Background：Chikungunya virus (CHIKV) is a re-emerged mosquito-borne alphavirus that can cause musculoskeletal disease and impose a substantial threat to public health globally. It would be desirable to develop a high-affinity antibodies for the diagnosis and therapy of CHIKV infection. As potential diagnostic and therapeutic agents, multivalent nanobodies hold a significant promise towards nanomedicine. Here, we developed the highly potent multivalent nanobodies from an alpaca naïve phage display library targeting the E2 glycoprotein of CHIKV virus. Multivalent nanobodies play an important role in the promotion of high-affinity binding to E2 protein.Results: In the present study, we generated 20 nanobodies using a naïve phage display library for binders to the CHIKV E2 glycoprotein. Of which, multivalent nanobodies of Nb-2E8 and Nb-3C5 had specific high-affinity binding to E2 protein with nanomolar range, showing the equilibrium dissociation constant (KD) of 2.59-20.7 nM, which is 100-fold stronger than monovalent nanobodies’ affinity. Moreover, epitope mapping showed that the 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 the 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 that it can be used for the quantitative detection of E2 protein.Conclusions: Multivalent nanobodies Nb-2E8 and Nb-3C5 exhibit functional features and high affinity distinct from monovalent nanobodies, showing new candidate diagnostic applications to detect sera binding protein and/or virions.

Background 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. Results 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 OD 450 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. Conclusions 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. Graphical Abstract

Chikungunya fever, caused by Chikungunya virus (CHIKV), is an Aedes mosquito-borne disease present worldwide, and millions of CHIKV infections have been reported. Treatment for CHIKV includes supportive care and anti-inflammatory medications, but there are currently no antiviral treatments or vaccines. Nonstructural protein 2 (nsP2) of CHIKV is the most important functional protein mediating virus replication and amplification, making it an ideal antiviral target for CHIKV. In this study, we determined the CHIKV nsP2 Epitope Rich Region, expressed recombinant nsP2 protein, and isolated 5 nsP2-specific nanobodies (Nb-A2, Nb-A9, Nb-D7, Nb-D12 and Nb-E12) from a phage display library comprising variable domains of Camellidae heavy chain-only antibodies (VHH). We subsequently established a stable Nbs-expressing HEK293T cell line to explore antiviral function. The results showed that Nb-A9 inhibited CHIKV replication at the early stage of CHIKV infection in HEK293T cells, and protected cells against CHIKV-induced cytopathic effect (CPE). This is possibly the first report of an Nbs-based strategy against CHIKV nsP2, Nb-A9 has great potential for developing a novel antiviral drug to treat CHIKV infection. The acquisition of antibodies has laid a foundation for further research on the function of CHIKV nsP2 and the development of therapeutic drugs.