Human Heavy Chain Antibody Genes Elicited in Thai Dengue Patients during DENV2 Secondary Infection.

Dengue is one of the most serious mosquito-borne viral diseases occurring in humans. To combat the complexity of 4 antigenically distinct serotypes, the ideal vaccine for dengue should be able to stimulate cross-neutralizing antibodies. Recently, genetics-based immune responses have been studied to guide vaccine design against several viral pathogens. Despite a recent approval of dengue vaccine, information on genetics-based immune responses against dengue virus (DENV) is still limited. Consequently, we aimed to determine the profiles of immunoglobulin heavy chain genes from DENV2 infected patients. The immunoglobulin heavy chain variable region genes (IGHV) were amplified from peripheral blood mononuclear cells of DENV2 secondary infected patients in the acute, convalescence, and recovery phases. Antibody heavy chain genes were sequenced using next-generation sequencing, and analyzed to identify correlations with neutralizing and enhancing activities of the serum samples. IGHV1-69, 3-23, and 3-30 were frequently discovered in our Thai DENV2 infected patients. Our findings provide new data on the human B cell response during secondary DENV2 infections in Thai dengue patients that offer supportive information for dengue vaccine design and therapeutics development.

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

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 by site-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 FcγR 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 of DENV envelope 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-B3B9 rIgG 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.