JAIDS Journal of Acquired Immune Deficiency Syndromes:
Basic and Translational Science
Characterization of Peripheral and Mucosal Immune Responses In Rhesus Macaques on Long-Term Tenofovir and Emtricitabine Combination Antiretroviral Therapy
Jasny, Edith PhD*; Geer, Suzanne BSc*; Frank, Ines PhD*; Vagenas, Panagiotis PhD*; Aravantinou, Meropi MSc*; Salazar, Andres M. MD†; Lifson, Jeffrey D. MD‡; Piatak, Michael Jr PhD‡; Gettie, Agegnehu BSc§; Blanchard, James L. DVM, PhD‖; Robbiani, Melissa PhD*
*Center for Biomedical Research, HIV/AIDS Program, Population Council, New York, NY
†Oncovir, Washington, DC
‡AIDS and Cancer Virus Program, SAIC-Frederick, National Cancer Institute, Frederick, MD
§Aaron Diamond AIDS Research Center, Rockefeller University, New York, NY
‖Tulane National Primate Research Center, Tulane University, Covington, LA
Correspondence to: Melissa Robbiani, PhD, Center for Biomedical Research, HIV and AIDS Program, Population Council, 1230 York Avenue, New York, NY 10065 (e-mail: firstname.lastname@example.org).
Supported by the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research Grant DE018293 and in part with federal funds from the National Cancer Institute, NIH, under Contract No. HHSN261200800001E. Partial support was provided to the Tulane National Primate Research Center by base Grant RR000164 and NIH construction Grants 1G20RR016930-01, 1G20RR018397-01, 1G20RR019628-01, 1G20RR013466-01, 1G20RR019607-01, 1G20RR21381, 1G20RR22760, and 1CO6RR012112-01. M. Robbiani is a 2002 Elizabeth Glaser Scientist.
The authors have no conflicts of interest to declare. None of the material in this manuscript has been published or is under consideration elsewhere.
Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's Web site (www.jaids.com).
Received March 15, 2012
Accepted June 25, 2012
Background: The goal of antiretroviral therapy (ART) is to suppress virus replication to limit immune system damage. Some have proposed combining ART with immune therapies to boost antiviral immunity. For this to be successful, ART must not impair physiological immune function.
Methods: We studied the impact of ART (tenofovir and emtricitabine) on systemic and mucosal immunity in uninfected and simian immunodeficiency (SIV)–infected Chinese rhesus macaques. Subcutaneous ART was initiated 2 weeks after tonsillar inoculation with SIVmac239.
Results: There was no evidence of immune dysregulation as a result of ART in either infected or uninfected animals. Early virus-induced alterations in circulating immune cell populations (decreased central memory T cells and myeloid dendritic cells) were detected, but normalized shortly after ART initiation. ART-treated animals showed marginal SIV-specific T-cell responses during treatment, which increased after ART discontinuation. Elevated expression of CXCL10 in oral, rectal, and blood samples and APOBEC3G mRNA in oral and rectal tissues was observed during acute infection and was down regulated after starting ART. ART did not impact the ability of the animals to respond to tonsillar application of polyICLC with increased CXCL10 expression in oral fluids and CD80 expression on blood myeloid dendritic cells.
Conclusion: Early initiation of ART prevented virus-induced damage and did not impede mucosal or systemic immune functions.
Antiretroviral therapies (ARTs) need to control virus replication, thereby limiting associated pathogenesis, which could be combined with immune therapies to boost immunity while replication is suppressed. Thus, ART must not interfere with immune functions. The similarities in disease pathogenesis, immunology, and physiology (ie, drug metabolism) between experimental simian immunodeficiency (SIV) infection in nonhuman primates and HIV infection in humans provide an excellent model to study the biology of HIV infection, including potential adverse effects of ART on immune function.1–3
ART is effective in nonhuman primates.3–8 The nucleotide reverse transcriptase inhibitors (NRTIs) tenofovir and emtricitabine are highly effective and generally well tolerated, and the combination of both is used as the NRTI backbone of numerous ART regimens to treat HIV.9–14 The tenofovir/emtricitabine regimen can control SIV replication in macaques.4,8,15–21
We investigated whether long-term ART impacts immunity. To minimize SIV-associated effects, we initiated ART 14 days after tonsillar inoculation with SIVmac239.22–24 Treated uninfected animals and uninfected and infected animals not receiving ART were included as controls. There was no evidence of immune dysfunction as a result of ART, but virus-induced changes that likely contribute to the onset and spread of infection were apparent. In primary infection via the tonsillar route, CXCL10 expression was elevated in oral, rectal, and blood samples and increased APOBEC3G (A3G) levels were detected in mucosal tissues. CXCL10 and A3G decreased on ART. We observed early infection-related loss of central memory CD4+ and CD8+ T cells and myeloid dendritic cells (mDCs) in blood, which normalized after initiation of ART. ART did not impair the animals' ability to respond to polyICLC applied to the tonsils. Thus, tenofovir/emtricitabine ART does not seem to adversely affect mucosal and systemic immune functions in macaques, and reinforces the idea that commencing ART early in infection can limit virus-induced damage to the immune system.
MATERIALS AND METHODS
Animals and Treatment
Adult male Chinese rhesus macaques (Macaca mulatta) were housed at the Tulane National Primate Research Center (TNPRC, Covington, LA). All animal studies were performed in accordance with federal laws and regulations and institutional policies, including the approval by the Animal Care and Use Committee of the TNPRC (OLAW Assurance #A4499-01), which has received continued full accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC #000594). Animals were housed and cared for in compliance with the regulations detailed under the Animal Welfare Act.25 All animals received environmental enrichment and were clinically monitored daily. Animals were pair housed or group housed when possible. For surgical and sampling procedures, animals were anesthetized with ketamine hydrochloride (10 mg/kg, intramuscularly) followed by appropriate analgesics for pain and discomfort (buprenorphine 0.25 mg/kg, intramuscularly), which was carefully monitored. On study termination or when signs of advanced stages of simian AIDS were present (IACUC-approved endpoint criteria), animals were euthanized using methods consistent with the recommendation of the American Veterinary Medical Association Guidelines on Euthanasia.
