538
Human Immunodeficiency Virus (HIV)–Specific Cytotoxic T Lymphocyte
Activity in HIV-Exposed Seronegative Persons
Nicole F. Bernard, Christina M. Yannakis,
Jimmy S. Lee,1 and Christos M. Tsoukas
Immunodeficiency Treatment Center, Montreal General Hospital,
Montreal, Canada
Repeated exposure to human immunodeficiency virus (HIV) does not always result in seroconversion. Understanding the conditions that permit or protect against progressive infection
with HIV is important for vaccine development. Nineteen subjects at risk for HIV infection
were CCR-5 genotyped and screened for virus-specific memory cytotoxic T lymphocytes
(CTL). None had the D32CCR-5/D32CCR-5 genotype associated with HIV resistance. HIVspecific CTL were detected in 7 (41.1%) of 17 exposed uninfected subjects versus 0 of 14
seronegative subjects with no HIV risk factors (P 5 .006 , x2 test). Recognition of virus by
CTL in exposed uninfected subjects was major histocompatibility complex class I–restricted
and multispecific, and specificity could change with time. Activity could persist up to 34
months after the last virus exposure. The presence of HIV-specific CTL in a greater proportion
of seronegative HIV-exposed versus unexposed subjects supports the notion that in some
cases, virus exposure induces HIV immunity without seroconversion or disease progression.
As observed for other human pathogens, individuals differ
in their susceptibility to and outcome after infection with the
human immunodeficiency virus (HIV). Some persons appear
to be resistant to this virus. Elucidation of the mechanisms
underlying their resistance has implications for the development
of effective prophylactic vaccines for HIV.
Resistance to HIV infection in exposed uninfected persons
(EUs) has been observed in sex partners of HIV-infected persons [1–8], infants born to HIV-infected mothers [9–12], health
care workers occupationally exposed to HIV-contaminated
body fluids [13, 14], and commercial sex workers [15, 16]. Resistance may result from either inherited or acquired factors.
Homozygosity for a 32-bp deletion mutation at the CCR-5
locus (D32CCR-5) is a genetically acquired HIV resistance factor [17–21]. The wild type allele at this locus encodes a functional coreceptor for entry of macrophage-tropic HIV into CD4
Received 5 May 1998; revised 12 October 1998.
Presented in part: Fifth Conference on Retroviruses and Opportunistic
Infections, Chicago, 1–5 February 1998 (abstract 92); Canadian Society for
Immunology meeting, Ste. Adele, Canada, 13–16 March 1998; Canadian
Association for AIDS Research meeting, Quebec, 30 April–3 May 1998.
Informed consent was obtained from all subjects who participated in this
study, and this research conformed with all ethical guidelines of the Montreal
General Hospital Research Ethics Committee.
Financial support: National Health Research Development Program of
Canada (6605-4162-AIDS). N.F.B. was a Senior Research Scholar of the
Fonds de Recherche en Santé du Québec (FRSQ).
1
Present affiliation: University of British Columbia Medical School, Vancouver, Canada.
Reprints or correspondence: Dr. Nicole F. Bernard, Immunodeficiency
Treatment Center, McGill University Hospital Center, Montreal General
Hospital, Room A5 140, Montreal, Quebec H3G 1A4, Canada (mctl@
musica.mcgill.ca).
The Journal of Infectious Diseases 1999; 179:538–47
q 1999 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/99/7903-0002$02.00
cells [22–27]. The D32CCR-5 protein is not expressed at the
cell surface [17, 18]. People homozygous for the deleted allele
are substantially, although not absolutely, resistant to HIV
infection in vivo [17–19, 28, 29]. About 1% of Caucasians are
homozygous for D32CCR-5 [17–19, 21]. Studies in highly exposed seronegative Caucasian cohorts suggest that the
D32CCR-5/D32CCR-5 genotype accounts for some but not all
instances of HIV resistance despite high levels of exposure [5,
18, 19].
