A human monoclonal antibody cocktail as a novel component of rabies postexposure prophylaxis.

The currently recommended treatment for individuals exposed to rabies virus is the combined administration of rabies vaccine and rabies immune globulin (RIG). This review sets out the criteria used to guide development of a cocktail of human monoclonal antibodies as a replacement for RIG. Using this process as a model, the general requirements for development of safe and efficacious monoclonal antibody alternatives to currently used polyclonal serum products are discussed.

Isolation and characterization of human monoclonal antibodies from individuals infected with West Nile Virus.

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 mug/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.

Novel human monoclonal antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants.

The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.

The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries.

Antibody phage display technology was used to identify human monoclonal antibodies that neutralize rabies virus (RV). A phage repertoire was constructed using antibody genes harvested from the blood of vaccinated donors. Selections using this repertoire and three different antigen formats of the RV glycoprotein (gp) resulted in the identification of 147 unique antibody fragments specific for the RV gp. Analysis of the DNA sequences of these antibodies demonstrated a large variation in the heavy- and light-chain germ-line gene usage, suggesting that a broad antibody repertoire was selected. The single-chain variable fragment (scFv) antibodies were tested in vitro for RV neutralization, resulting in 39 specificities that neutralize the virus. Of the scFv clones, 21 were converted into full-length human IgG(1) format. Analysis of viral escape variants and binding competition experiments indicated that the majority of the neutralizing antibodies are directed against antigenic site III of the RV gp. The obtained specificities expand the set of human anti-RV antibodies eligible for inclusion in an antibody cocktail aimed for use in rabies post-exposure prophylaxis.

Novel rabies virus-neutralizing epitope recognized by human monoclonal antibody: fine mapping and escape mutant analysis.

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein.

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.

Lipopolysaccharide regions involved in the activation of Escherichia coli outer membrane protease OmpT.

OmpT is an integral outer membrane protease of Escherichia coli. Overexpression of OmpT in E. coli and subsequent in vitro folding of the produced inclusion bodies yielded protein with a native-like structure. However, enzymatically active protease was only obtained after addition of the outer membrane lipid lipopolysaccharide (LPS). OmpT is the first example of an enzyme that requires LPS for activity. In this study, we investigated the nature of this activation. Circular dichroism analysis showed that binding of LPS did not lead to large structural changes. Titration of OmpT with LPS and determining the resulting OmpT activity with a fluorimetric assay yielded a dissociation constant of 10-4 m for E. coli K-12 LPS. Determining the dissociation constants for different LPS chemotypes revealed that a fully acylated lipid A part is minimally required for activation of OmpT. The heptose-bound phosphates in the inner core region were also important for activation. The affinity for LPS was not dependent on the concentration of substrate, neither was affinity for the substrate influenced by the concentration of LPS. This indicated that LPS most likely does not act at the level of substrate binding. We hypothesize that LPS induces a subtle conformational change in the protein that is required for obtaining a native active site geometry.