Immunology and Cell Biology (2000) 78, 586–595
Research Article
HIV-1-specific cellular immune responses among HIV-1-resistant
sex workers
K E I T H R F OW K E , 1 RU P E RT K AU L , 2 K E N N E T H L RO S E N T H A L , 3 J U L I U S OY U G I , 2
J O S H UA K I M A N I , 2 W J O H N RU T H E R F O R D, 1 N I C O J D NAG E L K E R K E , 2
T B L A K E BA L L , 1 J O B J B WAYO, 2 J N E I L S I M O N S E N, 2 G E N E M S H E A R E R 3 a n d
FRANCIS A PLUMMER2
1
Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, 3Immunology Program,
Department of Pathology, McMaster University, Hamilton, Ontario, Canada, 2Department of Medical Microbiology,
University of Nairobi, Nairobi, Kenya and 4Experimental Immunology Branch, National Cancer Institute, National
Institutes of Health, Bethesda, Maryland, USA
Summary The goal of the present study was to determine whether there were HIV-1 specific cellular immune
responses among a subgroup of women within a cohort of Nairobi prostitutes (n = 1800) who, despite their intense
sexual exposure to HIV-1, are epidemiologically resistant to HIV-1 infection. Of the 80 women defined to be resistant, 24 were recruited for immunological evaluation. The HIV-1-specific T-helper responses were determined by
IL-2 production following stimulation with HIV-1 envelope peptides and soluble gp120. Cytotoxic T lymphocyte
responses were determined by lysis of autologous EBV-transformed B cell lines infected with control vaccinia virus
or recombinant vaccinia viruses containing the HIV-1 structural genes env, gag and pol. Resistant women had
significantly increased HIV-1 specific T-helper responses, as determined by in vitro IL-2 production to HIV-1
envelope peptides and soluble glycoprotein 120, compared with low-risk seronegative and HIV-1-infected controls
(P ≤ 0.01, Student’s t-test). Seven of the 17 (41%) resistant women showed IL-2 stimulation indices ≥ 2.0. HIV-1specific CTL responses were detected among 15/22 (68.2%) resistant women compared with 0/12 low-risk controls (Chi-squared test, P < 0.001). In the two resistant individuals tested, the CTL activity was mediated by CD8+
effectors. Many HIV-1-resistant women show evidence of HIV-1-specific T-helper and cytotoxic responses. These
data support the suggestion that HIV-1-specific T-cell responses contribute to protection against HIV-1 infection.
Key words: Africa, AIDS, cellular immunity, cytotoxic T lymphocyte, exposed uninfected, HIV, resistance, T helper
cell, vaccine.
Introduction
The continued global spread of HIV-1, despite the efforts of
public health programs, and the inaccessibility of new treatment options to more than 90% of the world’s infected1 make
the development of HIV-1 vaccines an urgent priority. Efforts
to develop HIV-1 vaccines are hampered by the absence of
models of naturally occurring protective immunity to HIV-1.
Characterization of immune responses to HIV-1 that protect
against infection would help to provide a focus for HIV-1
vaccine research and could speed the development of
effective vaccines.
With the recognition of the type 1/type 2 dichotomy in the
T-helper cell response to infection,2,3 the concept that cellular effector mechanisms may mediate protective immunity to
HIV-1, in the absence of systemic humoral immunity, has
gained credence over the past several years.4–6 Several groups
Correspondence: Dr Keith R Fowke, Laboratory of Viral
Immunology, Department of Medical Microbiology, University of
Manitoba, Room 539, 730 William Avenue, Winnipeg, Manitoba
R3E 0W3, Canada. Email: [email protected]
Received 8 February 2000; accepted 15 May 2000.
of HIV-1 exposed uninfected and seronegative individuals
with evidence of T-cell responses to HIV-1 antigens have
been reported.7–9 Clerici et al. have described a group of gay
men who developed T-cell responses to HIV-1 after several
unprotected sexual exposures to HIV-1 infected partners.7
MHC Class I-restricted CTL to HIV-1 have been reported in
uninfected children of HIV-1-infected mothers,8–10 exposed
health-care workers,11 uninfected heterosexual partners
of HIV-1-infected individuals12 and HIV-1- and HIV-2uninfected prostitutes in the Gambia.13 These findings have
led to the suggestion that cellular rather than systemic
humoral immunity may be important in protection against
HIV-1 infection.14–16 According to this hypothesis, a systemic
humoral or type 2 T helper response represents an ineffectual
immune response, whereas the generation of a cellular, type
1 immune response is effective in eliminating the virus
and preventing the establishment of detectable infection.
Evidence for the ability of the cellular immune response to
control virus levels comes from the recent observation that,
among long-term non-progressors, HIV-1-specific T helper
responses are inversely correlated with viral load.17 The
detection of HIV-1-specific cellular immune responses in
HIV-1-exposed but uninfected individuals suggests that these
individuals have had exposures to HIV-1 sufficient to induce
HIV cellular immune responses in HIV-1 resistance
cellular immune responses but insufficient to induce antibody. This could result from exposure to defective virus or
HIV-1 antigens. A second possibility is that these individuals
have been infected with HIV-1 and cellular immune
responses have been responsible for clearance of infection, as
has been documented in children born to HIV-1-infected
mothers.10,18 However, the studies reported to date do not
provide evidence that these responses protect on subsequent
exposure. Rather, they only suggest that some individuals are
able to clear an HIV-1 infection. To examine whether immune
responses can contribute to protection against infection, a
study would be required in which these responses are
detected in a setting where high levels of exposure to HIV-1
provide sufficient opportunity for HIV-1 infection, and yet
there is evidence that the individuals are not infected.
