1 antigens at mucosal sites that stimulates and maintains T-cell responses in the gut may confer protection from infection by quickly eliminating cells infected by the founder virus at the portal of entry. This study establishes that the Indian rhesus macaque model for HTLV-2 infection is a suitable model to test this hypothesis, as it demonstrates that HTLV-2 targets systemic, lymphoid, as well as mucosal tissues of rhesus macaques. HTLV-2 infection induces humoral as well as cell-mediated immune responses, and importantly, T- cell responses can be found at both systemic and mucosal sites. In this study, we demonstrate that the viral and T-cell dynamics of macaques dually infected with HTLV-2 and SIV mac251 are similar to those of macaques singly infected with SIV mac251. MATERIALS AND METHODS Experimental HTLV-2 and SIV mac251 infection. The 11 Indian rhesus macaques (RMs) used in this study were housed and cared for under the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care International. RMs were housed at the Advanced Biosciences Laboratory in Rockville, MD. Seven RMs were inoculated with 10 8 HTLV-2-infected lethally irradiated cells of human (Mo-T) or rhesus (M304) origin. Mo-T cells are an HTLV-2-producing cell line that was derived from a male patient with hairy cell leukemia (29). To create an HTLV-2-expressing rhesus macaque cell line, we cocultured cells from macaque M304 with gamma-irradiated Mo-T cells at a 1:1 ratio. Cells were maintained in RPMI 1640 complete medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% penicillin-streptomycin, and 20 U/ml recombinant human interleukin 2 (IL-2). HTLV-2 infection was documented by measuring extracellular p19 Gag production by an antigen capture assay and by intracellular p19 Gag staining. The cell line derived from this culture was designated RhM304. Animals M304 and L900 were inoculated with HTLV-2-infected cells from animal M304, i.e., an autologous or allogeneic infection, respectively, while animal M214 received a heterologous infection with Mo-T cells. Both heterologous and autologous infections produced a persistent HTLV-2 infection; therefore, the remaining HTLV-2 infections were performed using irradiated Mo-T cells. Four additional animals (M893, M897, M905, and M906) were infected with HTLV-2; 10 months after HTLV-2 infection, these four animals and four naïve macaques were infected intrarectally with SIV mac251 ( % tissue culture infective dose [TCID 50 ]). All SIV-infected macaques were haplotyped, two macaques were MamuA01 (animals M905 and P160), one was an HTLV-2 preinfected animal, and the other a naïve control. All of the animals were B08 negative, and one of the HTLV-2 preinfected animals was positive for B17 (animal M893). Blood and tissue collection. Mononuclear cells were isolated from blood, rectal, jejunal pinch biopsy, lymph node biopsy, and bone marrow aspirate specimens. Mononuclear cells were separated from whole blood and bone marrow aspirate specimens by density gradient centrifugation (Ficoll). Lymph nodes were homogenized and passed through a 100- m cell strainer, and mononuclear cells were purified by density gradient centrifugation (Ficoll). Rectal and jejunal pinch biopsy specimens were treated with 1 mm ultrapure dithiothreitol (Invitrogen, Carlsbad, CA) for 20 min followed by incubation in 0.1 M EDTA solution in calcium-magnesium-free HBSS with penicillin-streptomycin for 60 min to remove the epithelial layer. Lamina propria lymphocytes were separated, following the removal of the intraepithelial lymphocytes, by incubating with collagenase D (400 U/ml; Boehringer Mannheim, Mannheim, Germany) and DNase (1 g/ml; Invitrogen, Carlsbad, CA) for 2hat37 C in Iscove s modified Dulbecco s medium supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. The dissociated mononuclear cells were then placed over 42% Percoll (General Electric Healthcare, Piscataway, NJ) and centrifuged at 2,000 rpm for 30 min at 4 C. Immunophenotypic studies were performed by polychromatic flow cytometry (as detailed below) on mononuclear cells derived from all tissues. All cells were stained on the day of sampling, except for cells from the jejunum and rectum, which were allowed to rest overnight and then stained. Immunophenotyping and intracellular cytokine assay. Four- and seven-color flow cytometric analysis was performed on mononuclear cells from blood and tissues. Surface staining was performed for 30 min at room temperature with antibodies to CD3 (clone SP34-2), CD4 (L200), chemokine (C-C motif) receptor 5 (CCR5) (clone 3A9), and CD95 (clone DX2), all obtained from BD Biosciences (San Diego, CA). Anti-CD28 (clone CD28.2) was obtained from ebiosciences (San Diego, CA), and anti-cd8 (clone 3B5) was obtained from Invitrogen (Carlsbad, CA). Following surface staining, the cells were