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Most Highly Exposed Seronegative Men Lack HIV-1-Specific, IFN--Secreting T Cells ¹

Florian Hladik^*, Anthony Desbien^*, Jean Lang, Lei Wang, Yan Ding^*, Sarah Holte, Aaron Wilson^*, Younong Xu^*, Micky Moerbe^*, Steve Schmechel and M. Juliana McElrath^2,*,,

^* Program in Infectious Diseases, Clinical Research Division, and Program in Biostatistics, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and Departments of Laboratory Medicine and Medicine, University of Washington School of Medicine, Seattle, WA 98195

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Naturally acquired cellular immunity in individuals who have been exposed to HIV-1 but have remained uninfected may hold clues for the design of an effective HIV vaccine. To determine the presence and nature of such an HIV-1-specific immune response, we evaluated the quantity and fine specificity of HIV-1-reactive IFN--secreting T cells in a group of highly exposed seronegative men having sex with men. All 46 ES reported frequent unprotected anal sex with known HIV-1-infected partners at enrollment, and high risk activities continued in at least one-half of the volunteers for up to >6 years of observation. Despite the high frequency of unprotected anal intercourse and potential HIV-1 exposure, the vast majority of individuals demonstrated no or very low numbers of HIV-1-specific, IFN--secreting T cells. Even when HIV-1 epitopes were presented by peptide-pulsed autologous dendritic cells in 15 of the highest risk volunteers, HIV-1-specific T cells remained infrequent, and the proportion of responders was not significantly different from that in a lower risk seronegative control cohort. Only PBMC from two individuals who have remained uninfected to date exhibited distinctly positive responses. However, these responses rarely persisted over time, single epitope specificities were identified in only one volunteer, and HIV-1-specific memory T cell clones did not expand in vitro. HIV-1-specific, IFN--secreting T cells are thus unlikely to substantially contribute to resistance against infection in most exposed seronegative men having sex with men.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Understanding strategies to prevent the transmission of and reduce susceptibility to HIV-1 infection is paramount in controlling the pandemic. Some insights have emerged from epidemiological and population-based studies. For example, the probability of acquiring HIV-1 infection depends on the route and nature of exposure as well as the size of the viral inoculum, which relates to the viral load of the source partner (1). In addition, the inheritance of certain HIV-1 coreceptor polymorphisms and HLA alleles is associated with a more favorable course of HIV-1 disease and in some instances with a reduced susceptibility to infection (2, 3, 4, 5, 6). However, there is no evidence as yet that natural immunity to HIV-1 infection exists and can protect against infection. If such immunity could be identified, the findings would have major relevance to vaccine development.

Steps toward understanding potential mechanisms of natural immunity have focused on observational studies of persons who resist infection despite repeated viral exposure. The most remarkable findings have emerged from a group of highly exposed commercial sex workers (CSW)³ in Nairobi who have remained free of infection for at least 3 and up to 12 years (7, 8, 9). Over time, we and others have described additional cohorts with similar apparent abilities to resist infection. The presence of HIV-1-specific cellular immunity (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) and in some cases humoral immunity (18, 19, 20, 21, 22, 23, 24, 25, 26, 27) has been demonstrated in these individuals, and it was not linked to the inheritance of protective HIV-1 coreceptor mutations. To date, the most consistent finding in exposed seronegative (ES) cohorts has been the presence of HIV-1-specific CD8⁺ CTLs, detected by either cytolytic activities or secretion of antiviral mediators such as IFN-.

Studies in HIV-1-infected individuals have demonstrated that HIV-1-specific CTL are critical for the control of viremia (28, 29, 30, 31, 32, 33), making the possibility that CTL assume a similar role in protecting ES individuals against overt HIV-1 infection an attractive hypothesis. However, linking CTL to protective immunity in ES individuals has been difficult. Frequencies of HIV-1-specific CTL are generally lower in ES than in HIV-1-infected individuals, and often the responses are intermittent and/or not persistent (7, 34). Although HIV-1-specific CD8⁺ T cell lines and CD4⁺ T cell clones from ES blood have been demonstrated by two groups in 1995 and 1997, respectively (15, 35), this has not been possible in most cases, which has created skepticism about their very existence. In addition, there has been no direct demonstration that CTL decline before certain ES fail to remain protected and seroconvert (8).

Most CTL studies in ES individuals have been conducted in female CSW in Africa (7, 8, 9, 11, 12) or heterosexual couples discordant for HIV-1 infection (14, 20). Since the per-contact probability of HIV-1 transmission through the genital mucosa is only 0.001 (1), a distinction between heterosexual ES who remain uninfected on the basis of protective CTL or chance alone requires very large cohorts and/or extremely high exposure rates. In contrast, the per-contact infectivity of anal intercourse is 1 log higher (1), and men having sex with men (MSM) who frequently practice unprotected sex with HIV-1-infected partners are potentially more suitable to address the role of HIV-1-specific CTL in protection.

To understand the factors contributing to the induction and persistence of CTL in ES, we initiated a study to first define the quantity, fine specificities, and breadth of CD8⁺ CTL and CD4⁺ Th cell responses in a well-characterized cohort of MSM followed longitudinally up to 6 years in Seattle, WA. The 46 MSM reported frequent unprotected anal sex with known HIV-1-infected partners. We initially employed a standard IFN- ELISPOT assay to assess responses to HIV-1 Env, Gag, Pol, and Nef epitopes. This procedure had undergone vigorous quality control analysis and validation for implementation into phase I and II vaccine trials (36). To amplify responses in cases of low precursor frequencies just below the level of detection, we also analyzed selected very high risk individuals by optimized ELISPOT and intracellular IFN- flow cytometric protocols using autologous dendritic cells (DCs) for Ag presentation. Despite continued and frequent high risk behavior, we observed minimal to absent frequencies of IFN--secreting, HIV-1-specific T cells in the majority of individuals tested, even with DC stimulation. Clearly positive responses were present in only three individuals, one of whom seroconverted 6 mo after ELISPOT testing. Our findings suggest that in the majority of our highly exposed cohort neither CD8⁺ CTL nor CD4⁺ Th cells were critical for protection against HIV-1 infection.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study population

Healthy HIV-1 seronegative men, 18 years old, who reported high risk sexual activity with known HIV-1-infected men were recruited within metropolitan Seattle for longitudinal study at the Fred Hutchinson Cancer Research Center HIV-1 Vaccine Trials Unit. Enrollment criteria were unprotected anal receptive and/or insertive intercourse with a known HIV-1-infected man six times or more in the previous 6 mo or an average of twice weekly for 4 mo within 2 years of enrollment. Prior receipt of an HIV-1 vaccine was an exclusion criterion. Volunteers entering the study were designated ES. A medical history, physical examination, complete blood cell count, T cell subset analysis, and HIV-1 testing were performed at the screening visit to confirm eligibility. HIV-1 infection was excluded by the following tests performed at the screening visit and on study day 0: HIV-1/2 ELISA and Western blot (37), HIV-1 plasma RNA RT-PCR (Amplicor HIV-1 Monitor; Roche, Branchburg, NJ) (38), and PBMC DNA PCR (39). HIV-1/2 ELISA and Western blot were repeated every 3 mo. HIV-1 PBMC DNA PCR was performed again 4 and 8 wk after enrollment and then repeated yearly. Clinicians performed HIV-1 risk reduction counseling during each visit. The volunteers completed a questionnaire concerning risk behavior and provided blood during visits scheduled every month over the initial 3 mo and every 3–6 mo thereafter.