Acquired immunity may be another mechanism that plays a
role in resistance to HIV infection. T helper type 1 (Th1) responses to HIV, such as the secretion of interleukin-2 (IL-2)
after stimulation with HIV peptides, have been observed more
frequently in EUs than in those at low risk for HIV exposure
[1, 3, 5, 10, 13, 14]. HIV-specific IgA antibody in the mucosa
has also been observed in a larger proportion of EUs than in
those at low risk for infection with HIV [5]. HIV-specific cytotoxic T lymphocytes (CTL) [4, 9, 11, 12, 14, 15] and suppression
of HIV replication by CD8 T cells [30, 31] are other manifestations of immunity to HIV reported in some EUs.
We report here on a cohort of 19 EUs, 14 of whom have
been in stable relationships with HIV-infected sex partners.
Methods
Patient Population
Nineteen HIV EUs were studied. Characteristics of the EU study
population, including the length of the relationship of each EU
with the seropositive partner and the last date of exposure, when
applicable, are shown in table 1. Documentation of HIV seronegativity was done by ELISA and confirmed by Western blotting.
For EUs for whom a last date of exposure to HIV was known,
serologic HIV testing was done at least within 1 month of the last
exposure date as well as ∼3 months after the first negative serologic
JID 1999;179 (March)
Immunity to HIV in Seronegative Persons
Table 1. Characteristics of HIV-exposed uninfected study subjects.
Patient
no.
Sex/age
(years)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
F/39
F/32
M/32
F/46
F/33
M/40
M/39
F/51
F/40
F/25
M/36
F/35
M/62
M/53
M/33
M/35
M/31
F/34
M/28
NOTE.
Risk
group
HIVinfected
partner
known
Duration
of exposure
(years)
Date of last
exposure
(month/last
2 digits of year)
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Sexual
Needlestick
Needlestick
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
6
1
3
18
2.2
3
1
1.9
15
0.7
2
16
1.5
16
NA
NA
NA
4 exposures
1 exposure
7/89
4/96
2/95
Ongoing
2/97
7/95
8/96
10/97
Ongoing
9/97
1/98
2/98
Ongoing
11/97
9/95
NA
NA
4/92, 9/94, 11/94, 9/95
11/96
539
CCR-5 Genotyping
CCR-5 genotyping was done by a modified version of the method
described by Samson et al. [17]. Briefly, 50-CCTGGCTGTCGTCCATGCTG-30 and 50-CTGATCTAGAGCCATGTGCACAACTCT-30 forward and reverse oligonucleotide primers, respectively,
were used to amplify a region of genomic DNA that spanned the
32-bp deletion differentiating the D32CCR-5 allele from its wild
type counterpart at the CCR-5 locus. The PCR reaction was carried
out in 30 mL of 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 0.75 mM
MgCl2, 0.2 mM each dATP, dCTP, dTTP, and dGTP, 0.01% gelatin,
5% dimethyl sulfoxide, 200 ng of DNA, 60 ng of each oligonucleotide primer, and 1.5 U of Taq DNA polymerase. The PCR
conditions were 937C for 2 min 30 s, followed by 30 cycles of 937C
for 1 min, 607C for 1 min, and 727C for 1 min and a 6-min extension
at 727C. The resulting product was digested with 10 U of EcoRI
at 377C for 60 min. Digested amplification products were loaded
onto a 2% agarose gel and electrophoresed for 1 h at 90 V. Amplified
DNA bands were visualized by ethidium bromide staining and sized
by comparison with a 1-kb ladder standard.
NA, not available.
Assay for Cytotoxic T Lymphocyte Activity
HIV test. For EUs with no known last date of exposure (either
because exposure is ongoing or because they are gay men not involved in stable relationships with partners of known HIV status),
HIV testing was done at least within 1 month of the time at which
samples for CTL analysis were obtained as well as ∼3 months after
this first negative serologic HIV test. All serologic tests done on
EUs were negative. After the last CTL analysis reported here, cell
samples were obtained from EUs to screen for HIV-1 DNA proviral
sequences by a qualitative polymerase chain reaction (PCR) assay
(Amplicor, HIV-1 PCR-EIA; Roche Diagnostics, Mississauga,
Canada). In no case was it possible to amplify HIV proviral sequences from the peripheral blood mononuclear cells (PBMC) of
any of the EUs in this cohort. Fourteen seronegative persons with
no risk factors for HIV infection were also studied as controls.