There is evidence for protection against HIV-1 infection
from observational epidemiological studies. Early studies of
risk factors for sexually acquired HIV-1 infection have suggested that the risk of becoming infected is independent of
exposure. Among Nairobi prostitutes, the mean duration of
prostitution is considerably shorter in HIV-1-infected women
compared to uninfected women.19 Similarly, in the sex partners of HIV-1-infected haemophiliacs and the sexual partners
of persons infected through blood transfusions, the risk of
HIV-1 infection is, to some degree, independent of sexual
exposure.20 Travers et al. have reported that HIV-2-infected
prostitutes are partially protected against HIV-1 infection.21
We have reported epidemiological evidence of resistance to
HIV-1 infection among a small group of prostitutes in
Nairobi who, despite prolonged and continuing exposure to
HIV-1 through their occupation, remain uninfected.22 Here,
we report studies of potential mechanisms mediating resistance and show that the majority of HIV-1-resistant women
tested have detectable HIV-1-specific cellular immune
responses.
Materials and Methods
Study subjects
Female sex workers enrolled in the Pumwani Sex Worker Cohort in
Nairobi, Kenya were the source of HIV-1-resistant and HIV-1infected subjects for the studies described here. All participants
in this study, including controls, gave informed consent and this
study conformed to all ethical guidelines from the University of
Manitoba and the University of Nairobi. Procedures for enrolment
and follow up and the HIV-1 and sexually transmitted disease (STD)
interventions offered through the clinic have been previously
described.19,23 Studies of the epidemiology, immunobiology and prevention of sexually transmitted infections are nested within the
overall cohort of 1800 women. As previously described, women were
defined as resistant to HIV-1 infection if they had been enrolled in
the cohort for three or more years, were HIV-1/2 seronegative and
HIV-1 polymerase chain reaction (PCR) negative with normal CD4+
counts. Women meeting the epidemiological definition of resistance
to HIV-1 infection were invited to participate in this study of mechanisms of resistance to HIV-1 infection and gave their informed
consent. Due to the fact that many of the women do not permanently
reside in Nairobi, we were only able to contact and recruit 24 of the
80 resistant women to participate in this particular arm of the study.
The HIV-1-seropositive women from the Pumwani Sex Worker
Cohort were also approached about their participation. The HIV-1
587
seronegative controls, who were at low risk of HIV-1 exposure, were
selected from Kenyan women enrolled as negative controls in a study
of mother-to-child transmission of HIV-1.24 In the latter subjects, the
last pregnancy was more than 1 year prior to participation in this
study. Additional low-risk seronegative controls included Kenyan
laboratory workers and some North American laboratory workers
(all of whom had resided in Nairobi for 6 months or more).
Detection of HIV-1
All individuals in the study were tested for the presence of HIV-1/2
antibodies with commercial enzyme immunoassays (HTLV-III
ELISA, DuPont, Wilmington, PE, USA from 1985 to 1988; Vironostika, Organon Technika, Durham, NC, USA from 1988 to 1990;
Detect HIV, IAF Biochem, Laval, Quebec, Canada from 1990 to
1992; and Enzygnost HIV-1/2 EIA, Behring, Marburg, Germany
from 1991 to present). These serology systems are capable of detecting infection resulting from all of the HIV-1 clades predominant in
East Africa and HIV-2, which has not been reported in that region.25,26
All women defined as resistant also had HIV-1 immunoblots performed on one or more occasions (Novapath Immunoblot, BioRad,
Hercules, CA, USA).
Methods for HIV-1 PCR have been previously described.22,27 In
brief, conserved regions of the env, vif and nef genes were targeted
for amplification. Because two of the three primer sets are in regulatory genes, which are not involved in defining HIV-1 clades, this
amplification strategy is capable of detecting HIV-1 variants from
multiple clades, including those most prevalent in East Africa. All
women classified as resistant had tested negative for HIV-1 PCR on
one or more occasions.
In vitro studies
Blood was drawn by venipuncture into sodium heparin vacutainers
(Becton Dickinson, Mississauga, Ontario, Canada) or into citrate,
phosphate, dextrose and adenine (CPDA) blood collection bags (Red
Cross Society International, Nairobi, Kenya). Peripheral blood
mononuclear cells were purified by Ficoll-Hypaque (Sigma, St
Louis, MO, USA) density gradient centrifugation. All cell cultures
were performed in RPMI-1640 supplemented with 10 mmol/L
HEPES and L-glutamine pH 7.2 (Gibco BRL, Gaithersburg, MD,
USA), 50 mmol/L β-mercaptoethanol (Sigma), 10 U/mL penicillin,
0.1 mg/mL streptomycin, 0.25 mg/mL amphotericin B and 10% fetal
calf serum (Intergen, Houston, TX, USA), which had been heat
inactivated at 56°C for 40 min.
