Neutralizing Monoclonal Antibodies Cross-React with Fusion Proteins Encoded by 129L of the Ectromelia Virus and A30L of the Variola Virus

Abstract

PCR fragments containing the fusion protein genes 129L of the ectromelia virus (EV) and A30L of the variola virus (VARV) were cloned in pQE32. The expression products, recombinant prA30L and pr129L, were isolated from Escherichia coli cell lysates by metal-chelate affinity chromatography. The recombinant proteins retained the capability of oligomerization, characteristic of their natural analogs. ELISA and immunoblotting were used to test 22 monoclonal antibodies (mAbs) to orthopoxviruses (19 mAbs to EV, 2 mAbs to the vaccinia virus (VACV), and 1 mAb to the cowpox virus (CPXV)) for interaction with prA30L, pr129L, and orthopoxviruses. Twelve species-specific epitopes were found in the EV fusion protein 129L and its recombinant analog. Ten cross-reacting epitopes were found in the EV, CPXV, and VACV fusion proteins. Of these, nine epitopes were present both in prA30L and in the VARV fusion protein. Five mAbs interacting with cross-reacting epitopes were capable of efficient neutralization of VACV; two of these mAbs neutralized VARV. It was demonstrated that there are species-specific epitopes in EV 129L and cross-reacting epitopes in the EV, VARV, CPXV, and VACV fusion proteins, including epitopes that induced synthesis of virus-neutralizing antibodies against VACV and VARV.