1412
Cervicovaginal Secretory Antibodies to Human Immunodeficiency Virus Type 1
(HIV-1) that Block Viral Transcytosis through Tight Epithelial Barriers
in Highly Exposed HIV-1– Seronegative African Women
Laurent Bélec,1,2 Peter D. Ghys,3,4 Hakim Hocini,1
John N. Nkengasong,3 Juliette Tranchot-Diallo,1,2
Mamadou O. Diallo,3 Virginie Ettiègne-Traore,3
Chantal Maurice,3 Pierre Becquart,1 Mattieu Matta,2
Ali Si-Mohamed,2 Nicolas Chomont,1,2
Issa-Malick Coulibaly,4 Stefan Z. Wiktor,3,5
and Michel D. Kazatchkine1
1
Unité INSERM U430, Immunopathologie Humaine, Hôpital
Broussais, and Université Pierre et Marie Curie Paris VI
and 2Laboratoire de Virologie, Hôpital Européen Georges Pompidou,
Paris, France; 3Projet RETRO-CI and 4Programme National de Lutte
contre le SIDA, les Maladies Sexuellement Transmissibles et la
Tuberculose, Abidjan, Ivory Coast; 5Centers for Disease Control
and Prevention, Atlanta, Georgia
Antibodies to human immunodeficiency virus (HIV) of the IgA, IgG, and IgM isotypes and
high levels of the HIV suppressive b-chemokine RANTES (regulated on activation, normally
T cell expressed and secreted) were found in the cervicovaginal secretions (CVSs) of 7.5% of
342 multiply and repeatedly exposed African HIV-seronegative female sex workers. The antibodies are part of a local compartmentalized secretory immune response to HIV, since they
are present in vaginal fluids that are free of contaminating semen. Cervicovaginal antibodies
showed a reproducible pattern of reactivity restricted to gp160 and p24. Locally produced
anti-env antibodies exhibit reactivity toward the neutralizing ELDKWA epitope of gp41.
Study results show that antibodies purified from CVSs block the transcytosis of cell-associated
HIV through a tight epithelial monolayer in vitro. These findings suggest that genital resistance
to HIV may involve HIV-specific cervicovaginal antibody responses in a minority of highly
exposed HIV-seronegative women in association with other protecting factors, such as local
production of HIV-suppressive chemokines.
The large majority of human immunodeficiency virus (HIV)
infections worldwide occur as a consequence of unprotected
heterosexual encounters with an HIV-seropositive person. Several pathways for viral entry through the cervicovaginal mucosa
have been proposed, including direct access of HIV to submucosal target cells secondary to the rupture in genital epithelial
integrity, transmucosal transport of HIV by Langerhans cells
[1], and transmucosal passage of the virus through monostratified epithelia [2–4]. In addition to epithelial integrity, mucosal
immunity constitutes a key element of the local defense against
infection [5, 6]. Locally produced antibodies to HIV of the IgA
Received 23 February 2001; revised 5 July 2001; electronically published 13
November 2001.
Presented in part: XIIth World AIDS Conference, Geneva, 28 June to 3 July
1998 (abstract 23); 39th Interscience Conference on Antimicrobial Agents and
Chemotherapy, San Francisco, 26– 29 September 1999 (abstract 17130).
Informed consent was obtained from all subjects. The study was approved by
ethics committees of the Ministry of Health, Ivory Coast, the Institute of
Tropical Medicine, Antwerp, Belgium, and the Centers for Disease Control and
Prevention.
Financial support: Agence Nationale de Recherches sur le SIDA; European
Community Program on Science and Technology for Development; National
Fonds voor Wetenschappelijk Onderzoek.
Reprints or correspondence: Prof. Laurent Bélec, Laboratoire de Virologie,
Hôpital Européen Georges Pompidou, 75 908 Paris, Cedex 15, France (laurent
[email protected]).
The Journal of Infectious Diseases
2001;184:1412–22
q 2001 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2001/18411-0007$02.00
and IgG isotypes and HIV-specific cytotoxic CD8 T cells are
found in the cervicovaginal secretions (CVSs) and mucosa of
HIV-seropositive women [7–12]. HIV-specific cytotoxic CD8
T cells also have been characterized within the vaginal mucosa
of chronically infected female rhesus macaques inoculated with
SIVmac251 by the vaginal route [13].
A small number of persons are resistant to HIV infection,
despite repeated sexual exposures to the virus [14], and systemic
and mucosal HIV type 1 (HIV-1)–specific cytotoxic T lymphocytes have been detected in sexually exposed HIV-seronegative
persons [12, 15, 16]. HIV-specific antibodies of the IgA and
IgG isotypes have been reported in CVSs of exposed seronegative African women [17–21] and of HIV-seronegative female
partners of HIV-seropositive males in serodiscordant couples
[22– 25]. There are reports of HIV-1–specific IgA in the genital
tracts of HIV-1–resistant Kenyan sex workers [12, 26, 27] and
in persistently seronegative Thai female sex workers [28].
Here, we describe the presence and characterization of HIV antibodies of the IgA, IgG, and IgM isotypes, including secretory (s)
IgA and sIgM, in cervicovaginal fluids obtained from multiply
and repeatedly exposed African HIV-seronegative female sex
workers.
Methods
Study population and clinical sample processing. African
women of childbearing age (range, 18– 45 years) were recruited on
JID 2001;184 (1 December)
HIV-1 Antibodies in Seronegative Women
a volunteer basis in a confidential clinic for female sex workers in
Abidjan, Côte d’Ivoire, as reported elsewhere [21, 29]. Female sex
workers in Abidjan are heterosexual; oral and anal sex and injection
drug use are extremely rare in this population [30]. The D32 deletion
in the CC chemokine receptor– 5 gene was not found in a similar
population of female sex workers in Abidjan [31]. Menstruating
women and those with genital bleeding were excluded.
