Human immunodeficiency virus type 1 envelope epitope-specific CD4(+) T lymphocytes in simian/human immunodeficiency virus-infected and vaccinated rhesus monkeys detected using a peptide-major histocompatibility complex class II tetramer

A tetrameric recombinant major histocompatibility complex (MHC) class II-peptide complex was used to quantitate human immunodeficiency virus type 1 (HIV-1) envelope (Env)-specific CD4(+) T cells in vaccinated and in simian/human immunodeficiency virus (SHIV)-infected rhesus monkeys. A rhesus monkey MHC class II DR molecule, Mamu-DR*W201, and an HIV-1 Env peptide (p46) were employed to construct this tetrameric complex. A p46-specific proliferative response was seen in sorted, tetramer-binding, but not nonbinding, CD4(+) T cells, directly demonstrating that this response was mediated by the epitope-specific lymphocytes. Although staining of whole blood from 10 SHIV-infected Mamu-DR*W201(+) rhesus monkeys failed to demonstrate tetramer-binding CD4(+) T cells (<0.02%), p46-stimulated peripheral blood mononuclear cells (PBMCs) from 9 of these 10 monkeys had detectable p46 tetramer-binding cells, comprising 0.5 to 15.2% of the CD4(+) T cells. p46-stimulated PBMCs from 7 of 10 Mamu-DR*W201(+) monkeys vaccinated with a recombinant canarypox virus-HIV-1 env construct also demonstrated p46 tetramer-binding cells, comprising 0.9 to 7.2% of the CD4(+) T cells. Thus, Env p46-specific CD4(+) T cells can be detected by tetrameric Mamu-DR*W201-p46 complex staining of PBMCs in both SHIV-infected and vaccinated rhesus monkeys. These epitope-specific cell populations appear to be present in peripheral blood at a very low frequency.     

Use of Major Histocompatibility Complex Class I/Peptide/β2M Tetramers To Quantitate CD8+ Cytotoxic T Lymphocytes Specific for Dominant and Nondominant Viral Epitopes in Simian-Human Immunodeficiency Virus-Infected Rhesus Monkeys

To evaluate the impact of the diversity of antigen recognition by T lymphocytes on disease pathogenesis, we must be able to identify and analyze simultaneously cytotoxic T-lymphocyte (CTL) responses specific for multiple viral epitopes. Many of the studies of the role of CD8(+) CTLs in AIDS pathogenesis have been done with simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys. These studies have frequently made use of the well-defined SIV Gag CTL epitope p11C,C-M presented to CTL by the HLA-A homologue molecule Mamu-A*01. In the present study we identified and fine mapped two novel Mamu-A*01-restricted CTL epitopes: the SIVmac Pol-derived epitope p68A (STPPLVRLV) and the human immunodeficiency virus type 1 (HIV-1) Env-derived p41A epitope (YAPPISGQI). The frequency of CD8(+) CTLs specific for the p11C,C-M, p68A, and p41A epitopes was quantitated in the same animals with a panel of tetrameric Mamu-A*01/peptide/beta2m complexes. All SHIV-infected Mamu-A*01(+) rhesus monkeys tested had a high frequency of SIVmac Gag-specific CTLs to the p11C,C-M epitope. In contrast, only a fraction of the monkeys tested had detectable CTLs specific for the SIVmac Pol p68A and HIV-1 Env p41A epitopes, and these responses were detected at very low frequencies. Thus, the p11C,C-M-specific CD8(+) CTL response is dominant and the p41A- and p68A-specific CD8(+) CTL responses are nondominant. These results indicate that CD8(+) CTL responses to dominant CTL epitopes can be readily quantitated with the tetramer technology; however, CD8(+) CTL responses to nondominant epitopes, due to the low frequency of these epitope-specific cells, may be difficult to detect and quantitate by this approach.     

