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Keywords:

  • ClpP;
  • infection;
  • protein;
  • Streptococcus pneumoniae;
  • vaccine

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Invasive pneumococcal diseases incur significant mortality, morbidity and economic costs. The most effective strategy currently available to reduce the burden of these diseases is vaccination. In this study, we evaluated the protective efficacy of immunizing mice with caseinolytic protease (ClpP) protein antigen whose gene sequences were shown to be highly conserved in different strains of Streptococcus pneumoniae in an invasive-disease model (intraperitoneal infection model), and protection against invasive challenge with 12 different serotypes of S. pneumoniae was assessed in two murine strains. Our findings demonstrated that active immunization with ClpP and passive immunization with antibodies specific for ClpP could elicit serotype-independent protection effectively against invasive pneumococcal infection. Therefore, to our knowledge, this study is the first report that immunization with single pneumococcal ClpP protein antigen could protect against such broad-range pneumococal strains, which thus supports the development of ClpP as a human penumococcal vaccine.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Streptococcus pneumoniae is the leading cause of life-threatening invasive diseases, including meningitis, septicaemia and pneumonia [1,2]. Invasive pneumococcal infection kills more than 1·5 million children each year [3]. Successful implementation of anti-infective therapy has become increasingly difficult because of widespread multiple-antibiotic resistance [4–6]. Therefore, the most effective strategy currently available to reduce the burden of invasive peumococcal diseases is vaccination.

The current use of 23-valent capsular polysaccharide vaccines is effective in adults but fails to protect children under 2 years of age, who suffer the highest rates of invasive pneumococcal infection, and immunocompromised patients show a severely impaired antibody response upon this vaccination [3]. In addition, polysaccharide vaccines do not produce a T cell-dependent immune response that implicates the absence of memory B cells and thus they have a limited period of protection [7]. The conjugate vaccines, with seven to 11 serotypes, could induce a memory immune response and they are useful against invasive infection caused by the vaccine-type strains. However, their protective effects are restricted to a certain number of serotypes, and too much carrier antigen may impair the antibody response to the polysaccharides by antigen competition or carrier-mediated epitope suppression [8,9]. Notably, because of the limited coverage of circulating pneumococcal strains by the conjugate vaccines, the remaining non-vaccine serotype strains will actually benefit from this selective immunological pressure and serotype replacement has occurred in diseases [10–12]. Therefore, a large-scale vaccination with conjugate vaccines may cause serious problems [5]. Besides those above, formulations of the pneumococcal conjugate vaccines must be according to the epidemiology of invasive pneumococcal diseases in different areas, and the high cost of conjugate vaccines prohibits their delivery and application in developing countries [13–16].

The emerging and potential shortcomings of polysaccharide-based vaccines prompt researchers to develop a new generation of pneumococcal vaccines. At present, it is considered that use of protection-eliciting pneumococcal proteins could be an alternative and feasible approach. The candidate protein vaccines against penumococcal infection are mainly their virulence factors, such as pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumococcal surface adhesion A (PsaA), pneumolysin, pneumococcal histidine triad (Pht) proteins PhtB and PhtE, neuraminidases A and B, Pilus subunits, etc. Despite their successes, these protein vaccines have not been ideal. For example, PspAs are divided by three classes and six clades and thus present antigen variability [17–19,20]; PspCs are also variable among pneumococcal strains and there are 11 main groups of PspC [21,22]; PsaA is undetectable on pneumococcal surface and immunization with PsaA could not protect efficiently against invasive pneumococcal infection [23]. Taken together, although these proteins have been shown to be able to elicit a significant level of protection in animal models [24–28], their protective effects are still limited to a subset of pneumococcal strains, and different levels of protection induced by the mixture of these protein vaccines against various pneumococcal strains have been achieved [26,28,29]. Thus, an effective protein vaccine which could protect against broad-range pneumococal strains, based on invariable and conserved antigen, is needed urgently.