All animals were antibody (Ab)-negative to simian type D retrovirus, simian T-lymphotropic virus type 1, and SIV at study enrollment. Twelve animals were inoculated with 2000 TCID50 SIVmac239 on the tonsils (TNPRC stock virus propagated in Staphylococcus Enterotoxin B (SEB)-stimulated rhesus peripheral blood mononuclear cells (PBMCs); “SIVmac239 RhPBMC 7/29/94” dribbled across the tonsils in 0.2 mL). ART was administered for 34 weeks between 2 and 36 weeks post challenge and consisted of a daily subcutaneous injection of tenofovir (PMPA, 9-[2-(Phosphonomethoxy)propyl]adenine, 20 mg kg−1 day−1) and emtricitabine (beta-2′,3′-dideoxy-3′-thia-5-fluorocytidine, FTC, 40 mg kg−1 day−1). PolyICLC treatment (0.5 mL, 1 mg/day on 2 consecutive days; Hiltonol, Oncovir, Washington, DC) was applied over the palatine tonsils and the back of the tongue. All animals received 2 polyICLC treatments during ART (weeks 28 and 32 post challenge) and 2 treatments after ART (weeks 44 and 48 post challenge). The animals and treatments were listed in Supplemental Digital Content 1 (see Table, http://links.lww.com/QAI/A344).
Sample Collection and Cell Isolation
Immune responses were monitored in blood, mucosal fluids, and oral and rectal biopsies. Peripheral lymph nodes (LNs) and tonsils were collected at necropsy. Samples were transported by overnight courier service (blood at room temperature, fluids and tissue samples on ice) and processed immediately after arrival. Cells from peripheral blood, LNs, and tonsils were isolated as described.26 Plasma samples were collected and stored as described.26
Mucosal fluids were collected by insertion of a foam pad (approximate size 1 × 0.5 cm) in the oral or rectal cavity for 5 minutes, after which the swab was placed into a tube containing 1 mL of phosphate-buffered saline/1% fetal calf serum/penicillin–streptomycin. After overnight shipment, the mucosal fluids were spun at 1100g, 4°C for 10 minutes, and the supernatant was aliquoted and stored at −80°C until analysis.
For the collection of oral biopsies, the oral cavity was exposed by placing gauze behind both the upper and lower canines with retraction of the gauze. Alligator forceps were used to obtain 1-mm pinch biopsies of the buccal mucosa. Rectal biopsies were sampled by placing sterile lubricant on the distal end of a vaginal speculum, which was then gently advanced through the anus into the rectum to visualize the mucosa. Alligator forceps were used to collect 1.5-mm pinch biopsies of rectal mucosa. Up to 20 mucosal biopsies were taken at one time point from the oral or rectal site. After overnight shipment, mucosal pinch biopsies were washed twice in phosphate-buffered saline (Invitrogen, Carlsbad, CA), incubated overnight at 4°C in RNAlater (Qiagen, Valencia, CA) then stored at −80°C until isolation of RNA.
Viral Load and SIV Ab Determination
Plasma SIV RNA was determined by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR),27 and SIV-specific Abs were measured by ELISA.28 Neutralizing Ab (nAb) activity against SIVmac251 was determined in the plasma collected at baseline and 52 weeks post infection (w.p.i.).17
Leukocytes in blood and tissues were characterized by polychromatic flow cytometry. T-cell subsets were identified as described.17 DC subsets were identified within the Lin−HLA-DR+ population using fluorescein isothiocyanate–conjugated anti-lineage Abs (CD3, clone SP34; CD14, clone M5E2; CD20, clone 2H7, all BD Biosciences, San Jose, CA) with HLA-DR-PerCP-Cy5.5 (clone G46-6), CD123-PE (clone 7G3), CD11c-PE-Cy7 (clone 3.9, all BD Pharmingen, San Jose, CA), CD80-biotin (clone L307.4, BD Pharmingen) Abs followed by streptavidin-APC-Alexa650 (Invitrogen). Isotype Ig controls were included in all experiments and typically gave mean fluorescence intensities (MFIs) of <1 log. All samples were acquired on a BD LSRII (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Antigen-specific T cells were detected using intracellular cytokine staining.29,30 Aldrithiol-2 (AT-2)-inactivated SIVmac239 (300 ng/mL p27, lot #P4148, AIDS and Cancer Virus Program, NCI-Frederick, Frederick, MD) was used to stimulate SIV-specific T cells.31 No-virus microvesicle preparations and 50 nM of phorbol 12-myristate 13-acetate and 1 mg/mL of ionomycin (both Sigma, St Louis, MO) were used as controls. Candida albicans (ATCC, strain SC5413) was maintained at room temperature on yeast–peptone–dextrose agar plates (Sigma), and Candida yeast (which induce CD4+ and CD8+ responses32) were amplified in Sabouraud dextrose broth (Sigma) overnight at 30°C. Viable yeast were counted by trypan blue exclusion and used in a Candida:PBMC ratio of 1:1. Amphotericin B (5 μg/mL, Sigma) was added to prevent Candida overgrowth. IL-17-Alexa Fluor 647 Abs (clone eBio64CAP17, eBioscience, San Diego, CA) was added to the published Ab panel.30 Data were acquired (200,000 events in the CD3+ lymphocyte gate) using BD LSRII and analyzed using FlowJo software.
