JOURNAL OF VIROLOGY, July 2005, p. 8121–8130
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 13
Longitudinal Assessment of Human Immunodeficiency Virus Type 1
(HIV-1)-Specific Gamma Interferon Responses during the
First Year of Life in HIV-1-Infected Infants
Barbara L. Lohman,1,2* Jennifer A. Slyker,7Barbra A. Richardson,3,5Carey Farquhar,2,4
Jenniffer M. Mabuka,1Christopher Crudder,1Tao Dong,7Elizabeth Obimbo,1
Dorothy Mbori-Ngacha,1Julie Overbaugh,5,6Sarah Rowland-Jones,7
and Grace John-Stewart1,2,4
Department of Paediatrics, University of Nairobi, Nairobi, Kenya1; Departments of Epidemiology,2Biostatisics,3and
Medicine,4University of Washington, Seattle, Washington; Divisions of Public Health Sciences5and
Human Biology,6Fred Hutchinson Cancer Research Center, Seattle, Washington; and Human
Immunology Unit, Weatherall Institute of Molecular Medicine,
Oxford University, Oxford, England7
Received 3 December 2004/Accepted 13 March 2005
Human immunodeficiency virus type 1 (HIV-1) infection results in different patterns of viral replication in
pediatric compared to adult populations. The role of early HIV-1-specific responses in viral control has not
been well defined, because most studies of HIV-1-infected infants have been retrospective or cross-sectional. We
evaluated the association between HIV-1-specific gamma interferon (IFN-?) release from the cells of infants of
1 to 3 months of age and peak viral loads and mortality in the first year of life among 61 Kenyan HIV-1-infected
infants. At 1 month, responses were detected in 7/12 (58%) and 6/21 (29%) of infants infected in utero and
peripartum, respectively (P ? 0.09), and in ?50% of infants thereafter. Peaks of HIV-specific spot-forming
units (SFU) increased significantly with age in all infants, from 251/106peripheral blood mononuclear cells
(PBMC) at 1 month of age to 501/106PBMC at 12 months of age (P ? 0.03), although when limited to infants
who survived to 1 year, the increase in peak HIV-specific SFU was no longer significant (P ? 0.18). Over the
first year of life, infants with IFN-? responses at 1 month had peak plasma viral loads, rates of decline of viral
load, and mortality risk similar to those of infants who lacked responses at 1 month. The strength and breadth
of IFN-? responses at 1 month were not significantly associated with viral containment or mortality. These
results suggest that, in contrast to HIV-1-infected adults, in whom strong cytotoxic T lymphocyte responses in
primary infection are associated with reductions in viremia, HIV-1-infected neonates generate HIV-1-specific
CD8?-T-cell responses early in life that are not clearly associated with improved clinical outcomes.
CD8?cytotoxic T lymphocytes (CTL) are responsible for
clearing acute viral infections such as cytomegalovirus (CMV)
and measles virus and play variable roles in chronic viral in-
fections, depending on the site and degree of ongoing viral
replication (reviewed in references 38 and 64). The CD8?CTL
response to human immunodeficiency virus (HIV) has been
extensively studied in humans and in the rhesus macaque-
simian immunodeficiency virus (SIV) model for association
with virus levels and disease progression. HIV-1- and SIV-
specific CD8?-T lymphocyte numbers rise during acute infec-
tion, and the peak number of CD8?-T cells coincides with the
decline in plasma viremia (30, 31, 48). In the SIV model, the
depletion of CD8?-T lymphocytes in either acute or chronic
infection leads to an increase in viral replication which is cur-
tailed by the regeneration of CD8?-T cells (26, 56). The con-
clusion that HIV-1-specific CTL are an important component
of the host immune response to infection is supported by
several notable findings. First, antiviral CTL, frequently gag-
specific, are associated with control of HIV-1 viral replication
in adults and children of ?10 years old (10, 13, 19, 39, 40, 45).
Second, levels of circulating HIV-1-specific CTL are main-
tained in long-term nonprogressors (24, 51). Finally, in both
acute and chronic HIV-1 infections, HIV isolates have evolved
mutations allowing escape from CTL recognition, indicating
immune pressure on viral replication (6, 22, 47).
The study of HIV-1-specific CD8?-T-cell responses in ver-
tically infected infants is complicated by several factors absent
in horizontal HIV-1 transmission. The patterns of HIV-1 peak
and set-point plasma viral loads are very different in adults and
infants (49). In infants, the levels of HIV-1 plasma viremia are
persistently high, with declines not seen until the second year
of life (17, 18, 37). In the absence of antiretroviral therapy,
vertically infected infants have a bimodal distribution of dis-
ease progression, with approximately 25% progressing to
AIDS within 1 year of life (reviewed in reference 35). Factors
that may influence the levels of viral replication and disease
progression in infants include the phenotype of the transmitted
virus, the high number of target cells available for HIV-1
infection, and an immature immune system. Infants are likely
infected with a viral variant modified by maternal immune
pressure due to the half-match in major histocompatibility
complex alleles (21, 55). In addition, infants have high levels of
thymic output, and their immune systems are predominantly
naı ¨ve (8, 15, 57), although the role of the thymus in the disease
* Corresponding author. Mailing address: IARTP, 325 Ninth Ave.,
Box 359909, Seattle, WA 98104. Phone: (206) 543-4278. Fax: (206)
543-4818. E-mail: email@example.com.
