CD8+-Cell-Mediated Suppression of Virulent Simian Immunodeficiency Virus during Tenofovir Treatment

JOURNAL OF VIROLOGY, May 2004, p. 5324–5337 Vol. 78, No. 100022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.10.5324–5337.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

CD8�-Cell-Mediated Suppression of Virulent Simian ImmunodeficiencyVirus during Tenofovir Treatment

Koen K. A. Van Rompay,1* Raman P. Singh,1 Bapi Pahar,1 Donald L. Sodora,2 Casey Wingfield,3Jonathan R. Lawson,1 Marta L. Marthas,1 and Norbert Bischofberger4

California National Primate Research Center, University of California, Davis,1 Bayer Diagnostics Reference Testing Lab,Bayer Diagnostics, Berkeley,3 and Gilead Sciences, Foster City,4 California, and Department of

Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas2

Received 1 October 2003/Accepted 6 January 2004

The ability of tenofovir to suppress viremia in simian immunodeficiency virus (SIV)-infected macaques foryears despite the presence of virulent viral mutants with reduced in vitro susceptibility is unprecedented in thisanimal model. In vivo cell depletion experiments demonstrate that tenofovir’s ability to suppress viremiaduring acute and chronic infection is significantly dependent on the presence of CD8� lymphocytes. Contin-uous tenofovir treatment was required to maintain low viremia. Although it is unclear whether this immune-mediated suppression of viremia is linked to tenofovir’s direct antiviral efficacy or is due to independentimmunomodulatory effects, these studies prove the concept that antiviral immune responses can play a crucialrole in suppressing viremia during anti-human immunodeficiency virus drug therapy.

Although highly active antiretroviral therapy (HAART) reg-imens provide significant progress in the clinical managementof human immunodeficiency virus (HIV) infection, their long-term use is often limited by problems such as incomplete virussuppression and drug resistance. To alleviate this problem, it isbelieved we need to invoke the help of the immune system tohelp limit virus replication and/or the virus-induced immunedysfunction.

Immune responses, especially cell-mediated immune re-sponses (CD4�-T-helper cells and CD8� T lymphocytes) arethought to play an important role in controlling virus replica-tion and determining disease-free survival. Evidence for thiscomes from studies in untreated long-term nonprogressors (fora review, see reference 2). In addition, macaque data haveshown conclusively that CD8� lymphocytes play an importantrole in limiting lentivirus replication because in vivo CD8�-celldepletion of untreated macaques resulted in an increase inviremia (23, 34, 67).

A number of dilemmas exist, however, for patients receivingHAART. HAART initiated during acute HIV infection canoften preserve or increase antiviral immune responses, butepisodes of transient viremia may be required to maintainthese responses (54, 63). In contrast, when HAART is initiatedduring chronic HIV infection, HIV-specific immune responsesare more difficult to restore (for a review, see reference 2).

The goal of immunotherapeutic strategies is to augmentthese antiviral immune responses to prevent the emergenceand/or the replication of drug-resistant mutants during HAARTor to allow simplified regimens or periods without drug treat-ment. Although different approaches are investigated to stim-ulate antiviral immune responses (e.g., structured treatmentinterruptions, active immunizations, and cytokines), the results

have been poor or success has been limited mainly to patientswith acute infection with often transient results (2, 3, 25, 63).Progress in this area may depend on a better understanding ofthe contribution of antiviral immune responses to the reduc-tion of viremia during HAART and of the complex in vivointeractions of viral replication, drug resistance, immune re-sponses, and drug pharmacokinetics.

To gain further insights in the viral and host immune factorsthat determine successful therapy, animal models can be veryuseful because they allow controlled experimental approachesthat are not feasible in humans. Infection of macaques withsimian immunodeficiency virus (SIV) is a good animal modelfor such studies because it shares many similarities in diseasepathogenesis with AIDS and has also been used to study theefficacy of antiviral compounds, including the emergence, vir-ulence, and clinical implications of drug-resistant mutants. Thereverse transcriptase (RT) inhibitor tenofovir {9-[2-(phospho-nomethoxy)propyl]adenine} has been used extensively in thismacaque model of AIDS because of its unprecedented efficacy,compared to other compounds, to prevent infection or sup-press viremia (33, 55, 64, 69–71, 75–79, 80, 82, 85, 88). Mostother commonly used antiviral drugs that have been tested inthe macaque model were usually able to delay or reduce thepeak of primary viremia if given early during infection, but incontrast to tenofovir, these other drugs were not very efficientin suppressing viremia once virus dissemination was alreadywell established or the emergence of drug-resistant viral mu-tants inevitably led to increased virus replication and disease(22, 30, 35, 47, 70, 84, 87, 90, 102).

The reasons for this high efficacy of tenofovir in the macaquemodel have been unclear but warrant further investigation, asthis could lead to the development of additional antiviral strat-egies. The experiments described here demonstrate that sup-pression of viremia during tenofovir treatment of SIV-infectedmacaques requires CD8�-cell-mediated antiviral immune re-sponses. Our data provide proof of concept for a model ofantiviral drug therapy in which the combined forces of antiviral

* Corresponding author. Mailing address: California National Pri-mate Research Center, University of California, Davis, CA 95616.Phone: (530) 752-5281. Fax: (530) 754-4411. E-mail: [email protected].

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immune responses and antiviral drugs are the key to success,especially in the presence of drug-resistant viral mutants. Theavailable evidence suggests that the reason for tenofovir’s suc-cess in this animal model is because tenofovir treatment allowsthe host to mount strong CD8�-cell-mediated immune re-sponses that complement tenofovir’s antiviral effects.

MATERIALS AND METHODS

Animals. All rhesus macaques (Macaca mulatta) were from the type D-retro-virus-free and SIV-free colony at the California National Primate ResearchCenter (CNPRC). The newborn and infant macaques were hand reared in aprimate nursery and were housed in accordance with American Association forAccreditation of Laboratory Animal Care standards. We adhered to the stan-dards outlined in the Guide for Care and Use of Laboratory Animals (50). Forblood collections, animals were immobilized with 10 mg of intramuscular ket-amine-HCl (Parke-Davis, Morris Plains, N.J.) /kg of body weight.

Virus inoculation. Animals 29045 and 29003 were inoculated orally with wild-type uncloned SIVmac251 at birth and animal 29276 was inoculated at birthintravenously with the K65R mutant isolate SIVmac385; these animals werestarted on tenofovir treatment 3 weeks later, as described previously (80, 81). Asindicated in Table 1, all other animals were juvenile animals (12 to 17 months ofage) at the time of virus inoculation; they were inoculated orally twice (on 2consecutive days) with 1 ml of uncloned SIVmac251 (with internal referencenumber 5/98). This virus was grown on rhesus peripheral blood mononuclearcells (PBMC), had a titer of 105 50% tissue culture infectious doses and 1.4 � 109

RNA copies/ml (as measured by SIV branched DNA [bDNA] assay), and waspathogenic for infant and adult macaques (43, 83). Our SIVmac251 isolates arevery difficult to control in vivo. Of the more than 180 rhesus macaques (including54 previously vaccinated animals) that became persistently infected after inocu-lation with these SIVmac251 virus stocks over the past decade at CNPRC, nonesuppressed viremia to persistently low or undetectable levels (19, 43, 65, 79, 80,82, 83, 85, 87, 88, 90–92).

