Orally exposed uninfected individuals have systemic anti-HIV responses associating with partners' viral load.

OBJECTIVES: To determine whether oral HIV-1 exposure incites a persistent systemic anti-HIV-1 response in exposed uninfected individuals of discordant couples of men who have sex with men, and whether this response associates with HIV-1 exposure measured by viral load in the HIV-positive partners. METHODS: Plasma were collected from exposed uninfected individuals (n = 25), HIV-positive partners (n = 25) and low-risk controls (n = 22). A peripheral blood mononuclear cells-based neutralization assay was used to test these samples against three primary HIV-1 isolates. Self-reported questionnaires described routes of HIV-1-exposure, and clinical records documented viral loads in HIV-positive partners. RESULTS: At enrollment, plasma samples from seven of 25 exposed uninfected individuals neutralized at least two of the three HIV-1 isolates. No samples from the 22 controls neutralized any HIV-1 isolate (P = 0.01). Of these seven exposed uninfected individuals, six retained neutralization capacity during follow-up. Neutralization capacity among exposed uninfected individuals associated with the highest measured viral load of their respective partners (P = 0.01) and also time since highest viral load (P = 0.02). Purified plasma immunoglobulin (Ig) A1-mediated neutralization was observed in six of the seven samples, whereas none of the IgA1-depleted plasma samples neutralized HIV-1. The neutralizing IgA1 was not HIV envelope specific as detected by ELISA and western blot. CONCLUSION: Orally exposed uninfected men who have sex with men can mount neutralizing anti HIV-1 activity in plasma, mediated primarily by non-HIV envelope-specific IgA1. Neutralization was associated with previous measured highest viral load in the HIV-positive partner, as well as time elapsed since the peak viral load. Neutralization also persisted over time in spite of a continuous low viral exposure.

Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8.

7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris-HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3(2)21, with unit-cell parameters a = b = 100.1, c = 196.8 A, and diffracted to 2.7 A resolution.

Oral HIV-exposure elicits mucosal HIV-neutralizing antibodies in uninfected men who have sex with men.

OBJECTIVE: To determine whether oral sexual exposure to HIV-1 (HIV) results in HIV-neutralizing activity in saliva of uninfected men who have sex with infected men? DESIGN: Saliva samples were collected from HIV IgG seronegative men (n = 25) whose male partners were HIV infected and from low-risk healthy controls (n = 22) and analyzed for HIV-neutralizing capacity. METHODS: The presence of neutralizing activity in saliva was tested in a peripheral blood mononuclear cell-based assay using primary HIV isolates. Self-reporting questionnaires described the individuals' sexual behaviors and routes of possible HIV exposure. RESULTS: Of 25 exposed, uninfected individuals (EUI), 21 reported receptive unprotected oral intercourse, whereas three of the 25 reported unprotected anal receptive intercourse. Whole saliva from both EUI and low-risk healthy controls contained HIV-neutralizing activity. However, a significant difference was seen when analyzing the salivary IgA1 fraction: 13 of 25 EUI neutralized HIV, whereas none of the 22 controls had this capacity. The neutralizing capacity of the EUI males persisted during 2 years of follow-up. CONCLUSION: Unprotected oral sex evokes a salivary IgA1-mediated HIV-neutralizing response that persists over time during continuous exposure in uninfected male partners of infected men.

Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals.

The mechanisms behind the resistance to human immunodeficiency virus type 2 (HIV-2) infection are still not fully understood. In the present study, we explored the HIV-2-specific humoral serum immunoglobulin A (IgA) immune response in HIV-2-exposed IgG-seronegative (EGSN) individuals. Serum samples from heterosexual EGSN individuals and their known HIV-2-infected partners, as well as controls originating from Guinea-Bissau in Africa, were studied. Antibody reactivity to native and recombinant envelope glycoproteins was investigated, and the capacity of purified serum IgA to neutralize HIV-2(SBL6669) was tested. Our results showed that 16 of 25 EGSN samples exhibited reactivity against whole HIV-2 antigen, 6 of 25 samples reacted with recombinant gp36 (rgp36), and 3 of 25 samples were positive against HIV-2 rgp105; no reactivity to native HIV-2 gp125 was detected. Purified serum IgA antibodies from both EGSN and HIV-2-positive individuals, but not that from the negative controls, exhibited neutralization of HIV-2(SBL6669). The most potent neutralization activity was exhibited by IgA purified from EGSN compared to infected individuals' IgA. The antigenic pattern of the HIV-2-positive partners showed that all serum IgA samples were reactive to whole HIV-2 antigen, and 14 of 15 reacted with rgp36. For rgp105 and gp125, 5 of 15 and 4 of 15 samples exhibited binding, respectively. The serum of the EGSN group had a higher mean IgA concentration than that of the negative controls (P < 0.05). Thus, we describe HIV-2-specific serum IgA antigen reactivity and show a more potent serum IgA-mediated HIV-2-neutralizing activity in EGSN individuals than in HIV-2-infected patients.

Serum immunoglobulin A (IgA)-mediated immunity in human immunodeficiency virus type 2 (HIV-2) infection.

In the present study, we sought to define the importance of serum IgA (sIgA)-mediated immunity in HIV-2 infection. Serum samples from a total of 29 HIV-2-infected patients from Guinea-Bissau (n = 20) and Portugal (n = 9) were studied. Samples from seronegative individuals were used as controls. Antibody reactivity to native and recombinant envelope glycoproteins as well as peptides representing various regions of the envelope glycoproteins was investigated. Furthermore, the capacity of purified IgA to neutralize the HIV-2(SBL6669) strain was tested. All serum samples showed IgA reactivity against whole HIV-2 antigen. Twenty-eight out of 29 IgA samples (96%) reacted with native HIV-2 gp125, 26/29 (90%) with recombinant gp105, and 29/29 (100%) with recombinant gp36. When using peptides, the most prominent IgA reactivity was seen against the peptide representing aa 644-658 of the transmembranous protein gp36, to which 72% of the sera reacted. Purified sIgA antibodies showed neutralizing effects against HIV-2(SBL6669) in 17/29 cases (59%). This work describes the HIV-2-specific sIgA antigenic response. Moreover, our findings show, for the first time, that sIgA may play a role in the in vitro neutralization of HIV-2.

Human neutralizing human immunodeficiency virus type 2-specific Fab molecules generated by phage display.

A panel of human immunodeficiency virus type 2 (HIV-2)-neutralizing, recombinant Fab fragments was generated by using the phage display technique. The combinatorial library was derived from an asymptomatic, HIV-2-seropositive individual and constructed on the surface of filamentous phage by using the pComb3 phagemid vector and then screened against native HIV-2 envelope glycoprotein (gp125). Ten of 30 Fab fragments generated displayed strong reactivity in an ELISA and were therefore selected for further study. Six of these possessed neutralizing capacity, with titres varying from 20 to 80 against the homologous HIV-2 strain, and one also had a weak neutralizing capacity against a heterologous HIV-2 isolate, K135. Sequencing of the heavy chain CDR3 regions showed that the gp125-specific Fabs represented individual clones. These reagents may be useful for studies on the conformational structures of the HIV-2 envelope antigens and their immunogenicity, which may help in vaccine design. Furthermore, the cloned Fab genes may be transformed into whole IgG for eukaryotic expression, and as such used for therapeutic and immunoprophylactic studies in HIV-2-infected macaques and, possibly, for human immunoprophylaxis against HIV-2.