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Center for Neurovirology, Department of Neurology, and Department of
Obstetrics and Gynecology, and
School of Public Health, MCP Hahnemann University, Philadelphia, PA 19102;
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Section of Immunology, Department of Pediatrics, St. Christopher’s Hospital for Children, Philadelphia, PA 19134;
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Department of Obstetrics and Gynecology, Temple University School of Medicine, Philadelphia, PA 19140; and Divisions of
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General Pediatrics,
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Immunologic and Infectious Diseases, and
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Neonatology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is generally considered that HIV-specific CTLs play a critical role in controlling the spread of viral infection (7). We and others have reported previously that HIV-specific CTL precursors (CTLp)4 are present in asymptomatic children with vertically transmitted HIV infection (8, 9, 10, 11, 12). Although HIV-specific CTL responses are not commonly detected in HIV-infected infants younger than 6 mo (9, 10, 11, 12), their presence in uninfected infants of HIV+ mothers (10, 13) and in cord blood mononuclear cells (CBMC) of one HIV-infected newborn (14) suggests that the capacity to generate virus-specific CTL responses might develop in fetal life. Additional studies that demonstrated HIV-specific IL-2 responses in CBMC of infants born to HIV+ mothers who subsequently became seronegative (15) raised the possibility that the presence of HIV-specific cellular immunity during fetal development might be protective against mother-to-infant transmission of HIV (1, 15).
It has been established that Th cells appear to be essential for the
maintenance of effective immunity during chronic viral infections
(16, 17, 18). They can be divided, based on their pattern of cytokine
production, into Th1, Th2, and Th0 subsets (19, 20, 21). Th1 cells produce
IFN-
, IL-2, and TNF-ß, and represent a principal effector
mechanism of cell-mediated immunity against intracellular and
extracellular pathogens. Th2 cells produce IL-4, IL-5, and IL-10, which
influence B cell development. Th0 cells produce all of these types of
cytokines. Because the development of CTL responses might be influenced
by the ability of HIV-exposed infants to generate Th1 cytokines (8, 22), it is possible that in utero development of Th1 responses to HIV
Ags may be important in establishing HIV-specific CTL activity in the
fetus or the newborn. Furthermore, activated Th cells are a rich source
of ß-chemokines (23), and they could therefore mediate nonlytic
inhibition of infection with macrophage-tropic HIV isolates (24, 25).
This, together with the recent findings that vigorous HIV-specific
CD4+ T cell responses are associated with control of
viremia in HIV-infected adults (26), prompted us to examine the
protective role of Th cells in the vertical transmission of HIV. Our
results showed that HIV-specific Th and CTL responses were either
undetectable or at low levels in cord blood and early specimens of PBMC
derived from HIV-infected newborns. In contrast, the majority of
uninfected newborns of HIV+ mothers exhibited IL-2
responses to env-specific peptides that were associated with
enhanced expression of ß-chemokines, but not with HIV-specific CTL
activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Umbilical cord and/or sequential specimens of peripheral blood samples were obtained during the first months of life from nine uninfected and seven HIV-infected infants born to HIV+ mothers. Additional specimens of CBMC and PBMC were obtained from 12 infants born to HIV- mothers. Twenty-one infants were African-Americans, three were Caucasians, and four were Hispanics. Absence of HIV infection was confirmed by repeated negative results in HIV PCR and/or in HIV cultures during the first months of life, and by subsequent seroreversion. One of the HIV-infected infants (121) was presumably breast-fed, but the others were not. Oral zidovudine was prescribed alone, or in combination with lamivudine, for all HIV+ mothers of the uninfected infants (except infant 135) and all of the HIV-infected infants (except infants 121, 366, and 818). Zidovudine was given i.v. during labor and delivery to the same mothers except that of infant 914. A 6-wk course of zidovudine was prescribed for all newborns of HIV+ mothers except infants 135, 219, 611, 307, and 366. Among HIV-infected infants, patients 121, 914, and 818 tested negative for plasma HIV RNA during the first weeks of life. The other infants were already symptomatic at the time of diagnosis of HIV infection.
HIV infection was diagnosed in infant 219 at 3 mo of age during
evaluation for thrombocytopenia, failure to thrive, and severe
hepatosplenomegaly, and in infant 611 at 4.5 mo of age during
evaluation for laryngeal and esophageal candidiasis. These two infants,
along with patient 307 who developed esophageal candidiasis at 5 mo of
age, were considered to have a rapidly progressive disease. The other
four infants had a slowly progressive disease. Further characterization
of the infected infants appears in Table I
. Our investigation had prior approval
of the institutional review board on human experimentation.