Luminex and ELISA
Chemokine and cytokine levels were measured in cell-free mucosal fluids using the monkey-reactive Beadlyte human 14-plex Detection System (Invitrogen). Data were acquired on a Luminex 200 instrument (Luminex, Austin, TX) and analyzed using StarStation software version 2.0 (Applied Cytometry Systems, Sacramento, CA). IFN-α, IFN-β (PBL Interferon Source, Piscataway, NJ; detection limit 25 pg/mL), and CXCL10 (R&D, Minneapolis, MN; detection limit 15.1 pg/mL) were detected by ELISA.
Detection of Immune Markers by Real Time PCR
Tissue samples were homogenized using a FastPrep bead mill homogenizer and lysing matrix D (MP Biomedicals, Irvine, CA). Total RNA was isolated with the RNeasy Mini Kit (Qiagen) and quantified on a Nanodrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). Immune marker mRNA levels were determined,17 with expression levels being calculated from normalized ΔCT values using the following formula: fold change in gene expression = 2−ΔΔCt. Test groups were compared with the uninfected controls collected at the same time points, rather than to the respective baselines or different sample collection time for each animal, to control for the quality of RNA that might have been affected by long-term storage.
Sequencing the SIV-RT Gene
Viral RNA was extracted from the plasma of animals EL42 and P427 collected at 35 w.p.i. (1 week before stopping ART), using the Qiagen Viral RNA Isolation Kit. The RT gene was sequenced33 using primers RTamp5′ (5′-TACTAAAGAATACAAAAATGTAGA-3′) and RTamp3′ (5′-CTCTGTGGATTGTATGGTACCCC-3′) for first round PCR product amplification and SIVmacRT5′ (5′-TGGAAAAGGATGGTCAGTTGGAGGA-3′) and SIVmacRT3′ (3′-CCGTGGCTTCTAATGGCTTGCCT-5′) for nested PCR reaction.
All statistical calculations were performed using GraphPad Prism software (San Diego, CA), version 5.02 for Windows. Nonparametric tests were used because of small sample size. When comparing 2 groups, 2-tailed Mann–Whitney U test was used, whereas comparison of more than 2 groups was performed using the Kruskall–Wallis test with Dunn multiple comparison test. P values were 2-sided and considered significant when <0.05.
Impact of Early ART on Viral Loads and Adaptive Immunity
The effect of a continuous tenofovir/emtricitabine ART regimen on immune parameters was evaluated in infected and uninfected macaques (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A344). To limit cumulative and progressive immune damage due to infection, ART was initiated 2 w.p.i. All animals reached peak viremia 2 weeks post tonsillar SIVmac239 inoculation (Fig. 1A). Peak viral loads were comparable in infected animals that did (6.0 ± 2.9 × 106 RNA copies/mL) or did not (2.8 ± 0.9 × 106 RNA copies/mL) receive ART (P = 0.91). All 5 treated animals initially responded to ART with viral loads dropping significantly between weeks 3 and 6 compared with untreated controls (Figs. 1B, C). Three of the animals continued to respond to ART with plasma viral loads being maintained below 100 copies/mL during treatment (Fig. 1C), while viral loads in the other 2 animals reached set point values comparable to untreated controls (Fig. 1A). Because of the small numbers of animals, we were unable to make statistical comparisons between the ART responders and transient responders. On stopping ART, 2 of the 3 responding animals had an initial rebound in viremia (<104 copies/mL), which returned to <400 copies/mL until euthanasia. The third ART responder maintained plasma viral RNA levels below threshold from the time of ART discontinuation through to euthanasia. Viremia in the 2 transient ART responders seemed unaltered on ART discontinuation. Virus from the transiently responding animals carried the RT-sequence of the parental challenge virus as detected in the plasma collected at 35 w.p.i., and there were no amino acid changes at positions known to confer resistance to NRTIs (see Table, Supplemental Digital Content 2, http://links.lww.com/QAI/A344).3,34,35 Mamu-A*01, -A*02, -A*08, -A*11, -B*01, -B*03, -B*04, -B*08, and -B*17 haplotypes were tested in all infected monkeys. Only FH22 expressed Mamu-A*01, but showed no evidence of enhanced virus control or delayed disease progression. There were no differences between the CD4 counts of treated and untreated animals (Fig. 1D).
All infected animals developed SIV-specific plasma IgG (see Table, Supplemental Digital Content 1, http://links.lww.com/QAI/A344). Anti-SIV nAbs were detected in all infected animals except for animals AA47 (fast progressor), EB50 (high viral load), and EL42 (transient responder) (see Figure A, Supplemental Digital Content 3, http://links.lww.com/QAI/A344), with similar titers in untreated and ART-treated animals (see Figure B, Supplemental Digital Content 3, http://links.lww.com/QAI/A344), SIV-specific TNF-α- and IFN-γ-producing T cells were most frequent in infected nontreated animals, whereas the ART-receiving group had low responses during ART treatment (Fig. 2A). SIV-specific IL-2 responses were most prominent earlier after infection, but SIV-specific IL-17 responses were not consistently detected (independent of ART) and were even detected in uninfected animals (Fig. 2A). Comparable Candida-specific responses were seen (independent of SIV infection and ART), with responses decreasing over time (Fig. 2B). Additionally, ART did not seem to significantly impact multifunctional T-cell responses in SIV-infected animals (Fig. 2C).