T helper assay
Interleukin-2 and IL-6 production in response to different stimuli
were used to assay T helper cell function. The methods for detection
of IL-2 production by PBMC in response to HIV-1 and control antigens were based on those of Clerici et al.7 Briefly, PBMC were isolated and cryopreserved until all samples could be assayed
simultaneously. In a 96-well plate, 3 × 105 PBMC were incubated
with test solutions (triplicate wells per test solution) in the presence
of 25 µL of a 1:100 dilution of anti-Tac monoclonal antibody (clone
322a, specific for the alpha chain of the IL-2 receptor, was provided
by the Experimental Immunology Branch, National Cancer Institute,
National Institutes of Health, Bethesda, MD, USA). Test solutions
used were media, recombinant soluble gp120 (Intracel Corporation,
Issaquah, WA, USA) 2 µg/mL, each of five different HIV-1 env
peptides (AIDS Research and Reference Reagent Program, AIDS
Program, NIAID, NIH, Bethesda, MD, USA) 2.5 µg/mL and
588
KR Fowke et al.
phytohaemagglutanin-P (PHA-P, Sigma) 5 µg/mL as a positive
control. These peptides were chosen because they have been previous shown to elicit IL-2 responses in HIV-infected and exposed
uninfected individuals.7,28 In these experiments, the media control
also served as an irrelevant antigen control, because the fetal calf
serum in the media contains many non-HIV-1 proteins and peptides.
Peptides have been listed by NIH catalogue number with the corresponding peptide name used in the previous studies7,28 in parentheses
and the amino acid location within HIV-1MN gp120 (env): 1929 (T2)
env 101–120, 864 (P18) env 306–327, 1991 (P23) env 361–380, 2007
(T1) env 421–440 and 1589 (T1) env 418–441. Samples were incubated at 37°C for 7 days and the supernatants were collected and
frozen at – 70°C and shipped on dry ice to the University of
Manitoba where they were thawed and assayed. These same culture
supernatants were used for IL-6 determinations. Production of IL-2
and IL-6 was measured using commercial enzyme immunoassays
performed according to the manufacturer’s instructions (Quantikine
Immunoassays, R & D Systems, Minneapolis, MN, USA). The
PBMC from all groups (resistant, HIV infected and low risk) were
handled similarly and assayed on the same plates.
Cytotoxic T cell assay
Cytotoxic T cell assays were performed as described by Grant et al.29
Both fresh (assayed in Nairobi) and cryopreserved cells (assayed at
the University of Manitoba) were used in these assays and the results
proved reproducible. Briefly, 3-day-old PHA blasts were infected
with HIV-1IIIB, inactivated with mitomycin C and used as autologous
stimulators for bulk autologous PBMC (effectors), which were
cultured for 7 days with recombinant human IL-2 (5 U/mL, Roche
Diagnostics, Laval, Quebec, Canada). On day 10 of the assay, autologous EBV-transformed B cells (targets) were infected with one of
four recombinant vaccinia/HIV-1 viral vectors. The recombinant
vaccinia/HIV-1 viruses included vSC8 (wild-type vaccinia with
the β-galactosidase gene),30 vDK1 (HIV-1 gag inserted into vSC8),
vCF21 (HIV-1 pol inserted into vSC8)31 and vPE16 (HIV-1 env
inserted into vSC8).32 Viral vectors were obtained through the
AIDS Research and Reference Reagent Program, AIDS Program,
NIAID, NIH. The following day, [51Cr]-labelled target cells were
washed and plated, in triplicate, with the effectors, at effector to
target ratios of 50:1, 25:1 and 12.5:1. Wells containing only target
cells or target cells with 1% SDS were used to determine the
spontaneous (Min) and maximal release (Max), respectively, of 51Cr
from the labelled cells. After 4 h incubation, 100 µL of supernatant
were removed and added to 1 mL of scintillation cocktail (Ecolume,
ICN, Costa Mesa, CA, USA) and the released 51Cr detected using
a liquid scintillation counter. Percent spontaneous release was calculated as Min/Max × 100. Percent specific lysis was calculated by
((Exp – Min)/(Max – Min)) × 100. Lysis of the targets infected with
the recombinant vaccinia/HIV-1 (vDK1, vCF21 and vPE16) viruses
was considered HIV-1 specific if lysis was 10% or greater than the
lysis of the vaccinia control (vSC8) and the results were titratable or
were repeatable.
CD8+ lymphocyte-depleted cultures were prepared by a 30 min
exposure of the PBMC cultures to immunomagnetic beads specific
for the CD8+ molecule (Dynal, Inc., Lake Success, NY, USA) and
elution of the CD8+ T cell-depleted PBMC for use in CTL assays.
Data analysis
Data were analysed using the Statistical Package for the Social
Sciences (SPSS) version 7.1 microcomputer program. Chi-squared
test and Student’s t-test were used for comparison of sample proportions and means. Linear discriminant analysis33 was used to analyse
the relationship between T-helper responses in the resistant and
control groups. Student’s t-tests were used to evaluate the relationship between T-helper and CTL responses. Discriminant analysis
discriminates two populations by a linear set of variables (for
instance IL-2 secretion at baseline and IL-2 secretion in response to
peptide stimulation). The distributions of these variables in the two
populations are assumed to be normal and have the same (co) variance structure. It is similar to logistic regression, but more efficient
for small samples. The conclusions drawn from discriminant analysis are not dependent on asymptotic approximations (which may be
moot in small samples such as ours). Discriminating variables were
logarithmically transformed in order to make their distributions
approximate to the above assumptions.