permeabilized and stained with anti-ki67 (clone B56 from BD Biosciences). To monitor SIV- and HTLV-2-specific immune responses, lymphocytes were resuspended at 10 6 cells per ml in RPMI 1640 complete medium with 10% heat-inactivated FCS in combination with anti-cd28 (Biosource International, Camarillo, CA), anti- CD49d (Becton Dickinson, San Jose, CA), and monensin (BD Biosciences). The cells were incubated with medium only or with peptide pools of 15-mers overlapping by 11 amino acids at a concentration of 1 g/ml derived from the entire HTLV-2 Tax, HTLV-2 Gag, SIV mac251 Gag, and SIV mac251 Env protein sequence. Stimulation with either phorbol myristate acid (PMA) and ionomycin A23187 (Sigma-Aldrich, St. Louis, MO) or staphylococcal endotoxin B (SEB) (Toxin Technologies, Sarasota, FL) was used as a positive control. Cells were stimulated for 5 h, washed, and stained for CD3, CD4, and CD8 for 30 min at room temperature. Following surface staining, lymphocytes were permeabilized with FACS Perm/Wash solution (BD Biosciences), and stained intracellularly for gamma interferon (IFN- ) (clone B27), tumor necrosis factor alpha (TNF- ) (clone MAB11), macrophage inflammatory protein 1 (MIP1 ) (clone 11A3), and IL-2 (clone MQ1-17H12) (all from BD Biosciences), in addition to IL-17 (clone ebio64dec17) from ebioscience. All cells were fixed with 1% paraformaldehyde, and at least 100,000 events were acquired on either a FACSCalibur or LSRII. Data analysis was performed with Flowjo (Treestar, CA). Infection of primary dendritic cells. Plasmacytoid dendritic cells (pdcs) were isolated from the peripheral blood mononuclear cells (PBMCs) of uninfected rhesus macaques. Non-pDC lineages were first removed using a commercially available kit (Miltenyi Biotec, Auburn, CA) followed by positive selection of BDCA-4 (NP-1) cells. A portion of the cells were fixed immediately, permeabilized, and stained for the presence of Tax-2. The remaining pdcs were infected with HTLV-2 harvested from the supernatant of Mo-T cells by incubating the virus with pdcs for 3 h, washing, and replating in six-well plates at /well. The pdcs were confirmed to be CD123 by flow cytometry using an anti-cd123 antibody (clone 7G3; BD Bioscience). These pdcs were cultured in RPMI 1640 medium supplemented with 10% human AB serum and IL-3 (10 ng/ml), and the cells were harvested, permeabilized, and stained for Tax-2 either 2 or 6 days later. Serological analysis and intracellular staining for HTLV-2 antigens. The presence of antibodies to viral antigens was determined by Western blotting the sera collected from infected animals. Blot strips were obtained from Zepto- Metrix Corporation (Buffalo, NY). The blots contain multiple viral proteins, including HTLV Gag (p19), a unique HTLV-1 envelope recombinant protein rgp46-i, and GD21, a common, yet specific HTLV-1 and HTLV-2 gp41 epitope of the envelope protein. Each strip also includes an internal sample control to minimize the risk of false-negative results due to operational errors. The assay was carried out according to the manufacturer s instructions. Intracellular staining for viral antigens was performed using a mouse anti-p19 Gag antibody (Zeptometrix, Buffalo, NY) or a rabbit anti-tax2 antibody (40). Briefly, cell pellets were fixed in 100 l IC fixation buffer (ebioscience). Intracellular staining was performed in 100 l of permeabilization buffer using a 1:250 dilution of the anti-p19 Gag antibody or 1:100 dilution of the anti-tax2 antibody and incubated for 30 min in the dark at room temperature. The cells were washed twice and stained with Alexa Fluor 488-labeled anti-mouse IgG antibody or Alexa Fluor 488-labeled anti-rabbit antibody (Invitrogen, Carlsbad CA) for 30 min in the dark at room temperature. Next, the cells were washed and resuspended in 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA)

3 VOL. 84, 2010 HTLV-2 AND SIV mac251 COINFECTION MODEL IN MACAQUES 3045 in phosphate-buffered saline (PBS). Four-parameter flow cytometric analysis was performed using a FACSCalibur with CELLQuest software. Viral load. SIV RNA in plasma samples and SIV DNA in the blood and tissue samples of macaques was quantified by nucleic acid sequence-based amplification (NASBA) as previously described (51). Quantification of HTLV-2 DNA in blood and tissue samples from animals L900, M214, and M304 was performed by extracting genomic DNA with the DNeasy tissue kit (Qiagen, Valencia, CA) according to the manufacturer s protocol, except for the DNA elution step. DNA was eluted with 10 mm Tris (ph 8.0). Five hundred nanograms of genomic DNA was subjected to real-time PCR. The TaqMan probe and PCR primers were designed within the gag gene of HTLV-2 (53). The TaqMan probe and primer sequences are as follows: probe, 5 -FAM-AGGCGTGGACACCCAAGGACA AAAC-TAMRASp-3 (FAM is 