In this study, we describe the heat shock protein (HSP) caseinolytic protease (ClpP), whose gene sequences in different serotypes of S. pneumoniae were highly conserved. We also confirmed that immunization with ClpP could protect against invasive challenge with 12 different serotypes of S. pneumoniae, and this protection was assessed in two murine strains (BALB/c and CBA/N mice) by active immunization with ClpP protein antigen and passive immunization with antibodies specific for ClpP.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains and growth conditions

Escherichia coli DH5α (Invitrogen, Carlsbad, CA, USA) was used as the host for routine plasmid cloning. Recombinant proteins were expressed in E. coli BL21(DE3) (Novagen, Darmstadt, Germany). E. coli were cultured in Luria broth supplemented with ampicillin antibiotics. Virulent S. pneumoniae strains (Table 1) were used for challenge experiments. S. pneumoniae was grown routinely on Trypticase soy agar plates supplemented with 5% sheep blood (blood agar) or in C+Y medium. Cultures in the exponential phase were frozen and stored at –80°C in C+Y medium containing 10% glycerol. The viability of bacterial stocks was analysed prior to challenge. Serotype-specific capsule production was confirmed by Quellung reaction.

Table 1.  Mouse-virulent strains of pneumococci used in study of effectiveness of caseinolytic protease (ClpP) as immunogen.
StrainHuman site of originCapsular type
  1. D39 isolate NCTC7466 (National Collection of Type Cultures, UK) was purchased from the NCTC (London, UK), and the other pneumococcal strains were obtained from the CMCC (National Center for Medical Culture Collections, China). CSF, cerebrospinal fluid.

CMCC(B)31011Unknown5
CMCC(B)31109CSF1
CMCC(B)31203Unknown3
CMCC(B)31207Unknown6B
CMCC(B)31216Unknown9V
CMCC(B)31446CSF4
CMCC(B)31507Blood7F
CMCC(B)31614Sputum14
CMCC(B)31687Middle ear18C
CMCC(B)31693Urine19F
CMCC(B)31759CSF23F
D39Unknown2

Production of hyperimmune mouse sera against ClpP

The cloning, expression and purification of ClpP from TIGR4 pneumococci have been described previously [29]. Hyperimmune mouse sera specific for ClpP (anti-ClpP) were generated by immunization of BALB/c mice intraperitoneally with recombinant ClpP protein. Each mouse was primed with 10 µg of ClpP, emulsified in complete Freund's adjuvant (1:1 ratio, v/v) on day 0, and boosted with the same concentration of recombinant protein emulsified in incomplete Freund's adjuvant (1:1 ratio, v/v) on day 14, and on day 28 each mouse received the last dose of 10 µg of antigen in sterile phosphate-buffered saline (PBS). Pooled sera from blood collected 14 days after the final immunization were stored at –20°C for future assays.

Detection of ClpP gene expression in different S. pneumoniae strains

Polymerase chain reaction (PCR) was used to demonstrate the presence of ClpP gene in these 12 different strains of S. pneumoniae. For this purpose, genomic DNA prepared from each of 12 pneumococcal strains according to the manufacturer's instructions were used as templates for PCR amplification by gene-specific primers (5′-CGA ATT CATGAT TCC TGT AGT TAT-3′and 5′-CGA GCT CTT AGT TCA ATG AATTGT TG-3′). The subsequent PCR products were cloned into PMD-18T vectors (Takara, Dalian, China), and then the recombinant plasmids were transformed into E. coli DH5α. The derived constructs were sequenced to confirm the correct insertion of ClpP gene and followed by further detailed sequence analysis

Isolation of S. pneumoniae cell-wall-associated proteins

The pneumococcal cell-wall-associated proteins were isolated according to the methods described by Yother and White [30]. S. pneumoniae were grown to the mid-exponential phase in C+Y medium. The bacteria were then harvested into PBS containing sucrose 20% w/v and pelleted by centrifugation at 6000 g for 10 min. The pellet was resuspended in PBS containing sucrose 20% w/v and centrifuged again as above. The bacteria were then resuspended in 2 ml of 50 mM glycine–NaOH (pH12) containing sucrose 20% w/v. Alkaline extraction of cell-wall-associated proteins was allowed to proceed for 30 min at room temperature with gentle shaking. The suspension was then centrifuged at 15 000 g for 20 min, and the supernatant was collected, adjusted to pH 7 with 1 M HCl, precipitated with acetone and analysed by sodium dodecyl sulphate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE).

Western blot analysis for ClpP protein expression in different S. pneumoniae strains

The pneumococcal cell-wall-associated proteins were subjected to SDS-10% PAGE and transferred electrophoretically to polyvinylidene difluoride membranes (Bio-Rad, Richmond, CA, USA) for Western blot analysis. Individual blots were reacted with hyperimmune mouse sera specific for ClpP diluted 1:500. Detection of ClpP protein expression was performed by an indirect antibody immunoassay using horseradish peroxidase-labelled anti-mouse immunoglobulin G (Sigma, St Louis, MO, USA) diluted 1:5000 in PBS-T and 3′3′-diaminobenzidine staining.