SIV-Induced Changes In Blood T-Cell and DC Subsets
No significant differences in the frequencies of T-cell subsets (see Figures, Supplemental Digital Content 4 and 5, http://links.lww.com/QAI/A344), mDCs, or plasmacytoid DCs (pDCs) (see Figure, Supplemental Digital Content 6, http://links.lww.com/QAI/A344) were found between uninfected ART-naive and -treated animals. To simplify the analyses and enhance the statistical power, the uninfected animals (ART treated and untreated) were consolidated into one control group. SIV-infected animals showed a significant decrease of central memory T cells in acute infection (Fig. 3, 40 ± 9% decrease for CD4+ and 30 ± 11% for CD8+, 2 w.p.i. compared with baseline). Trends toward lower numbers of CD4+ central memory T cells (Fig. 3) and higher frequencies of CD25+Foxp3+CD4+ Tregs were detected in untreated chronic infection (7.6 ± 2.1-fold increase at 54 w.p.i. compared with baseline vs 2.9 ± 0.5 in the controls and 4.3 ± 0.7 in infected ART-receiving group). Animals with the highest frequencies of CD4+ Tregs had the highest viral loads (see Figure, Supplemental Digital Content 4, http://links.lww.com/QAI/A344).
The percentages of Lin−HLA-DR+ DCs remained stable during acute and chronic treated and untreated infection (Fig. 4). However, CD11c+ mDCs decreased in acutely infected animals (25 ± 5% decrease at 2 w.p.i. compared with uninfected animals, P = 0.009), returning to control levels shortly after initiation of ART but persisting in ART-naive animals up to 8 w.p.i. (Fig. 4). CD80 expression on mDCs was not altered by infection (Fig. 4). Elevated CD123+ pDC percentages were detected in acute infection (2.01 ± 0.29-fold increase at 2 w.p.i. compared with uninfected animals, P = 0.004), which persisted in untreated infection up to 32 w.p.i. (Fig. 4). ART-receiving animals also showed higher pDC frequencies in chronic infection (not significant).
We used polyICLC to evaluate the innate responsiveness of the differently treated groups. No changes in viral loads were observed after polyICLC treatment when given during or after ART (Fig. 1A). There was variability in responsiveness to polyICLC (ie, not consistently responsive after each dose), but there were no significant differences between the groups. There were no changes in the frequencies of naive, effector and central memory T cells, mDCs, and pDCs in the blood (not shown), but there was transiently elevated expression of CD80 on mDCs 24 hours after the second and fourth polyICLC treatment (see Figure A, Supplemental Digital Content 7, http://links.lww.com/QAI/A344). Additionally, CXCL10 protein levels in the oral fluids were increased 24–72 hours after the first and fourth treatments (see Figure B, Supplemental Digital Content 7, http://links.lww.com/QAI/A344). IFN-α and -β protein levels in oral fluids and blood were unaltered. PolyICLC treatment did not affect antigen-specific T-cell responses (Fig. 2).
ART Shuts Down Virus-Induced Mucosal Immune Responses
We also compared the mRNA expression of 10 innate immune modulators in the oral cavity versus the rectum and blood during acute and chronic infection (±ART for the latter). No significant differences between uninfected ART-naive and -treated animals were detected in the expression levels of the tested parameters in the oral, rectal, or blood samples (data not shown). Hence, data from the ART-treated and untreated uninfected animals were consolidated as the control group to increase the power of the statistical comparisons to the infected groups.
SIV-infected animals showed elevated levels of CXCL10 (85.1 ± 28.8-fold increase, P = 0.04) and A3G (33.0 ± 22.0-fold increase, P = 0.006) mRNA and a reduction of type I IFN mRNA expression (1.69 ± 0.14-fold reduction of IFN-α2, P = 0.001 and 2.27 ± 0.42-fold reduction of IFN-β, P = 0.006) in oral tissues at 2 w.p.i. (Fig. 5A). Oral CXCL10 and A3G mRNA levels declined with ART, while persistently elevated levels were observed in untreated chronic infection (Fig. 5B). TNF-α and IL-10 mRNA levels increased in oral samples of acutely infected animals (Fig. 5A, not significant). This trend was maintained in chronic untreated infection, but levels dropped to within normal ranges under ART (Fig. 5B). Responses in blood paralleled the oral tissues. Elevated levels of CXCL10 mRNA were detected in acute infection in rectal tissue (Fig. 5A) and CXCL10 protein expression in the blood plasma as detected by ELISA at 1 w.p.i. (40 ± 8 pg/mL in the uninfected vs 127 ± 34 pg/mL in the SIV infected; 3.18 ± 0.85-fold increase compared with uninfected, P = 0.005). CXCL10 protein production in plasma was rapidly abrogated by initiation of ART and further augmented in the absence of ART (2.66 ± 0.83-fold increase in ART-treated animals vs 18.75 ± 8.39 in ART-naive infected animals compared with uninfected controls, P = 0.003).
Different responses were detected in rectal (vs oral) tissues: (1) CCL4 mRNA expression was significantly increased and returned to control levels under ART, (2) TNF-α and IL-10 mRNA expression was not increased in acute or chronic infection, and (3) A3G mRNA expression increased only minimally during acute infection.
Given the widespread use of Truvada (tenofovir/emtricitabine) and Atripla (tenofovir/emtricitabine/efavirenz) as first line regimens to treat HIV11,36 and promising results from studies using Truvada for preexposure prophylaxis to prevent HIV transmission,37 understanding the interactions of these drugs with the immune system is of increasing importance.