6-carboxyfluorescein); forward primer, 5 -GGG AGATGCTCCGGACATG-3 ; reverse primer, 5 -CGTGGTTGGACCACAAG GA-3. Reaction conditions were as follows. The 25- l PCR mixture for HTLV-2 and macaque albumin DNA consisted of 500 ng of DNA extracted from tissues or PBMCs, 200 nm primers, 100 nm probe, and 2 TaqMan Universal PCR Mastermix (Applied Biosystems, Carlsbad, CA). Amplification was performed using an ABI Prism 7500 sequence detector system (Applied Biosystems). The normalized value of the HTLV-2 proviral DNA load was calculated as HTLV-2 DNA copy number/macaque albumin gene copy number and expressed as the number of HTLV-2 proviral DNA copies per 10 6 cells (15). The HTLV-2 proviral DNA load on animals M893, M897, M905, and M906 was determined in quadruplicate for each sample using real-time quantitative PCR with SYBR green intercalation as described elsewhere (33). This assay had excellent amplification efficiency, as determined by diluted DNA standards included on each plate, had a lower limit of detection of a single copy per reaction mixture, and had good interassay reliability with an R 2 of 0.94 for HTLV-2- infected subjects. Serial samples from the same animal were included in the same testing batch to minimize interassay effects on serial viral loads. Cell input was determined by HLA-DQA quantification in the same aliquot (33). A nested quantitative PCR was also used to amplify a 128-nucleotide sequence in the HTLV-2 Tax gene. DNA was extracted from blood and lymph node samples. DNA extraction was performed using the DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer s instructions. The PCR mixture included 250 ng of DNA, the Platinum High Fidelity Supermix (Invitrogen, Carlsbad, CA), and primers specific for either -actin (forward, 5 -CGGT TGGCCTTGGGGTTCAGGGGG-3 ; reverse, 5 -ATCGTGGGGCGCCCCAG GCACCA-3 ) or the HTLV-2 tax gene (forward,5 -TGGATACCCCGTCTAC GTGT-3 ; reverse,5 -GAGCTGACAACGCGTCCATC-3 ; nested forward, 5 - GTGTTTGGCGATTGTGTACA-3 ; nested reverse, 5 -CCATCGATGGGGT CCCA-3 ). The primers were designed using Primer3 software. Immunohistochemistry and quantitative image analysis. All slides were stained using the Dako autostainer (Dako Inc., Carpinteria, CA). Slides were visualized with epifluorescence illumination using a Zeiss Axioplan 2 microscope (Carl Zeiss) and appropriate filters. Digital images were captured and analyzed by using a Zeiss Axiocam System and Openlab software (Inprovision) as previously described (14, 19). The primary antibodies used included monoclonal anti-cd4 mouse serum (clone IF6; Vector, Burlingame, CA), monoclonal anti- CD8 mouse serum (clone IA5; Leica Microsystems, Bannockburn, IL), and polyclonal anti-ki67 rabbit serum (Lab Vision, Fremont, CA). For all primary antibodies, slides were subjected to an antigen retrieval step consisting of incubation in AR10 (Biogenex Inc., San Ramon, CA) for 2 min at 123 C in the digital decloaking chamber (Biocare Medical Inc., Concord, CA) followed by cooling to 90 C before rinsing in running water and a final buffer rinse. Primary antibodies were replaced by normal (healthy) rabbit IgG (Zymed Inc., South San Francisco, CA) or mouse IgG (Dako Inc., Carpinteria, CA) and included with each staining series as a negative control. Binding of CD4 and Ki-67 was detected simultaneously using Alexa Fluor 488-labeled polyclonal goat anti-rabbit IgG (Molecular Probes, Eugene, OR) and Alexa Fluor 568-labeled polyclonal goat antimouse IgG (Molecular Probes, Eugene, OR) for 1 h. For CD8 and Ki67 staining, Envision mouse and rabbit polymer (Dako Inc., Carpinteria, CA) were used. Binding of CD8 and Ki-67 was detected by diaminobenzidine (DAB) (CD8) and vector SG (Ki67) (Dako Inc., Carpinteria, CA). All the control experiments gave appropriate results with minimal nonspecific staining (data not shown). Slides were visualized with epifluorescence illumination using a Zeiss Axioplan 2 microscope (Carl Zeiss Inc., Thornwood, NY) and appropriate filters. Digital images were captured and analyzed by using a Zeiss Axiocam System and Openlab software (Inprovision Inc., Waltham MA). Only clearly positive cells were considered positive. The number of positive cells is presented as the number of cells per square millimeter. ELISA. An HTLV-1/2 enzyme-linked immunosorbent assay (ELISA) kit from Advanced Biosciences Laboratories (Kensington, MD) was used to quantify TABLE 1. Inoculation of macaques a Animal code HTLV-2 cell line Type of inoculation L900 RhM304 Allogeneic M304 RhM304 Autologous M214 Mo-T Heterologous M893 Mo-T Heterologous M897 Mo-T Heterologous M905 Mo-T Heterologous M906 Mo-T Heterologous a Each macaque was inoculated intravenously with 10 8 RhM304 or Mo-T cells in 20 ml of PBS. HTLV-2 p24 in serum. Briefly, plates were coated with affinity-purified HTLV-2 p24. Standards and samples were added