JAIDS Journal of Acquired Immune Deficiency Syndromes:
The Clinical Benefits of Tenofovir for Simian Immunodeficiency Virus–Infected Macaques Are Larger Than Predicted by its Effects on Standard Viral and Immunologic Parameters
Van Rompay, Koen K. A. DVM, PhD*; Singh, Raman P. BS*; Brignolo, Laurie L. DVM*; Lawson, Jonathan R. BS*; Schmidt, Kimberli A. AA*; Pahar, Bapi PhD*; Canfield, Don R. DVM*; Tarara, Ross P. DVM, PhD, DACVP*; Sodora, Donald L. PhD†; Bischofberger, Norbert PhD‡; Marthas, Marta L. PhD*§
From *The California National Primate Research Center, §Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, CA; †University of Texas Southwestern Medical Center, Dallas, TX; and ‡Gilead Sciences, Foster City, CA.
Received January 27, 2004; accepted for publication March 31, 2004.
Supported by Gilead Sciences, E. Glaser Pediatric AIDS Foundation grants PG-50757 and PG-51014 (K.V.R); NIH/NIAID grant R01 AI46320-01 (M.L.M), DE12926 (D.L.S.); Public Science Health grant RR00169 from the National Center for Research Resources; E. Glaser Scientist Award 97-8 (M.L.M.) from the E. Glaser Pediatric AIDS Foundation.
Reprints: Koen K. A. Van Rompay, California National Primate Research Center, University of California Davis, Davis, CA 95616 (e-mail: email@example.com).
Summary: Previous studies have demonstrated that tenofovir (9-[2-(phosphonomethoxy)propyl]adenine; PMPA) treatment is usually very effective in suppressing viremia in macaques infected with simian immunodeficiency virus (SIV). The present study focuses on a subset of infant macaques that were chronically infected with highly virulent SIVmac251, and for which prolonged tenofovir treatment failed to significantly suppress viral RNA levels in plasma despite the presence of tenofovirsusceptible virus at the onset of therapy. While untreated animals with similarly high viremia developed fatal immunodeficiency within 3–6 months, these tenofovir-treated animals had significantly improved survival (up to 3.5 years). This clinical benefit occurred even in animals for which tenofovir had little or no effect on CD4+ and CD8+ lymphocyte counts and antibody responses to SIV and test antigens. Thus, the clinical benefits of tenofovir were larger than predicted by plasma viral RNA levels and other routine laboratory parameters.
To help a growing number of HIV-infected people, there is a need for simple and safe drug regimens that provide long-term clinical benefits. Because human clinical trials are very time-consuming and expensive, animal models can be useful, as they allow rapid identification of those strategies that are most promising and deserve to enter clinical trials first. While there is no ideal animal model of HIV infection, simian immunodeficiency virus (SIV) infection of macaques has been an appropriate model, because of its many similarities in disease pathogenesis, immunology, and physiology (including drug metabolism; reviewed by Van Rompay et al 1,2). The reverse transcriptase (RT) inhibitor tenofovir (9-[2-(phosphonomethoxy)propyl]adenine; PMPA) has been used extensively in this animal model because of its high efficacy. 3–12 In our first studies, initiation of tenofovir monotherapy for SIVmac251-infected infant macaques during the acute stage of infection generally induced a rapid and strong suppression of viremia and improved disease-free survival. 5,7,13 During prolonged tenofovir treatment, the emergence of viral mutants with reduced in vitro susceptibility to tenofovir, associated with a lysine-to-arginine mutation at codon 65 of RT (K65R), was not always associated with an increase in viremia, as some animals maintained low or undetectable viremia for >7 years. 7,13,14
In subsequent studies in which a different experimental design was used, however, we identified animals that did not show the desired reduction in viremia, despite the presence of drug-susceptible virus at the onset of tenofovir treatment. To better understand this phenomenon and drug therapy in general, the present report focuses on the viral, immunologic, and clinical findings in all these animals that did not show a significant reduction in viremia following tenofovir treatment. Our observations suggest that the efficacy of tenofovir therapy in suppressing viremia is diminished particularly in immunodeficient animals with high viremia. Most importantly, however, even with a poor virologic response, animals treated continuously with tenofovir had significantly improved survival.
MATERIALS AND METHODS
Animals and Parameters to Monitor Infection
All rhesus macaques (Macaca mulatta) were from the type D-retrovirus-free and SIV-free colony at the California National Primate Research Center. The newborn and infant macaques were hand reared in a primate nursery. Animals were housed in accordance with American Association for Accreditation of Laboratory Animal Care standards. We adhered to the Guide for Care and Use of Laboratory Animals. 15 For blood collections, animals were immobilized with 10 mg/kg intramuscular ketamine-HCl (Parke-Davis, Morris Plains, NJ). Ethylenediamine tetra-acetic acid–anticoagulated blood samples were collected for monitoring immunologic and viral parameters and complete blood cell counts according to methods previously described. 16
To monitor the development of immune responses to nonviral, nonreplicating antigens, infants were immunized subcutaneously with 0.1 mg of cholera toxin B subunit (List Biologic Laboratories, Campbell, CA) and intramuscularly with 0.5 mL of diphtheria and tetanus toxoids in Adsorbed USP (for pediatric use; Aventis Pasteur, Inc., Swiftwater, PA).
