Screening Of Protective Antigens Against Streptococcus Suis Serotype 2

Streptococcus suis serotype 2 (SS2) infection has become a public health concern due to its zoonotic capability to cause severe infections in slaughterhouse workers and those who handle infected pork. Two outbreaks have occurred in humans in China with high mortality (19%-56%). One that happended in 1998 invovled 14 humans who died out of 25 infected cases. Another one that happended in 2005 included 215 reported cases and 39 deaths. In these outbreaks, toxic shock syndrome (TSS) was a major etiological and mortality factor.The mechanisms involved in the pathogenesis and virulence of S. suis are not completely understood, and attempts to control the infection are hampered by the lack of an effective vaccine. The known virulence factors and protective antigens of S. suis includes CPS, muramidase-released protein (MRP), extracellular factor (EF), suilysin (SLY),Enolase, 6-phosphogluconate-dehydrogenase (6PGD), Sao, etc. Some of these proteins had been proved to be able to provide certain degree of protection against the challenge of S. suis but not good enough yet.In this study, using an immunoproteomic approach, we identified 32 cell wall proteins (CWPs) and extracellular proteins (ECPs) with high immunogenicity in SS2. Among these proteins, eight known cell wall attached proteins, including 6 proteins containing the LPXTG motif, were identified in this work. The other proteins includes 5 non-cytoplasm proteins, and 19 cytoplasm proteins. Some differentially expressed proteins were identified by comparative proteomic analysis between the highly pathogenic SS2 and the moderately pathogenic SS2, and the differences were confirmed in the following study.Ten selected proteins were expressed in E. coli, and the amplified gene sequences were inserted into pET-32a(+) and transformed into E. coli strain BL21 (DE3). At the same the time, MRP was expressed as the positive control for further study. The polyclonal antisera against each recombinant protein were obtained from Wistar rats, and the immunized rats were bled 7 days after the final immunization. To determine the prevalence of those selected proteins in different SS2 strains, CWPs of 16 different SS2 strains were analyzed by Western blot with specific antiserum against each of the 10 recombinant proteins. Five proteins (SSU98₀171, SSU98₁094, SSU98₁549, SSU98₁819 and SSU1664) were detected in all 16 strains tested. SSU98₀201 were not detected in S. suis 1330. SSU98₀156, SSU98₀197, SSU98₀267, SSU98₁675 were defined as differentially expressed proteins between differently pathogenic SS2. SSU98₁675 could be used to distinguish the domestic highly pathogenic SS2 and foreign pathogenic SS2, while SSU98₀197 and SSU98₀267 size variants were expressed in differently pathogenic SS2.The recombinant antigens except SSU98₀156 were further evaluated. We tested the reactivity of 6 patient sera as well as 5 healthy individuals to the recombinant antigens. Besides MRP, patients showed significantly higher antibody levels against 4 antigens (SSU98₀197, SSU98₁094, SSU98₁549 and SSU1664). Furthermore, we assessed the specific antibody responses to the recombinant proteins by using sera isolated from both infected piglet and piglets immunized with formaldehyde-killed bacteria. After experimental infections, protein SSU98₀197, SSU98₁094, SSU98₁549, SSU98₁675 and SSU1664 induced significant antibody responses. In comparison to the sera before immunization, specific antibodies against protein SSU98₀197, SSU98₀267, SSU98₁094, SSU98₁549 and SSU1664 were significantly increased in the immunized piglets. Opsonophagocytic killing abilities of specific antibodies against the recombinant antigens were evaluated by the standard bactericidal assay. Antisera against 6 proteins (SSU98₀171, SSU98₀197, SSU98₀267, SSU98₁094, SSU98₁819 and SSU1664) enhanced the inhibition of S. suis growth in human blood.Taken together, 7 antigens were selected for futher screening protective antigens. Addtionally, to avoid the interference of TRX in the protective capacity assay, we expressed 7 antigens using pET-28a(+) expression vector, and 5 antigens were expressed and purified successfully. CD-1 mice were immunized with recombinant antigens mixed with QS-21. The mice were challenged with 1.5×LD50, respectively, the survival rates of groups immunized with SSU98₀197, SSU98₁094, SSU98₁675, MRP were 70%, 67%, 60%, 70%. When the mice were challenged with higher dose, the survival rates decreased to 20%, 20%, 50%, 20%. In both protection experiments, the statistical differences between recombinant antigens vaccinated groups and QS-21 vaccinated group were significant, while SSU1664 had no protective capacity.Then SSU98₀197, SSU98₁094, SSU98₁675 were assembled to achieve better protective effects. The mice were challenged with 6.6×LD50, and the survival rates of groups immunized with SSU98₀197, SSU98₁094, SSU98₁675, SSU98₀197 plus SSU98₁675, SSU98₁094 plus SSU98₁675, SSU98₀197 plus SSU98₁094, 3 proteins combined vaccine were 30%,30%,30%,50%,40%,30%,60%, respectively. And the statistical difference between 3 proteins combined vaccinated group and SSU98₀197 vaccinated group was significant.Furthermore, the protective capacity of 3 proteins combined vaccine and SSU98₀197 plus SSU98₁675 combined vaccine was evaluated. When the mice were challenged with 4.8×LD50, respectively, the survival rates of groups immunized with 3 proteins combined vaccine, SSU98₀197 plus SSU98₁675 combined vaccine were 60% and 40%, respectively. The statistical difference was not significant. When the mice were challenged with 7×LD50, respectively, the survival rates decreased to 50% and 20%, and the statistical difference was significant. This indicated that the 3 proteins combined vaccine had the better protective capacity against high dose challenge.The protective antigens SSU98₀197, SSU98₁094, SSU98₁675 were named SIP1, Lpp36, SIP2. Two deletion mutants 05ZYΔLpp36 and 05ZYΔSIP2 were constructed successfully.