The Study On The Protective Effect Of Immunization With Pneumococcal Candidated Protein Vaccine ClpP

ObjectiveStreptococcus pneumoniae is the major pathogen causing invasive and localized diseases including sepsis, meningitis and pneumoniae and otitis media. The 23 valent capsular polysacchride vaccine is useful in adults but fails to protect those at highest risk of disease at the youngest and oldest ages, and although the current 7-valent conjugate vaccines are effective against invasive disease caused by the vaccine-serotype strains, but vaccine coverage is limited, the cost of production is very high and this conjugate vaccines are not applicable in the developing countries. Futhermore, replacement by invasive nonvaccine-serotypes in vaccinated individuals is a grim problem confronting us. The development of a vaccine against Streptococcus pneumoniae has been complicated by the existence of at least 90 antigenically distinct capsular serotypes. Common protein-based vaccines could represent the best strategy to prevent pneumococcal infections, regardless of serotype. ClpP, a member of HSP family, which was a novel protein vaccine candidate, is important for S. pneumonia transferation from nasalpharynx mucosa to blood and further survival. Besides, it has been shown that ClpP is also crucial in the process of modulating the expression of other virulence protein factors in S. pneumoniae. Now, we evaluate its conservation and immunogenicity and provide evidences for further using it as a vaccine protein against pneumococcal invasive infections independening of serotype.Methods1. Nucleotide sequence computer analysis. Genomic DNA prepared from each of 12 pneumococcal strains according to the manufacture instructions were used as templates for PCR amplification using ClpP gene-specific primer. The subsequent PCR products were cloned into PMD-18T vectors, and the recombinant plasmids were transformed into E.coli DH5α. The derived constructs were sequenced to confirm the inserted ClpP genes and analyze their Sequences. Sequence homology searches were performed with gene and protein sequences using BLAST.2. Western blot analysis for ClpP protein expression in different S. pneumoniae strains. 12 pneumococcal isolates were harvested from in C+Y culture, washed in sterile PBS, resuspended in 1×load buffer, and incubated for 5 minutes in 100℃. And then these whole bacterial lysates were subjected to sodium dodecyl sulfate–12% polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes for Western blot analysis. Individual blots were reacted with hyperimmune mouse serum specific for ClpP diluted 1:400. Detection of antigens was performed by an indirect antibody immunoassay using HRP-labeled anti-mouse IgG diluted 1:5000 in PBS-T and DAB staining.3. Pneumococcal challenge of actively immunized mice. Pneumococcal antigens were purified from recombinant Escherichia coli expressing ClpP cloned gene. 6- to 8-week-old female BALB/c mice were immunized intraperitoneally with either ClpP or PBS plus alum. Each mouse received three doses of 20μg antigen or PBS in 100 mg of alum adjuvant at 14d intervals. Sera were collected from mice and analyzed by enzyme-linked immunosorbent assay (ELISA) 1 week after the third immunization. Intraperitoneal-challenge experiments with 12 different serotypes of Streptococcus pneumoniae were carried out 2 weeks after the third immunization of mice, and we compared their median survival times and survival rates respectively by Mann Whitney U test and Fisher exact test.Days that the specific antibody titer in mice remained constant for were recorded.4. Passive transfer of protein-specific Abs confers protection. In order to evaluate the role of Abs in protection, Abs purified from sera from immunized rabbits prior to a lethal challenge. 12 stains were cultured in C+Y culture. 2ml bacterial solution of each stain mixed with anti-ClpP serum, equal volum bacterial solution of same stain mixed with non-specific serum. All incubated in 4℃overnight. After mixture was observed through microscope, every kind of mixture challenged 12 BALB/c mice, and we compared their median survival times and survival rates respectively by Mann Whitney U test and Fisher exact test.Results1. Presence of selected ClpP gene in different S. pneumoniae isolates. PCR amplification was used to demonstrate the presence of genes encoding the pneumococcal ClpP proteins in 12 different S. pneumoniae strains. The results revealed that band corresponding to ClpP was detected in all strains of S. pneumoniae analyzed. Using specific primers, PCR amplification exhibited single bands of identical size (591 bp for ClpP gene) in all strains. Then these PCR products were cloned into PMD-18T vectors and DNA sequencing showed that ClpP genes among different pneumococcal strains were highly consistent and ClpP proteins exhibited 99.5 % amino acid sequence identity.2. Characterization of ClpP protein expression in pneumococcal isolates. The whole-cell lysates of representative strains of different serotypes were separated on SDS-PAGE and electrotransferred onto PVDF membranes. Antisera specific for ClpP reacted with a single band of molecular mass 21 kDa in fractions of all of the strains of S. pneumoniae tested. Our observation that the ClpPs of different strains are of the same sizes suggests that different peumococcal strains could express ClpP without heterogeneity at protein level.3. Protection of BALB/c against 12 strains of S. pneumoniae by immunization with ClpP. ELISA analysis of sera from groups of mice immunized with the purified ClpP or PBS plus alum shows that strong antigen-specific antibody responses were generated in BALB/c immunized with the purified ClpP plus alum. Mock immunized mice had relatively undetectable titers of cross-reactive antibody. Groups of BALB/c mice immunized with ClpP were challenged with 12 strains of serotype-different pneumococcus respectively. The median survival times for mice that received ClpP plus AlPO4 were significantly longer than those for mice that received the corresponding AlPO4 alone (P <0.05 in all cases and P <0.002 in most cases), and the survival rates for mice that received ClpP in AlPO4 were significantly greater than those for mice received the corresponding AlPO4 alone (P <0.01 in all cases and P < 0.0004 in most cases). There was no mouse that could survive after invasive challenge with indicated virulent strains in control group, more than one half of 12 mice in each group could survive in all cases after the treatment of ClpP antigen.This studies shows active immunization with ClpP could elicit serotype-independent and effective protection against fatal invasive pneumococcal infection .In addition, the specific antibody titer in mice kept constant for at last 98 days.4. Passive protection by immunization of ClpP-specific antibodies. To confirm that protection was antibody mediated, As observed for active immunization, protected mice were those that received the mixture containing anti-ClpP. Abs reacted with the surface-accessible pneumococcal ClpP and resulted in agglutination. Passive immunization with anti-ClpP sera could elicit effective protection against fatal invasive pneumococcal infection with these 12 virulent pneumococcal strains of different serotypes which further confirmed the protection effect of active treatment with ClpP antigen.Conclusion1. ClpP gene sequences among different strains of S. pneumoniae were highly consistent.2. Different serotype pneumococcal strains could express ClpP without heterogeneity at protein level.3. Active immunization with ClpP could elicit serotype-independent and effective protection against fatal invasive pneumococcal infection. Besides that, the specific antibody titer in mice remained constant for longer time.4. Passive immunization with anti-ClpP sera could elicit effective protection against fatal invasive pneumococcal infection regardless of serotype, further confirmed the protection effect of active treatment with ClpP antigen.