Freshly obtained serum and EDTA-processed plasma samples
were frozen at 280 C until use. Simultaneously, CVS samples
were collected by a standardized nontraumatic 60-s vaginal wash
with 10 mL of PBS and immediately were placed on ice. After centrifugation at 1000 g for 10 min, the cellular pellets and supernatants
were aliquoted and frozen at 280 C. The dilution of native CVS
introduced by the wash procedure was 1 : 30 [32]. Serum samples
were screened for antibodies to HIV-1 and HIV type 2, as described
elsewhere [29]. HIV-1 RNA was detected in plasma by a domestic
reverse-transcription (RT)– nested polymerase chain reaction
(PCR), with primers adapted to African clades [33].
Detection of contaminating semen and of proviral HIV-1 DNA
in CVS samples. We detected the prostatic specific antigen
(PSA) and prostatic acid phosphatase (PAP) in the supernatant of
CVS samples by use of commercial EIAs (Abbott Laboratories).
The cutoffs of positivity for PSA and PAP antigens in cervicovaginal fluid were determined as the mean plus 2 SD of the values
obtained with a 1 : 30 dilution in PBS of native CVS samples
obtained from 30 healthy childbearing-aged HIV-seronegative
white women who claimed not to have had sexual intercourse for
>5 days (control subjects). For both markers, the cutoff was
0.4 ng/mL. A nested PCR for the Y chromosome was further performed on DNA extracted from the cellular pellet of CVS, as
described elsewhere [34]. The lack of contaminating semen in
CVS samples was defined as the lack of detectable PSA, PAP,
and Y gene; contamination of CVS samples with semen was
defined by the presence of >1 of these markers. For the detection
of proviral DNA in CVS samples , multiple HIV-1 gene sequences
were amplified from DNA extracted from the cellular pellets of
CVSs by use of a seminested PCR for the pol gene and a nested
PCR for the env gp120/V3 gene, as described elsewhere [35, 36].
Antibodies to HIV in CVS samples. Antibodies reactive with
gp160, p68, and p24 were detected in cervicovaginal lavage
(100 mL) by in-house indirect ELISAs, as described elsewhere
[37]. Indirect ELISA that used horseradish peroxidase (HRPO)–
labeled heterologous antibodies to human Fca, Fcg, Fcm, F(ab0 )2,
and secretory component (SC) were used to identify the isotypes of
anti-gp160 antibodies, the F(ab0 )2 moiety, and the secretory forms
of antibodies (sIg) to gp160 in CVSs. sIgA to gp160 was detected
following depletion of CVSs in IgG and IgM; sIgM to gp160 was
detected after preabsorbing IgG and IgA in CVSs. For absorption,
vaginal samples were incubated with 25% (vol/vol) protein
G– Sepharose for 1 h at 37 C. After centrifugation, the supernatant
was used to absorb IgM by using 15% (vol/vol) of anti– human Fcm
immobilized on agarose beads or to absorb IgA by using beads
coated with anti– human Fca. The depletion of CVS in IgG, IgM,
and IgA was confirmed by ELISA. sIgA was detected in IgG- and
IgM-depleted CVS by an indirect ELISA that used recombinant (r)
gp160 as antigen and HRPO-labeled antibodies to human Fca as
revealing conjugate. sIgM was detected in IgG- and IgA-depleted
1413
CVS by a similarly designed ELISA that used HRPO-labeled antibodies to human Fcm. The cutoff of positivity was determined as
the mean plus 2 SD of reactivities obtained by testing 100 mL of
cervicovaginal lavage fluid of 30 HIV-seronegative women.
Cervicovaginal immunoglobulins were separated by molecular
mass chromatography on 3 serial 1:5 100-cm columns of Sephacryl S300 (Pharmacia), as described elsewhere [38]. The reactivity of antibodies with rgp160 was further tested in each fraction
by ELISA by using goat biotinylated antibodies to human immunoglobulin and streptavidin HRPO as conjugates. We assessed
the patterns of reactivity of cervicovaginal anti-HIV antibodies
with HIV-1 antigens by use of commercial Western blot strips
(New LAV-BLOT I; Sanofi-Diagnostics Pasteur) [8].
The epitopic specificities of IgA, IgG, and IgM antibodies to envencoded envelope glycoproteins were characterized by an indirect
ELISA by using peptides derived from the gp41 and gp120 molecules of HIV-1 LAI and SF2 strains. The ELISAs used biotinylated
antibodies to human Fca, Fcg, and Fcm. The positivity cutoff was
defined as the mean (+ 2 SD) reactivity of 100 mL of cervicovaginal
lavage fluid of 30 HIV-seronegative women.