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The P18 and T1 env peptides correspond to amino acid residues 315–329 and 428–433, respectively, of gp160 from the HIV-IIIB isolate (27). The peptides were synthesized on solid phase using a Milligen 9050 automated peptide synthesizer by the standard Fmoc methodology at the Wistar Institute (Philadelphia, PA).
Monoclonal antibodies
The mAbs comprised the following: OKT3 (anti-CD3), OKT4 (anti-CD4), OKT8 (anti-CD8), 3G8 (anti-CD16), B4 (anti-CD19), and W6/32 (anti-MHC class I). Abs OKT3, OKT4, OKT8, and W6/32 were produced in Dr. G. Trinchieri’s laboratory (The Wistar Institute); mAb 3G8 was produced in the laboratory of J. Unkeless (Mount Sinai School of Medicine, New York, NY); and mAb B4 was purchased from Coulter Immunology (Hialeah, FL).
Lymphocyte separation and in vitro stimulation assays
The umbilical cord blood specimens of the majority of infants were obtained by venipuncture of an umbilical vein. Only with infants 145 and 175 were the cord blood specimens obtained by dripping. When the cord blood specimens were collected by venipuncture, the site of the puncture was cleaned and the umbilical vein was punctured with a catheter. The blood was drawn into a syringe, until flow could no longer be maintained, at which time the specimen was transferred into a collecting tube. When the cord blood was obtained by dripping, the placenta was cleaned first, and then the blood was allowed to drain into the collecting tube.
CBMC and PBMC were separated from EDTA-treated blood by Ficoll-Hypaque centrifugation (density, 1.077 g/cm3). For the Ag-specific responses, unseparated or CD4-enriched populations of CBMC or PBMC were used, and the experiments were conducted according to previously described protocols (28, 29, 30). Briefly, the CD4-enriched cells were obtained by removal of CD8+, NK, and B cells by the panning technique by first incubating the cells with the mixture of specific mAbs, then washing and incubating for 2 h at 4°C in petri dishes precoated with the F(ab')2 fragment of goat anti-mouse Ig (Organon Teknika, West Chester, PA). Nonadherent cells were collected, washed, and adjusted to a concentration of 3 x 106 cells/ml for Ag stimulation. CD4-enriched or total PBMC or CBMC were cultured in flat-bottom wells of 96-well tissue culture plates (3 x 105 cells/well) in RPMI 1640 supplemented with 10% FCS alone or in the presence of the following: HIV env peptides T1 or P18 (2.5 µM), or tetanus toxoid (TT) (10 µg/ml; Connaught Laboratories, Ontario, Canada). Stimulation with TT was performed on CBMC, and PBMC was derived from HIV-infected mothers to determine the potential contamination of cord blood specimens with maternal PBMC. The levels of Ag-specific responses were evaluated by measuring expression of IL-2- and RANTES-specific mRNA by a quantitative RT-PCR assay after 6 h of stimulation, as described (23, 31).
Antiviral ß-chemokines RANTES, MIP-1
, and MIP-1ß in supernatants
derived from 24-h env peptide-stimulated cultures were
quantified by ELISA using commercially available kits (R&D Systems,
Minneapolis, MN). The detection limit of the ELISA assays for
ß-chemokine production was 30 pg/ml.
Preparation of RNA and PCR amplification
Total cellular RNA was isolated from PBL, as previously described (31), using RNAzol B (Cinna/Biotecx Laboratories, Houston, TX). A constant amount of cDNA (equivalent of 104 cells adjusted to the amount of ß-actin) was amplified with known amounts of synthetic templates and primers specific for IL-2 or RANTES, as described (23, 31). ß-actin was amplified from each sample as quality control cDNA and to adjust for losses in the amount of cellular RNA isolated from the same number of cells. Amplification without RT in the RT mixture and with distilled water instead of cDNA in the PCR reaction mixture served as controls for possible contaminants. Bands corresponding to cellular and synthetic templates specific for the analyzed cytokine were excised from the agarose gels after staining with ethidium bromide, and radioactivity was determined by Cherenkov counting. The amount of cytokine-specific mRNA, expressed in attomoles (10-18 mol) per 106 cells, was determined at the point of equivalence of the PCR products from a plot of the ratio of the relevant PCR product pairs versus the concentration of the competitor template added to the PCR reaction (31). Each data point represents the average of two determinations that varied within 5% of the average. The detection limits for expression of IL-2- and RANTES-specific mRNA were 0.10 amol/106 cells and 0.05 amol/106 cells, respectively.
