The cocktail of mAbs being considered by the WHO as a potential alternative to current RIG therapy, E559 and 62-7-13 have been shown in general to be able to neutralise RABV viruses (Section 6.2 and (Muller et al., 2009)) however there have been instances where certain phylogroup I viruses have been demonstrated to escape neutralisation by these mAbs (Muller et al., 2009). Data from Section 6.2 suggested that mutations to CVS antigenic site I inhibited neutralisation by mAb 62-7-13 and mutations to site II influence E559 neutralisation, thus the sequences of antigenic sites I and II in a number of viruses known to escape neutralisation were examined in order to identify any
175 specific residues which may determine neutralisation. The viruses and their antigenic site sequences can be seen in Table 6.3.
A critical role for residue Lys 245 in 62-7-13 neutralisation was identified (Both et al., 2013) and it was found that an escape mutant of E559 had mutations of Leu to Arg at 57 and Lys to Glu at 217 (Muller et al., 2009). All three of these residues are located in antigenic sites: 245 in site I and 57 and 217 in site II. It was therefore postulated that any mutations at these positions would affect neutralisation by the respective mAbs. It can be seen from Table 6.3 that both PV and EBLV-1 were not neutralised by 62-7-13 and it was clear that both contain mutations at position 245 with Glu and Arg residues in place of Lys, respectively. This confirms the critical role of Lys 245 as well as explaining the lack of neutralisation of PV, EBLV-1 and all PTs containing LBV antigenic I.
Kelev is a strain of RABV and has been repeatedly shown to escape neutralisation by E559 (Muller et al., 2009). From Table 6.3 it was clear that Kelev contains mutations in site II at the same positions as the E559 escape mutant. This suggests that these residues may have a critical role in E559 neutralisation, however the USA Bobcat isolate had the same sequence in site II as both CVS and PV which were neutralised by E559. This suggested that there may be epitopes outside of antigenic site II which play a role in E559 neutralisation and it may be that these mutations affect the conformation of site II as it is the only discontinuous epitope on the lyssavirus glycoprotein so is likely to be the most susceptible to be affected by mutations resulting in conformational changes.
Despite the Bobcat isolate neutralisation profile against E559, the significance of the residues at positions 57 and 217 in the Kelev RABV strain was further investigated. An isolate of Kelev was available in the laboratory so a series of PCR products were
176
Table 6.3 : Sequence of antigenic sites and neutralisation profiles of a panel of viruses which escape neutralisation by the current mAb cocktail being considered by WHO as an alternative to current serological therapies. Residues in red differ from CVS- 11 which is neutralised by both mAbs in the cocktail.
Virus Site I (245-250) 62-7-13 Neutralised Site IIa (217- 291) Site IIb (53-61) E559 Neutralised CVS-11 KLCGVL Y KRA GCTNLSGFS Y PV QLCGVL N KRA GCTNLSGFS Y
Kelev, Israel KLCGVL Y TRA GCTNRSEFS N
Bobcat, USA KLCGVL Y KRA GCTNLSGFS N
EBLV-1,
177 generated from different sources of Kelev: from infected mouse brain homogenate and from an infected tissue culture cell pellet. Previously next generation sequencing (NGS) using the 454 platform had identified four different variants of Kelev, each with a different combination of aa at positions 57 and 59 (Denise Marston – unpublished data). The variation at these sites was of interest due to their presence in antigenic site IIb so in order to determine whether the same variants were present in the mouse brain
homogenate or the cell pellet PCR reactions were set up to generate the entire G ORF as product. These 1.5 kb PCR products were sequenced using the MiSeq platform
(Illumina) in house at the APHA Central Sequencing Facility and the results were analysed using Tablet (version 1.14.10.20). The consensus sequence used to map the reads was the glycoprotein ORF of rabies virus isolate RV2324 (KF154998.1).
A number of nucleotide (nt) variants were identified in the cell pellet product whereas only one nt variant was found in the mouse brain PCR products. The variant identified in all PCR products was a T to G at position 170. This nt variant was identified in 99.9% of all mouse brain reads and 72.4% of all cell pellet reads suggesting that this variation is under substantial selective pressure. In addition to the nt 170 variant in the cell pellet, a variant consisting of a C to T substitution at position 169 was identified in 32.2% of all reads. There were also a limited number of variants with a G to A substitution at position 175 and some with an A to G substitution at position 176. All of these variations are within antigenic site IIb and result in amino acid changes in the glycoprotein sequence.
The significance of the substitutions at positions 169 and 170 is that these determine which aa will be present at position 57 in the glycoprotein; the precise residue which appears to influence E559 neutralisation. The C to T substitution at 169 in combination
178 with the T to G substitution at 170 results in a tryptophan residue at aa 57 whereas the 170 substitution alone results in an arginine residue at aa 57. The consensus sequence of Kelev along with CVS, PV and EBLV-1 contain a leucine residue at position 57 whereas those viruses which escape neutralisation by E559; the Kelev isolate from this
investigation along with MOKV and IKOV (David Selden – unpublished data) contain arginine, glutamate and glycine residues at position 57, respectively. This therefore led to the hypothesis that leucine 57 plays a critical role with regard to mAb E559
neutralisation.