to the plate, any antibodies in the sera bound to the HTLV-2 antigen, the plates were washed, and the quantity of HTLV-2 p24 is detected by an enzymatic reaction. A value is considered positive if its optical density is at least twice the value of a known negative macaque serum sample read at 450 nm. A MIP1 ELISA kit from R&D Systems (Minneapolis, MN) was used to quantify MIP1 ; this assay uses a quantitative sandwich assay technique, where a monoclonal antibody specific to MIP1 is precoated on the plate. Standards and plasma samples from the macaques were added to the plates. If MIP1 is present, the immobilized antibody binds, the plate is then washed, and a polyclonal antibody is used to sandwich the MIP1. The quantity of MIP1 is detected using an enzymatic reaction. The minimum MIP detectable by the kit is 10 pg/ml. Statistical analyses. Comparisons between groups were performed using the Wilcoxon rank sum test for continuous factors or a repeated-measure analysis of variance with a Dunn s multiple comparison posttest. Graphical analysis was performed using GraphPad Prism, and error bars on graphs represent standard errors of the means. RESULTS HTLV-2 establishes a persistent infection in rhesus macaques and replicates in lymphoid and mucosal tissue. To determine the optimum method for establishing an HTLV-2 infection in rhesus macaques, we inoculated three monkeys with 10 8 lethally irradiated, HTLV-2-producing cells of either human (Mo-T) or rhesus (RhM304) origin. We performed a heterologous infection in one animal (M214), which was given the HTLV-2 cell line Mo-T (Table 1). This cell line was originally generated from cells obtained from a male patient with a T-cell variant of hairy cell leukemia (29). It contains a replication-competent genome of HTLV-2 and two defective HTLV-2 genomes. To perform an autologous infection, we cultured PBMCs from one macaque (M304) with lethally irradiated Mo-T cells and generated a HTLV-2-expressing macaque cell line, named RhM304. HTLV-2 infection and gene expression in RhM304 and Mo-T cells were confirmed by flow cytometric staining for Gag and by PCR (Fig. 1A and B). RhM304 cells were confirmed to be cells of nonhuman primate origin with a diploid set of 22 chromosomes. RhM304 cells were lethally irradiated and used to infect animal M304 and a third macaque, L900. Two of the animals, M214 and M304, became persistently infected and seroconverted; antibodies to HTLV-2 p24 were detected by ELISA (Fig. 1C). Eighteen months after inoculation, the animals were sacrificed, and blood, bone marrow, spleen, lymph nodes, intestinal, vaginal, and brain tissue samples were collected along with spinal fluid. HTLV-2 proviral DNA was amplified from the spleen, ileum, and jejunum (Fig. 1D), whereas proviral DNA was not detected in the brain or in the spinal cord of any of the animals

4 3046 GORDON ET AL. J. VIROL. Downloaded from 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 10 6 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). on July 24, 2018 by guest (data not shown). HTLV-2 was also demonstrated to infect primary rhesus macaque dendritic cells. Plasmacytoid dendritic cells were isolated from blood from a naïve rhesus macaque and infected with cell-free HTLV-2 in vitro. Figure 1E shows Tax staining in macaque CD123 dendritic cells after 6 days in culture. Altogether, these findings demonstrate that HTLV-2 infects rhesus macaques and replicates in lymphoid and mucosal tissues. HTLV-2 induces humoral and cell-mediated immune responses in rhesus macaques. As both autologous and heterologous HTLV-2-expressing cells were infectious in rhesus macaques, we inoculated 4 additional animals (M893, M897, M905, and M906) with the irradiated Mo-T cell line (Table 1) and monitored T-cell dynamics and humoral and cellular immune responses to HTLV-2 in these animals. The presence of antibodies specific for viral antigens was evaluated by Western blotting in all the HTLV-2-infected macaques. All four animals mounted antibody responses against HTLV-2 antigens within 30 days of infection, which persisted for over 6 months (Fig. 2A). HTLV-2 proviral DNA was measured for the first 18 weeks after HTLV-2 infection (Table 2). HTLV-2 tax DNA was amplified by nested PCR from T cells obtained from the lymph nodes of all four animals at 3 months postinfection (Fig. 2B), but not in the DNA of uninfected macaques used as

5 VOL. 84, 2010 HTLV-2 AND SIV mac251 COINFECTION MODEL IN MACAQUES 3047 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 -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 , while the CD4 cells show a trend toward significance with a P value of (D and E) Representative flow cytometric pseudocolor plots showing the frequency of IFN- and/or TNF- 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- /TNF- or IL-2.

