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Mucosal immunization with purified ClpP could elicit protective efficacy against pneumococcal pneumonia and sepsis in mice
Microbes and Infection 10 (2008) 1536e1542 www.elsevier.com/locate/micinf
Original article
Mucosal immunization with purified ClpP could elicit protective efficacy against pneumococcal pneumonia and sepsis in mice Ju Cao a,b, Tingmei Chen a, Dairong Li a, Chun K. Wong b, Dapeng Chen a,b, Wenchun Xu a, Xuemei Zhang a, Christopher W.K. Lam b, Yibing Yin a,* a
Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing Medical University, #1 Yixueyuan Road, ChongQing 400016, PR China b Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Received 2 June 2008; accepted 15 September 2008 Available online 1 October 2008
Abstract Caseinolytic protease (ClpP) has been found to be highly conserved among different strains of Streptococcus pneumoniae and intraperitoneal immunization with ClpP could elicit protection against invasive pneumococcal infections. In this study, mucosal immunization with ClpP antigen induced both systemic and mucosal antibodies, and in this way reduced lung colonization in an invasive pneumococcal pneumonia model and also protected mice against death in an intraperitoneal-sepsis model. Surface localization of ClpP was confirmed using flow cytometry analysis. Furthermore, characterization of human sera for anti-ClpP IgG antibody levels demonstrated that ClpP protein was immunogenic in healthy children and was expressed during disease based on the elevated antibody levels in infected individuals. Finally, we describe that in vitro functional anti-ClpP antibody could kill streptococcus pneumoniae by polymorphonuclear leukocytes in a complement-dependent assay. To our knowledge, this is the first study about the protective efficacy of mucosal immunization with ClpP as a promising pneumococcal protein antigen. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: ClpP; Vaccine; Mucosal immunization
1. Introduction Infections with Streptococcus pneumoniae are a major cause of human diseases [1]. The surface of the nasal mucosa is the major reservoir for pneumococcus, and at this site it resides primarily in a commensal relationship with its human host [2]. Under predisposing conditions, especially following infections with respiratory viruses and blockage of the estuation tube, the pneumococci colonizing the upper airways are able to cause symptomatic diseases [3]. Pneumococcal infections result in an estimated 1.5 million deaths per year worldwide [4]. The rapid emergence of multidrug-resistant pneumococcal strains throughout the world is leading to
* Corresponding author. Tel./fax: þ86 23 6848 5658. E-mail address: [email protected] (Y. Yin). 1286-4579/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2008.09.007
increased emphasis on the prevention of pneumococcal infections by vaccination [5]. The current 23-valent capsular polysaccharide vaccines classified as type 2 T-cell-independent antigens are poorly immunogenic in young children who suffer the highest rates of pneumococcal infections [7,8]. The conjugate vaccines with 7e11 serotypes could induce a memory immune response and are useful against invasive diseases caused by the vaccine-type strains [4]. However, there are shortcomings associated with the current capsular conjugate vaccines including high cost, serotype-specific protection, limited serotype coverage, and the likelihood of a concomitant increase in carriage and subsequent infection by nonvaccine serotypes [11]. At present, the new strategy tends to develop novel protein vaccines which may be highly immunogenic, T-cell dependent, antigenically conserved, and cross-protective among pneumococcal strains [3]. Protein antigens of S. pneumoniae
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have been evaluated for protective efficacy in animal models of pneumococcal infections. These include pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumococcal surface adhesion A (PsaA), pneumolysin (Ply), pneumococcal histidine triad (Pht) proteins, neuraminidases A and B, Pilus subunits, iron ABC transporters PiaA and PiuB, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP) [6,8,10,12,13]. Recent studies from several different research groups described a novel heat shock protein caseinolytic protease (ClpP) and immunization with ClpP has been suggested to possibly elicit protection against invasive pneumococcal infection [14e16]. Since the mucosal epithelium of the nasopharynx is the primary site of pneumococcal colonization and the natural route of infection with S. pneumoniae starts with colonization, which then progress to invasive diseases, mucosal immune responses may play a significant role [8]. Recent work from Richard Malley’s laboratory demonstrated that multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear could be achieved by killed nonencapsulated cells given intranasally with a nontoxic adjuvant [21], and they further showed that protection against nasopharyngeal colonization by S. pneumoniae is mediated by antigen-specific CD4þ T cells and intranasal immunization with a mixture of pneumococcal proteins protects against intranasal colonization in an antibody-independent, CD4þ T-cell-dependent manner [9,20]. However, mucosal vaccination with ClpP protein antigen against invasive pneumococcal infection has not been exploited. This study aimed to determine in detail whether or not the mucosal site would be efficient for induction of systemic and mucosal antibody responses after application of protein antigen ClpP. Protection elicited by intranasal immunization with ClpP was also evaluated in pneumonia and lethal sepsis models. Furthermore, in vitro opsonophagocytic killing activity induced by ClpP-specific antibody was investigated. 2. Materials and methods 2.1. Bacteria S. pneumoniae strain TIGR4 (ATCC BAA-334) was used for challenge experiments and the viability of bacterial stocks was analyzed prior to challenge. Escherichia coli DH5a (Invitrogen) was used as the host for routing plasmid cloning. Recombinant proteins were expressed in E. coli BL21(DE3) (Novagen). 2.2. Mice Six- to eight-week-old BALB/c mice were used for production of hyperimmune anti-ClpP sera. In pneumococcal pneumonia and sepsis model, we used 6- to 8-week-old CBA/N mice who have the Btk (XID) immune-response defect and fail to make natural antibodies to pneumococcal polysaccharides.
