HIV-1–Specific Cytotoxic T Lymphocytes and Breast Milk HIV-1Transmission

Grace C. John-Stewart1,2,a, Dorothy Mbori-Ngacha1,a, Barbara Lohman Payne1,2, CareyFarquhar2, Barbra A. Richardson2,3, Sandra Emery3, Phelgona Otieno1, ElizabethObimbo1, Tao Dong4, Jennifer Slyker4, Ruth Nduati1, Julie Overbaugh3, and Sarah Rowland-Jones41Department of Paediatrics, University of Nairobi, Nairobi, Kenya2Departments of Biostatistics, Medicine, and Epidemiology, University of Washington, Seattle3Fred Hutchinson Cancer Research Center, Seattle4Oxford University, Oxford, United Kingdom

AbstractBackground—Breast-feeding by infants exposed to human immunodeficiency virus type 1(HIV-1) provides an opportunity to assess the role played by repeated HIV-1 exposure in elicitingHIV-1–specific immunity and in defining whether immune responses correlate with protection frominfection.

Methods—Breast-feeding infants born to HIV-1–seropositive women were assessed for HLA-selected HIV-1 peptide–specific cytotoxic T lymphocyte interferon (IFN)–γ responses by means ofenzyme-linked immunospot (ELISpot) assays at 1, 3, 6, 9, and 12 months of age. Responses weredeemed to be positive when they reached ⩾50 HIV-1–specific sfu/1 × 106 peripheral bloodmononuclear cells (PBMCs) and were at least twice those of negative controls.

Results—A total of 807 ELISpot assays were performed for 217 infants who remained uninfectedwith HIV-1 at ∼12 months of age; 101 infants (47%) had at least 1 positive ELISpot result (median,78–170 sfu/1 × 106 PBMCs). The prevalence and magnitude of responses increased with age (P = .01 and P = .007, respectively); the median log10 value for HIV-1–specific IFN-γ responses increasedby 1.0 sfu/1 × 106 PBMCs/month (P < .001) between 1 and 12 months of age. Of 141 HIV-1–uninfected infants with 1-month ELISpot results, 10 (7%) acquired HIV-1 infection (0/16 withpositive vs. 10/125 [8%] with negative ELISpot results; P = .6). Higher values for log10 HIV-1–specific spot-forming units at 1 month of age were associated with a decreased risk of HIV-1infection, adjusted for maternal HIV-1 RNA level (adjusted hazard ratio, 0.09 [95% confidenceinterval, 0.01–0.72]).

Conclusions—Breast-feeding HIV-1–exposed uninfected infants frequently had HIV-1–specificIFN-γ responses. Greater early HIV-1–specific IFN-γ responses were associated with decreasedHIV-1 acquisition.

An estimated 80% of breast-feeding infants born to HIV-1–seropositive women escape HIV-1infection despite ingesting hundreds of liters of HIV-1–infected breast milk [1]. Thus, continual

© 2009 by the Infectious Diseases Society of America. All rights reserved.Reprints or correspondence: Dr. Grace John-Stewart, 325 Ninth Ave., Box 359909, Seattle, WA 98104 (gjohn@u.washington.edu).aG.C.J.-S. and D.M.-N. contributed equally to this work.Potential conflicts of interest: none reported.

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Published in final edited form as:J Infect Dis. 2009 March 15; 199(6): 889–898.

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exposure to HIV-1 does not invariably lead to transmission. There are at least 2 models thatmay explain this outcome. The first is that infants escape infection because they areinsufficiently exposed to HIV-1; the other is that they receive an immunizing, but not infective,dose of HIV-1 that protects them from subsequent infection. HIV-1–specific cytotoxic Tlymphocyte (CTL) interferon (IFN)–γ secretion has been reported in several small studies ofHIV-1–exposed uninfected infants [2–5]. Legrand et al. [3] demonstrated HIV-1 nef– andHIV-1 gag–specific dual IFN-γ and tumor necrosis factor–α secretion by CD8+ T cells in 16exposed uninfected infants; this study provided important evidence confirming the presenceof polyfunctional HIV-1–specific CD8+ T cell responses in exposed uninfected infants.

A key foundation for several HIV-1 vaccines is the concept that HIV-1–specific CTL responsesprovide protection against infection, but evidence to support this hypothesis has been limitedand conflicting. HIV-1–specific CTL responses have been detected in exposed uninfectedindividuals [6]. However, such studies have not discerned whether HIV-1–specific immuneresponses are markers of exposure or correlates of protection. In animal models, exposeduninfected sex workers, and HIV-1–infected individuals, HIV-1 infection or superinfectionoccurs despite the presence of CTLs, suggesting that simply having a cellular immune responseis insufficient for protection [7–9]. The recent disappointing results of the STEP trial, whichnoted no protection with a vaccine (Merck V520) designed to induce HIV-1–specific CTLs,confirms the need to define protective immune correlates [10].