We studied uninfected and SIVmac239-infected macaques to investigate the effect of tenofovir/emtricitabine ART on systemic and mucosal immune parameters. Based on studies showing that initiation of ART during acute HIV infection can preserve or increase antiviral immunity,38,39 ART was initiated early (14 days post infection) after tonsillar challenge with SIVmac239. Initiation of ART around the peak viremia poses a greater challenge and effective suppression of viral load may take longer compared to initiation of therapy during early chronic infection.17 Inclusion of a protease or integrase inhibitor to the regimen should increase the effectiveness of the treatment.40
Although all 5 treated animals initially responded to ART, 2 monkeys showed increasing viral loads that reached set point values comparable to untreated controls. Although the animal numbers are very small, we did examine the raw data of the ART responding versus poor-responding animals, and there was no difference in the parameters being measured. As a result, we included them in the analyses, because (although the virus did not respond to the drugs) the animals' immune systems were exposed to the drugs.
It is unclear why, despite persistent viremia and drug therapy, no mutations were observed in the poor ART responding animals. Previous studies examining tenofovir resistance in macaque models showed that prolonged treatment of SIV-infected animals with tenofovir monotherapy lead to the emergence of K65R RT mutants,18,41,42 which often coincided with or was followed by the development of additional compensatory mutations in the RT (ie, K64R, N69S, I118V, and S211N). The emergence of K65R viral mutants did not always lead to an increase in viremia, as some animals were able to suppress K65R viremia to low or undetectable levels for many years because of the development of strong CD8+ cell-mediated immune responses.41 Because we did not monitor the levels of tenofovir in the plasma during the ART-treatment, we cannot exclude the possibility that the drugs were cleared more rapidly in the 2 poor ART responders, rendering doses suboptimal, or the PMPA prodrug was not efficiently phosphorylated into the active form.
One limitation of the present study was the wide range of viremia in untreated animals, thereby possibly making statistically significant differences even more difficult to observe. Of note, when we looked at the immune parameters of each animal relative to viral loads, there were no patterns of differences evident.
The examination of uninfected animals treated or not with ART revealed no indication of peripheral or mucosal immune dysfunction as a result of ART, even at necropsy (see Table, Supplemental Digital Content 8 and Figure, Supplemental Digital Content 9, http://links.lww.com/QAI/A344). However, we observed several virus-induced changes in acute infection, that is, loss of central memory T cells and mDCs, and mobilization of pDCs, which were restored by ART to levels similar to those seen in control animals. Other studies also reported the early loss of memory T cells43,44 and mDCs.45–48 Barratt-Boyes et al49 observed a mobilization of pDCs into the blood at 3 days post infection followed by a significant loss within 14 days after intravenous inoculation with SIVmac251, despite evidence of a profound mobilization of pDCs into blood and recruitment to LNs. We detected increased levels of pDCs from 2 w.p.i., which remained elevated in untreated infection. Different routes of infection (tonsillar vs intravenous), virus strain (SIVmac239 vs SIVmac251) and methods of pDC quantification (frequency vs absolute counts) could account for the differences seen between the studies.
To obtain a better understanding of the unique attributes of mucosal immunity induced by virus and how long-term ART can affect them, we compared the expression of innate markers in the oral cavity as the site of viral inoculation to those at distal sites. We observed differences in the mRNA expression of innate mediators in acutely infected animals between the oral and rectal site. Type I IFN mRNA expression was reduced in oral tissue but was slightly elevated in rectal samples. In infant macaques infected orally with SIV, several IFN-α subtypes were rapidly induced in lymphoid tissues but only slightly in oral and gastrointestinal mucosal surfaces50 indicating that there are differences between distinct anatomical sites in the innate response to virus. The tissue-specific variation can be partly explained by the different cellular composition at each site. Similar to our results, data from a larger and more detailed study of early innate immune responses in the infant macaque model of oral SIV infection showed the induction of pro-inflammatory cytokines, and the relative lack of antiviral type I IFN responses in oral (gingiva) and mucosal (esophagus and colon) tissues.51 Interestingly, despite the lack of IFN-α response in mucosal tissues, the IFN-inducible genes Mx and CXCL10 were markedly increased in the gingiva and the esophagus of animals with detectable virus replication.51 In our study, CXCL10 up-regulation was driven by acute SIV replication in multiple anatomical sites (oral, rectal, and peripheral blood). Several reports showed elevated levels of CXCL10 after oral inoculation of macaques with pathogenic SIV51–53 indicating that CXCL10 could have an important role in pathogenesis. CXCL10 is a potent chemotactic factor for multiple cell types54,55 and increased levels of CXCL10 at the site of inoculation may enhance recruitment of and viral spread to target cells. Furthermore, CXCL10 stimulates HIV-1 replication in vitro56 and systemically heightened levels of CXCL10 could contribute to enhanced viral replication in blood. ART down-regulated CXCL10 expression to levels seen in uninfected controls indicating that active viral replication was responsible for the enhanced expression. We detected increased expression of A3G in acute infection in oral and blood samples, which persisted in untreated infection but remained at control levels in ART-treated animals. A study examining ART-responsive genes in HIV-infected individuals also showed that CXCL10 and A3G expression is abrogated after successful ART.57
In addition, we detected increased expression of CCL4 mRNA in rectal tissue. The production of beta-chemokines by CD8+ T cells was reported in naive and vaccinated macaques, the largest number of beta chemokine-secreting cells being in the rectal mucosa.58 CCL4 is one of the major HIV-suppressive factors produced by non–cytotoxic CD8+ T cells,59 and the increase in the rectal site (after tonsillar challenge) may contribute to the overall innate antiviral response.