Control subjects were genotyped for CCR-5 and screened for HIVspecific CTL activity. Eleven of these controls were screened for
HIV-specific CTL activity on three occasions, 1 was tested on two
occasions, and 2 were screened on one occasion.
DNA Preparation
Genomic DNA was prepared from fresh whole blood, fresh or
cryopreserved PBMC, or Epstein-Barr virus (EBV)–transformed B
cell lines. Fresh or thawed frozen samples were pelleted by centrifugation at 400 g for 10 min. Cell pellets were osmotically lysed
by sucrose–Triton X-100 buffer (0.32 M sucrose, 10 mM Tris, pH
7.5, 5 mM MgCl2, 1% Triton X-100). Nuclei were pelleted by centrifugation and then lysed by incubation at 567C for 60 min in a
buffer containing 50 mM KCl, 10 mM Tris, pH 8.3, 2.5 mM MgCl2,
0.5% Tween 20, 0.1 mg/mL gelatin, and 200 mg/mL proteinase K.
After this incubation, proteinase K was inactivated at 957C for 10
min. Genomic DNA samples were centrifuged at 13,000 g for 1
min to clear supernatant of debris and were stored frozen at 2207C.
Preparation of effectors.
Effector cells were HIV antigen–
driven cell lines generated from freshly isolated or frozen PBMC
by stimulation with fixed autologous PBMC expressing HIV gene
products. PBMC were obtained by ficoll-hypaque density centrifugation of heparinized blood. Stimulators were prepared by infecting PBMC with vaccinia virus vectors encoding HIV Env, Gag,
reverse transcriptase (RT), and Nef at an MOI of 5:1 vaccinia virus
to PBMC. After a 16-h incubation at 377C in 5% CO2, stimulators
were fixed for 45 s with 0.1 mL of 0.05% glutaraldehyde (Sigma,
St. Louis). Fixation was stopped by the addition of 0.1 mL of 2
M L-lysine (Sigma). Stimulators infected separately with the four
vaccinia virus constructs were washed three times and pooled at a
ratio of 1:1:1:1. Uninfected PBMC and autologous stimulators
were then mixed at a ratio of between 5:1 and 10:1 and cultured
in RPMI 1640 (Gibco BRL, Burlington, Canada), supplemented
with 15% fetal calf serum (Gibco BRL), 2 mM L-glutamine (ICN
Biomedicals, Costa Mesa, CA), 50 IU/mL penicillin (ICN), 50 mg/
mL streptomycin (ICN), 50 mM 2-mercaptoethanol (Sigma), and
200 U/mL IL-2 (Chiron, Emeryville, CA) (R-15-200). Twice a week,
cell cultures were adjusted to 5 3 10 5 cells/mL in fresh R-15-200.
Cell lines were tested for CTL activity between days 14 and 21 of
culture.
Vaccinia virus–HIV constructs. Five vaccinia virus–HIV constructs were used to infect autologous EBV-B cell lines for use as
targets; four of these were also used to infect autologous PBMC
for use as stimulators. vMN462 expressing the full-length gp160
Env of HIV-1MN, vDK1 expressing Gag of the HXB2 subclone of
HIV-1, vCF21 expressing all but the last 22 residues of RT of the
HXB2 subclone of HIV-1, and vSC8 expressing the LacZ gene of
Escherichia coli (a non-HIV gene product used as a negative specificity control) were obtained from the AIDS Research and Reference Reagent Program (Division of AIDS, National Institute of
Allergy and Infectious Diseases, NIH, Bethesda, MD). vP1218 expressing Nef of HIV-1MN was a gift of E. Paoletti (Virogenetics,
Troy, NY). Each of these constructs was added to PBMC (to be
540
Bernard et al.
used as stimulators) or EBV-B cells (to be used as targets) at a
vaccinia virus–to-cell ratio of 5:1 for 30 min with occasional shaking. EBV-B cells to be used as targets were then labeled with 100
mCi of [51Cr]NaCrO4 (New England Nuclear, Boston) per 5 3 10 5
EBV-B cells and incubated at 377C in 5% CO2 for 16 h. Labeled
targets were washed three times before assay.