Inhibition of transcytosis of cell-associated HIV-1 through a
tight epithelial barrier. We investigated inhibition by sIgA and
IgG of transcytosis of cell-associated HIV-1 in an in vitro system
involving a polarized monolayer of endometrial epithelial cell
line HEC1 (American Type Culture Collection), as described elsewhere [3, 4, 10]. HEC1 expresses the polymeric immunoglobulin
receptor (pIg-R) basolaterally. For purification of IgG from CVS,
cervicovaginal fluids were interacted with protein G– Sepharose
at 37 C before elution with glycine (0.1 M [pH 2.5]) and subsequent neutralization with Tris-HCl (1 M [pH 9.0]). The purity
of IgG was confirmed by the lack of detectable IgA and IgM by
sandwich ELISA. For purification of sIgA from CVSs, we used a
similar procedure with anti– SC-coated Sepharose beads with confirmation of purity by ELISA. The assay for inhibition of transcytosis has been described in detail elsewhere [3, 4, 10]. Sup T1
cells infected with HIV-1 subtype A (2 106 cells) were incubated with RPMI 1640 containing 10% fetal calf serum alone,
with whole vaginal wash fluid corresponding to 1mg of unpurified
total immunoglobulin or with 1 mg of sIgA or IgG purified from
CVS. The HIV-1 subtype A strain used is a T cell–adapted R5
strain. HIV transcytosis was monitored by measuring the concentration of p24 antigen in the basolateral chamber by using an
immunocapture ELISA (DuPont). Inhibition of transcytosis was
expressed as the percentage of p24 antigen recovered in the basolateral chamber in the presence of the immunoglobulin to be tested
by comparison with the amount of p24 antigen recovered in the
presence of irrelevant HIV-seronegative IgG.
Dimeric IgA (dIgA; 1 mg) purified from the 330– 410-kDa peak
obtained from the CVS pool chromatographed on Sephacryl s300
was internalized from the basolateral side of the HEC1 monolayer
for 25 min, a time sufficient to load the epithelial transcytotic pathway with pIgR-dIgA complexes [4]. Sup T1 cells infected with
HIV-1 subtype A (2 106 cells) then were added to the apical surface of the epithelial monolayer to trigger HIV transcytosis. After
5 min of apical contact, we replaced the remaining dIgA in the basolateral chamber with fresh medium free of antibody and allowed the
coculture to proceed for 3 h at 37 C.
1414
Bélec et al.
Cytokines. Interleukin (IL)– 1b, IL-2, IL-6, interferon (IFN)– g,
and RANTES were quantitated in the acellular fraction of CVS by
ELISA (Immunotech; R&D Systems Europe), as described elsewhere [39].
Statistical analysis. Quantitative data were compared by the
nonparametric Mann-Whitney U test for unpaired samples. Prevalences were compared by Fisher’s exact test. P , :05 was considered
to be statistically significant.
Results
Cervicovaginal HIV-specific antibodies in HIV-seronegative
women. In total, 1078 female sex workers were tested for
HIV seropositivity and were evaluated for sexually transmitted
diseases (STDs). Of these, 657 tested positive. We obtained cervicovaginal fluid and paired serum samples from 342 of the 421
HIV-seronegative women. Demographic data on the study
population have been reported elsewhere [21]. In brief, the
median age of the 342 women was 26 years (interquartile range
[IQR], 21–33 years). The median duration of sex work was 24
months (IQR, 10–48 months). Of the women, 84% reported
no consistent condom use during sexual intercourse. A high
JID 2001;184 (1 December)
prevalence of STDs was observed: Trichomonas vaginalis,
27%; Neisseria gonorrhoeae, 14%; Chlamydia trachomatis,
5%; genital ulcers, 3%; and positive serology for syphilis, 20%.
The acellular fraction of cervicovaginal fluid of the 342
women described above was assessed by ELISA for the presence
of anti-env–encoded gp160 immunoglobulin. Twenty-five
women (7.5%) tested positive (table 1). All cervicovaginal
samples were tested for the presence of contaminating semen,
to differentiate between antibodies that could have been transmitted passively by donor semen and antibodies generated by
the female genital mucosa. Cervicovaginal samples of 10 of the
25 women had no trace of contaminating semen, as shown by
the lack of detectable PSA, PAP, and Y chromosome in CVSs.
At least 1 of the semen markers was present in the CVS samples
of the 15 remaining women. Because false-positive results may
be obtained in corporal fluids when tested for the presence of
specific antibodies, we confirmed that the observed reactivities
corresponded to anti-gp160 immunoglobulins by demonstrating
that the ELISA reactivities were inhibited with excess rgp160
and with anti–human F(ab0 )2 antibody in a dose-dependent
fashion (figure 1). The anti-gp160 antibodies detected in the cervicovaginal fluids did not exhibit the polyspecific patterns of
Table 1. Characteristics of 25 human immunodeficiency virus (HIV)–seronegative female sex
workers with HIV antibodies in their cervicovaginal secretions (CVSs).
Age,
years
Duration of sex
work, months
205
429
587
693
756
849
1096
1174
1420
1614
138
177
196
376
738
867
946
1025
33
19
16
18
20
30
20
17
27
25
18
27
26
40
19
24
29
22
48
24
24
24
36
48
3
3
12
12
12
24
36
60
18
96
120
36
1116
1240
1313
1425
1602
1746
39
39
22
20
35
18
3
72
36
1
12
24
Subject
Sexually transmitted disease
None
Trichomonas vaginalis
None
Neisseria gonorrhoeae
None
T. vaginalis
T. vaginalis
None
T. vaginalis
None
N. gonorrhoeae
None
T. vaginalis, TPHA+
TPHA+
None
TPHA+
None
T. vaginalis, Chlamydia
trachomatis, Haemophilus ducreyi
T. vaginalis, TPHA+
T. vaginalis, TPHA+
None
None
None
T. vaginalis
Semen in
CVSs
HIV DNA
in CVSsb
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
2
2
2
2
2
2
2
2
2
2
2
+
+
2
2
+
2
+
Yes
Yes
Yes
Yes
Yes
Yes
2
2
2
2
+
+
NOTE. TPHA+, positive results for the Treponema pallidum hemagglutination assay (Fujirebio).
