Identification And Experimental Verification Of Protective Antigens Against Streptococcus Suis Serotype 2 Based On Genome Sequence Analysis

Streptococcus suis (S.suis) is an important cause of meningitis, endocarditis, septicaemia, polyarthritis, polyserositis, pneumonia and acute death among swine, which inflicts heavy losses to the industry of pig breeding in the world every year. Of the known 35 serotypes, serotype 2 is most pathogenic, prevalent, and liable to infect people. In the recent outbreak of the serious public health incident, the large-scale human infection of S.suis 2, there emerged the new clinical manifestation of toxic shock syndrome, which prompts us to strengthen the prevention and control of the infection of S.suis 2. As is proved by practice, vaccine is one of effective means to control and eradicate infectious diseases. However, the research and development of subunit vaccine of S.suis 2 is seriously hampered by the lack of clear understanding of virulent-related factors and pathogenesis mechanisms of S.suis 2. Therefore, by now there has been no clinically applicable vaccine available for effective prevention of S.suis 2 infection.The development of microbial genomics and bioinformatics has brought with it the new revolution in vaccine research, with the emergence of vaccine development strategy based on genome. Pizza et al reported that through the analysis of genome sequence of serogroup B Neisseria meningitidis (MenB) with bioinformatics tools, 570 genes encoding secreted proteins and surface-exposed proteins were selected, later cloned and expressed one by one, and then a total of 350 candidate antigens successfully expressed in Escherichia coli were purified and used to immunize mice, finally 25 candidate antigens that could induce a bactericidal antibody response were identified. Such work has laid foundation for the development of effective vaccine against MenB. Currently, the research strategy of reverse vaccinology has already been successfully applied to the study of vaccines for Bacillus anthracis,Chlamydia pneumoniae, Streptococcus pneumoniae, etc.In this study, we screen the novel vaccine candidates from the whole genome sequence of S. suis 2 through prediction of surface-exposed proteins, secreted proteins and putative virulence-related proteins in silico software, and then express interest genes in prokaryote system, finally evaluate the vaccine efficacy of the purified molecules in animal infection model to lay a foundation for the development of subunit vaccine of S. suis 2.The experimental contents and findings are as follows:1. Surface-exposed proteins, secreted proteins and virulence-related proteins are promising candidates for subunit vaccine, so they are considered as the targets of bioinformatics selection in this study. The procedures are as follows:①The subcellular localizations of 2194 putative proteins of strain 05ZYH33 are predicted via Cell-Ploc package, and secreted proteins are selected. The result shows that, of the 2194 putative proteins, there are 75 secreted proteins.②Cell wall-attached proteins are main surface proteins of gram-positive bacteria, which generally contain cell wall anchoring motifs, like cell wall sorting signal motifs (LPXTG, IPXTG, NPKTG, NXZTN, LPXAG, FPXTG, LPXTN and LPXTS) and lysin motif (LysM). 76 cell wall anchoring proteins are obtained from 2194 putative proteins through the search of BioEdit software and via Search Pfam at the website of Sanger institute.③10 putative virulent-related factors (3 known virulent factors of S. suis 2) and 15 proteins homologous to protective antigens previously characterized in other bacterium species or similar to immunogens in database are obtained via BLAST search. In all, 155 different candidate genes are identified from the genome of strain 05ZYH33 of S.suis 2. To shorten targets, we collect a number of references in order to pick out genes encoding possible protective antigens in S. suis or other bacterium species such as Streptococcus pneumoniae, Group B Streptococcus and so on from 155 genes. In this way, we initially pay attention to 25 molecules, and then we select 10 molecules with the greatest possibility of protective antigens from 25 vaccine candidates (RfeA,IBP,GDH,CWSP,ESA,SIP,SLY,FBP,VaA and SerP).2. Cloning, expression and purification of candidates.