A prospective study of exposed uninfected individuals is necessary to determine both theincidence of CTL responses and whether CTLs protect from infection. An ideal setting for thisapproach is breast-feeding infants of HIV-1–infected mothers; for these infants, repeatedexposure is common, measurable, and consistent, which is not the case for sexually exposedadults. Infants repetitively exposed to HIV-1 may provide a natural instance of vaccinationwith live virus, which can inform vaccine design based on mimicking a natural model ofeffective immune response. We hypothesized that exposed uninfected infants experiencesufficient viral exposure to induce systemically detectable HIV-1–specific immune responsesand that these early immune responses could protect against subsequent infection. Thus, wedesigned a prospective cohort study of infants born to HIV-1–seropositive mothers todetermine the prevalence, magnitude, and effect of HIV-1–specific cellular immune responses.

METHODSThe study was reviewed and approved by the Institutional Review Board of the University ofWashington and the Ethical Review Committee of Kenyatta National Hospital.

Clinical proceduresHIV-1–seropositive pregnant women were enrolled after provision of written informedconsent. Eligibility criteria included age ⩾18 years, gestation ⩽32 weeks, and willingness toadhere to scheduled infant blood samplings for at least 1 year. Mothers were counseledregarding breast milk HIV-1 transmission and were supported in their feeding choice. Mothersreceived short-course zidovudine (300 mg twice daily from 34 weeks of gestation and every3 h during labor) to prevent HIV-1 infection in their infants [11]. Within 48 h of birth, bloodwas collected from infants for molecular HLA typing and HIV-1 detection. Blood was collectedagain from the infants at 1, 3, 6, 9, and 12 months of age for HIV-1 DNA, RNA, and IFN-γenzyme-linked immunospot (ELISpot) assays.

HLA typingMolecular class I HLA typing was performed using DNA from infant peripheral bloodmononuclear cells (PBMCs). HLA alleles were determined using amplification-refractory

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mutation system polymerase chain reaction (PCR), with primers designed for East Africanalleles [12].

ELISpot assaysIFN-γ ELISpot assays were performed on freshly isolated PBMCs, as described elsewhere[13–15]. Briefly, PBMCs from infants were added to 96-well Millipore plates (Millipore)coated with 1-DIK monoclonal antibodies at 15 µg/mL (Mabtech) in duplicate at 2 × 105

PBMCs/well and stimulated with either phytohemagglutinin (Murex Biotech) at 20 µg/mL(positive control), medium alone (negative control), or HLA-selected peptide at 20 µg/mL (1peptide per well, except for clade variants, for which equivalent amounts were used for eachvariant). Spot-forming units were defined as the average number of spots in duplicate wells,and the number of HIV-1–specific spot-forming units was calculated as the total number minusthe average number in negative control wells.

Peptides for ELISpot assays were selected using a predefined algorithm based on infant HLA(table 1). The median number of peptides tested per infant per visit was 11 (range, 1–29). Spotswere counted by eye in plates until January 2001, after which an automated ELISpot assayreader was used (Autoimmun Diagnostika). In assays with both counting methods conductedconcurrently, κ concordance was 1.0 (P < .001), and correlation was 0.94 (P < .001). Eye-counted results were used before machine counting was instituted, and machine results wereused thereafter. Spot counts were entered into a database without links to HIV-1 status, andHLA-matched assays were computed as positive or negative on the basis of a predeterminedcomputer algorithm using published criteria (⩾50 HIV-1–specific sfu/1 × 106 PBMCs, withexperimental values at least twice those of negative control wells) [16,17]. Assays wereconducted blinded to infant HIV-1 status.

HIV-1 DNA and RNA assays and determination of infant HIV-1 statusHIV-1 DNA PCR filter paper assays were conducted using methods with 98% specificity and99% sensitivity [18]. HIV-1 RNA levels were quantified using the Gen-Probe transcription-mediated assay. Infants were deemed to be HIV-1 infected if 2 consecutive assays were HIV-1DNA or RNA positive or if a single HIV-1 assay was positive and it was the last availableassay.

Control studyThe specificity of ELISpot assays was determined in a control study conducted among 20infants who had been born to HIV-1–seronegative mothers who were determined to beuninfected by ELISA and HIV-1 RNA assay. HIV-1 DNA and ELISpot assays were performedin these infants.

Depletion of CD8+ cellsPBMCs were depleted of CD8+ T cells by means of anti-CD8 monoclonal antibody–coatedmagnetic beads (Dynal). In each case, 98% of CD8+ T cells were depleted from the population.

