A novel highly specific and sensitive mAb against capsid protein VP1 to study the involvement of Enteroviruses in type 1 diabetes.

Background: Enteroviruses (EVs) have been linked to pathogenesis of type 1 diabetes (T1D), but their causality in beta cell autoimmunity remains debated. EV presence in beta cells from subjects with recent-onset T1D has been shown by immunostaining with the monoclonal antibody (mAb) 5D8/1 against the VP1 capsid protein of a broad spectrum of EVs. However, the availability of additional antibodies against distinct EV epitopes may be very valuable to corroborate such findings, and thus the viral hypothesis of T1D pathogenesis.

Methods: VP1 from Coxsackievirus B5 (CVB5) Faulkner and MCA strains were expressed fused with GST in E. Coli and used for mouse immunization. mAbs from several hybridomas were screened for recombinant VP1 detection. Reactivity against various EV-infected cells was assessed by immunoblotting, ELISA, immuno-histo/cytochemistry and -fluorescence.

Results: One of the anti-VP1 mAb (termed Veronica) showed strong reactivity against recombinant VP1 and several EV serotypes, including CVA9, CVB1-6, Echo-9-Barty, -Hill and -DM, -11 and -30, and Polioviruses 1-3 by ELISA. Independent tests on formalin-fixed paraffin-embedded (FFPE) EV cell microarrays demonstrated that Veronica mAb recognized Echoviruses -3, -4, -6, -9, -11 and CVB6 more strongly than 5D8/1 mAb. Unlike 5D8/1 mAb, however, it did not recognize CVA2, 5, 6, and 10, and only weakly CVA4 and AdenoC. No background was observed in uninfected A549, HPeV1, Vero and Hela and RD cells. Analysis of FFPE uninfected and CVB4-infected neonatal heart tissue confirmed the specific reactivity of Veronica mAb with CVB4. No background was detected in uninfected tissue.

Conclusion: Differences in their ability to detect various EVs suggest that Veronica and 5D8/1 mAbs recognize distinct VP1 epitopes. Hence, their combined use for staining of tissue specimens from T1D could be valuable to validate the correlation between EV infection of beta cells and their autoimmune destruction.