Near pan-alphavirus neutralization by an antibody-nanobody bispecific

Alphaviruses represent a global concern, with some such as chikungunya virus currently infecting thousands in China and others such as Venezuelan equine encephalitis virus inducing neurological symptoms in 4-14% of infected individuals. A single antibody capable of protecting from all human-infecting alphaviruses, by achieving sufficient potency against each virus individually, may have high utility, as only a single therapeutic would then need to be stockpiled against this entire class of emerging pathogens. We previously identified an antibody, SKT05, with broad encephalitic alphavirus recognition, and we also identified nanobodies capable of neutralizing both arthritogenic and encephalitic alphaviruses. Here, we employ structure-based design to engineer a SKT05-nanobody bispecific, which has near pan-neutralization of alphaviruses that infect humans. Twenty-three designed SKT05-nanobody bispecifics broadly neutralized pseudotyped viruses expressing envelope proteins of encephalitic alphaviruses. With 10 of these bispecifics, we further assessed live arthritogenic alphavirus neutralization for Mayaro, O'nyong'nyong, Ross River, and Una viruses, observing potent neutralization for all of them with four bispecifics. The best of these four, V2C3-30-SKT05H, comprising the V2C3 nanobody genetically fused through a 30-amino acid linker to the heavy chain of SKT05, potently neutralized 9 of 11 tested alphaviruses that were phylogenetically proximal to all 26 alphaviruses that infect and cause symptoms in humans. Further, therapeutic administration of V2C3-30-SKT05H reduced viral loads in chikungunya virus-infected mice. Electron microscopy revealed that V2C3-30-SKT05H induced disorder of E1E2 spikes on the viral surface, suggesting E1E2 disassembly as a potential neutralization mechanism. Collectively, these results demonstrate near pan-neutralization of human-infecting alphaviruses by an antibody-nanobody bispecific.