6 3048 GORDON ET AL. J. VIROL. Animal code TABLE 2. HTLV-2 proviral load in the infected macaques Time after HTLV-2 infection (no. of wks) HTLV-2 proviral DNA load/10 6 PBMCs M M M ND a M ND a ND, not detected. negative controls. Interestingly, both CD8 and CD8 cells contained HTLV-2 tax, which suggests that HTLV-2 can replicate in both T-cell types in macaques (12). Importantly, HTLV-2 infection did not induce a loss of CD4 or CD8 T cells in vivo. A repeated-measure analysis of variance with square root-transformed counts over time demonstrated that the number of CD8 T cells increased significantly over time (P 0.007), while the number of CD4 T cells displayed a trend toward significance (P 0.049) (Fig. 2C). HTLV-2- specific T-cell responses in blood and the gastrointestinal tract were measured by intracellular cytokine staining for IFN-, TNF-, and IL-2 in response to peptide pools of 2 major antigenic proteins of HTLV-2 Gag and Tax. Representative flow cytometric plots of IFN- and/or TNF- staining in CD8 T cells isolated from the blood and gut before and after stimulation with HTLV-2 Gag or Tax are shown in Fig. 2D and E, respectively. Flow cytometric analysis of the average frequency of CD8 T cells producing cytokines demonstrated low levels of antigen-specific responses in the blood and gut (Fig. 2F and G). HTLV-2-specific responses peaked 3 to 4 months postinfection, and very low level responses were detectable up to 10 months postinfection (data not shown). Comparable viral and cellular dynamics in blood from dually HTLV-2/SIV mac251 infected or SIV mac251 singly infected macaques. The effects of preexisting HTLV-2 infection on the acute phases of HIV-1 infection in humans are unknown. We therefore studied the effect of HTLV-2 infection on SIV mac251 replication and the SIV mac251 -specific immune response in macaques coinfected with HTLV-2 and SIV mac251 to address these questions. As summarized in the study design (Fig. 3A), 10 months after HTLV-2 infection, we challenged macaques with SIV mac251 intrarectally. The persistence of HTLV-2 infection was confirmed by nested PCR in blood, before and after SIV mac251 infection in the macaques preinfected with HTLV-2 (Fig. 3B). HTLV-2 tax DNA was amplified from all four persistently infected macaques, while the uninfected control animal P182 (not infected with HTLV-2) was negative (Fig. 3B). Serum antibodies to HTLV-2 proteins were also present before and after SIV infection (Fig. 3C). The viral burden and T-cell dynamics in HTLV-2-infected macaques, superinfected with SIV mac251, were characterized and compared to SIV mac251 singly infected controls. SIV RNA in the plasma was quantified by reverse transcription-pcr (RT-PCR) (Fig. 4A). Peak viremia occurred at 14 days postinfection, with an average of copies/ml for the HTLV-2/SIV mac251 group and copies/ml in the controls. Set point viremia occurred by 60 days postinfection with an average of copies/ml in the HTLV-2/SIV mac251 group and copies/ml in the controls. Neither peak viral load nor set point viremia was significantly different between the two groups. These data support the clinical findings in HTLV-2/HIV coinfected patients, where HTLV-2 infection was not found to influence HIV viral loads (56). SIV mac251 infection caused a sharp decline in the number of CD4 T cells in blood in the animals of both groups; this decline stabilized by 30 days postinfection (Fig. 4B). Absolute CD4 T-cell counts were not significantly different between the groups. Memory CD4 T cells, particularly those that express CCR5, are the primary targets of HIV-1 and SIV mac251 infection. These target T cells are rapidly lost in the acute phase of the infection. HTLV-2-infected CD8 T cells secrete MIP1, a natural ligand for CCR5. The production of MIP1 by HTLV-2-infected CD8 T cells isolated from coinfected individuals has been shown to inhibit HIV-1 replication (12, 13). Thus, MIP1 secretion by HTLV-2-infected cells and its binding to CCR5 on circulating or mucosal CD4 T cells may theoretically protect this cell type from the typical rapid depletion observed during SIV mac251 and HIV-1 infections. To examine whether this is the case, we determined the frequency of CD4 CCR5 T cells in blood. To control for animal-toanimal variation, the frequency of CD4 CCR5 T cells is displayed as a percentage of baseline value over the course of the infection. A severe depletion of CD4 CCR5 cells was observed in both groups by 21 days postinfection (Fig. 4C), and no difference in the rate of decline of CD4 CCR5 cells was observed