The gag-specific cytotoxic T lymphocytes in rhesus monkeys infected with the simian immunodeficiency virus of macaques

The simian immunodeficiency virus of macaques (SIVmac) is a lentivirus which induces an AIDS-like disease in rhesus monkeys. We have explored the virus-specific cellular immune response in SIVmac-infected rhesus monkeys. Con A-activated, IL-2 expanded PBL of some SIVmac-infected rhesus monkeys lyse autologous B lymphoblastoid cell lines infected with a recombinant vaccinia virus that carries the SIVmac gag gene. This lysis is mediated by CD8+ lymphocytes and is MHC class I restricted. Moreover, these effector lymphocytes do not express the NK cell-associated molecules NKH1 or CD16. These cells are, therefore, CTL. In a limited prospective study of SIVmac-infected rhesus monkeys, the presence of the SIVmac gag-specific CTL activity in PBL correlated with both a reduced efficiency in isolating SIVmac from PBL of these monkeys and their extended survival. This method for assessing SIVmac gag-specific cellular immunity in rhesus monkeys will be important not only in investigating the immunopathogenesis of SIVmac-induced disease, but also in evaluating the capacity of candidate AIDS vaccines to elicit a cell-mediated immune response in this animal model.     

The TCR repertoire of an immunodominant CD8+ T lymphocyte population

The TCR repertoire of an epitope-specific CD8(+) T cell population remains poorly characterized. To determine the breadth of the TCR repertoire of a CD8(+) T cell population that recognizes a dominant epitope of the AIDS virus, the CD8(+) T cells recognizing the tetrameric Mamu-A*01/p11C(,CM) complex were isolated from simian immunodeficiency virus (SIV)-infected Mamu-A*01(+) rhesus monkeys. This CD8(+) T cell population exhibited selected usage of TCR V beta families and complementarity-determining region 3 (CDR3) segments. Although the epitope-specific CD8(+) T cell response was clearly polyclonal, a dominance of selected V beta(+) cell subpopulations and clones was seen in the TCR repertoire. Interestingly, some of the selected V beta(+) cell subpopulations and clones maintained their dominance in the TCR repertoire over time after infection with SIV of macaques. Other V beta(+) cell subpopulations declined over time in their relative representation and were replaced by newly evolving clones that became dominant. The present study provides molecular evidence indicating that the TCR repertoire shaped by a single viral epitope is dominated at any point in time by selected V beta(+) cell subpopulations and clones and suggests that dominant V beta(+) cell subpopulations and clones can either be stable or evolve during a chronic infection.     

Contribution of T-cell receptor repertoire breadth to the dominance of epitope-specific CD8+ T-lymphocyte responses

Dominant epitope-specific CD8(+) T-lymphocyte responses play a central role in controlling viral spread. We explored the basis for the development of this focused immune response in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys through the use of two dominant (p11C and p199RY) and two subdominant (p68A and p56A) epitopes. Using real-time PCR to quantitate T-cell receptor (TCR) variable region beta (Vbeta) family usage, we show that CD8(+) T-lymphocyte populations specific for dominant epitopes are characterized by a diverse Vbeta repertoire, whereas those specific for subdominant epitopes employ a dramatically more focused Vbeta repertoire. We also demonstrate that dominant epitope-specific CD8(+) T lymphocytes employ TCRs with multiple CDR3 lengths, whereas subdominant epitope-specific cells employ TCRs with a more restricted CDR3 length. Thus, the relative dominance of an epitope-specific CD8(+) T-lymphocyte response reflects the clonal diversity of that response. These findings suggest that the limited clonal repertoire of subdominant epitope-specific CD8(+) T-lymphocyte populations may limit the ability of these epitope-specific T-lymphocyte populations to expand and therefore limit the ability of these cell populations to contribute to the control of viral replication.     

Simian immunodeficiency virus (SIV)-specific CTL are present in large numbers in livers of SIV-infected rhesus monkeys

The immunopathogenesis of AIDS-associated hepatitis was explored in the SIV/rhesus monkey model. The livers of SIV-infected monkeys showed a mild hepatitis, with a predominantly CD8+ T lymphocyte infiltration in the periportal fields and sinusoids. These liver-associated CD8+ T cells were comprised of a high percentage of SIV-specific CTL as defined by MHC class I/Gag peptide tetramer binding and Gag peptide epitope-specific lytic activity. There was insufficient viral replication in these livers to account for attracting this large number of functional virus-specific CTL to the liver. There was also no evidence that the predominant population of CTL were functionally end-stage cells trapped in the liver and destined to undergo apoptotic cell death in that organ. Interestingly, we noted that liver tetramer-binding cells showed an increased expression of CD62L, an adhesion molecule usually only rarely expressed on tetramer-binding cells. This observation suggests that the expression of specific adhesion molecules by CTL might facilitate the capture of these cells in the liver. These results demonstrate that functional SIV-specific CD8+ T cells are present in large numbers in the liver of chronically SIV-infected monkeys. Thus, the liver may be a trap for virus-specific cytotoxic T cells.     