Preparation and administration of tenofovir. Tenofovir (Gilead Sciences) wassuspended in distilled water, dissolved by the addition of NaOH to a final pH of7.0 at 60 mg/ml, filter sterilized (0.2-�m pore size; Nalgene), and stored at 4°C.Tenofovir was administered subcutaneously into the back of the animal. Thedosage was adjusted weekly based on weight. Tenofovir was initially given ac-cording to a once-daily regimen of 30 mg/kg of body weight, with dosage reduc-tions described in Table 1.

Administration of cM-T807. CD8� cells were depleted by using the previouslydescribed cM-T807 antibody (67, 68); a total of 20 mg/kg of body weight was

administered in 3 doses: 10 mg/kg subcutaneously on day 0 and 5 mg/kg intra-venously 3 and 7 days later. The specific role of CD8� cells on viremia of SIV andother viral infections has been well demonstrated with this cM-T807 antibody(34, 57, 67). CD8�-cell depletion during acute SIV viremia did not affect peaklevels of viremia but inhibited the subsequent reduction in viremia (67). Becauseit is theoretically possible that administration of cM-T807 could induce virusreplication through immune activation, others have previously performed controlexperiments and found that administration of a nondepleting control antibody ora B-cell-depleting antibody had no effect on SIV viremia (34, 57, 59, 66, 67, 73).In addition, the CD8�-cell depletion does not affect CD4�-T-cell counts. Thus,the increase of viremia following cM-T807 antibody administration and thereturn of viremia to baseline levels which coincides temporally with the repopu-lation of CD8� cells can be considered the best proof of CD8�-cell-mediatedsuppression of virus replication. In our studies, no allergic reactions occurred andno lymphadenopathy or splenomegaly was observed as a result of the cM-T807administration.

Quantitation of plasma viral RNA. Viral RNA in plasma was quantified by abDNA signal amplification assay specific for SIV, versions 2.0, 3.0, and 4.0, whichhave lower quantitation limits of 1,500, 500 and 125 copies per ml, respectively(P. J. Dailey, M. Zamroud, R. Kelso, J. Kolberg, and M. Urdea, Abstr. 13th Ann.Symp. Nonhum. Primate Models AIDS, abstr. 99, 1995; J. Booth, L. Sawyer, E.McNelley, D. Tayama, C. Wingfield, D. Cox, and K. Leung, Abstr. 18th Ann.Symp. Nonhum. Primate Models AIDS, abstr. 129, 2000; C. Wingfield, J. Booth,P. Sheridan, J. Detmer, and J. Turczyn, Abstr. 20th Ann. Symp. Nonhum. Pri-mate Models AIDS, abstr. 135, 2002). These assays are similar to the QuantiplexHIV RNA assay, except that target probes were designed to hybridize with thepol region of the SIVmac group of strains including SIVmac251 and SIVmac239.

Virus isolation. Infectious virus was isolated in cultures of PBMC withCEMx174 cells and subsequent p27 core antigen measurement, according tomethods previously described (86). Levels of infectious virus in PBMC weredetermined by a limiting dilution assay (86).

Drug susceptibility assays. Phenotypic drug susceptibilities of SIVmac isolateswere characterized by a previously described assay based on a dose-dependentreduction of viral infectivity. This assay is able to detect SIV mutants withdecreased susceptibility to several antiviral drugs (80, 90).

Sequence analysis of SIV RT-encoding region. Because many plasma sampleshad insufficient viral RNA to allow sequencing, proviral DNA obtained fromCEMx174 infected with PBMC isolates was used. In a previous study, thismethod gave sequence results that were similar to those of plasma viral RNA(49). Infected CEMx174 and PBMC cocultures were harvested as soon as culturesupernatants were positive by antigen-capture enzyme-linked immunosorbentassay. For the DNA sequence analyses of codons 0 to 320 of RT, genomic DNAwas extracted and used for nested PCR according to methods and with primers

TABLE 1. Summary of tenofovir-treated SIV-infected macaques used for CD8�-cell depletion experiments

Animalno.

Presence ofMHC I allelea: Age (mo) at virus

infectionbStart of tenofovirc

(wk after SIV infection)CD8�-cell depletiond

(time after SIV infection)Temporary tenofovir

interruptione

MamuA*01 MamuB*01

29045 � � 0 (birth) 3 7 yr None29276 � � 0 (birth) 3 6 yr None32137 � � 17 2 39 wk None32186 � � 17 2 39 wk Wk 64–7132993 � � 12 2 2 wk None33088 � �/� 12 2 2 wk Wk 32–3933091 �/� � 12 2 2 wk Wk 32–39

a The presence of the MHC type I alleles MamuA*01 and MamuB*01 is indicated as � (present but unknown whether homozygous or heterozygous), �/�(homozygous for the presence of the alleles), �/� (heterozygous based on known haplotypes of parents), and � (homozygous for the absence of the particular allele).The frequency of the MamuA*01 and Mamu B*01 alleles in the CNPRC rhesus macaque colony is approximately 25%.

b All animals were inoculated orally with wild-type SIVmac251, with the exception of animal 29276, which was inoculated with a K65R SIV isolate (SIV mac385)derived from tenofovir-treated SIVmac251-infected animals, as described previously (80, 81). K65R mutants became dominant in animal 29045 at 14 weeks of age (80).Genotypic drug resistance data are summarized in Table 4.

c Tenofovir was initially given to all of these animals at a once-daily regimen of 30 mg/kg of body weight, administered subcutaneously. Tenofovir regimens for animals29045 and 29276 had been reduced over time to a low maintenance regimen of 2.5 mg/kg at the time of the CD8�-cell depletion (79a). The tenofovir regimen for theother animals listed in this table was reduced from 30 to 20 and then to 10 mg/kg after 10 and 14 weeks of treatment, respectively. Animal 32186’s dose was reducedfurther to 5 and then to 2.5 mg/kg at weeks 30 and 51, respectively. Thus, at the time of the CD8�-cell depletion, the 4 long-term-treated animals (29045, 29276, 32137,and 32186) (Fig. 2) had been on a stable tenofovir monotherapy maintenance regimen of 2.5, 2.5, 10, or 5 mg/kg (once daily, subcutaneously) with no changes in dosagefor 7, 9, 5, or 2 months, respectively.

d Animals 32137 and 32186 were first used as undepleted tenofovir-treated animals for comparison with the CD8�-cell-depleted tenofovir-treated animals (32993,33088, and 33091) for the acute viremia experiment (Fig. 1). Animals 32137 and 32186 were subsequently depleted of CD8� cells during chronic infection (Fig. 2).

e Tenofovir treatment was interrupted for a 7-week period for 3 animals at the indicated times after infection (Fig. 3) and then restarted at the same dosage regimen.At that time, the maintenance dosages were 10 mg/kg for animals 33088 and 33091 and 2.5 mg/kg for animal 32186 (subcutaneously, once daily).

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described previously (87). Each round of the nested PCR was carried out underthe following conditions: samples were incubated at 94°C for 45 s; followed by 30cycles at 94°C for 1 min, 57°C for 40 s, 72°C for 120 s; and followed by anextension at 72°C for 5 min. Amplicons were sequenced by Davis Sequencing,Davis, Calif., with primers 239-2786 and SIV-RT3 (87). Data were compared topublished sequences of SIVmac251 and SIVmac239 (accession numbers M19499and M33262, respectively). This method can detect the presence of a 20%subpopulation. Thus, if only K65R was detected, this means that �80% of theproviral DNA population had this mutation.

