Sexual transmission of HIV accounts for most of the new infections worldwide [1,2], and has been on the focus of prevention strategies to reduce its incidence [3,4] and experimental research to identify mechanisms and new potential targets for intervention . It is well known that susceptibility to sexual HIV transmission is highly variable among individuals , and that despite multiple and repeated exposure to HIV, some individuals remain uninfected as determined by its serological and virological status [7,8]. Although data on the susceptibility to HIV infection suggest that this is a multifactorial phenomenon, in which genetic factors, innate and adaptive immunity may participate [7,8]. The study of protective factors is of capital interest to improve preventive and immunomodulatory approaches aimed to protect HIV-exposed individuals .
In this regard, special attention has been paid to the role of adaptive immunity potentially generated by continuous HIV exposure in individuals engaged in stable and sexually active relationships with HIV-infected partners . The study of these HIV-exposed seronegative (HESN) individuals has revealed the presence of HIV-specific CD8 and CD4 T cells [10–15] and the association of protection from HIV acquisition by antibodies either recognizing specific epitopes in the HIV envelope glycoprotein [16,17] or autoantibodies against CD4 or CCR5 interfering with the HIV entry into host cells [18–20]. Interestingly, humoral responses against HIV have been identified in peripheral blood, reflecting a systemic immunoglobulin (Ig)G and IgA response [16,17], and are not restricted to mucosal secretions, in which IgA isotype has a major role [19–22]. Despite the well documented protective role of mucosal IgA in animal models of HIV transmission , the elicitation of adaptive immunity by HIV exposure is unclear in animal models  and its long-term durability seems to be compromised in longitudinal follow-up of HESN cohorts .
Recently, the role of T-cell responses and T-cell activation has been analyzed in a Spanish cohort of HESN; the results suggest that low but persistent sexual HIV exposure is able to induce virus-specific T-cell responses and immune activation in these individuals . The main objective of this work was to better define in the same cohort the role of adaptive immunity by analyzing systemic humoral anti-HIV immune responses. We have shown the presence of HIV envelope-specific IgG and IgA in plasma samples from HESN as compared with healthy unexposed individuals. Our data suggest that these seronegative individuals may have developed antibodies against specific epitopes that are not detected in routine ELISA-based or western-blot assays.
Materials and methods
Serodiscordant couples were recruited at Centro Sandoval, a sexually transmitted disease (STD) clinic located in Madrid (Spain) and have been previously described . The main criterion for inclusion was HIV seronegativity (routine HIVAb testing) and stable sexual partnership with an HIV-infected individual during at least 12 months prior to the inclusion. Participants were examined for STDs and asked to complete a structured questionnaire that included sexual behavior information as well as data concerning risk practices. The level of exposure to HIV in HESN individuals was regularly evaluated at each visit. Accordingly, HESN individuals were classified into two different levels of exposure to HIV, very low/low and moderate-high, as described previously .
Eleven healthy HIV-seronegative individuals with no risk behavior for acquiring HIV infection were identified at the Hospital Germans Trias i Pujol and recruited as healthy controls. Written informed consent was given by all participants before enrollment and the study protocol was approved by the Hospital ethical committee.
Sample processing and serology tests
Blood was collected by venipuncture in EDTA vacutainer tubes (BD Bioscience). PBMC were obtained by density gradient and cryopreserved until use as described . Plasma was obtained by centrifugation of blood at 1200g for 10 min and stored at −80°C. All samples were tested for the presence of anti-HIV antibodies using the Architect HIV Ag/Ab Combo (Abbott Laboratories) and the NEW LAV BLOT I kit (Bio-Rad).
293T cells (ATCC) and TZM-bl cells (NIH AIDS Research and Reference Reagent Program) were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% heat inactivated fetal bovine serum (FBS) with selection antibiotics when required. MOLT-4/CCR5 cells uninfected or chronically infected with the NL4-3 or the BaL isolates (>90% producing HIV particles) have been previously described [27,28] and were maintained in RPMI-1640 supplemented with 10% heat inactivated FBS. All media were from Life Technologies (Barcelona, Spain).