Experimental Animal Groups
Animals are presented as part of 2 cohorts. In the first cohort (experiments performed from 1996–1998), all animals, none of which had received any SIV vaccines, were inoculated orally with uncloned SIVmac251 within 3 days of birth, and 1 random animal (number 31042) was started on tenofovir treatment (see below). In the second cohort (experiments performed in 2001 and 2002), infant macaques were inoculated with uncloned SIVmac251 at 4 weeks of age. In this 2nd cohort, a total of 4 SIVmac251-infected infant macaques, none of which had received any SIV vaccines, were started on tenofovir treatment during chronic infection (see below); results for all 4 animals are included in the presented data (ie, no tenofovir-treated animals were excluded from the analysis).
In the first cohort, newborn macaques were inoculated orally with undiluted SIVmac251 (of a stock designated 8/95, containing 105 tissue culture infectious doses 50% [TCID50] per mL), as described previously. 5,16–18 In the second cohort, animals were inoculated orally at 4 weeks of age with 15 diluted doses (each dose containing 104 TCID50 of an SIVmac251 stock designated 5/98), according to a 5-day inoculation regimen described elsewhere. 19
Preparation and Administration of Tenofovir
Tenofovir (Gilead Sciences, Foster City, CA) was suspended in distilled water, dissolved by the addition of NaOH to a final pH of 7.0 at 60 mg/mL, filter sterilized (0.2 μm; Nalgene), and stored at 4°C. Tenofovir was administered subcutaneously into the back of the animal. The dosage was adjusted weekly based on the weight. Starting 3 weeks after virus inoculation, animal 31042 (cohort 1) was treated with tenofovir at a regimen of 10 mg/kg once daily for the rest of her life. Tenofovir treatment of animals 32989, 32990, 33093, and 33109 (cohort 2) was started at 14 or 16 weeks of age (30 mg/kg/d subcutaneously); to prevent the renal toxicity that occurs following prolonged dosing at 30 mg/kg, 20 animals were monitored with standard serum chemistry panels and urine analysis, and the dose of tenofovir was reduced to 20 mg/kg at 7 months of age, then to 10 mg/kg (at 1 year of age for animals 32989 and 33109; at 14 months of age for animal 33093).
Quantitation of Plasma Viral RNA
Viral RNA in plasma was quantified using a branched DNA signal amplification assay specific for SIV, versions 2.0, 3.0, and 4.0, which have lower quantitation limits of 1500, 500, and 125 copies per mL of plasma, respectively.
Infectious virus was isolated in cultures of peripheral blood mononuclear cells (PBMCs) with CEMx174 cells and subsequent p27 core antigen measurement via an enzyme-linked immunosorbent assay (ELISA), according to methods previously described. 21
Drug Susceptibility Assays
Phenotypic drug susceptibilities of SIVmac isolates were characterized by a previously described assay based on a dose-dependent reduction of viral infectivity. 7,22
Sequence Analysis of SIV RT-Encoding Region
DNA sequence analyses of codons 0–320 of RT were performed on proviral DNA obtained from CEMx174 cells infected with virus isolated from the SIV-infected animals. Infected cells were harvested as soon as culture supernatants were positive by p27 antigen capture ELISA. Genomic DNA was extracted and used for nested polymerase chain reaction, according to methods and with primers described previously. 14,23 This method can detect the presence of a 20% sub-population.
Detection of IgG Antibodies to SIV and Test Antigens
The ELISA to detect SIV-specific immunoglobulin G (IgG) was performed as described previously, 17 except that Costar EIA/RIA plates (Fisher Scientific, Santa Clara, CA) were used for the experiments of the second animal cohort. The antigen-specific IgG ELISAs for the other antigens were performed in a similar manner, by coating the ELISA plates with purified tetanus toxoid (10 Lf/mL; Connaught Laboratories, Ontario, Canada), diphtheria toxin (2.5 μg/mL), or cholera toxin (2 μg/mL; both from List Biologic Laboratories).
ELISPOT Assay for SIV-Specific Interferon-γ Secreting Cells
The number of antigen-specific interferon-γ (IFN-γ)-producing cells was measured using an ELISPOT assay described previously, 24 using a pool of 15-mer peptides with 10-amino-acid overlapping of the entire p24 gag region of SIVmac239.