PCR of genomic DNA for detection of the CCR-5 deletion in HIV-infected and uninfected infants of HIV+ and HIV- mothers was performed as described for cDNA, except that 0.5 µg of genomic DNA was used instead of RT products. The amplification was performed with the flanking primers (5'-CTTCATTACACCTGCAGCTCT-3' and 5'-CACAGCCCTGTGCCTCTTCTTC-3'), as described (32).
Recombinant vaccinia virus (vv) constructs
The vv constructs expressing HIV IIIB/gp160 (amino acids 42–60 deleted; vAbT299), the full-length gag (vAbT141), nef (vT23), or pol (vAbT204), and vaccinia virus (vac), which contains no part of the HIV genome (12), were provided by Dr. G. Mazzara (Therion Biologicals, Cambridge, MA).
Cytotoxicity assays
Standard 51Cr release assays were performed as described (8). Briefly, B-LCL were infected overnight with 5 multiplicity of infection of vac alone or of vv-expressing HIV gene products and labeled with Na251CrO4 (DuPont NEN, Boston, MA) for 12 h. After washing four times, 104 target cells were combined with autologous T cell lines established from each patient at various E:T ratios. After 4 h, supernatants were harvested and radioactivity was measured in a 1470 Wizard gamma counter (Wallac, Gaithersburg, MD). Spontaneous 51Cr release was always <15% of maximum release. Specific lysis was calculated as 100 x ([experimental release - spontaneous release]/[maximum release - spontaneous release]). Percent HIV-specific lysis was calculated by subtracting the percent specific lysis against vac-infected target cells from percent specific lysis against HIV Ag-infected target B cells. HIV-specific responses were considered positive if they exceeded the mean lysis of cells infected with vac alone by 3 SD (8, 12).
Limiting dilution assay of CTLp
Frequencies of gag, env, nef, and pol (GENP)-specific CTLp were determined using limiting dilution analysis (33, 34). Briefly, PBMC were diluted from 18,000 to 667 cells/well in 24 replicate wells of 96-well microtiter plates along with 2.5 x 104 irradiated (6000 rad) allogeneic PBMC from HIV-uninfected individuals and mAb against CD3 (100 ng/ml). On day 1 of culture, fresh medium containing rIL-2 (National Institutes of Health AIDS Research and Reference Program) (50 U/ml) was added, and on day 7, the cultures were restimulated with anti-CD3 mAb and 104 irradiated PBMC. On day 16, they were split and tested for cytotoxicity. Target cells consisted of 51Cr-labeled and vv-infected autologous B-LCLs. 51Cr-labeled target cells (5 x 103) were added to each well, and supernatants were harvested after 4 h. The fraction of nonresponding wells was defined as the number of wells in which 51Cr release did not exceed the mean plus 3 SD of the spontaneous release of the 24 control wells. The CTLp frequency and 95% confidence limits were calculated using the maximum likelihood method (34). Split well analysis was used to examine the cross-reactivity of HIV-specific and vac-specific CTLp. For presentation of data, HIV-specific CTLp frequencies were computed as differences between CTLp frequencies determined on HIV Ag-expressing LCLs versus background values from vac-infected targets. CTLp frequencies were expressed as numbers of CTLp per 106 PBMC or CBMC. CTL responses to HIV Ags that were higher than those directed to vac were considered positive.
Statistical analysis
Mixed model analyses of variance (35) were used to compare mean values of the env peptide-stimulated IL-2- and ß-chemokine-specific responses, as well as frequencies of HIV-specific CTLp between uninfected and HIV-infected infants of HIV+ and HIV- mothers. The Wilcoxon rank sum test (36) was used to compare expression of RANTES-specific mRNA in the env peptide-stimulated cultures derived from HIV-infected and uninfected infants of HIV+ mothers. All computations were performed using SAS version 6.10 (SAS Institute, Cary, NC) on a Windows-based platform.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CBMC and sequential specimens of PBMC obtained from nine
uninfected infants born to HIV+ mothers were analyzed for
the presence of env peptide-specific IL-2 responses. To
determine possible contamination of the cord blood with maternal
leukocytes, we analyzed IL-2-specific mRNA expression in PBMC and CBMC
derived from each mother and her infant. The analysis was conducted by
competitive RT-PCR after 6 h of stimulation with TT. As shown in
Table II, the expression of IL-2-specific
mRNA in TT-stimulated cultures derived from HIV-infected mothers ranged
from 0.96–2.38 amol/106 PBMC. In contrast, IL-2 responses
to TT were detected only in two specimens of CBMC derived from infants
175 and 135. The levels of IL-2 expression in these cultures were
10-fold lower than those detected in the TT-stimulated PBMC of the
mothers. In all of the other infants, the levels of IL-2 expression in
TT-stimulated CBMC did not exceed those detected in the unstimulated
cultures.