Pneumococcal challenge of actively immunized mice

All animal experiments were approved by the Ethical Committees of ChongQing Medical University. Twenty-four groups of 6-week-old female BALB/c mice (12 per group) were used for this study. Each mouse received intraperitoneally three doses of 10 µg of ClpP protein antigen in formulation with aluminophosphate (AlPO4) at 12–14-day intervals. Mice injected with AlPO4 alone served as negative controls. The recombinant ClpP protein and AlPO4 solutions for animal experiments did not contain any detectable lipopolysaccharide, as determined by the Limulus amoebocyte lysate assay (sensitivity limit 12 pg/ml; BioWhittaker, Walkersville, MD, USA). In another experiment, we used the CBA/N mouse model. CBA/N mice have the Btk (XID) immune-response defect and fail to make natural antibodies to pneumococcal polysaccharides, which make these mice highly susceptible to pneumococcal infections and much more reproducible in their susceptibility than most other strains [31]. Six-week-old CBA/N mice were treated with ClpP under the same conditions as BALB/c mice, and sera were then collected from BALB/c and CBA/N mice by retro-orbital bleeding 1 week after the third immunization. The sera were pooled on a group-by-group basis and assayed for ClpP-specific antibodies by enzyme-linked immunosorbent assay (ELISA).

Intraperitoneal challenge experiments were carried out 2 weeks after the third immunization using 12 different serotypes of S. pneumoniae. Before challenge, the bacteria were grown at 37°C overnight on blood agar and then inoculated into C+Y medium. Bacteria were harvested in early stationary phase at 1500 g for 5 min and suspended in sterile PBS. The bacterial concentration was estimated from the absorbance at 600 nm and confirmed by viable counts on blood agar. For challenge studies, the optimized challenge dose was two logs higher than the 50% lethal dose (LD50) for each strain. The mice were then monitored for death over 21 days and the survival time of each mouse was recorded.

Pneumococcal challenge of passively immunized mice

In order to investigate further whether the protective effects of ClpP protein antigen were antibody-dependent, we tested mouse anti-sera raised against recombinant ClpP antigen for their protective abilities by passive sera transfer in this experiment. The groups of 12 BALB/c or 12 CBA/N mice to be challenged were immunized passively with 100 µl of hyperimmune sera specific for ClpP by intraperitoneal injection. Control mice were injected intraperitoneally with 100 µl of pooled sera from normal non-immunized mice. At 24 h after passive immunization, each mouse was challenged intraperitoneally. The optimal colony-forming units for the challenge studies were calculated after LD estimation in a previous pilot study with these S. pneumoniae strains in BALB/c and CBA/N mice. The survival time of each mouse was then monitored for 21 days.

Statistical analysis

The Mann–Whitney U-test was performed to evaluate differences between the median survival times for groups of mice. Fisher's exact test was used to compare overall survival rates for groups of mice. Values were considered statistically significant at a P-value of < 0·05 (two-tailed).

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Presence of selected ClpP gene in different S. pneumoniae isolates

The PCR amplification was used to demonstrate the presence of gene encoding ClpP in 12 different strains of S. pneumoniae. The results revealed that bands corresponding to ClpP were detected in all strains of S. pneumoniae analysed (Fig. 1a). Using specific primers, PCR amplification exhibited single bands of identical size (591 base pairs for ClpP gene) in all strains. These PCR products were then cloned into PMD-18T vectors and subsequent DNA sequencing showed that ClpP genes in different pneumococcal strains were highly conserved (data not shown).