progression of HIV-1-infected infants is not well understood
(7). The ability of the neonate to respond effectively to infec-
tion is thought to be limited by the number of circulating
mature T and antigen-processing cells (50, 54). Cellular im-
mune responses in HIV-1-infected infants have been inconsis-
tently detected in infants younger than 6 months (33, 34, 36, 46,
61). The paucity of CTL responses in infants less than 1 year
old has been suggested (i) to be due to diminished Th1 re-
sponses, in particular a deficiency in gamma interferon
(IFN-?) secretion (58, 62, 63) or (ii) to be influenced by age,
CD4 counts, and antigen processing (53). The interpretation of
the earlier reports is limited by the lack of longitudinal data
and the imprecise detection of the timing of infection in the
We had the opportunity, with a prospective observational
cohort of infants born to HIV-1-infected women, to identify
infants infected before 1 month of life and to measure HIV-
1-specific CD8?-T-cell responses together with viral loads over
the first year of life. We hypothesized that sustained high
HIV-1 viral loads observed in perinatal transmission were con-
sistent with a deficiency in virus-specific CD8?-T-cell re-
sponses. We examined the HIV-1-specific IFN-? release from
CD8?-T cells at one to five time points during the first year of
life of 61 Kenyan infants diagnosed with HIV-1 infection at or
before the first month of life and investigated the relationship
between the timing and presence of early anti-HIV-1 CD8?-
T-cell responses and peak viral loads and mortality in the
MATERIALS AND METHODS
Patient cohort. As part of a larger cohort of infants born to HIV-1-infected
women between 1999 and 2003, 61 infants with HIV-1 RNA or DNA detected in
blood obtained during the first month of life were included in this cohort. Details
of the larger cohort of infants born to HIV-1-infected women have been pre-
sented elsewhere (20, 41). Written informed consent was obtained from all
mothers on behalf of themselves and their infants. Mothers were recruited
during pregnancy and were provided with zidovudine beginning between the 34th
and 36th weeks of gestation for the prevention of infant HIV-1 infection (59).
Infants were breast or formula fed as per maternal preference. Infants were
examined by clinicians, and 1 to 3 ml of peripheral blood was obtained at birth
and at months 1, 3, 6, 9, and 12. Seven infants born to HIV-1-seronegative
women were included as controls and were bled at 3 months of life. This research
complied with all U.S. and Kenyan guidelines and policies.
Blood collection. Freshly collected EDTA-anticoagulated blood was centri-
fuged for 10 min at 1,800 rpm to separate plasma, which was aliquoted and stored
at ?80°C. The remaining blood was then diluted with an equal volume of RPMI
1640 (Gibco-BRL), layered on a Ficoll gradient (Lymphocyte Separation Me-
dium; Organon Teknika, West Chester, PA), and centrifuged for 30 min at 2,000
rpm. Peripheral blood mononuclear cells (PBMC) at the interface of the gradi-
ent were isolated, washed in RPMI 1640, and used directly in the enzyme-linked
immunospot (ELISPOT) assay. The mean numbers ? standard errors of the
means of PBMC recovered at months 1, 3, 6, 9, and 12 were (13.6 ? 1.0) ? 106,
(17.7 ? 1.1) ? 106, (18.6 ? 1.7) ? 106, (16.8 ? 1.6) ? 106, and (15.5 ? 1.5) ?
HIV-1 diagnosis and determination of timing of infection. Infant HIV-1 in-
fection status was determined by PCR amplification of HIV-1 gag DNA se-
quences from dried blood spotted on filter paper (44) or by quantitative analysis
of infant plasma HIV-1 RNA using a transcription-mediated amplification
method sensitive for the detection of multiple HIV-1 subtypes (Gen-Probe
HIV-1 viral load assay) (43). Results relative to HIV RNA copies/ml plasma
were considered positive if there were ?100 copies per ml or, in cases where less
than 500 ?l was available for testing, if there were ?50 RNA copies per reaction.
The timing of an infection was categorized as in utero if the specimen collected
within the first 48 h of life was positive for either HIV-1 DNA or HIV-1 RNA.
The timing of infection was defined as peripartum if plasma HIV-1 RNA was
undetectable within the first 48 h of life and positive at 1 month of age. Quan-
titative plasma viral loads were determined using the Gen-Probe assay.
HLA typing. Molecular HLA typing was performed on DNA extracted from 5
? 106infant PBMC using amplification refractory mutation system PCR with
sequence-specific primers designed for East African populations (9).
Peptides. Sixty-eight peptides spanning five regions of HIV-1 were synthesized
by Fmoc (9-fluorenylmethoxy carbonyl) chemistry at the Peptide Core Facility at
the Weatherall Institute of Molecular Medicine (Tao Dong, Oxford University).
The peptides were chosen based on predefined CTL epitopes of the prevalent
HIV-1 clades in Kenya (A and D). Twenty-seven peptides were from gag, 18 were
from pol, 19 were from env, 13 were from nef, and 1 was from rev (Table 1).
Epitopes were chosen based on responses previously reported in HIV-1 infection
and included those present in acute infection, long-term nonprogressors, and
those associated with viral control (29). These peptides bind 29 common HLA
class 1 alleles (12 HLA-A, 15 HLA-B, and 2 HLA-C) representative of East
African populations. The identities of peptides tested for each individual were
based on the infant’s HLA type. In the event of a limited number of cells,
peptides were prioritized to test a complete panel for each HLA allele.
