J Gen Virol. the animals were intravenously challenged with a genotype 3 mammalian HEV, and necropsied at 4 weeks post-challenge. Viremia, fecal virus shedding, and liver histological lesions were compared to assess the protective and cross-protective abilities of these antigens against HEV challenge in pigs. The results indicated that pigs vaccinated with truncated recombinant capsid antigens derived from three animal strains of HEV induced a strong IgG anti-HEV response in vaccinated pigs, but these antigens confer only partial cross-protection against a genotype 3 mammalian HEV. The results have important implications for the efficacy of current vaccines and for future vaccine development, especially against the novel zoonotic animal strains of HEV. [31]. The objective of this study was to determine if the recombinant capsid antigens derived from swine, rat, and avian HEV strains can induce cross-protection against a genotype 3 HEV challenge in a pig model. Materials and Methods Expression and purification of truncated recombinant capsid proteins derived from swine, rat, and avian HEV strains The N-terminal truncated capsid proteins from swine, rat, meso-Erythritol and avian HEVs were expressed in a bacterial expression Rabbit Polyclonal to RAB34 system to generate the immunogenic ORF2 capsid proteins lacking the 111 N-terminal amino acid residues [32, 36]. Briefly, the truncated ORF2 genes were amplified from respective plasmids containing avian, genotype 3 swine, and rat HEV ORF2 genes with strain-specific primers (Table 1). Each amplified PCR product was gel-purified, digested with restriction enzymes, and subsequently cloned into the pRSET-A bacterial expression vector (Invitrogen). The authenticity of each ORF2 gene insert was verified by sequencing. Each recombinant pRSET-A plasmid was transformed into BL21(DE3)pLysS (Novagen) cells and then grown in an auto-inducing expression system using Overnight Express Instant TB Medium (Novagen) essentially as described [12]. To facilitate the purification of the resulting recombinant capsid proteins, six histidine residues were incorporated into the proteins. Table 1 Oligonucleotides used for amplification of truncated ORF2 proteins and for the detection of HEV RNA (Excel, Microsoft Inc.). A em P /em -value below 0.05 was considered significant. Table 2 Hepatic histological lesion scores at 4 weeks post-challenge in vaccinated and control pigs challenged with a genotype 3 HEV thead th align=”center” rowspan=”1″ colspan=”1″ Vaccine br / br / group /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Pig br / ID# /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Challenge br / inocula /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Liver histological br / lesion score a /th th align=”center” valign=”top” rowspan=”1″ colspan=”1″ Mean histological liver lesion br / Score /th /thead PBS11PBS00.17131140160170290Swine HEV2Genotype 300.33ORF210HEV0121150180401Rat HEV19Genotype 300.33ORF220HEV0221240260311Avian HEV4Genotype 300.00*ORF28HEV0210230250320PBS3Genotype 300.67*5HEV190301391421 Open in a separate window aLymphoplasmacytic scores: 0 = no inflammation, 1=1 to 2 lymphoplasmacytic infiltrates/10 hepatic lobules, 2 = 3 to 5 5 focal infiltrates/10 hepatic lobules, 3 meso-Erythritol = 6 to 10 focal infiltrates/10 hepatic lobules, and 4 = 10 focal infiltrates/10 hepatic lobules [39]. *statistical significance (P .05) RESULTS Characterization of the truncated recombinant capsid antigens of swine, rat, and avian HEV expressed in em E. Coli /em The truncated ORF2 proteins derived from swine, avian and rat HEV were each expressed as insoluble inclusion bodies with an approximate meso-Erythritol predicted size of 58 kDa [41]. Following nickel affinity chromatography purification, the his-tag fused to each protein was properly identified on each of the recombinant fusion proteins using Western blot with IRDy800 (LI-COR Biosciences) conjugated antibodies to poly-histidine tags (Fig. 1A). Further Western blot analyses using anti-HEV ORF2 hyperimmune swine antiserum raised against a genotype 1 human HEV confirmed the expression of swine, rat, and avian ORF2 proteins (Fig. 1B). The antiserum raised against the genotype 1 human HEV cross-reacted to the truncated recombinant capsid antigens from genotype 3 swine HEV, avian HEV and rat HEV (Fig. 1B), further confirming the existence of a single HEV serotype. Open in a separate window Figure 1 Western blot analyses of truncated bacterial-expressed recombinant swine, rat, and avian HEV ORF2 capsid antigensEach lane was loaded with truncated recombinant capsid proteins derived from swine HEV (1), rat HEV (2), or avian HEV (3) HEV. Panel A: A PVDF membrane containing the separated proteins was incubated with IRDye800 conjugated antibody against 6-His residue tags to detect the respective recombinant fusion capsid proteins. Panel B:.