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, Table III). The responses were also present in
sequential specimens of PBMC obtained from five of these newborns, and
in the first available specimens of PBMC obtained from infant 135 at 3
mo of age. The env peptide-stimulated responses ceased by 6
mo of life. The levels of env peptide-induced IL-2
transcripts ranged from 0.2–0.8 amol/106 cells, and were
usually induced by only one peptide, except for infant 163, who
responded to both T1 and P18 peptides. In the uninfected infants of
HIV- mothers, the env peptide-specific IL-2
responses were either undetectable or at the detection level (i.e.,
0.10 amol/106 CBMC).
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). In patient 818, the
development of the env peptide-specific IL-2 responses was
associated with a rapid decrease in viral load (from 1,000,000 to
25,100 HIV RNA copies/ml of plasma) and gradual increases in the number
of CD4+ T cells shortly after onset of antiretroviral
therapy at 1.5 mo of age (Table I). The ability of infant 818 to
develop HIV-specific IL-2 responses, in spite of the initial high level
of the virus, is consistent with previous results in adults, which
showed that an aggressive treatment of primary infection could
facilitate the generation of HIV-specific responses (26). Rapid
increases in the env peptide-specific IL-2 responses during
the first month of life were also demonstrated in infant 121. However,
in contrast to infant 818, this patient was capable of controlling the
viremia in the absence of antiretroviral therapy until 1 yr of age.
At this time, the viral load increased from 1,000 to 50,200 RNA
copies/ml of plasma and coincided with the onset of antiretroviral
therapy. In two other infants with a slowly progressive disease (914
and 366), the env peptide-specific IL-2 responses appeared
at the age of 3 and 6 mo, respectively. The delay in the development of
the env peptide-specific IL-2 activities in these infants,
and subsequent decreases in these responses, might be attributed to
relatively high viral loads (199,500 and 19,900 RNA copies/ml of plasma
in patients 914 and 366, respectively) at the time of diagnosis of HIV
infection, consistent with the reported CD4+ T cell
dysfunction in untreated or minimally treated patients experiencing
acute HIV infection (26). It is noteworthy that in both uninfected and infected infants of HIV+ mothers, responses to the T1 peptide were more frequent than those directed to the P18 peptide. These differences could be related to the heterogeneity of MHC-restricted recognition of T cell epitopes since both peptides appear to be promiscuous in their ability to be recognized in association with many HLA class II molecules (37). Additionally, because P18 corresponds to an immunodominant region within the hypervariable V3 loop of the HIV-1IIIB env glycoprotein (27), the mutation rate of the autologous viral sequences within the P18 region in vivo could be higher than that within the conserved T1 epitope.
Frequencies of HIV-specific CTLp in uninfected and HIV-infected infants
A standard limiting dilution assay with autologous LCLs infected
with recombinant vaccinia viruses expressing HIV Ags was performed to
estimate frequencies of HIV-specific CTL in CBMC and PBMC derived from
uninfected and HIV-infected infants of HIV+ and
HIV- mothers. Target cells infected with vac
were included in each assay to determine the level of nonspecific
killing, possibly mediated by EBV- or vac-specific CTL,
and/or expanded NK cells. Cytotoxic activity against
vac-infected autologous LCLs was detected in all infants.
The responses were low (2–38 CTLp/106 cells; Table IV), and did not correlate with the
levels of IL-2 expression in the env peptide-stimulated
cultures or with the frequencies of HIV-specific CTLp.