In the studies performed from 1998–2001, 3-color flow cytometry techniques were used. T-lymphocyte antigens were detected by direct labeling of whole blood with peridinin chlorophyll protein (PerCP)-conjugated antihuman CD8 (clone SK1; Becton Dickinson Immunocytometry, Inc., San Jose, CA), phycoerythrin (PE)-conjugated anti-human CD4 (clone M-T477; Pharmingen, San Jose, CA), and fluorescein isothiocyanate (FITC)-conjugated antihuman CD3 (clone SP34; Pharmingen). A separate aliquot of blood was labeled with FITC-conjugated antihuman CD3 and PerCP-conjugated anti-human CD20 (clone L27; Becton Dickinson). Starting in 2002, 4-color flow cytometry was used, consisting of a single tube containing the same antibody clones as described here, except that the antihuman CD20 antibody was conjugated to allophycocyanin. Red blood cells were lysed and the samples were fixed in paraformaldehyde using the Coulter Q-prep system (Coulter Corp., Hialeah, FL). Flow cytometry was performed on a FACSCalibur flow cytometer (Becton Dickinson). Lymphocytes were gated by forward and side light scatter and were then analyzed with Cellquest software (Becton Dickinson). CD4+ T lymphocytes, CD8+ T lymphocytes, B lymphocytes, and natural killer (NK) cells were defined as CD3+CD4+, CD3+CD8+, CD3−CD20+, and CD3−CD8+ lymphocyte populations, respectively.
At select time points, intracellular Ki67 expression was measured using PE-conjugated anti-Ki67 monoclonal antibody (clone B56; Becton Dickinson) or control antibody, in combination with the anti-CD3, anti-CD4, and anti-CD8 antibodies described here, with the exception that the anti-CD4 antibody was conjugated to FITC; intracellular staining procedures were performed according to methods described previously. 25 The PE-conjugated anti-CD28 antibody was clone L293 (Becton Dickinson).
Measurement of T-Cell Receptor Rearrangement Excision Circle Levels in Sorted Cells
Quantitation of T-cell receptor rearrangement excision circles (TREC) was performed as described previously. 26
Genetic Assessment of Major Histocompatibility Complex Class I Alleles
DNA extracted from lymphoid cells (with QIAamp DNA minikit, QIAgen, Valencia, CA) was used to screen for the presence of the major histocompatibility complex (MHC) class I alleles MamuA*01 and MamuB*01, using a polymerase chain reaction–based technique. 27,28
Criteria for Euthanasia and Animal Necropsies
Euthanasia of animals with simian AIDS was indicated by ≥3 of the following clinical observations that indicated a severe life-threatening situation for the animal: weight loss of >10% in 2 weeks or >30% in 2 months; chronic diarrhea unresponsive to treatment; infections unresponsive to treatment; inability to maintain body heat or fluids without supplementation; persistent, marked hematologic abnormalities, including lymphopenia, anemia, thrombocytopenia, or neutropenia; and persistent, marked splenomegaly or hepatomegaly. A complete necropsy with a routine histopathologic examination of tissues was performed. Tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 6 μm, stained with hematoxylin and eosin, and examined by light microscopy.
Statistical analysis of disease-free survival was done using a logrank test. Statistical analysis of antibody titers (after log transformation) according to viral RNA set points was performed with a 2-way analysis of variance with Bonferroni post-tests (Prism Version 3.0 for Mac, GraphPad Software, Inc., San Diego, CA).
SIVmac251 Infection of Infant Macaques: Correlation of Viremia, Survival, and Immunologic Parameters in Untreated Animals
In the first cohort, infant rhesus macaques were inoculated orally within 3 days of birth with highly virulent un-cloned SIVmac251. In the second cohort, infant macaques were inoculated orally at 4 weeks of age with uncloned SIVmac251. Animals were euthanized when life-threatening immunodeficiency had developed. In both animal cohorts, the viral RNA set point, defined as the highest viral RNA level in plasma at 6–8 weeks after virus inoculation, was found to be a good predictor of the rate of subsequent disease progression. When animals were stratified according to this viral RNA set point, a significant difference in survival curves was observed (cohort 1:P = 0.04; cohort 2:P < 0.0001; logrank test;Figs. 1 and 2). Similar results were obtained if instead of viral set point, the time-weighted AUC (ie, area under the curve divided by the time of infection) was used for the survival analysis (data not shown).
The untreated animals of cohort 1 have been described previously. 5,16–18 For cohort 2, to determine whether the level of viremia and survival correlated with the degree of virus-induced immunosuppression, we compared several immunologic parameters among these animal groups and age-matched, uninfected control animals. The status of humoral immunity in these animals was evaluated by measuring their ability to generate and maintain antibody responses to 3 nonreplicating test antigens. SIV-infected animals with a viral set point >7 log RNA copies/mL plasma had reduced antibody responses to the test antigens even early during infection; in contrast, the SIVmac251-infected infant macaques with a viral set point of 5–7 log RNA copies per mL had relatively normal antibody responses the first 2 months of life but immunosuppression became apparent later (Fig. 3).