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All animal experiments were approved by the respective ethics committees of ChongQing Medical University. 2.3. Human sera samples and anti-ClpP immunoglobulin G (IgG) characterization Sera from 87 children not suffering from any infectious diseases at the time of sampling and sera from 12 children patients between 5 and 10 years of age with acute pneumococcal pneumonia were obtained at the Children’s Hospital of ChongQing Medical University in a study approved by the Ethical Committees of ChongQing Medical University and Children’s Hospital of ChongQing Medical University. These sera samples were analyzed for anti-ClpP IgG levels by ELISA using recombinant ClpP protein as coating antigens as described later. 2.4. Protein vaccines and adjuvants The cloning, expression, and purification of ClpP from TIGR4 pneumococci were done according to the procedures described previously [14]. Cholera toxin (CT) was obtained from Calbiochem Corporation. The recombinant ClpP protein and CT solutions for animal experiments did not contain any detectable LPS, as determined by the quantitative Limulus amoebocyte lysate assay (sensitivity limit 12 pg/ml; Biowhittaker Inc., MD, USA). 2.5. Production of hyperimmune mouse sera against ClpP in BALB/C mice Hyperimmune mouse sera specific for ClpP (anti-ClpP) were generated by intraperitoneal immunization of BALB/c mice with recombinant ClpP protein. Each mouse was primed with 10 mg of ClpP, emulsified in complete Freund’s adjuvant (CFA,1:1 ratio, v/v) on Day zero, and boosted with the same concentration of recombinant protein emulsified in incomplete Freund’s adjuvant (IFA,1:1 ratio, v/v) on Day 14 and on Day 28, each BALB/C mouse received the last dose of 10 mg 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. 2.6. Active immunization Six- to eight-week-old female CBA/N mice (12 per group) were immunized intranasally with recombinant ClpP twice a week for three consecutive weeks. The following conditions were used: CT, ClpP (5 mg), ClpP (5 mg) þ CT. The dose of antigen solution corresponded to a mixture of 25 ml of the ClpP and 5 ml (1 mg/ml) of CT. The intranasal immunization was carried out with the CBA/N mouse held in a supine position with the head down while 30 ml of the antigen solution was delivered slowly with a micropipette onto the nares so that the CBA/N mouse could sniff it in.
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2.7. Blood and saliva sampling for antibody measurements The CBA/N mice were bled from the retro-orbital sinus 15 days after the last immunization. Sera were then isolated and stored at 70 C. Saliva was collected from each CBA/N mouse by the insertion of absorbent sticks (Polyfiltronics) to the mouth. After 5minutes, the sticks were transferred to phosphate-buffered saline, pH 7.4 (PBS), containing 10.0 mg of protease inhibitor (Sigma) per ml to prevent the proteolysis of antibodies. The dissolved saliva was pooled for each group of CBA/N mice and was stored at 70 C. Anti-ClpP specific IgG and immunoglobulin A (IgA) from sera and saliva were determined by enzyme-linked immunosorbent assay (ELISA) as described below.
