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Journal of Virology, July 2001, p. 6279-6291, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6279-6291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Relative Dominance of Epitope-Specific Cytotoxic T-Lymphocyte Responses in Human Immunodeficiency Virus Type 1-Infected Persons with Shared HLA Alleles

Cheryl L. Day,1 Amy K. Shea,1 Marcus A. Altfeld,1 Douglas P. Olson,1 Susan P. Buchbinder,2 Frederick M. Hecht,3 Eric S. Rosenberg,1 Bruce D. Walker,1 and Spyros A. Kalams1,*

Partners AIDS Research Center, Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 021141; AIDS Office, Department of Public Health, Division of AIDS, San Francisco, California 941202; and Positive Health Program, University of California at San Francisco, San Francisco, California 941433

Received 13 March 2001/Accepted 24 April 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cytotoxic T lymphocytes (CTL) target multiple epitopes in human immunodeficiency virus (HIV)-infected persons, and are thought to influence the viral set point. The extent to which HLA class I allele expression predicts the epitopes targeted has not been determined, nor have the relative contributions of responses restricted by different class I alleles within a given individual. In this study, we performed a detailed analysis of the CTL response to optimally defined CTL epitopes restricted by HLA class I A and B alleles in individuals who coexpressed HLA A2, A3, and B7. The eight HIV-1-infected subjects studied included two subjects with acute HIV infection, five subjects with chronic HIV infection, and one long-term nonprogressor. Responses were heterogeneous with respect to breadth and magnitude of CTL responses in individuals of the same HLA type. Of the 27 tested epitopes that are presented by A2, A3, and B7, 25 were targeted by at least one person. However, there was wide variation in the number of epitopes targeted, ranging from 2 to 17. The A2-restricted CTL response, which has been most extensively studied in infected persons, was found to be narrowly directed in most individuals, and in no cases was it the dominant contributor to the total HIV-1-specific CTL response. These results indicate that HLA type alone does not predict CTL responses and that numerous potential epitopes may not be targeted by CTL in a given individual. These data also provide a rationale for boosting both the breadth and the magnitude of HIV-1-specific CTL responses by immunotherapy in persons with chronic HIV-1 infection.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Cytotoxic T lymphocytes (CTL) play an important role in immune control of acute and chronic viral infections. Analyses of immune responses in individuals infected with human immunodeficiency virus type 1 (HIV-1) indicate that HIV-1-specific CTL are critical in controlling the initial viremia following acute infection and in suppressing viral replication during the chronic phase of infection. Studies of untreated individuals with acute HIV-1 infection show that the decline in viremia in acute infection is associated with the appearance of HIV-1-specific CTL (6, 7, 22, 38). In animals infected with simian immunodeficiency virus, antibody-mediated depletion of CD8 cells and CTL leads to an increase in viremia, which returns to baseline values when CTL reappear (31, 48). During chronic infection a negative association between HLA A2 Gag-specific CTL and viral load has been indicated (42), but other HLA alleles have not been similarly tested. Despite the clear antiviral activity of CTL, these cells fail to eliminate virus and numerous studies indicate that these responses actually decline with disease progression (reviewed in reference 61).

An increasing amount of data has been generated identifying viral peptides that are targeted by CTL, and these peptides largely conform to predicted motifs for HLA binding. CTL recognize infected cells through interaction of the T-cell receptor (TCR) with 8- to 11-amino-acid antigenic peptides complexed with major histocompatibility (MHC) class I molecules. The MHC-peptide complexes arise from intracellular processing of endogenously synthesized viral proteins. Although a large number of peptide epitopes may be generated, T-cell responses are focused to a select number of epitopes, a phenomenon known as immunodominance (reviewed in reference 62). The factors that determine which epitopes will be immunodominant in a given individual have not been clearly defined. Studies of alleles such as B14, B60, and A2 have shown that the majority of persons with these alleles recognize defined epitopes (10, 25, 36). However, no studies have examined the magnitude and specificity of responses in persons with multiple shared HLA alleles or the relative contributions of specific alleles to the total CTL response.

