A Recombinant Novirhabdovirus Presenting at the Surface the E Glycoprotein from West Nile Virus (WNV) Is Immunogenic and Provides Partial Protection against Lethal WNV Challenge in BALB/c Mice

Abstract

West Nile Virus (WNV) is a zoonotic mosquito-transmitted flavivirus that can infect and cause disease in mammals including humans. Our study aimed at developing a WNV vectored vaccine based on a fish Novirhabdovirus, the Viral Hemorrhagic Septicemia virus (VHSV). VHSV replicates at temperatures lower than 20°C and is naturally inactivated at higher temperatures. A reverse genetics system has recently been developed in our laboratory for VHSV allowing the addition of genes in the viral genome and the recovery of the respective recombinant viruses (rVHSV). In this study, we have generated rVHSV vectors bearing the complete WNV envelope gene (EWNV) (rVHSV-EWNV) or fragments encoding E subdomains (either domain III alone or domain III fused to domain II) (rVHSV-DIIIWNV and rVHSV-DII-DIIIWNV, respectively) in the VHSV genome between the N and P cistrons. With the objective to enhance the targeting of the EWNV protein or EWNV-derived domains to the surface of VHSV virions, Novirhadovirus G-derived signal peptide and transmembrane domain (SPG and TMG) were fused to EWNV at its amino and carboxy termini, respectively. By Western-blot analysis, electron microscopy observations or inoculation experiments in mice, we demonstrated that both the EWNV and the DIIIWNV could be expressed at the viral surface of rVHSV upon addition of SPG. Every constructs expressing EWNV fused to SPG protected 40 to 50% of BALB/cJ mice against WNV lethal challenge and specifically rVHSV-SPGEWNV induced a neutralizing antibody response that correlated with protection. Surprisingly, rVHSV expressing EWNV-derived domain III or II and III were unable to protect mice against WNV challenge, although these domains were highly incorporated in the virion and expressed at the viral surface. In this study we demonstrated that a heterologous glycoprotein and non membrane-anchored protein, can be efficiently expressed at the surface of rVHSV making this approach attractive to develop new vaccines against various pathogens.

Complete Protection against Influenza Virus H1N1 Strain A/PR/8/34 Challenge in Mice Immunized with Non-Adjuvanted Novirhabdovirus Vaccines

“…Recombinant rIHNV-HA and rVHSV-HA were generated as described previously [ 10 ]. Briefly, KpnI unique restriction enzyme sites were introduced by site-directed mutagenesis in the non-coding regions between N and P genes.…”

“…The incorporation efficiency of foreign antigens at the surface of recombinant Novirhabdoviruses with an expression cassette between N and P genes has been previously demonstrated [ 10 ]. The HA antigen could not be clearly visualized on SDS-PAGE after Coomassie blue staining as it co-migrated with the G glycoproteins of IHNV and VHSV platforms (data not shown).…”

“…The recombinant cassette integrated into VHSV was constructed as previously described [ 10 ]. For rIHNV-HA, a unique KpnI restriction enzyme site was integrated by site-directed mutagenesis (QuikChange Site-directed mutagenesis Kit, Stratagene) using specific primers ( Table 1 ) in between nucleoprotein N and polymerase-associated P genes of the full-length genome pIHNV [ 13 ].…”

“…For the first animal experiment (experiment #1), groups of 6 week-old female BALB/c mice (n = 8) (Janvier Labs) were immunized subcutaneously in the back neck with 30 μg of purified rIHNV-HA, rVHSV-HA or rVHSV-wt resuspended in 100 μL of TEN buffer supplemented with Freund’s adjuvant (complete form for the first immunization and incomplete form thereafter) at a 1:2 ratio [ 10 ]. In this first animal experiment, two mice died before the first immunization during the pre-immunization blood sampling at day -3 because of human mistake.…”

“…Reverse genetics systems for novirhabdovirus have been developed in the past, allowing the viral genome manipulation and the recovery of viable recombinant viruses. Furthermore, the immunogenic potential of novirhabdovirus as a recombinant vaccine platform has already been demonstrated in mice with a recombinant West Nile Virus antigen [ 10 ]. Novirhabdovirus platforms offer numerous advantages: 1) they are easy and fast to construct, 2) they grow to high titer in fish cell lines, 3) they can incorporate a large panel of foreign antigens at their surface (glycoproteins or protein domains) by adding the signal peptide and the transmembrane domain derived from the novirhabdovirus glycoprotein to the antigen of interest 4) they are self-adjuvanted and 5) they are naturally inactivated over 20°C and thus safe to use in mammals [ 11 , 12 ].…”