Lymphocyte phenotyping. Up until 1997, two-color flow cytometry was used asdescribed previously to detect CD4� and CD8� cells (80). From 1997 to 2001,three-color flow cytometry techniques were used to detect CD3, CD4, CD8, andCD20 with fluorochrome-conjugated antibodies described previously (82, 87)Starting in 2002, four-color flow cytometry was used, consisting of a single tubecontaining peridinin chlorophyll protein (PerCP)-conjugated anti-human CD8(clone SK1; Becton Dickinson Immunocytometry Inc., San Jose, Calif.), fluores-cein isothiocyanate-conjugated anti-human CD3 (clone SP34; Pharmingen), phy-coerythrin-conjugated anti-human CD4 (clone M-T477; Pharmingen), and allo-phycocyanin-conjugated anti-human CD20 (clone L27; Becton Dickinson). Redblood cells were lysed, and the samples were fixed in paraformaldehyde by theCoulter Q-prep system (Coulter Corporation, Hialeah, Fla.). Flow cytometry wasperformed on a FACSCalibur flow cytometer (Becton Dickinson). Lymphocyteswere gated by forward and side light scatter and were then analyzed withCellquest software (Becton Dickinson). CD4� T lymphocytes and CD8� Tlymphocytes were defined as CD3�CD4� and CD3�CD8� lymphocyte popula-tions, respectively. B lymphocytes were defined as CD3�CD20� lymphocytes.NK cells were defined as CD3�CD8� lymphocytes. For the CD8�-cell-depletionexperiment, as suggested in the cM-T807 protocol (67), the anti-CD8 antibodywas replaced by the DK25 clone (DAKO, Carpinteria, Calif.) conjugated tofluorescein isothiocyanate (and combined with anti-CD3-PerCP, anti-CD4-phy-coerythrin, and anti-CD20-allophycocyanin).

ELISPOT assay for SIV-specific IFN-�-secreting cells. The number of anti-gen-specific gamma interferon (IFN-�)-producing cells in rhesus monkeys wasmeasured with an enzyme-linked immunospot (ELISPOT) assay. This assay wasperformed as previously described (83), with the exception that a pool of 15-merpeptides with 10 amino acids overlapping of the entire p24 gag region ofSIVmac239 was used (56). Results were considered positive if the number ofspot-forming cells (SFC) for 2 � 105 cells was �10 per well and greater than theaverage of the negative-control (medium only) wells plus 2 standard deviations.

Genetic assessment of MHC class I alleles. DNA extracted from lymphoidcells (with QIAamp DNA mini kit; Qiagen, Valencia, Calif.) was used to screenfor the presence of the major histocompatibility complex (MHC) class I allelesMamuA*01 and MamuB*01 by a PCR-based technique (15, 28). The frequencyof MamuA*01 and Mamu B*01 alleles in the CNPRC rhesus macaque colony isapproximately 25%.

Statistical analysis. Statistical analyses were performed with Prism 3 andInstat 3 (GraphPad Software, Inc., San Diego, Calif.).

RESULTS

Role of CD8�-cell-mediated immune responses during sup-pression of acute viremia by tenofovir. The acute stage ofinfection consists of a fierce battle between the replicatingvirus and emerging antiviral immune responses, and relativelysmall disturbances can have a great impact. Cell depletionexperiments demonstrated previously that the reduction ofacute SIV viremia is determined mainly by CD8�-cell-medi-ated immune responses instead of humoral immunity (66, 67).

To test the hypothesis of whether tenofovir alone is sufficientto suppress acute viremia, we designed an experiment in whichCD8� cells were depleted simultaneously with the start oftenofovir treatment. The week-2 time point of infection wasselected for these interventions because this is approximatelythe time (i) of peak viremia, (ii) when antiviral CD8�-cell-mediated responses can be detected in blood and mucosaltissues of SIV-infected macaques (29, 60, 66, 93, 98), and (iii)when the number and activity of NK cells in the blood havebeen reported to be high (18). Others have already demon-strated that CD8�-cell depletion of untreated animals during

acute SIV viremia did not affect peak levels of viremia butinhibited the subsequent reduction in viremia so that viremiaremained relatively unchanged (67). In our experiments, 11juvenile macaques (�1 to 2 years of age) were divided intothree groups and infected orally with highly virulent wild-typeSIVmac251. At 1 and 2 weeks after infection, virus levels inplasma were not different among the 3 groups (P � 0.4 byanalysis of variance) (Fig. 1; Tables 2 and 3), and virus hadwild-type susceptibility to tenofovir. Six animals remained un-treated and maintained high viremia (virus set point � 6 to 7log RNA copies per ml), confirming the high virulence of thisSIVmac251 stock (Fig. 1). The other 5 animals were started ontenofovir treatment (30 mg/kg subcutaneously once daily) 2weeks after infection; 3 of these 5 animals were simultaneouslydepleted of their CD8� lymphocytes through administration ofthe well-characterized monoclonal antibody cM-T807 (67).

In contrast to the untreated animals, the SIVmac251-infected animals that received tenofovir had a 12- to 39-folddecrease in viral RNA levels after 1 week of tenofovir treat-ment (P � 0.05) (Fig. 1; Tables 2 and 3), indicating a half-lifeof productively infected cells of �1.3 to 2 days. The magnitudeof this reduction of viremia in the tenofovir-treated animals isconsistent with many previous observations of a rapid reduc-tion of viral RNA and infectious titers after the start of teno-fovir treatment (�20- to 40-fold reduction in 1 week; half-life,�1.3 0.2 days) (52, 76, 80, 82). In contrast, the three CD8�-cell-depleted animals (which had no detectable CD8� cells inthe blood at days 3 and 7 after the first injection of cM-T807)had only a two- to threefold decrease in viral RNA levels after1 week of tenofovir treatment (Fig. 1), indicating that in theabsence of CD8� cells, productively infected cells have a half-life of 4 to 6 days. Thus, for these CD8�-cell-depleted tenofo-vir-treated animals, the change in viral RNA between weeks 2and 3 of infection was significantly different from those of thetenofovir-treated undepleted animals (P � 0.01) but indistin-guishable from those of the untreated SIVmac251-infectedanimals (Tables 2 and 3). In these tenofovir-treated CD8�-cell-depleted animals, by week 4 of infection (i.e., 2 weeks afterthe start of tenofovir treatment and CD8�-cell depletion),CD8� cells had become detectable again and this return ofCD8� cells was associated with a 10- to 30-fold decrease inviral RNA between weeks 3 and 4 (indicating a half-life ofproductively infected cells of 1.4 to 2 days). This sudden de-crease in virus levels was significantly different from the rela-tively stable viremia of the untreated SIVmac251-infected an-imals (P � 0.01) but was indistinguishable from the declinerate of the tenofovir-treated undepleted animals (Fig. 1; Ta-bles 2 and 3). The CD4�-T-cell counts did not differ signifi-cantly among the different groups.