Negativity was defined as the lack of detectable prostatic acid phosphatase, prostatic specific antigen, and Y
gene in CVSs. Positivity was defined by presence of >1 of these markers in CVSs.
b
HIV-1 proviral DNA was positive (+) if at least the seminested polymerase chain reaction (PCR) for the pol
gene or the nested PCR for the env gp120/V3 gene was positive. 2, Negative.
a
JID 2001;184 (1 December)
HIV-1 Antibodies in Seronegative Women
1415
Figure 1. Inhibition of binding of anti-gp160 antibodies (IgA, IgG, and IgM) in cervicovaginal secretions (CVSs) by soluble recombinant (r)
gp160 and affinity-purified sheep antibodies to human F(ab0 )2 fragments. In all, 100 mL of vaginal lavage fluid from human immunodeficiency
virus (HIV)– seronegative subjects (nos. 376 [B], 693 [X], and 1174 [O]) and from a chronically infected HIV-1– seropositive woman (K) were
incubated with increasing amounts of rgp160 (A) or anti-F(ab0 )2 antibodies (B) for 30 min at room temperature before quantitation of anti-gp160
titer by indirect ELISA with biotin-streptavidin amplification. Optical density readings in the absence of competitor were 1.3–2.0. Results are
expressed as the percentage of inhibition of reactivity in the ELISA.
reactivity of natural antibodies. Indeed, the immunoglobulins
did not recognize actin, tubulin, and myosin when tested at
1mg/mL, unlike the natural polyreactive IgG antibodies purified
from colostrum tested at a similar concentration and used as a
positive control in the ELISA (data not shown). None of the 25
HIV-1–seronegative women who had anti-gp160 antibodies in
their CVS samples had detectable HIV-1 RNA in plasma by
RT– nested PCR (table 2).
Proviral HIV DNA in cervical and vaginal cells. HIV-1
DNA sequences were detected in 7 (47%) of 15 semen-containing CVS samples but in no semen-free CVS samples (P ¼ :020;
table 1). In the semen-containing samples, PCR results were
positive for the pol and env genes for 1 woman (subject no. 177)
and positive for only 1 of these genes in the remaining 6 samples
(data not shown).
Isotype and secretory form of cervicovaginal anti-HIV antibodies. We determined the isotype of anti-gp160 antibodies
in the cervicovaginal fluid of the 25 women discordant for the
presence of anti-HIV antibodies in CVS samples and serum by
EIA. These cervicovaginal lavage samples contained IgA,
IgG, and IgM at concentrations (mean ^ SEM) of 2:710 ^
0:503 mg/mL (range, 0.743– 5.420 mg/mL), 8:901 ^ 0:910
mg/mL (range, 0.231–18.613 mg/mL), and 1:521 ^ 0:102
mg/mL (range, 0.363–6.030 mg/mL), respectively. The isotypic pattern of anti-gp160–specific antibodies included IgA
and IgG antibodies, together with a weaker reactivity for IgM
(figure 2). The specific activities of cervicovaginal IgA and IgG
to gp160 (calculated as ½OD492nm 100= ½immunoglobulinmg=mL )
were 72:3 ^ 19:5 and 69:1 ^ 46:3 arbitrary units for IgA and
IgG, respectively.
The presence of sIgA and sIgM was shown immunochemically (figure 2) and by molecular analysis of the antibodies in
effluents of chromatography on Sephacryl S300 from pooled
material of 5 cervicovaginal fluid samples (figure 3). Anti-gp160
antibodies of the IgA, IgG, and IgM isotypes, including sIgA and
sIgM, also were found in the CVSs of the 15 women in whom
contaminating semen was detected in cervicovaginal fluid
(figure 2). The mean level of reactivity of IgG to gp160 in the
latter group was significantly higher than in the cervicovaginal
fluid of the 10 women with no detectable contaminating semen
(data not shown). The mean avidity for gp160 of antibodies in
CVSs of HIV-seronegative women exhibiting cervicovaginal
anti-HIV antibodies was significantly lower than that of antibodies in CVSs of 25 HIV-seropositive women (figure 4). The
affinity of anti-gp160 antibodies in CVSs without semen of
HIV-seronegative women was lower than that of antibodies in
CVSs contaminated with semen.
Antigen specificity and epitope mapping of cervicovaginal
HIV-specific antibodies. The Western blot pattern of reactivity
Table 2. Levels of cytokines in acellular fraction of cervicovaginal
secretions (CVSs) of human immunodeficiency virus (HIV)–seronegative African female sex workers with and without HIV antibodies in
their CVSs.
CVS
Cytokine
With HIV antibodies
(n = 10)
Without HIV antibodies
(n = 15)
Pa
IFN-g
IL-2
IL-6
IL-1b
RANTES
50.6 ^ 11.8
22.1 ^ 3.6
19.1 ^ 1.4
26.7 ^ 1.4
11.5 ^ 2.2
25.0 ^ 3.1
14.5 ^ 1.8
11.2 ^ 1.1
21.4 ^ 2.8
1.4 ^ 0.8
.045
.08
.001
NS
.0007
NOTE. Data are mean ^ SEM (pg/mL). Cytokines were quantitated by
ELISA. All women were from Abidjan, Côte d’Ivoire. CVSs contained no traces
of contaminating semen. IFN, interferon; IL, interleukin; NS, not significant.
a
Mann-Whitney U test.
1416
Bélec et al.
JID 2001;184 (1 December)
restricted pattern of reactivity was observed in 8 of 15 anti–
HIV-positive semen-containing CVS samples from HIV-negative women; the 7 remaining CVS samples of this group showed
a complete pattern of reactivities with rgp160, rp68, and rp24
(figure 2).