Results
Infection status of the study subjects
Twenty-four women who were classified as resistant to
HIV-1 were available to participate in these studies. All were
HIV-1 antibody negative by both enzyme immunoassay and
immunoblot. Negative serology on each individual was
performed 8–56 times (mean ± SD, 21 ± 13) over 4–10 years.
All women were HIV-1 PCR negative on specimens obtained
simultaneously with the studies reported here. None of the
HIV-1-resistant women participating in the present study
have subsequently seroconverted to HIV-1 in an additional
24 months of follow up, despite continued exposure to HIV-1
through sex work.
The HIV-1 systemic antibody status of the positive and
negative control groups was confirmed by enzyme immunoassay.
CD4 Levels
CD4 levels were not available for the same day that the test
subjects were bled for this study. However, as an estimate we
took the CD4 values for time points that were < 3 months
earlier and later that this study date. A Student t-test analysis
showed no difference between the earlier and later values.
The average of the two points was taken for each individual
and means generated for each group (low risk 758 CD4/mm3,
resistant 1019 CD4/mm3 and HIV-infected 434 CD4/mm3).
Student’s t-test analysis showed that while the CD4 values for
the low-risk and resistant groups did not significantly differ
(P = 0.16), both had significantly higher CD4 levels than the
HIV-infected group (P = 0.04 and P = 0.001, respectively).
HIV-1-specific T-helper responses
To determine whether the resistant women exhibit HIV-1
specific immune responses, PBMC from 17 resistant, seven
HIV-1-infected and six low-risk seronegative subjects (five
Kenyan women and one North American laboratory worker)
were cultured in the presence of sgp120 and each of the
HIV-1 envelope peptides separately. After 7 days of culture,
the supernatants were assayed for IL-2 levels by enzyme
immunoassay. Table 1 and Fig. 1 show the median IL-2
responses to media and HIV-1 antigens. The majority of
women in the resistant group exhibited spontaneous IL-2
secretion. Twelve of 17 resistant women, no low-risk seronegative subjects and no HIV-1-infected women showed
HIV cellular immune responses in HIV-1 resistance
Table 1
589
Interleukin-2 secretion following stimulation of PBMC with media and HIV-1 antigens
Study
Number
Status
Media
sgp120
T1(2007)
T2(1929)
p18
p23
T1(1589)
NA
MCH
MCH
MCH
MCH
MCH
1
180
5451
7007
8404
12499
Low risk
Low risk
Low risk
Low risk
Low risk
Low risk
Median
9 (1)
6 (0)
10 (0)
7 (3)
6 (0)
6 (0)
6.5
9 (0)
6 (0)
19 (2)
10 (1)
6 (0)
6 (0)
7.5
8.5 (3)
6 (0)
7 (2)
10 (1)
6 (0)
6 (0)
6.5
6 (0)
6 (0)
6 (0)
6 (0)
6 (0)
6 (0)
6.0
6 (0)
6 (0)
21 (13)
6 (0)
6 (0)
6 (0)
6.0
10 (4)
6 (0)
7 (2)
6 (0)
Not done
6 (0)
6.0
6 (0)
6 (0)
10 (4)
6 (0)
Not done
6 (0)
6.0
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
ML
466
857
858
887
889
893
935
1025
1250
1260
1275
1356
1358
1362
1371
1376
1490
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Resistant
Median
7 (1)
90 (22)
112 (39)
15 (5)
34 (7)
129 (40)
34 (12)
36 (9)
32 (20)
6 (0)
18 (2)
7 (1)
12 (6)
19 (8)
9 (3)
64 (6)
21 (9)
21.0
16 (2)
104 (31)
70 (3)
12 (6)
64 (10)
159 (17)
46 (2)
53 (37)
47 (5)
8 (2)
46 (12)
14 (3)
–
15 (1)
10 (4)
196 (18)
23 (1)
46.0
7 (1)
95 (23)
71 (4)
8 (2)
56 (9)
73 (4)
47 (3)
36 (12)
24 (2)
–
42 (8)
6 (0)
31 (2)
16 (8)
8 (2)
245 (7)
30 (1)
31.0
6 (0)
68 (12)
51 (11)
14 (9)
31 (7)
97 (41)
49 (14)
41 (6)
45 (3)
6 (1)
37 (4)
6 (0)
43 (2)
11 (5)
15 (8)
182 (8)
31 (3)
37.0
10.5 (5)
92 (24)
124 (26)
8 (3)
35 (2)
93 (29)
50 (5)
24 (19)
38 (12)
–
33 (11)
13 (6)
16 (11)
9 (4)
8 (3)
149 (20)
27 (5)
30.0
6 (0)
65 (22)
51 (8)
11 (4)
165 (85)
77 (23)
53 (6)
43 (19)
37 (9)
6 (1)
37 (14)
17 (8)
13 (7)
15 (1)
8 (3)
108 (20)
22 (12)
37.0
8 (0)
106 (31)
81 (1)
10 (2)
–
175 (53)
75 (21)
14 (5)
47 (2)
9 (4)
32 (6)
9 (4)
14 (9)
11 (2)
17 (8)
188 (36)
25 (1)
25.0
ML
ML
ML
ML
