HIV-1-exposed uninfected individuals are of interest to identify factors that can be protective against HIV-1 infection, as recently reviewed by Miyazawa et al. . Genetic factors that may account for protection from HIV acquisition include the homozygous delta chemokine (C–C motif) receptor 5 (ΔCCR5) deletion , certain human leukocyte antigen types  or chemokine (C-C motif) ligand 3-like 1 (CCL31) copy numbers  (reviewed in ). Other investigators [6,7] discuss the possible protection of HIV-1-specific immunity induced by the HIV-1 exposure itself. The durability of such responses is not known, but results from several studies suggest a correlation with continuous exposure to HIV-1 .
Exposed uninfected individuals along with long-term nonprogressors or so called ‘elite controllers’  are two distinct populations that might hold clues as to host-mediated factors that protect from HIV infection or disease progression, respectively. However, examining exposed uninfected individuals such as commercial sex workers does not offer the possibility of analyzing the extent of exposure measured by viral loads. Such measurements are feasible in discordant couples, as HIV-1 concentrations in blood have been correlated with those in semen , although surprisingly, few researchers [6,10] have used this opportunity. Therefore, this approach might provide insights into whether the exposed uninfected individuals status is predestined or acquired.
Studies of exposed uninfected individuals have previously been performed mostly in HIV-1-exposed heterosexual individuals . In this paper, we describe a cohort of discordant couples of men who have sex with men (MSM) followed during 2 years of consecutive samplings. We previously showed that the exposed uninfected individuals in this group were exposed mainly through oral unprotected sex and only rarely via the anal route. Further, their salivary immunoglobulin (Ig) A1 had a significant capacity to neutralize HIV-1 primary isolates, a capacity that persisted throughout the study period . We have now investigated whether this exposed uninfected individuals cohort, although engaged in ‘low-grade’ mucosal exposure, is also capable of a systemic immunological response to HIV-1.
Participants and methods
The study was approved by the ethical committee at Karolinska Institute. All participants gave their informed consent.
Exposed uninfected MSM were recruited via the HIV-positive partner attending the Gay Men's Health Clinic (Venhälsan) in Stockholm. The uninfected partners were followed up to five visits every 6 months. Inclusion criterion was HIV IgG-seronegative homosexual man who had at least 6 months relationship with a HIV-positive partner. Every individual was physically examined at the first visit by the responsible physician (P.S.). At all visits, the study participant received a questionnaire regarding demographical data, sexual behaviors, attitudes towards condoms and further psychosocial questions (summarized in ), and was tested for HIV (regular plasma screening), Chlamydia (throat, urine and rectum) and gonorrhea (throat, urethra and rectum). Plasma was purified from whole blood (100 ml) collected from each participant by venous puncture; samples were stored at −80°C.
The control participants were recruited by advertisement at a blood donor clinic. Samplings of blood were collected in the same way as from the exposed uninfected individuals, however, only on one occasion. As MSM according to Swedish legislation is a group considered at risk of HIV infection and not allowed to become blood donors, these low-risk healthy controls were heterosexual men. The HIV-positive partners were not personally involved in the study; however, clinical data from their files as well as plasma samples were available for the study in a fashion that did not reveal their identity to the study personnel.
Chemokine (C–C motif) receptor 5 delta 32 genotyping
CCR5Δ32 genotyping was essentially performed by pyrosequencing, as described elsewhere . Briefly, genomic DNA was purified from 200 μl of blood by use of a QiaAmp 96 DNA Blood Kit, in accordance with the manufacturer's instructions (Qiagen, Venlo, The Netherlands). DNA was eluted into 200 μl of Adams–Evans buffer (Qiagen) and stored frozen at −20°C until analyzed. Patient DNA (10 μl) was used to amplify a 132-bp-long fragment of CCR5, including the deletion with primers 5′-CACCTGCAGCTCTCATTTTCC-3′ (forward) and 5′-BIOTIN-GTTTTTAGGATTCCCGAGTAGCA-3′ (reverse). Amplified PCR products were then sequenced by pyrosequencing as described elsewhere  using the sequencing primer 5′-CAGCTCTCATTTTCCAT-3′ and the dispension order GACAGTC (instrument PSQ96 MA; Biotage AB, Uppsala, Sweden).
