title: Alanine scanning of the rabies virus glycoprotein antigenic site III using recombinant rabies virus: implication for post-exposure treatment.
abstract: The safety and availability of the human polyclonal sera that is currently utilized for post-exposure treatment (PET) of rabies virus (RABV) infection remain a concern. Recombinant monoclonal antibodies have been postulated as suitable alternatives by WHO. To this extent, CL184, the RABV human antibody combination comprising monoclonal antibodies (mAbs) CR57 and CR4098, has been developed and has delivered promising clinical data to support its use for RABV PET. For this fully human IgG1 cocktail, mAbs CR57 and CR4098 are produced in the PER.C6 human cell line and combined in equal amounts in the final product. During preclinical evaluation, CR57 was shown to bind to antigenic site I whereas CR4098 neutralization was influenced by a mutation of position 336 (N336) located within antigenic site III. Here, alanine scanning was used to analyze the influence of mutations within the potential binding site for CR4098, antigenic site III, in order to evaluate the possibility of mutated rabies viruses escaping neutralization. For this approach, twenty flanking amino acids (10 upstream and 10 downstream) of the RABV glycoprotein (G) asparagine (N336) were exchanged to alanine (or serine, if already alanine) by site-directed mutagenesis. Analysis of G expression revealed four of the twenty mutant Gs to be non-functional, as shown by their lack of cell surface expression, which is a requirement for the production of infectious RABV. Therefore, these mutants were excluded from further study. The remaining sixteen mutants were introduced in an infectious clone of RABV, and recombinant RABVs (rRABVs) were recovered and utilized for in vitro neutralization assays. All of the viruses were effectively neutralized by CR4098 as well as by CR57, indicating that single amino acid exchanges in this region does not affect the broad neutralizing capability of the CL184 mAb combination.
authors.affiliation: Department of Microbiology and Immunology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
authors.forename: Amy B
authors.initials: AB
authors.lastname: Papaneri
authors.forename: Christoph
authors.initials: C
authors.lastname: Wirblich
authors.forename: Wilfred E
authors.initials: WE
authors.lastname: Marissen
authors.forename: Matthias J
authors.initials: MJ
authors.lastname: Schnell
mesh_terms: D000409:Alanine / Q000235:genetics; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000627:therapeutic use; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000627:therapeutic use; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology; D001666:Binding Sites, Antibody; D002460:Cell Line; D006224:Cricetinae; D006023:Glycoproteins / Q000235:genetics* / Q000276:immunology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009500:Neutralization Tests; D056990:Post-Exposure Prophylaxis; D011818:Rabies / Q000628:therapy*; D011819:Rabies Vaccines / Q000627:therapeutic use; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D006023:Glycoproteins; D011819:Rabies Vaccines; D014759:Viral Envelope Proteins; D000409:Alanine
keywords: B-cell epitope; Lyssavirus; Monoclonal antibodies; Post exposure treatment; Rabies virus