Highly potent multivalent VHH antibodies against Chikungunya isolated from an alpaca naïve phage display library

Chikungunya virus (CHIKV) is a re-emerged mosquito-borne alphavirus that can cause musculoskeletal diseases, imposing a substantial threat to public health globally. High-affinity antibodies are need for diagnosis and treatment of CHIKV infections. As a potential diagnostic and therapeutic agent, the multivalent VHH antibodies is a promising tookit in nanomedicine. Here, we developed potent multivalent VHH antibodies from an alpaca naïve phage display library targeting the E2 glycoprotein of the CHIKV virus.

In the present study, we generated 20 VHH antibodies using a naïve phage display library for binders to the CHIKV E2 glycoprotein. Of these, multivalent VHH antibodies Nb-2E8 and Nb-3C5 had specific high-affinity binding to E2 protein within the nanomolar range. The equilibrium dissociation constant (KD) was between 2.59-20.7 nM, which was 100-fold stronger than the monovalent antibodies' affinity. Moreover, epitope mapping showed that Nb-2E8 and Nb-3C5 recognized different linear epitopes located on the E2 glycoprotein domain C and A, respectively. A facile protocol of sandwich ELISA was established using BiNb-2E8 as a capture antibody and HRP-conjugated BiNb-3C5 as a detection antibody. A good linear correlation was achieved between the OD₄₅₀ value and the E2 protein concentration in the 5-1000 ng/mL range (r = 0.9864, P < 0.0001), indicating its potential for quantitative detection of the E2 protein.

Compared to monovalent antibodies, multivalent VHH antibodies Nb-2E8 and Nb-3C5 showed high affinity and are potential candidates for diagnostic applications to better detect CHIKV virions in sera.