Virus isolated from PBMC of the tenofovir-treated unde-pleted animals at 4 weeks of infection (i.e., 2 weeks after thestart of tenofovir treatment) was wild type, but by 6 weeks ofinfection, viral mutants with �5-fold-reduced in vitro suscep-tibility to tenofovir and a lysine-to-arginine mutation at codon65 in RT (K65R) were the only detectable virus populations(Table 4). This rapid emergence of K65R mutants after only 4weeks of treatment demonstrates that tenofovir is a highlyeffective inhibitor of reverse transcription of wild-type SIV andexerts strong selection pressure for the K65R mutation (51).Despite the initial immunological disturbance induced by the

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CD8�-cell depletion and the emergence of K65R mutants, twoof the three tenofovir-treated depleted animals obtained un-detectable viremia by 32 weeks after infection, similar to one ofthe tenofovir-treated undepleted animals. The two remainingtenofovir-treated animals had a virus set point of �104 to105 RNA copies per ml of plasma but were clinically healthy

at �19 months of infection. In contrast, the 6 untreatedSIVmac251-infected animals maintained high viremia (�106 to107 copies/ml) and 5 animals had to be euthanized due tosevere immunodeficiency at 14 to 36 weeks after infection. Allof the tenofovir-treated animals had detectable levels of SIV-specific IFN-�-producing cells in the blood, although no cor-

FIG. 1. Effect of CD8�-cell depletion on efficacy of tenofovir during acute SIV viremia. Eleven animals were inoculated orally with wild-typeSIVmac251. Six animals were untreated, and 5 animals were started on tenofovir treatment at 2 weeks of infection (arrow). Three of thesetenofovir-treated animals were simultaneously depleted of CD8� cells by 3 injections of cM-T807 (on days 14, 17, and 21 after SIVmac251infection). Data presented are averages ( standard errors of the means) for each group (left side) and individual values (right side). (A and B)Viral RNA levels; (C and D) CD8�CD3� T lymphocytes; (E and F) absolute CD8� CD3� NK cell counts. The CD8�-cell depletion had no effecton CD4�-T-lymphocyte counts (data not shown). Five of the 6 untreated SIVmac251-infected animals had to be euthanized at 14, 22, 25, 32, and36 weeks. In panel B, the first time points at which virus isolated from PBMC predominantly had the K65R mutation in RT are indicated bysquares. More-detailed RT sequence data are given in Table 4.


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relation was found between their absolute level and the plasmavirus RNA set point (data not shown). In summary, this CD8�-cell depletion experiment demonstrated that tenofovir’s effi-cacy to suppress acute viremia of wild-type virus was signifi-cantly reduced in the absence of CD8� cells.

Long-term suppression of viremia in tenofovir-treated SIV-infected macaques. In the experiment described above and inpreviously published studies, when macaques were inoculatedwith the highly virulent SIVmac251 isolate, initiation of teno-fovir treatment near the time of peak viremia rapidly sup-pressed viremia (80). Prolonged treatment led to the emer-gence of viral mutants with a K65R mutation in RT (followedby other, presumably compensatory, mutations). Remarkably,however, the emergence of these K65R mutants was for mostanimals not associated with an increase in viremia (80). Ap-proximately half of the animals showed a progressive decline ofviral RNA levels in plasma to undetectable levels (�125 to 500RNA copies/ml) after �6 to 12 months of continued tenofovirtreatment. A similar gradual decline in viremia was also ob-served in an animal inoculated at birth with a virulent K65Rmutant and started on tenofovir treatment 3 weeks later: viruslevels, initially high (�6 log RNA copies/ml), declined gradu-ally to undetectable levels after approximately 4 years of teno-fovir treatment (Fig. 2, animal 29276). For these animals, viralRNA levels remained undetectable, with the exception of anoccasional transient episode of low-level viremia (125 to 600RNA copies/ml), throughout the observation period (up to 7years), and the frequency of infected PBMC (as determined byvirus isolation) decreased to less than 1 infected cell per 107 to�108 PBMC. Immune activation through booster immuniza-tions of these animals with tetanus toxoid did not result indetectable increases in viremia. This tenofovir-induced long-term suppression of viremia was observed in animals regard-less of the presence of MamuA*01 and Mamu B*01 alleles.

Those tenofovir-treated animals with low or undetectableviremia had detectable cell-mediated immune responses, as

measured by gag-specific IFN-� ELISPOT assay (Table 5).Thus, we decided to perform 2 sets of experiments to detectwhether the low viremia in these tenofovir-treated animals wasdue to CD8�-cell-mediated antiviral immune responses (bydepleting CD8� cells) and/or direct suppression of K65R virusreplication by tenofovir (by removing tenofovir).

CD8�-cell-mediated suppression of viremia during pro-longed tenofovir treatment. To determine the contribution ofcell-mediated immune responses to the low viremia, 4 tenofo-vir-treated animals with chronic SIV infection were depleted ofCD8� cells (Fig. 2). All 4 animals had been started on teno-fovir treatment 2 to 3 weeks after virus inoculation, had K65RSIV mutants, and had been on continuous tenofovir treatmentfor 9 months (2 animals) to 6 to 7 years (2 animals) (Table 1).Three of these animals had undetectable viremia (�125 RNAcopies/ml of plasma), and one had moderate viremia (animal32137; �29,000 copies RNA/ml of plasma). In all 4 animals,CD8� cells were undetectable in peripheral blood within 3days after the first cM-T807 antibody dose (Fig. 2). Viral RNAlevels increased rapidly and reached peak levels 3 days to 3weeks after the first cM-T807 dose. This increase in viral RNAlevels in plasma was accompanied by an increase in infectioustiter in PBMC (Fig. 2). The increase in viremia was highest(�3,000- to 5,000-fold) for the two oldest animals, which hadbeen on tenofovir for 6 to 7 years and had undetectable viralRNA levels for �20 months (Fig. 2, animals 29045 and 29276).The increase in viral RNA was relatively smaller (�20- to210-fold) for the �2-year-old animals, which had only beeninfected and on tenofovir treatment for 9 months (Fig. 2,animals 32137 and 32186). Virus isolated during this resurgingviremia had the expected genotypic and phenotypic resistanceto tenofovir that had previously been detected in these animals(Table 4). Because a stable tenofovir treatment regimen wascontinued throughout the CD8�-cell depletion experiment,this sudden increase in viremia demonstrates that K65R SIVmutants can replicate well in the presence of tenofovir. Vire-

TABLE 2. Effect of CD8�-cell depletion on ability of tenofovir to suppress acute viremiaa

Animal group (n),treatment

Mean (SD) log-transformed viral RNA copies/mlof plasma at wk (after virus inoculation):

Mean (SD) difference in log-transformed viral RNA copies/ml of plasma between wk (after virus inoculation):

2 3 4 3 and 2 4 and 3 4 and 2

A (2), tenofovir 7.39 (0.14) 6.06 (0.23) 4.90 (0.91) �1.34 (0.37) �1.16 (0.68) �2.50 (1.05)B (3), tenofovir, anti-CD8 7.38 (0.42) 6.98 (0.35) 5.75 (0.39) �0.40 (0.10) �1.23 (0.26) �1.63 (0.20)C (6), none 7.56 (0.35) 6.88 (0.38) 6.72 (0.61) �0.68 (0.29) �0.15 (0.38) �0.83 (0.36)

a All animals were inoculated with SIVmac251. Tenofovir treatment and CD8�-cell depletion were started 2 weeks after infection (Fig. 1).

TABLE 3. Statistical comparison of viral RNA levels of the three animal groups of Table 2a

Test Groups

P value at wk: P value for difference between wk:

2 3 4 3 and2 4 and 3 4 and 2

One-way ANOVA A, B, C 0.72 0.045 0.015 0.015 0.009 0.007Posttest A, B ND �0.05 �0.05 �0.01 �0.05 �0.05

A, C ND �0.05 �0.05 �0.05 �0.05 �0.01B, C ND �0.05 �0.05b �0.05 �0.01 �0.05

a The posttest comparison was the Bonferroni test for selected pairs. Groups are defined based on treatment and are as given in Table 2. ANOVA, analysis ofvariance; ND, not done.

b The P value was �0.05 with the two-tailed unpaired t test.