IFN-? ELISPOT assay. An ELISPOT assay was used to detect HIV-1-specific
IFN-? release from PBMC following overnight incubation with peptides. Briefly,
96-well Millipore plates (MAIP45; Millipore SA, Molsheim, France) were coated
with 7.5 ?g monoclonal antibody to IFN-? (1-DIK; Mabtech Ab, Nacka, Swe-
den) for 2 h at 37°C. Excess antibody was removed by washing six times with
RPMI 1640 and blocked with RPMI 1640 containing L-glutamine and supple-
mented with 10% fetal calf serum (all from Gibco-BRL), designated R10, for 30
min at room temperature before cells were added. Duplicate wells containing 2
? 105PBMC/well were stimulated with 20 ?g/ml peptide, 10 ?g/ml phytohe-
magglutinin (PHA) (positive control) (Murex Biotech Limited, Dartford, United
Kingdom), or R10 media alone (negative control). The mean numbers ? stan-
dard errors of the means of PBMC used per assay at months 1, 3, 6, 9, and 12
were (6.5 ? 0.5) ? 106, (6.4 ? 0.4) ? 106, (6.9 ? 0.4) ? 106, (6.7 ? 0.5) ? 106,
and (6.8 ? 0.4) ? 106, respectively. After overnight stimulation in a humidified
incubator at 37°C and with 5% CO2, cells were removed from the plates by
washing with phosphate-buffered saline containing 0.05% Tween 20 (Sigma, St.
Louis, MO), followed in sequence by the application of biotinylated anti-IFN-?
antibody (1:1,000; 7-B6-1 biotin; Mabtech) for 3 h at room temperature, washing,
and the application of streptavidin alkaline phosphatase (1:1,000; Mabtech) for
1.5 h at room temperature. After the final washing, spot-forming units (SFU)
were visualized by the addition of alkaline phosphatase (Bio-Rad Laboratories,
Hercules, CA) for approximately 10 min or until an intense blue reaction was
visible in the wells stimulated with PHA. Additional color development was
prevented by washing the plates under running water. Plates were allowed to dry
overnight before reading. Plates were read visually until January 2001, after
which an automated ELISPOT reader was used (Autoimmun Diagnostika,
Strassberg, Germany). The number of HIV-specific SFU per 106cells was de-
fined as the average number of SFU per 106cells from peptide-stimulated wells
minus the average number of SFU per 106cells in wells containing R10 (back-
ground control). The following criteria were used to determine a positive assay:
(i) a response to PHA of ?100 SFU after the subtraction of the background, (ii)
the number of HIV-specific SFU per 106cells being greater than or equal to 50,
and (iii) the number of SFU in peptide-stimulated wells being greater than or
equal to two times the number of background control SFU (4). The qualities of
the ELISPOT responses were compared in two ways: relative to the mean of
positive responses and relative to the peak of positive responses.
Detection of antigen-specific CD8?-T cells. PBMC from an infant expressing
HLA-A2 and -B8 were stained with phycoerythrin-conjugated-HLA class I tet-
rameric complexes refolded with either HIV-1 nef-FLKEKGGL (B8-nef) or
CMV pp65-NLVPMVATV (A2-CMV) (Tao Dong, Oxford University). Briefly,
for each stain, 150 ?l of whole blood was incubated with phycoerythrin-conju-
gated tetramer for 15 min at 37°C, followed by the addition of peridin chlorophyll
protein-labeled anti-CD8 and fluorescein isothiocyanate-labeled anti-CD45R0
(both from Becton Dickenson, San Diego, CA) for an additional 15 min. Three
milliliters of FACSLysis solution (Becton Dickenson) was added per tube, and
the tubes were left for 5 min at room temperature, after which time the tubes
were centrifuged for 5 min at 1,500 rpm. The cell pellet was washed once in
phosphate-buffered saline containing 0.5% fetal calf serum and 0.5 mM EDTA,
suspended in 170 ?l of FACSfix (Becton Dickenson), and stored overnight in the
dark at 4°C prior to analysis with CellQuest software (Becton Dickenson).
Statistical analysis. To compare differences in the IFN-? responses of infants
infected with HIV in utero to the responses of those infected peripartum, dif-
ferences in the distributions of background SFU, numbers of mean and peak
positive HIV-specific SFU, numbers of peptides tested, and numbers of peptides
8122LOHMAN ET AL.J. VIROL.
recognized were determined using Mann-Whitney U tests. Pearson’s chi-square
statistic was used for the comparison of the proportion of infants with positive
HIV-1-specific ELISPOT results who were infected in utero to that of those who
were infected peripartum. Data was log transformed for analysis of viral loads
and changes in the numbers of HIV-specific SFU over time. Differences between
the mean and peak log10s of the HIV-1 RNA plasma viral loads of (i) infants
infected with HIV-1 in utero and those of the infants infected peripartum or (ii)
between infants who had and those who lacked HIV-1-specific ELISPOT re-
sponses were assessed using t tests for independent samples. To model the
changes in peak numbers of HIV-specific SFU over the first year of life of
infected infants, a linear mixed-effect model was used. For determination of the
change in viral load over time, a linear mixed-effect model with a compound
symmetric covariance structure was used with the log10of the HIV RNA viral
load between month 1 and month 12 as the outcome variable. Linear regression
analysis was used to compare peak plasma HIV-1 RNA loads in infants who had
or lacked HIV-1-specific IFN-? responses at 1 month of age, controlling for viral
load at 1 month of age after testing for and excluding nonsignificant effect
modification. Cox regression analysis was used to model the association between
mortality and (i) the presence of either HIV-1-specific IFN-? responses at 1
month of age or (ii) weak HIV-specific IFN-? responses (?500 HIV-specific
SFU), controlling for viral load at 1 month of age and in utero infection.
