Generation and characterization of monoclonal antibodies to hemaggutinin/ humagglutinin-neuraminidase proteines of rinderpest and peste des petits ruminanats viruses: mapping of B-cell epitops and development of a sero- surveillance kit

Abstract

Generation and Characterization of Monoclonal Antibodies to Hemagglutinin / Hemagglutinin Neuraminidase Proteins of Rinderpest and Peste des Petits Ruminants Viruses: Mapping of B Cell Epitopes and Development of a Sero Surveillance Kit ________________________________________ Introduction Rinderpest (“cattle plague”) is a highly contagious and often fatal disease primarily affecting large ruminants such as cattle and buffaloes. Clinical features include fever, erosive stomatitis, gastro enteritis, dehydration, and death. It is caused by rinderpest virus (RPV), a member of the genus Morbillivirus (family Paramyxoviridae). Morbilliviruses are enveloped viruses with a single stranded, negative sense RNA genome. The envelope contains two major glycoproteins: 1. Hemagglutinin (H), which recognizes host cell receptors, and 2. Fusion (F), which mediates fusion of viral and host membranes. Both are highly immunogenic and can confer complete protection. Recombinant vaccinia and capripox viruses expressing H and/or F glycoproteins have provided complete protection; however, the B cell epitope repertoire of antibodies elicited against H and F has not been mapped in detail. A closely related morbillivirus causes Peste des Petits Ruminants (PPR) (“goat plague”) in small ruminants (goats and sheep). The tissue culture rinderpest vaccine and recombinant vaccines (vaccinia/capripox expressing RPV H and/or F) protect goats against PPR. Nonetheless, the B cell epitopes recognized on HN (hemagglutinin neuraminidase) and/or F proteins following immunization have also not been comprehensively mapped. ________________________________________ Aim of the Present Investigation This study aimed to generate monoclonal antibodies (mAbs) against recombinant H (RPV) and HN (PPRV) glycoproteins and to characterize them extensively. The main objectives were to: • Map antigenic stretches harboring B cell epitopes on RPV H and PPRV HN using overlapping gene deletion constructs expressed and purified from E. coli, alongside synthetic peptides. • Functionally delineate regions on RPV H and PPRV HN required for biological activities via various inhibition assays using the characterized mAbs. • Identify unique and cross reactive B cell epitopes on RPV H and PPRV HN to enable development of serological diagnostics for disease status assessment and vaccine efficacy monitoring. ________________________________________ Generation of Monoclonal Antibodies to Baculovirus Expressed H (RPV) and HN (PPRV) Baculovirus expressed secretory H (SecH) protein of RPV and purified PPRV HN extracellular virus (ECV) were used as immunogens for hybridoma production. • RPV H mAbs: From previously generated parental hybridomas, three subclones were selected-D2F4, E2G4, E2B6. These mAbs were characterized further. One non cross reactive clone (no reactivity with PPRV HN) was chosen to develop a sero surveillance kit. • PPRV HN mAbs: Splenocytes from PPRV HN ECV immunized mice were fused with Sp2/0 cells. Parental clones were screened using lysates of baculovirus infected insect cells (PPRV HN) and Vero cells infected with PPRV Nig 75/1 and TN 87/1 strains. Four parental clones positive in indirect ELISA (A6E9, C10A1, D2E4, F10E7) were subcloned by limiting dilution; one subclone from each was used for subsequent work. ________________________________________ Characterization of RPV H and PPRV HN Monoclonal Antibodies Each mAb was typed for isotype and cross reactivity with RPV H/PPRV HN. • RPV H mAbs: None neutralized virus, but the two cross reactive H mAbs inhibited hemagglutination (HA) of PPRV in the HI test. • PPRV HN mAbs: All four neutralized PPRV, and three also neutralized RPV. All exhibited HI activity but lacked hemolysis inhibition. • In competitive binding assays, two mAbs competed with each other. • Neuraminidase (NA) inhibition: One mAb inhibited NA using fetuin as substrate; two mAbs inhibited NA using N acetyl neuraminolactose. • Kinetics: Association with antigen was appropriate, but rapid dissociation indicated reduced epitope affinity. ________________________________________ Mapping of B Cell Epitopes on RPV H and PPRV HN Overlapping gene deletion constructs for RPV H and PPRV HN were expressed in E. coli as N terminal His tagged proteins. After narrowing epitope containing fragments, synthetic peptides were employed to precisely locate epitopes/epitopic domains. RPV H • Western blotting of deletion constructs mapped the mAb reactive domain to aa 569–609 (C terminal 41 aa). • Two 15 mer Measles virus H peptides (aa 563–587, 5 aa overlap; 67% identity to RPV H) were used in peptide ELISA: o One mAb reacted with 563–577, another with 573–587. • Two RPV H specific 9 mers spanning 569–583 (3 aa overlap) were then used: o The two peptide reactive mAbs bound 569–577 and 575–583; the third mAb mapped to 588–609 (21 aa domain). PPRV HN • Immunoreactivity to both RPV H and PPRV HN deletions indicated two discontinuous regions: aa 263–368 and aa 538–609. • Using four RPV H synthetic peptides (two 6 mers, two 7 mers), two mAbs recognized constituents of discontinuous epitopes at aa 270–276 and 527–540; another recognized aa 270–276 and 527–554. The non cross reactive mAb’s site could not be narrowed further. • PPRV HN mAbs were mapped to B cell epitopic domains on the PPRV HN protein accordingly. ________________________________________ Development of Recombinant H Competitive ELISA (C ELISA) for Rinderpest Sero Surveillance Using the RPV H non cross reactive mAb D2F4 as the competing antibody, a C ELISA kit was developed. • Cut off determination: Screening of 100 known negative, unvaccinated bovine sera (1–2 years, heifers) yielded a positive/negative (P/N) cut off of 44% inhibition (PI) (Mean PI + 2 SD). • Field evaluation: 1,519 sera from Karnataka, Kerala, Tamil Nadu were tested. o H C ELISA (this study): 30.8% positive for RPV antibodies. o Commercial C ELISA (Pirbright, BDSL): 31.2% positive. These comparable results validate the newly developed kit for field sero surveillance. ________________________________________ Conclusions • Monoclonal antibodies to RPV H and PPRV HN were generated and extensively characterized. • B cell epitopes on these glycoproteins were mapped using overlapping deletion constructs and synthetic peptides, identifying unique and cross reactive determinants. • A recombinant H C ELISA using a non cross reactive mAb was developed and validated against a commercial kit, supporting its utility for sero surveillance and vaccine efficacy assessment.