ML
ML
ML
566
1018
1075
1359
1382
1563
1728
HIV+
HIV+
HIV+
HIV+
HIV+
HIV+
HIV+
Median
6 (0)
6 (0)
8 (3)
6 (0)
6 (0)
6 (0)
9 (0)
6.0
6 (0)
6 (0)
7 (2)
6 (0)
6 (0)
6 (0)
6 (0)
6.0
6 (0)
6 (0)
6 (0)
6 (0)
6 (0)
6 (0)
6 (0)
6.0
6 (0)
6 (0)
8 (2)
8 (2)
6 (0)
6 (0)
6 (0)
6.0
6 (0)
6 (0)
7 (1)
7 (1)
6 (0)
6 (0)
6 (0)
6.0
6 (0)
6 (0)
8 (3)
6 (0)
6 (0)
14 (7)
6 (0)
6.0
6 (0)
6 (0)
8 (3)
6 (0)
6 (0)
7 (1)
6 (0)
6.0
0.007
0.002
–
0.003
< 0.001
< 0.001
0.004
0.001
< 0.001
0.001
0.001
< 0.001
0.004
0.001
< 0.001
0.007
0.002
< 0.001
0.010
0.002
< 0.001
P value,* Low risk versus resistant
P value,* HIV+ versus resistant
P value, discriminant analysis†
For HIV-1 resistant women (resistant), low risk controls (low risk) and HIV-1-infected sex workers (HIV+), mean IL-2 secretion in pg/mL
(with SEM in parentheses) is shown for each stimulus. The median IL-2 production for each group is listed. The limit of IL-2 detection was
6 pg/mL. *Two-tailed Student’s t-test, †baseline media responses were entered into the discriminant model for each antigen and were < 0.001 in
each instance. Bold type indicates ≥ twice IL-2 production of media alone. ML, samples derived from the Pumwani Sex Worker Cohort; MCH,
samples are HIV negative controls from the mother-to-child HIV-1 transmission study; NA, HIV negative North American control.
spontaneous IL-2 responses of greater than threefold the
lower limit of detection in the assay with the media control
(Chi-squared test, P < 0.001). Interleukin-2 responses to all
stimuli were significantly greater in the HIV-1-resistant
group compared with both HIV-1-infected and low-risk
controls. However, because of the significantly higher baseline responses in the resistant group we performed a discriminant analysis of the responses to HIV-1 antigens,
controlling for the potential confounding effects of IL-2
responses to media, by including it as an additional discriminating variable. This confirmed that, after controlling for
IL-2 responses to media, resistant women have statistically
significant higher responses to the HIV-1 antigens (t-test
P < 0.01 for each peptide and sgp120). As another way of
controlling for baseline responses, we calculated stimulation
indices for responses to the HIV-1 peptides and sgp120.
Among the groups who have studied T-helper responses in
HIV-infected and exposed seronegatives,7,11,17,28,34–36 various
arbitrary stimulation index cut-off values have been selected.
Seven of the 17 (41%) resistant women tested had stimulation
indices (SI) of ≥ 2.0 against the HIV-1 antigens. Four of those
seven responded to two or more HIV-1 antigens. One of the
six low-risk negative controls and one of the seven HIV-1positive subjects had a single HIV-1 antigen that had an SI of
≥ 2.0. When an SI of ≥ 2.5 was used to define responders,
there were 0/6 low risk, 0/7 HIV-1 positive and 4/17 resistant
women who responded, two of whom did so to two or more
antigens. Three of the 17 resistant women had SI of ≥ 3.0
with one responding to two or more antigens. To better illustrate the trends in responses to the various HIV-1 antigens, a
590
KR Fowke et al.
Figure 1 Interleukin-2 production by PBMC from six low-risk
women, 17 HIV-1-resistant prostitutes and seven HIV-1 infected
prostitutes after stimulation with
envelope peptides and soluble
glycoprotein 120 (sgp120). The
IL-2-values represent the raw data
and are not corrected for by subtracting the unstimulated background. Dark lines indicate the
medians.
summary of the IL-2 experiments is presented in Fig. 1. The
IL-2 production (pg/mL) in response to stimulation by the
HIV-1 peptides and soluble gp120 for the resistant women is
significantly greater than either the low-risk (t-test, P < 0.01)
or HIV-1-infected individuals (t-test, P < 0.002). Interleukin6 production at baseline and in response to HIV-1 peptides
and PHA was not different between the three study groups
(data not shown).