Concordance between peptide-stimulated IFN-? secretion
and detection of antigen-specific CD8?-T cells. The presence
of antigen-specific CD8?-T cells and the relationship between
the detection of these cells by major histocompatibility com-
plex-tetrameric complexes and detection by IFN-? ELISPOT
assay were confirmed in three individuals. Figure 1 illustrates
representative results obtained from one HIV-1-infected in-
fant at 9 and 12 months of age, demonstrating the presence of
both A2-CMV and B8-nef-specific CD8?-T cells in this infant
together with peptide-specific ELISPOT assay results. At 9
months of age, we detected tetramer-positive A2-CMV-spe-
cific and B8-nef-specific CD8?-T cells, comprising 0.68% and
0.27%, respectively, of total lymphocytes. In the concordant
ELISPOT assay, the frequency of IFN-?-producing cells re-
sponding to A2-CMV peptide was 1,497 per 106PBMC, and
the frequency of cells responding to B8-nef-peptide was 303
TABLE 1. Panel of HIV-1 peptides, HLA restriction, and frequency of positive individualsa
No. of positive
No. of positive
B8-D/EIYKRWII.......................................... 2/2 (100)
B35-PPIPVGDIY.......................................... 2/3 (67)
B57-I/LSPRTLNAWL.................................. 2/4 (50)
B8-GGKKKYR/KL...................................... 1/2 (50)
B57/B5801-KAFSPEVIPMF...................... 3/7 (43)
A3-KIRLRPGGK........................................ 1/3 (33)
B14/Cw8-RAEQAS/TQEV ........................ 1/3 (33)
B14/Cw8-DRFF/YKTLRA......................... 1/3 (33)
A2-TLNAWVKVI/V................................... 4/13 (31)
Cw4-QASQEVKNW................................... 2/7 (29)
B57/B5801-TSTLQEQIG/AW................... 2/7 (29)
B7/B42-GPGHKARVL............................... 3/12 (25)
B18/B49-FRDYVDRFY/FK...................... 1/5 (20)
B53-QATQEVKNW................................... 1/5 (20)
B7/B8101-ATPQDLNTM........................... 1/6 (17)
A2-SLF/YNTVATL .................................... 2/13 (15)
B53-TPQDLNM/TML................................ 2/13 (15)
B53-AS/TQEVKNWM ............................... 0/5 (0)
B53 VKNWMTETLL................................. 0/5 (0)
A24/B44-RDYVDRFY/FKTL................... 0/9 (0)
Cw4-KYRLKHLVW................................... 0/9 (0)
A26-YVDRFFKTL ..................................... 0/1 (0)
B7-SPRTLNAWV....................................... 0/4 (0)
A1-GSEELRSLY ........................................ 0/5 (0)
A30-RSLYNTVATLY................................ 0/16 (0)
B14/Cw8-DLNM/TMLNI/TV..................... 0/3 (0)
A25/B53-D/ETINEEAAEW ...................... 0/6 (0)
B35-H/NPDIVIYQY..................................... 2/3 (67)
Cw8-VTDSQYALGI..................................... 1/2 (50)
B8-GPKVKQWPL ....................................... 1/2 (50)
A6802/A74-ITLWQRPLV.......................... 6/13 (46)
B45-GAETFYVDGA ................................. 3/7 (43)
B35-EPIVGAETFY.................................... 2/5 (40)
A6802/A74-ETFYVDGAAN..................... 5/13 (38)
B57-KITTESIVIW ...................................... 1/3 (33)
A6802-ETAYFILKL................................... 3/9 (33)
aExpressed as the number of babies with a positive peptide response over the number of infants tested with that peptide, followed by the percent. Assays were
conducted at multiple ages per infant, and thus responses to a given peptide were counted once per infant if there was ever detection of a response with a particular
peptide. Peptides that stimulated positive responses in ?50% of infants are indicated in boldface.
bHLA restriction of each peptide precedes the sequence. Clade variants are indicated by a slash within the amino acid sequence.
A2-VIYQYMDDL .................................... 3/12 (25)
A3/A11/A33-A/SIFQSSMTK ................... 1/4 (25)
A6802-DTVLEEMNL............................... 2/9 (22)
A30-KLNWASQIY ................................... 2/13 (15)
B57/B5801-IVLPEKDSW......................... 1/7 (14)
A2-ILKE/DPVHGV.................................. 1/13 (8)
A30-KQNPDIVIYQY............................... 1/15 (7)
A3/A11/A31/A33-DLEIGQHRTK.......... 0/5 (0)
A6802-DVTLEDINL ................................ 0/9 (0)
B14-ERYLKDQQL.................................... 1/1 (100)
A29-FNCGGEFFY ..................................... 5/8 (63)
Cw8-NCSFNISTSI..................................... 1/2 (50)
B35-TA/NPWNA/SSW.............................. 1/3 (33)
A24/B8-YLR/KDQQLL............................ 2/7 (29)
A3/A31-RLRDLLLIVTR......................... 1/4 (25)
B15/Cw4-SFNCGGEFF............................ 3/17 (18)
A30-IVNRVRQGY................................... 1/16 (6)
A3-TVYYGVPVWK................................. 0/3 (0)
A30-KYCWNLLQY.................................. 0/16 (0)
B7-IPRRIRQGL........................................ 0/4 (0)
B35-VPLRPMTY........................................ 3/3 (100)
Cw8-KAAVDLSMFL ................................. 2/2 (100)
A3-QVPLRPMTYK.................................... 2/3 (67)
B42-TPQVPLRPM..................................... 5/10 (50)
B7/B42/B8101-TPGPGI/VRYPL............... 2/4 (50)
B18/B49-YPLTFGWCY/F........................ 2/5 (40)
A2-PLTFGWCYKL .................................. 3/11 (27)
A3-DLSHFLKEK...................................... 1/5 (20)
B8-FLKEKGGL......................................... 2/11 (18)
A2-VLEWRFDSRL.................................. 1/11 (9)
A2-ALKHRAYEL..................................... 1/12 (8)
B7-FPVTPQVPLR.................................... 0/4 (0)
A24-DSRLAFHHM.................................. 0/5 (0)
revA1-ISTERILSTY....................................... 1/5 (25)
VOL. 79, 2005CD8?IFN-? ELISPOT RESPONSES IN HIV-1-INFECTED INFANTS 8123
per 106PBMC. When the assays were repeated 3 months later,
the frequencies of tetramer-positive CD8?-T cells were 0.35%
and 0.38% of total lymphocytes specific for A2-CMV and B8-
nef, respectively. The corresponding frequencies of A2-CMV
and B8-nef peptide-specific IFN-?-producing cells were 1,025
per 106PBMC and 450 per 106PBMC, respectively. Differ-
ences between the percentage of CD8?-T cells detected by
tetramer and the corresponding functional response following
cognate peptide stimulation have been observed before (3,
29a), and the detection of IFN-? ELISPOT responses is likely
representative of the presence of CD8?antigen-specific T cells
HIV-1-specific ELISPOT responses in infants infected in
utero or peripartum. We measured HIV-1-specific IFN-? se-
cretions from PBMC isolated from infants infected with HIV-1
in utero or peripartum and followed those responses over the
first year of life. The peptides were used either singly or paired
by clade variants and are presented by decreasing frequency of
response in Table 1.
