A pan genome reverse vaccinology approach to prevent Streptococcus agalactiae infection in farmed tilapia

Abstract

Streptococcus agalactiaen(Group B Streptococcus; GBS) is a Gram-positive pathogenic bacterium that causes disease in a wide range of animals including fish. Outbreaks of GBS in farmed fish have substantial economic impacts on the aquaculture industry, especially in tilapia production, a staple food product in tropical and subtropical low- and middle-income countries and the second most widely farmed fish after carp. Although vaccination can effectively prevent disease caused by GBS in fish it often fails after relatively short periods of use. Capsular polysaccharide (CPS) is the major protective antigen in GBS vaccines and a critical virulence factor. There are currently ten CPS serotypes of GBS and, whilst vaccination against one serotype confers protection in fish, it is not cross-protective against other serotypes. In addition, under the strong selective pressure of vaccination-driven adaptive immunity, evolution of novel serotypes can occur. To improve the serotype cross-protective efficacy of GBS vaccines for aquaculture, I employed a pan-genome reverse vaccinology approach to design candidate serotype-independent protective vaccines based on conserved proteins in fish-pathogenic GBS serotypes. Reverse vaccinology exploits recent advances in genomics to predict antigens in the genome sequence. Reverse vaccinology combined with pan-genome can identify the antigens that are conserved across a diversity of strains of a pathogen.nTo identify potential vaccine candidate antigens, a pan-genome was constructed for GBS comprising 82 genomes including representative isolates from wild marine fish and stingrays, farm and companion animals and from human clinical cases in Australia, along with isolates from farmed tilapia in Honduras and representative genomes from all current serotypes available via the NCBI Genbank database. Core genome single nucleotide polymorphism (SNP) analysis indicated dissemination of the aquatic host-adapted ST-261 lineage in wild Australian fish from an ancestral tilapia strain, which is congruent with several introductions of tilapia into northern Australia from Israel and Malaysia during the 1970s and 1980s.nAquatic serotype Ib GBS strains have lost many virulence factors during adaptation, but six adhesins, named ST-261 adhesins, were well-conserved across the aquatic isolates and might be critical for virulence in fish and good targets for vaccine development. Three of six ST-261 adhesins were also conserved across all strains including terrestrial isolates and were chosen as vaccine candidates. Expressions of ST-261 adhesin genes were inversely co-regulated with CPS expression dependent upon carbon source and metal ions in the medium. However, gene transcription was not reflective of surface expression as serum antibodies derived in tilapia against recombinant adhesins did not detect similar variation in the level of ST-261 adhesins in whole cell ELISA or Western blot. This may be due to post-transcriptional processing causing a disconnect between gene and protein expression.nTo evaluate the efficacies of recombinant ST-261 adhesins as vaccines in tilapia, a vaccination and challenge experiment was conducted. All recombinant ST-261 adhesin vaccines resulted in strong antibody response detected by ELISA against the recombinant adhesins. However, no protection from disease was elicited by any of the recombinant vaccines in the challenge model. This may be due to antigenic masking by CPS. It may also be that adhesins are not expressed during the critical stages of proliferation in the host and are required at the mucosal surfaces during pathogen entry via the gastrointestinal tract. Future work should elucidate the role of these ST-261 adhesins in pathogenicity in fish, including where and when they are expressed on GBS during the infection.nPrevention of disease in aquaculture caused by highly antigenically diverse pathogens such as GBS might be achieved through deployment of autogenous (custom-made) vaccines supported by careful surveillance to enable incorporation of variants as they arise. Use of rapid, low cost genomics as employed in this thesis will aid in accurate surveillance and inform vaccine modification as required. Genomics may also aid in defining the epidemiological unit over which an autogenous formulation might be employed, increasing the cost-effectiveness of custom-made autogenous vaccines. In the era of lone healthr, with global antimicrobial use in animal production far exceeding use in human medicine, it is critical that affordable disease prevention methods are promoted in the worldrs fastest growing food production sector.n