2.10. Pneumococci surface staining by flow cytometry The abilities of hyperimmune sera specific for ClpP from BALB/C mice to bind to the surface of live S. pneumoniae cells were determined using flow cytometry. TIGR4 from the early logarithmic growth phase were collected and washed twice in PBS. Bacterial suspensions were aliquoted in 300-ml samples, and primary serum antibodies were added for a final dilution of 1:100. The bacterial suspensions were incubated for 1 h at 4 C and washed twice before the addition of fluorescein isothiocyanate-conjugated goat antibodies to mouse (R & D systems) for 30 min at room temperature. After two additional washes, the bacteria were fixed with 0.25% (v/v) formaldehyde for 18 h at 4 C, and surface staining was detected by a flow cytometry (Beckman Coulter). 2.11. Challenge studies
2.8. Determination of ClpP-specific antibody titers in saliva and sera by ELISA Ninety-six-well ELISA plates were coated with recombinant ClpP (10 mg/ml, 100 ml per well in PBS) overnight at 4 C and blocked with 200 ml of 10% fetal calf serum (Sigma) in PBS for 3 h at room temperature. Individual sera or saliva from immunized CBA/N mice were tested in duplicate. Sera and saliva samples (100 ml) were added and serially diluted in PBS. After 1 h incubation at 37 C, plates were washed three times with 250 ml of PBS containing 0.05% Tween 20 (PBS-T) and titers of IgG and IgA were determined by addition of 100 ml of peroxidase conjugated goat anti-mouse IgG (Sigma) and peroxidase-conjugated goat anti-mouse IgA (Sigma), respectively. Following incubation for an additional hour at 37 C, the plates were washed six times and the color reaction for this ELISA was developed by adding 100 ml of tetramethylbenzidine (Sigma). The ELISA was allowed to react for 20 min and stopped with 50 ml 2% H2SO4. The plates were read spectrophotometrically at 405 nm and the endpoint titers were calculated for all samples. For human sera anti-ClpP IgG assays, antigen-specific antibodies in sera of children were analyzed by recombinant ClpP protein coated ELISA plates and peroxidase-conjugated goat anti-human IgG secondary antibodies (Sigma).
2.9. Western blot analysis Whole-cell lysates were prepared from S. pneumoniae strain TIGR4 by boiling cell suspensions containing SDSe PAGE sample buffer (Ambresco) for 5 min. After electrophoresis in 10% polyacrylamide gels, the proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes for immunoblotting with CBA/N hyperimmune mouse sera diluted 1:800. Detection of antigens was performed by an indirect antibody immunoassay using HRPlabeled anti-mouse IgG (Sigma) diluted 1:5000 in PBS-T and DAB staining.
In the sepsis model, intraperitoneal-challenge experiments were performed. The CBA/N mice were challenged 1 week after the last intranasal immunization with a challenge dose 1 105 CFU of the TIGR4. The challenged CBA/N mice were observed twice daily by an experienced person, and signs of sickness and dead CBA/N mice were recorded. CBA/N mice still alive after 21 days were considered to have survived the infection. In the pneumonia model, CBA/N mice were lightly anesthetized with 3% (v/v) halothane over oxygen by using a methacrylate box connected to Fluovac 240 (Anaesthetizing Systems). Following anesthetization, suspensions of 40 ml of PBS containing 1 106 bacteria were introduced into the nares of the CBA/N mice to induce aspiration pneumonia and the challenged CBA/N mice were euthanized on day 3 after challenge, and the lungs were removed and homogenized in PBS. Samples were serially diluted and plated on blood agar plates, and viable counts were determined after overnight incubation. 2.12. Passive protection studies CBA/N mice were injected in the tail vein with 150 ml of hyperimmune mouse sera specific for ClpP from BALB/C mice, and control CBA/N mice were injected with 150 ml of pooled preimmune sera from non-immunized BALB/C mice. CBA/N mice received a second dose of sera 24 h after intranasal infection as described above, under the same conditions as the first dose. 2.13. Antibody-dependent killing of S. pneumoniae by complement and human polymorphonuclear leukocytes (PMN) The ability of hyperimmune sera from BALB/c mice immunized with ClpP to opsonize pneumococcus for uptake and killing by human PMN was determined using an opsonophagocytosis assay as previously described [12,26]. TIGR4 were grown at 37 C overnight on blood agar and then inoculated into CþY medium. Bacteria were harvested in early
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stationary phase at 1500 g for 5 min, resuspended in sterile PBS, and diluted to 1 105 CFU/ml in RPMI 1640 medium (Invitrogen) with 10% FBS. PMN were isolated from 30 ml heparinized blood by Percoll gradient separation. Recovered PMNs were washed in HBSS without calcium chloride, magnesium chloride, magnesium sulfate, or phenol red (Life Technologies), and resuspended in RPMI 10% FBS to a concentration of 2 107cells/ml. The killing assays were performed by using BALB/c mice hyperimmune (HI) sera at a dilution of 1:10 or 1:100 and with their respective preimmune (PI) sera as control. A total of 100 ml of bacterial preparation (1 104 CFU), 50 ml of diluted sera, 200 ml of PMNs, and 40 ml of human sera obtained from an agammaglobulinemic patient as the complement source were combined (ratio of PMN to bacteria ¼ 400:1). The final volume was brought to 400 ml with RPMI 10% FBS and incubated at 37 C for 1 h with shaking. Aliquots (100 ml) from each tube were diluted in RPMI with 10% FBS, and 10 ml of each dilution was plated in duplicate on TSA II plates with 5% sheep blood. After overnight incubation at 37 C, CFU were counted and calculated for each original reaction tube, and data were expressed as the percentage of bacterial survival relative to the CFU at time 0.