between the coinfected animals and controls. Activation and proliferation drive T-cell differentiation from naïve to memory and effector cells. The phenotype of T cells in HTLV-2/SIV mac251 coinfected animals was compared to the phenotype of T cells in SIV mac251 singly infected controls. Expression of CD28 along with CD95 was used to determine the differentiation status of CD4 and CD8 T cells. T cells that express only CD28 were considered naïve, cells dually positive for CD28 and CD95 were characterized as central memory cells, and cells positive for CD95 only were categorized as effector/effector memory cells (48). A sharp decline in the fraction of CD4 central memory T cells was observed in both groups (similar to CD4 CCR5 T cells), which then stabilized between day 30 and day 60 (Fig. 4D). An increase in the absolute number of CD8 T cells was observed following SIV mac251 infection in both groups (Fig. 4E); this increase was mainly due to an expansion of CD8 effector cells (graphed as a fraction of the baseline level in Fig. 4F). The CD8 T-cell expansion coincides with the postpeak decline in viremia, suggesting a contribution of CD8 T cells to the partial control of acute viremia, as observed during HIV infection (9, 30). The acute increase in the percentage of CD8 effectors peaked at 21 days postinfection and then declined transiently, only to

7 VOL. 84, 2010 HTLV-2 AND SIV mac251 COINFECTION MODEL IN MACAQUES 3049 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 -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. continue to increase in the chronic phase; this is likely due to the continued generalized immune activation observed during chronic HIV-1 and SIV mac251 infections. Altogether, there were no significant differences between the two groups of CD8 T cells (Fig. 4E and F). SIV mac251 replicates and induces CD4 T-cell loss in the lymphoid organs and gastrointestinal tracts of HTLV-2 coinfected macaques. The tropism of HTLV-2 for T cells and its potential ability to activate T cells in lymphoid and mucosal sites raised the possibility that HTLV-2 infection may increase SIV mac251 replication by creating more viral targets. We compared the number of viral DNA copies and CD4 T-cell depletion in lymphoid and mucosal sites in HTLV-2/SIV mac251 coinfected macaques and SIV mac251 singly infected controls. Bone marrow aspirate specimens, along with lymph node, jejunum, and rectum biopsy specimens were collected before and 10 and 30 days after SIV mac251 infection. SIV mac251 DNA was amplified by real-time PCR on DNA extracted from lymph nodes

8 3050 GORDON ET AL. J. VIROL. and the gastrointestinal tract at both 10 and 30 days postinfection (Fig. 5A). The level of SIVmac251 viral DNA in the tissues, including the gastrointestinal tract, was not significantly different between the two groups. Similar to the measurements in blood, we monitored the frequency of CD4 T cells in the lymphoid organs (bone marrow and lymph nodes; Fig. 5B) and the gastrointestinal tract (jejunum and rectum; Fig. 5C). Both macaques coinfected with HTLV-2 and SIVmac251 and macaques singly infected with SIVmac251 showed a decline in the frequency of CD4 T cells in both lymphoid organs at 30 days postinfection, but there was no significant difference between the groups. A significant depletion of lamina propria CD4 T cells was observed in the jejunum and rectum (Fig. 5C). However, the extent of the depletion was severe in both groups; these observations were confirmed by immunohistochemical staining which enumerated the absolute number of CD4-ex- FIG. 4. Similar viral and cellular dynamics in HTLV-2/SIVmac251 coinfected and SIVmac251 monoinfected macaques. (A) SIVmac251 viral load in HTLV-2 coinfected macaques (shown in black) and controls (shown in red) measured by the number of SIVmac251 RNA copies/ml of plasma. (B) Average CD4 T-cell count in blood following SIVmac251 infection in HTLV-2/SIVmac251-infected macaques (black) and SIVmac251-infected macaques (red). (C) Percentage of baseline CD4 CCR5 T-cell count following SIVmac251 infection in HTLV-2/SIVmac251-infected macaques (shown in black) and SIVmac251-infected animals (shown in red). (D) Percentage of baseline CD4 memory T-cell count in blood following SIVmac251 infection. Memory cells are defined as cells that express both CD28 and CD95. (E) Average CD8 T-cell count following SIVmac251 infection. (F) Percentage of baseline CD8 effector/effector memory T-cell count in blood after SIVmac251 infection. CD8 effector/effector memory T cells are CD8 CD28 CD95.