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). In the uninfected infants of
HIV- mothers, the frequencies of HIV-specific CTLp did not
exceed 10 CTL/106 cells. Among the uninfected infants of
HIV+ mothers, low levels of HIV-specific CTL were detected
in cord blood specimens derived from two uninfected infants, 082 and
180 (Fig. 2, Table IV). The responses were transient and disappeared
shortly after birth in the absence of antigenic stimulation. In infant
082, a low level of pol-specific cytotoxic activity was
detected in CBMC and the 1-mo-old specimen of PBMC. In infant 180,
cytotoxic responses directed toward gag, env, and
pol were detected in CBMC and in the 1-day-old specimen of
PBMC. At 3 mo of age, frequencies of gag-,
env-, and pol-(GEP) specific CTLp
in this infant continued to be higher than those directed to
vac-infected LCL; however, there appears to be no difference
at the 95% confidence limit between these responses (Table IV).
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and Fig. 2). They were either absent or did not exceed
30 CTL/106 cells throughout the entire study in infants
with a rapidly progressive disease (219, 611, and 307). In infants with
slow disease progression, the HIV-specific CTL activity varied in terms
of antigenic specificity and intensity. Consistent with the profile of
the env peptide-stimulated IL-2 responses, the highest
frequencies of HIV-specific CTLp frequencies were detected in patients
818 and 121. In infant 818, the frequency of HIV-specific CTLp
increased rapidly after initiation of antiretroviral therapy, and the
responses were primarily directed toward the pol and
gag Ags. A similar profile of HIV-specific CTL activity was
measured in infant 121, in which the CTL responses developed shortly
after birth, with the highest activity against the gag Ag
(Table IV). Increases in CTL responses to env,
nef, and pol were detected at about 8 mo of age.
In the other infants with a slowly progressive disease (366 and 914),
low levels of HIV-specific CTL responses were detected in older
specimens obtained after initiation of the antiretroviral therapy.
The small amounts of blood available from the HIV-infected infants
precluded the determination of CTLp frequencies in the CD4- or
CD8-depleted population of T cells. To examine whether HIV-specific CTL
responses were mediated by CD8+ T cells, cytotoxic assays
were performed on CD8-enriched cultures after stimulation with
anti-CD3 mAb. Among the infants analyzed, the highest CTL activity
against the pol Ag was detected in CD8+ cultures
derived from HIV-infected infant 121, collected at 12 mo of age (23%
specific lysis at an E:T ratio of 50:1). The CTL responses were
inhibited by mAbs to MHC class I and CD3, demonstrating MHC class I
restriction and TCR-mediated recognition (Fig. 3). Similar experiments performed on
uninfected infants 180 and 082 failed to delineate the phenotype of the
effector cells in cultures established from CBMC after stimulation with
anti-CD3 mAb (Fig. 3).
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To further characterize the differences in HIV-specific cellular
responses in HIV-infected and uninfected infants, we examined
expression of ß-chemokines in cultures stimulated with the
env peptides. The analysis was conducted on CBMC and the
first available specimens of PBMC. Given the demonstrated importance of
ß-chemokines in inhibition of infection with macrophage-tropic HIV
isolates, we examined env peptide-stimulated cultures for
evidence of ß-chemokine production. Consistent with previous studies
that activated Th cells are a source of ß-chemokines (23, 24),
significant increases in RANTES-specific transcripts were detected in
the majority of env peptide-stimulated cultures established
from uninfected infants of HIV+ compared with those derived
from age-matched HIV-infected and control infants (Table III). The
highest expression of RANTES-specific mRNA was detected in the
uninfected infants who were capable of eliciting the env
peptide-specific IL-2 responses (Fig. 4A). On the other hand, the
levels of RANTES-specific mRNA in the env peptide-stimulated
CBMC or PBMC derived from HIV-infected infants shortly after birth were
comparable or slightly higher than those measured in the unstimulated
cultures (Fig. 4B). A similar profile of responses was also
detected with RANTES, MIP-1ß, and MIP-1 proteins. As shown in
Table V, the levels of these
ß-chemokines in supernatants of the env peptide-stimulated
cultures of the uninfected infants of HIV+ mothers were
significantly higher than those detected in cultures established from
HIV-infected or control infants.
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). To establish the effect of CD4+ T cells on
IL-2 and RANTES expression, we compared the levels of IL-2- and
RANTES-specific mRNA in T1-stimulated CD4-enriched and total PBMC from
HIV-infected infants. In infants with AIDS, T1-stimulated IL-2 and
RANTES responses in unseparated and CD4-enriched PBMC were comparable
with each other and with those measured in unstimulated cultures,
suggesting that the observed differences in response to the T1 peptide
among the HIV-infected infants reflect their intrinsic inability to
elicit the env peptide-specific responses (Table VI). In infants with a slowly progressive
disease, the levels of IL-2 were increased in CD4-enriched cultures
compared with those detected in unseparated PBMC, consistent with the
results of previous studies showing that CD4+ T lymphocytes
are a major contributor to the synthesis of IL-2 (38). On the other
hand, the expression of RANTES-specific mRNA was less affected by
depletion of CD8+ lymphocytes (Table VI), suggesting that
both CD4+ and CD8+ T cells contribute to
production of this ß-chemokine.
