A reverse vaccinology approach to identify subunit proteins for use in vaccines against Brachyspira pilosicoli infections in humans and animals

sciences, Murdoch University, Western Australia movahedi.ar@gmail.com The weakly β-haemolytic anaerobic spirochaete Brachyspira pilosicoli is the causative agent of “intestinal spirochaetosis” (IS), a disease which is defined by one cell end attachment to the colorectal epithelium of humans and a number of species of animals. IS has been reported in adults and children worldwide but the prevalence in people living in poor hygienic conditions, indigenous populations, homosexual males, and in immunocompromised people is much higher than in other populations. IS is also widespread in pigs and chickens with significant economic impact in pigs and poultry industries. So far there have been no successful attempts to develop a vaccine against B. pilosicoli to protect humans and animals. In this study, using a reverse vaccinology approach, 24 putative open reading frames (ORFs) derived from a partial genome sequence of B. pilosicoli were subjected to in silico and laboratory screening processes to identify potential efficacious antigens. In silico analysis of the ORFs by several bioinformatics algorithms assigned 12 out 24 ORF products as periplasmic, outer membrane, or secretory proteins with high priority to be selected as potential vaccine candidate rather than remaining 12 products with cytoplasmic and innermembrane subcellular localisations. Meanwhile 4 out of the 24 ORFs failed to be well distributed amongst 25 genomic DNA of different B. pilosicoli strains using distribution study by PCR analysis. Results obtained from both in silico and distribution studies resulted in selection of 12 ORFs for further screening steps. The 12 selected ORFs were amplified from a human strain of B. pilosicoli (Wes-B), and cloned where 9 of their products were successfully overexpressed in an Escherichia coli expression system followed by purification using affinity chromatography. In in vitro immunogenicity trail using Western immunoblot, all the nine recombinant proteins except NAV-P27 protein strongly recognised by a mouse serum raised against B. pilosicoli strain WesB and a subset of convalescent sera from pigs naturally and experimentally infected with B. pilosicoli. In in vivo immunogenicity, the post- immunisation mouse sera raised against each recombinant protein had strong reactivities with each specific proteins and also mouse sera were recognised strongly by native protein extract of B. pilosicoli strain WesB using Western immunoblot. Conservation study using sequencing analysis on four random selected ORFs as the representees of the whole subset of the ORFs proved that these ORFs were highly conserved amongst the genomes of different human and swine strains of B. pilosicoli. Evaluation of data obtained from dry and wet screening steps of the reverse vaccinology approach resulted in selection of four ORF products including NAV-P3, NAV-13, NAV-22 and NAV-31 as potential protective antigens to be analysed for their further efficacy. Four recombinant proteins (NAV-P3, NAV-13, NAV-22 and NAV-31) were assessed for their efficacy in a mouse model of human IS, where the animals were challenged with a human strain of B. pilosicoli. The proteins all induced systemic and local antibody responses, and tended to reduce bacterial colonisation. These proteins used individually or in combination now have the potential to be further developed into a new vaccine to prevent B. pilosicoli infections.