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mia was reduced to baseline values as soon as the CD8� Tlymphocytes and NK cells became detectable again. The steep-est interval of the viral RNA decay curve (Fig. 2) suggests ahalf-life of productively infected cells of �1 day, similar to theshortest half-life reported for cytotoxic-T-lymphocyte (CTL)-mediated clearance of acute viremia in untreated macaques(72). Following the return of CD8� cells, the number of SIV-specific IFN-�-producing PBMC (by ELISPOT assay) in-creased again to near baseline values (i.e., prior to the CD8�

cell depletion) (Table 5). In summary, these CD8�-cell deple-tion studies in chronically infected animals indicate that thesustained suppression of K65R SIV replication during pro-

longed tenofovir treatment required CD8�-cell-mediated im-mune responses, and tenofovir alone was not sufficient.

Continued tenofovir treatment is required to sustain lowviremia. Because the long-term efficacy of tenofovir to sup-press viremia is determined by the induction of strong antiviralimmune responses, an important question is whether contin-ued tenofovir treatment is required to maintain low viremiaonce these antiviral immune responses exist. Four SIVmac251-infected macaques, including three animals from the CD8�-cell depletion experiment discussed above, had K65R virus andeventually reached undetectable viremia (�125 RNA copies/ml). When treatment was interrupted, a slow increase in vire-

TABLE 4. Mutations in RT of SIV isolates from tenofovir-treated macaquesa

Animal no. Time point Mutation(s) in RT sequence

29045 9 mo P-SIV K65R, N69S, I118V3 yr P-SIV K65R, N69S, I118V7 yr P-SIV, day 35 P-CD8 K65R, N69S, I118V.

29276 9 mo P-SIV K64R, K65R, N69S, Y115F, I118V, F171Y, S211N4 yr P-SIV K65R, N69S, S211N6 yr P-SIV; wk 1, 3, and 6 P-CD8 K65R, N69S, S211N

32137 2 and 4 wk P-SIV WT6 wk P-SIV K65R8 wk P-SIV K65R, S211N12 wk P-SIV K65R, I118V, S211N28 wk P-SIV K65R, N69S, I118V, S211N40 wk P-SIV, 1 wk P-CD8 K65R, N69S, I118V, S211N55 wk P-SIV K64R, K65R, N69S, I118V, I145V, S211N

32186 2 and 4 wk P-SIV WT6 wk P-SIVI K65R12 wk P-SIV K65R, I118V28 wk P-SIV K65R, N69S, I118V40 wk P-SIV, 1 wk P-CD8 K65R, N69S, I118V64 wk P-SIV L21V, K65R, N69S, I118V71 wk P-SIV, 7 wk P-TI L21V, K65R, N69S, I118V

32993 2 and 4 wk P-SIV WT8 wk P-SIV T88A10 wk P-SIV K65R, R82K12 wk P-SIV K65R, N69S, S211N32 wk P-SIV K65R, N69S, I118V, S211N43 wk P-SIV K65R, N69S, R82G, Y115F, S211N

33088 2 wk P-SIV WT4 wk P-SIV S211N8 wk P-SIV K65R, S211N10 and 12 wk P-SIV K65R35 wk P-SIV, 3 wk P-TI K65R, N69S, R82K, S211N39 wk P-SIV, 7 wk P-TI K65R, N69S42 wk P-SIV K65R, N69S

33091 2, 4, and 8 wk P-SIV WT12 wk P-SIV K65R32 wk P-SIV K65R, N69S39 wk P-SIV, 7 wk P-TI K65R, N69S, S211N43 wk P-SIV K65R, N69S, I118V, S211N

a DNA from CEMx174 cells infected with SIV isolated from PBMC of SIV-infected animals was used for sequence analysis of RT (codons 1 to 320). The RT aminoacid sequence was compared with that of our uncloned SIVmac251 isolates, which is similar to that of the molecular clone SIVmac251, with the exception that at codon11 alanine is found instead of threonine. For each animal, selected isolates with the wild-type and K65R genotypes were tested for phenotypic drug susceptibility andfound to have wild-type and approximately 5-fold-reduced susceptibility to tenofovir, respectively. RT sequences of early time points for animals 29045 and 29276 havebeen reported previously (80, 81). For more details on the timing and virus levels during the CD8�-cell depletion experiments and the 7-week tenofovir treatmentinterruption experiment, see Table 1 and Fig. 1, 2, and 3. Although we did not sequence viral RNA in plasma, another study found that when SIVmac251-infectedjuvenile macaques (inoculated with the same SIVmac251 stock as used here) were started on tenofovir treatment (30 mg/kg per day, subcutaneously), the K65Rmutation could often be detected in viral RNA in plasma after 1 week of treatment by using a high-sensivity heteroduplex tracking assay; at 2 weeks of treatment, allviral RNA could already have the K65R mutation (40). Abbreviations: WT, wild type; P-SIV, post SIV infection; P-CD8, after the start of the CD8�-cell depletionexperiment; P-TI, after tenofovir treatment interruption.


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FIG. 2. CD8�-cell depletion during chronic tenofovir treatment. Four animals with chronic SIV infection and prolonged tenofovir treatment(started 2 or 3 weeks after infection) (Table 1) were depleted of CD8� cells through administration of the cM-T807 antibody (3 doses). All animalshad developed K65R SIV mutants. No viral RNA data were available for animal 29045 for the first 6 weeks of infection, but infectious PBMC titerswere available for the whole period. At the time of the CD8�-cell depletion, these 4 long-term-treated animals were on a stable tenofovirmonotherapy maintenance regimen. Please note the discontinuous x axes. Abbreviations: TCID50, 50% tissue culture infective dose; m, months;y, years.


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mia was observed. Upon reinitiation of tenofovir treatment,viral RNA levels returned to undetectable levels (Fig. 3). Theabsence of a rapid rebound of viremia following tenofovirwithdrawal suggests that effective antiviral immune responsesinitially limited virus replication in vivo, but continued tenofo-vir treatment was required to maintain optimal suppression ofK65R SIV viremia.

DISCUSSION

While currently available models of viral kinetics duringHAART focus mainly on direct inhibition of virus replicationby the antiviral drugs, the contribution of antiviral immuneresponses to the success of HAART is less understood. Ourdata support viewing antiviral drug therapy and immunologyfrom a perspective in which there is synergistic interaction.Thus, we propose that models that describe viral kinetics dur-ing drug therapy must incorporate an important variable,namely the strength of antiviral immune responses. In otherwords, antiviral drugs require immune responses to reach fulleffectiveness. This way of thinking may seem provocative atfirst, but it helps to explain why tenofovir has been more effectivethan many other antiviral drugs in the SIV-macaque model.

Tenofovir has potent direct antiviral effects against SIV invivo, as demonstrated by the rapid selection for the K65Rmutation in RT. The controlled animal experiments describedhere provide further insights into the role of immunologicalevents during successful tenofovir therapy. In particular,CD8�-cell-mediated antiviral immune responses were impor-tant in reducing viremia (i) of wild-type virus at the start oftenofovir therapy and (ii) of K65R mutants during prolongedtenofovir treatment. In other words, the ability of tenofovir tosuppress viremia relied on the presence of antiviral CD8�-cell-mediated immune responses. Both these antiviral effects ofCD8� cells and tenofovir treatment complement each otherand are required, as neither tenofovir nor CD8�-cell-mediatedimmune responses alone were sufficient to induce and main-tain low or undetectable viremia. As predicted by mathemati-cal models (11), strong antiviral immune responses can lowerthe viral burst and reduce the basic reproductive ratio, so thateven in the presence of drug-resistant mutants, a low virus setpoint can be achieved.