A commonly recognized simian immunodeficiency virus Nef epitope presented to cytotoxic T lymphocytes of Indian-origin rhesus monkeys by the prevalent major histocompatibility complex class I allele Mamu-A*02

The ability to monitor vaccine-elicited CD8(+) cytotoxic T-lymphocyte (CTL) responses in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys has been limited by our knowledge of viral epitopes predictably presented to those lymphocytes by common rhesus monkey MHC class I alleles. We now define an SIV and SHIV Nef CTL epitope (YTSGPGIRY) that is presented to CD8(+) T lymphocytes by the common rhesus monkey MHC class I molecule Mamu-A*02. All seven infected Mamu-A*02(+) monkeys evaluated demonstrated this response, and peptide-stimulated interferon gamma Elispot assays indicated that the response represents a large proportion of the entire CD8(+) T-lymphocyte SIV- or SHIV-specific immune response of these animals. Knowledge of this epitope and MHC class I allele substantially increases the number of available rhesus monkeys that can be used for testing prototype HIV vaccines in this important animal model.     

In vivo T-lymphocyte activation and transient reduction of viral replication in macaques infected with simian immunodeficiency virus

While it is well established that cellular activation can increase human immunodeficiency virus (HIV) replication in T lymphocytes, it is also clear that both activated CD8+ and CD4+ T lymphocytes mediate anti-HIV activity. To assess the relative importance of these contrary effects on HIV replication in vivo, we evaluated the consequences of Mycobacterium bovis BCG and staphylococcal enterotoxin B (SEB) inoculation in vivo in rhesus monkeys chronically infected with simian immunodeficiency virus of macaques (SIVmac). BCG inoculation induced as much as a 2.5-log reduction of plasma and intracellular SIV RNA in SIVmac-infected monkeys. This down-regulation of virus replication persisted as long as 4 weeks after BCG inoculation. Similarly, SEB injection resulted in up to a 3-log decrease in plasma and intracellular SIV RNA in SIVmac-infected macaques. Interestingly, the short-term reduction of viremia in these monkeys correlated with the peak in vivo production of SEB- and BCG-induced cytokine responses. However, no long-term clinical benefit was observed in the SIVmac-infected macaques. These studies provide in vivo evidence that potent T-cell stimulation driven by antigens other than the virus itself can, under some circumstances, mediate short-term reduction of viremia in AIDS virus-infected individuals.     

Simian immunodeficiency virus-specific cytotoxic T lymphocytes are present in the AIDS-associated skin rash in rhesus monkeys.

An infiltration of CD8+ lymphocytes in the dermis and epidermis underlies the skin rash that commonly occurs as a primary manifestation of an AIDS virus infection. These cutaneous lymphocytes were characterized in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. Skin rash-associated lymphocytes exhibited greater lysis of SIVmac-expressing target cells and a higher cloning efficiency for SIVmac-specific effector T cells than PBL. Moreover, both SIVmac envelope- and gag-specific CTL could be readily cloned from these skin rash-associated lymphocytes. In fact, the skin rash-associated CTL exhibited the same MHC restriction and epitope specificity as those CTL derived from PBL. These studies, therefore, demonstrate that the cutaneous infiltrating CD8+ lymphocytes in SIVmac-infected rhesus monkeys include SIVmac-specific CTL. Thus, whereas virus-specific CTL are likely to represent an important mechanism for controlling AIDS virus infections, they also may play a role in the pathogenesis of the skin lesions that occur after this infection.     

Mutations in a dominant Nef epitope of simian immunodeficiency virus diminish TCR:epitope peptide affinity but not epitope peptide:MHC class I binding