The reproducible pattern of epitopic reactivities of the antienv antibodies in semen-free CVS samples, as opposed to the
more heterogeneous pattern observed in semen-containing
CVS samples, is further documented in figure 6. Semen-free
CVS samples contained antibodies of IgA and IgG isotypes and
to a lesser degree to IgM, reactive with linear epitopes of gp41
and gp120, with the predominant reactivity being directed to
the gp41/K2 peptide. Nine of 15 semen-containing CVS samples
had patterns of epitopic specificity similar to semen-free CVS
samples, with a predominant reactivity directed to the gp41/K2
peptide (figure 6). All but 1 sample (subject 738) did not show
cervicovaginal antibody to rp68 by ELISA (figure 2). The 6
remaining semen-containing CVS samples exhibited high
levels of IgA or IgG directed to the gp41/Id peptide and antibodies to rgp160, rp68, and rp24 (figure 2 and figure 6).
Nine of the 15 women with CVS samples contaminated by
semen (subjects 177, 376, 738, 946, 1025, 1116, 1240, 1425,
and 1739) exhibited cervicovaginal IgA or IgG antibodies,
Figure 2. Human immunodeficiency virus (HIV) antigens recognized and isotypes of anti-HIV antibodies (Ab) in cervicovaginal
secretions (CVSs) of HIV-seronegative women with anti-gp160 antibodies in their CVSs: 10 with no contaminating semen (left) and 15
with contaminating semen (right). Reactivity of antibodies in 100 mL
of cervicovaginal lavage fluid was tested by indirect ELISA with
immobilized recombinant gp160, p68, and p24 as antigens. Bound
antibodies were revealed by use of horseradish peroxidase–labeled heterologous antibodies to human Fca, Fcg, Fcm, F(ab0 )2, and secretory
component. Secretory (s) IgA to gp160 was detected in CVSs after
depletion of IgG and IgM. sIgM was detected after preabsorption of
IgG and IgA. Intensity of reactivity is shown by solid (OD . 0:5) and
hatched (cutoff ½CO , OD , 0:5) areas. Blank areas indicate lack of
reactivity in ELISA corresponding to optical density below cutoff.
Subject nos. are at left.
of cervicovaginal antibodies was assessed by ELISA in samples
from 4 of the 25 HIV-seronegative women who exhibited the
highest levels of anti-HIV antibodies in CVSs (figure 5). A
reproducible pattern of reactivity restricted to gp160, p24, and
p18 was observed for IgA, IgG, and IgM antibodies in CVS
samples that were free of contaminating semen (subjects 849
and 1614) and in 1 CVS sample with traces of semen (subject
738). A more complete pattern of reactivities was observed in
the other semen-containing CVS sample (subject 138). We confirmed a restricted pattern of reactivities with rgp160 and rp24,
but not with rp68, in 9 of 10 anti–HIV-positive semen-free
CVS samples from HIV-negative women (figure 2). A similar
Figure 3. Presence of secretory (s) IgA and IgM in pooled cervicovaginal secretions (CVSs) of 5 human immunodeficiency virus
(HIV)–seronegative women with anti-gp160 antibodies in their CVSs,
in the absence of contaminating semen. The CVS pool was chromatographed on Sephacryl S300 (Pharmacia) sizing column in PBS-azide
solution containing 0.5 M NaCl. By comparing results of antiFca/anti–secretory component (SC) asymmetric ELISA and those of
anti-Fca/anti-F(ab0 )2 ELISA, the 330–410-kDa peak was found to
mainly consist of sIgA, with a small proportion (10%) of dimeric
(d) IgA. By comparing results of anti-Fcm/anti-SC ELISA and those
of anti-Fcm/anti-F(ab0 )2 ELISA, the 450-kDa peak was found to consist
of sIgM. Proteins in effluent were revealed by use of heterologous antiFca, anti-Fcg, or anti-Fcm antibodies. mIgA, monomeric IgA.
JID 2001;184 (1 December)
HIV-1 Antibodies in Seronegative Women
1417
Figure 4. Avidity of anti-gp160 antibodies in cervicovaginal secretions (CVSs) of HIV-seropositive women (Sab+, CVSab+), HIV-seronegative
women with anti-HIV antibodies in their CVSs, with no detectable contamination of CVSs with semen (Sab2, CVSab+, Se2), and HIV-seronegative women with anti-HIV antibodies in their CVSs, with semen traces (Sab2, CVSab+, Se+). Avidity of anti-gp160 antibodies in CVSs was
assessed by the method of Pullen et al. [40], which is based on the dissociation of antigen-antibody complexes by chaotropic ions. gp160–antigp160 complexes were dissociated with sodium thiocyanate (NaSCN) [41]. A, Mean ratio of absorbance values at 492 nm obtained in the presence
of NaSCN to those obtained in the absence of NaSCN. B, Avidity index of cervicovaginal anti-gp160 antibodies (mM; mean ^ SEM). Avidity
index was defined as the molarity of NaSCN equivalent to the interpolation point corresponding to 50% of control absorbance value obtained in
the absence of NaSCN.
with a predominant reactivity directed to the gp41/K2 peptide.
Six women with CVSs contaminated by semen had high levels
of IgA or IgG directed to the gp41/Id peptide (subjects 138,
196, 867, 1313, 1602, and 1746). HIV-1 proviral DNA sequences were detected more frequently in CVS samples with
high levels of antibodies to the gp41/Id peptide than in CVS
samples exhibiting predominant reactivity to the gp41/K2 peptide (3 [16%] of 19 vs. 4 [67%] of 6; P ¼ :032).