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32 allele in uninfected
and HIV-infected infants of HIV+ mothers and
HIV- mothers
A failure to synthesize functional cell surface coreceptor caused
by alteration of the CCR-5 coding sequences was suggested to account
for the resistance to infection with M-tropic HIV isolates in vitro
(24, 32). Furthermore, the finding of an increased frequency of the
CCR532 homozygotes found among high risk, uninfected adults (32, 39, 40) suggested that the CCR-5 gene defect might reduce risk of
transmission with common strains of the virus. This defect involves a
deletion from nucleotides 794–825 in a region corresponding to the
second extracellular loop of CCR-5, and encodes a severely truncated
molecule that fails to reach the cell surface (32). Amplification of
cellular DNA derived from the group of nine uninfected newborns (eight
African-Americans and one Hispanic) revealed the presence of the
predicted fragment of 182 bp for the wild-type (WT) allele in all
analyzed specimens (Fig. 5). Only one of
the twins (163), born to an African-American mother, expressed both the
WT and a lower 150-bp fragment corresponding to the CCR532 allele.
The cohort of HIV-infected infants, which consisted of five
African-Americans and two Hispanics, expressed exclusively WT CCR-5
alleles, and only one uninfected infant of HIV- mother
(Caucasian) was heterozygous for CCR-5 expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transient expression of low levels of HIV-specific CTL responses in
only two of nine uninfected infants of HIV-infected mothers suggests
that it is unlikely that they represent a major proportion of
HIV-specific suppressor activity during perinatal HIV infection.
Rather, the early development of HIV-specific Th responses associated
with ß-chemokine production may play a protective role against
vertical transmission of HIV. The absence of HIV-specific CTL activity
in the majority of uninfected newborns of HIV+ mothers who
exhibited env peptide-induced IL-2 expression may reflect
differences in priming of fetal T cells as well as intrinsic immaturity
of the immune system of the fetus at the time of exposure to the HIV
Ags. This is supported by the earlier development of the env
peptide-induced IL-2 responses than HIV-specific CTL activity in
HIV-infected infants with slow disease progression. The differences in
the kinetics of HIV-specific IL-2 versus CTL activity in the
HIV-infected, asymptomatic infants are also consistent with our earlier
studies, which showed an age-dependent expression of IFN- during the
first 2 yr of life, which was significantly lower in HIV-infected
infants than in their uninfected counterparts (28). Because IFN-
plays an important role in induction of cytotoxic activity (43, 44),
the differences in the ability of vertically infected infants to
generate HIV-specific Th1 responses could contribute to the delay in
development of CTL activity, which, in turn, may lead to more rapid
disease progression. Further prospective studies are required to
determine how changes in the profile of IFN- and/or IL-2 expression
correlate with HIV-specific CTL responses during different stages of
infection.
It is also noteworthy that although HIV-specific CTL activity could have contributed to low viremia in patient 121 during the initial stage of infection, the effect was generally transient. It is likely that the apparent increases in viral load in these infants in the face of mounting HIV-specific CTL activity might be due to expanding HIV variants that have escaped CTL recognition (45). It is also possible that CTL clones specific for new variants might have been generated, but their function was inhibited in vivo. Furthermore, although the presence of HIV-specific CTL activity was associated with asymptomatic or mildly symptomatic infection, the responses were generally lower than those detected in HIV-infected children with a slowly progressive disease (8, 9, 10, 11). One possible explanation is that type-specific CTL responses to the env gene products might have predominated during early infancy (14), but they were undetected in our assay using prototype HIV sequences. This is consistent with the notion that the use of target cells expressing laboratory isolate gene products might limit the detection of HIV-specific CTLs in newborns (14). Additionally, the vAbT299 construct expressing the env glycoprotein has a deletion in the C1 region of gp120, which contains several CTL epitopes and may therefore underestimate frequencies of env-specific CTLp (46).
