Generation of Anti-HIV-1 envelope monoclonal antibodies using B-cells from HIV-1 sub-type C infected individuals with high levels of neutralizing antibodies
Date
2006-11-01T13:25:40Z
Authors
Nhlapo, Jabulani
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Abstract
The generation of human monoclonal antibodies (mAbs) that are able to block HIV-1 infection in
vitro would be useful reagents for studying virus neutralization, and assist in identifying
neutralizing antibody (NAb) epitopes of HIV-1 envelope glycoprotein. This may provide
important information for designing HIV-1 vaccine that aim to induce NAbs. HIV-1 subtype C
individuals with high levels of NAb titres were identified, and peripheral blood mononuclear
cells (PBMC) from these individuals were isolated and B-cells transformed with Epstein-Barr
virus (EBV). Clones specific to HIV-1 gp120 using cell lysate preparations derived from HIV-1
subtype C infected cell lines were generated by performing limiting dilutions. Transformation
efficiencies were estimated at over 80% by evaluating EBV-transformation cultures by
microscopic visualization. Of these approximately 5% were HIV-1-specific. Five clones derived
from the Du23 (1) sample secreting anti-HIV-1 antibodies were generated: 2.3C, 2.9D, 3.2C,
4.12E, and 1.5D. The 1.5D mAb could not be confirmed as anti-HIV-1 clone and it was probably
lost during the process of subculturing. The remaining four Du23 mAbs were determined to be
of IgG1 isotype lambda (λ) light chain. These mAbs bind to gp120, and 2.9D is probably a
polyreactive clone. Clones 2.3C, 3.2C and 4.12E appear to be A32-like, but do not share the
same epitope. We have determined that the binding sites for all four Du23 mAbs require at least
the C1 region, and they also showed binding sites overlapping with F91 and 1.5E. All four Du23
mAbs required intact gp120 proteins for their binding, and soluble CD4 enhance their binding.
Thus, their binding site is discontinuous and conformational. These mAbs are non-neutralizing
as they showed limited activity of 30-59% when tested using T-cell line grown viruses or 0-30%
when tested against pseudovirions. This activity is rather low when compared to over 80%
shown by broadly neutralizing mAbs that have been described in the literature. The challenge in
generating mAbs, in particular subtype C-derived, is to find those antibodies capable of suppressing viral replication in vivo and be capable of preventing infection. These reagents could
be used to identify epitopes to guiding the design of HIV-1 subtype C envelope immunogens or
vaccines. It is also envisaged that neutralizing antibodies used in therapeutic setting or in
combination with antiviral drug therapy could reduce viral load and retard disease progression in
infected people.
Description
Student Number : 9000987E -
PhD thesis -
School of Medicine -
Faculty of Health Sciences
Keywords
monoclonal antibodies, HIV-1 sub C, neutralizing antibodies, anti-HIV-1 mAbs, Du23 mAbs