Present PEP treatment of rabies virus-infected people involves the use of HRIG. Blood-derived products, like HRIG, have potential health risks normally associated with those products. Also, HRIG can display batch-to-batch variation and may be limited in availability in case of sudden mass exposure (5
). An alternative to HRIG would be to use human monoclonal antibodies produced according to industrial standards. The concern regarding blood-derived products could thereby be circumvented, and consistent batches of antibodies could be produced in large quantities. Therefore, we initiated a study to identify and characterize human MAbs to be included in such a cocktail. We focused on the human MAbs previously described by Prosniak et al. (25
), which displayed potent neutralization of several rabies virus strains.
For expression of the antibodies, we used the human cell line PER.C6, which produces consistently high levels of antibody and can easily be adapted for industrial use. This method contrasts with that of the rhabdoviral antibody system (22
), which involves the error-prone rhabdoviral RNA polymerase and is therefore less suitable as an industrial standard for the production of a consistent, high-quality antibody. Reformatting of the antibodies SOJA, SOJB, and SO57 into the PER.C6 expression system resulted in improved neutralizing potency of the antibodies, with the exception of CRJA. We decided to exclude CRJA from further experiments due to its low potency. The low potency would result in exorbitant protein content if CRJA were to be used in a future cocktail of MAbs. Therefore, CR57 and CRJB were further investigated.
Earlier data suggested that CR57 and CRJB show a distinct pattern of coverage of different lyssavirus genotypes, which may be based on qualitative or quantitative characteristics of the respective MAbs (25
). Competition experiments using both immobilized rabies virus glycoprotein and glycoprotein expressed on cells showed that CR57 and CRJB recognize competing, overlapping epitopes and cannot bind simultaneously to the rabies virus glycoprotein (Fig. ). CR57 was tested in a PEPSCAN ELISA to determine the epitope and was shown to recognize a linear peptide of the rabies virus glycoprotein (Fig. ), which agreed with the finding that CR57 is able to bind to reduced denatured rabies virus glycoprotein on Western blotting. Detailed analysis of the binding region of CR57 revealed a minimal binding region of 6 amino acids (KLCGVL, aa 226 to 231) critical for CR57 binding, with K-CGV as key residues (Fig. ). This finding was confirmed by analysis of the amino acid sequences of the escape viruses generated with CR57. Specific nonsilent point mutations (K226E, K226N, and G229E) were detected in this minimal binding region (Fig. , Table ). This underlines the importance of these residues for binding of CR57 to the rabies virus glycoprotein. Analysis of the glycoprotein open reading frame of EJB viruses showed point mutations in two different spots of the glycoprotein, including mutations (G229E and L231P) within the minimal binding region of CR57, indicating that amino acids within this epitope are important for binding of CRJB to the rabies virus glycoprotein. Furthermore, these data suggest that CRJB recognizes a nonlinear, conformational epitope that partially overlaps with the minimal binding region of CR57. This is also in agreement with the competition data described above.
Another goal of the escape virus studies was to mimic the theoretical event of escape in vivo. Antibody escape viruses could occur from naturally existing quasispecies present in the virus inoculum as well as from antibody-dependent selection of a neutralization-resistant variant as a consequence of a humoral immune response against the virus. However, the latter is not likely to occur, as was demonstrated by the observed conservation of the antigenic sites on rabies virus glycoprotein (2
The antigenic structure of the rabies virus glycoprotein was initially defined by Lafon et al. (18
). The antigenic sites were identified using a panel of mouse MAbs and their respective MAb-resistant virus variants. Since then, the antigenic sites have been mapped by identification of the amino acid mutations in the glycoprotein of MAb-resistant variants (3
). The majority of rabies virus-neutralizing MAbs are directed against antigenic site II (3
), which is a discontinuous conformational epitope comprising aa 34 to 42 and aa 198 to 200 (24
). Antigenic site III is a continuous conformational epitope at aa 330 to 338 and harbors two charged residues, K330 and R333, that affect viral pathogenicity (7
). The conformational antigenic site I was defined by only one MAb, 509-6, located at aa 231 (3
). Antigenic site IV is known to harbor overlapping linear epitopes (4
). Benmansour et al. (3
) also described the presence of a minor site located at position 342 to 343, which is distinct from antigenic site III despite its close proximity.