Tenofovir’s efficacy in suppressing acute viremia of wild-typevirus was reduced significantly in the absence of CD8� lym-phocytes (P � 0.01). This observation is consistent with amathematical model that proposed that the initial phase ofrapid viral RNA decline (with a half-life of 1 to 2 days) fol-lowing initiation of HAART in HIV-infected individuals maybe due to immune-mediated killing or inhibition of produc-tively infected cells (e.g., CTL, apoptosis, and noncytotoxicinhibition) (7). In the absence of immune-mediated killing orinhibition of productively infected cells, the decline of viralRNA induced by a completely effective drug regimen wouldrepresent the natural death rate of productively infected cells(as determined by the cytopathogenicity of the virus) (7). Al-though some evidence suggested a role of antiviral immuneresponses in the virological response to HAART, other studiesdid not confirm this (37, 58, 61). This question has been diffi-cult to answer conclusively in human studies due to manyuncontrollable variables (12, 53). In contrast, the SIV-macaquemodel allows us to better distinguish whether the decay ofproductively infected cells after initiation of drug therapy isdue to host immune responses or to viral cytopathogenicity. Inour animal study, we controlled many variables (the virulenceof the virus strain and the timing of drug treatment) and weperformed the experiment that was proposed by mathemati-cians to answer this question (7): we used a very drastic ap-proach (in vivo CD8�-cell depletion), which is not feasible inhumans, to maximally eliminate CD8�-cell-mediated immuneresponses. The very slow decline in viral RNA levels in theCD8�-cell-depleted SIV-infected macaques during the firstweek of tenofovir treatment suggests that, in the absence ofCD8� cells, productively infected cells during acute viremiahad a longer half-life (�4 to 6 days), suggesting that virulentSIVmac251, during concomitant tenofovir treatment, may notbe as cytopathic as expected.

The initial rapid decline of SIV RNA levels in plasma and ofinfectious titers of PBMC (shown in previous studies) (80, 82)with a half-life of 1 to 2 days, which is generally observed afterthe start of tenofovir treatment, suggests that during tenofovirtreatment strong CD8�-cell-mediated antiviral immune re-sponses rapidly destroy or inhibit productively infected cells. Amodel to fit these data is proposed in Fig. 4. In contrast, the

TABLE 5. IFN-� ELISPOT data in tenofovir-treated animals withchronic SIV infection undergoing CD8�-cell depletiona

Animal no. Time point Tissue SFC/million cells

29045 3 mo pre-CD8 PBMC 2902 mo pre-CD8 PBMC 3501 wk post-CD8 PBMC �505.5 wk post-CD8 PBMC �50

Axillary lymph node 145

29276 3 mo pre-CD8 PBMC 1401 mo pre-CD8 PBMC 803 wk post-CD8 PBMC 558 wk post-CD8 PBMC 6510 wk post-CD8 PBMC 385

32186 2 mo pre-CD8 PBMC 601 mo pre-CD8 PBMC 1751 wk post-CD8 PBMC �503 wk post-CD8 PBMC 6512 wk post-CD8 PBMC 180

32137 1 mo pre-CD8 PBMC 801 wk post-CD8 PBMC �503 wk post-CD8 PBMC 5712 wk post-CD8 PBMC 210

a Data presented in this table are from the 4 tenofovir-treated macaquespresented in Fig. 2, which had been chronically infected but had low or unde-tectable viremia at the time of the CD8�-cell depletion experiment. Enumera-tion of virus-specific IFN-�-producing cells was performed by ELISPOT assay.The cutoff value was 50 SFC per million cells. Prior to the in vivo CD8�-celldepletion experiment, PBMC collected from these animals were also enrichedfor CD4� and CD8� cells in vitro (by negative selection with anti-CD8 andanti-CD4 Dynabeads). It was found that for animals 29276 and 32186, theIFN-�-producing activity was approximately equally divided among CD4� andCD8� enriched populations, whereas for animals 29045 and 32137, the IFN-�production resided almost exclusively in the CD8�-cell population. Similar re-sults were obtained by intracellular IFN-� flow cytometry assays (56). For animal29045, IFN-�-producing cells were detected readily in lymphocytes derived froman axillary lymph node at a time when no SFC could be detected in PBMC, sug-gesting that PBMC data may not reflect the antiviral activity that occurs in lym-phoid tissues and that SIV-specific IFN-�-producing antiviral CD8� cells mayaccumulate in lymphoid tissues rather than in PBMC (similar to observationswith humans) (4). This finding explains why the timing of reappearance of IFN-�-producing cells in PBMC did not necessarily coincide exactly with the clear-ance of viremia (Fig. 2). pre- and post-CD8, before and after, respectively, theadministration of the first dose of the CD8�-cell-depleting cM-T807 antibody.


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inability of most other antiviral drugs to rapidly suppress es-tablished viremia of highly virulent virus in this animal modelsuggests that even though these drugs may have strong directantiviral effects (as indicated by the rapid detection of drug-resistant mutants) (87), the antiviral immune responses wereprobably not sufficient to kill productively infected cells. Be-cause untreated macaques infected with virulent SIV isolatessuch as SIVmac251 have higher viremia, lower CTL responses,

and more rapid disease than HIV-infected humans (for a re-view, see reference 44), this model could explain why mostantiviral drugs that are effective in HIV-infected people havebeen less effective in SIV-infected macaques, with the excep-tion of tenofovir, which must somehow allow or rescue thedevelopment of such antiviral immune responses. In the ab-sence of CD8� cells, tenofovir’s effects on viremia resembledthose of these other drugs. Accordingly, further research is

FIG. 3. Effect of tenofovir treatment interruption on viremia. (A) As previously described (80), animal 29003 had been inoculated at birth withwild-type SIVmac251 and started 3 weeks later on chronic tenofovir treatment (30 mg/kg once daily subcutaneously). Even after the emergenceof K65R viral mutants, virus levels decreased gradually. At 22 months of age (time zero, indicated by an arrow), when virus levels were undetectable(�500 copies RNA/ml) (81), tenofovir treatment was withdrawn. Tenofovir therapy was restarted 18 months later (large arrow and shaded area).The dotted line represents the 1,500-copies/ml cutoff value of the bDNA assay, version 2.0. (B) Some animals which had previously been CD8�-celldepleted had subsequently reached undetectable virus levels (�125 RNA copies/ml; cutoff value of bDNA, version 4.0) upon return of the CD8�

cells (Fig. 1, 2; Table 1). For 1 animal (no. 29276), tenofovir was continued and virus levels remained undetectable throughout the observationperiod (�12 months after the CD8�-cell depletion). For 3 other animals that had reached undetectable viremia (no. 32186, 33088, and 33091),tenofovir treatment was interrupted and restarted 7 weeks later (shaded area) at the same dosage regimen. Virus isolated during the interruptionhad the expected genotypic (K65R) and phenotypic resistance to tenofovir that had been detected previously in isolates of these 3 animals (Table4). The reason for the transient spike of viral RNA for animals 33088 and 33091 at 2 and 3 weeks after restarting tenofovir (which was associated withan increase in infectious titer in PBMC) is not clear but may represent transient replication of an immune escape mutant which was subsequentlycontrolled. No significant changes in CD8� or CD4� lymphocyte counts were observed during this period.