Inhibition of viral transcytosis through tight epithelial barriers by cervicovaginal HIV-specific antibodies. As shown
in figure 7, immunoglobulins from both semen-free and semencontaining CVSs of 5 of 6 unselected women inhibited trancytosis of HIV. Unfractionated CVSs were more potent in inhibiting
transcytosis than were purified IgG and sIgA, when tested at
similar concentrations of immunoglobulin. Transcytosis of
HIV-1 derived from Sup T1 cells also was inhibited by dIgA
purified from pooled CVS samples (figure 7). No inhibition of
transcytosis was observed after preincubation of HIV-infected
Sup T1 cells with IgG from the serum of an HIV-seronegative
donor (irrelevant serum IgG) and with total immunoglobulin
purified from a CVS sample of an HIV-seronegative unexposed
woman living in Europe (irrelevant cervicovaginal Ig). The
results demonstrate that anti-HIV antibodies in CVSs of sexually exposed HIV-seronegative women exhibit blocking properties toward HIV transcytosis in vitro.
Transcytosis of HIV-1 also was inhibited by dIgA purified
from pooled CVS samples of HIV-seronegative women exhibiting cervicovaginal anti-HIV deposited in the basolateral side of
the tight monolayer of HEC1 cells (figure 7). In the control
experiment, preincubation of dIgA with anti-SC antibodies
restored HIV transcytosis.
Cytokines in CVS samples. The mean levels of the Th1-type
cytokines IFN-g and IL-2 were higher in CVSs of women with
cervicovaginal anti-HIV antibodies than in the CVSs of HIVseronegative sex workers with no evidence of anti-HIV antibodies in CVSs, although the difference only reached statistical
significance for IFN-g (table 2 and figure 8). There were
also significantly higher levels of IL-6 and RANTES in CVS
samples of women with cervicovaginal anti-HIV antibodies
than in the CVS samples of women with no local anti-HIV
immunity.
Discussion
We found significant levels of anti-HIV antibodies of the IgA,
IgG, and IgM isotypes in the CVSs of 7.5% of 342 multiply and
repeatedly exposed African HIV-seronegative sex workers. In
half these women, the antibodies represented a local compartmentalized secretory immune response to HIV, since they were
present in vaginal fluids free of contaminating semen. Antibodies in CVSs of HIV-seronegative women exhibited a homogeneous pattern of reactivity restricted to gp160 and p24 resembling that described in serum during the primary invasion
phase of HIV infection. Locally produced anti-env antibodies
exhibited a predominant reactivity toward the neutralizing
ELDKWA epitope of gp41 for the linear epitopes tested. We
also found that the antibodies purified from CVSs block the
transcytosis of cell-associated HIV through a tight epithelial
monolayer in vitro. Although the cervicovaginal anti-HIV
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Bélec et al.
JID 2001;184 (1 December)
Figure 5. Reactivity with human immunodeficiency virus (HIV) antigens of antibodies in cervicovaginal secretions (CVSs) of 4 HIV-seronegative women who had high levels of anti-HIV antibodies in their CVSs, shown by isotype-specific Western blot analysis. Cervicovaginal lavage fluid
from HIV-seronegative subjects 849 and 1614 had no traces of semen (Semen2); those from subjects 138 and 738 had contaminating semen
(Semen+). The left strip in each set of blots is a positive control that corresponds to CVSs of an HIV-seropositive woman for anti-HIV IgA and
IgG and to the serum of an HIV-1 seroconverter for anti-HIV IgM.
humoral response was seen in a minority of exposed HIV-seronegative women, our observations suggest that there is a protective role for the cervicovaginal anti-HIV antibody response
in these women.
The HIV-specific antibody response characterized was
restricted to the lower genital tract compartment. The antibodies
originated from the immune effector sites of the cervicovaginal
mucosa, since they comprised sIgA and sIgM and were found
in women with no traces of contaminating semen in their CVS
samples. For women whose CVS samples contained traces of
semen, a similar local secretory immune response likely
occurred, although it could not be distinguished from passively
transferred heterologous anti-HIV antibodies from the semen
[43]. The presence of contaminating semen (assessed by
the detection of PAP, PSA, or Y chromosome) in 60% of the
HIV-seronegative women exhibiting high titers of cervicovaginal anti-HIV antibodies, demonstrates that the women practiced
unprotected sex and emphasizes the need to search for evidence
of contaminating semen when investigating local immunity in
exposed women [44]. Locally produced anti-gp160 antibodies
included monomeric IgA, dIgA, sIgA, IgG, and sIgM.
Our observations extend those of Mazzoli et al. [24] and of
Kaul et al. [26, 27] on the presence of HIV-specific IgA in the
genital tract of HIV-seronegative partners of HIV-seropositive
persons and of HIV-resistant Kenyan female sex workers. We
found that high amounts of IgG to gp160 was produced locally,
as was sIgA. Mazzoli et al. [24] also reported on the presence of
HIV-specific IgG in 18% of CVS samples of HIV-seronegative
women who were sex partners of HIV-seropositive men.
Several factors may account for the differences between our
data and those of Mazzoli et al. [24] and Kaul et al. [26], including differences in the study populations (particularly with
regard to the duration of exposure in the women studied) and
the fact that we excluded samples with traces of semen from
our calculation of the prevalence of genital antibody responses
in HIV-seronegative women. Also, Kaul et al. [26] investigated
endocervical secretions, which contain higher levels of IgA
than the CVS samples [6] used in the present study. Antibodies
of the IgG isotype are predominant in normal CVSs and originate both from plasma and from local synthesis by submucosal
IgG-committed plasma cells [38]. A compartmentalized genital
IgG anti-HIV antibody response was documented previously in
chronically infected women. Cervicovaginal HIV-specific IgG
differs from serum anti–HIV IgG in specific activity and
epitopic specificity [9, 41]. A predominance of IgG over IgA
anti-HIV antibodies also has been reported in other corporeal
fluids of chronically infected persons [45], including tears,
mixed saliva, duodenal fluid, breast milk, and seminal fluid.