A control group of 13 HIV-1-seronegative, monogamous, and sexually active individuals, 18 years of age, were also recruited for comparison of T cell responses to HIV-1. These subjects, designated ES controls, were all Caucasian and consisted of nine homosexual men, one heterosexual man, and three heterosexual women. They reported at least one episode of unprotected intercourse with an HIV-1-seronegative partner in the 6 mo before screening and no previous history of HIV-1 exposure. In addition, the HIV-1-infected partners of the ES were recruited as previously described (10). They all tested positive by HIV-1 ELISA and Western blot and thus provided confirmation of high risk exposure.

Each volunteer provided written consent before enrollment. All aspects of the study were approved by the institutional review board at the University of Washington and the Fred Hutchinson Cancer Research Center.

CCR5 genotype analysis

To determine whether volunteers inherited the 32-bp deletion within the trans-membrane region of CCR5, DNA was isolated from PBMC and amplified by PCR with primers and conditions previously described (2, 3). The amplicons were subjected to agarose gel electrophoresis, and the size was assessed for the absence or the presence of a 32-bp deletion as previously described (10).

IFN- ELISPOT assay

To evaluate T cell responses recognizing HIV-1 epitopes, IFN- ELISPOT assays were performed either on whole PBMC or selected T cell subsets. For the latter, PBMC were reacted with anti-CD8 magnetic microbeads (Miltenyi Biotec, Auburn, CA) and sorted into CD8⁺ and CD8^- cells on a paramagnetic separation column according to the manufacturer’s instructions. On the average, 70–90% of PBMC were recovered after magnetic bead selection, the ratio of the purified CD8⁺ and CD8^- fractions was 1:2.3, and purity of the CD8⁺ T cell fraction was >95%.

Synthetic 20-mer peptides overlapping by 10 aa and synthetic 15-mer peptides overlapping by 11 aa, each set spanning the entire HIV-1 subtype B (HXB2) Gag, Env, Pol, and Nef region, were obtained through the National Institutes of Health AIDS Research and Reference Program (Bethesda, MD). Optimal 8- and 9-mer peptides with known HLA class I restriction patterns were synthesized by either Chiron (Emeryville, CA) or the Proteomics Facility of the Fred Hutchinson Cancer Research Center. The peptides were reconstituted at a concentration of 2 mg/ml in 100% DMSO (Sigma-Aldrich, St. Louis, MO) and were used at a final concentration of 2 µg/ml. A pool of five irrelevant peptides derived from actin and a highly conserved region of HLA class I -chain precursor were used as negative controls (Mimotopes, Clayton, Australia).

Cryopreserved PBMCs were thawed, allowed to rest in complete medium overnight at 37°C in 5% CO₂, and plated in 96-well, hydrophobic, polyvinylidene difluoride-backed plates (Millipore, Bedford, MA) previously coated with 50 µl of 10 µg/ml anti-IFN- mAb (clone 1-D1K; Mabtech, Nacka, Sweden) overnight at 4°C. The cells were added to the wells in either duplicate or triplicate at 2 x 10⁵ cells/well in 100 µl of complete medium. HIV-1-specific peptides and negative control peptides were added to the wells at a final concentration of 2 µg/ml. Cells stimulated with PHA (1 µg/ml; Sigma-Aldrich), staphylococcal enterotoxin B (SEB; 1 µg/ml; Sigma-Aldrich), or an immunodominant HLA-A2-restricted CMV p65 9-mer peptide (NLVPMVATV, 2 µg/ml; Mimotopes) served as positive controls. Plates were incubated overnight (16–20 h) at 37°C in 5% CO₂, washed with PBS containing 0.05% Tween 20, and incubated at room temperature for 2 h with biotinylated anti-IFN- mAb at 1 µg/ml (7-B6-1; Mabtech). The avidin-biotinylated enzyme complex from the Vectastain ABC Elite Kit (PK-6100; Vector Laboratories, Burlingame, CA) was added at room temperature for 1 h, followed by the Vectastain 3-amino-9-ethyl-carbazole peroxidase substrate. Spot-forming cells (SFC) were counted using an automated ELISPOT reader (Immunospot; Cellular Technology, Cleveland, OH).

Use of DCs as APCs in the IFN- ELISPOT assay

To generate autologous DCs in vitro, PBMCs were enriched for DC precursors and monocytes by plastic adherence for 90 min at 37°C in 5% CO₂ (40). The nonadherent fraction was discarded, and the adherent cells were cultured for 8 days with 800 U/ml GM-CSF and 800 U/ml IL-4 (PeproTech, Rocky Hill, NJ) in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM HEPES (BioWhittaker, Walkersville, MD), and 10% heat-inactivated FBS (Gemini, Calabasas, CA; complete medium). The cytokines were added to the cultures on days 0, 3 and 6. On day 6, cultures were supplemented with monocyte-conditioned medium (MCM) at 25% (v/v) to induce maturation (41). Mature DCs were collected on day 8.

The DC-ELISPOT was conducted similarly to the standard overnight ELISPOT assay with the following changes: 10⁴ mature DCs/well were first added in duplicate or triplicate to the anti-IFN- mAb-coated, 96-well, hydrophobic, polyvinylidene difluoride-backed plates in 50 µl of complete medium. The DCs were pulsed with the HIV-1-specific 15-mer and negative control peptides at a final concentration of 4 µg/ml for 1 h at 37°C in 5% CO₂. CD8-enriched and CD8-depleted PBMC fractions were then added in 50 µl of complete medium at a final concentration of 10⁵ cells/well for overnight incubation and subsequent IFN- detection.

Statistics and criteria for a positive HIV-1-specific ELISPOT assay

Absolute SFCs are reported per 10⁵ mononuclear cells and after subtraction of background IFN- secretion in the cultures containing the control peptides. Criteria for a positive culture are based on a likelihood ratio statistic assuming the numbers of SFCs per well approximate a simple Poisson distribution (36, 42).⁴ This allows conversion of data into a binomial distribution by calculating, for each peptide pool tested, the proportion of total SFCs attributable to the experimental wells (with the total SFCs being the sum of all SFCs in the experimental and control wells). We then applied an exact one-sided test to determine the likelihood that this proportion is significantly greater than would be expected if the SFCs were randomly distributed across the experimental and control wells. Results with a likelihood of 0.01 of having occurred merely by chance were considered positive. Since multiple peptide pools were tested per assay in each individual, this level was adjusted for multiple comparisons by the Bonferroni correction. Differences between the ES and the control cohort were tested for statistical significance using Fisher’s exact test. With 15 subjects in the ES and 13 subjects in the control cohort, a difference of 50% in the proportion of responders between the two groups can be detected by a one-sided test with 0.05 and a power of >80%.