image

Figure 1. (a) Polymerase chain reaction (PCR) analysis of Streptococcus pneumoniae strains. Molecular mass markers are indicated at the left. Serotypes (1–23F) of the 12 isolates (Table 1) from which genomic DNA was amplified are indicated. M, DNA markers. Arrows at the right indicate the open reading frames (ORF) of ClpP by using high-fidelity thermostable DNA polymerase, PrimeStar (Takara), which were cloned in PMD-18T vectors (Takara) for subsequent sequencing analysis. (b) Purification of His6-tagged ClpP protein from recombinant Escherichia coli. Lanes: 1, lysate of recombinant E. coli expression construct before induction; 2, 100 000 g supernatant of recombinant E. coli lysate after a 4-h induction with Isopropyl β-D-1-thiogalactopyranoside (IPTG) before being loaded onto Ni-NTA resin; 3, purified His6-tagged ClpP protein (approximate mass, 42 kDa) after elution from Ni-NTA with imidazole; M, protein markers. MM, molecular mass (in kilodaltons). (c) Western blot analysis of S. pneumoniae strains. Sodium dodecyl sulphate-10% polacrylamide gels were loaded with cell wall protein fractions obtained from the strains described in Table 1 and indicated by their serotypes. Lysates from Staphylococcus aureus (S) and untransformed E. coli (E) expression strain from which the recombinant protein was purified were used as a negative control. Apparent molecular mass of ClpP in kilodaltons is indicated by arrow.

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Characterization of ClpP protein expression in pneumococcal isolates

The ClpP recombinant protein (42 kDa) containing a plasmid-encoded S-tag, a Trx-protein and a polyhistidine tag was produced and recovered in the soluble fraction of the E. coli expression strains. The purified recombinant protein was > 95% pure, as judged by SDS-PAGE, after being stained with Coomassie brilliant blue R250 (Fig. 1b) and was confirmed by amino acid N-terminal acid sequencing. BALB/c mice were immunized with the purified ClpP protein emulsified in Freund's adjuvant for production of hyperimmune sera. Western blot analysis demonstrated that hyperimmune anti-sera specific for ClpP reacted with a single band of molecular mass 21 kDa in cell-wall-associated fractions isolated from each of 12 pneumococcal strains tested (Fig. 1c). The anti-sera did not react with a lysate of the untransformed E. coli expression strain from which the recombinant protein was purified and a lysate from Staphylococcus aureus, although S. pneumoniae ClpP is highly homologous (approximately 66%) to S. aureus ClpP using the National Center for Biotechnology Information database. 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.

Protection of BALB/c and CBA/N mice against 12 strains of S. pneumoniae by immunization with ClpP

In order to assess the protection afforded by immunization of mice with ClpP, two separate mouse models were carried out. ELISA analysis of pooled sera from groups of mice immunized with the purified ClpP showed that strong, antigen-specific antibody responses were generated in both BALB/c and CBA/N mice (Table 2). Unimmunized mice had relatively undetectable titres of cross-reactive antibody. In the first series of experiments, groups of BALB/c mice immunized with ClpP were challenged with 12 strains of serotype-different pneumococci respectively. Table 3 shows the results obtained from challenge studies, the median survival times for mice that received ClpP in 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 that received the corresponding AlPO4 alone (P < 0·01 in all cases and P < 0·0004 in most cases). Nomouse could survive after invasive challenge with indicated virulent strains in all control groups.

Table 2.  Antibody titres obtained from mice immunized with caseinolytic protease (ClpP).
Immunization groupAntibody titre (ELISA) (mean ± s.e.m.)
BALB/cCBA/N
  1. Antibody titres obtained from mice prior to challenge with live pneumococci. Groups of 12 mice were immunized with indicated antigens and sera were collected from all mice by retro-orbital bleeding 1 week after the third immunization. Antibody titres were determined as the reciprocal of the dilution of sera yielding 50% of the maximum A405 above the background, and data represent the standard error of the mean (s.e.m.) from the range of titres from the individual mice in the experimental groups. ELISA, enzyme-linked immunosorbent assay.

Preimmune< 100< 100
AlPO4< 100< 100
ClpP + AlPO461 000 ± 690032 000 ± 5600
Table 3.  Effect of active treatment with caseinolytic protease (ClpP) or aluminophosphate (AlPO4) on subsequent infection with Streptococcus pneumoniae in a BALB/c mouse model.
Challenge strainChallenge dose (CFU)Days to death, medianSurvival, %
ClpPControlClpPControl
  1. Statistical differences (P-value) between mice immunized with ClpP in AlPO4 and control mice immunized with AlPO4 for groups (12 mice per group) challenged with indicated virulent pneumococal strains. Differences in median survival times between two groups were analysed using the Mann–Whitney U-test; differences in survival rates between two groups were analysed with Fisher's exact test. *P < 0·0004. P < 0·002. P < 0·05. §P < 0·01. CFU, colony-forming units.