Using this panel, we estimated the prevalence of the detec-
tion of HIV-1-specific IFN-? responses during the first year of
life in HIV-1-infected infants (Table 2). The number of infants
tested at each time point varied because of clinic attendance
and mortality. We were able to test all HLA-restricted peptides in
our panel on 29/33 (88%) infants tested at month 1 and on 43/45
(96%), 40/41 (97%), 26/29 (90%), and 24/26 (92%) infants at
months 3, 6, 9, and 12, respectively. At 1 month of age, there was
a trend for more-likely detection of HIV-1-specific IFN-? re-
sponses in infants infected in utero compared to infants infected
peripartum. Seven of 12 (58%) infants infected in utero had
detectable responses versus 6 (29%) of 21 infants infected peri-
partum (P ? 0.09). Although the differences did not reach signif-
icance due to small sample sizes, this trend is likely explained by
of HIV-1-specific IFN-? responses was approximately 50% in
either group and, at this and subsequent ages, there was no sig-
in infants with respect to the timing of infection with HIV-1.
To determine if the timing of HIV-1 infection affected the
quality of the cellular immune response, we compared the
breadths and strengths of IFN-? responses in infants infected
in utero versus peripartum (Table 3). We chose the month 3
time point for comparisons because the greatest number of
infants (45/61 [75%]) were tested at that time. To determine
HIV-1 specificity, we compared the prevalence of positive as-
says in HIV-1-infected infants with that in HIV-1 unexposed,
uninfected infants. None of seven 3-month-old HIV-1 unex-
posed, uninfected infants had positive ELISPOT responses,
suggesting a high specificity for the assay. We observed trends
for infants infected peripartum to have stronger HIV-1-specific
IFN-? responses detected at 3 months of age, although the
data did not reach significance. For infants infected in utero,
the median magnitudes of mean and peak responses were 151
and 155 HIV-specific SFU per 106PBMC, respectively, versus
384 and 458 per 106PBMC, respectively, in infants infected
peripartum (P ? 0.06 for both).
HIV-1-specific IFN-? responses over the first year of life. In
a subset of 18 infants, IFN-? responses and plasma viral load
measurements were conducted at every time point up to month
12 or death. These results are shown in Fig. 2a and b, respec-
tively, and demonstrate a diverse pattern of immune recogni-
tion and viral replication. Three infants were completely lack-
ing in detectable responses (B1-276, B1-160, and B1-005). Two
infants demonstrated strengthening and broadening responses
to infection over time (B1-454, B1-473). Two infants lost early
responses, but the loss was not associated with mortality (B1-
093, B1-259). In one infant, the loss of early responses pre-
ceded the infant’s death (B1-424). To interpret the changes in
HIV-1-specific IFN-? responses over the first year of life in all
the infants, we employed a model that adjusted for age, in-
complete and repeated measures, and the timing of infections.
Log10-transformed peak HIV-specific SFU numbers were used
to model the responses over time. We found a significant
change in peak HIV-1-specific IFN-? responses in infants dur-
FIG. 1. Assays of CMV- and HIV-specific tetramer staining and
IFN-? ELISPOT responses. Responses detected from an HIV-1-in-
fected infant at 9 (upper panels) and 12 (lower panels) months of age.
PBMC were stained with anti-CD8 monoclonal antibody (y axes) and
with the relevant HLA class I/peptide tetramer (x axes) or stimulated
with peptides in an overnight ELISPOT assay to detect IFN-? secre-
tion. A2-CMVpp65-NLVPMVATV (left panels) and B8-HIV-1 nef-F
LKEDGGL (right panels) responses were detected by both assays. The
tetramer-positive CD8?T cells, as percentages of total lymphocytes,
are indicated adjacent to each plot. The antigen-specific IFN-? ELI-
SPOT assay results are shown above each plot.
TABLE 2. Prevalence of detection of HIV-1-specific IFN-?