Cytotoxic T cells to HIV-1 antigens
Twenty-two HIV-1-resistant prostitutes, eight low-risk
Kenyan women and four laboratory workers were tested,
some on multiple occasions, for HIV-1-specific CTL. As
shown in Fig. 2a, 15 of 22 resistant women showed levels of
env-specific lysis that were equal to or greater than 10%
above that seen in targets infected with vaccinia alone. Of
nine women tested with the vaccinia-gag construct, three
were positive (Fig. 2b). Two of seven women tested had
≥ 10% specific lysis of the vaccinia-pol target (Fig. 2b). All
22 women were tested for env responses and of the nine
women who had sufficient numbers of effector cells to test
for gag and/or pol responses, four tested positive to more
than one HIV-1 antigen, with subject 1376 being positive to
all three targets. Overall, 15 of 22 resistant prostitutes showed
significant levels of CTL to one or more of the vaccinia/
HIV-1 vectors. The HIV-1 specific cytotoxic activity was not
detected in seven of the resistant prostitutes. None of the 12
HIV-1 seronegative low-risk test subjects showed significant
specific lysis of the vaccinia/HIV-1 constructs, which is
significantly fewer responders than the resistant women (0/12
versus 15/22; Chi-squared test, P < 0.001). The depletion of
CD8+ lymphocytes from the effector PBMC by immunomagnetic beads, performed on two subjects, reduced the high
levels of cytotoxic activity achieved with the vaccinia/env
construct to that of the vaccinia construct alone (Fig. 2c). A
comparison of various attributes of women with and without
HIV-1-specific CTL responses showed no differences
between the two groups. Values for CTL+ (n = 15) and CTL–
subjects (n = 7) and P values, respectively, for various parameters were as follows: age, 37.7 years, 36.1 years, 0.6;
CD4/mm3, 970, 1088, 0.6; CD8/mm3 732, 863, 0.4; CD4/CD8
ratio 1.4, 1.3, 0.3; clients/day 3.9, 5.0, 0.3; condom use 85.5%,
79.1%, 0.6; duration of commercial sex work (years) 10.7,
10.2, 0.9.
Comparison of tested versus non-tested resistant women
The 24 resistant women who participated in this immunological study were unselected and did not differ significantly
(by Student’s t-test) from the 56 untested resistant women in
terms of age, proportion of visits with gonorrhea, episodes of
genital ulcers, number of sex partners per day and duration of
prostitution prior to cohort entry (Table 2). Studied women
had a longer duration of follow up and, because condom use
had increased over the period of observation, reported a
slightly greater average condom use (over the period of
observation). The women tested were also similar to those not
tested for HLA class I alleles, which are associated with an
increased or decreased risk of HIV-1 seroconversion in
epidemiological studies.37 Although only 24 women were
available at the time of the present study, the other 56 women
have subsequently been seen in the clinic and remain HIV
uninfected despite continued high-risk activity.
Association between cytotoxic T cells and T-helper
responses
Cytotoxic T lymphocyte assays and T helper cell assays were
both performed on 15 resistant women. Eleven of these
women had significant CTL to one or more HIV-1 antigens.
Student’s t-test confirmed an association between the presence of CTL and IL-2 secretion to the env peptides T1-1589
(P = 0.035), T1-2007 (P = 0.051), T2 (P = 0.024), p18
(P = 0.049), p23 (P = 0.001) and sgp120 (P = 0.014).
HIV cellular immune responses in HIV-1 resistance
Figure 2 (a) Cytotoxic T-cell
responses among 22 resistant
prostitutes to autologous B lymphoblastoid targets infected with
vaccinia/env constructs. The data
are plotted as percentage specific
lysis, which is defined as the lysis
of targets infected with the control
vaccinia subtracted from the lysis
of vaccinia/env-infected targets.
Three effector to target (E:T)
ratios were used (12.5:1, 25:1 and
50:1), although for clarity only
50:1 is shown, except for patient
1490 in which the 25:1 dilution is
shown. (b) Cytotoxic T cell
responses to autologous B lymphoblastoid targets infected with
vaccinia/gag and vaccinia/pol
constructs. The data are plotted as
percentage specific lysis. Three
E:T ratios were used: 12.5:1 (h),
25:1 ( ) and 50:1 (j). (c) Cytotoxic T cell responses of two resistant women to autologous B
lymphoblastoid targets infected
with vaccinia and vaccinia/env
constructs following CD8+ depletion of effector cells. The percentage lysis is plotted. Three E:T
ratios were used: 12.5:1 (h), 25:1
( ) and 50:1 (j). VAC, targets
infected with vaccinia; VAC ENV,
targets infected with vaccinia/env;
–CD8, CD8-depleted PBMC used
as effectors.
591
592
Table 2
KR Fowke et al.