Viruses like HIV and SIV escape from containment by CD8(+) T lymphocytes through generating mutations that interfere with epitope peptide:MHC class I binding. However, mutations in some viral epitopes are selected for that have no impact on this binding. We explored the mechanism underlying the evolution of such epitopes by studying CD8(+) T lymphocyte recognition of a dominant Nef epitope of SIVmac251 in infected Mamu-A*02(+) rhesus monkeys. Clonal analysis of the p199RY-specific CD8(+) T lymphocyte repertoire in these monkeys indicated that identical T cell clones were capable of recognizing wild-type (WT) and mutant epitope sequences. However, we found that the functional avidity of these CD8(+) T lymphocytes for the mutant peptide:Mamu-A*02 complex was diminished. Using surface plasmon resonance to measure the binding affinity of the p199RY-specific TCR repertoire for WT and mutant p199RY peptide:Mamu-A*02 monomeric complexes, we found that the mutant p199RY peptide:Mamu-A*02 complexes had a lower affinity for TCRs purified from CD8(+) T lymphocytes than did the WT p199RY peptide:Mamu-A*02 complexes. These studies demonstrated that differences in TCR affinity for peptide:MHC class I ligands can alter functional p199RY-specific CD8(+) T lymphocyte responses to mutated epitopes, decreasing the capacity of these cells to contain SIVmac251 replication.     

Impairment of Gag-Specific CD8+ T-Cell Function in Mucosal and Systemic Compartments of Simian Immunodeficiency Virus mac251- and Simian-Human Immunodeficiency Virus KU2-Infected Macaques

The identification of several simian immunodeficiency virus mac251 (SIV(mac251)) cytotoxic T-lymphocyte epitopes recognized by CD8(+) T cells of infected rhesus macaques carrying the Mamu-A*01 molecule and the use of peptide-major histocompatibility complex tetrameric complexes enable the study of the frequency, breadth, functionality, and distribution of virus-specific CD8(+) T cells in the body. To begin to address these issues, we have performed a pilot study to measure the virus-specific CD8(+) and CD4(+) T-cell response in the blood, lymph nodes, spleen, and gastrointestinal lymphoid tissues of eight Mamu-A*01-positive macaques, six of those infected with SIV(mac251) and two infected with the pathogenic simian-human immunodeficiency virus KU2. We focused on the analysis of the response to peptide p11C, C-M (Gag 181), since it was predominant in most tissues of all macaques. Five macaques restricted viral replication effectively, whereas the remaining three failed to control viremia and experienced a progressive loss of CD4(+) T cells. The frequency of the Gag 181 (p11C, C-->M) immunodominant response varied among different tissues of the same animal and in the same tissues from different animals. We found that the functionality of this virus-specific CD8(+) T-cell population could not be assumed based on the ability to specifically bind to the Gag 181 tetramer, particularly in the mucosal tissues of some of the macaques infected by SIV(mac251) that were progressing to disease. Overall, the functionality of CD8(+) tetramer-binding T cells in tissues assessed by either measurement of cytolytic activity or the ability of these cells to produce gamma interferon or tumor necrosis factor alpha was low and was even lower in the mucosal tissue than in blood or spleen of some SIV(mac251)-infected animals that failed to control viremia. The data obtained in this pilot study lead to the hypothesis that disease progression may be associated with loss of virus-specific CD8(+) T-cell function.     

Emergence of CTL coincides with clearance of virus during primary simian immunodeficiency virus infection in rhesus monkeys

The CTL response was characterized during primary SIV/macaque (SIVmac) infection of rhesus monkeys to assess its role in containing early viral replication using both an epitope-specific functional and an MHC class I/peptide tetramer-binding assay. The rapid expansion of a single dominant viral epitope-specific CTL population to 1.3-8.3% of circulating CD8+ peripheral blood and 0. 3-1.3% of lymph node CD8+ T cells was observed, peaking at day 13 following infection. A subsequent decrease in number of these cells was then demonstrated. Interestingly, the percent of tetramer-binding CD8+ T cells detected in the lymph nodes of all evaluated animals was smaller than the percent detected in PBL. These epitope-specific CD8+ T cells expressed cell surface molecules associated with memory and activation. Early clearance of SIVmac occurred coincident with the emergence of the CTL response, suggesting that CTL may be important in containing virus replication. A higher percent of annexin V-binding cells was detected in the tetramer+ CD8+ T cells (range, from 33% to 75%) than in the remaining CD8+ T cells (range, from 3.3% to 15%) at the time of maximum CTL expansion in all evaluated animals. This finding indicates that the decrease of CTL occurred as a result of the death of these cells rather than their anatomic redistribution. These studies provide strong evidence for the importance of CTL in containing AIDS virus replication.     