Statistical methodsAnalyses were restricted to infants whose mothers reported any breast-feeding. Categoricaldata were compared using χ2 and Fisher’s exact tests, and continuous data were comparedusing the Mann-Whitney U test. For paired comparisons, the Wilcoxon signed-rank test wasused for continuous outcomes, and McNemar’s test was used for categorical outcomes. Linearregression analysis was used to determine the change in magnitude of HIV-1–specificresponses with age for each infant; the Wilcoxon signed-rank test was used to determinewhether the median slope differed from 0. For Kaplan-Meier and Cox regression analyses

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among infants who were HIV-1 uninfected at 1 month of age, the following time intervals wereused: the time to the midpoint between the last HIV-1–negative and the first HIV-1–positiveresult for infants who became HIV-1 infected between 1 and 12 months of age; the time to thelast visit for uninfected infants who were lost to follow-up or died before 12 months of age;and 12 months for infants who remained uninfected at 12 months of age.

RESULTSRecruitment and follow-up

From July 1999 through October 2002, 36,059 women were offered testing for HIV-1 at 8clinics, of whom 88% accepted testing. Among HIV-tested women, 4512 (14%) were HIV-1seropositive, 3190 (71%) of whom received results and were referred to the study clinic. Of1539 women who came to the study clinic, 510 (33%) were eligible, interested, and enrolled.Delivery information was available for 476 (93%) of the infants, including 474 (99.6%)singleton or first-born infants who were followed up (7 second-born twins were excluded); 465(98%) had HIV-1 testing at least once. By 1 month of age, 72 infants (15%) had acquired HIV-1infection, 9 HIV-1–uninfected infants (2%) were lost to follow-up, and 10 uninfected infants(2%) had died, with 374 HIV-1–uninfected infants remaining in follow-up, of whom 284 (76%)were breastfed (figure 1).

Prevalence, magnitude, and longitudinal changes in HIV-1–specific CTL responses in breast-feeding HIV-1–uninfected infants who remained uninfected at 1 year of age

Among 217 exposed HIV-1–uninfected infants who remained uninfected at >11.5 months ofage, filter paper HIV-1 DNA assays were serially negative for an average of 5.7 time points(range, 3–7). In addition, 195 (90%) of these infants had at least 1 confirmatory negative HIV-1RNA assay result (mean, 1.8; range, 1–7). Of these breast-feeding uninfected infants, 101(47%) had at least 1 time point with a positive ELISpot result.

The prevalence of positive ELISpot assays increased over time and ranged from 12% of 112infants at age 1 month to 22% of 194 infants at age 12 months (table 2). The magnitude ofHIV-1–specific responses ranged from 50 to 4535 HIV-1–specific sfu/1 × 106 PBMCs, andthe number of peptides that elicited a positive response ranged from 1 to 11 in infants withpositive assays. The prevalence of positive ELISpot results increased with age (P = .01), andthe number of HIV-1–specific spot-forming units (maximal response to a single epitope) alsoincreased with age (P = .007) in paired comparisons between the ages of 1 and 12 months. Themedian change in magnitude per month was 1.02 HIV-1–specific sfu (interquartile range, −2.02to 7.23; P < .001). Positive assays had a higher median number of peptides tested than negativeassays (12 vs. 10 peptides; P = .009).

Of 62 infants with ELISpot assays conducted at all 5 time points, 26 (42%) had 1 positiveassay, and 13 (21%) had >1 (2 positive assays for 6 infants, 3 for 5 infants, and 4 for 2 infants).Of 9 HIV-1–uninfected infants with positive ELISpot results at month 1 of age and assaysresults at all 4 subsequent time points, 3 infants had positive ELISpot results once, some hadrepeated responses to consistent epitopes, and some had diversified epitope recognition (figure2).

Immune response: comparison with HIV-1–infected infants, specificity, and phenotypeIn a previous study, we determined HIV-1–specific immune responses at the same time pointsin 61 infants derived from the same cohort who acquired HIV-1 infection before 1 month ofage [14]. These infected infants were excluded from the current analysis, which focuses onHIV-1–uninfected infants and later HIV-1 acquisition. HIV-1–specific responses in age-matched infants with early HIV-1 infection in the previous study were 2.5–9-fold higher in

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prevalence and 2–5-fold higher in magnitude than those the uninfected exposed infantsanalyzed here.

To determine the specificity of responses, exposed uninfected infants were compared with 20control infants born to HIV-1–uninfected mothers. None of the 20 HIV-1–unexposed controlinfants (median age, 3 months; age range, 1–9 months) had a positive ELISpot result,suggesting high specificity (100% [95% confidence interval {CI}, 84%–100%]). Becauseexposed uninfected and infected infants underwent sampling at multiple time points and controlinfants were assessed only once, each time point was compared with the entire group of controlinfants. In exposed uninfected infants at months 1, 3, 6, 9, and 12 of age, the prevalence ofELISpot responses was higher than that among the 20 control infants (P = .2, P = .08, P = .08,P = .02, and P = .02, respectively). Among infants with early HIV-1 infection from the previousstudy, the prevalence of positive results was significantly higher than that in unexposed controlinfants at all time points (P < .001).

