Identification of a Cytotoxic T-Cell Epitope on the Recombinant Nucleocapsid Proteins of Rinderpest and Peste des petits ruminants Viruses Presented as Assembled Nucleocapsids

Abstract

The nucleocapsid protein (N) of morbilliviruses is not only a major structural protein but also the most abundant protein made in infected cells. We overexpressed the N proteins of Rinderpest virus and Peste des petits ruminants virus in E. coli, which assemble into nucleocapsids in the absence of viral RNA that resemble nucleocapsids made in the virus-infected cells. Employing these assembled structures resembling subviral particles, we studied the induction of both the antibody response and the cytotoxic T-lymphocyte (CTL) response in a murine model (BALB/c). A single dose of the purified recombinant nucleocapsids of both viruses in the absence of an adjuvant induces a strong CTL response. The CTLs generated are antigen specific and cross-reactive with respect to each virus and, furthermore, this CTL response is MHC class I restricted. Based on the prediction for H-2(d)-restricted T-cell motifs we tested the lysis of transfected P815 (H-2(d)) cells expressing a nine amino acid potential CTL epitope, by splenic T cells in vitro restimulated with bacterially expressed RPV or PPRV N proteins. We extended our study to the bovine system both to analyze the immunogenicity of these recombinant proteins in the natural hosts and to show that PBMC from cattle vaccinated with Rinderpest vaccine proliferate in vitro, in response to restimulation with soluble nucleocapsid proteins. Furthermore, the murine CTL epitope functions in the bovine system as a cytotoxic T-cell epitope. This sequence, which is conserved in the N proteins of morbilliviruses, conforms well to the predicted algorithm for some of the most common BoLA CTL antigenic peptides.

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

“…Rabbit polyclonal antibodies against RPV P (Kaushik & Shaila, 2004), N (Mitra- Kaushik et al, 2001) and L proteins were made previously in the laboratory. Horseradish peroxidase-conjugated goat anti-rabbit IgG was obtained from Sigma.…”

Antigenic and Immunogenic Investigation of B-Cell Epitopes in the Nucleocapsid Protein of Peste des Petits Ruminants Virus

“…There is a growing threat for the emergence of PPR in countries free of the disease, especially ones neighboring areas where PPR is endemic. PPR is caused by an enveloped RNA virus known as PPR virus (PPRV), which belongs to the Morbillivirus genus in the family Paramyxoviridae (2,32). Other members of the genus Morbillivirus include rinderpest virus (RPV), measles virus (MV), canine distemper virus (CDV), phocine distemper virus (PDV), and dolphin morbillivirus (DMV) (2,13).…”

Current Advances in Serological Diagnosis of Peste des Petits Ruminants Virus

“…However, the passive protection of kids born to vaccinated or exposed mothers is relatively short compared to other species, showing a declining trend from 60-90 days onwards and a complete loss at 120 days (Libeau et al 1995). Consequently, the presence of antibodies, whether they are neutralizing or not, can serve as surrogate markers of protection when detected with serological tests, despite the fact that cellular immunity is implicated in the control of PPRV replication (Mitra-Kaushik et al 2001). …”