Virus isolation and HIV-1 neutralization assay
Viruses used in the HIV-1 neutralization assays were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (USA): BZ-167 (X4R5 virus, Brazil), 92/US/727 and QZ-4589 (R5 virus, USA). Viruses were chosen with the criteria of being primary isolates of subtype B (the HIV-positive partners were infected with this subtype) and having R5 tropism (the predominant virus tropism in mucosally transmitted infections). BZ-167 virus was used as a screening virus, and all neutralizing samples were further tested against one R5 tropic virus for confirmation. The virus was collected from phytohemagglutinin (PHA) and interleukin 2-stimulated peripheral blood mononuclear cells (PBMCs). Fifty percent tissue culture infectious dose (TCID50) values were determined for each isolate, and three virus dilutions (ranging from TCID50 5–20×) were always tested at each collection time to compensate for PBMCs donor variability. Plasma samples were diluted 1: 20; IgA1 fractions (through the purification process already diluted 1: 5) were diluted 1: 2 and IgA1-depleted fractions (through the purification process already diluted 1: 4) diluted 1: 2.5 in Rosewell Park Memorial Institute (RPMI) 1640 medium (developed by Moore et al.) when tested for neutralization capacity.
The neutralization assays were performed according to previous studies [14–16] by our group and others. In brief, duplicate wells of each virus dilution and each sample fraction were incubated for 1 h at 37°C, followed by the addition of 1 × 105 PHA-stimulated PBMCs pooled from two different donors. The cells were washed at days 1 and 4, respectively, and on day 6 of incubation at 37°C, supernatant from each well was collected and analyzed with an ELISA of p24 antigen (Vironostika HIV-1 Antigen Kit; Biomérieux, Boxtel, The Netherlands). Neutralization capacity was defined as a more than 67% reduction in the supernatant as compared with p24 antigen content in samples from low-risk healthy controls, a cut-off shown to be biological relevant in several previous studies [12,14,15,17].
Immunoglobulin A1 purification and quantification
IgA1 was purified from plasma as previously described . Briefly, plasma was diluted 1: 4 in phosphate buffer solution (PBS), pH 7.4, and subsequently purified with jacaline–agarose (Vector Labs, Burlingame, California, USA). A total of 800 μl was added to 200 μl jacalin/agarose beads and mixed for 2 h at 4°C followed by centrifugation. The supernatant (i.e., the IgA1-depleted fraction) was collected. The jacalin–agarose beads were washed five times with PBS, pH 7.4, after which the bound IgA1 was eluted overnight at 4°C by adding 1 ml 0.8 mol/l D-galactose, pH 7.4. The supernatant was subsequently collected; all fractions were stored at −80°C. An in-house ELISA was used for total IgA1 quantification .
Investigating HIV specificity of immunoglobulin A1 antibodies
Plasma samples from 25 exposed uninfected individuals and their 25 HIV-positive partners were sent blindly to the laboratory of Dr Lucia Lopalco, Milan, Italy. The 22 low-risk controls were labeled to serve as controls. The purification of IgA1 and subsequent binding of IgA1 to recombinant gp41 envelope (env) proteins is described elsewhere . Briefly, IgA1 was purified from plasma using sepharose (Pharmacia, Uppsala, Sweden) coupled with rabbit antihuman IgA (Aldrich, Milan, Italy). Plasma (96 μl) was incubated on columns containing 2400 μl sepharose–anti-IgA. After washing, the columns were eluted with glycine/NaCl. The purified IgA was concentrated on Ultrafree-15 Biomax30 membranes (Millipore, Bedford, Massachusetts, USA).
The samples were then tested in ELISA using microwell plates coated with gp41 recombinant proteins at 0.1 μg/well. The plates were saturated with PBS and bovine serum albumin. IgA was added and incubated for 1 h at 37°C, and binding was demonstrated with horseradish peroxidase (HRP)-conjugated rabbit antihuman IgA (Dako, Santa Barbara, California, USA). The reaction was read at 492 nm.
