Generation of Monoclonal Antibodies against Variable Epitopes of the M Protein of Rabies Virus.

Liu Liu,Wang Wang,He He,Zhou Zhou,Chen Chen,Zhao Zhao,Su Su,Ji Ji

doi:10.3390/v11040375

Abstract

Rabies virus (RABV), the causative agent of rabies, is highly neurovirulent for warm-blooded animals with a mortality rate of up to 100%. The RABV matrix protein (M) is required for virus particle assembly and budding. However, little is known about antigenic differences in the M protein. In this study, five monoclonal antibodies (mAbs), designated 3B9, 4A1, 2B11, 2C1, and 4B11, against the RABV M protein were generated using a recombinant M protein. All five mAbs reacted with the CVS-11 strain but showed no reactivity against the HEP-Flury strain in indirect immunofluorescence and western blotting. The epitope targeted by these mAbs was further identified by peptide scanning using GST-fused peptides. The 25PPYDDD30 peptide was defined as the minimal linear epitope. Alignment of amino acid sequences and phylogenetic analysis of different RABV strains indicated that the variable epitope 25PPDGDD30 is only present in the HEP-Flury and variant Flury strains of clade III, while the other strains resembling ERA and SRVA9 within the clade had another variable epitope, 25PLDDDD30. A Y27D mutation within the epitope was found among the rest of the RABV strains distributed in different clades. However, a single D28G mutation eliminated the reactivity of these five mAbs. In addition, the mAbs were able to recognize wildtype RABV strain in indirect immunofluorescence and western blotting and detect RABV-infected brain tissue using immunohistochemistry. The newly established mAbs and identified epitope may facilitate future investigations in the structure and function of the M protein and the development of diagnostic methods for the detection of different RABV strains worldwide. Most importantly, the epitope recognized by the mAbs against M protein might serve as a novel target for the development of a vaccine targeting RABV virulent strains.

Highlights

Rabies, caused by the rabies virus (RABV), is an acute progressive zoonotic disease attacking the central nervous system
The full-length M gene (GenBank accession: GQ918139) of the Rabies virus (RABV) Challenge virus standard 11 (CVS-11) strain was amplified by reverse transcription-PCR (RT-PCR) using total RNA extracted from CVS-11-infected SK-N-SH cells as template
Western blotting (WB) analysis showed that the purified His-M protein could react with mouse anti-His monoclonal antibodies (mAbs) showed that the purified His-M protein could react with mouse anti-His mAbs (Figure 1C)

Summary

Introduction

Rabies, caused by the rabies virus (RABV), is an acute progressive zoonotic disease attacking the central nervous system. Viruses 2019, 11, 375 threat to human health [3] with more than 59,000 deaths worldwide caused by rabies each year (https://www.who.int/). RABV belongs to the genus Lyssavirus of the Rhabdoviridae family. It contains a negative-sense and single-stranded linear RNA genome that encodes five proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L) [4]. The M protein, as a connector between the viral nucleocapsid and envelope [7], determines the budding and bullet shape of the virus [8,9]. Recombinant RABV lacking M protein are unable to be effectively packaged into the typical bullet-shaped virus particles [10].

Methods

Results

Conclusion