Detection of intracellular IFN- production by flow cytometry

Staining for intracellular IFN- was performed as described by Bitmansour et al. (43). In brief, thawed PBMCs were stimulated with anti-CD28 mAb (L293; BD Biosciences, San Diego, CA) and anti-CD49d at 5% CO₂ in 37°C (HP2/1; Coulter, Miami, FL; both 1 µg/ml final concentration), followed by the HIV-1-specific 15-mer, the negative control peptide, or SEB as the positive control. After incubation for 1.5 h, brefeldin A (10 µg/ml; BD Biosciences) was added for an additional 5 h. Cells were then washed and stained with fluorochrome-conjugated mAbs for surface markers (anti-CD4-PE, RPA-T4; anti-CD8-PE-Cy5, HIT8a; anti-CD3-allophycocyanin, SK7) and intracellular IFN- (anti-IFN--FITC, 25723.11; all from BD Biosciences) according to the manufacturer’s protocol. In some experiments a combination of anti-IFN--FITC, ethidium monoazide bromide (EMA; Molecular Probes, Eugene, OR), anti-CD14-PE (RMO52; Coulter), anti-DC-SIGN-PE (120507; R&D Systems, Minneapolis, MN), anti-CD4-PE-Cy5 (RPA-T4), and anti-CD8-allophycocyanin (RPA-T8; both from BD Biosciences) was used to exclude EMA⁺ dead cells, CD14⁺ monocytes, and DC-SIGN⁺ DCs, all falling in the FL2 channel, from the analysis. Cells were fixed in 0.5% paraformaldehyde (JT Baker, Phillipsburg, NJ), and analyzed on a Calibur flow cytometer (BD Biosciences) for four-color fluorescence. Cells incubated with the negative control peptide pool and/or stained with matching isotype control mAbs (BD Biosciences) were used to define background staining and quadrant markers. Cells stained with only one specific mAb were used to compensate for overlapping signals between different fluorescence channels. Absolute IFN--positive cells are reported per 10⁵ mononuclear cells and after subtraction of background IFN- secretion in the cultures containing the control peptide pool. Cultures were considered positive if the counts of IFN- positive cells were 2.5 times the negative control.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study population

The ES study population consisted of 46 MSM with an identified HIV-1-infected sexual partner who met the enrollment criteria and had no evidence of HIV-1 infection by serology and routine PCR testing. Forty-four (92%) reported unprotected anal intercourse six times or more during the 6 mo before enrollment, and two (4%) engaged in these activities a minimum of twice weekly for 4 mo within the past 2 years. Seven of the 46 MSM also acknowledged i.v. drug use. The median age at study entry was 38 years (range, 24–60). The majority (96%) of the study participants were Caucasian; two ES were Hispanic. Two ES individuals were homozygous (ES7 and ES61), and 12 were heterozygous for the CCR532 mutation, which were higher, but not statistically different, frequencies than those reported for the general population (3).

The ES MSM were followed for a median of 280 wk (range, 30–340 wk) or 5.4 years. The majority continued to report unprotected anal intercourse with known HIV-1-infected partners (70% during the first 6 mo after recruitment), although this activity tended to decline over time (Table I and Fig. 1). At present, 18 of 39 ES individuals (46%) who were followed for 1 year still practice unprotected anal intercourse with known HIV-1-infected partners. At the same time, high risk sexual activity with partners of unknown HIV-1 status increased nearly 2-fold, from 13 of 46 (28%) ES individuals practicing unprotected anal sex during the first 6 mo after recruitment to 20 of 39 ES individuals (51%) at present (Table I). The total number of sexual partners also increased during the study period (Table I). The number of individuals reporting five or more different partners per 6-mo period nearly doubled from 14 during the first 6 mo of the study to 22 at present. At the most recent visit, only 13 ES reported sex exclusively with the same HIV-1-infected partner as at the time of enrollment.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Reported high risk sexual activities of 46 ES homosexual men since recruitment into the cohort. Each dot indicates that the individual reported unprotected anal intercourse with a known HIV-1-infected partner since his last visit in the clinic. Individuals are sorted by the density of high risk activity over the observation period. The color of a dot categorizes the frequency of high risk activities since the last visit (red dot, more than five high risk events per month; yellow dot, one to five high risk events per month; blue dot less than one high risk event per month). June 2002 is marked with a vertical line, the seroconversion date of the six seroconverters is marked by an s. Individuals with bold ID numbers were analyzed for HIV-1-specific T cell responses using the DC-ELISPOT assay. The initial DC-ELISPOT assay dates are indicated by a filled black triangle for positive tests and an open black triangle for negative tests. Additional DC-ELISPOT assays performed subsequently are indicated by a filled blue triangle for positive tests and an open blue triangle for negative tests. Some individuals were also analyzed for intracellular IFN- by flow cytometry at additional time points. Positive tests are marked by a gray triangle with a continuous border; negative tests are indicated by a gray triangle with a dashed border. The tips of all triangles are pointing downward and, if unprotected intercourse with a known HIV-1-infected partner was recorded at this clinic visit, they touch the colored dot to which they apply.

HIV-1-specific T cell responses by standard IFN- ELISPOT assay

To determine whether HIV-1-specific, IFN--secreting T cells are present in the peripheral blood of the ES MSM, we tested PBMC from 26 ES individuals at one visit (n = 19) or two or more visits (n = 6) using an overnight ELISPOT assay. These analyses were focused on fresh specimens and cryopreserved PBMC from known CTL responders and nonresponders (10). The expression of HIV-1 epitopes was accomplished by pulsing PBMC with pools containing 10 20-mer peptides overlapping by 10 aa and spanning the complete HIV-1 Env, Gag, Pol, and Nef region. This assay and the statistical criteria used to determine positive HIV-1-specific responses were validated in previous experiments. In comparisons between uninfected unvaccinated controls and either HIV-1-seropositive individuals (36) or recipients of a recombinant canarypox ALVAC-HIV vaccine (vCP 1452) in a large phase II vaccine trial (H. Horton et al., manuscript in preparation), the test approached a specificity of 100%. As shown in Fig. 2, this assessment allows robust discrimination of positive responses based on a constant probability of false positive responses 0.01 regardless of varying numbers of IFN- SFCs in the negative control wells.

View larger version (16K): [in this window] [in a new window]   FIGURE 2. IFN- ELISPOT responses in 26 ES homosexual men. IFN- production of bulk PBMCs to peptide pools spanning the complete HIV-1 Gag, Env, Pol, and Nef region was measured by standard overnight ELISPOT assay. The horizontal axis is the total number of SFCs in the experimental and control wells. The vertical axis is the proportion of the total number of SFCs attributable to the experimental wells. The jagged line is the cutoff between negative and positive results based on a set false positive rate of 0.01 and 26 different peptide pools tested. The cutoff was determined as described in Materials and Methods and was adjusted for multiple comparisons by the Bonferroni correction. Each peptide pool was tested either in duplicate or triplicate wells. Each individual symbol represents one peptide pool, and the shape of the symbol signifies each of the 26 study participants. For individuals tested more than once (n = 6), only one visit is shown. In the legend, only the symbols of the four positive responders are specified.

Amplification of HIV-1-specific IFN- production following Ag presentation by autologous DCs

The low frequency of HIV-1-specific, IFN- secreting T cells in this highly exposed cohort suggested that HIV-1-specific CTL were mostly absent or their detection required more sensitive techniques. Thus, we considered alternative approaches to identify these responses if present. Since DCs are extremely efficient at Ag presentation and activation of T cells, the use of DCs to present the HIV-1 peptides could potentially reveal low level HIV-1-specific responses (45, 46, 47, 48). We therefore established an ELISPOT protocol that employed autologous DCs for peptide presentation (DC-ELISPOT). The assay conditions were delineated first in six chronically HIV-1-infected individuals and then employed in the ES and control groups.