To detect SIV-specific cell-mediated immune responses, PBMCs collected during the first 12 weeks of infection were tested by SIV-specific IFN-γ ELISPOT assay. Twenty-three PBMC samples collected from 7 of the SIVmac251-infected infant macaques with high viral set point (>7 log RNA copies/mL) were tested, and only 1 sample had detectable IFN-γ-producing cells (95 spot-forming cells [SFC]/million PBMCs). Of the 31 samples from 9 infants with intermediate viral set point (5–7 log RNA copies/mL) that were tested, only 5 samples (from 3 animals) were positive (60–240 SFC/million PBMCs). The difficulty of detecting anti-SIV cell-mediated immune responses by ELISPOT assay is similar to what we have found previously in SIVmac251-infected infants. 29
Similar to our previously described studies, 29 absolute lymphocyte counts in infant macaques were highly variable (Fig. 4). The percentages of lymphocyte subpopulations were more reliable parameters for monitoring SIV infection. SIVmac251-infected infant macaques with a high viral RNA set point (>7 log RNA copies per mL) showed in general a reduction in CD4+/CD8+ T-lymphocyte ratio to values <2 (due to a decrease and increase in the percentages of CD4+ and CD8+ T lymphocytes, respectively) and more variable percentages of B lymphocytes and NK cells in comparison with uninfected animals (Fig. 4).
Prolonged Tenofovir Treatment of SIVmac251-Infected Infant Macaques: Benefits on Survival Despite High Viremia
In animal cohort 1, one randomly selected infected animal (number 31042), which had peak viremia of ≈108 RNA copies/mL, was started at 3 weeks of age on low-dose tenofovir treatment (10 mg/kg once daily, subcutaneously;Table 1). The rationale for using a 10-mg/kg daily dose (instead of the 30-mg/kg regimens of previous studies 5,7) was that this regimen in a 3-week old infant is pharmacokinetically similar to a subcutaneous ≈25- to 30-mg/kg regimen for juvenile macaques. 20
Tenofovir treatment of animal 31042 had minimal effect on viremia: the lowest RNA level that was observed during the first 3 weeks of treatment was only 2.5-fold lower than the baseline value at the start of treatment (Fig. 2). Despite the minimal effect on viremia, tenofovir treatment selected for the emergence of K65R viral mutants (with 5-fold reduced in vitro susceptibility to tenofovir), which were detected after 7 weeks of tenofovir therapy (Table 2). In addition to K65R, secondary RT mutations gradually accumulated, similar to our previous observations. 7,13 Following the detection of K65R mutants, viremia increased to ≈108 RNA copies per mL plasma by 20 weeks of age and then showed a slow decline. Animal 31042 mounted a strong and persistent IgG response to SIV (ELISA titer > 1:102,400 from week 5 onwards). Following immunizations with cholera toxin B subunit at 2 and 10 weeks of age, the primary and secondary antibody responses (titers of 1:25,600 and 1:102,400, respectively) were only ≈4-fold lower than the average responses in uninfected animals (data not shown). This is in contrast to untreated animals with high viremia, which had generally low or transient antibody responses, as reported previously. 5,16–18 At ≈3 years of age, this animal had SIV-specific T-cell responses (≈85–170 SFC/million PBMCs by IFN-γ ELISPOT). Although the % CD4+ T lymphocytes in peripheral blood of animal 31042 was at some time points slightly reduced (<30%) compared with uninfected animals, the % CD8+ T lymphocytes, the % NK cells, the absolute CD4+ and CD8+ T-lymphocyte and NK cell counts, and the CD4+/CD8+ T-lymphocyte ratio remained generally within the range of uninfected age-matched animals throughout the observation period (data not shown). Thus, these data show the relative preservation of immunologic function in this animal in the presence of high viremia, which could explain its prolonged survival. Starting at approximately 2 years of age, this animal developed intermittent diarrhea, which temporarily responded to standard treatments but eventually progressed and the animal required euthanasia at 3.5 years of age. Pathologic findings were consistent with terminal stages of SIV infection (Table 1). At that time, the % CD4+ T lymphocytes, CD4+/CD8+ T-cell ratio, and absolute CD4+ T-lymphocyte counts were in the normal range (44%, 2.2, and 687 per μL, respectively). Although this was a single case study in cohort 1, the prolonged survival of animal 31042 was remarkable when compared with the untreated SIVmac251-infected infant macaques with similarly high virus levels (P = 0.06; logrank test;Figs. 1A and 2A), or with infected infant macaques that were started on other RT inhibitors (lamivudine, emtricitabine) at 3 weeks of age. 23
This unexpected observation of animal 31042 in cohort 1 prompted larger studies in cohort 2, but this time, tenofovir treatment was started at a later stage of infection. Four infant macaques (numbers 32989, 32990, 33093, 33109) were started on high-dose tenofovir treatment (30 mg/kg subcutaneously once daily) at 14 or 16 weeks of age (ie, 10–12 weeks after SIVmac251 infection), when virus had wild-type phenotypic susceptibility to tenofovir and wild-type RT sequence (Tables 1 and 2). Based on their virus levels and clinical condition at the start of tenofovir treatment, these animals are categorized in 2 groups for the purpose of summarizing their findings.
