Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison.
Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag-pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40% of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100% concordance with FAVN and strongly correlated with neutralization titres (r2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or beta-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 microl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies.
Wright
Edward
E
MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland Street, London W1T 4JF, UK.
Temperton
Nigel J
NJ
MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland Street, London W1T 4JF, UK.
Marston
Denise A
DA
Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency (Weybridge), WHO Collaborating Centre for the Characterisation of Rabies and Rabies-related Viruses, New Haw, Addlestone KT15 3NB, UK.
McElhinney
Lorraine M
LM
Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency (Weybridge), WHO Collaborating Centre for the Characterisation of Rabies and Rabies-related Viruses, New Haw, Addlestone KT15 3NB, UK.
Fooks
Anthony R
AR
Rabies and Wildlife Zoonoses Group, Veterinary Laboratories Agency (Weybridge), WHO Collaborating Centre for the Characterisation of Rabies and Rabies-related Viruses, New Haw, Addlestone KT15 3NB, UK.
Weiss
Robin A
RA
MRC/UCL Centre for Medical Molecular Virology, Division of Infection and Immunity, University College London, 46 Cleveland Street, London W1T 4JF, UK.