In this study we analyzed CTL responses in a cohort of subjects matched at the HLA A2, A3, and B7 alleles. These alleles were chosen because they are represented at high frequencies in the population studied (50), and the HIV-1 epitopes have been well characterized and optimally defined (9). The breadth, magnitude, and relative immunodominance of HIV-1-specific CTL responses in these individuals to 27 previously defined optimal A2-, A3-, or B7-restricted epitopes was determined by bulk stimulation of peripheral blood mononuclear cells (PBMC) as well as by enzyme-linked immunospot (Elispot) assay and intracellular cytokine staining (ICS). Furthermore, CTL responses to previously defined optimal epitopes restricted by the unshared HLA class I B allele were determined in each subject in order to more completely assess which epitopes are immunodominant in the context of HLA A and B alleles in each individual and to determine the relative contributions of these class I alleles to the total HIV-1-specific CTL response.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Subjects. Eight HIV-1-infected subjects were included in this study. Subjects were enrolled through the University of California at San Francisco, the San Francisco City Clinic, and the Massachusetts General Hospital. Subjects 11324, 11841, 13070, 16732, and 221L are individuals with chronic HIV infection receiving highly active antiretroviral therapy (HAART). Subjects AC-03 and OP337 are individuals with acute HIV-1 infection who were treated with HAART within 6 months of seroconversion. Subject 161j is a long-term nonprogressor who has been infected >20 years. The viral loads of the subjects ranged from <50 to 5,390 RNA copies per ml of plasma. All subjects gave written informed consent for these studies. The individual parameters (CD4 count in cells per cubic millimeter/viral load in RNA copies per milliliter of plasma/antiviral therapy) for each of the subjects were as follows: 161j, 1,400/<50/none; 221L, 1,184/478/stavudine, lamivudine, and indinavir; 13070, 358/5,390/zidovudine and lamivudine; 11841, 562/<250/didanosine and stavudine; 11324, 576/697/lamivudine, stavudine, and nelfinavir; 16732, 489/876/zidovudine, saquinavir, and stavudine; AC-03, 947/<50/zidovudine, lamivudine, and nelfinavir; and OP337, 800/1,599/stavudine, didanosine, and nelfinavir.

HLA typing. HLA typing was performed by the San Francisco Department of Public Health and/or by the Massachusetts General Hospital Tissue Typing Laboratory by standard serological and molecular techniques (13).

Synthetic peptides. The 9- to 11-amino-acid peptides used in this study have been reported in the Los Alamos Immunology Database (9). All were synthesized as COOH-terminal free acids on a Synegy 432A peptide synthesizer (Applied Biosystems, Foster City, Calif.). Lyophilized peptides were reconstituted at 2 mg/ml in sterile distilled water with 10% dimethyl sulfoxide (Sigma) and 1 mM dithiothreitol (Sigma).

Bulk stimulation of peripheral blood mononuclear cells. Cryopreserved PBMC (4 × 106) were stimulated with 106 autologous, peptide-pulsed PBMC. PBMC were incubated with each peptide (10 µg/ml) for 1 h at 37°C and then washed three times in R10. Irradiated feeder cells (15 × 106 allogeneic PBMC) were added to the culture in a 25-cm2 culture flask (Costar, Cambridge, Mass.). Recombinant interleukin-2 (25 U/ml) was added on day 4 and twice a week thereafter. After 10 to 14 days, the cells were assayed on 51Cr-labeled peptide pulsed autologous B-lymphoblastoid cell lines (B-LCL) in a standard 51Cr release assay (35).

Elispot assay. Cryopreserved PBMC were thawed in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U of penicillin per ml, and 50 µg of streptomycin per ml. Ninety-six-well nitrocellulose plates (Millipore, Bedford, Mass.) were coated with 0.5 µg of human anti-gamma interferon (IFN-gamma ) monoclonal antibody (MAb) (Mabtech, Stockholm, Sweden) per ml overnight at 4°C. Plates were washed with phosphate-buffered saline (PBS) plus 1% fetal calf serum. PBMC were added at 0.5 × 105 and 0.25 × 105 per well in duplicate wells. For detection of peptide-specific CD8+ T cells, synthetic peptides (10 µM) corresponding to defined optimal epitopes were added to PBMC. The peptides used are listed in Table 1. Following an overnight incubation at 37°C and 5% CO2, the plates were washed with PBS before 100 µl of biotinylated anti-IFN-gamma MAb (1 µg/ml; Mabtech) was added and incubated at room temperature for 90 min. After the plates were washed again with PBS, 100 µl of 1:2,000-diluted streptavidin-alkaline phosphatase conjugate (Mabtech) was added per well and the plates were incubated at room temperature for 45 min. Wells were washed again with PBS, and individual IFN-gamma -producing cells were detected as dark spots after a 30-min color reaction with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium using an alkaline phosphatase-conjugated substrate (Bio-Rad Laboratories, Hercules, Calif.). Spots were counted by direct visualization and are expressed as numbers of spot-forming cells (SFC) per 106 cells. The number of specific IFN-gamma -secreting T cells was calculated by subtracting the negative control value from the established SFC count. Wells with greater than 20 SFC/million PBMC were considered positive, based on analysis of seronegative controls (data not shown).

