Plasmid DNA encoding neutralizing human monoclonal antibody without enhancing activity protects against dengue virus infection in mice
Surachet Benjathummarak1, Atsushi Yamanaka2, Thanyaluk Krasae3, Chonlatip Pipattanaboon4, Subenya Injampa5, Pannamthip Pitaksajjakul1, Pongrama Ramasoota1
1 Department of Social and Environmental Medicine; Center of Excellence for Antibody Research (CEAR), Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
2 Mahidol-Osaka Center for Infectious Diseases (MOCID), Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Mahidol-Osaka Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
3 Laboratory Animal Science Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand
4 Department of Microbiology, Faculty of Medicine, Khon Kaen University, Thailand
5 Faculty of Medicine, King Mongkut’s Institute of Technology Ladkrabang, Bangkok, Thailand
Department of Social and Environmental Medicine; Center of Excellence for Antibody Research (CEAR), Faculty of Tropical Medicine, Mahidol University, Bangkok
Source of Support: This research was supported by the Faculty of Tropical Medicine, Mahidol University, Thailand, Research Fund through a Research and Researcher for Industry (RRi, Grant Number PHD59I0063 for SB), TRF Grant for New Researcher (TRG, Grant Number TRG5980015 for CP) and the Office of the National Research Council of Thailand-Japan Society for the Promotion of Science (JSPS) or NRCT-JSPS, Conflict of Interest: None
Objective: To evaluate the expression of DNA plasmid-harboring modified antibody gene that produces neutralizing human monoclonal antibodies against four serotypes of dengue virus (DENV) without enhancing activity in BALB/c mice.
Methods: We constructed pFUSE-based vectors (pFUSE_1G7C2_ hVH and pFUSE_1G7C2_hVL) containing genes encoding the variable domains of the heavy or light chain of the anti-dengue virus antibody 1G7C2, a human IgG1 that has been characterized for its neutralizing activity to DENV-1-4. Leucine (L) at positions 234 and 235 on the Fc CH2 domain in pFUSE_1G7C2_hVH was mutated to alanine (A) (LALA mutation) by site direct mutagenesis, and the new plasmid was termed pFUSE_1G7C2_hVH_LALA. An equal amount of pFUSE_1G7C2_hVL and 1G7C2_hG1-LALA plasmids were co-transfected into Chinese hamster ovary cells (CHO-K1) and a single dose of 100 μg 1G7C2_hG1-LALA plasmid was intramuscularly injected, followed by electroporation in BALB/c mice. The secreted 1G7C2_hG1-LALA antibodies in cell culture supernatant and mouse serum were examined for their biological functions, neutralization and enhancing activity.
Results: The co-transfection of heavy- and light-chain 1G7C2_ hG1-LALA plasmids in CHO-K1 cells produced approximately 3 900 ng/mL human IgG and neutralized 90%-100% all four DENV, with no enhancing activity. Furthermore, the modified human IgG was produced more than 1 000 ng/mL in mouse serum on day 7 post plasmid injection and showed cross-neutralization to four DENV serotypes. Subsequently, antibody production and neutralization decreased rapidly. Nevertheless, the secreted neutralizing 1G7C2_ hG1-LALA in mouse serum demonstrated complete absence of enhancing activities to all DENV serotypes.
Conclusions: These findings reveal that a new modified 1G7C2_ hG1-LALA expressing plasmid based on gene transfer is a possible therapeutic antibody candidate against DENV infection.