Chromium-release assays. Cytolytic activity of antigen-driven
effectors present in bulk cultures established from PBMC was measured in a 4-h 51Cr-release assay. Targets were autologous EBV-B
cells or EBV-B cell lines unmatched or matched with effectors for
single HLA class I alleles. EBV-B cells were generated for each
subject by use of B95-8 marmoset line supernatant [32]. Assays
were done in triplicate in round-bottomed 96-well microtiter plates
at effector to target (E:T) ratios of 10:1, 5:1, and 2.5:1 or as indicated for individual experiments. Chromium release was measured in a gamma counter (5500B; Beckman Instruments, Fullerton, CA). The percentage of specific lysis was determined as
follows: 100 3 (experimental release 2 spontaneous release)/(maximal release 2 spontaneous release). For each target, spontaneous
release was determined from wells containing medium only, and
maximal release was calculated from wells containing 1% Triton
X-100. Spontaneous release ranged from 15% to 30% of maximal
release. CTL activity was defined as present if the percentage of
specific lysis of test wells was 110% above background lysis (cytotoxicity directed at vSC8, a non–HIV antigen–expressing target)
at at least two E:T ratios or in more than one experiment. Vaccinia
virus–specific cytolysis also present in bulk cultures was reduced
by adding to replicate cultures, for each E:T, a 30-fold excess of
unlabeled vSC8-infected targets. All of the results reported for CTL
activity of antigen-stimulated bulk cultures are those corresponding
to wells seeded with cold targets to inhibit recognition of non-HIV
specificities.
HLA Typing
PBMC were typed for major histocompatibility complex (MHC)
class I antigens by standard complement-mediated cytotoxicity assay with 72-well plates purchased from the Canadian Red Cross
Society (Ottawa).
Statistical Analysis
The x2 test was used for comparison of sample proportions.
P ! .05 was considered significant.
Results
CCR-5 genotyping. The expected size of EcoRI-digested
fragments amplified from the genomic DNA of persons homozygous for the CCR-5 locus wild type allele determined by
use of the primer set described in Methods was 332 and 403
bp. Fragment sizes expected from heterozygotes were 332, 371,
and 403 bp. Fragment sizes expected from homozygotes for
the deletion mutant were 332 and 371 bp. Of the 19 HIV EUs
genotyped for CCR-5, 15 (79%) were homozygous wild type
and 4 (21%) were heterozygous at this locus. None were
D32CCR-5 homozygous (table 2). No differences in allele or
JID 1999;179 (March)
Table 2. CCR-5/D32CCR-5 heterozygotes in exposed uninfected and
HIV-infected subjects.
Subpopulation
All
Couples
Couples (Caucasian only)
Exposed
uninfected persons
Infected persons
P
4/19 (21)
3/14 (21.4)
3/13 (23)
10/52 (19)
2/13 (15.3)
2/12 (16.7)
.9
.7
.7
a
NOTE. Data are no. of CCR-5/D32CCR-5 heterozygotes/total no. of subjects
in group (%).
a
Between-group comparison of CCR-5 allele frequency by x2 test.
genotype frequencies were detected between these 19 EUs and
52 HIV-infected patients (table 2) or between these 19 EUs and
33 seronegative persons with no risk factors for HIV infection
(not shown) by the x2 test. Furthermore, no differences in the
proportion of CCR-5 heterozygotes were detected between the
14 EUs who were in HIV infection status–discordant couples
and their HIV-seropositive partners, whether all subjects or only
Caucasians were included in analyses (table 2).
HIV-specific memory CTL activity. To detect potentially
low-frequency CTL clones specific for HIV in EUs, an in vitro
stimulation step with autologous cells expressing HIV-1 antigens was used to amplify HIV-specific memory CTL. Bulk cultures prepared from HIV-infected persons by this method display HIV-specific CTL responses (figure 1A, 1B), whereas those
prepared from HIV-seronegative subjects do not (figure 1C,
1D). HIV-specific CTL activity was measured in bulk cultures
established from the PBMC of 17 EUs and 14 uninfected controls at low risk for HIV infection. Figures 1E and 1F show
examples of HIV-specific CTL activity detected in 2 EUs. Table
3 summarizes the specificity of HIV-specific CTL derived from
the 17 EUs and 14 low-risk seronegative controls screened by
this method. Seven (41.1%) of 17 EUs, compared with none of
the low-risk seronegative controls, exhibited above-background
levels of CTL activity directed at HIV. A significantly greater
proportion of EUs versus low-risk seronegative persons had
HIV-specific memory CTL (P 5 .006 , x2 test).