                              
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  		TABLE 1.   Description of optimal HLA class I-restricted HIV-1 epitopes testeda

Intracellular IFN-gamma staining. ICS was performed as described previously (21, 43). Briefly, 1.0 × 106 PBMC were incubated with 4 µM peptide and anti-CD28 and anti-CD49d MAbs (1 µg/ml each; Becton Dickinson) at 37°C and 5% CO2 for 1 h before the addition of GolgiPlug (1 µl/ml; Becton Dickinson). The cells were incubated for an additional 5 h at 37°C and 5% CO2. PBMC were then washed and stained with surface antibodies, antigen-presenting cell-conjugated anti-CD3 and phycoerythrin-conjugated anti-CD8 (Becton Dickinson) at room temperature for 20 min. Following the washing, the PBMC were fixed and permeabilized (Caltag, Burlingame, Calif.), and the fluorescein isothiocyanate-conjugated anti-IFN-gamma MAb (Becton Dickinson) was added. Cells were then washed and analyzed on a FACS-Calibur flow cytometer using CELLQuest software (Becton Dickinson).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Qualitative analysis of HLA class I A2-, A3-, and B7-restricted CTL responses in HIV-1-infected individuals by bulk stimulation of PBMC. To begin to address the relative contribution of single HLA class I alleles to the overall CTL response, we recruited three persons matched at the HLA A2, A3, and B7 alleles. CTL responses to 12 optimal A2-, A3-, and B7-restricted epitopes known at this time were analyzed by bulk stimulation of PBMC. These and subsequent epitopes chosen for use in this study (Table 1) contain motifs important for binding the relevant class I HLA allele (20) and were defined as optimal epitopes using truncated peptides at limiting concentrations (9). All epitopes were shown to be processed endogenously in infected cells, using vaccinia virus recombinants to express antigen and CTL clones specific for the epitopes in Table 1 (data not shown).

PBMC were pulsed with the A2, A3, or B7 peptides indicated, expanded for 10 days, and then assayed for the ability to lyse autologous B-LCL pulsed with the relevant peptides. Significant heterogeneity in the dominance and breadth of responses was demonstrated to these epitopes in the three individuals analyzed (Fig. 1). The dominant A2-restricted response in subject 161j is clearly directed against the Gag epitope p17 77-85, whereas no clearly dominant A2-restricted response could be identified in subjects 13070 and 221L (Fig. 1A). For the A3-restricted epitopes (Fig. 1B), subject 161j targeted two codominant epitopes (p17 18-26 and gp41 770-780) in addition to two other subdominant responses (p17 20-28 and Nef 73-82). In contrast, the dominant epitope for subject 13070 (Nef 73-82) was recognized least in subject 161j. The strongest A3-restricted response in subject 221L was directed against the p17 18-26 epitope, which was similarly targeted as a dominant epitope by subject 161j. Of the B7-restricted epitopes tested, the dominant responses in subject 161j were directed against gp41 843-851 and Nef 128-137. The most dominant epitope targeted by both 13070 and 221L was Nef 128-137 (Fig. 1C). These results indicate significant heterogeneity in the dominance of responses in persons of the same HLA type, as measured following in vitro stimulation of PBMC.


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  		FIG. 1.   HLA-A2-, A3-, and B7-restricted HIV-1-specific CTL responses in 161j, 13070, and 221L as determined by bulk stimulation of PBMC, followed by chromium release assay using B-LCL pulsed with the indicated peptides and nonpulsed B-LCL. (A) CTL responses to HLA-A2-restricted HIV-1 epitopes; (B) CTL responses to HLA-A3-restricted HIV-1 epitopes; (C) CTL responses to HLA-B7-restricted HIV-1 epitopes.

