Shirley Lam(a), Min Nyo(a), Patchara Phuektes(a), Chow Wenn Yew(b), Yee Joo Tan(b),(c) and Justin Jang Hann Chu(a),(b)
(a) Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore. Yong Loo Lin School of Medicine, MD4 Level 5, 5 Science Drive 2, Singapore 117597.
(b) Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore.
(c) Hepatitis Viruses and Newly Emerging Viruses, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore. Yong Loo Lin School of Medicine, MD4 Level 5, 5 Science Drive 2, Singapore 117597.
Published in mAbs in August 2015.
Chikungunya virus (CHIKV) is a medically important human viral pathogen that causes Chikungunya fever accompanied with debilitating and persistent joint pain. Host-elicited or passively-transferred monoclonal antibodies (mAb) are essential mediators of CHIKV clearance. Therefore, this study aims to generate and characterize a panel of mAbs for their neutralization efficacy against CHIKV infection in a cell-based and murine model.
To evaluate their antigenicity and neutralization profile, indirect ELISA, immunofluorescence assay and plaque reduction neutralization test were performed on mAbs of IgM isotype. CHIKV escape mutants against mAb 3E7b neutralization were generated, followed by using reverse genetics techniques to create infectious CHIKV clone with single mutation. 3E7b was also administered to neonate mice prior or after CHIKV infection. Survival rate, CHIKV burden in tissues and histopathology of the limb muscles were evaluated. Both IgM 3E7b and 8A2c bind strongly to native CHIKV surface and potently neutralizes CHIKV replication. Further analyses on 3E7b binding and neutralization to CHIKV single mutant clones revealed that N218 of CHIKV E2 protein is a potent neutralizing epitope. In pre-binding neutralization assay, 3E7b blocks CHIKV attachment to permissive cells, possibly by binding to the surface-accessible E2-N218 residue. Prophylactic administration of 3E7b to neonate mice markedly reduced viremia and protected against CHIKV pathogenesis in various mice tissues. Given therapeutically at 4 h post-infection, 3E7b conferred 100% survival rate and similarly reduced CHIKV load in most mice tissues except the limb muscles. Collectively, these findings highlight the usefulness of 3E7b for future prophylactic or epitope-based vaccine design.
Figure legend: Structural localization of 3E7b escape epitopes on Chikungunya virus E1/E2 heterodimer modeled on CH122508 strain (GenBank Accesion number: FJ445502). A top-down view is shown with E1 (orange), and E2- domain A (yellow), domain B (violet), C (cyan), beta-ribbon connectors (green) and transmembrane helix (grey). Arrow denotes the direction of E1/E2 protein complex relative to the viral membrane. Side-chain of escape residues, E24, N218 and D223, are presented as stick structures that show carbon and hydrogen atoms (white), oxygen atom (red) and nitrogen (blue). Amino acid distance is measured in angstroms (1Å = 0.1 nm) and figure is prepared using Pymol software.
For more information on Justin CHU's laboratory, please click here.
For more information on Yee Joo TAN's laboratory, please click here.