FIG. 4. Proposed model of tenofovir’s effects on virus replication. (A) Without drug treatment, virulent virus can replicate to high titers becauseof high infection rates of CD4�-T-helper cells and antigen-presenting cells and insufficient CD8�-cell-mediated immune responses. (B) Tenofoviracts (i) as an inhibitor of RT, reducing the number of CD4�-T-helper cells and antigen-presenting cells that become newly infected and (ii) possiblyalso as an immunomodulator. Potent CD8�-cell-mediated immune responses help to reduce the half-life (T1/2) and thus the burst of viral progenyfor those cells that became infected. Both tenofovir and antiviral CD8� cells are required to induce and maintain low viremia. The role of the CD8�

cells becomes even more pronounced after the emergence of drug-resistant K65R viral mutants. (C) During artificial CD8�-cell depletion,productively infected cells survive longer and produce more progeny virus, resulting in an increase in viremia.


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needed to confirm whether this positive interaction we ob-served between tenofovir and the development of CD8�-cell-mediated immune responses is unique or more pronounced fortenofovir than for other drugs. Repeating the present CD8�-cell depletion experiments with other antiviral drugs, however,is unlikely to provide further insights, because these drugs arenot very effective in suppressing established viremia of veryvirulent SIV isolates, so the effect of CD8�-cell depletion ondrug efficacy would be difficult to detect unless attenuated virusisolates were used.

The ability of chronic tenofovir treatment to persistentlysuppress SIVmac251 viremia to low or undetectable levels insome animals for more than 6 to 7 years despite the emergenceof drug-resistant viral mutants is remarkable and has not beenachieved with other antiviral drugs (see the introduction). Withpathogenic simian-HIV isolates, short-term drug- or vaccine-induced suppression of acute viremia is often sufficient to pro-mote strong antiviral immune responses that induce a low virusset point and provide protection against rapid disease, al-though the emergence of CTL escape mutants in some animalsstill leads to disease (9, 16, 32). In contrast, with highly viru-lent isolates such as SIVmac251, sustained control of viremiaand disease has rarely been achieved (16, 32). In this regard,SIVmac251 infection closely mimics HIV infection but is morevirulent. Although there is variation in the virulence of dif-ferent SIVmac251 stocks, the high viremia induced by theSIVmac251 stocks used in the experiments described here hasbeen very difficult to control (see Materials and Methods).

Tenofovir-treated animals can show a progressive and sus-tained suppression of viremia even after the emergence ofK65R viral mutants. At first thought, potential explanations forthis observation would include the possibility of strongly re-duced fitness and virulence of K65R mutants and drug phar-macokinetics resulting in continued suppression of K65Rmutants which have fivefold-reduced levels of in vitro suscep-tibility to tenofovir. However, the available evidence, includingthe results of the present experiments, points against each ofthese explanations as the sole factor. K65R SIV mutants alsoaccumulate other mutations in RT, which are believed to becompensatory and improve replication fitness in vivo (80, 81).These K65R SIV mutants, when inoculated in infant or juve-nile macaques in the absence of tenofovir treatment, werefound to replicate to high levels and be as virulent as wild-typevirus with similar time to disease without reversion to the wildtype (46, 81, 89). Persistent suppression of virulent K65R SIVmutants has only been observed in tenofovir-treated animals.The gradual decline in virus levels in these animals could notbe explained by progressive accumulation of tenofovir levelsbecause the dosage had been reduced from a high inductionregimen (30 mg/kg) to a low maintenance regimen (2.5 to 10mg/kg) and pharmacokinetic studies confirmed that plasmalevels at these low regimens were within the expected range(79a). The CD8�-cell depletion experiments reported hereconfirmed that the tenofovir regimen by itself was insufficientto suppress K65R viremia in the absence of CD8� cells (Fig.2). Instead, while continuous tenofovir was still required,CD8�-cell-mediated immune responses played a major role insuppressing K65R viremia (Fig. 4). In other words, the emer-gence of K65R viral mutants is not associated with virological

failure of tenofovir therapy as long as there are good antiviralimmune responses.

When animals were started on tenofovir treatment at peakSIVmac251 viremia (�2 to 3 weeks after infection), approx-imately half of them were able to gradually suppress viremiato undetectable levels. Persistent suppression of virulentSIVmac251 viremia has been more difficult to achieve whenanimals were started on tenofovir monotherapy during inter-mediate-to-late-stage infection, and the emergence of K65Rviral mutants is then usually associated with a gradual increasein viremia (92a). Because virulent SIV infection induces im-mune dysfunction at many levels, the reduced ability of teno-fovir to persistently suppress viremia in such immunocompro-mised animals could be due to a defect in any events that makeit more difficult to rescue the generation, maturation, andmaintenance of strong antiviral CD8�-lymphocyte responsesto complement tenofovir’s direct antiviral effects (20, 26, 27,39, 101). Without proper T-helper-cell function and antigenpresentation, CD8�-cell-mediated immune responses may notremain active at low levels of antigen (24, 31, 45) and are thusnot able to suppress viremia to low or undetectable levels,especially once drug-resistant mutants emerge. However,chronic viremia was suppressed more efficiently during teno-fovir treatment when less-virulent SIV isolates were used (52,76, 80) or when tenofovir treatment was combined with otherantiviral drugs and/or immunotherapy strategies (17, 21, 36, 95).

Because the cM-T807 antibody depletes both the CD3�

CD8� T lymphocytes as well as CD3�CD8� NK cells, therelative contribution of these two cell populations to the im-mune-mediated suppression of viremia could not be deter-mined in the present experiments. CD8� T and NK lympho-cytes inhibit virus replication in vitro through a variety ofmechanisms, including cytotoxicity and noncytolytic pathways(secretion of antiviral factors) (31, 45). In macaques, most NKT cells (believed to be important immunoregulators) are alsoCD8� (48). Thus, further studies are needed to identify whichCD8� cells and antiviral mechanisms are most important insuppressing viremia. In another study, the number of expand-ing CD4�- and CD8�-T-cell clonotypes (as determined by Vchain CDR3 region DNA heteroduplex tracking assay) inSIVmac251-infected macaques did not correlate with their re-sponse to tenofovir treatment (9a). As suggested by data fromboth human and macaque studies (1, 10, 34), the present invitro assays, especially when PBMC are used, may not suffi-ciently capture the strength and breadth of the cell-mediatedimmune responses that are active in vivo, especially in lym-phoid tissues where most of the virus replication occurs. In ourlong-term tenofovir-treated macaques with undetectable vire-mia, the cell-mediated immune responses must be unusuallystrong and/or broad, as immune-escape mutants have notemerged in these animals even after 6 to 7 years of tenofovirtreatment (Fig. 2).

Why does tenofovir behave differently from other antiviraldrugs in the SIV macaque model? Several features of tenofovirthat may be dependent or independent of its direct antiviraleffects may contribute to the beneficial immunological eventsobserved in the macaque studies. It is possible that due to theintrinsic antiviral potency and favorable pharmacokinetics incomparison to other drugs, tenofovir treatment provides betterpreservation of virus-specific CD4�-T-helper-cell function in


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vivo, which is expected to lead to improved antiviral CD8�-cell-mediated immune responses (24). Early tenofovir treat-ment was also found to reduce CD8�-lymphocyte apoptosisduring acute SIVmac239 viremia (71). In vitro studies havedemonstrated that tenofovir is more active to inhibit wild-typeHIV type 1 (HIV-1) replication in monocytes/macrophages,dendritic cells, and Langerhans cells than in lymphocytes orlymphocytoid cell lines because of (i) a more efficient conver-sion to its active diphosphate metabolite, (ii) a longer intracel-lular half-life of these metabolites, and (iii) the lower dATPlevels in these cells (6, 8, 62). Tenofovir may thus be moreeffective in inhibiting replication, including that of K65R SIVmutants, in vivo in these antigen-presenting cells than in lym-phocytes. Relatively subtle improvements in the early steps ofan antiviral immune response are likely to be amplified in latersteps, as suggested by the beneficial effects of administration ofexogenously pulsed dendritic cells to SIVmac251-infected ma-caques (38).

