| ObjectiveStreptococcus pneumoniae is the major pathogen causing invasive disease in infants, the elderly, and the immunocompromised individuals, including sepsis, meningitis and pneumoniae.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-type strains, yet streptococcus pneumoniae can be divided into more than 90 serotypes according to the immunochemistry of their capsular polysaccharide, so vaccine coverage is limited, the cost of production is ver 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. Therefore, development of effective protein-based vaccines is pretty urgent.PspA is one of the most important virulence factors playing some crtical roles in the pathogenesis of pneumococcal infection, and it is one of the earlist pneumococcal candidate vaccines.CbpA is a surface protein is necessary for pneumococcal meningitis. Streptococcus pneumoniae encounters heat stress after penetration from the nasal mucosa(30℃to 34℃) into the blood and/or meninges (37℃) during the pathogenic process, and ClpP plays some important roles in helping S.pn adapt to the host environment and cause invasive diseases.In this study, we attempte to express those proteins and compare abilities of PspA, PspC and ClpP or serums containing their specific polyclonal antibodies to elicit protection against pneumococcal infection and examined the possibility that an immunization combining these proteins or their polyclonal antibodies might elicit better protection against pneumococcal infection.Methods1 The production of recombinant PspA, CbpA, and ClpP was achieved by PCR amplification of pneumococcal genes, with subsequent cloning into pET32a(+) plasmid. The plasmids were transformed into E.coli BL21. With IPTG induction the proteins were expressed. The recombinant proteins were identified by SDS-PAGE and purified with Ni-NTA column. The antigenicity of proteins was confirmed with anti-His monoclonal antibodies by Western-blot. Hyperimmune mouse sera specific for PspA (anti-PspA), CbpA (anti-CbpA),and ClpP (anti-ClpP) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 were to be identified with these serums by Western blot.2 Active immunization protection assays.96 BALB/C mice were divided 8 groups randomly.For this experiment, BALB/c mice (12 per group) were primed with either PspA, PspC, ClpP, PspA plus PspC, PspC plus ClpP, ClpP plus PspA, or a combination of PspA, PspC, and ClpP, each in CFA(1:1 ratio [vol/vol]) and each mouse received 10 ug of each protein on day zero ,and boosted with the same concentration of each protein in IFA (1:1 ratio [vol/vol]) on day 14,and on day 28,mice received the last dose of each protein in PBS. Mice injected with respective adjuvants in PBS served as negative controls. All vaccines were administered i.p., and sera were collected from all mice by retro-orbital bleeding 1 week after the third immunization. The sera were pooled on a group-by-group basis and assayed for anti-PspA, anti- PspC and anti-ClpP specific antibodies by ELISA prior to challenge with live pneumococci. After challenging with TIGR4, The mice were then monitored for death over 21 days, and the median survival time of each mouse were recorded.3 Active immunization protection assays, BALB/C mice were divided 8 groups randomly.For this experiment, the groups of 12 BALB/c mice to be challenged were passively immunized with 100ul of hyperimmune serums specific for PspA, PspC, and ClpP by i.p. injection and followed by challenging with TIGR4 24 hours later, the survival time of mice were monitored for 21 days.Results1 The results of didestion with restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene PspA had been cloned into the plasmid pET32a(+). The length of the PspA gene was 1329bp. Compared to the sequence of GeneBank, no mutation was found.A recombinant protein about 66kD was expressed in BL21(DE3) after induction of IPTG. SDS-PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 50% of total baterial protein and the purity of target protein was up to 90%. Western-blot suggested that the rP30 could be recognized by anti-His monoclonal antibodies. Hyperimmune mouse sera specific for PspA(anti-PspA) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 could be identified with these serums by Western blot.2 The results of didestion with restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene CbpA had been cloned into the plasmid pET32a(+). The length of the CbpA gene was 1369bp. Compared to the sequence of GeneBank, no mutation was found.A recombinant protein about 66kD was expressed in BL21(DE3) after induction of IPTG. SDS-PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 20% of total baterial protein and the purity of target protein was up to 90%. Western-blot suggested that the rCbpA could be recognized by anti-His monoclonal antibodies. Hyperimmune mouse sera specific for CbpA(anti-CbpA) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 could be identified with these serums by Western blot.3 The results of didestion with restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene ClpP had been cloned into the plasmid pET32a(+). The length of the ClpP gene was 1369bp. Compared to the sequence of GeneBank, no mutation was found.A recombinant protein about 66kD was expressed in BL21(DE3) after induction of IPTG. SDS-PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 20% of total baterial protein and the purity of target protein was up to 90%. Western-blot suggested that the rClpP could be recognized by anti-His monoclonal antibodies. Hyperimmune mouse sera specific for ClpP (anti-ClpP) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 could be identified with these serums by Western blot.4 Active immunization protection assays, In this experiment, the median survival times for mice that were immunized with single antigen or their combinations were significantly longer than that for mice that received adjuvants alone (p<0.0001), the PspA only delayed the time to death but failed to protect mice from death. Mice that received a combination of three antigens survived significantly longer than those that received single antigen or the combinations of two antigens(p<0.05). The highest survival rate of the various groups of mice was observed with the combination of three antigens, this rate was significantly different from that for mice that received either single antigens or two antigens (p<0.025)except the combination of ClpP and PspA.5 Passive immunization protection assays, mice that received either single serum containing specific antibody or their combinations lived significantly longer than mice that received serum from nonimmunized mice(p<0.001), Similarly, the anti-PspA serum could only elongate the survival time of the mice challenged intraperitoneally by S.pneumoniae TIGR4 but failed to protect these mice from death. the highest protection rate was observed with the combination of three serums containing anti-PspA, anti- PspC, and anti-ClpP polyclonal antibodies, it was significantly higher than those for mice treated with single serum(p<0.05). Conclusion1 The S.pn PspA, CbpA and ClpP genes were clone successfully into the plasmid pET32a(+). Sequencing and sequencing analysis were done and no mutation was found. The expressed and purified recombinant proteins were proved with immunogenicity and comtetence to induce protective immune response. These recombinant proteins can be candidate protein vaccines against S.pn.2 We successfully got the anti-PspA, anti-CbpA and anti-ClpP polyclonal antibodies and they could reacted with whole-cell lysates prepared from S.pneumonie strain TIGR4 specifically.3 Our active immunization protection assays showed that thses antigens could elicit protection against S.pn, and the combination of PspA, CbpA, and ClpP could enhance protection against S.pneumoniae TIGR4 challenged intraperitoneally, suggesting combining different pneumococcal virulence proteins to achive better protection is an applicable way.4 Our passive immunization protection assays showed that thses antibodies could elicit protection against S.pn, and the combination of anti-PspA, anti-CbpA, and anti-ClpP could enhance protection against S.pneumoniae TIGR4 challenged intraperitoneally, which proved our active immunization protection results.
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