The anaerobic intestinal spirochaete Brachyspira pilosicoli is the causative agent of “intestinal spirochaetosis” (IS), a disease of humans and a number of animal species. 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, and causes significant economic impact in the associated industries. To date attempts to develop a vaccine against B. pilosicoli to protect humans and animals have not been successful.
In this study a reverse vaccinology approach was used, in which 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 vaccine antigens. In silico analysis of the ORFs using a range of bioinformatics algorithms assigned 12 ORF products as periplasmic, outer membrane, or secretory proteins, and these were given a high priority as potential vaccine candidates. The 12 selected ORFs were amplified from a human strain of B. pilosicoli (Wes-B) and cloned. Products from nine ORFS were successfully over-expressed in an Escherichia coli expression system, and then purified using affinity chromatography.
In an in vitro immunogenicity trial all the recombinant proteins except for NAV-P27 were strongly recognised in Western immunoblots by a mouse serum raised against B. pilosicoli strain WesB, and by a subset of convalescent sera from pigs naturally and experimentally infected with B. pilosicoli. In an analysis of in vivo immunogenicity, the post-immunisation mouse sera raised against each recombinant protein reacted strongly with each specific proteins, and also recognised the native
protein in extracts of B. pilosicoli strain WesB. Sequence analysis of four randomly selected ORFs showed that these were highly conserved amongst the genomes of different human and swine strains of B. pilosicoli. Evaluation of all the data obtained in the reverse vaccinology approach resulted in selection of four ORF products (NAV-P3, NAV-13, NAV-22 and NAV-31) as being potentially protective antigens to be analysed for their further efficacy. These four recombinant proteins were assessed for their efficacy as vaccine components in a mouse model of 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 spirochaete colonisation following experimental infection. These proteins used individually or in combination now have the potential to be further developed into a new vaccine to prevent B. pilosicoli infections.
| Identifer | oai:union.ndltd.org:ADTP/222238 |
| Date | January 2008 |
| Creators | movahedi.ar@gmail.com, Abdolreza Movahedi |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Abdolreza Movahedi |
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