Comparison of the HIV-resistant women who participated in this immunological study and those resistant women who did not
Category of comparison
Age (years)
Condom use
Duration of follow up (years)
Proportion of visits with gonorrhea
Episodes of genital ulcers
Sex partners per day
Duration of prostitution prior to entry (years)
Not tested ± SD (n = 56)
Tested ± SD (n = 24)
P value (2 tailed)
31.90 ± 6.00
2.7 ± 0.9
4.50 ± 1.80
0.35 ± 0.28
4.10 ± 7.50
3.60 ± 2.20
4.90 ± 4.40
31.10 ± 5.30
3.20 ± 0.50
6.00 ± 2.00
0.24 ± 0.13
6.20 ± 5.80
3.40 ± 2.10
5.40 ± 6.60
0.580
0.013
0.001
0.070
0.225
0.700
0.690
Discussion
Over the past several years, evidence for heterogeneity in
susceptibility to HIV-1 infection has accumulated. Some of
the strongest epidemiological evidence for resistance to
HIV-1 infection comes from observational studies of women
in the Pumwani Sex Worker Cohort in Nairobi.22 These
women have intense exposure to HIV-1 through their occupation and, although condom use is frequent (> 80% of
sexual encounters), their risk of acquiring HIV-1 infection is
enormous. Despite this intense exposure of up to 500 unprotected sexual exposures to HIV-1-infected clients, a small
number (13% of initially HIV-1 seronegative women) remain
HIV-1 uninfected for prolonged periods (up to 13 years). The
risk of HIV-1 infection has been shown to be heterogeneous
and to decline with time. Women who remain HIV-1 uninfected after 3 years are at a substantially reduced risk of
subsequent HIV-1 infection. In previous studies, we have
shown that this plateau in HIV incidence is not a chance
phenomenon and is not related to safer sexual behaviour,
altered susceptibility to factors increasing the risk of HIV-1
infection or seronegative HIV-1 infection. Having excluded
these other possibilities, we have concluded that, on an
epidemiological basis, these women are resistant to HIV-1.22
The present study shows that a high proportion of resistant
women from the Pumwani Sex Worker Cohort have HIV-1specific cellular immune responses.
Several mechanisms of resistance to HIV-1 infection are
beginning to be understood. Polymorphisms in chemokine
receptors,38,39 which serve as HIV-1 coreceptors,40,41 mediate
cellular resistance to infection. As an example, cellular resistance to macrophage-tropic HIV-1 isolates is associated with
a polymorphism in the CCR5 receptor (∆32CCR5), which
results in the absence of its expression.38 The heterozygous
condition is found at a frequency of approximately 11% in
the Caucasian population, but not at all among 261 native
Africans (many of whom were Kenyan) studied.42,43 Several
soluble factors that inhibit HIV-1 replication could potentially be involved in resistance. It has been suggested that
increased production of β-chemokines, which compete with
HIV-1 for their cognate receptors,44,45 could mediate resistance. We have investigated a number of these potential
mechanisms in the Nairobi sex workers. We have determined
that altered in vitro cellular susceptibility to T cell- and
macrophage-tropic HIV-1 infection, CCR5 and CCR3 polymorphisms, altered expression of CCR5 and CXCR4 proteins
and overproduction of β-chemokines do not explain the resistance phenomenon.46 Soluble factors produced by CD8+
T cells that inhibit HIV-1 replication but are distinct from
chemokines47 are also found in individuals who are exposed
to HIV-1 but escape infection. We are currently investigating
the role of these CD8+ soluble factors in the Nairobi cohort.
Cellular immune responses (CTL48,49 and T helper cell50,51)
have been implicated in the control of viral infections. It is
important to define immune mechanisms that may mediate
resistance to HIV-1 infection and provide a basis for the
design of effective vaccines for HIV-1 containment. The
importance of HIV-1-specific T-helper responses in controlling the HIV-1 viraemia has been re-emphasized by Rosenberg et al.17 They have observed that, among long-term
non-progressors and patients on protease inhibitor therapy,
HIV-1-specific T-helper responses correlate with low viral
titres. In the present study, resistant women exhibited significantly greater production of IL-2 in response to HIV-1 antigens than either low-risk seronegative controls or infected sex
workers, suggesting that they have T helper cell memory for
HIV-1 antigens (Table 1; P < 0.01, Student’s t-test). An
important observation is that the resistant women had statistically higher IL-2 baseline responses (P < 0.01, Student’s
t-test) than the low-risk controls (Table 1). The reason for this
is not known. Because experiments in the three study groups
were performed simultaneously and under identical conditions, it seems unlikely that this is an experimental artefact.
Furthermore, because the spontaneous IL-6 secretion was not
increased in resistant women, this appears to be a phenomenon that is specific to IL-2. One possibility is that these high
IL-2 responses are essential to the resistant state and are
involved in protection against HIV-1 infection. Alternatively,
these responses may result from a highly activated immune
state associated with protection against HIV infection.
Further studies of cellular activation markers in conjunction
with cytokine production in this group of women are planned
and would help to clarify these possibilities.
Despite the higher spontaneous IL-2 levels, superimposed
HIV-specific responses were also detected among the resistant women but not in controls. We used two methods of
analysis to control for the higher IL-2 baselines. First, IL-2
responses were compared by discriminant analysis in the
three groups, controlling for baseline IL-2 responses. For
each test peptide, IL-2 responses were significantly greater in
resistant women than controls (Table 1, P < 0.001, discriminant analysis). We also compared all test peptide IL-2
responses relative to the unstimulated control, that is, the
stimulation index (SI), although this is a less than ideal
statistical technique. In previous studies of T-helper responses
among HIV-1-infected or exposed seronegatives, an arbitrary
HIV cellular immune responses in HIV-1 resistance
range of stimulation index cut-off values have been
used.7,11,17,28,34–36,52 Kelker et al. have compared proliferative
responses in HIV-infected individuals and their exposed
seronegative partners (who had been exposed a median of
600 times) using SI of 2.0, 2.5 and 3.0 and performed comparisons of multiple antigen responses using the 2.0 cut-off.36
Using an SI cut-off of 2.0,7,28,35,36 we have shown that 1/6
(17%) low risk seronegatives, 1/7 (14%) HIV positives and
7/17 (41%) resistant women responded to the HIV antigens.