Breadth and magnitude of CTL responses by Elispot assay to optimal HIV-1 epitopes restricted by HLA class I A and B alleles in HIV-1-infected individuals with shared HLA class I alleles. The above assays rely on in vitro expansion of PBMC and require large numbers of cells. Newer methods assessing cytokine production by antigen-specific CD8 cells allow for rapid, direct quantitation and require fewer cells. We therefore expanded our analysis to include five additional HLA A2-, A3-, and B7-positive HIV-1-infected individuals, using the IFN-gamma Elispot assay to evaluate CTL responses to 27 optimal A2-, A3-, or B7-restricted epitopes in all eight subjects. Subjects 11324, 11841, 13070, 16732, 221L, and 161j all have chronic HIV-1 infection. AC-03 and OP337 were both identified with acute HIV-1 infection. All subjects except for 161j were receiving antiretroviral therapy, although virus was still detectable (>50 RNA copies/ml of plasma) in four subjects.

CTL responses to eight optimal A2-restricted epitopes were evaluated in each subject. p17 77-85 was the dominant epitope recognized in all subjects with chronic HIV-1 infection, except for subject 11841 for which RT 33-41 appeared to be the dominant A2-restricted epitope targeted. Subject 11841 also had the broadest A2-restricted HIV-1-specific CTL response, with five of the eight A2-restricted epitopes tested being targeted. Three of the subjects (11324, 16732, and 221L) had detectable A2-restricted CTL responses directed against the p17 77-85 epitope only. Subjects 13070 and 161j had very weak responses to the A2-restricted RT 33-41 and RT 309-317 epitopes, respectively, in addition to the p17 77-85 epitope. In contrast, both subjects with acute HIV-1 infection failed to recognize the p17 77-85 epitope. No A2-restricted epitopes were detected in AC-03, whereas OP337 recognized only the gp41 813-822 epitope. The dominance of the p17 77-85 epitope in subjects with chronic HIV-1 infection and the lack of response in individuals with acute HIV-1 infection are consistent with previously published studies of A2-restricted responses (10, 21, 24, 26, 42, 57). Furthermore, the magnitude of responses to each epitope was highly variable among individuals. The sum total number of A2-restricted CTL responses differed over 40-fold, ranging from 70 SFC/million PBMC in subject 16732 to 2,820 SFC/million PBMC in subject 161j.

CTL responses to eight optimally defined A3-restricted HIV-1 epitopes were similarly evaluated in these same eight individuals (Fig. 2). All subjects recognized at least one A3-restricted epitope (range, one to eight), but there was no clearly dominant epitope targeted by all subjects as had been observed with the A2-restricted epitope p17 77-85. In subjects 11324 and 11841, RT 158-166 is clearly targeted as the dominant A3-restricted response. The Elispot results indicate that in subjects 13070 and 16732, Nef 73-82 was the dominant A3-restricted epitope targeted. However, three A3-restricted epitopes were recognized in 221L with no one dominant epitope clearly targeted. All eight A3-restricted epitopes targeted were recognized by 161j, with epitopes p17 18-26 and gp41 770-780 generating the strongest CTL responses in the Elispot assay. Furthermore, there was over a 100-fold difference in the overall total magnitude of CTL responses to A3-restricted epitopes among the subjects evaluated, ranging from 40 SFC/million PBMC in subject 16732 to 4,700 SFC/million PBMC in subject 161j.





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  		FIG. 2.   HIV-1-specific CTL responses in eight HIV-1-infected individuals as determined by IFN-gamma Elispot assay. All optimal HIV-1 epitopes defined (9) were tested for each individual's HLA class I A and B alleles. Only epitopes recognized with more than 20 SFC/million PBMC are shown. The number in parentheses next to each epitope indicates the number of SFC/million PBMC. Subject OP337 is homozygous for the HLA B7 allele.