Initial studies determined the specific number of days of DC differentiation and maturation to achieve optimal stimulation. Amplification of responses was observed at all three time points of DC maturation tested. However, the best overall enhancement of HIV-1-specific responses compared with stimulation in the absence of autologous DCs was achieved when DCs were used on day 8 of in vitro differentiation and after 2 days of maturation with MCM (Fig. 3a, representative donor). Under these conditions, autologous DCs mixed with PBMC at a ratio of 1:10 amplified responses to either HIV-1 peptide pools or single peptides an average of 3.2-fold (range, 1.69–7).

View larger version (25K): [in this window] [in a new window]   FIGURE 3. Development of the DC-ELISPOT assay. a, Responses to the HIV-1 Gag27 20-mer peptide in a chronically HIV-1-infected patient after overnight peptide stimulation with or without autologous DCs. Autologous DCs were used after 6, 8, or 10 days of in vitro differentiation. Day 8 and day 10 DCs were matured by addition of MCM on day 6 of culture. b, Responses to HIV-1 peptide pools and single peptides in six HIV-1-infected patients, five of whom were LTNP. MCM-matured day 8 DCs were used at a ratio of 1 DC per 10 responder cells. Responder cells were whole PBMC in all individuals except LTNP13 and LTNP15, in whom CD8+ T cells were tested. Peptide pools consisted of 15-mer peptides in all individuals except 0471, in whom pools containing 20-mer peptides were used. All single peptides were 20-mers. c, Responses to five different HIV-1 20-mer peptide pools in an ES MSM (ES40) after overnight peptide stimulation with or without autologous DCs. MCM-matured day 8 DCs were used. HIV-1-specific responses determined positive using our established criteria are indicated by a plus sign. SEB and a CMV peptide (see Materials and Methods) served as positive controls. Error bars are SEMs of triplicate cultures. Background responses in the control cultures treated with an irrelevant peptide pool are shown in b and c and were subtracted from the other responses.

Finally, we determined the ability of mature autologous DCs to trigger HIV-1-specific T cells to produce IFN- in ES40. As previously shown, ES40 exhibited low level responses in two of three visits by the standard ELISPOT assay (Fig. 2), and the DC-ELISPOT studies were performed with PBMC from an additional visit. Peptide presentation by autologous DCs sufficiently enhanced the sensitivity of detection to uncover low frequency, IFN--secreting memory T cells responsive to the HIV-1 Env_1–10 and Env_21–30 20-mer peptide pools, which would have been missed by standard ELISPOT assay (Fig. 3c). Responses to the other three HIV-1 peptide pools tested remained negative by our criteria. In addition, the responses to the irrelevant control peptides were similar for cultures with or without DCs and were <5 IFN- SFCs/10⁵ PBMC. These results indicate that peptide presentation by mature autologous DCs is a suitable method to reveal low frequencies of circulating HIV-1-specific, IFN--secreting memory T cells in ES, and thus we proceeded to evaluate the ES and the ES controls using this approach.

Frequency and magnitude of HIV-1-specific responses in ES and ES controls tested by DC-enhanced ELISPOT assay

To improve the distinction between true false positive responses due to recognition of non-HIV-1 cross-reactive epitopes and genuine responses to HIV-1 epitopes, we conducted the DC-ELISPOT in 13 HIV-1-uninfected adults who were monogamous with a known HIV-1-uninfected partner along with the studies in the ES. In addition to using DCs as APCs, we further increased the sensitivity for detection of HIV-1-specific T cells by employing peptides that were 15 aa in length (overlapping by 11 aa) instead of the 20-mers (36). Five of 13 controls exhibited reactivity to HIV-1 peptide pools, and the IFN--secreting responses were mediated by both CD8⁺ (n = 3) and CD8^- (n = 4) subsets (Figs. 4 and 5). The most striking responses were observed in one MSM (C1001; Fig. 4a), whose CD8⁺ T cells recognized epitopes within six peptide pools (median of 29 IFN- SFCs/10⁵ cells; range, 12–89) and whose CD4⁺ T cells recognized three pools (median of 10 IFN- SFCs/10⁵ cells; range, 9–11). Of the other four responders in the control group (Fig. 4a), three were MSM, and one was a heterosexual man. Responses in these four ES controls were directed against three or more peptide pools, and the mean SFC counts per peptide pool were low, ranging from 6–12/10⁵ CD8⁺ cells and from 27–138/10⁵ CD8^- cells.

View larger version (33K): [in this window] [in a new window]   FIGURE 4. IFN- ELISPOT responses of CD8+ and CD8- T cells to HIV-1 peptide pools presented by autologous DCs. a, Control group of 13 sexually active subjects at average risk for HIV-1 exposure; b, group of 15 ES MSM. ES MSM were chosen based on their persistent high risk behavior and the availability of frozen PBMCs. DCs were pulsed with pools of 15-mer peptides spanning the complete HIV-1 Gag, Env, Pol, and Nef region for 1 h before PBMCs selected for the CD8+ and CD8- subsets were added for overnight culture. The ELISPOT assay for detection of IFN- was performed as described in Materials and Methods. The plot design is identical with that in Fig. 2. The black jagged line is the cutoff between negative and positive results based on a set false positive rate of 0.01 and 26 different peptide pools tested. The gray jagged line is based on five different peptide pools tested and applies to ES40 only. The cutoff was determined as described in Materials and Methods and adjusted for multiple comparisons by the Bonferroni correction. Each peptide pool was analyzed in duplicate or triplicate wells. Only symbols of positive responders are specified in the legend. c, Frequencies of IFN--secreting CD8+ T cells responding to the individual HIV-1 peptide pools in ES1, ES15, and ES29. HIV-1 Gag-specific responses were not found and are not shown. SEB served as the positive control; an irrelevant peptide pool served as the negative control (see Materials and Methods). Error bars are SEMs of duplicate or triplicate cultures.

View larger version (17K): [in this window] [in a new window]   FIGURE 5. HLA class I serotypes, CCR5 genotypes, and frequencies of HIV-1-specific, IFN--secreting CD8+ and CD8- T cells in eight of 15 ES and five of 13 control individuals reactive by DC-ELISPOT assay. Selection of CD8+ and CD8- T cells, generation and peptide pulsing of mature DCs, and ELISPOT assays were performed as described in Materials and Methods. If more than two peptide pools were recognized within a gene region, the number of recognized pools is indicated.

In comparison with the controls and other ES MSM, three (ES15, ES29, and ES63) of the 15 ES demonstrated broad and strong responses to stimulation with more than three peptide pools (Figs. 4c and 5). The CD8⁺ T cells of ES15 responded to 13 pools (median, 148 IFN- SFCs/10⁵ cells; range, 12–306; Fig. 4c), and the CD8^- T cells recognized 10 pools (median, 191 IFN- SFCs/10⁵ cells; range, 16–377). CD8⁺ T cells of ES29 responded to seven peptide pools (median, 360; range, 117–375; Fig. 4c), and the CD8^- subset responded to four pools (median, 166; range, 147–200). ES63 demonstrated the broadest reactivity, with the CD8⁺ cells recognizing 13 peptide pools (median, 198; range, 49–380) and the CD8^- cells recognizing six pools (median, 47; range, 37–277). However, of note, ES63 seroconverted 26 wk later. At the time point that PBMC were examined by the DC-ELISPOT assay, ES63 tested HIV-1 seronegative, and HIV-1 DNA was not identified by PCR within PBMC, sorted peripheral blood CD14⁺ monocytes, and CD4⁺ T cells (T. Zhu, unpublished observations). Twelve weeks later, he remained seronegative, and no viral DNA was found by PCR of PBMC. The other 14 individuals have remained seronegative. Thus, we found no evidence for occult or very low level HIV-1 infection in these high risk MSM around the time points that T cell responses were detected.