ELISPOT responses during the first year of life of
61 HIV-1-infected infants
(% of cohort)
Prevalence of HIV-1-specific
responses [no. of positive
infants/no. of infants infected
aThe timing of infection of infants was categorized as in utero if HIV-1 DNA
or RNA was detected in dried blood spots or plasma collected within the first
48 h of life. Infants were categorized as infected peripartum if HIV-1 DNA or
RNA was undetectable within the first 48 h. of life and detectable at 1 month of
bP values determined by Pearson’s chi-square statistic.
8124LOHMAN ET AL. J. VIROL.
ing the first year of life, independent of the timing of infection
with HIV-1 (P ? 0.03) (Fig. 3). Peak HIV-specific SFU in-
creased twofold with increasing age, from a mean of 251/106
PBMC (2.4 log10) at 1 month of age to mean of 501/106PBMC
(2.7 log10) at 12 months of age. To address the potential bias of
survivor effect, we also limited the analysis to 30 infants who
survived to month 12. We found a similar increase in peak
IFN-? responses with age, but the change was no longer sta-
tistically significant (P ? 0.18). The strength of the peak HIV-
specific SFU increased 1.5-fold with increasing age in the in-
fants who survived to month 12, from a mean of 343/106PBMC
(2.53 log10) at 1 month of age to a mean of 540/106PBMC
(2.73 log10) at 12 months of age.
Early HIV-1-specific IFN-? responses and HIV-1 replication
kinetics. The emergence of HIV- and SIV-specific CD8?-T-
cell immune responses in primary infection has been shown to
correlate with declines in levels of viral replication. Therefore,
we sought to determine the relationship between early HIV-
1-specific IFN-? responses and outcomes of HIV-1 infection in
infants, as measured by peak viral loads, rates of decline of
viral replication, and risk of mortality. The patterns of HIV-1
RNA plasma viral replication over time in infants with or
without HIV-1-specific IFN-? responses at 1 month of age,
depicted by timing of infection, are shown in Fig. 4.
Peak HIV-1 plasma load is an important marker of disease
progression in HIV-1-infected infants (52). Peak viral load was
defined as the highest viral load detected within 6 months
postinfection. We found no significant difference in mean log10
peak viral loads for infants infected in utero versus those in-
fected peripartum. The mean peak viral load for 23 infants
with defined in utero infection was 6.59 log10? 0.84 versus
6.86 log10? 0.67 among 37 infants infected peripartum (P ?
0.2). Thirty-two infants had concurrent HIV-1-specific IFN-?
ELISPOT assays and HIV-1 RNA plasma viral loads measured
at 1 month of age. As the timing of HIV-1 infection was shown
not to significantly affect peak viral load, all 32 infants were
considered as one group for the subsequent analyses. There
was not a significant difference in mean log10peak viral loads
for infants with or without detectable HIV-1-specific IFN-?
responses at 1 month of life, after controlling for the baseline
month 1 viral load. The mean peak viral load for 12 infants
with detectable HIV-1-specific IFN-? responses at 1 month of
age was 6.82 log10? 0.18 versus 6.62 log10? 0.18 for 20 infants
who lacked detectable responses at 1 month of age (P ? 0.5).
There were no significant differences in peak viral loads for
infants with broad (?2 peptides) or strong (?500 HIV-1 SFU)
IFN-? responses at 1 month compared to those with narrower,
weaker, or negative responses (data not shown).
Having seen no difference in the peak viral loads, we next
investigated the relationship between the presence of HIV-1-
specific IFN-? responses at 1 month of age and the change in
viral load over time. The presence of detectable HIV-1-specific
IFN-? responses at 1 month of life was not associated with a
difference in the rate of change of viral load over the first year
of life compared to the rate for infants who lacked such re-
sponses at 1 month (decline of 0.04 versus 0.02 log10HIV-1
RNA copies/ml plasma/month, respectively; P ? 0.2).
We lastly investigated the relationship between the presence
and strength of HIV-1-specific IFN-? responses at 1 month of
age and infant mortality during the first year of life. There was
a trend correlating the presence of HIV-1-specific IFN-? re-
sponses at 1 month of life and increased mortality found with
univariate analysis (hazard ratio [HR] ? 2.72; 95% confidence
interval [CI], 0.89 to 8.36; P ? 0.08). There was also a trend for
higher mortality in those infants with IFN-? responses of ?500
HIV-1 SFU (HR ? 3.77; 95% CI, 0.94 to 15.13; P ? 0.06).
After controlling for the viral load at month 1 and the timing
of infection in multivariate analysis, there were no statisti-
cally significant relationships either between mortality and
the presence of month 1 HIV-1-specific IFN-? responses
(HR, 2.20; 95% CI, 0.62 to 7.87; P ? 0.2) or between
mortality and IFN-? responses of ?500 HIV-1 SFU (HR,
2.08; 95% CI, 0.70 to 6.17; P ? 0.2). Thus, the presence or
strength of HIV-1-specific IFN-? responses in the first
months of life had no effect on peak viral load, on the rate
of decline in HIV-1 plasma viral load, or on survival in this
cohort of HIV-1-infected infants.