Eleven previously defined B7-restricted optimal epitopes were also tested in the Elispot assay in the eight subjects included in this study. As shown in Fig. 2, all subjects with chronic HIV-1 infection recognized at least two B7-restricted epitopes (range, two to eight). Subject OP337, who is homozygous for the B7 allele, recognized five B7-restricted epitopes. However, the other subject with acute HIV-1 infection, AC-03, failed to recognize any of the 11 B7-restricted epitopes tested. Similar to the results seen with the A3-restricted epitopes, the B7-restricted CTL response was highly variable among all subjects and there was no clearly dominant epitope targeted in most individuals. Figure 3 summarizes the breadth of CTL responses to all the optimal A2-, A3-, and B7-restricted CTL responses in the eight subjects analyzed.


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  		FIG. 3.   Frequency of recognition of all optimal A2-, A3-, and B7-restricted HIV-1 epitopes among the eight HIV-1-infected individuals included in this study. Gray boxes represent a positive CTL response in a subject by Elispot to a given epitope. Responses greater than 20 SFC/million PBMC above background were considered to be positive, based on analysis of seronegative controls (data not shown).

Relative contributions of CTL responses restricted by the second B allele in persons coexpressing A2, A3, and B7. The above results included analyses of the CTL response in each subject to HIV-1 epitopes restricted by the three HLA class I alleles shared by all of the subjects. We also determined CTL responses to optimal epitopes defined for each subject's HLA class I unmatched B allele in order to gain a more comprehensive view of the total HIV-1-specific CTL response in each individual. These alleles, including B8, B27, B44, B60, B61, and B62, are less common in the population, and there are fewer optimally defined epitopes for these alleles than for the A2, A3, and B7 alleles (9). As shown in Fig. 2, no immunodominant epitopes were recognized in subjects 13070 and 11324 in response to the five B61- and four B62-restricted epitopes tested, respectively. However, in subject 221L, the B8-restricted epitope Nef 90-97 was the second highest response in magnitude compared to all the HLA class I A- and B-restricted epitopes tested in this individual. Moreover, B8 accounted for about one-third of the total CTL response. Five B60-restricted epitopes were tested in subject 161j, and responses were detected against all five epitopes. The strongest CTL response detected in subject 161j was directed against the B60-restricted epitope p24 44-52, and all the B60-restricted responses together contributed over one-third of the total CTL response. Overall, the unmatched HLA class I B allele contributed between 0 and 38% of the total magnitude of the response, and the numbers of epitopes targeted through this fourth allele ranged from 0 to 5. These studies provide additional quantitative evidence that the magnitude and breadth of CTL responses differ considerably in persons of similar HLA types and indicate that the assessment of responses to three alleles still significantly underestimates the breadth and magnitude of the HIV-1-specific CTL response.

The breadth and magnitude of the HIV-1-specific CTL responses in the above experiments were determined by IFN-gamma production in an Elispot assay. In order to obtain a more precise definition of both the phenotype and the quantity of responding cells, we performed a more comprehensive analysis of the HIV-1-specific CTL responses in subject 161j by flow cytometry-based ICS assays. Thirty-two A2-, A3-, B7-, or B60-restricted epitopes were tested by Elispot assay in subject 161j, of which 22 epitopes generated positive responses (Fig. 2). Eighteen epitopes generated responses with a magnitude of >100 SFC/million PBMC. These 18 epitopes were then tested in an ICS assay for IFN-gamma production. Representative ICS data for 10 immunogenic epitopes recognized by subject 161j are indicated in Fig. 4. Of the 18 epitopes tested by ICS, the sum total percentage of CD8+ T lymphocytes specific for HIV-1 in subject 161j was 10.9% (Fig. 4 and data not shown). These data confirm the magnitude and breadth of CTL responses and together with the Elispot data provide firm evidence that the immune response can be extremely broadly directed in some persons.


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  		FIG. 4.   Recognition of optimally defined A2-, A3-, B7-, and B60-restricted HIV-1 epitopes in subject 161j by ICS for IFN-gamma production. The percentage of CD8+ T cells expressing IFN-gamma (minus background IFN-gamma production) is indicated in the upper right-hand corner of each plot. The no-peptide control shows the percentage of IFN-gamma production by CD8+ T cells in the presence of costimulatory molecules only.