9 VOL. 84, 2010 HTLV-2 AND SIV mac251 COINFECTION MODEL IN MACAQUES 3051 Downloaded from 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/10 6 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. on July 24, 2018 by guest pressing cells in the rectum (Fig. 5D). Interestingly, immunohistochemical staining demonstrated a significantly greater number of CD4 cells in the rectum before SIV infection in animals preinfected with HTLV-2 (P 0.05). Overall, we observed no difference in the number of cells in the rectum expressing CD8 (Fig. 5E) or the frequency of CD8 T cells (data not shown) in the singly or dually infected animals before or after SIV infection. Thus, at least in rhesus macaques, coinfection with HTLV-2 did not attenuate SIV mac251 replication or the loss of CD4 T cells at mucosal sites (Fig. 5). Similar levels of spontaneous MIP1 production in HTLV- 2/SIV mac251 coinfected and SIV singly infected macaques. Early in HIV-1 infection, viruses that utilize the chemokine receptor CCR5 for entry into T cells and macrophages domi-

10 3052 GORDON ET AL. J. VIROL. FIG. 6. No difference in spontaneous MIP expression by T cells in blood from HTLV-2/SIV mac251 coinfected and SIV mac251 monoinfected animals. (A) Mean fluorescence intensity (MFI) of MIP1 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 MIP1 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. nate (52). HIV-1 and SIV mac251 entry can be inhibited by the binding of the natural ligands for CCR5; these CC chemokines include MIP1, MIP1, and RANTES (16, 35). HTLV-2-infected T cells have been shown to spontaneously secrete MIP1 ; the induction of MIP1 is likely the result of the viral protein Tax transactivating the chemokine promoter (36). To characterize the ability of CD4 and CD8 T cells to spontaneously produce MIP1, we cultured PBMCs for 5hinthe presence of monensin and performed intracellular cytokine staining for MIP1. Both the MIP1 expression per cell (mean fluorescence intensity [MFI]) and the frequency of MIP1 production (data not shown) decreased after SIV mac251 infection (Fig. 6A and B). However, no difference was observed in the levels of spontaneous MIP1 produced by CD4 or CD8 T cells from HTLV-2/SIV mac251 coinfected and SIV mac251 singly infected controls at any time point before or after SIV mac251 infection (Fig. 6A and B). In addition, no correlation was observed between the levels of spontaneous MIP1 produced and viral load in either coinfected macaques or controls. MIP1 was not detected in the plasma samples from all macaques by ELISA. HTLV-2 preinfection does not affect T-cell proliferation in SIV mac251 -infected macaques. Increased T-cell proliferation is a hallmark of HTLV infections (18). Thus, we monitored the level of proliferation by staining for the nuclear antigen Ki67 in CD4 and CD8 T cells from blood and tissue samples following SIV mac251 infection. An increase in the percentage of CD4 and CD8 T cells expressing Ki67 was observed in blood following SIV mac251 infection; this peaked between 21 and 30 days postinfection (Fig. 7A and B). The frequency of proliferating T cells declined between 30 to 60 days but continued to be elevated in the chronic phase. The magnitude of the increased acute proliferation was greater in CD8 T cells, and a significantly higher level of Ki67 expression remained at 90 days postinfection compared to the pre-siv mac251 infection levels (P ). However, there were no significant differences in the magnitude of proliferating CD4 or CD8 T cells in blood from HTLV-2 preinfected and control macaques (Fig. 7A and B). The frequency of CD4 and CD8 Ki67 cells increased in the bone marrow with kinetics similar to the kinetics observed in blood, and the lymph nodes also demonstrated an increase in activating and proliferating CD4 and CD8 T cells (Fig. 7C and D). Interestingly, the enumeration of CD4 cells expressing Ki67 in rectal tissues demonstrated significantly more proliferating cells in HTLV-2 preinfected animals compared with uninfected controls (i.e., before SIV infection; Fig. 7E). This increased turnover may account for the increased number of CD4 T cells measured by immunohistochemical staining in the rectum before SIV infection (Fig. 5D). SIV mac251 infection caused an increase in mucosal CD8 T-cell proliferation; however, the extent of this increase was similar in both HTLV-2 preinfected animals and controls measured by immunohistochemical staining for CD8 and Ki67 in the rectum (Fig. 7F). Overall, SIV mac251 infection was associated with a significant increase in the level of proliferating T cells in blood and tissues. HTLV-2 preinfection did not affect the magnitude or duration of this proliferation in most tissues. Limited polyfunctional SIV mac251 responses in infected animals despite HTLV-2 preinfection. HIV-1 infection induces measurable virus-specific T-cell responses; however, these