Generation and characterization of cross neutralizing human monoclonal antibody against 4 serotypes of dengue virus without enhancing activity

Abstract

Background. Dengue disease is a leading cause of illness and death in the tropics and subtropics. Most severe cases occur among patients secondarily infected with a different dengue virus (DENV) serotype compared with that from the first infection, resulting in antibody-dependent enhancement activity (ADE). Our previous study generated the neutralizing human monoclonal antibody, D23-1B3B9 (B3B9), targeting the first domain II of E protein, which showed strong neutralizing activity (NT) against all four DENV serotypes. However, at sub-neutralizing concentrations, it showed ADE activity in vitro. Methods. In this study, we constructed a new expression plasmid using the existing IgG heavy chain plasmid as a template for Fc modification at position N297Q bysite-directed mutagenesis. The resulting plasmid was then co-transfected with a light chain plasmid to produce full recombinant IgG (rIgG) in mammalian cells(N297Q-B3B9). This rIgG was characterized for neutralizing and enhancing activity by using different Fc R bearing cells. To produce sufficient quantities of B3B9 rIgG for further characterization, CHO-K1 cells stably secreting N297Q-B3B9 rIgG were then established. Results. The generated N297Q-B3B9 rIgG which targets the conserved N-terminal fusion loop ofDENVenvelope protein showed the same cross-neutralizing activity to all four DENV serotypes as those of wild type rIgG. In both FcγRI- and RII-bearing THP- 1 cells and FcγRII-bearing K562 cells, N297Q-B3B9 rIgG lacked ADE activity against all DENV serotypes at sub-neutralizing concentrations. Fortunately, the N297Q-B3B9rIgG secreted from stable cells showed the same patterns of NT and ADE activities as those of the N297Q-B3B9 rIgG obtained from transient expression against DENV2. Thus, the CHO-K1 stably expressing N297Q-B3B9 HuMAb can be developed as high producer stable cells and used to produce sufficient amounts of antibody for further characterization as a promising dengue therapeutic candidate. Discussion. Human monoclonal antibody, targeted to fusion loop of envelope domainII (EDII), was generated and showed cross-neutralizing activity to 4 serotypes of DENV, but did not cause any viral enhancement activity in vitro. This HuMAb could be further developed as therapeutic candidates. Subjects Biotechnology, Molecular Biology, Virology, Infectious Diseases.