Recognition of cell-surface expressed HIV envelope
MOLT/CCR5 cells either uninfected or chronically infected with the HIV isolate NL4-3 or BaL  were stained with one of 10 plasma dilutions for 30 min at room temperature, cells were extensively washed in PBS and cell-bound antibodies were revealed using phycoerythrin-labeled antihuman IgG or DyLight649-labeled antihuman IgA, both from Jackson ImmunoResearch Laboratories, (West Grove, Pennsylvania, USA). Negative controls were set up using only secondary antibodies; specific envelope recognition was calculated as percentage of positive cells by subtracting the staining background determined in uninfected cells to the staining obtained in infected cell lines. Potential staining by antibodies against other viral proteins was controlled by determining the absence of extracellular p24 gag protein on the surface of infected MOLT cells as described . Positive controls were performed using the antigp120 human monoclonal antibody b12 (4 μg/ml, Polymun, Vienna, Austria). In parallel, determination of cryptic envelope glycoproteins (Env) epitopes recognition was carried out by adding soluble CD4 protein (sCD4 2 μg/ml, AIDS Reagent and Reference Program) to MOLT cells before incubation with antibodies or plasma samples. In these incubations, the b12 human mAb was used as control to check for the blockade of the CD4 binding site in gp120, whereas the 17b mAb (AIDS Reagent and Reference Program) was used to confirm the exposure of CD4-induced epitopes.
ELISA assays were performed as described . Briefly, monomeric recombinant gp120 (BaL isolate) or trimeric gp140 (SF162 isolate), both from NIH AIDS Reagent Program, were used to coat Maxisorb Nunc plates (50 ng/well in PBS) washed with PBS 0.05% Tween-20, saturated with FBS 10% in PBS, and incubated with sCD4 (Progenics, NIH AIDS Reagent Program, 50 ng/well) or buffer, before addition of duplicate plasma samples (1/300 in PBS/FBS 1%) or control mAbs (IgGb12 or 17b, 0.5 ng/well). Bound antibodies were revealed using a peroxidase-labeled goat antihuman IgG (Jackson ImmunoResearch) and OPD as substrate.
Pseudoviruses were generated by cotransfecting 293T cells with envelope (NL4-3, BaL or AC10) expression plasmids and a HIV-1 backbone vector (pSG3ΔEnv). Culture supernatants were harvested 24 h after transfection, filtered (0.45 μm) and stored at −80°. The 50% tissue culture infective dose (TCID50) of the pseudoviruses was determined in TZM-bl cells, by infecting 10 000 cells with serial dilutions in the presence of 37.5 μg Diethylaminoethyl-dextran/ml. After 48 h, the cultures were analyzed using a luminometer (Labsystems, Waltham, Massachusetts, USA) with the Britelite Reagent (Britelite Luminescence Reporter Gene Assay System; Perkin Elmer Life Sciences, Madrid, Spain). The presence of neutralizing antibodies was assessed by incubating 200 TCID50 of pseudovirus with serially diluted heat-inactivated plasma in duplicate for 1 h at 37°, 5% CO2. The mixture was then used to infect TZM-bl cells as described above. Neutralizing activity was calculated as the percentage of inhibition achieved compared with the luciferase activity observed in a positive infection control carried out in quadruplicate .
HIV-specific T-cell responses
PBMC (1 × 106 cells) were stimulated with different pools of overlapping peptides (NIH AIDS Research and Reference Reagent Program, 2 mg/ml each) during 6 h at 37°C with 5% CO2 in the presence of Brefeldin A. Cells were harvested, washed with PBS and incubated with anti-CD4-PECy5 (Cytognos, Salamanca, Spain), anti-CD8-PECy7, and anti-CD3-ECD (Beckman Coulter, Fullerton, California, USA) for 30 min at 4°C. The cells were permeabilized using the Cytofix/Cytoperm kit (BD-Biosciences, San Jose, California, USA) and were then incubated with anti-MIP-1b-FITC (R&D, Minneapolis, Minnisota, USA), and anti-IFN-g-PE (BD Biosciences). IFN-g and MIP-1b expression defined positive responses in gated CD3+CD4+ or CD3+CD8+ cells .
Continuous variables were expressed as the median (interquartile range) and compared using nonparametric tests (Mann–Whitney). Discrete variables were described as percentages (number of individuals) and compared using the Fisher's exact test. Spearman correlation coefficient was calculated to assess the association between different variables. Positive responses were defined by cutoff values for each parameter using the Mean and two-fold the SD (2 × SD) of values obtained using the 11 plasma samples from healthy controls.
