Therapeutic Efficacy of Antibodies Lacking FcγR against Lethal Dengue Virus Infection Is Due to Neutralizing Potency and Blocking of Enhancing Antibodies

Mount Sinai School of Medicine, United States of America
PLoS Pathogens (Impact Factor: 7.56). 02/2013; 9(2):e1003157. DOI: 10.1371/journal.ppat.1003157
Source: PubMed


Author Summary
The four dengue virus serotypes (DENV1-4) cause the most prevalent mosquito-transmitted viral disease globally, infecting 50–100 million people annually in tropical and sub-tropical regions worldwide, yet no vaccine or therapy has been licensed to prevent or treat dengue. The greatest risk factor for severe dengue disease is a previous infection with a different serotype, which is thought to be due in part to a phenomenon known as antibody-dependent enhancement (ADE) whereby anti-DENV antibodies from a prior infection augment DENV infection of target Fcg receptor (FcgR)-expressing cells. We previously developed a mouse model that demonstrates antibody-enhanced lethal DENV disease and showed that genetically-modified antibodies incapable of interacting with the FcgR eliminate ADE in vitro and in vivo. In this study, we studied a larger panel of modified MAbs that recognize different regions of the DENV envelope protein. While all modified MAbs acted therapeutically to prevent a lethal, virus-only DENV infection, only certain MAbs effectively protected mice following an antibody-enhanced lethal infection. We determined that therapeutically effective MAbs following an ADE infection worked by competing for binding of enhancing antibodies on the DENV virion. Based on this, we designed an in vitro suppression-of-enhancement assay that predicted the ability of modified MAbs to act therapeutically in vivo.

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    • "Collectively, these results demonstrate the potential of plant-made MAbs and their variants to be used as therapeutics against ADE-prone viruses. Although it was previously reported that an aglycosylated version of a MAb can eliminate the risk of ADE [41], [42], the complete removal of N-linked glycans and the resulting abolishment of binding to C1q also may compromise the efficacy of a MAb that uses CDC activity. Plant-derived MAb glycolvariants that minimize ADE but retain C1q binding might have better efficacy than their aglycosylated counterparts. "
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    ABSTRACT: 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 (CH1-3) of human IgG (pHu-E16scFv-CH1-3) and a tetravalent molecule (Tetra pHu-E16) assembled from pHu-E16scFv-CH1-3 with a second pHu-E16scFv fused to the light chain (LC) constant region. pHu-E16scFv-CH1-3 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-CH1-3 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.
    PLoS ONE 03/2014; 9(3):e93541. DOI:10.1371/journal.pone.0093541 · 3.23 Impact Factor
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    ABSTRACT: Dengue viruses are the most common arthropod-transmitted viral infection, with an estimated 390 million human infections annually and similar to 3.6 billion people at risk. Currently, there are no approved vaccines or therapeutics available to control the global dengue virus disease burden. In this study, we demonstrate the binding, neutralizing activity, and therapeutic capacity of a novel bispecific dual-affinity retargeting molecule (DART) that limits infection of all four serotypes of dengue virus.
    Journal of Virology 05/2013; 87(13). DOI:10.1128/JVI.00327-13 · 4.44 Impact Factor
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    ABSTRACT: Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV serotypes 1, 2, and 3 (DENV-1, DENV-2, and DENV-3), few reports have assessed the activity of MAbs against DENV-4. Here, we evaluated the inhibitory activity of 81 new mouse anti-DENV-4 MAbs. We observed strain and genotype-dependent differences in neutralization of DENV-4 by MAbs mapping to epitopes on domains II (DII) and III (DIII) of the envelope (E) protein. Several anti-DENV-4 MAbs inefficiently inhibited at least one strain and/or genotype, suggesting that the exposure or sequence of neutralizing epitopes varies within isolates of this serotype. Remarkably, flavivirus cross-reactive MAbs, which bound to the highly conserved fusion loop in DII and inhibited infection of DENV-1, DENV-2, and DENV-3, more weakly neutralized five different DENV-4 strains encompassing the genetic diversity of the serotype after pre-incubation at 37°C. However, increasing the time of pre-incubation at 37°C or raising the temperature to 40°C enhanced the potency of DII fusion loop-specific MAbs and some DIII-specific MAbs against DENV-4 strains. Prophylaxis studies in two new DENV-4 mouse models showed that neutralization titers of MAbs after pre-incubation at 37°C correlated with activity in vivo. Our studies establish the complexity of MAb recognition against DENV-4, and suggest that differences in epitope exposure relative to other DENV serotypes affects antibody neutralization and protective activity.
    Journal of Virology 06/2013; DOI:10.1128/JVI.01314-13 · 4.44 Impact Factor
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