Identification and in vitro affinity maturation of HIV-1 envelope glycoprotein-specific early intermediate B cells isolated from immortalized human naïve B cell library

Abstract

Broadly neutralizing antibodies (bNAbs) against HIV-1 have been increasingly isolated since 2009. The therapeutic and prophylactic values of bNAbs have been well recognized because these antibodies confer full protection in animal models at relatively low concentrations that are achievable by vaccination. However, the development of effective vaccines to induce such antibodies remains a challenge. The bNAbs are highly mutated and often polyreactive. Moreover, the inferred germline antibodies of known bNAbs do not bind recombinant HIV-1 envelopes (Env). Based on these findings, we hypothesized that naïve B cells were activated by non-HIV-1 immunogens, which triggered the initial somatic mutations of germline antibody genes. The limited mutations authorized the B cells (intermediate B cells) the ability to bind HIV-1 envelopes. Upon HIV-1 infection or vaccination with Envs, antibody affinity maturation would be further induced, leading to the development of bNAbs. Although HIV-1 intermediate antibodies have recently been isolated from health donors, information about the statute of HIV-1 intermediate B cells in vivo is rare. The main objective of this study is to isolate HIV-1 intermediate B cells from healthy donors followed by characterization and in vitro maturation of the isolated intermediate B cells.

A highly efficient EBV transformation method was used to immortalize purified naïve B cells from healthy donors. The enrichment of HIV-1 envelope-specific B cells was observed after panning of library with magnetic beads. An HIV-1intermediate B cell line (LCL-P4) was generated which acquired the germinal center-like B cell phenotype, but did not express AID, an enzyme responsible for the initiation of somatic hypermutation in germinal center B cells. Interestingly, the variable region of LCL-P4 heavy chain shares the same VDJ recombination as PG16, a known HIV-1 bNAbs, according to IMGT analysis. Similar to PG16, LCL-P4 also shows preferential binding with gp140 trimers to gp120 or gp41 monomer. This paper is the first report of an HIV-1 intermediate B cell line generated by panning EBV-immortalized naïve B cell libraries.

To study the in vitro maturation of identified HIV-1 intermediate B cells, a full-length human antibody display platform was established by constructing two lentiviral expression plasmids with distinct antibiotic-resistant genes for the co-expression of IgH and IgLon mammalian cell surface. All the necessary gene fragments encoding the full-length IgG1, except the variable regions, were cloned to the two plasmids. Using a known bNAb b12 as a model Ab, an in vitro antibody affinity maturation system was further developed based on AID-induced somatic hypermutation. AID-induced mutations could be detected in both B and non-B cells. An HcRed reverse assay was conducted, which enabled the early detection of AID-induced mutations. This full-length human antibody display platform and in vitro antibody affinity maturation method may be used to study the mechanisms of B cell development in vivo, as well as for de novo discovery and engineering of human monoclonal antibodies.