Development of a highly protective combination monoclonal antibody therapy against Chikungunya virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans

Generation and analysis of novel plant-derived antibody-based therapeutic molecules against West Nile virus.

Previously, our group engineered a plant-derived monoclonal antibody (MAb) (pHu-E16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed several pHu-E16 variants to improve its efficacy. These variants included a single-chain variable fragment (scFv) of pHu-E16 fused to the heavy chain (HC) constant domains (CH(1-3)) of human IgG (pHu-E16scFv-CH(1-3)) and a tetravalent molecule (Tetra pHu-E16) assembled from pHu-E16scFv-CH(1-3) with a second pHu-E16scFv fused to the light chain (LC) constant region. pHu-E16scFv-CH(1-3) and Tetra pHu-E16 were efficiently expressed and assembled in plants. To assess the impact of differences in N-linked glycosylation on pHu-E16 variant assembly and function, we expressed additional pHu-E16 variants with various combinations of HC and LC components. Our study revealed that proper pairing of HC and LC was essential for the complete N-glycan processing of antibodies in both plant and animal cells. Associated with their distinct N-glycoforms, pHu-E16, pHu-E16scFv-CH(1-3) and Tetra pHu-E16 exhibited differential binding to C1q and specific Fcγ receptors (FcγR). Notably, none of the plant-derived Hu-E16 variants showed antibody-dependent enhancement (ADE) activity in CD32A+ human cells, suggesting the potential of plant-produced antibodies to minimize the adverse effect of ADE. Importantly, all plant-derived MAb variants exhibited at least equivalent in vitro neutralization and in vivo protection in mice compared to mammalian cell-produced Hu-E16. This study demonstrates the capacity of plants to express and assemble a large, complex and functional IgG-like tetravalent mAb variant and also provides insight into the relationship between MAb N-glycosylation, FcγR and C1q binding, and ADE. These new insights may allow the development of safer and cost effective MAb-based therapeutics for flaviviruses, and possibly other pathogens

Antigen binding of pHu-E16 variants to DIII of WNV E.

<p><b>A</b>. ELISA analysis. Serial dilutions of pHu-E16 variants were incubated on plates coated with WNV DIII and detected with a HRP-conjugated anti-human gamma chain antibody. Dilutions of pHu-E16 were used in parallel as reference standards. A commercial generic human IgG (Southern Biotech) was used as a negative control. One set of representative O.D. 450 nm readings from several independent experiments is presented. <b>B</b>. Binding of pHu-E16scFv-C_H^1-3 to domain III of WNV E displayed on the cell surface of yeast. Yeast cells displaying domain III of WNV E protein were stained with pHu-E16scFv-C_H^1-3, mHu-E16 (positive control), or a plant-produced humanized MAb against Ebola virus GP1 protein (negative control). Yeast cells were then processed by flow cytometry. Representative data from three independent experiments are shown.</p

Relative abundance in percentage of major glyco-structures detected on Hu-E16 variants.

<p>∑other complex: sum of glycoforms present at levels below 5%. The glycan structures are assigned using the ProGlycAn nomenclature (<a href="http://www.proglycan.com" target="_blank">www.proglycan.com</a>). HC: heavy chain, C_H^1-3: the constant regions 1–3 of HC, LC: light chain, C_L: constant region of LC.</p