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

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock

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

FcγR and C1q binding and antibody-dependent enhancement of pHu-E16 variants.

<p><b>A</b>. SPR analysis of FcγR and C1q binding. FcγRs including CD16A (CD16V¹⁵⁸, CD16F¹⁵⁸), CD32A (CD32A-R¹³¹ and CD32A-H¹³¹ fused to an aglycosylated Fc region of IgG2 (CD32A-R¹³¹-G2agl and CD32A-H¹³¹-G2agl)), CD32B (CD32B fused to an aglycosylated Fc region of IgG2 (CD32B-G2agl)) and CD64, and C1q were injected at the same concentration over the surfaces with Hu-E16 variants captured on immobilized WNV E protein. Buffer injection was subtracted as blank and responses were normalized to same level of captured antibodies. <b>B</b>. ADE of WNV infection with antibody variants. Serial dilutions of Hu-E16 variants were mixed with WNV RVP and added to CD32A-expressing K562 cells. Forty-eight hours later, cells were analyzed by flow cytometry for GFP expression.</p

Neutralization of WNV by pHu-E16 variants.

<p>WNV was incubated with serial dilutions of mHu-E16 (positive control 1), or pHu-E16 (positive control 2), pHu-E16scFv-C_H^1-3, or Tetra pHu-E16 and used to infect Vero cells. Cells were then fixed, permeabilized, analyzed by focus reduction assay and quantitated by Biospot analysis. Mean ± SEM is shown from one of several independent experiments. EC50 values are listed below the graphs.</p

Purification of pHu-E16 variants from N. benthamiana leaves.

<p>Leaf proteins were extracted on day 9 after agroinfiltration. pHu-E16 variants were purified and analyzed on a 4-20% gradient SDS-PAGE gel under reducing condition and visualized with Coomassie stain. Lane 1, pHu-E16 as a reference standard; lane 2, pHu-E16 HC/pHu-E16scFv-C_L; lane 3, Tetra pHu-E16; lane 4, pHu-E16scFv-C_H^1-3/pHu-E16 LC; lanes 5, pHu-E16scFv-C_H^1-3. Ten μg of purified protein was loaded in each lane of the gel. One representative of several independent experiments is shown.</p