For western blot analyses, we used a commercial kit normally used for detecting HIV-specific IgA in urine (Calypte Biomedical HIV-1 W Btest, Berkeley, California, USA). This test detects the following HIV-1 proteins (p) or glycoproteins (gp) on a nitrocellulose strip: p17, p24, p31, gp41, p51, p55, p66, gp120 and gp160 (the number refers to the apparent molecular mass in kDa). The kit was used according to the manufacturer's instruction, with the modification of adding IgA1 plasma samples (purified by jacaline as described above) instead of urine, the conjugate being changed to goat antihuman IgA–HRP (Southern Biotech, Birmingham, Alabama, USA) and the substrate being changed to Enhanced chemiluminescence (ECL) Advanced WB Detection System (Amersham Biosciences Uppsala, Sweden).
Preliminary tests for normality showed a skewed distribution of values for all parameters measured. Thus, all analyses of results were performed with nonparametric tests. Intergroup variations were analyzed in parallel by comparing a single parameter from one group of individuals against the corresponding parameter of each other group using the unpaired two-tailed Mann–Whitney test. For qualitative analyses, Fisher's exact test was used. Calculations were performed by using the GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, California, USA) and, for the questionnaires answered by the exposed uninfected individuals and control participants, SPSS version 16.0 (SPSS Inc., Chicago, Illinois, USA).
Chemokine (C–C motif) receptor 5 deletion
All exposed uninfected individuals were tested for the CCR5 deletion, as homozygosity protects against sexual HIV-1 transmission. Of 25 exposed uninfected individuals, one individual (#4B) had this homozygosity, explaining his uninfected status despite exposure. However, this individual was not excluded from the experiments, and his plasma did not neutralize HIV-1. Of the remaining exposed uninfected individuals, five of 25 were heterozygous for the CCR5 deletion, but these individuals were not excluded either, as such heterozygosity does not alter the outcome in this setting . The prevalence of CCR5 deletions in this exposed uninfected individuals cohort, either homozygous or heterozygous, was consistent with a previous study  in the Swedish general population.
Prevalence of other sexually transmitted infections
One individual (#1B) was tested positive for gonorrhea (pharyngeal sample) once, 18 months after enrolment. Another individual (#10B) tested positive for Chlamydia (urine sample) once, 6 months after enrolment. Another individual (#19B) was diagnosed (clinically, confirmed by serology) with syphilis 12 months after enrolment.
Determination of exposure to HIV-1 in the exposed uninfected individuals, and prevalence of sexual acts outside the partnership with persons of unknown HIV status
At enrolment and follow-up visits, all exposed uninfected individuals answered questions regarding the frequency and protection usage during oral and anal sex throughout the 2 weeks prior to the clinic visit. A summary of sexual frequency and condom use during the preceding 6 months were also collected and transformed into ordinal data (summarized in ). Unprotected receptive oral intercourse (ROI) had been practised with their HIV-positive partner by all but four individuals. Of these four exposed uninfected individuals, one did not respond to the question and the other three engaged in unprotected ROI with casual partners of unknown HIV status outside the longer term relationship. In total, nine of 25 exposed uninfected individuals engaged in sexual acts outside the relationship. Unprotected receptive anal intercourse (RAI) was practiced by three of 25 exposed uninfected individuals, and unprotected active anal intercourse (AAI) by five of 25 exposed uninfected individuals. In sexual relations outside the partnership, only one individual performed unprotected AAI with a person of unknown HIV status, and none performed unprotected RAI. Of these, owing to overlapping responders, a total of six partners with ‘anal risk,’ only two graded the frequency of unprotected RAI as ‘sometimes/often’ (the other four reporting more sporadic unprotected occasions), and none of these was among the individuals who had HIV-1 neutralizing activity in plasma. Thus, the oral mucosa was the major route of potential HIV-1 encounter in these individuals.
As a reference of mode of HIV transmission at Gay Men's Health Clinic (Venhälsan), a pilot study was performed during 1990–1992 at this clinic among newly HIV-infected (<1 year) individuals. In total, 28 newly infected individuals were questioned about their sexual behavior by a counselor and a physician, independently (when present, also the respective partner confirmed the patient's description). Six of 28 individuals (21%) reported exclusively oral sex with their partner/partners prior to infection (data were presented as an abstract by Grutzmeier et al. at the IX International AIDS Conference in Berlin, 1993), thus our finding of an MSM milieu were exclusive that oral sex is frequent is a confirmation of these earlier results.