Table 1 summarizes the main characteristics of 27 HIV-serodiscordant couples enrolled in the study. At the time of recruitment, the median (IQR) CD4 cell count in HIV-infected patients was 434 (204–666) cells/μl and the median length of HIV infection was 108 (30–198) months. Overall, 66% (18/27) had undetectable (<50 copies/ml) HIV-RNA copies/ml and the median in the remaining nine HIV-infected patients was 5821 (3507–52259) HIV-RNA copies/ml. All HESN had a seronegative test at the inclusion and remained seronegative in further follow-up tests. Fourteen of 27 (52%) HESN were men and the median number of sexual intercourses was five [4–8] per month and the median length of their relationship was 11 [6–18] years. Fifteen (56%) HESN reported never using condom, eight (30%) reported sporadic use, and three (11%) reported frequent use and only one HESN individual reported using a condom always. According to the frequency and protection of sexual intercourses and to the viral load of the HIV-infected partners, HESN individuals were classified into two HIV-exposure levels: very low/low (n = 18) and moderate/high (n = 9) HIV exposure level.
Recognition of cell surface expressed envelope
By definition, HESN do not show detectable plasmatic levels of IgG against HIV proteins by western-blot. However, several groups have identified systemic and mucosal HIV envelope reactive IgG and IgA antibodies [19,21,30,31]. We have, therefore, developed an assay to quantify the presence of IgG and IgA recognizing native (cell-surface expressed) HIV Env. This assay uses MOLT cell lines either uninfected or chronically infected with the isolates NL4-3 or BaL . HESN showed intermediate levels of recognition of native Env for both NL4-3 and BaL isolates. Recognition was significantly lower than the values observed for HIV-infected partners but significantly higher than those of healthy controls for the BaL isolate in both, the IgG and the IgA fractions (Fig. 1a and b). Samples were categorized as positive or negative according to a cutoff defined as the MEAN + 2 × SD values found in healthy control. Using this approach, eight HESN individuals (30%) showed IgG reactive against BaL Env, whereas only 8% showed IgG reactive against NL4-3. IgA positivity reached 26% for BaL recognition and 15% for NL4-3. A pooled analysis showed that 14 out of 27 HESN (52%) and two healthy control showed IgG or IgA reactivity against at least one tested envelope. However, only one HESN showed both IgG and IgA reactive against both isolates.
Env protein shows a complex trimeric conformational structure with an important set of epitopes masked inside of the protein that become accessible after the interaction with CD4 cell count. These epitopes are the target for antibodies blocking relevant phenomena in HIV transmission such as transcytosis  and have been correlated with protection in different cohorts . To analyze reactivity against these epitopes, we added sCD4 to MOLT cell lines before staining with plasma samples. This treatment significantly reduces the binding of the anti-CD4 binding site mAb b12 and efficiently exposes CD4-induced epitopes as demonstrated by the increased recognition of the 17b mAb (Fig. 2a). The addition of sCD4 significantly enhanced the binding of IgG from HESN to both BaL and NL4-3 envelopes. The effect was specific since sCD4 failed to modify the background level of Env recognition observed in healthy control samples (Fig. 2a and b). No significant increases were observed for the binding of systemic IgA (data not shown). Using the cutoff defined by the mean and two-fold the SD of values found in healthy control we could identify 30% (eight out of 27) positive samples for NL43 and 48% (13 out of 27) for BaL.
The analysis of IgG responses showed that four out of eight (50%) HESN plasma samples that recognized untreated Env increased reactivity after sCD4 treatment, whereas the remaining Env-reactive samples maintained or slightly reduced its recognition. Interestingly, 12 out of 19 (63%) HESN plasma samples that lacked detectable IgG against untreated Env become positive upon sCD4 addition, suggesting that the analysis of reactivity against CD4i epitopes greatly increased the sensitivity of our assay. Altogether, 85% (23 out of 27) of the HESN samples showed some level of humoral reactivity against cell-surface expressed Env proteins, whereas only two samples from the healthy control group (18%) were identified as positive in at least one of the assays (p = 3.5 × 10−5, Fisher's exact test). These data confirm the presence of detectable systemic humoral response in HESN that is directed against a broad range of Env epitopes.
To further characterize Env-reactive antibodies in HESN, we performed ELISA assays using two different recombinant Env proteins, a monomeric soluble gp120 and a trimeric uncleaved Env (gp140). Figure 2c illustrates that plasma samples from HESN and healthy control showed similar reactivity against gp120, with 7 and 10% of positive samples in each group (P = 0.45). The reactivity against trimeric gp140 tended to be higher in HESN, although no difference in the percentage of positive samples (11 and 10%, P = 0.44) or the absolute recognition levels were observed (Fig. 2c). The addition of sCD4 failed to specifically increase HESN reactivity against recombinant Env forms either assessed by the percentage of positive samples or by absolute values (Fig. 2d). Interestingly, one HESN sample (couple 21) showed a strong reactivity against trimeric gp140 but not gp120. Remarkably, this sample was positive for the recognition of CD4-induced epitopes in gp120 and showed a positive increase in CD4-induced epitopes in trimeric gp140. As a whole, these data suggest that difference in Env recognition between HESN and healthy control are better detected by cell-surface expressed Env than by recombinant Env forms.
