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In vitro production of novel HIV-1 specific IgA antibody variable heavy and light chains from HIV-1 resistant sex workers from Nairobi, Kenya
Presented by Caitlin Sarna, Canada.
Sarna C.1, Ball T.B.1, Gubbins M.J.2, Berry J.D.2, Plummer F.A.2
1University of Manitoba, Department of Medical Microbiology, Winnipeg, Canada, 2National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada
Objectives: HIV-1-specific mucosal IgA antibodies are important in the development and maintenance of protective immunity against HIV-1. In the Pumwani female sex worker cohort of Nairobi, Kenya, a subset of women remain HIV-1 seronegative despite repeated and prolonged exposure to HIV-1. It has been reported that 76% of these HIV-resistant women produce HIV-1-specific IgA at the genital mucosa; these antibodies may help explain the resistance phenomenon in this cohort. We hypothesize that HIV-1-specific IgA cloned from the genital tract of HIV-1 resistant women of the Pumwani cohort will efficiently recognize the HIV-1 envelope protein gp120, and in its natural form inhibit HIV-1 replication.
Methods: RNA was isolated from cervical B-cells collected from twelve HIV-1 resistant sex workers and the variable regions of one light and two heavy chain antibody genes were isolated. These novel genes were cloned into an IgA2 expression vector with variable genes from the potent HIV-1-neutralizing monoclonal antibody IgG1b12. Chinese hamster ovary cells were transfected with the constructs and cultured until monoclonal IgA production was detectable by ELISA. The culture supernatants were immunoaffinity purified and gp120 specificity was determined by means of ELISA analysis.
Results: Six antibody constructs, comprising of one b12-like control, three African-b12 hybrids, and two entirely African variants, were successfully produced in vitro. Several samples have been confirmed to bind gp120 with high affinity.
Conclusions: We have successfully cloned antibody genes from the cervical B-cells of HIV-1-resistant women and produced human monoclonal HIV-1-specific IgA in vitro. This cloning strategy enables the production of large quantities of IgA needed to conduct an in-depth analysis of the protective role of HIV-specific IgA at the genital tract, which is the first step towards designing safe and effective treatments to block HIV-1 transmission. Future directions of this study include viral neutralization and transcytosis assays, and MALDI-TOF mass spectroscopy epitope mapping.
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