Antigen-driven bulk cultures derived from 3 EUs were
screened for MHC class I antigen restriction by use of targets
matched or not for MHC alleles expressed by bulk CTL effectors (figure 2). CTL from all 3 EUs lysed MHC-matched
but not unmatched targets. Serologic MHC class I typing of
EU subject 8 (EU8) showed that this subject was HLA-A2,
B14, B62, Cw3, Cw5. HIV-specific CTL activity amplified from
the PBMC of EU8 recognized Gag presented on autologous
and HLA-A2– and HLA-B62–matched targets but not on
MHC class I–unmatched targets (figure 2).
PBMC from 3 EUs were tested several times after their last
exposure to HIV to assess persistence of HIV-specific CTL activity. EU6’s last exposure to HIV was in July 1995. In September 1995, EU6 had HIV RT– and Nef–specific CTL activity.
In January 1996, 6 months after the last exposure, it was still
possible to amplify RT- and Nef-specific CTL activity from
EU6’s PBMC. In addition, reactivity to HIV Gag was also
detected at this time (figure 3A). EU18 is a health care worker
JID 1999;179 (March)
Immunity to HIV in Seronegative Persons
541
Figure 1. HIV-specific cytotoxic T lymphocyte activity in 2 HIV-seropositive subjects (A, B), 2 seronegative subjects at low risk for HIV
infection (C, D), and 2 HIV-exposed seronegative subjects (E, F). * Positive (% specific lysis 110% above lysis of control targets expressing LacZ
[non-HIV gene product]). RT, reverse transcriptase. SDs were !10%.
who was exposed four times to HIV-infected body fluids by
needlestick injuries. The last exposure occurred in September
1995. This subject was tested three times: in September 1995,
18 months later in March 1997, and 34 months later in July
1998. In September 1995, EU18 had HIV-specific CTL to HIV
Table 3. HIV specificity of antigen-driven cytotoxic T lymphocyte
lines prepared from HIV-exposed uninfected (EU) subjects and lowrisk controls.
Subject ID
HIV specificity
EU1, EU2, EU3, EU4, EU5, EU10,
EU11, EU12, EU14, EU19
EU7, EU16
EU6
EU8
EU9
EU13
EU15
EU17
EU18
Low-risk-controls 1–14
NOTE.
RT, reverse transcriptase.
None
Not done
Gag, RT, Nef
Gag
Gag
Env
Env, Nef
Env, Gag, RT, Nef
Gag, RT, Nef
None
Env, Gag, RT, and Nef. Eighteen months later, this subject had
above-background CTL activity to Gag, RT, and Nef. Thirtyfour months after the final exposure, EU18 had above-background CTL activity to Nef at an E:T ratio of 10:1 (figure 3B).
EU15 is a gay man with a history of high-risk behavior. As of
September 1995, EU15 was involved in a stable relationship
with a seronegative partner and was not exposed to HIV during
the observation period. In September 1995, EU15 had HIVspecific CTL to HIV Nef. This HIV-specific reactivity broadened by November 1995 to recognize HIV Env and Nef. When
the subject was retested in December 1995, the same HIV gene
products were recognized (figure 3C).
Discussion
This report shows that a significant proportion of HIV-exposed seronegative subjects have HIV-specific precursor CTL
that can be expanded in vitro under conditions of antigenspecific stimulation. This activity can persist after the last HIV
542
Bernard et al.
JID 1999;179 (March)
Figure 1. (Continued).
exposure for up to 34 months. During this time, the specificity
of the response can change. Patterns of reactivity varied from
person to person, with recognition of up to four HIV gene
products by bulk cultures derived from EUs.