Contribution of CD8+ T cells to containment of viral replication and emergence of mutations in Mamu-A*01-restricted epitopes in Simian immunodeficiency virus-infected rhesus monkeys

Here, we investigated the containment of virus replication in simian immunodeficiency virus (SIV) infection by CD8(+) lymphocytes. Escape mutations in Mamu-A*01 epitopes appeared first in SIV Tat TL8 and then in SIV Gag p11C. The appearance of escape mutations in SIV Gag p11C was coincident with compensatory changes outside of the epitope. Eliminating CD8(+) lymphocytes from rhesus monkeys during primary infection resulted in more rapid disease progression that was associated with preservation of canonical epitopes. These results confirm the importance of cytotoxic T cells in controlling viremia and the constraint on epitope sequences that require compensatory changes to go to fixation.     

Epitope-specific CD8+ T lymphocytes cross-recognize mutant simian immunodeficiency virus (SIV) sequences but fail to contain very early evolution and eventual fixation of epitope escape mutations during SIV infection

Human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) evade containment by CD8(+) T lymphocytes through focused epitope mutations. However, because of limitations in the numbers of viral sequences that can be sampled, traditional sequencing technologies have not provided a true representation of the plasticity of these viruses or the intensity of CD8(+) T lymphocyte-mediated selection pressure. Moreover, the strategy by which CD8(+) T lymphocytes contain evolving viral quasispecies has not been characterized fully. In the present study we have employed ultradeep 454 pyrosequencing of virus and simultaneous staining of CD8(+) T lymphocytes with multiple tetramers in the SIV/rhesus monkey model to explore the coevolution of virus and the cellular immune response during primary infection. We demonstrated that cytotoxic T lymphocyte (CTL)-mediated selection pressure on the infecting virus was manifested by epitope mutations as early as 21 days following infection. We also showed that CD8(+) T lymphocytes cross-recognized wild-type and mutant epitopes and that these cross-reactive cell populations were present at a time when mutant forms of virus were present at frequencies of as low as 1 in 22,000 sequenced clones. Surprisingly, these cross-reactive cells became enriched in the epitope-specific CD8(+) T lymphocyte population as viruses with mutant epitope sequences largely replaced those with epitope sequences of the transmitted virus. These studies demonstrate that mutant epitope-specific CD8(+) T lymphocytes that are present at a time when viral mutant epitope sequences are detected at extremely low frequencies fail to contain the later accumulation and fixation of the mutant epitope sequences in the viral quasispecies.     

Two distinct lymphocyte populations mediate simian immunodeficiency virus envelope-specific target cell lysis

The relative contributions of the effector lymphocyte responses to the AIDS virus envelope glycoprotein (env) were explored in simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys. CD8+, MHC class I-restricted, env-specific CTL were cloned from PBL of SIVmac-infected monkeys, indicating that such cells constitute a component of the env-specific effector lymphocyte response. A limiting dilution 51Cr release assay was then established for quantitating the frequency of SIVmac-specific effector lymphocytes in PBL of rhesus monkeys. Using this assay we demonstrate that SIVmac env-specific effector lymphocytes are comprised of both CD16-, MHC class I-restricted and CD16+, MHC class I-unrestricted cells. We also demonstrate that the env-specific response is the predominant SIVmac-specific effector lymphocyte response in rhesus monkeys. These studies document the complexity of the effector lymphocyte response to the AIDS virus envelope glycoprotein and establish the role played by two distinct effector cell populations in this response.     

Trafficking of antigen-specific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination

A critical goal of vaccine development for a wide variety of pathogens is the induction of potent and durable mucosal immunity. However, it has been assumed that this goal would be difficult to achieve by systemic vaccination due to the anatomic and functional distinctness of the systemic and mucosal immune systems and the resultant compartmentalization of immune responses. In this study, we show that Ag-specific CD8(+) T lymphocytes traffic efficiently to mucosal surfaces following systemic vaccination. Intramuscular immunization with recombinant adenovirus (rAd) vector-based vaccines expressing SIV Gag resulted in potent, durable, and functional CD8(+) T lymphocyte responses at multiple mucosal effector sites in both mice and rhesus monkeys. In adoptive transfer studies in mice, vaccine-elicited systemic CD8(+) T lymphocytes exhibited phenotypic plasticity, up-regulated mucosal homing integrins and chemokine receptors, and trafficked rapidly to mucosal surfaces. Moreover, the migration of systemic CD8(+) T lymphocytes to mucosal compartments accounted for the vast majority of Ag-specific mucosal CD8(+) T lymphocytes induced by systemic vaccination. Thus, i.m. vaccination can overcome immune compartmentalization and generate robust mucosal CD8(+) T lymphocyte memory. These data demonstrate that the systemic and mucosal immune systems are highly coordinated following vaccination.     