The overall proportions of responders in the control cohort (38%) and the ES cohort (53%) were not statistically different (p = 0.48). Likewise, no statistically significant difference was observed when the proportions of responders were analyzed in the CD8⁺ (p = 0.68) and the CD8^- (p = 1.0) subsets. Thus, the majority of ES MSM in our cohort had minimal to absent HIV-1-specific IFN- ELISPOT responses, even when the sensitivity of the assay was optimized by presentation of peptides with autologous DCs. Only two individuals, who remained uninfected despite documented long term, high risk behavior, demonstrated responses to HIV-1 peptide pools whose breadth and magnitude clearly exceeded that observed in either the control cohort or the remaining uninfected ES MSM.

Risk behavior and HIV-1-specific T cell responses over time

Examination of HIV-1-specific, IFN--secreting T cells at a single visit could not reliably distinguish between the presence of a true HIV-1-specific memory T cell population in the ES group and nonspecific or cross-reactive responses in the control group, even when DCs were used for peptide presentation. Since risk behavior in the ES cohort changed over the course of study, as exemplified in Fig. 6a for ES15, we reasoned that repeated priming of HIV-1-specific T cells during phases of increased high risk activities may boost HIV-1-specific immunity. If such boosts were observed at additional visits and in correlation with intensified risk behavior, this would signify the presence of true HIV-1-specific T cell memory. We first analyzed three of the low responders, ES1, ES40, and ES43, at three additional visits that occurred during or shortly after a period of high risk activities for reactivity to the HIV-1 peptide pools previously recognized in the DC-ELISPOT assay. (These visits are depicted by gray triangles in Fig. 1). To clearly distinguish between CD4⁺ and CD8⁺ T cell responses, we chose intracellular IFN- detection and simultaneous CD4, CD8, and CD3 surface phenotyping by flow cytometry for this longitudinal analysis.

View larger version (27K): [in this window] [in a new window]   FIGURE 6. Frequencies of anal intercourse and HIV-1-specific, IFN--secreting T cells over time in ES15. a, Frequencies of unprotected anal-receptive and insertive intercourse with known HIV-1-infected partners since enrollment in the study. Arrows mark the time of an ELISPOT assay or intracellular cytokine staining by flow cytometry. Except when indicated by an asterisk next to the arrow, peptides were presented by autologous DCs. b, IFN--secreting, HIV-1-specific CD4+ and CD8+ T cells measured by intracellular cytokine staining at eight different time points. Peptide stimulation, staining with fluorochrome-conjugated Abs, and analysis by flow cytometry were performed as described in Materials and Methods. Except when indicated by an asterisk, peptide pools were presented by autologous DCs. To decrease background levels, dead cells, monocytes, and added DCs were excluded from the analysis (see Materials and Methods). Background responses in control cultures treated with an irrelevant peptide pool were subtracted. c, Representative four-color flow cytometry analysis at 32 mo. CD8+ (upper panel) or CD4+ (lower panel) T cells were gated and analyzed for intracellular IFN- in response to SEB (left panel), the negative control peptide pool (middle panel), or the HIV-1 Env151–175 peptide pool (right panel). EMA+ dead cells, CD14-PE+ monocytes, and DC-SIGN-PE+ DCs were excluded to reduce unspecific background. The percentages of IFN--positive cells are indicated in the upper right quadrant.

In contrast to the paucity of responses detected at one or more visits in the majority of ES individuals, the high frequencies of HIV-1-specific, IFN--producing T cells in ES15, ES29, and ES63 may indicate true HIV-1-specific immunity, and we sought to confirm this by longitudinal analysis. We therefore tested ES15, who regularly practiced unprotected anal intercourse with known HIV-1-infected partners over the whole study period of 72 mo (Fig. 6a), at eight additional visits by intracellular IFN- staining (Fig. 6, b and c). In initial experiments the frequencies of HIV-specific, IFN--producing T cells detected by flow cytometric intracellular staining were surprisingly high, but were accompanied by relatively high background levels (data not shown). Thus, we could not reliably distinguish between specific and unspecific binding of the FITC-conjugated anti-IFN- Ab. However, when dead cells, monocytes, and added DCs were strictly excluded from the analysis (see Materials and Methods), background IFN- staining of cells treated with the irrelevant peptide pool and background isotype staining were greatly reduced. We therefore repeated all time points with this approach, using the three peptide pools that stimulated the highest frequencies of IFN--producing T cells in our initial studies. Only Env_151–175 was recognized at more than one time point (32, 38, and 65 mo) and by both CD4⁺ and CD8⁺ T cells (Fig. 6, b and c). The Env_126–150 and Pol_101–125 peptide pools were recognized exclusively by CD8⁺ T cells at one visit each (9 and 32 mo, respectively). Overall, frequencies were mostly low (mean, 77 IFN-⁺ T cells/10⁵ T cells; range, 20–220), and four visits tested entirely negative.

Autologous DCs were used for Ag presentation in all of these intracellular IFN--staining experiments, except at 69 mo. However, we performed another DC-ELISPOT assay at this visit, testing not only the Pol and Env peptide pools, but also 15-mer peptides spanning the complete regulatory gene regions of HIV-1 and 11 8- or 9-mer peptides restricted to HLA haplotypes found in ES15. Of this comprehensive spectrum of HIV-1 peptides, PBMC from 69 mo were reactive only to the Env_151–175 pool (219 IFN- SFCs/10⁵ PBMC; data not shown). Of note, ES15 had also been tested initially by our standard ELISPOT assay (44 mo), and no responses were found in the absence of DCs. Taken together, in our longitudinal analysis, we could not corroborate the broad and strong DC-ELISPOT reactivity found at 44 mo. Rather, responses were infrequent and weak, comparable to those in ES1, ES40, and ES43, and a correlation to risk activity could thus not be established.

In ES29, the other strong responder in our initial DC-ELISPOT assay who has remained uninfected to date, only two additional time points were available for analysis. One time point tested entirely negative by DC-ELISPOT, and at the other visit PBMC of ES29 reacted only to the Env_151–175 peptide pool (23 IFN- SFCs/10⁵ PBMC; data not shown). Of note, no responses were observed to 11 optimal 9-mer peptides with known restriction to HLA haplotypes present in ES29 and to the peptide pools representing the regulatory HIV-1 genes. Thus, as in ES15, we could not substantiate the reactivity found in our initial DC-ELISPOT assay at two further time points. In sum, even if rare individuals such as ES15 and ES29 sporadically exhibited increased frequencies of HIV-1-specific, IFN--producing T cells, responses over time were mostly weak or absent.