To confirm the phenotype of lymphocytes secreting IFN-γ in ELISpot assays, responses ofCD8+ T cell–depleted and undepleted PBMCs were assessed in 2 separate assays of HIV-1–infected infants. In each case, after depletion of 98% of CD8+ T cells, positive responses wereabrogated, with levels falling to that of background stimulation or lower (data not shown).

To address the potential role of maternal microchimerism, maternal HLA type was determinedfor a subset of 23 HIV-1–exposed uninfected infants with HIV-1–specific immune responses.Twelve (52%) of the 23 HIV-1–exposed uninfected infants had responses to epitopes selectedby nonmaternal (i.e., paternal) HLA alleles, suggesting that these responses originated in theinfants.

HLA-selected peptide-stimulated ELISpot assays in breast-feeding infants who were HIV-1uninfected at month 1 of age

At 1 month of age, 141 HIV-1–uninfected infants underwent ELISpot testing and follow-up.During the subsequent follow-up, 10 acquired HIV-1 infection, 13 were lost to follow-up, 6died, and 112 never acquired HIV-1 infection. Characteristics of these 141 infants are outlinedin table 3. The median duration of zidovudine exposure was 27.0 days, and the median durationof labor was 9.0 h. The median maternal plasma HIV-1 RNA level was 4.6 log10 copies/mL;this decreased to 4.0 log10 copies/mL at delivery (after zidovudine), and returned to 4.7log10 copies/mL at 1 month post partum.

Cofactors and protective correlates for HIV-1 transmission via breast milkIn univariate analyses, maternal plasma and breast milk HIV-1 RNA levels at month 1 wereassociated with transmission (P = .004 and P = .03, respectively). None of the 16 infants whohad a positive ELISpot result at 1 month of age acquired HIV-1 infection, versus 10 (8%) of125 with negative ELISpot results at month 1(P = .6). In multivariate analyses, there was noassociation between positive ELISpot responses and protection when results were adjusted formaternal plasma HIV-1 RNA level (P = .9).

In analyses evaluating HIV-1–specific IFN-γ spot-forming units as a continuous variable, thisvariable showed a trend toward a protective effect in univariate analyses (P = .11) (table 4). Inmultivariate analyses, maternal plasma HIV-1 RNA levels and infant HIV-1–specific spot-forming units were each associated with significant independent effects on transmission.Maternal plasma HIV-1 RNA level was associated with increased transmission risk (adjustedhazard ratio [aHR], 9.1 [95% CI, 1.7–47.7]). Each log10 increase in infant HIV-1–specific spot-forming units was associated with a decreased risk of transmission (aHR, 0.09 [95% CI, 0.01–

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0.72]). In an alternate regression model with the follow-up time shortened for women whodiscontinued breast-feeding early, results were similar (aHR, 0.08 [95% CI, 0.01–0.66]).

DISCUSSIONThe present prospective analysis of this HIV-1–exposed infant cohort has provided severalimportant observations relevant to HIV-1 transmission and the mechanisms of immuneprotection. First, of >200 breast-feeding HIV-1–exposed uninfected infants who had nodetectable HIV-1 infection throughout the first year of life and were serially assessed forimmune responses, almost half had HIV-1–specific CTL IFN-γ responses despite the absenceof HIV-1 infection. Second, in breast-feeding HIV-1–exposed uninfected infants whoremained uninfected at ∼1 year of life, age was associated with significant increases in theprevalence and magnitude of IFN-γ responses during infancy. Finally, although detection ofHIV-1–specific immune responses per se was not associated with protection from HIV-1transmission, the magnitude of early HIV-1–specific IFN-γ responses was associated withdecreased breast milk HIV-1 transmission.

Detection of HIV-1–specific CTLs in an HIV-1–exposed uninfected infant was firstdocumented in 1992, using conventional chromium release assays with viral stimulation [2].Subsequently, HIV-1–specific CTLs have been detected in several small cohorts of HIV-1–exposed uninfected infants (most <30 infants), most recently by investigators usingintracellular cytokine staining assays [3,4,19]. Here, we provide the first population-basedestimate of the prevalence of HIV-1–specific immune responses after exposure to HIV-1 inbreast milk. We observed that 47% of HIV-1–uninfected infants had HIV-1–specific IFN-γresponses during serial evaluation over the course of 1 year. At any single time point, only12%–22% of infants had HIV-1–specific IFN-γ responses. Serial assessment enhanced ourability to detect responses in uninfected infants, who appeared to have intermittently detectableHIV-1–specific IFN-γ responses. Thus, smaller studies with assessment at a single time pointmaybe limited in their ability to detect responses.