We addressed a mechanism hypothesized to contribute to
the sustained high viral loads measured in perinatal HIV-1
TABLE 3. HIV-1-specific IFN-? responses in HIV-1 unexposed, uninfected infants and HIV-1-infected infants measured at 3 months of age
No. of individuals tested
No. of peptides tested (range of median)
Background SFU/106PBMC median (25th–75th quartile)
No. with positive responses (%)
No. of positive peptides (range of median)
Mean positive HIV SFU/106PBMC median (25th–75th quartile)
Peak positive HIV SFU/106PBMC median (25th–75th quartile)
aUnexposed infants were born to HIV-1-seronegative women. The infection status of an infant was confirmed by testing for HIV-1 DNA in dried blood spots
collected at the time of the visit, as described in Materials and Methods.
bInfants were categorized as infected in utero if HIV-1 DNA or RNA was detected in dried blood spots or plasma collected within the first 48 h of life. Infants were
categorized as infected peripartum if HIV-1 DNA or RNA was undetectable within the first 48 h of life and detectable at 1 month of life.
cThe P values for differences in responses between infants infected in utero and peripartum was determined by nonparametric test for differences in medians from
independent samples, except for comparisons of numbers of individuals with positive responses, in which the P values were calculated from the chi-square statistic.
dn.a., not applicable.
VOL. 79, 2005CD8?IFN-? ELISPOT RESPONSES IN HIV-1-INFECTED INFANTS8125
infection, the adequacy of the CD8?-T-cell immune response.
We were able to establish the timing of infection in the infants
as occurring either in utero or peripartum and to compare the
development of CD8?-T-cell effector function and viral loads.
Infants infected peripartum had a tendency to generate stron-
ger but not broader responses to HIV-1 peptides than did
infants infected in utero. Both groups had similar increases in
the magnitudes of HIV-1-specific IFN-? responses over their
first year of life, an indication that infants infected in utero are
not more immunosuppressed than infants infected peripartum
and that both groups possess the capacity for continuing im-
mune maturation. Infants infected in utero did not demon-
strate higher peak viral loads than infants infected peripartum,
as has also been described in the Abidjan ANRS 049 Ditrame
Study (52). A primary report describing the bimodal disease
progression in perinatally infected infants suggests that in
utero infection may interfere with the maturation of the im-
mune system and increase the rate of development of immu-
nosuppression (5), something we do not show with our data.
Despite a rapid and relatively robust immune response to
infection, induction of these early responses did not appear
beneficial to infants during the first year of life, regardless of
the timing of infection. Specifically, there was no relationship
between the detection of these responses and a reduction in
peak viral load, rate of decline of viral replication, or risk of
mortality (independent of viral load).
Previous studies have relied on cross-sectional studies in
older children, where there may be bias towards those who
survived. The approach here, using a longitudinal analysis of a
cohort of HIV-1-infected infants with well-defined timing of
FIG. 2. Spectrum of HIV-1-specific peptide responses in HIV-1-infected infants with measurements at every time point to 1 year (A) or death
(B). Individual graphs present HIV-1-specific SFU/106PBMC (stacked colored bars) and HIV-1 RNA copies/ml plasma (closed circles) as
functions of age. The numbers above each bar or above the horizontal axes indicate the numbers of peptides tested at each time point, while the
heights of each of the colored sections of the bars indicate the strengths of the peptide-specific responses. The number of peptides was the lowest
at month 1, due to limitations in cell numbers, and remained constant from month 3 to 1 year or death. The notation of death indicates the infant
died 1 to 3 months after the last measurement.
8126 LOHMAN ET AL.J. VIROL.
infection, has allowed us to address survivor bias and enabled
prospective characterization of HIV-1-specific immunity over
the first year of life. In our study, results from serial assays of
the same infants revealed a significant twofold increase in
HIV-1-specific IFN-? responses over the first year of life,
which became a trend for increasing responses when limited to
those infants who survived to 1 year. Thus, older infants had
stronger responses, which may be due in part to a survivor
effect and in part to age-related maturation.
We observed that neonates with in utero or peripartum
HIV-1 infection were able to generate IFN-? responses of
breadth and strength similar to those reported for adults with
primary HIV-1 infection, albeit weaker than responses re-
ported for adults with chronic infection (1). Two detailed stud-
ies with adults of CD8?-T-cell responses to primary HIV-1
infection both show a narrow response to approximately two
epitopes within the first year of infection and a lack of corre-
lation between the frequency of virus-specific IFN-? responses
and viral containment (11, 14). In our study, we find very
similar kinetics in the induction of HIV-1-specific responses,
with 52% of infants having detectable responses by 3 months of
age to a median of two peptides at a mean magnitude of 384
HIV-1-specific SFU. There are few studies that describe virus-
specific IFN-? responses in perinatally infected infants. The
results presented here indicate that neonatal CD8?IFN-?
responses to HIV-1 infection are more prevalent than previ-
ously reported. Scott and colleagues investigated IFN-? re-
sponses in a group of 13 infants of less than 6 months of age
and found that 2/13 (15%) infants had detectable HIV-1-spe-
cific responses before the initiation of antiretroviral therapy
(58). Wasik et al. observed an age-related increase in responses
in their cohort of children on antiretroviral therapy and de-
scribed two infants under 1 year of age who generated increas-
ing IFN-? responses in the setting of increasing viral loads (63).
Our findings based on a large number of infants in the longi-
tudinal cohort extend these reports and support the concept
that the human neonate appears quite capable of mounting
appreciable CD8?-T-cell-mediated IFN-? responses. The tim-
ing and strength of the responses suggest that the main factor
in the development of HIV-1-specific IFN-? responses is not
the age at the time of infection but rather the duration of
exposure to HIV-1.