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saliva. However, intranasal immunization with ClpP only or CT alone could not lead to systemic and mucosal anti-ClpP antibody responses. 3.2. Characterizing specific anti-ClpP antibodies in patients with pneumococcal pneumonia and healthy children In this part, 87 sera obtained from healthy children and 12 sera from pediatric patients with acute pneumococcal pneumonia were analyzed for anti-ClpP IgG levels. We detected a wide range of IgG titers from almost undetectable to high levels (Fig. 1). In the groups of healthy children, the ClpP-specific antibody levels showed a continuous increase, with peak median value in the 8e11 years old group. In sera from infants (<12 months old) and very young children (12e24 months old), the median ELISA values for ClpP were lower than those in older age groups. Further analysis of sera from the group of pediatric patients with acute pneumonia showed significantly (P < 0.05) higher ELISA activity than those from groups of healthy children, and the median value for anti-ClpP IgG ELISA unit in this group was about 1000. 3.3. Confirmation of surface localization of ClpP
2.14. Statistics Differences between the overall survival rates for groups of CBA/N mice were analyzed by Fisher’s exact test. A nonparametric test (ManneWhitney) was used to compare concentrations of anti-ClpP antibodies. Statistical comparisons of CFU levels were all carried out on log transformed data by the Wilcoxon tests. The limit statistical significance was a P value of 0.05. 3. Results 3.1. Anti-ClpP specific IgG and IgA in sera and saliva following immunization ELISA assays were performed in order to evaluate the ability of intranasal immunization with ClpP to induce antibody production in CBA/N mice. Results were shown in Table 1. It was evident from the results of this experiment that the nasal route, with CT as a mucosal adjuvant, was a way for the induction of serum IgG antibodies to purified ClpP, but IgG levels in saliva were not detectable in all groups. Notably, nasal immunization with ClpP in CT was clearly evident for induction of IgA antibodies to ClpP in sera, as well as in
Our previous work has shown that hyperimmune sera from BALB/C mice by immunization with the purified ClpP protein emulsified in Freund’s adjuvant could react with a single band of molecular mass 21 kDa in fraction of S. pneumoniae TIGR4 [14,15]. In this study, we demonstrated that antisera from CBA/N mice mucosally immunized with ClpP in CT also reacted with S. pneumoniae TIGR4 lysates and 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 (Fig. 2A). To further verify the surface location of ClpP, flow cytometry analysis was performed using anti-ClpP antisera obtained from immunized BALB/c mice. For bacteria stained with preimmune sera, geometric mean fluorescence intensity (GMFI) values were about 2.56; For bacteria stained with hyperimmune sera, GMFI values were about 4.86 (Fig. 2B). 3.4. Protection by ClpP in models of pneumococcal sepsis After analysis of anti-ClpP IgG, CBA/N mice underwent an intraperitoneal challenge with 1 105 CFU of S. pneumoniae strain TIGR4. Survival, observed up to 21 days after
Table 1 Serum and salivary antibody responses to mucosal immunization with ClpP Conditions
CT ClpP (5 mg) ClpP (5 mg) þ CT
IgG in serum
ND 1.8 (1.4e6.3) 2860 (2119e3890)
IgA Serum
Saliva
ND 0.1 (0.1e0.3) 120.1 (82.1e172.5)
ND ND 4.9 (3.4e10.9)
Median concentrations (ranges) of IgG and IgA antibodies to ClpP (kilounits per milliliter); ND, not detectable.