CMCC(B)310112·0 × 106> 211·075·00*0
CMCC(B)311092·5 × 105> 211·566·670
CMCC(B)312035·0 × 106> 211·566·670
CMCC(B)312076·0 × 106> 211·075·00*0
CMCC(B)312165·0 × 106> 21*2·0100·00*0
CMCC(B)314468·0 × 106> 211·066·670
CMCC(B)315072·5 × 106> 21*2·091·97*0
CMCC(B)316142·0 × 106> 211·075·00*0
CMCC(B)316871·0 × 106> 21*4·0100·00*0
CMCC(B)316931·5 × 106> 211·083·33*0
CMCC(B)317597·0 × 106> 21§2·066·670
D395·0 × 105> 211·058·33§0

In the second series of experiments, groups of CBA/N mice immunized with ClpP were challenged with indicated virulent pneumococccal strains. Because these mice are highly susceptible to pneumococcal infection, challenge doses for these experiments were about 1/100 of that of BALB/c mice. As observed in the BALB/c mouse model, the median survival times for mice that received ClpP in AlPO4 were significantly longer than those for mice that received the corresponding AlPO4 alone (P < 0·01 in all cases and P < 0·002 in most cases; Table 4), and the survival rates for mice that received ClpP in AlPO4 were significantly greater than those for mice treated with the corresponding AlPO4 alone (P < 0·05; Table 4) in all cases, except when they were challenged with D39. The reason may be that the virulence of this challenge strain was higher in CBA/N mice.

Table 4.  Effect of active treatment with caseinolytic protease (ClpP) or aluminophosphate (AlPO4) on subsequent infection with Streptococcus pneumoniae in a CBA/N mouse model.
Challenge strainChallenge dose (CFU)Days to death, medianSurvival, %
ClpPControlClpPControl
  1. Statistical differences (P-value) between mice immunized with ClpP in AlPO4 and control mice immunized with AlPO4 for groups (12 mice per group) challenged with indicated virulent pneumococal strains. Differences in median survival times between two groups were analysed using the Mann–Whitney U-test; differences in survival rates between two groups were analysed with Fisher's exact test. *P < 0·0004. P < 0·002. P < 0·05. §P < 0·01. CFU, colony-forming units.

CMCC(B)310111·0 × 104> 211·058·33§0
CMCC(B)311091·0 × 1033·0§1·041·670
CMCC(B)312032·0 × 1044·0§1·041·670
CMCC(B)312072·0 × 1046·01·050·000
CMCC(B)312162·0 × 1044·01·041·670
CMCC(B)314463·0 × 1044·0§1·050·000
CMCC(B)315071·5 × 104> 211·058·33§0
CMCC(B)316141·5 × 1046·01·041·670
CMCC(B)316871·0 × 104> 211·058·33§0
CMCC(B)316931·0 × 1045·0§1·041·670
CMCC(B)317593·0 × 1044·01·041·670
D391·0 × 1033·0§1·033·330

Passive protection by immunization of ClpP-specific antibodies

To confirm that this protection was antibody-mediated, mice were immunized passively with mouse hyperimmune sera raised against this recombinant ClpP protein prior to a lethal challenge. As observed for active immunization, mice that received sera containing specific anti-ClpP antibodies lived significantly longer than those treated with sera obtained from normal non-immunized mice in both BALB/c and CBA/N mouse models (P < 0·01 in all cases; Tables 5 and 6). The treatment of sera containing specific anti-ClpP antibodies could provide greater survival rate than that of control sera in BALB/c mouse model (P < 0·05 in all cases and P < 0·0004 in most cases; Table 5). In the CBA/N mouse model, the survival rates for mice that received anti-ClpP sera were also significantly higher than those for mice that received the control sera (P < 0·05; Table 6) in all cases except when they were challenged with relatively more virulent strains of D39 and CMCC(B)31109. These results from passive immunization with anti-ClpP sera confirmed further the protection of active treatment with ClpP antigen.

Table 5.  Effect of passive treatment with anti-caseinolytic protease (ClpP) or control sera on subsequent infection with Streptococcus pneumoniae in a BALB/c mouse model.
Challenge strainChallenge dose (CFU)Days to death, medianSurvival, %
Anti-ClpPControlAnti-ClpPControl
  1. Statistical differences (P-value) between mice immunized with anti-ClpP sera and control mice immunized with pooled sera from normal non-immunized mice for groups (12 mice per group) challenged with indicated virulent pneumococcal strains. Differences in median survival times between two groups were analysed using the Mann–Whitney U-test; differences in survival rates between two groups were analysed with Fisher's exact test. *P < 0·0004. P < 0·002. P < 0·05. §P < 0·01. CFU, colony-forming units.