Association of envelope glycoproteins recognition with the level of exposure and with cellular anti HIV responses
We correlated the presence of Env recognition with several parameters measuring the exposure to HIV in HESN. The magnitude of Env-reactive IgG or IgA was not associated with the length of relationship, the level of exposure (very low/low versus moderate/high) or the presence of STD during the relationship (data not shown). Similarly, IgG and IgA reactivity against exposed epitopes of BaL or NL4-3 isolate showed comparable values in those individuals that display detectable or undetectable HIV-specific T-cell responses assessed by MIP-1b and/or IFN-g production (Fig. 3) . Furthermore, the analysis of reactivity against the CD4i epitopes confirmed the lack of apparent association between humoral and cellular responses (Fig. 3). Therefore, although a strong association between the presence of anti-Env antibodies and the HESN condition has been identified, these antibodies seem to be independent of the level of HIV exposure and independent of the presence of T-cell responses.
Analysis of HIV-neutralizing activity
Plasma samples from HESN, HIV-infected partners and healthy control were also analyzed for neutralization capacity in a standard assay using three different viral isolates (NL4-3, BaL and AC10). Plasma samples were tested at one of 60 dilution assuming a low neutralization capacity in samples from HESN. The results shown in Fig. 4 identify neutralizing activity (more than 50% inhibition) in three out of 27 HESN tested. Two of them neutralized BaL isolate, whereas one modestly neutralized both BaL and NL4-3 isolates. None of the HESN plasma samples showed activity against the AC10 virus, consistent with the higher resistance to neutralization of this isolate . Conversely, HIV-infected samples showed significantly higher neutralizing responses, although in some cases neutralization was low, mainly for AC10 virus. As expected, healthy control showed no detectable neutralizing capacity (Fig. 4).
Although HESN showed higher Env recognition than healthy controls, the presence of Env-reactive antibodies showed a striking lack of association with neutralization capacity (supplementary Table 1, http://links.lww.com/QAD/A317). Samples with neutralizing capacity did not display higher levels of Env recognition in the MOLT assay. Moreover, no clear neutralizing capacity could be determined in purified IgG samples isolated from HESN plasma (data not shown), suggesting that direct neutralizing activity of systemic Env-specific IgG or IgA does not play a major role in protection from HIV infection in our HESN cohort.
HIV-exposed seronegative individuals (HESN) include a set of people maintaining an uninfected status after being exposed to HIV, even over a long time. The protective mechanisms associated with this status are not well understood but it is known that both genetic and immunological processes are involved. Recently, it has been reported that a low level of exposure to HIV can promote the development of a HIV-specific T-cell response in HESN . Therefore, the aim of the present study was to evaluate whether systemic anti-Env humoral responses exist in these individuals and play a relevant role in protection.
Generation of antibodies against HIV has been widely reported in HESN, despite the persistent negative standard serology [7,33]. In our cohort, the routine assays ARCHITECT HIV Ag/Ab Combo and NEW LAV BLOT I, which, respectively, detect anti-HIV antibodies by a gp41-based ELISA assay using recombinant proteins and synthetic peptides or by a western blot of, among other, recombinant gp160, gp120 and gp41, yielded negative results. These observations led us to hypothesize that HESN may show some level of antibodies recognizing native epitopes that are not detectable by routine assays. Our data confirm this hypothesis and show detectable levels of anti-Env antibodies for both the IgG and the IgA isotypes. Remarkably, plasma from HESN but not from healthy control individuals significantly and specifically increased Env recognition after exposure of CD4-induced epitopes, which include the 17b epitope in gp120 and some gp41 regions involved in the fusion process [35,36]. This observation points to the presence of anti-Env humoral responses with a wide epitope repertoire in HESN, and suggest that increasing the range of Env epitopes analyzed may help to identify and further characterize humoral responses in these individuals. Furthermore, data from ELISA assays suggest that only one HESN sample recognizes recombinant forms of Env (either gp140 or CD4-treated gp120) suggesting that most of the targeted epitopes by HESN are specific of native cell-surface expressed Env. However, the interpretation of these data is limited by several factors, such as Env maturation (gp140 is unprocessed) and Env sequence variation, which may contribute to the unexpected ELISA data. Indeed, BaL and NL4-3 show different behavior, being BaL Env much more sensitive to detect anti-Env responses, either directed to exposed or cryptic epitopes. In summary, our data suggest that development of anti-Env antibodies in HESN may mirror seroconversion events in HIV-infected individuals, in which multiple sequence-specific gp41 and gp120 epitopes are targeted by antibodies .