Three animals (32990, 33093, and 33109) had persistently high viremia (>107 RNA copies/mL plasma), were indistinguishable from the untreated animals with similar viremia, and had symptoms of opportunistic infections when tenofovir treatment was started. Following tenofovir treatment, these 3 animals showed little decrease in virus levels; their viral RNA levels during the first 2 weeks of treatment either increased (animal 32990) or were only 2-fold (33109) to 4-fold (33093) lower than the pretreatment baseline value (Fig. 2B). A 2- to 4-fold decrease in viral RNA during this period was observed in 2 of the 13 untreated infants that survived for >16 weeks of age (P = 0.14; Fisher exact test). K65R viral mutants (with ≈5-fold reduced in vitro susceptibility to tenofovir) became first detectable 4 weeks (animals 32990 and 33093) or 6 weeks (animal 33109) after the start of tenofovir treatment (Fig. 2B;Table 2).
Despite little change in viremia, these 3 tenofovir-treated animals showed clinical improvement, with a reduction in the frequency and severity of opportunistic infections. At the start of tenofovir treatment at 16 weeks of age, animal 32990’s condition was most critical, as it had weight loss, poor appetite, and had required daily subcutaneous fluids to maintain hydration for the 3 preceding days. Accordingly, this animal fit the criteria for euthanasia. One day after the start of tenofovir treatment, clinical improvement was noticed (improved appetite, activity, and hydration), and the animal did not require parenteral fluids anymore for 4 weeks until his condition deteriorated again at 20 weeks of age. This animal required euthanasia at 22 weeks of age (ie, 6 weeks after the start of tenofovir treatment). Histopathology revealed generalized lymphoid depletion and opportunistic infections (Table 1). Thus, despite the transient clinical improvement following initiation of tenofovir treatment, the immune dysfunction and opportunistic infections were too advanced to allow longer survival.
Animal 33093, which remained highly viremic (≈7–8 log RNA copies/mL plasma;Fig. 2B), had dermatitis and poor weight gain prior to tenofovir treatment. Following tenofovir treatment at 14 weeks of age, the animal remained stable, had slow growth, and had relatively few clinical problems, except for a few episodes of diarrhea and bacterial skin infections with abscess formation, but which resolved following standard antibiotic treatments. Although no TREC or Ki67 data were available from the untreated, highly viremic SIV-infected infants in this study at this 1-year time point (due to their early mortality), a comparison to historical data of SIV-infected juvenile macaques 26,30 suggests that animal 33093 had relatively preserved thymic function and little immune activation at 1 year of age despite this very high viremia and long-term infection, which is rather unusual (TREC levels: 1080 per 100,000 CD8+ cells; 0.3 and 0.5% of CD4+ and CD8+ T lymphocytes, respectively, were Ki67+; 61% of CD8+ T lymphocytes were CD28+). Animal 33093 eventually developed virus levels of ≈9 log RNA copies per mL plasma and was euthanized at 17 months of age due to declining health (Table 1). The third animal, number 33109, had Candida-positive cheilitis prior to tenofovir treatment, which resolved afterward. This animal, which maintained persistently high viremia (≈7 log RNA copies/mL plasma), developed intermittent diarrhea at 13 months of age, with variable responses to antibiotic treatments, but was able to maintain normal hydration (without the need for parenteral fluids) and good appetite and activity. At 1 year of age, the % of CD8+ T lymphocytes that expressed CD28+ (61%) was within normal range. The reduced level of TREC in CD8+ cells (370 per 100,000 CD8+ cells) at 1 year of age may be caused by slightly increased T-cell proliferation (2.5% of CD4+ T lymphocytes and 6.7% of CD8+ T lymphocytes were Ki67+), and thus it was difficult to evaluate thymic function in this animal. Animal 33109 eventually had to be euthanized due to progressive disease at 26 months of age (Table 1). Thus, the survival of these 3 tenofovir-treated SIVmac251-infected animals was significantly improved in comparison to the 30 untreated age-matched animals, which had a similarly high viral set point of >7 log RNA copies/mL plasma (Fig. 1B, P = 0.007 by logrank test) and which were raised in the same conditions (including similar exposure to opportunistic infectious agents in the environment).
A closer look at the antibody responses in these 3 tenofovir-treated animals with high viremia suggests that tenofovir treatment had some beneficial effect on their humoral immunity, but the effect was relatively small and the magnitude and duration were variable among individual animals and among the antigens (Fig. 3). Altogether, however, the antibody titers remained within the range from those observed in the untreated animals that had similarly high viremia but that succumbed rapidly to opportunistic infections. Thus, the humoral immune responses in these tenofovir-treated animals were not improved sufficiently to be able to account for the prolonged survival.