Relative contributions of HLA class I A- and B-restricted epitopes to the overall HIV-1-specific CTL immune response. The above studies indicate heterogeneity in the magnitude and breadth of CTL responses to individual known HIV-1 epitopes in persons sharing multiple MHC class I alleles. We next analyzed the relative contributions of the HLA class I A and B alleles to the total HIV-1-specific CTL response in the eight individuals included in this study. The sum of the CTL responses as determined by Elispot assay for each HLA class I A and B allele are shown in Fig. 5 for each subject. The overall highest magnitude of responses was seen in subject 161j, with combined CTL responses to all epitopes of >18,000 SFC/million PBMC. These responses are over 33-fold greater than those seen in subject 16732, who had the lowest total magnitude of responses.


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  		FIG. 5.   Total magnitude of HIV-1-specific CTL responses for the eight HIV-1-infected subjects included in this study. The sum of the individual epitopes for each HLA class I A and B allele is indicated as follows: black bars, A2 epitopes; white bars, A3 epitopes; gray bars, B7 epitopes; spotted bars, second B allele.

The percent contribution of each HLA class I A and B allele to the total HIV-1-specific CTL response is indicated for each subject in Fig. 6. The relative contributions of each A and B allele were highly variable among all subjects. In subject AC-03 with acute HIV-1 infection, no responses were detected to any A2- or B7-restricted epitopes. In subjects 11324 and 13070, all of the HIV-1 epitopes recognized were restricted by either the A2, A3, or B7 allele. The response restricted by A2, which has been the most commonly studied HLA class I allele in terms of analyzing CTL responses in HIV-1 infection, was not the dominant contributor to the total CTL response with respect to the other three class I A and B alleles in any of the eight individuals studied. Subject 11841 exhibited the highest percentage of A2-restricted CTL responses, with 30% of total CTL responses detected in this study attributable to A2-restricted epitopes. However, in all seven of the other subjects, the contribution of the A2-restricted epitopes to the total CTL response ranged from 0% in subject AC-03 to 15% in subjects 221L and 161j. Hence, analysis of A2-restricted epitopes alone does not allow for sufficient representation of the total HIV-1-specific CTL response in an individual. These findings are consistent with previous studies among HLA-A2- and HIV-1-positive individuals by Betts et al. (5).


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  		FIG. 6.   Relative contributions of each individual HLA A and B allele to the total HIV-1-specific CTL response in eight HIV-1-infected individuals, all expressing HLA A2, A3, and B7. Percentages were determined by dividing the total number of SFC/million PBMC for each allele by the sum total of the SFC/million PBMC for all four A and B alleles.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In this study we have analyzed HIV-1-specific CD8+ T-cell responses among a cohort of subjects matched at three common HLA class I alleles. Analysis of these CD8+ responses in eight HIV-1-infected individuals coexpressing the A2, A3, and B7 alleles revealed a wide range in both the breadth and the magnitude of responses. Although all but two of the optimal epitopes reported to be presented by these alleles were recognized by at least one person, the percentages of persons targeting each of the peptides differed over a wide range, as did the magnitude of the responses. The most frequently recognized epitope was A2-restricted p17 77-85, although the A2-restricted response was narrowly directed in most individuals. In addition, the CTL responses to A2-restricted epitopes were not the major contributors to the total HIV-1-specific CTL response by the HLA class I A and B alleles. The patterns of immunodominance also differed significantly among the persons tested, with HLA type not being a predictive factor of which epitopes will be targeted as a dominant response. The lack of recognition of some epitopes suggests that many potential epitopes are not being targeted, and that the immune response to HIV-1 might be broadened in infected persons.

This is the first study, to our knowledge, to analyze the breadth and magnitude of CTL responses to optimal epitopes from several HIV-1 gene products in multiple individuals matched at multiple class I alleles. There has been one previous study, by Goulder et al. (24), that investigated patterns of immunodominance in two HLA-identical HIV-1-infected siblings. Bulk CTL assays were done with eight optimal epitopes in each sibling and, consistent with our results presented in this paper, the CTL response profile was different for each sibling, in terms of the percent specific lysis for each epitope and which epitopes were targeted as the dominant response. Previous studies by Betts et al. (5) analyzed HIV-1-specific CTL responses in a cohort of A2-positive individuals and their recognition of the putative immunodominant A2-restricted epitope p17 77-85. Their results agree with ours in that recognition, or lack thereof, of this epitope is not representative of the total HIV-1-specific CTL response. Their study, however, compared individuals matched only at the A2 allele and did not assess similarities or differences of CTL responses to individual optimal A2-restricted epitopes other than the p17 77-85 epitope among the cohort studied.