We believe our report to be the first comprehensive study of
immune function in HIV-1-infected neonates. Our sample size
permitted multivariate analysis of factors including baseline
viral loads (month 1), the presence of detectable IFN-? re-
VOL. 79, 2005CD8?IFN-? ELISPOT RESPONSES IN HIV-1-INFECTED INFANTS8127
sponses, and the timing of infection in the model. The presence
of HIV-1-specific IFN-? responses was not associated with the
control of HIV-1 infection in neonates. One explanation for
this finding is the possibility that although IFN-? secretion is
widely used as a surrogate for CTL activity, it may not predict
CTL levels and may be an inadequate marker to use as a
measure of HIV-1-specific immunity. The ability of cells to
produce IFN-? in response to HIV-1 peptide stimulation has
been shown not to fully predict levels of functional CTL. The
quality of the CD8?-T-cell response is affected by perforin and
granzyme production (2, 23), T-cell receptor flexibility (32),
and the quality of CD4?T-cell help (27), and all these factors
are likely important for the control of HIV-1 replication. Ad-
ditionally, the repertoire of cytokine production from human
CD8?-T cells stimulated by either vaccination or natural in-
fection is broad and diverse, and limiting analyses to one cy-
tokine reduces the ability to detect associations (3, 16). Alter-
natively, IFN-? secretion may accurately reflect the CD8?-T-
cell responses to pediatric HIV-1 infection, but the immune
pressure may promote generation of CTL escape variants.
High levels of HIV-1 replication in the setting of antiviral CTL
responses have been linked to the rapid emergence of CTL
escape variants (6, 12, 22, 42). Additionally, viral variants
adapted to the maternal CTL responses may be transmitted to
the infant, limiting the capacity of the infant’s CTL response to
have an effect on viral replication. The appearance of viral
variants not recognized by the neonatal immune system may
contribute significantly to the high viral loads we observed in
this cohort. Delineation of viral and host factors in the patho-
genesis of pediatric HIV-1 infection will hopefully generate
better options for treatment and care of this population.
The lack of detectable responses in almost half of the indi-
viduals over the course of the study may not reflect a complete
absence of HIV-1-specific IFN-? responses, because of our use
of defined CD8?-T-cell epitopes rather than overlapping pep-
tides spanning the HIV genome. By its nature, the peptide
panel does not fully represent the HIV-1 viral genome or
recombinant variants, limiting our ability to detect responses to
undefined epitopes. Also, by testing individual peptides rather
than peptide pools, we maximized our chances of detecting
low-level responses (4), and we were able to test peptides
derived from HIV-1 subtypes A and D, previously known to
elicit responses in HIV-1-infected individuals from Kenya (28),
including many defined as being in a state of acute infection
We find no link between the detection of early HIV-1-spe-
FIG. 3. HIV-1-specific IFN-? responses increase with age in HIV-
1-infected infants. Individual peak HIV-1 SFU responses are plotted
on the y axis with respect to age of the infant, which is plotted on the
x axis. Infants infected with HIV-1 in utero are represented by trian-
gles; those infected peripartum are represented by circles. The linear
mixed-effect regression lines are shown for changes in peak HIV-1
SFU numbers over time for infants infected in utero (dotted) and
peripartum (solid) and are not significantly different from each other.
FIG. 4. HIV-1 RNA plasma viral loads during the first year of life in infants with or without HIV-1-specific IFN-? responses detected at 1
month of age. (A) Twelve infants infected with HIV-1 in utero: 7 with month 1 HIV-1-specific IFN-? early responses, 5 without. (B) Twenty infants
infected peripartum: 6 with HIV-1-specific IFN-? responses, 14 without. Open symbols/dashed lines represent individuals who lacked detectable
HIV-1-specific IFN-? responses at 1 month of life. Closed symbols/solid lines represent those who had month 1 HIV-1-specific IFN-? responses.
The mean log10s of HIV-1 RNA copies per ml plasma for infants with month 1 HIV-1-specific IFN-? responses are indicated by bold solid lines;
the mean log10s of HIV-1 RNA copies/ml plasma for infants without month 1-specific responses are represented by bold dashed lines.
8128 LOHMAN ET AL.J. VIROL.
cific immune responses and viral pathogenesis, but this lack of
apparent protective effect in the setting of infection does not
indicate vaccine-induced cellular responses will be nonprotec-
tive, as the outcome of neonatal exposure to antigen is likely
determined by the antigenic dose and the timing and route of
exposure. Our observation that neonates have the capacity to
mount antiviral immune responses in the first months of life
that are comparable to levels observed in adults with primary
infection supports the concept of newborn immunization strat-
egies. Immunization of newborn rhesus macaques results in
the emergence of SIV-specific immune responses, and immu-
nized infants demonstrated prolonged survival after challenge
virus (60). In the lymphocytic choriomeningitis model, DNA
immunization of newborn mice results in rapidly generated
CD8?-T-cell responses within the first weeks of life as well as
long-lived, fully functional responses detectable 1 year postim-
munization (25, 65). However, our data support a cautionary
approach with vaccines designed to mimic HIV-1-specific
IFN-? responses observed in infected individuals reliant on
IFN-? secretion as the sole immune correlate. These responses
may be ineffective in the control of early HIV-1 replication and
may not be representative of the spectrum of immune re-
sponses generated by vaccination.
We thank Sandy Emery for viral load measurements, Julius Oyugi
and Sammy Wambua for HLA typing, and Rose Bosire, Phelgona
Otieno, Dalton Wamalwa, Grace Wariua, and Christine Gichuhi for
clinical examinations. We are grateful to Tim Rostron for assistance
with HLA typing, training, and reagents. We thank Rupert Kaul and
Marie Reilly for helpful discussions and critical reading of the manu-
script. We acknowledge the women and infant participants in this study
and peer counselors for their contributions.
This work was supported by AIDS International Training and Re-
search Program, NIH Fogarty International Center grant D43 TW
00007, and NIH grants TW06080 (B.L.L.) and HD23412-14 (G.J.-S.).
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