PBMCs collected during the later stages of infection (animal 33093: 63 and 71 weeks; animal 33109: 63 and 79 weeks of age) did not have any detectable SIV-specific IFN-γ-producing cells by ELISPOT assay. Prior to the start of tenofovir treatment, these 3 SIVmac251-infected animals (32990, 33093, and 33109) with high viremia had the general changes in lymphocyte populations that are associated with high viremia, in particular a reduced CD4+/CD8+ T-lymphocyte ratio and increased % CD8+ T lymphocytes (Fig. 4). Tenofovir treatment did not have any significant effect on these parameters, and lymphocyte subpopulations were indistinguishable from the values observed in the untreated animals with high viremia and rapid disease progression. Throughout the rest of the observation period, the absolute CD4+ T-lymphocyte counts of these 3 tenofovir-treated animals were generally below the values of uninfected animals but remained >500 per μL.
The fourth tenofovir-treated infant macaque of this second cohort, number 32989, had a delayed viremia, as virus became detectable at 8 weeks of age. Viral RNA levels were still increasing at 16 weeks of age, when tenofovir treatment was initiated (Fig. 2C). Although there was no stable viral set point at that time, viral levels were ≈6 log RNA copies per mL, and therefore animal 32989 was compared with the animals that had a viral RNA set point of 5–7 log RNA copies/mL plasma (Figs. 2–4). At the start of tenofovir treatment, animal 32989 did not have any clinical signs of opportunistic infections but already had evidence of SIV-induced changes in lymphocyte populations in peripheral blood (Fig. 4). Thus, in contrast to the other 3 tenofovir-treated animals of this cohort (see above), animal 32989 had an intermediate stage of SIV disease. The most important finding in this animal (see “Discussion”) was that within 2 weeks of starting tenofovir treatment, there was a 180-fold reduction in viremia, associated with a normalization in the proportions of lymphocyte subpopulations (Figs. 2C and 4). Concomitantly with the detection of K65R viral mutants after 4 weeks of treatment (Table 2), viremia increased slowly to reach eventually 6–7 log RNA copies per mL, and lymphocyte populations gradually returned to pre-treatment levels. Animal 32989’s antibody responses were similar to those of untreated animals with similar RNA levels (5–7 log RNA copies/mL;Fig. 3). PBMCs collected at 63 and 79 weeks of age did not have detectable SIV-specific IFN-γ-producing cells by ELISPOT assay. Animal 32989 developed clinical symptoms (diarrhea, rapid weight loss) at approximately 60 weeks of age, which first responded well to standard treatments, but eventually progressed to chronic diarrhea, and the animal required euthanasia at 21 months of age (Table 1).
The goal of antiviral therapy for HIV-infected persons is to prolong disease-free survival. For practical reasons, the standard management of HIV-infected patients relies heavily on monitoring surrogate laboratory markers (virus levels, CD4+ T-lymphocyte counts), because of their strong predictive value with disease progression and survival. 31–33
The present data obtained in SIV-infected macaques provide further insights about the therapeutic benefits of prolonged tenofovir treatment. Our data demonstrate that tenofovir monotherapy delayed disease progression and prolonged survival even when there was little or no effect on commonly measured viral and immunologic parameters in peripheral blood. Such clinical benefits are logistically difficult to detect in human studies, as it requires years of follow-up, and without a reasonable virologic and immunologic response, drug regimens would probably be changed in meantime. We were able to detect clinical benefits of tenofovir monotherapy in an animal model due to our study design. The rapid disease course in untreated SIVmac251-infected infant macaques is relatively consistent and allows evaluation of disease progression in a relatively short time. 22 Most importantly, we did not use poor virologic or immunologic responses as criteria for changing treatment.
To explore possible explanations, this discussion will focus in sequential order on the 2 main observations of the present studies: the absence of a virologic response to tenofovir in some infant macaques, and the prolonged survival despite high viremia.
Tenofovir has been used in many studies with macaques because of its high efficacy in reducing viremia, which has not been observed with other drugs in this animal model. 4–8,10,12 In the animals described in this report, tenofovir treatment induced a rapid suppression of viremia (which was, however, transient once K65R viral mutants emerged) for animal 32989, which had moderate viremia (≈6 log RNA copies/mL). But tenofovir therapy did not induce a rapid suppression of viremia in the other animals that had high viremia (>7 log RNA copies/mL) despite the presence of drug-susceptible virus at the onset of treatment. Anecdotal cases of poor virologic responses to tenofovir have been described previously by others, particularly in chronically infected animals with relatively high virus levels and immunosuppression. 4,34,35 Our experimental design in cohort 2 somehow induced a poor virologic response in the majority of animals. Accordingly, these studies provide insights into the events that determine the efficacy of tenofovir therapy in suppressing viremia. The available data suggest that a combination of factors is responsible for these findings, including the virulence of the virus isolate, the age of the animals, the timing of tenofovir treatment, and the status of the immune system.