Despite exposure to HIV, in no case can persistent HIV seronegativity in the 19 subjects described herein be explained by
homozygosity for D32CCR-5, a known genetic resistance factor
[17–21]. The product of this mutant allele is a nonfunctional
form of the coreceptor for HIV entry into CD4 cells of the
monocyte/macrophage lineage [17, 18, 28]. Macrophage-tropic
viruses are the principal form of virus involved in person-toperson transmission [33]. The D32CCR-5 allele is found in
∼10% of Caucasians [17–19, 21]. People expressing the homozygous phenotype are substantially, although not absolutely,
resistant to HIV [17–19, 28, 29], and CD4 cells from these
persons are resistant to infection with macrophage-tropic HIV
[2, 18, 28, 34]. As many highly exposed seronegative persons,
including members of the cohort described here, are not
D32CCR-5 homozygotes, other factors likely play a role in
resistance to HIV [5, 18, 19, 34].
Hoffman et al. [35] found a higher frequency of D32CCR-5
heterozygotes in seronegative partners of HIV infection
status–discordant couples compared with their HIV-positive
partners. This effect was seen in heterosexual but not in homosexual couples. The results imply that heterozygosity at this
locus may confer partial protection from infection through a
heterosexual route. Although the sample sizes are too small for
a definitive conclusion, comparison of the proportion of CCR5 heterozygotes in the EU cohort described here with that in
52 HIV-infected subjects or with that in their HIV-infected
partners revealed no significant proportional differences.
The presence of memory HIV-specific MHC class I–restricted
CTL in these persons implies that they were exposed to live
replication-competent HIV. Induction of CTL likely results
from infection of CD4 cells by HIV followed by one or more
cycles of replication to produce immunogenic viral epitope–
JID 1999;179 (March)
Immunity to HIV in Seronegative Persons
543
Figure 1. (Continued).
MHC antigen combinations. Defective virus particles or virus
debris would not be efficient at inducing HIV-specific CTL [15].
Therefore, maintenance of seronegativity in EUs with HIVspecific CTL implies that, although they were exposed to live
virus, they were able to control viral replication and clear virus
before establishment of a progressive infection characteristic of
those who undergo HIV seroconversion.
The interest in immune mechanisms potentially mediating
resistance arises from the implications this would have for vaccine development. There is no evidence that systemic antibodies
to HIV mediate protection. On the contrary, presence of serum
anti-HIV antibodies is diagnostic for HIV infection. Cellular
immune responses to HIV are often observed in the absence
of infection [1, 3–5, 8–15]. Although proof is lacking as to
whether HIV-specific memory CTL activity in these persons
protects them from seroconversion on subsequent exposure,
information from several sources supports this possibility.
In mice, as in humans, infectious pathogens frequently induce
either a predominant cell-mediated or humoral immune response [36, 37]. The nature of the dominant response depends
on the type of T helper cell induced. Th1 cells secrete IL-2 and
interferon-g and support the differentiation delayed-type hypersensitivity, CTL, and macrophage activation [38]. Th2 cells
produce IL-4, IL-5, and IL-10 and provide help for B cell differentiation into antibody-secreting cells [38]. There is a tendency for either cell-mediated or antibody responses to predominate in any particular immune response [38]. This
tendency is thought to result from cross-regulation through the
cytokine network, such that Th1 cells and their products inhibit
induction of Th2 cells and vice versa [39]. In several animal
and human disease models, one or the other pole of the T helper
response is associated with resistance or susceptibility to infection or disease [40–46]. The genetic background of the host and
dose and route of antigen delivery influence which type of im-
544
Bernard et al.
JID 1999;179 (March)
Figure 2. HIV-specific cytotoxic T lymphocyte (CTL) activity in exposed HIV-seronegative (EU) subject is restricted by major histocompatibility
complex class I antigens. In A, CTL activity in bulk cultures from EU8 was tested on targets matched with effectors for HLA-A2, HLA-B62, or
no HLA alleles expressing either LacZ as non-HIV background control or HIV Gag. B shows CTL activity in bulk cultures from EU13 tested
on autologous targets or major histocompatibility complex (MHC)–unmatched targets expressing either LacZ as non-HIV background control
or HIV Env. C shows CTL activity in bulk cultures from EU18 tested on autologous targets or MHC class I–unmatched targets expressing either
LacZ as non-HIV background control or HIV Nef. * Positive (% specific lysis 110% above lysis of control targets). SDs were !10%.
mune response will predominate [47]. In macaques, low-dose
immunization with a simian immunodeficiency virus vaccine
results in induction of cellular immune responses without humoral immunity. Macaques immunized this way are protected
from challenge with infectious virus [48].