Our data suggest that, rather than completely escaping viral exposure, many HIV-1–uninfectedinfants born to HIV-1–infected mothers are exposed to cell-associated HIV-1 and elicit immunerecognition of HIV-1–infected cells. HIV-1–infected individuals harbor large populations ofreplication-incompetent virus [20]. Thus, such viruses may elicit cellular responses but belimited in their ability to cause productive infection. Sacha et al. [21] have demonstrated rapidinduction of CTL responses (within 2 h) that are capable of eliminating HIV-1–infected cellsbefore protein synthesis or productive infection ensue. An alternative possibility for ourobservation is that infants had restricted viral replication in oropharyngeal or esophageallymphoid tissue without systemic viral detection, similar to what has been observed in primatemodels after low-dose lentiviral exposure [22]. HIV-1–exposed uninfected infants had levelsas high as 4535 HIV-1–specificsfu/1 × 106 PBMCs in response to a single peptide epitope andresponded to as many as 11 epitopes in a single assay, levels comparable to those in adultsafter HIV-1 vaccination [23,24]. These responses were lower than those in infected infants[14]. Thus, the observed grading of responses—none in unexposed infants, intermediate inexposed uninfected infants, and the highest responses in infected infants—fits with a model ofexposure-mediated induction of HIV-1–specific responses. In this model, mucosal exposurein uninfected exposed infants elicits transient moderate responses, whereas infected infantsexposed to systemically circulating and replicating virus develop more-robust and sustainedimmune responses.

We observed that HIV-1–exposed uninfected infants were able to mount HIV-1–specificimmune responses within the first month of life, but these responses were less frequent and oflower magnitude than those in older infants. This difference could have been due to ongoing

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breast-feeding exposure to HIV-1. In addition, our observations are consistent with those ofstudies showing age-related enhancement of immune responses in HIV-1–infected infants[14,25,26]. In HIV-1–infected children with intrahost circulation of HIV-1, it is challengingto discriminate between the effects of age and the effects of time since exposure to HIV-1. Ina study of adults, HIV-1–exposed uninfected individuals were noted to have CTL responsesas late as 34 months after exposure [27]. Infants who were potentially exposed at delivery andagain via breast-feeding may be analogous to recipients of a prime-boost vaccination, whichresults in sustained immune responses long after exposure. The combination of enhancedimmune maturation and increased exposure may have contributed to our observation of age-mediated increased responses [8].

No infants with positive ELISpot results at 1 month of age acquired HIV-1 infection, in contrastto 8% of those with negative ELISpot results. Although the difference in transmission risk waslarge, power for the analysis was limited by the small number of infants with HIV-1–specificimmune responses at 1 month of age. Because the empiric cutoffs used to dichotomize spot-forming unit values are arbitrary (although widely used in vaccine trials) and do not have abiological basis, we also examined the HIV-1–specific immune response as a continuousparameter. This approach is similar to analyses of HIV-1 load and takes into account the factthat the assays currently do not have a defined threshold linked to the quantity of HIV-1–specific CTLs required for preventive efficacy. Indeed, when we analyzed HIV-1–specificIFN-γ spot-forming units as a log10-transformed continuous variable rather than empiricallycreating a cutoff, statistical power was increased, and there was evidence of a protective effect.Evaluation of log10 HIV-1 spot-forming units as a continuous variable rather than with arbitrarycutoffs may be useful in HIV-1 vaccine trials, both to increase power and to discern biologicallyrelevant cutoffs for the future.

We chose the ELISpot assay because it is the most sensitive assay for detection of HIV-1–specific CTLs, requires minimal quantities of blood, and, most importantly, is the main methodused to detect CTLs in vaccine studies [28,29]. HIV-1 CTL ELISpot assays have highsensitivity and specificity compared with conventional lysis assays, and their resultsspecifically correlate with HIV-1–specific CD8+ T cell recognition [28]. ELISpot results wereinterpreted blinded to infant HIV-1 status, using a predefined computerized algorithm frompublished criteria. These systems eliminated potential observer bias in determining a positiveassay. Lack of responses in HIV-1–unexposed infants suggests assay specificity, in additionto controls built into assays. Depletion of CD8+ T cells eliminated responses almost completely,consistent with CD8+ T cells being the origin of responses. In addition, the peptide size (8–9mer) used for HLA-selected ELISpot assay stimulation makes it unlikely that these were Thelper responses. Finally, the absence of HIV-1 infection was confirmed in infants byconducting serial assays for both HIV-1 DNA and RNA.