In this report, animals were inoculated with highly virulent SIVmac251. SIVmac251-infected infant macaques maintain higher viremia and develop immunodeficiency more rapidly than most juvenile or adult macaques. 22 The inability of tenofovir therapy to induce strong suppression of viral RNA levels cannot be explained solely by the high virus levels at the start of tenofovir treatment. In previous studies, when tenofovir treatment was initiated for infant or juvenile macaques during acute SIVmac251 viremia (≤3 weeks), similar pretreatment virus levels (≈7–8 log RNA copies per mL plasma) were reduced with ≥1.5–2 logs within 3 weeks of treatment. 5,7,14 Instead, the status of the immune system at the start of tenofovir treatment must also play an important role. Tenofovir treatment initiated during the chronic stage of infection was usually effective in reducing viremia for juvenile animals, especially when infected with less virulent isolates that induced lower viral RNA set points than SIVmac251. 4,7,12,36 In the present study, however, tenofovir treatment was started in several infant macaques at an advanced stage of SIVmac251 infection, which represents a worst case scenario. Under such conditions, tenofovir therapy no longer induced a strong reduction in viremia. Yet, the relatively rapid detection of K65R viral mutants suggests that tenofovir efficiently inhibited reverse transcription of wild-type virus and exerted strong selection pressure for the outgrowth of such K65R mutants.
In different studies, we observed that the efficacy of tenofovir therapy to suppress viremia was reduced by a depletion of CD8+ lymphocytes with monoclonal antibody. 14 Thus, a model has been proposed in which CD8+ cell-mediated antiviral immune responses contribute significantly to the antiviral effects of anti-HIV drugs, presumably by reducing the burst of viral replication in productively infected cells; without cooperation of effective antiviral immune responses, antiviral drugs face a more daunting task to control viremia as already infected cells survive longer and produce more viral progeny. 37 The data presented here provide further support for this model. Because SIV and HIV infection induces immune dysfunction at many stages of the immune response (including antigen presentation; CD4+ and CD8+ lymphocytes), the absence of reduced viremia in these tenofovir-treated SIVmac251-infected infant macaques may be due to any defect that impairs the generation, maintenance, or maturation of effective antiviral immune responses. 38–41 Evidence from human trials also suggests a role of the immune system in the efficacy of tenofovir treatment, as the reduction in viral RNA levels was larger in treatment-naive patients than in treatment-experienced patients, who generally had lower CD4+ T-cell counts. 42–44
The second important finding in the present studies was the effect of tenofovir treatment on survival. In untreated SIVmac251-infected infant macaques, a strong correlation was demonstrated between the viral RNA set point, their ability to make immune responses to test antigens, and disease-free survival, similar to what has been described for older SIV-infected macaques and HIV-infected patients. 31,32,45–49 During tenofovir treatment, however, SIVmac251-infected animals showed clinical improvement and prolonged survival despite persistently high viremia, even those animals of cohort 2 that had little improvement in the adaptive immune responses that were measured in this study. This delayed disease progression has not been observed in this animal model with other drugs (eg, zidovudine, lamivudine, and emtricitabine), as high viremia, associated with drug-resistant virus, led to disease within the expected time frame as predicted by the viral RNA levels. 23,50 Thus, unlike most other anti-HIV drugs, tenofovir therapy may preserve, induce, or rescue some immune responses (including innate immunity) that promote disease-free survival in the presence of high viremia. Immune responses were also found to play a major role in animals that maintain low viremia during years of tenofovir monotherapy even after the emergence of K65R viral mutants. 14
For some SIV isolates, it has been reported that the rhesus macaque MHC class I allele MamuA*01 is associated with a lower viral RNA set point or delayed disease progression. 51–54 We have not seen this effect in animals of our colony with our SIVmac251 stocks (unpublished data). In this study, the frequency of the MamuA*01 and MamuB*01 alleles in the untreated animals with high viral RNA set point (>7 log) was approximately 25%, which is similar to the frequency in the California National Primate Research Center (CNPRC) rhesus macaque colony. A similar frequency of these alleles was found in the tenofovir-treated animals (Table 1), and thus this does not explain the findings of prolonged survival.
Disease-free survival in the presence of high virus levels has been reported for African primate species that are persistently infected with naturally occurring SIV isolates, 55–57 and this lack of disease is associated with preserved CD4+ lymphocyte counts, lower levels of generalized immune activation and lymphocyte apoptosis, and lower antiviral cellular immune responses than what is observed during pathogenic infection of rhesus macaques. 58 Although some tenofovir-treated macaques in our study had poor antiviral immune responses and relatively little to moderate CD4+ lymphocyte depletion and immune activation (despite the long-term high viremia), further research is needed to determine if both models share some immunologic mechanisms to stay healthy in the presence of high viremia.
Because all SIVmac251-infected infant macaques in the present studies developed the K65R mutation following tenofovir treatment, could this mutation by itself play a direct role in delaying the disease course, because it reduces viral virulence? The K65R mutation in HIV-1 and SIV is associated with a ≈5-fold reduced in vitro susceptibility to tenofovir. 7,59 The K65R mutation in HIV-1 reduces replication capacity in vitro. 60 In tenofovir-treated SIV-infected macaques, the emergence of K65R is rapidly followed by the accumulation of secondary mutations in RT, which are believed to be compensatory mutations that improve replicative fitness. 7,13 When such K65R mutant SIV populations were inoculated into macaques in the absence of tenofovir treatment, they were found to be fully virulent in the absence of reversion, and the virus levels and disease course (including survival) were indistinguishable from those of wild-type virus. 13,61 Thus, these available data from untreated animals demonstrate that the intrinsic properties of the K65R viral mutants are not sufficient to fully explain the prolonged survival in the tenofovir-treated animals.