Four of those seven responded to two or more antigens. Using
higher SI cut-offs of 2.5 and 3.0 eliminates the positive
responses among the low-risk seronegatives and HIV positives and reduces the number of responders among the resistant women to 4/17 (24%) and 3/17 (18%), respectively. The
absence of HIV-1-specific T-helper responses in the HIV-1
infected group is not surprising, considering their advanced
immune dysfunction as indicated by their significantly lower
CD4 cell levels. Using an SI of ≥ 2.0, Kelker et al. have
found that, among the exposed seronegatives, 60% responded
to one or more antigens and 43% responded to two or more
antigens.36 It is worth noting that other studies have used
fresh cells, while we used cryopreserved PBMC. Because it
is known that freezing affects the ability of APC to efficiently
present antigens, it is highly probable that the use of cryopreserved PBMC contributed to our more modest responses.
In addition, our responses may have been lower because other
studies have used whole-protein antigens,17 which possess
multiple epitopes, whereas we used primarily single-epitope
HIV peptides. It is also likely that the MHC class II allelic
frequencies of our study population are significantly different from previously reported groups17,28,36 and therefore could
represent differences in peptide presentation rather than
strength of responses. Therefore, it is not unexpected that the
level of immune response is lower than that observed by
others. Significantly, the responses detected in the present
study were to clade B peptides, while the population has been
primarily exposed to clades A, C and D viruses.25,26 This
suggests that either these epitopes are conserved or that there
is significant cross-reactivity of these epitopes across clades.
This is supported by the recent observation of cross-clade
CTL among HIV-1-infected individuals53 and by other studies
in this cohort,54 in which resistant women have been shown
to have stronger CTL responses to A and D clade epitopes
than to B clade epitopes.
The specificity of the T-helper responses is suggested by
the observation that among the seven resistant women who
had SI ≥ 2.0 to the HIV-1 antigens, the pattern of HIV-1 antigens that they responded to was very different for each individual. This suggests that the responses are not due to a
non-specific increase in IL-2 production to every peptide, but
rather that each subject specifically responds to a different set
of antigens (Table 1). The present study is the first to demonstrate T-helper responses among seronegative highly exposed
sex workers who are epidemiologically resistant to HIV-1
infection.
Several groups have reported HIV-1-specific CTL in individuals who are exposed but not infected with HIV-1. MHC
class I-restricted CTL to HIV-1 have been reported in the
uninfected children of HIV-1-infected mothers,8,9 uninfected
regular heterosexual partners of HIV-1-infected individuals,12
health-care workers with occupational exposure to HIV-111
593
and HIV-1-uninfected prostitutes in the Gambia.13 The
women who constituted our study population have had high
HIV exposure, with up to 500 unprotected sexual exposures
to HIV-1-infected men over the period of up to 13 years.22
Mathematical modelling has shown that statistically these
women should be infected if all women were equally susceptible.22 Overall, we show that CTL to HIV-1 targets are
detectable in a high proportion (68%) of resistant women
tested. We found CTL activity mainly against env constructs,
but also some against gag and pol. The CD8+ cell depletion
experiments (Fig. 2c) suggest that, for those two individuals
tested, the cytotoxic activity observed in these CTL assays
was mediated by CD8+ T cells.
The present study is among the few11,52 that have reported
HIV-1-specific T-helper and CTL responses among highly
exposed seronegative individuals. In the present study, among
those individuals to whom both assays were performed, CTL
activity was significantly correlated (by Student’s t-test) with
all of the HIV-1 peptides used in the T-helper assays. It is possible that the IL-2 provided through the significantly higher
spontaneous and HIV-1-specific helper responses could be
important in the development and maintenance of HIV-1specific CTL. This suggests that it may be important for
future HIV-1 vaccines to incorporate both T-helper and CTL
epitopes into vaccines and to monitor those responses.55
Resistant women who do not have detectable HIV-1-specific
cellular responses may have CTL and T helper cells that
recognize HIV-1 antigens not tested in these studies or
may be protected by mechanisms other than systemic HIV-1
cellular immune responses, such as mucosal immune
responses.56–58
In conclusion, we have conducted a number of studies to
attempt to determine potential mechanisms responsible for
HIV-1 resistance observed among some of the women of the
Nairobi prostitute cohort.46 Many of the HIV-1-resistant
women tested exhibit evidence of HIV-1-specific T helper
and CTL responses. These findings support the concept that
cellular immune responses are important and may contribute
to providing protection against HIV-1 infection and should be
a central consideration when developing HIV-1 vaccine
strategies.
Acknowledgements
This work was supported by grants from the Medical
Research Council of Canada (Group grant-GR13301) and the
Rockefeller Foundation. KR Fowke was the recipient of a
fellowship from the National Health Research Development
Program (Canada). R Kaul is the recipient of a fellowship
from the Medical Research Council (MRC) of Canada
and the MRC/Glaxo-Wellcome/Canadian Infectious Disease
Association. FA Plummer is the recipient of a Senior Scientist award from the MRC of Canada. JN Simonsen was the
recipient of a scholarship from the Manitoba Health Research
Council and was an American Foundation for AIDS Research
Scholar.
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