HIV-1 neutralizing capacity
The functional activity of each baseline sample was tested in a neutralization assay against three primary HIV-1 isolates: one X4R5 virus and two R5 viruses. When whole plasma samples from exposed uninfected individuals at enrolment were compared with those from controls, the ability to neutralize differed significantly: seven of 25 versus none of 22, respectively, (P = 0.01 by Fisher's exact test) neutralized HIV-1 infection (Table 1). The seven exposed uninfected individuals neutralized the X4R5 virus with a median of 96% neutralization (range 81–100%), whereas the nonneutralizing exposed uninfected individuals had a median of 0% neutralization (range 0–39%), thus clustering well above or beneath the 67% cutoff determining neutralization capacity. Samples from the HIV-positive group, as expected due to presence of HIV-specific IgG antibodies, had a HIV-1 neutralization capacity in 21 of 25 plasma samples (data not shown). The seven exposed uninfected individuals with effective neutralization at enrolment were further tested throughout the follow-up period (Table 1). The 18 exposed uninfected individuals who were not able to neutralize at enrolment were also tested with at least one sequential sample each but did not develop a neutralizing capacity (data not shown). None of the neutralizing seven exposed uninfected individuals practiced unprotected anal sex at enrolment, although two reported sporadic occasions of unprotected anal sex during follow-up. The majority of these individuals retained their neutralizing capacity over time, with a few intermittent exceptions; six of seven exposed uninfected individuals were still able to neutralize at the last time point of sampling (Table 1). However, when analyzing the breadth of the neutralizing response, four of six exposed uninfected individuals now neutralized fewer viral strains as compared to the first time point (Table 1).
Mechanisms of neutralization in plasma samples
To establish the mediator of neutralization in these exposed uninfected individuals, we investigated mechanisms with known anti-HIV capacity. The contribution of HIV-specific IgG was unlikely, as all individuals tested negative in routine diagnostic ELISAs throughout the study period (data not shown). To test whether other immunoglobulin subclasses were involved in viral neutralization, IgA1 was isolated and tested in parallel with the corresponding IgA1-depleted fraction. No significant difference was detected when comparing the concentration of total plasma IgA1 in the three study groups: (mean) 2257, 2288 and 2919 μg/ml (HIV-1-positive, exposed uninfected individuals and low-risk controls, respectively) (data not shown). Also there was no significant difference between the total plasma IgA1 levels in neutralizing versus nonneutralizing exposed uninfected individuals (2712 and 2122 μg/ml, respectively) (data not shown). However when analyzing samples corresponding to those individuals in whom whole plasma could neutralize HIV-1, six of seven of the corresponding IgA1 fractions also neutralized HIV-1 (primary isolate 92/US/727) with the median of 80.5% neutralization (range 73–81%). In contrast, none of the corresponding IgA1-depleted fractions neutralized HIV-1 (data not shown). Thus, our data strongly suggest that IgA1 is the main mediator of neutralizing activity in the plasma of MSM discordant couples.
Neutralizing capacity by plasma immunoglobulin A1 is not due to HIV-1 envelope specificity
To determine whether the purified IgA1 plasma antibodies are able to neutralize HIV-1 due to env specificity, we evaluated the plasma samples in an ELISA testing IgA1 against recombinant HIV-1 env gp41 epitope. Exposed uninfected individuals and HIV-positive samples were run blindly and compared with low-risk controls. Twenty-two of 25 HIV-positive samples showed high titers (>1: 160) of reactivity against env-gp41, whereas one of 25 exposed uninfected individuals had a low titer of reactivity (1: 20) and 24 of 25 exposed uninfected individuals lacked detectable reactivity (data not shown). Further testing of some samples in a western blot assay confirmed these results; of 11 exposed uninfected individuals tested, none showed reactivity against any HIV-1 env epitopes (data not shown).
Clinical status of the HIV-positive partners
By the time of inclusion in the study, at the same time as the exposed uninfected individuals were sampled for the present study, 22 of 25 HIV-positive partners were on antiretroviral treatment (ART), and the remaining three remained treatment naive, with low virus levels and high CD4 cell counts. However 22 of 25 exposed uninfected individuals were exposed to viremia due to initial unawareness of HIV-positive status during the relationship, no ART or ART interruption due to side effects or low compliance, treatment failure, that is, viral load above 50 HIV-RNA copies/ml at two consecutive samplings in spite of continuous ART (Table 2). Only three of the partners had been on ART and maintained undetectable virus levels throughout their current relationships.