Several factors likely contribute to immunodominance, including efficiency of processing of peptides, binding of peptides to MHC class I molecules, affinity of T-cell receptors for peptide-MHC complexes, and development of immune escape. It is not clear from the results of this study why individuals of the same HLA type do not target the same epitopes. Immune escape alone cannot account for lack of recognition. Sequencing of autologous virus was performed to begin to address whether lack of response to a particular epitope was due to sequence variation in the autologous virus of the individual. Eight sequences of the B7-restricted Nef epitope 128-137 were obtained from each one of subjects 11841, 16732, and 13070 (data not shown). Subjects 11841 and 13070 both showed an autologous virus sequence identical to the consensus epitope sequence; however, subject 11841 did not recognize this epitope at detectable levels in the Elispot assay, whereas subject 13070 recognized this epitope at 2,980 SFC/million PBMC. Preliminary sequence data therefore indicate that lack of response to a given epitope is not due solely to immune escape and mutation of the epitope. It has been suggested that mutations in the amino acids of the flanking sequences of an epitope may affect efficient processing of the epitope such that it is not presented on the MHC class I allele and potentially resulting in CTL escape (18, 52, 55). Previous studies have failed to show this in HIV infection (11). The lack of recognition of specific epitopes thus remains to be explained.

Our data presented here demonstrate a marked degree of heterogeneity in CTL responses, but the degree of heterogeneity is likely to be even larger than shown here because of the expected contribution to the HIV-1-specific immune response of MHC class I C alleles expressed by these individuals, which was not evaluated in this study. Furthermore, this study analyzed CTL responses to epitopes that have been optimally defined for each individual's class I A and B alleles, and it is likely that CTL responses are present to epitopes that have yet to be defined. Studies are under way to define new CTL epitopes in the HIV-1 regulatory proteins, including Tat, Rev, VPR, and Vif (1, 8). Analysis of CTL responses to these viral proteins has not been included in this study; therefore, the data presented here in terms of the total magnitude and breadth of responses most definitely underestimate the total HIV-1-specific CTL response in these individuals.

It should be noted that many of the subjects were on antiviral therapy at the time of analysis. Although this may result in the diminution in responses over time (35, 42), established responses are not lost (3). Even though responses measured may in part be lower in magnitude due to therapy, the study still allows comparative analysis of responses driven by the viral load present in a given individual. Our data also provide additional evidence that the breadth and magnitude of the response can be immense in persons such as 161j who control viremia spontaneously.

In summary, we conclude that there are marked differences in the breadth and magnitude of the CTL response to optimal HIV-1 epitopes in individuals coexpressing the A2, A3, and B7 alleles. These studies thus indicate that HLA type alone is a poor predictor of which epitopes will be targeted in a given individual, and potential epitopes may not be targeted in some persons despite the presence of virus containing peptide sequences predicted to bind to the class I molecule. The identification of immunodominant epitopes targeted in HIV-1 infection has important implications in epitope-based vaccine development. None of the subjects studied targeted all of the potential epitopes that would be predicted to be targeted based on their HLA haplotype. These results indicate that a clear opportunity exists to broaden the repertoire of HIV-1-specific CTL responses and provide rationale for the development of immunotherapy to augment CTL responses in chronic HIV-1 infection.


    ACKNOWLEDGMENTS

This project was supported by the National Institutes of Health (AI39966, AI28568, AI42851, and AI50914) and the Doris Duke Charitable Foundation. B.D.W. is a Doris Duke Distinguished Clinical Science Professor.


    FOOTNOTES

* Corresponding author. Mailing address: Massachusetts General Hospital, AIDS Research Center, 149 13th Street, Rm. 5217, Charlestown, MA 02129. Phone: (617) 724-4958. Fax: (617) 726-4691. E-mail: kalams{at}helix.mgh.harvard.edu.


    REFERENCES
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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Journal of Virology, July 2001, p. 6279-6291, Vol. 75, No. 14
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.14.6279-6291.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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