HIV-specific CTL may be particularly well-suited to controlling virus spread. In vitro studies on HIV-specific CTL
clones revealed that HIV-specific CTL recognize and kill targets
infected with replication-competent virus before mature virus
can bud from the host cell and infect new targets [49]. Once
CTL clones have been induced to kill HIV-infected targets
through MHC-restricted recognition of HIV epitopes, they also
secrete factors that inhibit the replication of HIV in a non–
MHC-restricted manner [50].
Although 7 of 17 EUs had detectable HIV-specific CTL activity, 10 subjects did not. Several reasons may explain why no
HIV-specific CTL activity was detected in these persons. Several
steps must be completed successfully before HIV either establishes a progressive infection or induces a CTL response without
seroconversion in its host. HIV must pass through mucosal and
epithelial barriers, enter CD4 cells, replicate itself, and present
viral antigens to the immune system. HIV entry may be aborted
at any of these steps, such that the host, despite exposure to
HIV through high-risk behavior, never encounters the signals
that induce differentiation of virus-specific CTL. Another possibility is that HIV-specific CTL have been induced but are
directed at epitopes not present on the virus isolates used to
amplify and screen for CTL activity. Although this is a possibility, others have shown that HIV-specific CTL can tolerate
up to 3 amino acid changes within a recognized HIV epitope
[49, 50]. This permits a degree of cross-isolate and cross-clade
recognition of HIV isolates by HIV-specific CTL.
In summary, the study of HIV-exposed seronegative persons
is important because such persons hold the key to what con-
Figure 3. Persistence of HIV-specific cytotoxic T lymphocyte (CTL) activity in HIV-exposed uninfected (EU) subjects. In A, EU6 was tested
for presence of memory HIV-specific CTL activity twice. Assays were done on samples obtained 2 and 6 months after last exposure to HIV in
July 1995. In B, EU18 was tested for presence of memory HIV-specific CTL activity 3 times. Assays were done on samples obtained within 1
month, 18 months, and 34 months after last exposure to HIV in September 1995. In C, EU15 was tested for presence of memory HIV-specific
CTL activity twice. Last exposure date is unknown, but he was not exposed to HIV after September 1995. HIV-specific CTL activity was assessed
on samples obtained at 3 time points in September, November, and December 1995. Results were generated in 4-h 51Cr-release assay and are
mean of triplicate cultures at E:T ratios shown. Effectors were generated from peripheral blood mononuclear cells obtained at indicated times.
Effectors were stimulated with fixed autologous PBMC expressing HIV gene products and expanded for 14 days before assay. % specific lysis of
control targets expressing LacZ (non-HIV gene product) has been subtracted from that of each HIV gene product–expressing target for each E:
T ratio. % specific lysis 110% above background is considered positive. SDs were !10%.
546
Bernard et al.
stitutes protection from HIV infection. Identification of and
replication of acquired immunity factors that protect against
HIV infection is the principal goal for vaccine development.
Data presented here show that HIV-exposed seronegative subjects can be primed through their high-risk behavior to develop
HIV-specific CTL activity in the absence of seroconversion.
These persons seem to have encountered HIV as a limited infection and as a result have been able to clear it from the
circulation. These memory CTL responses can be multispecific,
can change over time, and can persist for up to 34 months after
the last exposure.
Acknowledgments
We thank Famane Chung for performance of many of the CTL
assays, Rick Pilon and Sharon Cassol (Ottawa General Hospital, Ottawa) for HIV quantitation, Galit Alter for CCR-5 genotyping, and
Nadine Ayoub for technical assistance. We are indebted to Jason Szabo,
Julian Falutz, Erwin Goldberg, Howard Turner, Vihn Kim Nguyen,
and Philip Joliot for referring EU patients to this study. We also thank
the members of the HIV EU cohort and their partners, whose cooperation has made this study possible.
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