As had been reported for short-term tenofovir monotherapy initiated very early (24 or 48 hours post infection) in SIVmac239-inoculated macaques,60,61 we observed low to background levels of SIV-specific T-cell responses during ART. However, SIV-specific CD4+, and to a lesser extent CD8+, T-cell responses rebounded after ART discontinuation suggesting that early ART intervention preserved antigen-specific T cells before they could be affected by the virus. Decrease of HIV-specific cytotoxic T-cell responses62–64 and decay of both CD4+ and CD8+ T memory-cell responses under ART has been reported,65 indicating that maintenance of HIV-specific memory T cells requires antigen persistence. Candida-specific T cells were not affected by ART or chronic infection, which is not surprising because the animals (except AA47) remained healthy overall. Connick et al66 detected an increase in Candida-specific lymphoproliferative responses in HIV-infected individuals after 48 weeks on ART initiated in acute/recent infection, followed by a decrease after treatment interruption. Candida-specific lymphoproliferative responses and cytokine secreting capacity of T cells might not be congruent, and it is likely that ART was initiated later than 2 w.p.i. with HIV, thereby not allowing ART to rescue virus-induced effects.
The limited sample sizes in the study were a limitation to potentially identifying changes in immune functions as a result of ART. However, although we might have missed more subtle (but significant) changes, it is clear that there were not dramatic differences in the parameters measured as a result of ART exposure. Additionally, because of limited oral and rectal sample collection that could be obtained under survival surgery without endangering the health of the animals, we performed more extensive analyses on blood samples. Further studies should include the assessment of mucosal CD4+ and CD8+ T-cell responses and DC subsets especially closer examination of oral and rectal CD4+ T cells (ie, α4β7 T cells), regulatory T-cell subsets, such as CTLA-4 and IDO-expressing cells and different DC subsets in ART-treated individuals.
In conclusion, tenofovir/emtricitabine ART does not adversely affect mucosal and systemic immune functions in uninfected and SIV-infected macaques. Early virus-induced changes, including loss of blood central memory T cells and mDCs and elevated oral, rectal, and blood CXCL10 expression were detected, which were rapidly restored by ART to control levels. These data highlight the fact that commencing ART early in infection can help avoid some virus-induced damage to the immune system that might allow immune therapies more chance at boosting potent immune responses to more effectively control virus replication.
The authors thank Julian Bess, William Bohn, Jeremy Miller, Terra Schaden-Ireland, Rodman Smith, Robert Imming, and Elena Chertova, at The National Cancer Institute at Frederick, for producing, inactivating, purifying, and characterizing AT-2 SIV and microvesicle preparations. They thank Norbert Bischofberger from Gilead Sciences for providing the antiretroviral drugs. The authors would like to acknowledge the Rockefeller University Flow Cytometry Resource Center for flow cytometry assistance and the veterinary staff at the TNPRC for their continued support. They thank members of their laboratory for the assistance in editing the manuscript and continued help during the course of this study and particularly, Nina Derby and Ariel Martinez for the assistance with PCR. Their additional thanks go to Evan Read for assistance with graphics.
1. Desrosiers RC. The simian immunodeficiency viruses. Annu Rev Immunol. 1990;8:557–578.
2. Desrosiers RC. Non-human primate models for AIDS vaccines. AIDS. 1995;9(suppl A):S137–S141.
3. Van Rompay KK. Evaluation of antiretrovirals in animal models of HIV infection. Antiviral Res. 2010;85:159–175.
4. Van Rompay KK. Antiretroviral drug studies in nonhuman primates: a valid animal model for innovative drug efficacy and pathogenesis experiments. AIDS Rev. 2005;7:67–83.
5. Franchini G, Nacsa J, Hel Z, et al.. Immune intervention strategies for HIV-1 infection of humans in the SIV macaque model. Vaccine. 2002;20(suppl 4):A52–A60.
6. Sellier P, Mannioui A, Bourry O, et al.. Antiretroviral treatment start-time during primary SIV(mac) infection in macaques exerts a different impact on early viral replication and dissemination. PLoS One. 2010;5:e10570.
7. Clements JE, Gama L, Graham DR, et al.. A simian immunodeficiency virus macaque model of highly active antiretroviral treatment: viral latency in the periphery and the central nervous system. Curr Opin HIV AIDS. 2011;6:37–42.
8. George MD, Reay E, Sankaran S, et al.. Early antiretroviral therapy for simian immunodeficiency virus infection leads to mucosal CD4+ T-cell restoration and enhanced gene expression regulating mucosal repair and regeneration. J Virol. 2005;79:2709–2719.
9. Hill A, Sawyer W. Effects of nucleoside reverse transcriptase inhibitor backbone on the efficacy of first-line boosted highly active antiretroviral therapy based on protease inhibitors: meta-regression analysis of 12 clinical trials in 5168 patients. HIV Med. 2009;10:527–535.
10. Sax PE, Tierney C, Collier AC, et al.. Abacavir-lamivudine versus tenofovir-emtricitabine for initial HIV-1 therapy. N Engl J Med. 2009;361:2230–2240.
11. Deeks ED, Perry CM. Efavirenz/emtricitabine/tenofovir disoproxil fumarate single-tablet regimen (Atripla®): a review of its use in the management of HIV infection. Drugs. 2010;70:2315–2338.
12. Smith KY, Patel P, Fine D, et al.. Randomized, double-blind, placebo-matched, multicenter trial of abacavir/lamivudine or tenofovir/emtricitabine with lopinavir/ritonavir for initial HIV treatment. AIDS. 2009;23:1547–1556.
13. Gay CL, Mayo AJ, Mfalila CK, et al.. Efficacy of NNRTI-based antiretroviral therapy initiated during acute HIV infection. AIDS. 2011;25:941–949.
14. Perry CM. Emtricitabine/tenofovir disoproxil fumarate: in combination with a protease inhibitor in HIV-1 infection. Drugs. 2009;69:843–857.
15. Shen A, Zink MC, Mankowski JL, et al.. Resting CD4+ T lymphocytes but not thymocytes provide a latent viral reservoir in a simian immunodeficiency virus-Macaca nemestrina model of human immunodeficiency virus type 1-infected patients on highly active antiretroviral therapy. J Virol. 2003;77:4938–4949.