Lack of maternal-to-infant transmission of HIV in this cohort of
uninfected infants of HIV+ mothers, which consisted of
African-Americans and Hispanics, could not be explained by the
homozygous deletion of the CCR-5 coreceptor, since no infant exhibited
this defect. Although our study involved a relatively small number of
infants, the results are consistent with those of much larger studies
that showed the absence or a very low frequency of the CCR532 allele
among non-Caucasians (39, 40). Further study of chemokine receptor
usage by the vertically transmitted HIV isolate(s) will help to
elucidate the potential mechanism of this selection, important for a
thorough understanding of the pathogenesis of vertical transmission of
HIV.
The finding that induction of nonlytic immune responses against HIV is
protective against HIV transmission is in agreement with recent studies
that demonstrated that vigorous HIV-specific CD4+ T cell
proliferative responses are associated with the production of IFN-,
antiviral ß-chemokines, and control of viremia (26). Although the
production of IFN- in the stimulated cultures has not been analyzed
in this study, the association between the env
peptide-mediated expression of IL-2 and ß-chemokine production in
uninfected infants of HIV+ mothers suggests that the
protection against HIV transmission might be mediated by factors
induced during T cell activation. This further supports the notion that
production of antiviral chemokines by leukocytes in exposed but
uninfected individuals may be operative in establishing relative
resistance to HIV infection (47). Whether this would be the result of
autocrine ligation of CCR-5 by overexpressed ß-chemokines or whether
the HIV isolate is sensitive to another factor(s) released by activated
T cells, has yet to be determined. Analysis of the relative resistance
of CD4+ lymphocytes of exposed versus unexposed infants to
infection with HIV in the presence of supernatants from stimulated
CD8+ cells will allow us to gain a deeper insight into the
mechanism of T cell-mediated protective immunity in vertical
transmission of HIV.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, Thomas Jefferson University, 1020 Locust Street, JAH 490, Philadelphia, PA 19107.
3 Address correspondence and reprint requests to Dr. Danuta Kozbor, Department of Microbiology and Immunology, Thomas Jefferson University, 1020 Locust Street, JAH 490, Philadelphia, PA 19107. E-mail address:
4 Abbreviations used in this paper: CTLp, CTL precursor; B-LCL, B lymphoblastoid cell line; CBMC, cord blood mononuclear cell; env, human immunodeficiency virus envelope glycoprotein; GENP, gag, env, nef, and pol; LCL, lymphoblastoid cell line; MIP, macrophage-inflammatory protein; RT, reverse transcriptase; TT, tetanus toxoid; vac, vaccinia; vv, recombinant vaccinia virus; WT, wild type.
Received for publication June 2, 1998. Accepted for publication January 13, 1999.
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  D. B. Schramm, S. Meddows-Taylor, G. E. Gray, L. Kuhn, and C. T. Tiemessen Low Maternal Viral Loads and Reduced Granulocyte-Macrophage Colony-Stimulating Factor Levels Characterize Exposed, Uninfected Infants Who Develop Protective Human Immunodeficiency Virus Type 1-Specific Responses Clin. Vaccine Immunol., April 1, 2007; 14(4): 348 - 354. [Abstract] [Full Text] [PDF]
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 S. Meddows-Taylor, S. L. Donninger, M. Paximadis, D. B. Schramm, F. S. Anthony, G. E. Gray, L. Kuhn, and C. T. Tiemessen Reduced ability of newborns to produce CCL3 is associated with increased susceptibility to perinatal human immunodeficiency virus 1 transmission J. Gen. Virol., July 1, 2006; 87(7): 2055 - 2065. [Abstract] [Full Text] [PDF]
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 O. Barabitskaja, J. S. Foulke Jr., S. Pati, J. Bodor, and M. S. Reitz Jr. Suppression of MIP-1{beta} transcription in human T cells is regulated by inducible cAMP early repressor (ICER) J. Leukoc. Biol., February 1, 2006; 79(2): 378 - 387. [Abstract] [Full Text] [PDF]