responses are not of sufficient quality and/or quantity to eliminate virus replication (7). The goal of any vaccination strategy for HIV-1 is to induce immune responses that can eliminate or at least reduce HIV-1 replication so that both transmission and disease progression are prevented. Live attenuated viruses with low levels of virus replication may prime and continually boost immune responses, creating a protective immune response and successfully immunizing the individual. In order to evaluate HTLV-2 as a potential platform for an HIV-1 vaccine, we sought to determine whether HTLV-2 preinfection impacted either the quantity or quality of the SIV mac251 -specific response. Previous studies have indicated that HTLV-2 infection and specifically, its transactivating Tax protein, can induce IFN- production (11, 12). The quality of an HIV-1- or SIV mac251 -specific immune response has been determined by monitoring the ability of CD4 and CD8 T cells to respond to viral antigens by secreting multiple cytokines and chemokines. Using polychromatic flow cytometry, we assessed the ability of CD4 and CD8 T cells to

11 VOL. 84, 2010 HTLV-2 AND SIV mac251 COINFECTION MODEL IN MACAQUES 3053 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 ). (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.

12 3054 GORDON ET AL. J. VIROL. Downloaded from 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- and/or TNF-, 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- stained along with TNF-. 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- /TNF- ), 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- and/or TNF-, IL-2, and IL-17). (B) Average total cytokine (IFN- and/or TNF-, 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- and/or TNF-, 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. on July 24, 2018 by guest simultaneously produce the cytokines IFN- and/or TNF-, IL-2, and IL-17 and evaluated the magnitude of the SIV mac251 - specific response. Overlapping peptide pools to SIV mac251 Gag and SIV mac251 Env were used to stimulate mononuclear cells from blood. The proportion of CD8 and CD4 T cells responding to SIV mac251 Env stimulation by concurrently producing multiple cytokines (3 or 2 functions) was not different in HTLV-2/SIV mac251 coinfected animals compared to SIV mac251 singly infected controls (Fig. 8A and 9A). Similarly, no difference in polyfunctional responses was observed in CD8 T cells stimulated with SIV mac251 Gag (data not shown). Figures 8B and C show the magnitude of the mean SIV mac251 -specific response following SIV mac251 infection. Overall, the immune response to SIV mac251 Env was of greater magnitude than the response to Gag; however, no clear differences in the magnitude of the response to either antigen was observed between HTLV-2/SIV mac251 coinfected animals and controls. Figure 9B and C show the magnitude of the mean SIV mac251 -specific response in CD4 T cells. Again, there were no differences between the groups. In all, these data demonstrated that preinfection with HTLV-2 neither enhances nor limits the SIV mac251 -specific immune responses.

13 VOL. 84, 2010 HTLV-2 AND SIV mac251 COINFECTION MODEL IN MACAQUES 3055 Downloaded from 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- stained along with TNF-. 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- /TNF- ), 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- and/or TNF- and IL-2 and IL-17). (C) Average total cytokine (IFN- and/or TNF-, 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- and/or TNF-, 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. on July 24, 2018 by guest DISCUSSION HTLV-2 infection is prevalent among intravenous drug users (IDU) coinfected with HIV-1 (56). The effects of HTLV-2 infection on progression to AIDS are controversial, as multiple studies have suggested a protective role of HTLV-2 in patients coinfected with HTLV-2 and HIV-1, while others have reported no beneficial effect. In the former studies, maintenance of CD4 T cells and lower levels of virus replication and immune activation have been reported in dually infected HTLV-2/HIV-1 individuals versus patients infected with HIV-1 only (4, 12, 23). Additionally, several IDU long-term nonprogressors with stable CD4 counts, in the absence of antiretroviral therapy, have been reported to be infected with HTLV-2 (56, 58). A possible mechanism underlying the milder disease course has been attributed to the increased expression of chemokines in HTLV-2-infected patients (12, 36, 47), particularly MIP1, a chemokine that inhibits viral entry by binding to CCR5. Despite the fact that it has been many years since the discovery of HTLV-2/HIV-1 coinfection, little is known about the effects of HTLV-2 on the HIV-specific response and on viral and T-cell dynamics in blood and tissues. Clinical studies of dually infected patients