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

Abstract

BackgroundDengue 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.MethodsIn 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.ResultsThe 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.DiscussionHuman 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.

Generation and therapeutic efficacy of the novel Fc-modified cross-neutralizing human monoclonal antibodies against dengue virus

“…The variable genes of the heavy (VH) and light (VL) chains of the B3B9 HuMAb were amplified separately via polymerase chain reaction (PCR) from hybridoma cells [22], and cloned into plasmid containing antibody constant region of pQCX plasmid backbone (Pitaksajjakul et al, 2014;Injampa et al, 2017). Antibody mutations were performed over the Fc portion of the heavy chain plasmid regions at positions 234 and 235 from leucine (L) to alanine (A) (L234A, L235A [LALA]) by site-directed mutagenesis with the In-Fusion Cloning System (In-Fusion® HD Cloning Plus; Clontech Laboratories Inc., Shiga, Japan) based on the manufacturer's protocol [13].…”

“…Our previous study revealed the glycosylation of human monoclonal antibody against DENV (N297Q-B3B9) [13]. This cross-neutralizing HuMAb is targeted to domain II of the envelope proteins, which is a major target epitope of human antibodies for neutralizing and enhancing activity [14,15].…”

“…This cross-neutralizing HuMAb is targeted to domain II of the envelope proteins, which is a major target epitope of human antibodies for neutralizing and enhancing activity [14,15]. This antibody exhibits cross-neutralization properties and eliminates ADE activity in all DENV serotypes with NT50, ranging from 12 to 0.125 g/mL [13]. However, a previous study found that N-glycans are essential for not only immune effector functions but also stability under physiological or low temperature conditions, pharmacokinetics, and biodistribution [16][17][18].…”

Antibody-Dependent Enhancement of Viral Infections

“…Use of serotype-specific mAbs or high dose administration of cross-reactive mAbs has demonstrated reduced risk of ADE in vitro and in animal models. Further, antibodies with modifications in the Fc region such as deletion of nine amino acids (at positions 231-239) in the N terminus, mutation of asparagine to glutamine at position 297 (N297Q), or mutation of leucine to alanine at positions 234 and 235 (LALA mutants) have also shown reduced risk of ADE and exhibited prophylactic and therapeutic efficacy in animal models (Balsitis et al 2010;Shi et al 2016;Xu et al 2017;Injampa et al 2017;Lu et al 2018). Fc region modifications that extend the antibody half-life also help minimize ADE by maintaining the mAbs at high levels.…”

Retrospective investigation of the origin and epidemiology of the dengue outbreak in Yunnan, China from 2017 to 2018

“…Three domains (EDI, EDII, and EDIII) are formed in the spatial structure based on the determined crystal structure of the E protein. Among these, EDII, which is located at the amino terminus of the E protein, contains a fusion peptide structure that mediates the fusion of the virus and the target cell (fusion loop, 98-110 AA) (6). Meanwhile, EDIII facilitates the adsorption of viruses to target cells and is considered to contain the receptor binding region as well as the most important neutralizing epitope (7)(8)(9).…”