For women with no evidence of contaminating semen in their
CVSs, the pattern of the local antibody response was reminiscent
of that observed in serum during the primary invasion phase of
HIV infection. Thus, the antibodies exhibited a restricted anti-
JID 2001;184 (1 December)
HIV-1 Antibodies in Seronegative Women
1419
Figure 6. Epitopic specificity of anti-env antibodies of IgA, IgG, and IgM isotypes in cervicovaginal secretions (CVSs) of 25 human immunodeficiency virus (HIV)– seronegative women with anti-gp160 antibodies in their CVSs. Linear peptides used were gp41/Id (LLGIWGCSGKLIC 597– 609 LAI), gp41/K1 (LDKWASLWNWFNITNWL 668–684 LAI), gp41/K2 (EKNEQELLELDKWASLW 659– 675 LAI), gp41/Fu
(VGIGALFLGFLGAAG 518– 532 LAI), gp120/C2 (CTHGIRPVVSTQLLNGSLAE 252–272 LAI), gp120/V3 (CTRPNNNTRKSIYIGPGRAFHTTGRIIGDIRKA 301– 333 SF2), gp120/V4 (EGSDTITLPCRIKQFINMWQE 414–434 LAI), and gp120/C4 (ITLPCRIKQFINMWQEVGKAMYAPPI 419– 444 LAI). The linear sequence ELDKWA of gp41 residues 662–667 is recognized by monoclonal antibody 2F5
that neutralizes the majority of clade B primary isolates [42]. Left, Results with CVSs with no traces of contaminating semen (Semen2).
Right, Results in CVSs with semen traces (Semen+). Reactivity of antibodies in 100 mL of cervicovaginal lavage fluid was tested by indirect
ELISA with immobilized gp120 and gp41 peptides as antigens. Bound antibodies were revealed by use of anti–human Fcg, Fca, and Fcm biotinylated labeled heterologous antibodies and streptavidin– horseradish peroxidase conjugate. Intensity of reactivity is shown by solid
(OD . 0:15) and gray (cutoff ½CO , OD , 0:15) areas. Blank areas indicate lack of reactivity in ELISA (defined as optical density below
that of the cutoff, representing the mean plus 2 SD of reactivities from cervicovaginal lavage samples of 30 control HIV-seronegative
women not at risk for HIV).
env and anti-gag pattern of reactivity after Western blotting. In
addition, the anti-gp160 response comprised IgM antibodies
and was of low avidity, in contrast to antibodies in CVSs of
chronically HIV-infected women. The latter pattern of antibody
response clearly differs from that reported in CVSs of chronically infected women, which is characterized by a complete
env, pol, and gag pattern of antigenic reactivity and the dominance of high avidity IgG antibodies [8, 9]. It also differs from
the complete pattern of antigenic reactivity and strong predominance of the IgG isotype that we observed in 6 of 15 samples of
semen-contaminated CVS samples of HIV-seronegative women
in whom the detected antibodies could have originated from
HIV-infected male donors. It is unlikely that the local response
that we describe occurred within the context of a systemic primary invasion phase, since none of the women had evidence of
circulating plasma HIV RNA. We suggest that the restricted pattern of reactivity with HIV antigens of cervicovaginal antibodies
that we observed is the consequence of a local immunization in
the absence of systemic spread of the virus.
Cervicovaginal antibodies in highly exposed persistently
seronegative women recognize several epitopes on HIV-1 gp41
that differ from epitopes recognized in HIV-infected persons
[46]. Among the linear epitopes of gp41 and gp120 that we
tested, cervicovaginal antibodies of the IgA, IgG, and IgM isotypes in semen-free CVS samples of HIV-seronegative women
predominantly recognized the ELDKWA sequence contained
within the gp41/K2 peptide. The latter pattern of reactivity was
in contrast with the broader pattern of epitopic recognition of
gp41 and gp120 that was observed with cervicovaginal antibodies of women with contaminating semen in CVSs. The linear
ELDKWA sequence is part of determinant III of the gp41 molecule that is conserved among many HIV-1 isolates [42]. It is
recognized by the IgG monoclonal antibody 2F5 [42], which
exhibits neutralizing activity against the majority of clade B pri-
1420
Bélec et al.
JID 2001;184 (1 December)
We demonstrated that sIgA and IgG purified from CVSs of
HIV-seronegative women inhibited the transcytosis of cellassociated HIV when deposited on the apical surface of a tight
monolayer of HEC-1 cells. The antibodies exhibit functional
properties in vitro that are suggestive of an inhibitory capacity
toward transepithelial passage of HIV. Genital and salivary
purified IgA from HIV-exposed uninfected persons inhibit the
transcytosis of cell-associated primary HIV when deposited on
the apical surface of a tight monolayer of CaCo-2 cells [20]
and neutralize infection of peripheral blood mononuclear cells
by a nonsyncytium-inducing HIV-1 primary isolates [27]. The
mechanism of transcytosis inhibition remains to be established
[3, 4, 10] and may differ from those involved in conventional
in vitro neutralization assays of infectivity. dIgA purified from
CVSs of HIV-seronegative women also inhibited apical to
basal transcytosis of HIV when deposited at the basolateral surface of the monolayer, suggesting that these antibodies may
Figure 7. Inhibitory activity of whole cervicovaginal secretions
(CVSs; black bars), purified secretory (s) IgA (white bars) and IgG
(hatched bars) fractions from CVSs of human immunodeficiency
virus (HIV)– seronegative women, and of dimeric IgA (gray bars) purified from pooled CVSs of HIV-seronegative women exhibiting cervicovaginal anti-HIV on transcytosis of Sup T1 cell–associated HIV-1
(subtype A) through tight HEC-1 cell monolayer. IgG purified from
serum pool from HIV-1– seropositive subjects was used as a positive
control. Control serum IgG from seropositive women was tested at
1mg/assay. Whole CVSs corresponded to 1 mg of total immunoglobulin
(IgA + IgG + IgM)/assay. Purified IgG and secretory (s) IgA from CVSs
of seronegative women were tested at 1 mg/assay. CVSs from subjects
693, 849, and 1096 were free of detectable semen (Semen2). CVSs of
subjects 138, 376, and 1313 contained traces of contaminating semen
(Semen+). Results are expressed as the percentage of inhibition of
transcytosis in the presence of 1 mg/assay of serum IgG of an HIV-seronegative subject (standard). The experiments were done in duplicate.