Anti-HIV-1 capacity associating with exposure
To address the issue whether the difference in neutralizing ability within exposed uninfected individuals is dependent on the grade of exposure, we investigated the partners of the exposed uninfected individuals who were able to neutralize HIV-1 in plasma (seven individuals) looking at the highest measured virus load during the time of the relationship. The highest measured level of HIV-RNA in partners of the exposed uninfected individuals who demonstrated neutralizing capacity in plasma had a median level of 373 000 copies/ml. Compared with the exposed uninfected individuals who lacked neutralizing activity in plasma, the median level of their partners highest measured level of HIV-RNA was significantly different 18 950 copies/ml, (P = 0.01, Mann–Whitney test) (Fig. 1). We further compared the time (in months) from the above-mentioned viral peak with the study start and subsequent first plasma sampling (Fig. 2). The seven partners of neutralizing exposed uninfected individuals had had their viral peaks closer (median 14 months) to the plasma sampling than the 18 partners of nonneutralizing exposed uninfected individuals (median 57.5 months), thus a significant difference was observed (P = 0.02, Mann–Whitney test).
We document here, for the first time, a functional anti-HIV response in plasma from exposed uninfected individuals that associates with HIV-1 exposure measured as highest observed level of HIV-RNA in the respective HIV-1-infected partner, as well as time since highest viral load. These data were observed in a MSM cohort engaged primarily in oral unprotected sexual practices, which is considered a lower risk route of exposure to HIV-1 than genital or anal exposure [21,22]. Because our ongoing investigation has been directed toward the oral route of viral exposure, we previously measured salivary IgA1 in these individuals and found a marked HIV-1 neutralizing response . In this present endeavor, most of the exposed uninfected individuals (four of seven) whose plasma samples neutralized HIV-1 also had a neutralizing response in salivary IgA1 but not the reverse, as 13 of 25 exposed uninfected individuals had salivary IgA1 that neutralized HIV-1. Possibly, the explanation for this discrepancy is that oral HIV-1 exposure more readily results in limited T-cell-independent, local mucosal anti-HIV-1 IgA1 production than a systemic involvement. Immunity against HIV-1 could, theoretically, exist due to genetic factors or from favorable HIV-1 exposure that generates an immunological response in the absence of productive infection . The latter hypothesis seems applicable in the setting described here, and our results would be in agreement with an earlier extensive study by Suy et al. . Those authors detected an altered T-cell subset in heterosexual exposed uninfected individuals correlating with viral load of the partner as well as HIV-specific response (in a few individuals) by both cellular and humoral immunity.
The mechanism of neutralization in plasma from the exposed uninfected individuals (who throughout the study period were tested negative for HIV-1 specific IgG) was here attributed to IgA1 in all but one sample; however, the IgA1 was not found to be directed towards HIV env epitopes as detected by ELISA or western blot. Limitations in our investigation are the inability to detect antibodies directed against epitopes exposed after env fusion. Also, the IgA1 purification is performed using different methods prior to the neutralization assays and the env gp41 ELISA, respectively. Further dilution of the IgA1 samples (1: 80) did not result in neutralizing responses; however, the present neutralizing titer of 1: 10 (including purification process) is in the same range as those neutralizing antibody titers shown in recent experiments with antibody-treated macaques with repeated low-dose simian HIV exposure . Viral lysis mediated by the complement system was excluded as a mechanism for the functional activity, as we heat inactivated all plasma samples before testing them for neutralization capacity. Neutralization can also occur by non-HIV-specific IgG , but this mechanism was less likely as IgG remained in the IgA1-depleted fractions, which were not able to mediate neutralization. The only exposed uninfected individual (#8B) whose whole plasma, but not isolated IgA1 or IgA1-depleted plasma, was able to neutralize HIV-1 may represent an episode of such neutralization through synergy between IgA1 and innate factors such as beta-chemokines.