Tenofovir may have immunopreserving or immunomodula-tory effects for macaques that are independent of its directantiviral effects. Immunomodulatory effects of tenofovir andrelated compounds have been extensively demonstrated in mu-rine models in vitro and in vivo, including enhancement of NKcell activity and secretion of cytokines and chemokines (13, 14,94, 99, 100). During in vitro experiments with PBMC of unin-fected rhesus macaques, tenofovir did not directly stimulateNK cell activity or cytokine production but primed cells formore rapid and enhanced interleukin-12 (IL-12) productionfollowing exposure to bacterial antigens (K. K. A. Van Rom-pay, M. L. Marthas, and N. Bischofberger, submitted for pub-lication). Considering the many biological effects of IL-12 (i.e.,activation of NK cells and CTLs, stimulation of IFN-� produc-tion and Th1 immune responses, and antiapoptotic effects) (fora review, see reference 74), further research is needed to de-termine its potential role in the observations with tenofovir inthe macaque model. Interestingly, studies performed with ex-ogenous IL-12 administration to SIV-infected macaques dur-ing acute and chronic SIV infection showed effects on viremiaremarkably similar to those of tenofovir therapy during theserespective stages (5, 96, 97).

Other observations provide additional evidence for immune-preserving or immunomodulatory effects of tenofovir therapyin SIV-infected macaques. Tenofovir therapy initiated duringlate-stage SIVmac251 infection significantly improved disease-free survival even in the presence of high viremia and sup-pressed acquired immune responses, which has not been ob-served with other drugs in this animal model (81, 92a).

Because CD8�-cell depletion experiments are not feasible inhumans, clinical studies are needed to investigate whether sim-ilar immune events occur also in drug-treated HIV-infectedhumans and are preferentially seen with tenofovir-containingregimens. Two pieces of evidence from tenofovir-treatedHIV-1 infected patients are consistent with our proposed syn-ergistic effects of CD8� cells and tenofovir in SIV-infectedmacaques. First, short-term studies comparing the early decaykinetics of viral RNA following the initiation of therapy havedemonstrated that a tenofovir-containing regimen induced afaster decline in viral RNA levels than other commonly usedcombination regimens without tenofovir (37, 42). This can beinterpreted as a reflection of the higher efficacy of the tenofo-

vir-containing regimen than the standard regimen to directlyinhibit virus replication. However, our experiments offer analternative explanation: when different regimens are equallyeffective in preventing infection of new cells, the decay rate ofvirus-producing cells is determined mainly by the potency ofCD8�-cell-mediated antiviral immune responses. Thus, thefaster decay rate during a tenofovir-containing regimen couldbe consistent with enhanced CD8�-cell-mediated inhibition ofvirus replication. The second observation is that K65R mutantsof HIV-1 are generally infrequently (�2%) detected afterlong-term tenofovir treatment as part of most HAART regi-mens, and their emergence is generally not associated witha rebound in viremia (41). These clinical observations withHIV-1 are consistent with our observations of reduced viremiaof K65R SIV mutants in macaques due to improved antiviralimmune responses during tenofovir treatment. Of note, effectsof improved antiviral immune responses are not mutually ex-clusive of effects of decreased replication capacity of the K65Rmutant virus and/or residual drug activity of tenofovir. In par-ticular, even a relatively minor decrease in intrinsic replicationfitness of K65R viral mutants during tenofovir treatment canhave a major impact on viremia if it provides more opportunityfor antiviral CD8� cells to kill productively infected cells priorto the major viral burst. Thus, multiple factors are likely tocontribute to reduced viremia in tenofovir-treated humanswho develop K65R mutants of HIV-1.

Recently, it has been reported that when treatment-naivepatients were started on a combination of tenofovir, lamivu-dine, and abacavir used once daily, approximately half of themshowed less-than-desired (i.e., �2 log) suppression of viremiaby week 8 or had evidence of an early viral rebound, which wasassociated with the detection of M184V mutants in all failurepatients and K65R mutants in approximately 50% of failurepatients (J. Gallant, Abstr. 43rd Intersci. Conf. Antimicrob.Agents Chemother., late-breaker abstract 1722a, 2003; C. Far-thing et al., Prog. Abstr. 2nd Int. AIDS Soc. Conf. Pathog.Treat., abstr. 43, 2003). While further investigations are cur-rently being done to explain these findings, an overall poorpotency of this investigational triple-nucleoside regimen withabacavir once daily is a likely explanation, and as a result,resistance emerged to one or more drugs in the regimen. Thefindings of this human study are consistent with our macaquedata, confirming the high selection pressure for the emergenceof K65R mutants during tenofovir treatment and the impact ofthese K65R mutants on reducing tenofovir’s direct efficacy insuppressing viremia. Our macaque studies demonstrated thateven after a relatively modest early virological response totenofovir or partial viral rebound due to the emergence ofK65R viral mutants, continued tenofovir treatment leads insome animals to a gradual suppression of viremia to low orundetectable levels over the course of months to years, due toa strengthening of antiviral CD8�-cell-mediated immune re-sponses. The human patients with a poor early response to thistenofovir-containing regimen were switched to a different reg-imen, so the long-term response was not determined.

If these interactions between antiviral immune responsesand antiviral drugs such as tenofovir are confirmed in humans,the clinical implications are multiple. First, early drug treat-ment is more likely to promote the induction and maintenanceof strong antiviral immune responses (63). Second, the detec-


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tion of drug resistance mutations such as K65R does not nec-essarily indicate that therapy is failing and is by itself not a validreason to stop tenofovir treatment unless better treatmentoptions are available. Finally, elucidation of the CD8�-cell-mediated antiviral immune responses that are elicited duringsuccessful tenofovir treatment in macaques and that are un-usually strong in suppressing virus replication for years may aidthe development of novel immunotherapeutic strategies. Asmentioned above, macaque studies have suggested that theefficacy of tenofovir during chronic infection can be improvedfurther by combining it with additional immunotherapeuticstrategies. Accordingly, further research in this area should beexplored, as this may result in finding long-term sustainableand affordable strategies that combine the strengths of antivi-ral drugs and the immune system to indefinitely delay diseaseprogression.

ACKNOWLEDGMENTS

For technical assistance, we thank D. Bennett; T. Dearman; L. Hirst;J. Li; A. Spinner; W. von Morgenland; the Veterinary Staff, ColonyServices, and Clinical Laboratory of the CNPRC; T. Matthews; and J.Murry (Center for Comparative Medicine, University of California,Davis). We also thank K. Abel, M. Miller, A. Muthukumar, T. North,K. A. Reimann, and G. Silvestri for assistance, useful suggestions, anddiscussions.

This work was supported by Gilead Sciences, E. Glaser PediatricAIDS Foundation grants PG-50757 and PG-51014 (K.V.R), NIH/NIAID grant R01 AI46320-01 (M.L.M), DE12926 (D.L.S.), and PublicScience Health grant RR00169 from the National Center for ResearchResources. The cM-T807 antibody used in this work was produced bythe National Cell Culture Center with funds provided by NIH grantRR16001.

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CD8+-Cell-Mediated Suppression of Virulent Simian Immunodeficiency Virus during Tenofovir Treatment

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