16. Murry JP, Higgins J, Matthews TB, et al.. Reversion of the M184V mutation in simian immunodeficiency virus reverse transcriptase is selected by tenofovir, even in the presence of lamivudine. J Virol. 2003;77:1120–1130.
17. Vagenas P, Aravantinou M, Williams VG, et al.. A tonsillar PolyICLC/AT-2 SIV therapeutic vaccine maintains low viremia following antiretroviral therapy cessation. PLoS One. 2010;5:e12891.
18. Van Rompay KK, Durand-Gasselin L, Brignolo LL, et al.. Chronic administration of tenofovir to rhesus macaques from infancy through adulthood and pregnancy: summary of pharmacokinetics and biological and virological effects. Antimicrob Agents Chemother. 2008;52:3144–3160.
19. Lifson JD, Rossio JL, Arnaout R, et al.. Containment of simian immunodeficiency virus infection: cellular immune responses and protection from rechallenge following transient postinoculation antiretroviral treatment. J Virol. 2000;74:2584–2593.
20. Rosenwirth B, ten Haaft P, Bogers WM, et al.. Antiretroviral therapy during primary immunodeficiency virus infection can induce persistent suppression of virus load and protection from heterologous challenge in rhesus macaques. J Virol. 2000;74:1704–1711.
21. Verhoeven D, Sankaran S, Silvey M, et al.. Antiviral therapy during primary simian immunodeficiency virus infection fails to prevent acute loss of CD4+ T cells in gut mucosa but enhances their rapid restoration through central memory T cells. J Virol. 2008;82:4016–4027.
22. Stahl-Hennig C, Steinman RM, Tenner-Racz K, et al.. Rapid infection of oral mucosal-associated lymphoid tissue with simian immunodeficiency virus. Science. 1999;285:1261–1265.
23. Tenner-Racz K, Hennig CS, Uberla K, et al.. Early protection against pathogenic virus infection at a mucosal challenge site after vaccination with attenuated simian immunodeficiency virus. Proc Natl Acad Sci U S A. 2004;101:3017–3022.
24. Suh YS, Park KS, Sauermann U, et al.. Prolonged survival of vaccinated macaques after oral SIVmac239 challenge regardless of viremia control in the chronic phase. Vaccine. 2008;26:6690–6698.
25. Animal Welfare Act and Regulation. Code of Federal Regulations. Chapter 1. In: Animals and Animal Products. Beltsville, MD: US Department of Agriculture.
26. Vagenas P, Williams VG, Piatak M Jr, et al.. Tonsillar application of AT-2 SIV affords partial protection against rectal challenge with SIVmac239. J Acquir Immune Defic Syndr. 2009;52:433–442.
27. Cline AN, Bess JW, Piatak M Jr, et al.. Highly sensitive SIV plasma viral load assay: practical considerations, realistic performance expectations, and application to reverse engineering of vaccines for AIDS. J Med Primatol. 2005;34:303–312.
28. Smith SM, Holland B, Russo C, et al.. Retrospective analysis of viral load and SIV antibody responses in rhesus macaques infected with pathogenic SIV: predictive value for disease progression. AIDS Res Hum Retrovir. 1999;15:1691–1701.
29. Gauduin MC. Intracellular cytokine staining for the characterization and quantitation of antigen-specific T lymphocyte responses. Methods. 2006;38:263–273.
30. Crostarosa F, Aravantinou M, Akpogheneta OJ, et al.. A macaque model to study vaginal HSV-2/immunodeficiency virus co-infection and the impact of HSV-2 on microbicide efficacy. PLoS One. 2009;4:e8060.
31. Frank I, Santos JJ, Mehlhop E, et al.. Presentation of exogenous whole inactivated simian immunodeficiency virus by mature dendritic cells induces CD4+ and CD8+ T-cell responses. J Acquir Immune Defic Syndr. 2003;34:7–19.
32. Vachot L, Williams VG, Bess JW Jr, et al.. Candida albicans-induced DC activation partially restricts HIV amplification in DCs and increases DC-to-T-cell spread of HIV. J Acquir Immune Defic Syndr. 2008;48:398–407.
33. Kenney J, Aravantinou M, Singer R, et al.. An antiretroviral/zinc combination gel provides 24 hours of complete protection against vaginal SHIV infection in macaques. PLoS One. 2011;6:e15835.
35. Van Rompay KK, Johnson JA, Blackwood EJ, et al.. Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy in rhesus macaques with chronic RT-SHIV infection. Retrovirology. 2007;4:25.
36. Volberding PA, Deeks SG. Antiretroviral therapy and management of HIV infection. Lancet. 2010;376:49–62.
37. Grant RM, Lama JR, Anderson PL, et al.. Preexposure chemoprophylaxis for HIV prevention in men who have sex with men. N Engl J Med. 2010;363:2587–2599.
38. Ortiz GM, Hu J, Goldwitz JA, et al.. Residual viral replication during antiretroviral therapy boosts human immunodeficiency virus type 1-specific CD8+ T-cell responses in subjects treated early after infection. J Virol. 2002;76:411–415.
39. Rosenberg ES, Altfeld M, Poon SH, et al.. Immune control of HIV-1 after early treatment of acute infection. Nature. 2000;407:523–526.
40. Lewis MG, Norelli S, Collins M, et al.. Response of a simian immunodeficiency virus (SIVmac251) to raltegravir: a basis for a new treatment for simian AIDS and an animal model for studying lentiviral persistence during antiretroviral therapy. Retrovirology. 2010;7:21.
