Antibody therapies for the prevention and treatment of viral infections

Salazar, Georgina; Zhang, Ningyan; Fu, Tong-Ming; An, Zhiqiang

[13] [22] [23] [24] [25] [26] [27] Since predefined antigens are required to identify the memory B cells then isolated by flow cytometry sorting, the abovementioned approach is not suitable for identification of novel neutralizing targets. To identify novel target epitopes, first, a large number of memory B cells are cultured, and then, the culture supernatant is screened against various antigens. This approach has been used for the discovery of broad and potent neutralizing antibodies against HIV and the discovery of a new HIV-1 vaccine target. 11 In this work, 11 surface IgG-expressing memory B cells were enriched from peripheral blood mononuclear cells (PBMCs) of a HIV-1 infected donor by negative depletion with antibodies to CD3, CD14, CD16, IgM, IgA, IgD on magnetic beads. Individual memory B cells were then seeded in microtiterplates in the presence of feeder cells and conditioned media generated from mitogen-stimulated human T cells from healthy donors. The supernatants were collected 8 days later and screened for antigenbinding reactivity. In another protocol, the addition of cytokines interleukin (IL)-2, IL-21, and irradiated 3T3-msCD40L feeder cells can successfully stimulate memory B cells to produce high concentrations of IgG in the supernatant in 2 weeks. 10 In a third approach, B cells are immortalized by Epstein-Barr Virus (EBV) in the presence of a toll-like receptor. This approach has been applied to the isolation of neutralizing antibodies against rabies, SARS-CoV, and other viruses. 13, 22, 27 Single-antibody-secreting plasma B cells. It has been reported that antigen-specific plasma cells account for up to 40-90% of total IgG-secreting cells in peripheral blood one week after boost immunization. 28 Methods for cloning IgG from human antibodysecreting cells have been developed (Fig. 3) . 15, [29] [30] [31] [32] This approach has been applied for isolating neutralizing antibodies from naturally infected and immunized human donors for dengue 33 and H1N1 influenza. 15, 34 Protocols for cloning mAbs from nonhuman primate plasma and memory B cells have also been reported. 16, 35, 36 In one report, a large panel of dengue targeting mAbs were cloned from single-antibody-secreting B cells of a rhesus macaque immunized with an experimental vaccine. 16 Single-antibody-secreting B cells were identified and isolated by flow cytometry, using a panel of phenotype markers. 16 To interrogate a large number of plasma B cells, a method was developed using microwell array chips. This method enables the analysis of live cells on a single-cell basis and offers rapid, efficient, and high-throughput (up to 234,000 individual cells) identification of antibody-secreting plasma cells. 37 In addition to direct cloning of antibody encoding genes from plasma B cells, single plasma cells can also be cultured on a monolayer of immortalized stromal cells or with IL-6 stimulation; the cultured cells would be able to produce enough antibody for screening assays of parallel viral binding and neutralization for identifying rare antibodies. 29 Proteomics-directed cloning of mAbs from serum. Antibodies cloned from single memory or plasma B cells represent the genetic antibody repertoire of an individual at a given time in response to a specific viral infection. However, this repertoire does not necessarily correspond to that of the antibodies present in circulation. Protocols have been developed to identify antigenspecific antibody sequences directly from