Dengue virus (DENV) is a significant public health concern in tropical and subtropical regions, causing severe illness and fatalities. One of the challenges in combating DENV is the phenomenon of antibody-dependent enhancement (ADE), where antibodies from a previous DENV infection can enhance the severity of a subsequent infection with a different serotype. To address this issue, a human monoclonal antibody (HuMAb) called B3B9 was previously developed, showing strong neutralizing activity against all four DENV serotypes but also exhibiting ADE activity at sub-neutralizing concentrations. In this study, a modified version of B3B9, termed N297Q-B3B9 rIgG, was engineered to prevent ADE while maintaining neutralizing efficacy.
The modification involved altering the Fc region of the antibody by mutating the asparagine residue at position 297 to glutamine, which eliminates a glycosylation site crucial for Fc receptor binding. The new N297Q-B3B9 rIgG was produced in mammalian cells and characterized for its neutralizing and enhancing activities against DENV. Stable CHO-K1 cell lines secreting the engineered rIgG were established to ensure consistent production for further studies. The engineered antibody retained its cross-neutralizing activity against all four DENV serotypes, without exhibiting ADE activity in vitro, unlike the original B3B9 HuMAb.
The wild-type and mutated B3B9 antibodies were compared for their ability to neutralize DENV in Vero cells. The N297Q-B3B9 rIgG showed comparable neutralizing efficacy to the wild-type rIgG against DENV2, DENV3, and DENV4, with a slightly higher concentration needed for DENV1. Importantly, the engineered antibody completely eliminated ADE activity across all DENV serotypes, a significant improvement over the original B3B9 HuMAb. The isotype analysis confirmed that B3B9 is an IgG1 antibody, a desirable characteristic for therapeutic antibodies due to their effector functions.
Further epitope mapping studies revealed that the binding site of B3B9 on the DENV envelope protein lies within the highly conserved N-terminal fusion loop of domain II. This information is crucial for understanding the specificity and mechanism of action of the engineered antibody. Additionally, phage display techniques identified a consensus peptide motif within this epitope, providing insights into the molecular interactions between B3B9 and the viral protein. The engineered N297Q-B3B9 rIgG produced from stable CHO-K1 cells demonstrated consistent neutralizing activity against DENV2 without any enhancing effects, highlighting its potential as a safer and more effective therapeutic candidate.
In conclusion, the development of the N297Q-B3B9 engineered antibody represents a significant advancement in the quest for a safe and potent therapeutic option against DENV. By specifically targeting the conserved fusion loop region of the DENV envelope protein and abolishing ADE activity, this modified HuMAb shows promise as a valuable tool in combating dengue infections. The successful engineering and characterization of this antibody pave the way for future studies and potential clinical applications in the fight against dengue fever and its severe manifestations.
Tags: monoclonal antibodies, secretion, cell culture
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