Limitations of the present study include the fact that it was conducted before the availabilityof comprehensive peptide epitopes spanning HIV-1. Because we used peptides derived fromHIV-1–infected individuals, we may have underestimated the prevalence of immuneresponses. It has been suggested that uninfected individuals respond preferentially to peptidesdistinct from those eliciting responses in infected individuals [30]. Conversely, stimulationwith a single peptide per pool in our study may have yielded higher sensitivity for relevantpeptides than the pooled peptide assays necessary for comprehensive epitope stimulation[31]. Our control cohort (20 infants), although small, was larger than or comparable to thosein other studies. In addition, all assays included internal controls with standard cut-offs [32].Imperfect specificity may lead to an overestimated prevalence but would bias age effects ortransmission analyses to the null. We did not use intracellular cytokine staining or tetramerassays. However, polyfunctional CD8+ T cell responses to HIV-1 in intracellular cytokine-staining assays has been demonstrated in HIV-1–exposed uninfected infants by others [3].

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HIV-1–specific CTLs are thought to play a pivotal role in containing primary HIV-1 infectionand may defer or attenuate infection in animal models [33–35]. However, observations inanimals, in highly exposed but persistently seronegative individuals, and in HIV-1 vaccinerecipients demonstrate incomplete protection by CTLs, and HIV-1 superinfection occurs inHIV-1–infected individuals despite robust CTL responses, as measured by ELISpot assay [7–9]. Thus, the utility of inducing HIV-1–specific CTL responses by protective vaccines has beendebated, particularly given the results of the recent STEP trial, which may reflect the generalinadequacy of HIV-1 vaccines that rely on the inducement of HIV-1–specific cellular immuneresponses [11]. Alternatively, the findings of the STEP trial may reflect the failure of a specificvaccine product, in which case it is possible that prime-boost vaccine approaches that inducehigher magnitudes or different specificities of cellular immune responses may be at leastpartially protective. The association we observed between levels of HIV-1–specific IFN-γresponses and protection from breast milk HIV-1 transmission lends some support for vaccinestrategies that include as a component inducing responses to HIV-1 to prevent breast milkHIV-1 transmission and possibly to prevent transmission in other settings.

AcknowledgmentsWe thank the women and children who participated in the study, the Nairobi City Council mother-child clinics, andKenyatta National Hospital. In addition, we thank the laboratory, data, and clinical teams involved in the study.

Financial support: US National Institute of Child Health and Human Development (grant R01 HD-23412). B.L.P.,C.F., P.O., and E.O. were scholars in the AIDS International Training and Research Program (funded by NationalInstitutes of Health research grant D43 TW000007, the Fogarty International Center, and the Office of Research onWomen’s Health).

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Figure 1.Participant flow from enrollment to follow-up, focusing on breast-feeding infants who wereHIV-1 uninfected at 1 month of age and subsequently followed up with HIV-1 and HLA-selected HIV-1 enzyme-linked immunospot (ELISpot) assays.

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Figure 2.Results of serial HIV-1 enzyme-linked immunospot (ELISpot) assays for 13 infants who hada positive HIV-1–specific immune response at 1 month of age. A, Results for 9 infants whohad ELISpot assays at all 5 visits. B, Results for 4 infants with incomplete follow-up ELISpotassays. Each box represents results for 1 infant, subdivided by visits. Each color bar representsa peptide epitope, specified at the bottom of each box. The Y-axes show log10-transformedHIV-1–specific spot-forming units per 1 × 106 peripheral blood mononuclear cells (PBMCs),and the X-axes show visits at 1, 3, 6, 9, and/or 12 months of age.

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Table 1

Peptide epitopes used for stimulation in enzyme-linked immunospot assays, by HLA type.

HLA Protein Sequence (5′→3′)