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challenge, was shown in Fig. 3. Partial protection was observed in CBA/N mice immunized with 5 mg of ClpP in CT (58.3%). This value was significantly higher than that for the mice treated with CT alone (P < 0.01). 3.5. Protection by ClpP in models of pneumococcal pneumonia
Fig. 1. ClpP protein antigen was immunologic in healthy children and antiClpP IgG could be elevated in children patients with acute pneumococcal pneumonia. Sera samples were analyzed for anti-ClpP IgG levels by ELISA using recombinant proteins as coating antigens. Each datum point represents an individual’s data. A horizontal line denotes the median anti-ClpP IgG titer for the group. Antibody titers are expressed as ELISA units (OD, 405 nm) at 1:1000 serum dilutions. m, months; y, years.
CBA/N mice were mucosally immunized with recombinant ClpP and challenged intranasally with TIGR4. Lung colonization of individual CBA/N mouse was shown at day 3 after challenge. ClpP in CT was capable of reducing the bacterial load in the lung about 100-fold relative to CBA/N mice immunized with either CT only or ClpP alone (P < 0.001, Table 2). In sera transfer experiments, anti-ClpP sera treatment was reproducibly as effective as intranasal immunization with ClpP in reducing lung infection by TIGR4 compared to control sera (P < 0.01) 3.6. Sera from ClpP-immunized BALB/c mice were efficiently opsonic for uptake and killing of pneumococcus by human PMN in a complement-dependent assay In this part, we used in vitro functional assay to measure the bactericidal activity of anti-ClpP hyperimmune sera. We were able to detect a dose-dependent opsonophagocytic killing activity for anti-ClpP sera (Fig. 4). Bacterial survival rate for the treatment of hyperimmune anti-ClpP sera diluted 1:10 was as low as 8%. For hyperimmune anti-ClpP sera diluted 1:100, there was an increase for bacterial survival rate which was about 40%. However, this bacterial survival rate was still much
Fig. 2. (A) Western immunoblot of pneumococcal lysates of TIGR4 by use of antisera from CBA/N mouse mucosally immunized with ClpP in CT. Lane 1 was developed with lysates from Staphylococcus aureus; lane 2 was developed with lysates from untransformed E. coli expression strain from which the recombinant ClpP protein was purified; lane 3 was developed with lysates from TIGR4 Streptococcus pneumoniae. Apparent molecular mass of ClpP in kilodaltons (KD) is indicated by arrow. (B) Confirmation of surface localization of ClpP. Flow cytometry analysis was performed using anti-ClpP hyperimmune sera obtained from immunized BALB/c mice. Shown are overlays of primary flow cytometry histograms demonstrating staining of TIGR4 with anti-ClpP hyperimmune (HI) or with their respective preimmune (PI) sera. Results are expressed as histograms of relative cell counts with fluorescence intensity. This figure is representative of three independent experiments with similar results.
Fig. 3. Protection by ClpP in model of pneumococcal sepsis. CBA/N mice (n ¼ 12) were intranasally immunized with recombinant ClpP and then intraperitoneally challenged with S. pneumoniae TIGR4. Control mice (n ¼ 12) were sham immunized with CT only. Mucosal immunization with ClpP significantly protected the CBA/N mice (P < 0.01) from an intraperitoneal lethal challenge with S. pneumoniae TIGR4.
J. Cao et al. / Microbes and Infection 10 (2008) 1536e1542 Table 2 Protection by ClpP in model of pneumococcal pneumonia Conditions
No. of mice
CFU/lung, mean log (standard error)
CT ClpP (5 mg) ClpP (5 mg) þ CT Preimmune sera Anti-ClpP sera
12 12 12 12 12
5.47 5.51 3.46 5.78 3.68
(0.34) (0.31) (0.20)* (0.29) (0.34)#
*
Value is significantly different from the value observed for CBA/N mice immunized with CT or ClpP alone (P < 0.001);# value is significantly different from the value observed for CBA/N mice treated with preimmune sera (P < 0.01).