Epitope mapping and T cell cloning

To confirm the presence of and further define HIV-1-specific T cells in ES15 and ES29, we repeatedly attempted to map T cell reactivities to single HIV-1 epitopes and to establish HIV-1-specific T cell clones in these individuals. In ES15, single epitope reactivities were not discernible, and HIV-1-specific T cell lines or clones did not grow out in vitro. In ES29, T cells reactive to one 15-mer peptide in the Env_151–175 pool (aa 649–663, KSQTQQEKNEQELLE, 70 IFN- SFCs/10⁵ PBMC) and to two Pol 15-mer peptides (aa 149–163, IGCTLNFPISPIETV, 22 IFN- SFCs/10⁵ PBMC; aa 909–923, YSAGERIVDIIATDI, 190 IFN- SFCs/10⁵ PBMC) were detected, although the corresponding Pol_26–50 and Pol_226–248 peptide pools, respectively, were not recognized (data not shown). However, despite intense efforts over extended time periods, these HIV-1 epitope-specific T cells did not expand in vitro. Of note, as mentioned above, T cells of neither ES15 nor ES29 reacted to a set of optimal 8- or 9-mer peptides, which were selected based on their reported restriction to HLA haplotypes present in these two individuals. In conclusion, the failure to map single HIV-1 epitopes in ES15 and to clone peptide-reactive T cells in either ES15 or ES29 provides additional support that HIV-1-specific, IFN--secreting T cells are unlikely to be a significant factor in helping these ES MSM resist HIV-1 infection.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This investigation provides evidence that IFN--secreting memory T cells recognizing HIV-1 epitopes are not consistently detectable in our ES individuals who continue to practice profoundly high risk activities for years. In fact, their T cell responses are not significantly different from those of a lower risk control group. Moreover, even when presenting HIV-1 epitopes through potent immunostimulatory DCs, most ES MSM tested exhibited minimal to absent numbers of HIV-1-specific, IFN--secreting T cells. Nevertheless, two individuals clearly demonstrated high frequencies of T cells secreting IFN- in response to several HIV-1 peptide pools presented by autologous DCs. Although these responses diminished in magnitude and breadth over time, it is possible that even low levels of HIV-1-specific, IFN--secreting T cells may have contributed to rapid containment of HIV-1 following exposure in these individuals. It is also conceivable that the frequencies of HIV-1-specific T cells are higher at the sites of viral exposure, such as the rectal mucosa, than in peripheral blood. Kaul et al. (12) have suggested this possibility in their study examining the cervical mucosa of highly exposed CSW in Nairobi. Although we did not examine mucosal responses by IFN- ELISPOT, in our previous unpublished studies we have examined mucosal CTL responses, and the frequencies were not greater than those of systemic CTL.

The scarcity of HIV-1-specific, IFN--secreting T cells in our cohort must be reconciled with the findings of other studies that have implicated cellular immunity to be an important factor of resistance in multiply exposed, uninfected individuals (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 48). The first consideration is the potential for HIV-1 exposure in our ES, who were MSM, compared, for example, to the Nairobi CSW cohort, whose risk activities are heterosexual. The route of exposure differs in the two, with penile-vaginal intercourse, as practiced in the Nairobi cohort, having an 10-fold lower chance of HIV-1 transmission than penile-anal intercourse (1), as practiced by our ES. However, due to the marked differences in HIV-1 seroincidence in the two geographic regions, the female CSW in Nairobi are likely to be exposed to greater numbers of HIV-1-infected partners in their clientele than our ES MSM in Seattle. We presume that the viral loads of the Nairobi CSW sexual partners are higher than those of our ES partners, in whom common antiretroviral therapy use may effectively decrease the likelihood of HIV-1 exposure. Of note, in our previous report we did compare the plasma HIV-1 RNA levels in our ES partners with those of a contemporary matched, HIV-1-infected population enrolled in clinical trials, and no significant differences were noted between these groups (10), indicating that the ES partners are not unusual in their virologic profiles. Despite these caveats, approximately half of our ES MSM and the ones chosen for further detailed analyses continued to regularly practice high risk MSM activities with the same HIV-1-infected or new partners up to 6 or more years of observation. At the same time, the seroconversion rate in this group was markedly lower than that in 100 ES MSM followed for the HIVNET Vaccine Preparedness Study (44) despite our risk criteria being significantly more stringent. Thus, although our ES MSM are probably exposed to fewer numbers of HIV-1-infected partners, and their partner viral loads may be lower than those of the African ES CSW cohort, the predominant route of exposure in our cohort may confer greater transmissibility. Moreover, the lower seroincidence rates in comparison with contemporary MSM suggests that our ES MSM may exhibit some degree of resistance that reduces the likelihood of transmission from their infected partners.

Our examination of the ES responses by the sensitive IFN- ELISPOT methodologies was thorough, and if responses existed, they were likely to be revealed by these studies. It should be noted that the standard ELISPOT assay and the statistical interpretation of ELISPOT data have undergone rigorous evaluation (7, 36, 49) for validation purposes in phase II vaccine trials. We determine positive results with a stringent likelihood ratio statistic that maintains a consistent false positive rate of 0.01 across all assays and also incorporates a Bonferroni correction for the testing of multiple peptide pools. Our method thus treats all ELISPOT results with identical stringency and tends to be more conservative for borderline responses than previously published criteria (10). Low IFN- SFC frequencies that would be considered positive using traditional cutoffs are thus more likely to be interpreted as negative by our statistical criteria. This in part may account for why our findings indicate that as a group the ES responses were no different from those of the control subjects.

We recognize that there remains the possibility that the effector response important in preventing overt infection in the ES may not be represented by the identification of IFN--secreting T cells. In this case, the results of the present study stand in contrast with our own findings (10) of HIV-1-specific cytolysis in approximately one-third of ES MSM. This discrepancy can be explained largely by the different nature of the assays used and the effector cells tested. Although peptide presentation by DCs increased the ELISPOT sensitivity 3-fold, the 16-h time frame of the assay precludes actual expansion of HIV-1-specific T cells. In contrast, cytolysis was measured 10 days after antigenic stimulation, which allows proliferation and differentiation of HIV-1-specific precursor T cells and thus increases the frequency of responding effector cells. The T cells from the ES have normal proliferative capacities, as they are derived from immunocompetent HIV-1-uninfected subjects (10, 50). Thus, in ES, the ability to detect very low frequency responses may be greater in the CTL precursor analysis than the ex vivo ELISPOT analysis, in contrast to more recent studies comparing these assays in HIV-1-infected individuals (51, 52, 53). Protocols measuring cytolytic activities may also be less prone to identifying weak cross-reactivities, because T cells have to pass two check points for specificity, one during initial stimulation and a second during lysis of target cells. Cytolysis assays may thus be more robust for identifying low frequency, but true HIV-1-specific, responses than ELISPOT assays. This can explain why we found a difference between the ES and the control cohort based on assessment of cytotoxicity in our earlier study, but detected no significant difference in the proportion of responders in the ES and the control cohort based on the DC-ELISPOT assay in our present study.

Difficulties in distinguishing true HIV-1 responses from cross-reactivities may also explain some of the discrepancies found between studies conducted in different ES cohorts. There is compelling evidence that memory CTL triggered by one virus can be cross-reactive to Ags from another virus (54, 55, 56, 57). The frequency of such cross-reactive responses is likely to increase with the number of exposures to virus-derived Ags, which may be reflected by the magnitude of overall immune activation. One can thus speculate that the higher levels of cellular activation found in people living in sub-Saharan Africa (58, 59, 60) as well as in promiscuous ES individuals (61) are accompanied by broader and stronger cross-reactivities.