CMCC(B)310112·0 × 106> 21§1·058·33§0
CMCC(B)311092·5 × 105> 211·558·33§0
CMCC(B)312035·0 × 106> 212·066·670
CMCC(B)312076·0 × 106> 211·075·00*0
CMCC(B)312165·0 × 106> 212·083·33*0
CMCC(B)314468·0 × 106> 211·058·33§0
CMCC(B)315072·5 × 106> 211·583·33*0
CMCC(B)316142·0 × 106> 211·083·33*0
CMCC(B)316871·0 × 106> 212·083·33*0
CMCC(B)316931·5 × 106> 211·075·00*0
CMCC(B)317597·0 × 1066·0§1·050·000
D395·0 × 1052·0§1·050·000
Table 6.  Effect of passive treatment with anti-caseinolytic protease (ClpP) or control sera on subsequent infection with Streptococcus pneumoniae in CBA/N mouse model.
Challenge strainChallenge dose (CFU)Days to death, medianSurvival, %
Anti-ClpPControlAnti-ClpPControl
  1. Statistical differences (P-value) between mice immunized with Anti-ClpP sera and control mice immunized with pooled sera from normal non-immunized mice for groups (12 mice per group) challenged with indicated virulent pneumococal strains. Differences in median survival times between two groups were analysed using the Mann–Whitney U-test; differences in survival rates between two groups were analysed with Fisher's exact test. *P < 0·0004. P < 0·002. P < 0·05. §P < 0·01. CFU, colony-forming units.

CMCC(B)310111·0 × 104> 211·058·33§0
CMCC(B)311091·0 × 1033·0§1·033·330
CMCC(B)312032·0 × 1046·0§1·041·670
CMCC(B)312072·0 × 1043·0§1·050·000
CMCC(B)312162·0 × 104> 211·058·33§0
CMCC(B)314463·0 × 1044·0§1·041·670
CMCC(B)315071·5 × 104> 211·066·670
CMCC(B)316141·5 × 1046·0§1·050·000
CMCC(B)316871·0 × 1043·0§1·050·000
CMCC(B)316931·0 × 1044·01·041·670
CMCC(B)317593·0 × 1046·01·041·670
D391·0 × 1033·01·033·330

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The S. pneumoniae is carried asymptomatically in the nasopharynx of healthy individuals, and the invasive pneumococcal infection process exposes S. pneumoniae to numerous stress conditions. Changes in temperature between the nasal mucosa (30–34°C) and the blood or meninges (37°C) may serve as a key trigger for a rapid, transient increase in the synthesis of a highly conserved set of proteins referred to as HSPs, including the adenosine triphosphate (ATP)-dependent ClpP, which consists of an ATPase specificity factor and a proteolytic subunit called ClpP [32–35]. ClpP represents a unique family of serine proteases by itself and ClpP-mediated proteolysis copes with protein folding, repair and degradation [36]. Therefore, ClpP plays a complex and central role in the pathogenesis of invasive pneumococcal infection. As ClpP is increased to protect S. pneumoniae from various insults during periods of stress caused by infection, this HSP is considered to be a prominent antigen in the humoral and cellular immune response mediated by antibodies and T cells respectively [27,29].

In this study, our PCR amplification and DNA sequencing studies demonstrated that pneumococcal ClpP genes were highly conserved among 12 different serotypes of S. pneumoniae. Subsequent Western blot analysis confirmed that antibodies to ClpP did not appear to cross-react with other pneumococcal proteins when ClpP anti-sera was used to probe pneumococcal cell-wall-associated proteins from these strains. Previous reports have shown that fractionation of S. pneumoniae revealed a substantial increase in the amount of ClpP in the cell wall and ClpP was the first Clp protein shown to be mobilized into the cell wall fraction after heat shock only in pneumococcal strains, but not in other bacterial strains [37].

