Isolation and Deep Sequencing of Monoclonal Antibodies Against HIV

National Institute Of Allergy And Infectious Diseases

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Abstract

Neutralizing antibodies (NAb) against HIV-1 are likely to be a major component of an effective vaccine-induced immune response. Cross-reactive NAbs commonly arise during HIV-1 infection, though only a small subset of infected patients produce NAbs with high breadth and potency. In contrast, the HIV-1 envelope glycoprotein (Env) vaccine immunogens tested to date have failed to elicit cross-reactive neutralizing antibodies. Thus, studying the development of broadly neutralizing antibodies (bNAbs) in infected individuals may provide important lessons for vaccine design. In addition, the isolation of bNAbs from selected donors has greatly aided our understanding of HIV-1 Env structure and vulnerability to neutralizing antibodies and such antibodies have potential for prevention or treatment of HIV-1 infection. For several years our lab has been a leader in the field of isolating and characterizing broadly neutralizing antibodies from HIV-infected donors. We have pioneered the development of reagents for isolating epitope-specific B cells, as well as a method for high-throughput screening of unselected B cells. After identification by one of these methods, we recover IgG from the B cells by single-cell PCR, subcloning, and expression in mammalian cells. The resulting antibodies are assayed for virus binding and neutralization, and their breadth, potency, epitopes, and modes of recognition analyzed. We also use next-generation deep sequencing to find clonal relatives of the antibodies and to understand their origins in B cell development. For the latter studies, donors for whom we have longitudinal samples from the time of HIV infection are particularly valuable. We also collaborate with labs within the VRC as well as extramural and within the pharmaceutical industry to analyze engineered versions of these antibodies that have been modified to improve potency, breadth, autoreactivity, and manufacturability.