The possibility that certain cohorts exhibit higher levels of cross-reactivities underscores the importance of a well-matched control cohort to distinguish true HIV-1-specific from cross-reactive responses. To better distinguish cross-reactivities from responses stimulated by actual contact with HIV, we enrolled control subjects whose life style patterns were not that dissimilar from those of ES individuals: nine were MSM, and all 13 were sexually active. The controls were distinguished from ES mainly by the absence of reported contact with known HIV-1-infected partners. The reported HIV-1 seroincidence of 0.83/ 100 person-years in the Seattle metropolitan area (44) indicates a very small overall risk of HIV-1 exposure in our controls. When we compared the DC-ELISPOT results between this control and the ES cohort, the proportion of subjects in each group who demonstrated HIV-1 cellular immunity was not significantly different. This suggests that for the most part we have observed true cross-reactive responses triggered by Ags other than derived from HIV-1. Alternatively, we may have recorded false positive responses due to the technical limitations of our assay. However, given the rigorous evaluation of the ELISPOT protocol, we think that this is unlikely.

The findings in this and our earlier study (10) lead us to believe that HIV-1-specific cellular immunity represented by IFN--secreting T cells is either absent or extremely weak in the majority of ES MSM, and that resistance to infection conferred by IFN--secreting T cells in our ES cohort is therefore relative at best. Of note, even in the few ES in whom we were able to detect such T cell responses, seroconversions did occur thereafter in two of them, which is consistent with other reports that some female CSW who have resisted for some time do go on to seroconvert (7, 8). Moreover, in the two individuals, ES15 and ES29, who demonstrated relatively consistent responses recognizing several HIV-1 peptide pools and who have remained uninfected to date, we were unable to identify HIV-1 single epitope-specific memory T cell clones. Although this failure could have been due to technical problems, we believe that this is unlikely, since we made repeated attempts and have extensive experience with T cell cloning (6, 62, 63), particularly from specimen sources containing extremely few HIV-1-specific T cells (62, 63).

In conclusion, the low frequency of HIV-1-specific, IFN--secreting T cells in the ES MSM cohort, the inability to demonstrate HIV-1-specific T cell clones in the few stronger responders, and the possibility that many of the low responses stem from cross-reactivities stimulated by viruses other than HIV argue against a prominent role of HIV-1-specific T cells that secrete IFN- in preventing infection in our cohort. It is clear that the ES represent a unique population in whom mechanisms of HIV-1 protection can be elucidated (2, 4, 64, 65), and that it is likely that multiple factors contribute to their relative resistance. Our findings emphasize the importance of considering the type and extent of risk activity as well as inclusion of appropriate controls in interpreting findings that may have a role in HIV-1 protection. Future studies comparing these methodologies in heterosexual ES from regions where the HIV-1 epidemic has hit hardest will be enlightening in understanding the role of IFN--secreting T cells and other relevant antiviral mediators that confer protection.

	Acknowledgments

 
We thank the study volunteers for their participation, and Paul J. Nelson for patient recruitment.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants AI47806, AI27757, and AI48017. F.H. was a Fellow of the James B. Pendleton Trust. M.J.M. is a recipient of the Burroughs Wellcome Clinical Scientist Award for Translational Research.

² Address correspondence and reprint requests to: Dr. M. Julianna McElrath, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, D3-100, Seattle, WA 98109. E-mail address: kd{at}u.washington.edu

³ Abbreviations used in this paper: CSW, commercial sex workers; ES, exposed seronegative; DC, dendritic cells; EMA, ethidium monoazide bromide; LTNP, long term nonprogressors; MCM, monocyte-conditioned medium; MSM, men having sex with men; SEB, staphylococcal enterotoxin B; SFC, spot-forming cells.

⁴ M. G. Hudgens, S. G. Self, Y. Vhiu, N. D. Russell, and M. J. Elrath. Statistical considerations of the ELISPOT assay in HIV-1 vaccine trials. Submitted for publication.