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 B. L. Lohman, J. A. Slyker, B. A. Richardson, C. Farquhar, J. M. Mabuka, C. Crudder, T. Dong, E. Obimbo, D. Mbori-Ngacha, J. Overbaugh, et al. 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 J. Virol., July 1, 2005; 79(13): 8121 - 8130. [Abstract] [Full Text] [PDF]
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 A. S. Goldman, L. R. Goldman, and D. A. Goldman What Caused the Epidemic of Pneumocystis Pneumonia in European Premature Infants in the Mid-20th Century? Pediatrics, June 1, 2005; 115(6): e725 - e736. [Abstract] [Full Text] [PDF]
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 L. Sun, S. F. Abdelwahab, G. K. Lewis, and A. Garzino-Demo Recall antigen activation induces prompt release of CCR5 ligands from PBMC: implication in memory responses and immunization Int. Immunol., November 1, 2004; 16(11): 1623 - 1631. [Abstract] [Full Text] [PDF]
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 F. O. TER KUILE, M. E. PARISE, F. H. VERHOEFF, V. UDHAYAKUMAR, R. D. NEWMAN, A. M. VAN EIJK, S. J. ROGERSON, and R. W. STEKETEE THE BURDEN OF CO-INFECTION WITH HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 AND MALARIA IN PREGNANT WOMEN IN SUB-SAHARAN AFRICA Am J Trop Med Hyg, August 1, 2004; 71(2_suppl): 41 - 54. [Abstract] [Full Text] [PDF]
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 E. T. Abrams, H. Brown, S. W. Chensue, G. D. H. Turner, E. Tadesse, V. M. Lema, M. E. Molyneux, R. Rochford, S. R. Meshnick, and S. J. Rogerson Host Response to Malaria During Pregnancy: Placental Monocyte Recruitment Is Associated with Elevated {beta} Chemokine Expression J. Immunol., March 1, 2003; 170(5): 2759 - 2764. [Abstract] [Full Text] [PDF]
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F. Buseyne, D. Scott-Algara, F. Porrot, B. Corre, N. Bellal, M. Burgard, C. Rouzioux, S. Blanche, and Y. Riviere Frequencies of Ex Vivo-Activated Human Immunodeficiency Virus Type 1-Specific Gamma-Interferon-Producing CD8+ T Cells in Infected Children Correlate Positively with Plasma Viral Load J. Virol., November 13, 2002; 76(24): 12414 - 12422. [Abstract] [Full Text] [PDF]
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B. L. Shacklett, K. E. S. Shaw, L. A. Adamson, D. T. Wilkens, C. A. Cox, D. C. Montefiori, M. B. Gardner, P. Sonigo, and P. A. Luciw Live, Attenuated Simian Immunodeficiency Virus SIVmac-M4, with Point Mutations in the Env Transmembrane Protein Intracytoplasmic Domain, Provides Partial Protection from Mucosal Challenge with Pathogenic SIVmac251 J. Virol., October 11, 2002; 76(22): 11365 - 11378. [Abstract] [Full Text] [PDF]
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L. Pedroza-Martins, W. J. Boscardin, D. J. Anisman-Posner, D. Schols, Y. J. Bryson, and C. H. Uittenbogaart Impact of Cytokines on Replication in the Thymus of Primary Human Immunodeficiency Virus Type 1 Isolates from Infants J. Virol., June 14, 2002; 76(14): 6929 - 6943. [Abstract] [Full Text] [PDF]
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 P. Hansasuta and S. L Rowland-Jones HIV-1 transmission and acute HIV-1 infection Br. Med. Bull., September 1, 2001; 58(1): 109 - 127. [Abstract] [Full Text] [PDF]
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 R. Kamin-Lewis, S. F. Abdelwahab, C. Trang, A. Baker, A. L. DeVico, R. C. Gallo, and G. K. Lewis Perforin-low memory CD8+ cells are the predominant T cells in normal humans that synthesize the beta -chemokine macrophage inflammatory protein-1beta PNAS, July 19, 2001; (2001) 161298998. [Abstract] [Full Text] [PDF]
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A. Garzino-Demo, R. B. Moss, J. B. Margolick, F. Cleghorn, A. Sill, W. A. Blattner, F. Cocchi, D. J. Carlo, A. L. DeVico, and R. C. Gallo Spontaneous and antigen-induced production of HIV-inhibitory beta -chemokines are associated with AIDS-free status PNAS, October 12, 1999; 96(21): 11986 - 11991. [Abstract] [Full Text] [PDF]
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R. Kamin-Lewis, S. F. Abdelwahab, C. Trang, A. Baker, A. L. DeVico, R. C. Gallo, and G. K. Lewis Perforin-low memory CD8+ cells are the predominant T cells in normal humans that synthesize the beta -chemokine macrophage inflammatory protein-1beta PNAS, July 31, 2001; 98(16): 9283 - 9288. [Abstract] [Full Text] [PDF]
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