41. Van Rompay KK, Cherrington JM, Marthas ML, et al.. 9-[2-(Phosphonomethoxy)propyl]adenine therapy of established simian immunodeficiency virus infection in infant rhesus macaques. Antimicrob Agents Chemother. 1996;40:2586–2591.
42. Van Rompay KK, Miller MD, Marthas ML, et al.. Prophylactic and therapeutic benefits of short-term 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA) administration to newborn macaques following oral inoculation with simian immunodeficiency virus with reduced susceptibility to PMPA. J Virol. 2000;74:1767–1774.
43. Veazey RS, Tham IC, Mansfield KG, et al.. Identifying the target cell in primary simian immunodeficiency virus (SIV) infection: highly activated memory CD4(+) T cells are rapidly eliminated in early SIV infection in vivo. J Virol. 2000;74:57–64.
44. Mattapallil JJ, Douek DC, Hill B, et al.. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature. 2005;434:1093–1097.
45. Wijewardana V, Soloff AC, Liu X, et al.. Early myeloid dendritic cell dysregulation is predictive of disease progression in simian immunodeficiency virus infection. PLoS Pathog. 2010;6:e1001235.
46. Macatonia SE, Lau R, Patterson S, et al.. Dendritic cell infection, depletion and dysfunction in HIV infected individuals. Immunology. 1990;71:38–45.
47. Pacanowski J, Kahi S, Baillet M, et al.. Reduced blood CD123+ (lymphoid) and CD11c+ (myeloid) dendritic cell numbers in primary HIV-1 infection. Blood. 2001;98:3016–3021.
48. Sabado RL, O'Brien M, Subedi A, et al.. Evidence of dysregulation of dendritic cells in primary HIV infection. Blood. 2010;116:3839–3852.
49. Barratt-Boyes SM, Wijewardana V, Brown KN. In acute pathogenic SIV infection plasmacytoid dendritic cells are depleted from blood and lymph nodes despite mobilization. J Med Primatol. 2010;39:235–242.
50. Easlick J, Szubin R, Lantz S, et al.. The early interferon alpha subtype response in infant macaques infected orally with SIV. J Acquir Immune Defic Syndr. 2010;55:14–28.
51. Abel K, Pahar B, Van Rompay KK, et al.. Rapid virus dissemination in infant macaques after oral simian immunodeficiency virus exposure in the presence of local innate immune responses. J Virol. 2006;80:6357–6367.
52. Milush JM, Stefano-Cole K, Schmidt K, et al.. Mucosal innate immune response associated with a timely humoral immune response and slower disease progression after oral transmission of simian immunodeficiency virus to rhesus macaques. J Virol. 2007;81:6175–6186.
53. Durudas A, Chen HL, Gasper MA, et al.. Differential innate immune responses to low or high dose oral SIV challenge in rhesus macaques. Curr HIV Res. 2011;9:276–288.
54. Groom JR, Luster AD. CXCR3 in T cell function. Exp Cell Res. 2011;317:620–631.
55. Farber JM. Mig and IP-10: CXC chemokines that target lymphocytes. J Leukoc Biol. 1997;61:246–257.
56. Lane BR, King SR, Bock PJ, et al.. The C-X-C chemokine IP-10 stimulates HIV-1 replication. Virology. 2003;307:122–134.
57. Boulware DR, Meya DB, Bergemann TL, et al.. Antiretroviral therapy down-regulates innate antiviral response genes in patients with AIDS in sub-Saharan Africa. J Acquir Immune Defic Syndr. 2010;55:428–438.
58. Bergmeier LA, Wang Y, Lehner T. The role of immunity in protection from mucosal SIV infection in macaques. Oral Dis. 2002;8(suppl 2):63–68.
59. Cocchi F, DeVico AL, Garzino-Demo A, et al.. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells [see comments]. Science. 1995;270:1811–1815.
60. Lifson JD, Piatak M Jr, Cline AN, et al.. Transient early post-inoculation anti-retroviral treatment facilitates controlled infection with sparing of CD4+ T cells in gut-associated lymphoid tissues in SIVmac239-infected rhesus macaques, but not resistance to rechallenge. J Med Primatol. 2003;32:201–210.
61. Kubo M, Nishimura Y, Shingai M, et al.. Initiation of antiretroviral therapy 48 hours after infection with simian immunodeficiency virus potently suppresses acute-phase viremia and blocks the massive loss of memory CD4+ T cells but fails to prevent disease. J Virol. 2009;83:7099–7108.
62. Jin X, Ogg G, Bonhoeffer S, et al.. An antigenic threshold for maintaining human immunodeficiency virus type 1-specific cytotoxic T lymphocytes. Mol Med. 2000;6:803–809.
63. Kalams SA, Goulder PJ, Shea AK, et al.. Levels of human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte effector and memory responses decline after suppression of viremia with highly active antiretroviral therapy. J Virol. 1999;73:6721–6728.
64. Casazza JP, Betts MR, Picker LJ, et al.. Decay kinetics of human immunodeficiency virus-specific CD8+ T cells in peripheral blood after initiation of highly active antiretroviral therapy. J Virol. 2001;75:6508–6516.
65. Sester U, Sester M, Kohler H, et al.. Maintenance of HIV-specific central and effector memory CD4 and CD8 T cells requires antigen persistence. AIDS Res Hum Retroviruses. 2007;23:549–553.
66. Connick E, Bosch RJ, Aga E, et al.. Augmented HIV-specific interferon-gamma responses, but impaired lymphoproliferation during interruption of antiretroviral treatment initiated in primary HIV infection. J Acquir Immune Defic Syndr. 2011;58:1–8.
antiretroviral therapy; mucosal immunity; peripheral immunity; SIV
Supplemental Digital Content
© 2012 Lippincott Williams & Wilkins, Inc.
Highlight selected keywords in the article text.