Although Env recognition by HESN was higher than in control individuals, suggesting its strong association with real exposure to HIV, the origin of these Env-recognizing antibodies still remain undefined. A relevant question is whether they were generated by HIV exposure or come from other mechanisms like cross-reactivity with other pathogens or autoantigens, or even from polyreactive nonspecific natural antibodies with low affinity for Env. To evaluate a direct link between specific responses and HIV exposure, we analyzed its association with the length of relationship or risk of exposure; however, no positive correlation could be detected. Similar results were found with the HIV-1 specific cellular response previously identified in these individuals . Therefore, in the absence of evidence for a causative relationship, it is plausible to think that natural polyreactive or cross-reactive antibodies that may recognize Env could be in the base of this response. Natural antibodies are present in the general population , have been associated with protection to infectious agents  and may be enriched in HESN. Alternatively, antibodies against other pathogens have been described to cross-react with retroviral serological tests providing an additional source for nonspecific responses . Consistently, two individuals in the healthy control group showed some level of reactivity against native Env, supporting the idea that these antibodies could have developed before and independently of HIV exposure. However, the high prevalence of Env recognition found in HESN strongly supports a contribution of HIV exposure in the priming or boosting of anti-Env humoral response.
Although we detected Env reactivity in the IgG and the IgA fractions, our data do not show a clear link with protection against HIV infection. This is first due to the cross-sectional design of this study, which do not allow for the definition of causative relationships, and second due to the identification of very poor neutralizing activity in HESN individuals, which avoid accurate statistical analysis  and suggest that neutralization would play only a residual role in protection. This result is consistent with previous data reporting a higher relevance for cytotoxic T-lymphocyte responses in HESN protection . However, despite the poor neutralizing capacity showed by the plasma samples of HESN, we cannot completely rule out that the anti-Env humoral response indentified in HESN could play an active role in HIV protection by other mechanisms like antibody-dependent cell-mediated cytotoxicity, activating complement, inhibiting HIV transcytosis or even indirectly, stimulating the secretion of protective chemokines by innate cells [32,43–45].
In summary, our data show that an anti-Env humoral response is detectable in a high proportion of persistently exposed but HIV-seronegative individuals. We have shown that these responses are directed against several Env regions including both exposed and cryptic CD4-induced epitopes. However, we cannot clearly define whether these responses were specifically elicited by HIV exposure or came from preexisting polyreactive natural antibodies boosted by exposure as reported for HIV-infected individuals . Irrespective of its origin, our data suggest that a direct HIV-neutralizing activity does not play a major role in protection from HIV infection. A more detailed analysis of antibody specificities and functionality in both peripheral blood and mucosal secretions may help to define this potential role.
We are grateful to all individuals participating in the study.
This work was supported by the HIVACAT Program and the Spanish AIDS network ‘Red Temática Cooperativa de Investigación en SIDA (RD06/0006)’ and Fondo de Investigaciones Sanitarias (grant number PI050283). J.B. is a researcher from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the ISCIII and the Health Department of the Catalan Government (Generalitat de Catalunya). J.M.B. is a researcher from Fundación Investigación Biomédica Hospital Carlos III supported by the ISCIII and the Health Department of the Community of Madrid. J.C. is supported by a ‘Sara Borrell’ grant from the Spanish Health Institute ‘ISCIII’. M.M. is supported by a predoctoral grant from Generalitat de Catalunya and European Social Fund. C.R. and NIR are supported by Fundación para la Investigación y Educación del SIDA en España (IES foundation).
Conflicts of interest
Unrelated to this work, V.C. has served as a consultant on advisory boards or participated in speakers’ bureaux or conducted clinical trials with Boehringer, Gilead, BMS, Merck; Roche, Janssen, Viiv. B.C. has served as a consultant on advisory boards or participated in speakers’ bureaus or conducted clinical trials with Boehringer-Ingelheim, GlaxoSmithKline, Gilead, Janssen, Merck, Pfizer and ViiV. J.B. has received research funding, consultancy fees or lecture sponsorships from GlaxoSmithKline, ViiV and Merck. All other authors declare no competing interests.
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