Remarkably, animals with high viremia of K65R SIV mutants show only improved survival when treated with teno-fovir, 13,61 which suggests that tenofovir therapy results in immunologic effects that provide some protection against opportunistic infections and immunopathology. Although our study was not able to pinpoint the immunologic mechanisms, the available information suggests that such effects can be caused by antiviral activity of tenofovir against K65R viral mutants or direct immunomodulatory effects.
Tenofovir treatment may still exert some antiviral activity against K65R mutants. The high plasma RNA levels in these animals suggest that tenofovir probably had minimal effect to inhibit replication of K65R mutants in productively infected CD4+ T lymphocytes, which are believed to be the main sources of plasma viral RNA. 62 However, in vitro studies have demonstrated that tenofovir inhibits wild-type HIV-1 more efficiently in monocytes/macrophages, dendritic cells, and Langerhans cells than in lymphocytes, 63–67 and the same may be true for inhibition of K65R viral mutants. These antigen-presenting cells and their precursors play a crucial role in innate immunity as well as in the induction and maintenance of effective acquired immune responses. 68,69 Relative preservation of the function of these cells in tenofovir-treated animals despite high viremia could potentially still increase the resistance to opportunistic infections, reduce chronic immune stimulation, and prolong survival. 70 Preservation of innate immunity has also been associated with disease-free survival in some HIV-infected patients who have very low CD4+ T-cell counts. 71,72
Tenofovir may also have immunomodulatory effects that are independent of its antiviral effects. Immunomodulatory effects of tenofovir and related compounds have been demonstrated extensively in murine models in vitro and in vivo, including enhancement of NK cell activity, secretion of cytokines and chemokines, and increased in vivo resistance against infection with viruses that are not directly sensitive to these drugs in vitro. 73–77 Tenofovir did not induce a detectable increase in NK cell activity in rhesus macaque PBMCs in vitro or in vivo, but primed rhesus macaque PBMCs in vitro for enhanced interleukin-12 (IL-12) secretion following exposure to bacterial antigens. 77a Given the diverse role of IL-12 in strengthening innate immunity (including NK cells), stimulating IFN-γ/TH1 responses, reducing apoptosis, and limiting immunopathology (reviewed by Trinchieri 78,79 and Potvin et al 80) and the demonstrated efficacy of IL-12 in primate models, 81–83 further research is needed to confirm whether IL-12 plays a role in tenofovir’s efficacy in vivo in the SIV macaque model. The measurement of plasma IL-12 levels in tenofovir-treated animals, however, was found to be little informative due to high temporal and individual variability. 77a
In the current studies, prolonged survival was observed in a very virulent animal model (high viremia with highly virulent virus in infant macaques), which represents a worst case scenario. Such clinical benefits of tenofovir are likely to exist also at lower virus levels and less advanced immunosuppression, as suggested by our previous studies. 5,7,13,14,61 Thus, by analogy, also for the majority of HIV-infected patients who respond to tenofovir treatment with large or modest reductions in viremia and increases in CD4+ T lymphocytes, the clinical benefits on disease progression may be larger than predicted by these changes in laboratory markers.
The tenofovir-treated animals with high viremia, despite having improved survival, eventually still developed fatal immunodeficiency. Thus, the ultimate goal of antiviral therapy remains to suppress virus replication to levels as low as possible and restore CD4+ T-cell counts, because this provides maximal benefits on improving disease-free survival. From a practical point of view, however, a relatively poor virologic or immunologic response to tenofovir may not be a valid reason per se to withdraw tenofovir from the patient’s regimen, unless more effective salvage regimens, which are also feasible in terms of cost, toxicity, and compliance, are available. These findings are also relevant for resource-poor countries, where trials can determine whether tenofovir monotherapy or simplified tenofovir-containing regimens are practical and offer clinical benefits, especially when due to financial or other constraints, there is less opportunity for frequent monitoring of laboratory parameters (viral levels, CD4+ T-cell counts, viral drug resistance). Information from such studies will aid the development of better and affordable therapies to indefinitely delay disease progression for a growing number of HIV-infected people.
The authors thank D. Bennett, T. Dearman, L. Hirst, A. Spinner, W. von Morgenland, the Veterinary Staff, Colony Services, and Clinical Laboratory of the California National Primate Research Center, and T. Matthews and J. Murry of the Center for Comparative Medicine (University of California, Davis) for expert technical assistance. The authors thank C. Wingfield (Bayer Diagnostics, Berkeley, CA) for bDNA analysis. The authors thank S. Deeks (University of California, San Francisco), M. Miller (Gilead Sciences), A. Muthukumar (University of Texas Southwestern Medical Center), and G. Silvestri (Emory University, Atlanta, GA) for useful discussions and suggestions.
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