A1 p17 GSEELRSLY

A1 Rev ISERILSTY

A2 RT ILKEPVHGV/ILKDPVHGV

A2 Nef ALKHRAYEL

A2 p17 SLFNTVATL/SLYNTVATL

A2 p24 TLNAWVKVI/TLNAWVKVV

A2 RT VIYQYMDDL

A2 Nef VLEWRFDSRL

A2 Nef PLTFGWCYKL

A3 p17 KIRLRPGGK

A3 Nef QVPLRPMTYK

A3 gp160 TVYYGVPVWK

A3/11/33 RT AIFQSSMTK/SIFQSSMTK

A3/31 gp160 RLRDLLLIVTR

A3/11/31/33 RT DLEIGQHRTK

A3 Nef DLSHFLKEK

A24 Gp160 YLRDQQLL/YLKDQQLL

A24/B44 p24 RDYVDRFYKTL/RDYVDRFFKTL

A24 Nef DSRLAFHHM

A25 p24 DTINEEAAEW/ETINEEAAEW

A26/B70/72 p24 YVDRFFKTL

A29 gp160 FNCGGEFFY

A30 RT KLNWASQIY

A30 p17 RSLYNTVATLY

A30 gp160 IVNRVRQGY

A30 RT KQNPDIVIYQY

A30 gp41 KYCWNLLQY

A6802 Protease DVTLEDINL

A6802 Int ETAYFILKL

A6802/74 RT ETFYVDGAAN

A6802/74 Protease ITLWQRPLV

A6802 Protease DTVLEEMNL

B7/8101/42 Nef TPGPGVRYPL/TPGPGIRYPL

B7 gp160 IPRRIRQGL

B7 p24 SPRTLNAWV

B7 p24 GPGHKARVL

B7/8101 p24 ATPQDLNTM

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HLA Protein Sequence (5′→3′)

B7 Nef FPVTPQVPLR

B8 Nef FLKEKGGL

B8 p24 DIYKRWII/EIYKRWII

B8 RT GPKVKQWPL

B8 gp160 YLKDQQLL/YLRDQQLL

B8 p17 GGKKKYRL/GGKKKYKL

B14/Cw8 p24 DRFFKTLRA/DRFYKTLRA

B14/Cw8 p24 DLNTMLNTV/DLNMMLNIV

B14/Cw8 p24 RAEQASQEV/RAEQATQEV

B14 gp160 ERYLKDQQL

B15/Cw4 gp160 SFNCGGEFF

B18/49 p24 FRDYVDRFYK/FRDYVDRFFK

B18/49 Nef YPLTFGWCY/YPLTFGWCF

B27 p24 KRWIILGLNK/KRWIIMGLNK

B35 p24 PPIPVGDIY

B35 Nef VPLRPMTY

B35 RT HPDIVIYQY/NPDIVIYQY

B35 gp160 TAVPWNASW/TNVPWNSSW

B35 RT EPIVGAETFY

B37/73 Nef YFPDWQNYT

B42 p24 GPGHKARVL

B42 Nef TPQVPLRPM

B45 RT GAETFYVDGA

B51 Pol EPIVGAETFY

B53 p24 ASQEVKNWM/ATQEVKNWM

B53 p24 TPQDLNMML/TPQDLNTML

B53 p24 DTINEEAAEW

B53 p24 QATQEVKNW

B53 p24 VKNWMTETLL

B57/5801 p24 TSTLQEQIGW/TSTLQEQIAW

B57/5801 p24 KAFSPEVIPMF

B57/5801 RT IVLPEKDSW

B57 p24 ISPRTLNAW/LSPRTLNAW

B57 Pol KITTESIVIW

B70/72 RT DVKQLTEVV/DVKQLAEAV

Cw4 p24 QASQEVKNW

Cw4 p17 KYRLKHLVW

Cw8 RT VTDSQYALGI

Cw8 gp160 NCSFNISTSI

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HLA Protein Sequence (5′→3′)

Cw8 Nef KAAVDLSMFL

NOTE. RT, reverse transcriptase.

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Tabl

e 2

Prev

alen

ce, m

agni

tude

, bre

adth

, and

dur

abili

ty o

f HIV

-1–s

peci

fic c

ytot

oxic

T ly

mph

ocyt

e re

spon

ses i

n H

IV-1

–exp

osed

infa

nts w

ho n

ever

bec

ame

HIV

-1in

fect

ed d

urin

g fo

llow

-up

(up

to ∼

12 m

onth

s of a

ge).

Age

Prev

alen

ce o

fH

IV-1

–spe

cific

resp

onse

s,apr

opor

tion

(%)

Mag

nitu

de o

f HIV

-1–s

peci

fic r

espo

nseb

Mag

nitu

de o

f the

sum

of H

IV-1

–spe

cific

resp

onse

s am

ong

infa

nts w

ith p

ositi

veE

LIS

pot r

esul

tsc

Mea

n (r

ange

) no.

of p

ositi

veep

itope

s/m

ean

(ran

ge) n

o.te

sted

am

ong

infa

nts w

ithpo

sitiv

e E

LIS

pot r

esul

tsA

ll in

fant

ste

sted

Infa

nts w

ithpo

sitiv

eE

LIS

pot r

esul

ts

1 m

onth

13/1

12 (1

2)13

(5–2

8) [n

= 1

12]

105

(58–

273)

[n =

13]

125

(71–

351)

1.3

(1–2

)/10.

4 (4

–18)

3 m

onth

s21

/146

(14)

16 (8

–35)

[n =

146

]78

(55–

114)

[n =

21]

80 (5

5–22

3)2.

1 (1

–10)

/12.

