Claudia Devito

Mucosal and Plasma IgA from HIV-1-Exposed Uninfected Individuals Inhibit HIV-1 Transcytosis Across Human Epithelial Cells

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate.

ObjectiveTo characterize functional properties of HIV-specific IgA in samples representing both systemic and mucosal compartments of HIV-1 highly exposed persistently seronegative (HEPS) individuals. MethodsIgA was purified from plasma and mucosal samples from HEPS individuals and tested for the ability to neutralize infection of peripheral blood mononuclear cells (PBMC) by a non-syncytium inducing HIV-1 (clade B) primary isolate. None of these individuals had measurable HIV-1-specific IgG. ResultsHIV-1-specific neutralizing activity of the purified IgA from plasma (n = 15), saliva (n = 15) and cervicovaginal fluid (CVF) (n = 14) were found in the majority of samples (73, 73 and 79%, respectively). In contrast, plasma, saliva and CVF samples of low-risk, uninfected HIV-seronegative individuals lacked neutralizing IgA, with the exception of two out of 34 (6%) saliva samples. ConclusionMucosal and plasma IgA from HEPS individuals can neutralize HIV-1 infection.

Cross-clade HIV-1-specific neutralizing IgA in mucosal and systemic compartments of HIV-1-exposed, persistently seronegative subjects

There is an urgent need for a universally effective HIV-1 vaccine, but whether a vaccine will be able to protect against HIV-1 of different clades is a significant concern. IgA from HIV-1-exposed, persistently seronegative (HEPS) subjects has been shown to neutralize HIV-1 and to block epithelial HIV-1 transcytosis, and it may target novel HIV-1 epitopes. We have tested the ability of plasma and mucosal IgA purified from HEPS subjects to neutralize HIV-1 primary isolates of different viral clades and phenotypes. IgA from two groups of HEPS subjects was tested: sex workers from Nairobi, Kenya, where clades A and D predominate, and the heterosexual partners of individuals infected by clade B virus. HIV-1-infected and low-risk uninfected individuals were included as controls. IgA purified from the blood, genital tract, and saliva of most HEPS sex workers demonstrated significant cross-clade HIV-1 neutralization, whereas a more clade-restricted pattern of neutralization was found in partners of clade B-infected individuals. IgA purified from HIV-1-infected individuals also mediated cross-clade neutralization, whereas IgA from uninfected controls lacked neutralizing activity. In conclusion, mucosal and plasma IgA from HEPS subjects neutralizes HIV-1 of different clades. This ability to induce HIV-1-specific systemic and mucosal IgA may be an important feature of an effective prophylactic HIV-1 vaccine.

Functional HIV-1 specific IgA antibodies in HIV-1 exposed, persistently IgG seronegative female sex workers

Although HIV-specific cellular immune responses are found in a number of HIV highly-exposed, persistently seronegative (HEPS) cohorts, late seroconversion can occur despite pre-existing cytotoxic T lymphocytes (CTL), suggesting that a protective HIV vaccine may need to induce a broader range of HIV-specific immune responses. Low levels of HIV-specific IgA have been found in the genital tract and plasma of the majority of Nairobi HEPS sex workers and appeared to be independent of HIV-specific cellular responses. IgA purified from genital tract, saliva and plasma of most HEPS sex workers were able to neutralize infection of PBMC by a primary (NSI) clade B HIV isolate, as well as viral isolates from clades A and D, which predominate in Kenya. In addition, these IgA were able to inhibit transcytosis of infective HIV virions across a transwell model of the human mucosal epithelium in an HIV-specific manner. Preliminary work in other HEPS cohorts has suggested the recognition of different gp41 epitopes in HEPS and HIV-infected subjects. Although present at low levels, these IgA demonstrated cross-clade neutralizing activity and were able to inhibit HIV mucosal transcytosis, suggesting an important functional role in protection against HIV infection.

Serum IgA of HIV-exposed uninfected individuals inhibit HIV through recognition of a region within the α-helix of gp41

BackgroundHIV-specific IgA is present in HIV-exposed uninfected individuals (EU) and neutralizes primary strains of HIV-1 in vitro. ObjectivesTo analyse the antigenic correlates of HIV-1 neutralization using HIV epitopes and IgA from EU and HIV-seropositive individuals. MethodsSera from six heterosexual couples discordant for HIV serostatus, six age-matched HIV-infected subjects and six healthy controls (HC; as negative controls) were analysed. IgA binding on HIV Env recombinant proteins was assayed. Serum IgA was affinity purified on specific Env peptides and tested in HIV neutralization using resting and activated peripheral blood mononuclear cells as target. Monoclonal antibody 2F5 was used as neutralizing positive control. BALB/c mice were immunized with specific gp41 peptide and anti-sera were tested in syncytia formation and in HIV viral replication. ResultsIgA of EU exclusively bound an epitope within gp41; this epitope was restricted to residues 582–588 (QARILAV) and corresponded to the leucine zip motif in the α-helical region. IgA of HIV-positive patients recognized epitopes expressed both in gp120 and gp41; these epitopes were in the N-terminal portion of the extramembrane region. Additionally, IgA of EU and antisera of QARILAV-immunized Balb/C mice blocked syncytia formation and viral replication. The dose-dependent neutralization behaviour of specific QARILAV-purified IgA was very similar to that obtained with monoclonal antibody 2F5. ConclusionThese results have important implications for the development of vaccines and therapeutical strategies against HIV infection.

Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity.

An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1–9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse’s life span.

Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents.

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.

DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant

Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.

Human IgM monoclonal antibodies block HIV-transmission to immune cells in cervico-vaginal tissues and across polarized epithelial cells in vitro

The importance of natural IgM antibodies in protection against infections is still emerging and these antibodies have a potential role in the maintenance of homeostasis through clearance of apoptotic bodies, complement-dependent mechanisms, inflammation and exclusion of misfolded proteins. Natural IgM act as a first line of defence against unknown hazardous factors and are present in most vertebrates. We investigated the functional capacity of anti-HIV-1 IgM monoclonal antibodies, from a combinatorial Fab library derived from healthy individuals, and evaluated their protective role in inhibiting HIV-1 in vitro when passing across the human mucosal epithelial barrier. Primary HIV-1 isolates were efficiently transmitted over the tight polarized epithelial cells when added to their apical surface. Efficient inhibition of HIV-1 transmission was achieved when anti-HIV-1 IgM monoclonal antibodies were added to the basolateral side of the cells. Two of these human IgM MoAbs had the ability to neutralize HIV and reduced infection of dendritic cells in primary cervico-vaginal tissue biopsies in vitro. This indicates a potential role of natural IgM antibodies in the reduction of HIV-1 transmission in mucosal tissues and improve our understanding of how natural IgM antibodies against a neutralizing epitope could interfere with viral transmission.