Vertical dashed line, threshold of significant transcytosis inhibition.
dIgA, dimeric IgA; SC, secretory component.
mary isolates and by several non–clade B HIV-1 strains [47]. A
marked antibody response to the ELDKWA epitope is found in
most colostrum samples of chronically infected women, suggesting that it may be a strong inducer of mucosal antibody
responses [48]. Furthermore, recent evidence suggests that the
ELDKWA sequence is one of the target epitopes recognized by
HIV-specific dIgA and pentameric IgM serum antibodies capable
of intracellular neutralization of HIV [4].
Of note, we tested for reactivity of anti-gp160 antibodies from
CVSs with linear peptides but not for reactivity with conformational epitopes of envelope protein. In addition, the lack of
reactivity of the antibodies with peptides of the V3 loop of the
SF2 strain does not exclude the possibility that the antibodies
would be reactive with V3 peptides of the particular strain of
HIV to which the women had been exposed.
Figure 8. Distribution of cervicovaginal concentrations of cytokines in acellular fraction of cervicovaginal secretions (CVSs) of
human immunodeficiency virus (HIV)–seronegative female sex workers with no traces of semen (Se2) in their CVSs: 10 with anti-HIV
antibodies in CVS (CVS+) and 15 without HIV antibodies (CVS2).
Horizontal bars, mean values of distribution. IFN, interferon; IL,
interleukin; S2, HIV-negative serum.
JID 2001;184 (1 December)
HIV-1 Antibodies in Seronegative Women
neutralize HIV intracellularly [4]. Anti-HIV antibodies in CVS
of chronically infected women may also inhibit viral transcytosis in vitro [10, 49].
Recently, protection in highly exposed persistently seronegative women in the absence of HIV-1–specific vaginal IgA or
IgG with no neutralizing activity in vaginal secretions was
observed in female sex workers in The Gambia, suggesting that
resistance is not based solely on HIV-1–specific humoral immunity [50]. Although the present study focused on the local
secretory antibody response, our observations of local production of the Th1 cytokines IL-2 and IFN-g, in addition to that
of IL-6 and RANTES, support the hypothesis of genital immune
activation, as described elsewhere for HIV-exposed seronegative women [51]. Furthermore, these findings suggest that the
cervicovaginal immunity to HIV also involves specific T cells,
as reported elsewhere for HIV-1– resistant sex workers in Nairobi [12], and the local production of HIV-suppressive chemokines. Genital produced b-chemokines, including RANTES,
likely participate in the inhibition of simian immunodeficiency
virus transmission in nonhuman primates [52]. Taken together,
these data suggest that both humoral and cellular immunity
may play a role in protecting the genital tract from mucosal
HIV-1 infection in a minority of exposed women. Because
female sex workers in Abidjan are exposed to HIV genitally
only, the induction of HIV-specific immunity occurred in the
cervicovaginal mucosa. Langerhans cells may have transported
HIV to the draining lymph nodes to elicit HIV-specific T and B
cell responses. HIV-specific IgA, IgG, and IgM plasma cells
then could have migrated back from the genital lymph nodes to
the genital mucosa. Whether genital immunization is secondary
to active infection of the genital mucosa or merely to the mucosal
deposition of HIV antigens with subsequent capture by mucosal
antigen-presenting cells remains unknown.
Cervicovaginal immunization with passively deposited
proteins has elicited little or no specific antibody production in
experimental animal models [53, 54]. However, healthy women
may respond to vaginal immunization with inert antigens [55].
We speculate that inert HIV antigens could elicit local immune
responses in women with fragile cervicovaginal mucosa. Alternatively, the mucosal response to HIV of female sex workers
may occur after cervicovaginal infection. Repeated sexual
exposure to HIV may serve as recall immunization for HIVspecific mucosal immunity. We found no proviral DNA in the
samples of CVS that were free of contaminating semen. Proviral
DNA sequences were found in a few CVS samples containing
semen, either representing passively deposited HIV from
semen or corresponding to local viral replication. Thus, in the
absence of evidence of systemic infection and in the absence of
detectable proviral DNA in cervicovaginal cells, our results
raise the possibility that the genital anti-HIV immune response
cleared the virus efficiently at the mucosal level and prevented
systemic infection. The elucidation of the mechanisms involved
in the generation of a protective HIV-specific immunity in the
1421
genital tract of exposed seronegative women may be of major
relevance for the design of a vaccine against HIV.
Acknowledgments
We thank the Abidjan female sex worker community and the peer
educators’ network at Clinique de Confiance for their cooperation.
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