2 (5

–19)

6 m

onth

s25

/169

(15)

20 (8

–33)

[n =

169

]85

(64–

285)

[n =

25]

125

(70–

371)

2.1

(1–7

)/10.

6 (2

–17)

9 m

onth

s40

/186

(22)

25 (8

–58)

[n =

186

]10

3 (7

3–26

5) [n

= 4

0]18

5 (8

6–49

5)2.

6 (1

–10)

/12.

7 (2

–24)

12 m

onth

s43

/194

(22)

d25

(10–

58)e

[n =

194

]17

0 (8

0–44

5) [n

= 4

3]31

0 (1

03–1

003)

3.0

(1–1

1)/1

1.7

(2–2

8)

NO

TE

. All

infa

nts i

n th

is ta

ble

wer

e fo

llow

ed u

p fo

r at l

east

11.

5 m

onth

s, an

d m

othe

rs re

porte

d br

east

-fee

ding

dur

ing

follo

w-u

p. E

LISp

ot, e

nzym

e-lin

ked

mm

unos

pot;

IQR

, int

erqu

artil

e ra

nge;

PB

MC

s,pe

riphe

ral b

lood

mon

onuc

lear

cel

ls.

a Posi

tive

resp

onse

s wer

e de

fined

as ⩾

50 H

IV-1

–spe

cific

sfu/

1 ×

106

PBM

Cs,

with

exp

erim

enta

l val

ues a

t lea

st tw

ice

thos

e of

neg

ativ

e co

ntro

l wel

ls (b

ackg

roun

d)

b Dat

a ar

e th

e m

edia

n (I

QR

) of m

axim

um re

spon

ses t

o a

sing

le e

pito

pe, g

iven

in H

IV-1

–spe

cific

sfu/

1 ×

106

PBM

Cs

c Dat

a ar

e th

e m

edia

n (I

QR

) of s

umm

ed p

ositi

ve re

spon

ses,

give

n in

HIV

-1–s

peci

fic sf

u/1

× 10

6 PB

MC

s.

d Sign

ifica

ntly

hig

her p

reva

lenc

e at

mon

th 1

2 th

an a

t mon

th 1

(P =

.01)

e Sign

ifica

ntly

hig

her m

agni

tude

at m

onth

12

than

at m

onth

1 (P

= .0

07)

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Table 3

Maternal and infant characteristics for 141 breast-feeding infants with HIV-1–negative enzyme-linkedimmunospot results at 1 month of age and at follow-up for subsequent HIV-1 risk.

Characteristic

Subset withHLA-selected

peptide-stimulatedassay at 1 month of age

Delivery

Cesarean section, no. (%) 21/140 (15)

Duration of labor, h 9.0 (6–12)

Duration of ruptured membranes, h

0.5 (0.1–2.3)

Duration of zidovudine treatment, days

27.0 (17–37)

Episiotomy, no. (%) 14/139 (10)

Maternal characteristics

Maternal age, years 24 (21–28)

Maternal plasma HIV-1 RNA level, log10 copies/mL

At 32 weeks of gestation 4.6 (4.2–5.1)

At delivery 4.0 (3.4–4.7)

At 1 month after delivery 4.7 (4.2–5.2)

Breast milk HIV-1 RNA level at month 1, log10 copies/mL

2.3 (1.9–3.1)

Potential cofactors for HIV-1 exposure after delivery

Mastitis in first month, no. (%) 10/111 (9)

Infant thrush in first month, no. (%) 12/141 (9)

NOTE. Data are median (interquartile range) values, unless otherwise indicated.

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Table 4

Cofactors for postnatal breast milk HIV-1 transmission at 1 month post partum.

Cofactora

HR (95% CI) for infant HIV-1 infection

Univariate Multivariateb

Maternal plasma HIV-1 RNA level 5.4 (1.7–17.2) [P= .004] 9.1 (1.7–47.7) [P= .009]

Breast milk HIV-1 RNA level 2.5 (1.1–5.6) [P = .03] 1.7 (0.7–4.4) [P = .24]

Infant HIV-1–specific response in ELISpot assay at 1 month of age

0.4 (0.2–1.2) [P = .11] 0.09 (0.01–0.7) [P = .02]

NOTE. Ten of 141 infants acquired HIV-1 between 1 and 12 months of age. CI, confidence interval; ELISpot, enzyme-linked immunospot; HR,hazard ratio.

aMaternal plasma and breast milk HIV-1 RNA levels were measured in log10 copies per milliliter, and the infant HIV-1–specific ELISpot assay

response was measured in log10 HIV-1–specific spot-forming units/1 × 106 peripheral blood mononuclear cells.

bIn the multivariate analysis, the HR for each cofactor was adjusted for the other 2 cofactors.

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