Received for publication February 14, 2003. Accepted for publication June 24, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Royce, R. A., A. Sena, W. Cates, Jr., M. S. Cohen. 1997. Sexual transmission of HIV. N. Engl. J. Med. 336:1072.[Free Full Text]
2. Liu, R., W. A. Paxton, S. Choe, D. Ceradini, S. R. Martin, R. Horuk, M. E. MacDonald, H. Stuhlmann, R. A. Koup, N. R. Landau. 1996. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86:367.[Medline]
3. Samson, M., F. Libert, B. J. Doranz, J. Rucker, C. Liesnard, C. M. Farber, S. Saragosti, C. Lapoumeroulie, J. Cognaux, C. Forceille, et al 1996. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 382:722.[Medline]
4. Dean, M., M. Carrington, C. Winkler, G. A. Huttley, M. W. Smith, R. Allikmets, J. J. Goedert, S. P. Buchbinder, E. Vittinghoff, E. Gomperts, et al 1996. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene: Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science 273:1856.[Abstract/Free Full Text]
5. Carrington, M., G. W. Nelson, M. P. Martin, T. Kissner, D. Vlahov, J. J. Goedert, R. Kaslow, S. Buchbinder, K. Hoots, S. J. O’Brien. 1999. HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage. Science 283:1748.[Abstract/Free Full Text]
6. Malhotra, U., S. Holte, S. Dutta, M. M. Berrey, E. Delpit, D. M. Koelle, A. Sette, L. Corey, M. J. McElrath. 2001. Role for HLA class II molecules in HIV-1 suppression and cellular immunity following antiretroviral treatment. J. Clin. Invest. 107:505.[Medline]
7. Kaul, R., T. Dong, F. A. Plummer, J. Kimani, T. Rostron, P. Kiama, E. Njagi, E. Irungu, B. Farah, J. Oyugi, et al 2001. CD8⁺ lymphocytes respond to different HIV epitopes in seronegative and infected subjects. J. Clin. Invest. 107:1303.[Medline]
8. Kaul, R., S. L. Rowland-Jones, J. Kimani, T. Dong, H. B. Yang, P. Kiama, T. Rostron, E. Njagi, J. J. Bwayo, K. S. MacDonald, et al 2001. Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8⁺ responses. J. Clin. Invest. 107:341.[Medline]
9. Rowland-Jones, S. L., T. Dong, K. R. Fowke, J. Kimani, P. Krausa, H. Newell, T. Blanchard, K. Ariyoshi, J. Oyugi, E. Ngugi, et al 1998. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV-resistant prostitutes in Nairobi. J. Clin. Invest. 102:1758.[Medline]
10. Goh, W. C., J. Markee, R. E. Akridge, M. Meldorf, L. Musey, T. Karchmer, M. Krone, A. Collier, L. Corey, M. Emerman, et al 1999. Protection against human immunodeficiency virus type 1 infection in persons with repeated exposure: evidence for T cell immunity in the absence of inherited CCR5 coreceptor defects. J. Infect. Dis. 179:548.[Medline]
11. Rowland-Jones, S., J. Sutton, K. Ariyoshi, T. Dong, F. Gotch, S. McAdam, D. Whitby, S. Sabally, A. Gallimore, T. Corrah, et al 1995. HIV-specific cytotoxic T-cells in HIV-exposed but uninfected Gambian women. Nat. Med. 1:59.[Medline]
12. Kaul, R., F. A. Plummer, J. Kimani, T. Dong, P. Kiama, T. Rostron, E. Njagi, K. S. MacDonald, J. J. Bwayo, A. J. McMichael, et al 2000. HIV-1-specific mucosal CD8⁺ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi. J. Immunol. 164:1602.[Abstract/Free Full Text]
13. Clerici, M., J. V. Giorgi, C. C. Chou, V. K. Gudeman, J. A. Zack, P. Gupta, H. N. Ho, P. G. Nishanian, J. A. Berzofsky, G. M. Shearer. 1992. Cell-mediated immune response to human immunodeficiency virus (HIV) type 1 in seronegative homosexual men with recent sexual exposure to HIV-1. J. Infect. Dis. 165:1012.[Medline]
14. Langlade-Demoyen, P., N. Ngo-Giang-Huong, F. Ferchal, E. Oksenhendler. 1994. Human immunodeficiency virus (HIV) nef-specific cytotoxic T lymphocytes in noninfected heterosexual contact of HIV-infected patients. J. Clin. Invest. 93:1293.
15. Pinto, L. A., J. Sullivan, J. A. Berzofsky, M. Clerici, H. A. Kessler, A. L. Landay, G. M. Shearer. 1995. ENV-specific cytotoxic T lymphocyte responses in HIV seronegative health care workers occupationally exposed to HIV-contaminated body fluids. J. Clin. Invest. 96:867.
16. Bernard, N. F., C. M. Yannakis, J. S. Lee, C. M. Tsoukas. 1999. Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte activity in HIV-exposed seronegative persons. J. Infect. Dis. 179:538.[Medline]
17. Makedonas, G., J. Bruneau, H. Lin, R. P. Sekaly, F. Lamothe, N. F. Bernard. 2002. HIV-specific CD8 T-cell activity in uninfected injection drug users is associated with maintenance of seronegativity. AIDS 16:1595.[Medline]
18. Mazzoli, S., D. Trabattoni, S. Lo Caputo, S. Piconi, C. Ble, F. Meacci, S. Ruzzante, A. Salvi, F. Semplici, R. Longhi, et al 1997. HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals. Nat. Med. 3:1250.[Medline]
19. Mazzoli, S., L. Lopalco, A. Salvi, D. Trabattoni, S. Lo Caputo, F. Semplici, M. Biasin, C. Blé, A. Cosma, C. Pastori, et al 1999. Human immunodeficiency virus (HIV)-specific IgA and HIV neutralizing activity in the serum of exposed seronegative partners of HIV-seropositive persons. J. Infect. Dis. 180:871.[Medline]
20. Skurnick, J. H., P. Palumbo, A. DeVico, B. L. Shacklett, F. T. Valentine, M. Merges, R. Kamin-Lewis, J. Mestecky, T. Denny, G. K. Lewis, et al 2002. Correlates of nontransmission in US women at high risk of human immunodeficiency virus type 1 infection through sexual exposure. J. Infect. Dis. 185:428.[Medline]
21. Beretta, A., S. H. Weiss, G. Rappocciolo, R. Mayur, C. De Santis, J. Quirinale, A. Cosma, P. Robbioni, G. M. Shearer, J. A. Berzofsky, et al 1996. Human immunodeficiency virus type 1 (HIV-1)-seronegative injection drug users at risk for HIV exposure have antibodies to HLA class I antigens and T cells specific for HIV envelope. J. Infect. Dis. 173:472.[Medline]
22. Kaul, R., F. Plummer, M. Clerici, M. Bomsel, L. Lopalco, K. Broliden. 2001. Mucosal IgA in exposed, uninfected subjects: evidence for a role in protection against HIV infection. AIDS 15:431.[Medline]
23. Devito, C., J. Hinkula, R. Kaul, L. Lopalco, J. J. Bwayo, F. Plummer, M. Clerici, K. Broliden. 2000. Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate. AIDS 14:1917.[Medline]
24. Lopalco, L., C. Pastori, A. Cosma, S. E. Burastero, B. Capiluppi, E. Boeri, A. Beretta, A. Lazzarin, A. G. Siccardi. 2000. Anti-cell antibodies in exposed seronegative individuals with HIV type 1-neutralizing activity. AIDS Res. Hum. Retroviruses 16:109.[Medline]
25. Pinto, L. A., S. Sharpe, D. I. Cohen, G. M. Shearer. 1998. Alloantigen-stimulated anti-HIV activity. Blood 92:3346.[Abstract/Free Full Text]
26. Brown, L., B. E. Souberbielle, J. B. Marriott, M. Westby, U. Desselberger, T. Kaye, M. L. Gougeon, A. Dalgleish. 1999. The conserved carboxy terminal region of HIV-1 gp120 is recognized by seronegative HIV-exposed people. AIDS 13:2515.[Medline]
27. Devito, C., K. Broliden, R. Kaul, L. Svensson, K. Johansen, P. Kiama, J. Kimani, L. Lopalco, S. Piconi, J. J. Bwayo, et al 2000. Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells. J. Immunol. 165:5170.[Abstract/Free Full Text]
28. Ogg, G. S., X. Jin, S. Bonhoeffer, P. R. Dunbar, M. A. Nowak, S. Monard, J. P. Segal, Y. Cao, S. L. Rowland-Jones, V. Cerundolo, et al 1998. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279:2103.[Abstract/Free Full Text]
29. Koup, R. A., J. T. Safrit, Y. Cao, C. A. Andrews, G. McLeod, W. Borkowsky, C. Farthing, D. D. Ho. 1994. Temporal association of cellular immune responses with the initial control of viremia in primary human immunodeficiency virus type 1 syndrome. J. Virol. 68:4650.[Abstract/Free Full Text]
30. Borrow, P., H. Lewicki, B. H. Hahn, G. M. Shaw, M. B. Oldstone. 1994. Virus-specific CD8⁺ cytotoxic T-lymphocyte activity associated with control of viremia in primary human immunodeficiency virus type 1 infection. J. Virol. 68:6103.[Abstract/Free Full Text]
31. Kuroda, M. J., J. E. Schmitz, W. A. Charini, C. E. Nickerson, M. A. Lifton, C. I. Lord, M. A. Forman, N. L. Letvin. 1999. Emergence of CTL coincides with clearance of virus during primary simian immunodeficiency virus infection in rhesus monkeys. J. Immunol. 162:5127.[Abstract/Free Full Text]
32. Schmitz, J. E., M. J. Kuroda, S. Santra, V. G. Sasseville, M. A. Simon, M. A. Lifton, P. Racz, K. Tenner-Racz, M. Dalesandro, B. J. Scallon, et al 1999. Control of viremia in simian immunodeficiency virus infection by CD8⁺ lymphocytes. Science 283:857.[Abstract/Free Full Text]
33. Jin, X., D. E. Bauer, S. E. Tuttleton, S. Lewin, A. Gettie, J. Blanchard, C. E. Irwin, J. T. Safrit, J. Mittler, L. Weinberger, et al 1999. Dramatic rise in plasma viremia after CD8⁺ T cell depletion in simian immunodeficiency virus-infected macaques. J. Exp. Med. 189:991.