①The genes encoding target antigens were respectively amplified by PCR, and then were ligated into the prokaryotic expression vector pET-30b(+).②Recombinant plasmids were transformed into Escherichia coli Rosetta for expression of target proteins. Eight of ten candidate molecules except VaA and SerP were successfully expressed in E.coli Rosetta.③The recombinant fusion proteins harboring 6-histidine tag were abundantly induced for expression, and then were purified by affinity chromatography. The purified recombinant proteins showed a single protein band upon SDS-PAGE after being stained with Coomassie brilliant blue R250. Finally, purified proteins were dialyzed gradually to promote refolding or desalting, and then were freezed drying.3. Protective evaluation of expressed antigens in mouse infection model.①Eight candidate antigens were used to immunize mice, and antibody titer was determined by ELISA. The eight molecules could all stimulate mice to produce immune responses of varying degrees. Among them, anti-SIP serum presented the lowest antibody titer (1∶12800), while RfeA and ESA stimulated mice to produce the most powerful immune response, of which antibody titers were both 1∶819200. The antibody titers generated by immunization with IBP or CWSP were respectively 1∶102400 and 1∶204800. GDH and FBP stimulated mice to produce the same level of antibody as did SLY (1∶25600).②One week after the last time of immunization, 5LD50 dose of strain 05ZYH33 was used to challenge mice. Mice were observed for mortality in control group and immunization groups, of which survival rates were calculated. Differences of the overall survival rates between mice in control group and that in immunized groups were analyzed by Statistics software. The survival rates of mice in the groups of RfeA (9/10), ESA (7/10), SLY (6/10) and IBP (6/10) are signifinantly higher than those of mice in the control group (1/10) (P<0.029). There exist no significant differences between the survival rates of the mice in the groups of GDH (2/10), CWSP (1/10), SIP (3/10) and FBP (4/10) and that of the mice in the control group (P>0.05). These results indicate that RfeA, ESA, SLY and IBP are capable of effectively stimulating BALB/c mice to produce protection against the lethal challenge with high virulent strain 05ZYH33 of S.suis 2.③IgG isotypes of antisera that were specific to 4 antigens conferring protection against S.suis 2 were assessed. The results indicate that RfeA, ESA and IBP induce Th2-like response, whereas SLY stimulates a mixed Th1-Th2 type pattern.4. Cellular localization of protective antigens in S.suis 2. 96-well microplate coated with culture supernatant of Strain 05ZYH33 was used for detecting preimmune serum, antisera specific to RfeA, ESA, SLY or IBP. The results indicated that supernatant of S.suis 2 could react with anti-RfeA serum, anti-ESA serum and anti-SLY serum respectively, namely RfeA, ESA and SLY were secreted proteins of S.suis 2, whereas those could not react with anti-IBP serum, suggesting that IBP was not secreted into supernatant by S.suis 2. Subsequently, fluorescence activated cell sorter (FACS) was used to analyze IBP localized on surface of S.suis 2. The result revealed that anti-IBP serum could not combine with the surface protein of S.suis 2, in other words, IBP was not surface protein of S.suis 2. Therefore, IBP was of possible cytoplasmic protein.In a word, 155 different genes encoding surface-exposed proteins, secreted proteins and virulent-related factors were identified from the genome of strain 05ZYH33 of S.suis 2 using bioinformatics softwares and web tools. To shorten targets, after collecting a number of references, we selected 10 genes encoding molecules with the greatest possibility of protective antigens from 155 genes for expression in prokaryote system. Eight proteins successfully expressed were used for immunization in BALB/c mice for evaluation on vaccine efficacy. RfeA, ESA, SLY and IBP afford protection against lethal challenge of highly pathogenic strain 05ZYH33 of S.suis 2. Immunoprotection function of SLY is consistent with reports in other references. However, it is in this study that the immunological competences of RfeA,ESA and IBP have been explored for the first time. Identification of the novel protective antigens lays a foundation for the development of subunit vaccine of S. suis 2.