The mucosal antibody repertoire of the cervical mucosa in commercial sex workers from Nairobi, Kenya, who are highly sexually exposed to human immune deficiency virus type-1 (HIV-1) but remain persistently IgG seronegative (HEPS), may represent a novel source of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1. Mucosal IgA specific for HIV-1 envelope (Env) subunit gp41 has been suggested as a correlate of protection in HEPS individuals. The in depth studies at both the gene and function level required to confirm their role in HIV-1 resistance are possible only using recombinant monoclonal IgAs. Human mAbs have traditionally been selected from libraries displayed on the surface of microorganisms (phage, yeast). However, due to inherent limitations, such techniques may not be optimal for isolating such rare mAbs from a pool of cervical B cells. We have developed an antibody selection system based on surface display on mammalian cells and used this technology to isolate four novel monoclonal antibodies, against linear epitopes on gp41, from the IgA repertoire of the cervical mucosa in Kenyan HEPS. Furthermore, three of the four mAbs were shown to bind with surface expressed consensus clade B and clade C Env on mammalian cells. Characterization of the variable region cDNA of the two strongest binding mAbs reveals extensive somatic mutations with a bias of replacement mutations clustering in the complementary determining regions (CDR) indicating antigen-driven affinity maturation had occurred. Affinity matured monoclonal IgAs, such as these, may play a role in the identification of new, vulnerable epitopes on HIV-1, or act as a component in a topical microbicide.

The mucosal antibody repertoire of the cervical mucosa in commercial sex workers from Nairobi, Kenya, who are highly sexually exposed to human immune deficiency virus type-1 (HIV-1) but remain persistently IgG seronegative (HEPS), may represent a novel source of broadly neutralizing monoclonal antibodies (mAbs) against HIV-1. Mucosal IgA specific for HIV-1 envelope (Env) subunit gp41 has been suggested as a correlate of protection in HEPS individuals. The in depth studies at both the gene and function level required to confirm their role in HIV-1 resistance are possible only using recombinant monoclonal IgAs. Human mAbs have traditionally been selected from libraries displayed on the surface of microorganisms (phage, yeast). However, due to inherent limitations, such techniques may not be optimal for isolating such rare mAbs from a pool of cervical B cells. We have developed an antibody selection system based on surface display on mammalian cells and used this technology to isolate four novel monoclonal antibodies, against linear epitopes on gp41, from the IgA repertoire of the cervical mucosa in Kenyan HEPS. Furthermore, three of the four mAbs were shown to bind with surface expressed consensus clade B and clade C Env on mammalian cells. Characterization of the variable region cDNA of the two strongest binding mAbs reveals extensive somatic mutations with a bias of replacement mutations clustering in the complementary determining regions (CDR) indicating antigen-driven affinity maturation had occurred. Affinity matured monoclonal IgAs, such as these, may play a role in the identification of new, vulnerable epitopes on HIV-1, or act as a component in a topical microbicide.

The female genital tract is a mucosal epithelium that has an array of factors contributing to the cervicovaginal immune environment. Like with systemic immunity, innate and adaptive immune mediators are integrated in the efficiency for fighting infections in the female genital tract. Our study addresses the role of pregnancy and HIV infection on concentrations of cytokines in genital tract fluid that play a role in HIV sexual transmission. We focused on two pathways: The NF-KB inflammation pathway that has been implicated in susceptibility to HIV infection, and two interferon pathways that have been associated with antiviral immune defense. We hypothesized that pregnant women have increased proinflammatory cytokines in genital secretions, potentially putting them at increased risk for HIV infection, and that HIV-infected women could have upregulated Type 1 interferon pathways that may regulate HIV replication in the genital tract, and infection by other viruses. This study compared the immune mediator profiles in genital secretions of women between the ages of 18 and 40 years old belonging to four groups: HIV Negative/ Non Pregnant, HIV Negative/Pregnant, HIV Positive/ Non Pregnant, and HIV positive/ pregnant. Cytokine concentrations in cervicovaginal lavage fluid were measured using ELISA and MAGPIX assays. A number of statistical methods were used to characterize cytokine pathways and to link pathway associated cytokines to HIV serostatus and/or pregnancy. The study showed that HIV positive women had higher levels of proinflammatory cytokines including IL-1α, IL-2RA, IL-4, IL-5, IL-6, IL-7, IL-12, IL-17, MIF, MIG, MIP-1ß, SCGF, TNF-α, and TRAIL. Only the antimicrobial peptide lysozyme was significantly decreased in HIV positive women. However, lysozyme was significantly increased in pregnant women where as the following cytokines were significantly decreased in pregnant women: ß-NGF, G-CSF, GM-CSF, GRO-α, HGF, IGN-α2, IFN-γ, IL-2RA, IL-3, IL-4, IL-6, IL-7, IL-12, IL-13, IL-16, IL-17, TNF-α, TRAIL. The correlation analysis showed that HIV positive nonpregnant women could have upregulated: NFκB, type I interferon and interferon-γ pathways. The correlation analysis showed that the NFκB pathway could be upregulated in pregnant HIV negative women and these findings suggest that vaginal inflammation may play a role in HIV transmission from HIV-infected women to uninfected pregnant women. Moreover, upregulated interferon pathways could help understand how the body regulates genital viral infections in HIV-infected women.