Vaccine-induced CD8+ T lymphocytes of rhesus monkeys recognize variant forms of an HIV epitope but do not mediate optimal functional activity

The sequence diversity of HIV-1 presents a challenge for the development of an effective HIV-1 vaccine, because such a vaccine must confer protection against diverse forms of the virus. The present studies were initiated to explore how vaccine-induced clonal populations of CD8(+) T lymphocytes of rhesus monkeys recognize variants of an HIV-1 envelope epitope sequence. Evaluating a subset of variants of a selected epitope peptide that retain their binding to the MHC class I molecule of rhesus monkeys that presents this epitope peptide, we show that vaccine-elicited CD8(+) T lymphocytes comparably recognize the wild-type and a number of variant epitope peptides as determined by tetramer binding assays. In fact, the same clonal populations of CD8(+) T lymphocytes recognize the wild-type and variant epitope peptides. However, functional assays show that many of these variant epitope peptides stimulate suboptimal cytokine production by the vaccine-elicited CD8(+) T lymphocytes. These findings suggest that vaccine-induced CD8(+) T lymphocyte populations may recognize diverse forms of a viral epitope, but may not function optimally to confer protection against viruses expressing many of those variant sequences.     

Immunodomination in the evolution of dominant epitope-specific CD8+ T lymphocyte responses in simian immunodeficiency virus-infected rhesus monkeys

Because the control of HIV-1 replication is largely dependent on CD8+ T lymphocyte responses specific for immunodominant viral epitopes, vaccine strategies that increase the breadth of dominant epitope-specific responses should contribute to containing HIV-1 spread. Developing strategies to elicit such broad immune responses will require an understanding of the mechanisms responsible for focusing CD8+ T lymphocyte recognition on a limited number of epitopes. To explore this biology, we identified cohorts of rhesus monkeys that expressed the MHC class I molecules Mamu-A*01, Mamu-A*02, or both, and assessed the evolution of their dominant epitope-specific CD8+ T lymphocyte responses (Gag p11C- and Tat TL8-specific in the Mamu-A*01+ and Nef p199RY-specific in the Mamu-A*02+ monkeys) following acute SIV infection. The Mamu-A*02+ monkeys that also expressed Mamu-A*01 exhibited a significant delay in the evolution of the CD8+ T lymphocyte responses specific for the dominant Mamu-A*02-restricted SIV epitope, Nef p199RY. This delay in kinetics was not due to differences in viral load kinetics or magnitude or in viral escape mutations, but was associated with the evolution of the Mamu-A*01-restricted CD8+ T lymphocyte responses to the highly dominant SIV epitopes Gag p11C and Tat TL8. Thus, the evolution of dominant epitope-specific CD8+ T lymphocyte responses can be suppressed by other dominant epitope-specific responses, and this immunodomination is important in determining the kinetics of dominant epitope-specific responses.     

Viral escape from dominant simian immunodeficiency virus epitope-specific cytotoxic T lymphocytes in DNA-vaccinated rhesus monkeys

Virus-specific cytotoxic T lymphocytes (CTL) are critical for control of human immunodeficiency virus type 1 replication. However, viral escape from CTL recognition can undermine this immune control. Here we demonstrate the high frequency and pattern of viral escape from dominant epitope-specific CTL in SIV gag DNA-vaccinated rhesus monkeys following a heterologous simian immunodeficiency virus (SIV) challenge. DNA-vaccinated monkeys exhibited initial effective control of the SIV challenge, but this early control was lost by serial breakthroughs of viral replication over a 3-year follow-up period. Increases in plasma viral RNA correlated temporally with declines of dominant SIV epitope-specific CD8(+) T-lymphocyte responses and the emergence of viral mutations that escaped recognition by dominant epitope-specific CTL. Viral escape from CTL occurred in a total of seven of nine vaccinated and control monkeys, including three animals that initially controlled viral replication to undetectable levels of plasma viral RNA. These data suggest that CTL exert selective pressure on viral replication and that viral escape from CTL may be a limitation of CTL-based AIDS vaccine strategies.