Alignment of the CR57 epitope with the presently known linear and conformational neutralizing epitopes on rabies virus glycoprotein (Fig. ) revealed that the CR57 epitope is located in the same region as the conformational antigenic site I, defined by the single MAb 509-6 (3
). Competition experiments described within this study revealed that CR57 and 509-6 competed for binding to ERA G in both ELISA and fluorescence-activated cell sorting (data not shown). Interestingly, neutralization of E57 escape viruses by 509-6 could be demonstrated (data not shown), which agreed with earlier data by Dietzschold et al. (8
) showing neutralization of an SO57 escape variant by the 509-6 antibody. Coverage of 509-6 escape viruses by CR57 could not be performed, as this escape virus was not available. However, based on literature, it is expected that CR57 does neutralize 509-6 escape viruses. CR57 neutralizes silver-haired bat rabies viruses (SHBRV) (25
), which all harbor a proline at position 231 (9
). This proline was identical to the mutation L231P observed in the 509-6 escape virus (3
). Thus, SHBRV resemble the 509-6 escape virus with respect to antigenic site I, suggesting that CR57 is capable of neutralizing 509-6 escape viruses. Overall, antigenic site I might be redefined as a region harboring both conformational (MAbs CRJB and 509-6) and linear epitopes (CR57), as indicated in Fig. .
FIG. 4. Neutralizing epitopes on rabies virus glycoprotein. A schematic drawing of the rabies virus glycoprotein is shown depicting the antigenic sites including the novel CR57 epitope. The signal peptide (19 aa) and transmembrane domain are indicated by black (more ...)
We analyzed the glycoprotein sequence of a large set of rabies virus isolates to assess the occurrence of quasispecies that might have a mutation in the CR57 epitope. We observed a high level of conservation within the CR57 epitope, which was underlined by experiments showing that a panel of rabies street viruses were all neutralized by CR57 in a standard RFFIT (data not shown). This may be explained in part by the presence of the central cysteine residue, which is structurally involved in glycoprotein folding and is conserved among all lyssaviruses (2
). From the 229 analyzed naturally occurring rabies virus isolates, only 3 isolates (AF346857, AF346861, and U72050) contained nonconserved amino acid changes at key residues within the epitope that would eliminate antibody binding. Hence, approximately 99% of the rabies viruses that can be encountered are predicted to be recognized by the CR57 antibody.
In two bat virus isolates (AF346857 and AF346861), the amino acid changes within the CR57 epitope were identical to those observed in some of the EJB viruses (i.e., KLCEVP). However, none of the 229 rabies virus isolates contained an aspartic acid at position 182 of the mature glycoprotein, as was observed in the EJB viruses. Apparently, such genotype 1 rabies viruses do not exist in nature. In contrast, Mokola and Lagos bat viruses do contain an aspartic acid at position 182 in combination with a proline at position 231. In vitro analysis of neutralizing activity of CR57 towards representative viruses of genotypes 2 to 7 revealed that CR57 neutralizes genotypes 4 to 7 but not genotypes 2 and 3 (data not shown). The results contradict earlier reports showing that CR57 only covers genotype 1 viruses (8
). This might be explained by the mutations we observed in the rhabdoviral pSPBN SO57 vectors compared to the originally described MAb 57 sequence (16
), which potentially could result in altered antigen binding properties. Because the PER.C6-produced CR57 completely matches the MAb 57-amino-acid sequence, the studies formally cannot be compared because different MAbs were used. In addition, it could be explained by the use of different virus isolates as representatives of lyssavirus genotypes 2 to 7. Apparently, mutation of the key residue K to R in genotypes 4 and 5 (Table ) at position 1 of the minimal binding region of CR57 is tolerated as well as an isoleucine at position 5 (genotypes 4, 6, and 7). In contrast, changing key residues K to T at position one and V to K at position five eliminates neutralization of Mokola and Lagos bat viruses by CR57.
Our study showed that one of the most potent rabies virus-neutralizing human MAbs recognizes a novel highly conserved epitope on the glycoprotein of rabies viruses. This epitope was shown to be linear and to contain four key residues for binding of the antibody. All rabies virus variants escaping neutralization by this MAb had nonsilent mutations in the key residues of the epitope. The replacement of the wild-type amino acids within a peptide that encompasses the minimal binding region with those observed in the glycoprotein of the escape eliminated antibody binding. The second antibody that we characterized, CRJB, recognized a conformational epitope that partially overlapped with the CR57 epitope, identifying a neutralization epitope complex. Rabies virus mutants escaping from CRJB completely escaped CR57 neutralization. These results argue for the inclusion of only one of these two antibodies in a cocktail product aimed at replacing HRIG.