Alphavirus neurovirulence: monoclonal antibodies discriminating wild-type from neuroadapted Sindbis virus

Abstract

Wild-type Sindbis virus strain AR339 (SV) and a neurovirulent mutant (NSV), derived by neonatal and weanling mouse brain passage, both cause acute fatal encephalitis in neonatal mice, but NSV alone kills adult mice. NSV cannot be distinguished from SV by immune sera or simple biochemical tests. To localize the molecular changes associated with neuroadaptation, we used a new array of 30 anti-SV monoclonal antibodies to probe for differences between SV and NSV in four tests: immunoprecipitation, enzyme-linked immunosorbent assay binding, neutralization, and hemagglutination inhibition. Seventeen monoclonal antibodies detected differences. Both El and E2 glycoprotein gene products were altered during neuroadaptation, but the preponderance of changes was clustered on E2. The capsid protein C was not measurably altered. Mapping of both viruses with these monoclonal antibodies showed that during neuroadaptation SV topography substantially shifted, masking and unmasking biologically important neutralization and hemagglutination inhibition sites. These conformational rearrangements, predominantly on E2, coincided with the acquisition of increased neurovirulence and new lethality for adult mice.

Novel packages of viral and self-antigens are generated during apoptosis.

“…1). Immunofluorescence of Sindbis-infected cells was performed with a panel of defined polyclonal and mAbs recognizing SV capsid protein or surface glycoproteins (4,11). Infected islands of cells, 1-10 in number, could be identified at 20 h after infection.…”

“…Ro and U1-70-kD RNP were stained with human sera of defined specificities (8) (diluted 1:160) and were visualized with fluorescein-conjugated goat anti-human F(ab')2 (Organon-Teknika Corp./Cappel, Durham, NC). Sindbis virus proteins were visualized with affinity-purified rabbit antibodies to SV (4) or mAb 4F directed against Sindbis capsid protein (11). Visualization was with Texas red-conjugated goat anti-rabbit or anti-mouse antibodies (The Jackson Laboratory, Bar Harbor, ME).…”

The role of antibody in recovery from alphavirus encephalitis

“…MAbs lo both the El and E2 glycoproteins can neutralize SV infectivity in vitro, but anti-E2 antibodies are generally more potent. The mechanism(s) by which MAbs to these glycoproteins reduce SV infectivity for cells is not clear (17,18). Antibodies to both the E1 and E2 glycoproteins can also protect against subsequent NSV challenge (19-21) and promote recovery after virus infection is established (19.…”

Sindbis virus as a model for studies of conformational changes in a metastable virus and the role of conformational changes in in vitro antibody neutralisation

“…This finding is consistent with current knowledge about the metastable nature of the virus and the structural associations of the E1/E2 heterotrimer and supports the contention that purified E1 protein is not in the same conformation as E1 within intact virus. Much of the work done identifying neutralising epitopes of Sindbis virus is based on the mAbs produced in the laboratories of A. Schmaljohn et al [93,94], R. Johnston [95][96][97] and D. Griffin [98]. Less well characterised mAbs to Sindbis virus [99,100] have been reported by others with one mAb described to recognise a discontinuous epitope found on the E1/E2 dimer [100].…”