The association between the peak viral load, and the time elapsed since it occurred, and HIV-1 neutralization suggests that this earlier viral exposure for the exposed uninfected individuals was important to elicit a neutralizing response. The exposure was, however, declining as measured by partner's viral loads already when the study began due to effective ART and counseling that focused on sexual behavior as a risk factor of HIV transmission. This information, which is routinely given to HIV-infected patients, was now reinforced by also informing the corresponding uninfected partner as part of this study's design. Still, in almost all the exposed uninfected individuals described here, a HIV-1 neutralizing capacity persisted over time, in spite of a continuous low viral exposure during the study period. One can speculate that the highest exposure occurred at times of peak viral load, at treatment failure as well as before knowledge of the partner's seropositive status (which may have included acute primary infection). Thereafter, a lower grade of exposure may have maintained the acquired antiviral immune responses. However, the expression ‘low-grade exposure’ should be used carefully, as HIV transmission has been documented from a HIV-positive partner with an undetectable viral load to the discordant partner, although this is probably rare in the absence of other sexually transmitted diseases . Still, a previous study  showed diminishing IgA anti-HIV-1 activity in exposed uninfected female sex workers due to lesser degree of exposure. Additionally, anti-HIV-1 responses may wane over time due to ART-suppressed viral load in seropositive partners, as shown in the T-cell compartment of another MSM group . Accordingly, our study also showed an association of time since high viral load and neutralization capacity. Although the total number of study participants was low in the present study, it is further noteworthy that the breadth of the neutralizing response (defined as reactivity against different HIV-1 isolates and illustrated in Table 1) decreased in five of seven exposed uninfected individuals over time. Thus, future studies may focus on not only the presence of functional humoral activity but also the quality (breadth and magnitude) of the response.
The quite remarkable finding of anti-HIV-1 capacity in plasma in foremost orally exposed uninfected individuals can be recognized as a product of acquired immunity, whether it is a correlate of protection against HIV or not is yet another question. Notably, two of the exposed uninfected individuals in this study's cohort seroconverted after the study ended. One of these had initial anti-HIV-1 capacity in his plasma, mediated by IgA1 (subject #19B), but he was the only one of the seven exposed uninfected individuals who lost that capacity completely during follow-up. Furthermore, he subsequently became infected not by his regular HIV-infected partner but with a viral strain from a different clade (CRF-01AE). In such a situation, one might question whether the initial anti-HIV-1 capacity was limited to the regular partner's virus clade, whether that response declined also in vivo and whether it was protective at all. Our research group  previously described a broader cross-clade neutralization capacity in highly HIV-1-exposed sex workers but a lower degree of cross-clade neutralization in less HIV-1-exposed individuals. The even lower degree of exposure in exposed uninfected individuals described here may stem from the relatively low risk attributed to oral sexual practice. Further studies, for example, using the respective partner's viral isolate in the neutralizing assay, are needed to address these alternatives.
As research increases for the purpose of identifying candidate substances for anti HIV vaccines, acquired as well as innate humoral and cellular immunity must be considered as potentially necessary key players [28,29]. Here, we have shown an association of HIV-1 exposure, measured as viral load in sex partners, to the humoral immunity, a response that was maintained during continuous mucosal (oral) exposure. As the mucosal route of viral exposure significantly strengthens the development of both mucosal and systemic anti HIV-1 responses , the overall implications for vaccine development are considerable.
This study was funded by the Swedish Research Council, Swedish Physicians against AIDS Research Foundation and SIDA/Sarek.
We thank all study participants. Lena Persson, research nurse at Venhälsan, for collecting samples. Douglas Nixon, MD, PhD, for valuable discussions. Elin Kindberg, MSc, PhD student, for the CCR5 genotyping assays. Magnus Backheden for statistical advices.
K.H. provided the hypothesis, data generation, analysis and interpretation, as well as drafting and editing the manuscript. G.B. provided data on all clinical records, as well as editing the manuscript. T.H. provided analysis and interpretation, as well as editing the manuscript. P.S. established the cohort, performed all clinical examinations of the study participants and collected samples. M.E. provided data generation, analysis and interpretation. L.L. provided data generation, analysis and interpretation. E.S. established the cohort and provided editing of the manuscript. K.B. provided analysis and interpretation, as well as editing the manuscript. All authors reviewed and approved the final version of the paper.
There are no conflicts of interest.
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Keywords:© 2010 Lippincott Williams & Wilkins, Inc.
exposed uninfected individuals; HIV-1; immunoglobulin A; men who have sex with men; neutralization