In order to examine the possibility that this highly conserved ClpP antigen could elicit serotype-independent protection against invasive pneumococcal infection, we took advantage of two murine strains in an intraperitoneal model of invasive infection. The challenge peumococcal strains we used covered the most prevalent invasive strains, and we determined their respective LD50 (data not shown) and optimized the best challenge dose to cause invasive pneumococcal infection for each strain. In the challenge experiments, large infecting doses were employed to produce sufficient concentrations of the diffusible pathogenic molecules and to detect protection more effectively. These challenge doses to establish invasive pneumococcal infection were significant for us in evaluating the protective effects of protein-based vaccines.

The results of active immunization with ClpP antigen indicated that the protection afforded by single ClpP protein was challenge strain- and mouse strain-independent. Even for some highly virulent strains, the protective effects were still promising. Because antibody-initiated complement-dependent opsonization, which activates the classical complement pathway, is believed to be the major immune mechanism protecting the host against invasive infection with pneumococci [38–40], we evaluated the protective effects elicited by passive immunization with hyperimmune sera containing polyclonal antibodies specific for ClpP. In these experiments, passive immunization with anti-ClpP sera could also achieve protective effects as those observed from active immunization with ClpP. The putative mechanism for protection of ClpP maybe that specific anti-ClpP antibodies promote phagocytosis of pneumococci by opsonization and activation of complement. Alternatively, inhibition of ClpP by specific antibodies may damage the function of ClpP-mediated proteolysis, leading to the aggregates of unfavourable stress-induced damaged proteins to an extent which then could attenuate virulence of pneumococci, or antibodies may also promote the internalization of cell-wall-associated ClpP and thus reduce the invasiveness of S. pneumoniae. Recent work from Richard Malley's laboratory [41–43] has suggested that CD4+ T cells mediated antibody-independent acquired immunity to pneumococcal colonization, and they demonstrated further that intranasal immunization with a mixture of pneumococcal proteins protected against intranasal colonization in an antibody-independent, CD4+ T cell-dependent manner. More interestingly, a new lineage of effector CD4+ T cells, T helper 17 (Th17), also mediated pneumococcal immunity in mice and probably in humans. Therefore, the clarification of the role of CD4+T cells in ClpP-elicited protection against pneumococcal infection may help in the development of candidate pneumococcal vaccines.

Previous strategies have proved that immunization with selective pneumococcal protein virulence factors could elicit protection against peumococcal infection [38]. However, their protection was limited. This can be explained by the occurrence of allelic variation within most individual proteins. S. pneumoniae is a highly transformable bacteria in vitro and in vivo, and antibodies raised against a single protein may not recognize allelic variants [44]. A combination of proteins with distinct roles may be a strategy to broaden the protection [28,29], but which combination of proteins should be chosen remains a difficult problem to deal with because the composition, expression and exposure of cell-wall-associated proteins vary in different pneumococcal strains, and little is known about the differential expression patterns of the multiple proteins and how those might account for particular tissue tropism, invasivity and disease outcomes [45]. In fact, gene expressions of pneumococcal virulence factors differ in the progress of invasive pneumococcal infection for one specific pneumococcal strain, and they would be much more complicated for the full range of pneumococcal strains [46,47]. In this regard, searching for a set of conserved proteins that play critically constituent roles in invasive pneumococcal infection by different strains would be an appropriate way to solve this problem.

In summary, to out knowledge, this is the first report that immunization with single pneumococcal ClpP protein antigen could protect against such broad-range pneumococal strains. Previous studies excluded the use of protein antigens with 40% homology to human proteins in vaccine development, and S. pneumoniae ClpP had < 40% similarity to proteins from humans [27]. These findings support the development of ClpP as a human penumococcal vaccine. However, because of potential sequence similarity to other human proteins, the use of pneumococcal ClpP to induce human immune responses must be evaluated carefully. Moreover, it has been proposed that HSPs are particularly suited to be carriers for peptides in immunization protocols because of their high affinity of binding to certain peptides and their involvement in various steps in antigen processing, which could improve the immunogenicity of defined epitopes in immunization experiments [48]. Use of ClpP as carrier molecules for other pneumococcal antigen determinants to augment protective effects may provide a basis for applying ClpP in conjugate vaccines, and this work will be continued. Finally, we believe that future studies will improve ClpP protein vaccine efficacy further by use of more efficient adjuvants or a variety of host molecules potentially able to enhance the immunogenicity of selected target antigens such as cytokines and complement component 3d [49,50].

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grants from National NaturalScience Foundation grants of China (numbers 30400376, 30471838 and 30371275).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References