lower than that for the treatment of preimmune sera which had a bacterial survival rate of about 91%. 4. Discussion Streptococcus pneumoniae is a major pathogen which enters the body through the respiratory mucosa and may cause serious infections [11]. Intranasal vaccination may offer an alternative approach to current strategies since it induces mucosal as well as systemic immune responses and systemic immunization does not induce mucosal immune responses [3,18]. Mucosal immunization would be a more effective route for immunizing young children, the elderly and the age groups most at risk of pneumococcal infections, as the mucosal immune system develops earlier in infants and lasts longer in the elderly than the systemic immune system [8,17].Mucosal delivery of pediatric vaccines has become an explicit goal of the NIH as well as of WHO from economical, logistical and safety standpoints [19] ClpP represents a unique family of serine proteases by itself and ClpP-mediated proteolysis copes with protein folding, repair, and degradation [15]. Accordingly, ClpP plays a complex and central role in the pathogenesis of pneumococcal infections. In this study, we could detect surface
Fig. 4. ClpP-immunized hyperimmune (HI) mouse sera at a dilution of 1:10 or 1:100 is opsonic for killing of pneumococcus by human PMN in the presence of complement, while their respective preimmune (PI) sera are much less opsonic. Data are expressed as the percentage of bacterial survival relative to the initial CFU at time 0. The average of three experiments is shown; error bars represent standard deviations.
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staining with BALB/C mouse hyperimmune sera specific for ClpP by FACS analysis and then demonstrated that mucosal immunization with ClpP could induce both systemic and mucosal antibodies when applied CT as a mucosal adjuvant. Besides that, intranasal immunization with ClpP protected CBA/N mice against death caused by S. pneumoniae TIGR4 in an intraperitoneal-sepsis model. This way could also reduce lung colonization in an invasive pneumococcal pneumonia model, and passive immunization with anti-ClpP sera demonstrated that this protection was antibody mediated. The induction of protective efficacy to pneumococci via the nasal route suggests that the nasopharyngeal mucosa possesses the necessary structures to make mucosal immunization a realistic alternative to the use of ClpP protein vaccines. Furthermore, we detected natural antibody responses against ClpP in healthy children and they were followed by the development of specific antibody levels as a function of age. Our results showed that anti-ClpP IgG titer was elevated in pediatric patients with acute pneumococcal pneumonia, so ClpP antigen is expressed during infection in the human host and may form part of the normal repertoire of protective antibodies induced by prior infection. Host defenses against S. pneumoniae depend largely on phagocytosis following opsonization by specific antibodies and complement [11]. Our sera transfer experiments suggested that protective efficacy of ClpP was antibody mediated. We further established in vitro assays to investigate opsonophagocytic killing activity of functional anti-ClpP antibody. In these assays, we used TIGR4 pneumococcus which is a virulent strain with less capsule [27]. At this stage, we still do not know whether or not functional anti-ClpP antibody could kill other encapsulated strains. However, previous studies have demonstrated that most of the polysaccharide capsule was shed on S. pneumoniae adhesion to the epithelial cell [28]. Therefore, shedding of the polysaccharide capsule may reveal cell wall-associated proteins that are otherwise masked so that functional antibodies would interact with these protein antigens [29]. To our knowledge, this is the first report that in vitro functional anti-ClpP antibodies could kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent assay, which hints that anti-ClpP sera may have therapeutic utility, particularly in immunocompromised patients. Based on the results presented in this study, mucosal immunization with ClpP appears therefore as a novel, noninvasive vaccine approach that could protect mice against invasive pneumococcal pneumonia and sepsis. The findings of the present study should stimulate attempts to extend the development of ClpP as a promising pneumococcal protein vaccine component. However, it is also apparent that elicitation of protection by mucosal immunization with ClpP is dependent on CT, and CT is not an acceptable adjuvant for humans due to toxicity [21]. There are other potential mucosal adjuvants including mycoplasma-associated lipoprotein (MALP-2), porcine lung surfactant (PLS), Bordetella pertussis monophosphoryl lipid A (MPLA) and cholera toxin binding B subunit (CTB) which has been given safely by the intranasal route to humans and shown to be effective as an adjuvant by
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this route [22e25]. Therefore, our next goal is to identify a mucosal adjuvant that could be used safely and economically to develop ClpP-based pneumococcal protein vaccines for human use. Acknowledgment This work was supported by grants from National Natural Science Foundation grants of China (Nos. 30400376, 30471838, 30371275). References [1] H.K. Parsons, S.C. Metcalf, K. Tomlin, R.C. Read, D.H. Dockrell, Invasive pneumococcal disease and the potential for prevention by vaccination in the United Kingdom, J. Infect. 54 (2007) 435e438. [2] J. Casal, D. Tarrago´, Immunity to Streptococcus pneumoniae: Factors affecting production and efficacy, Curr. Opin. Infect. Dis. 16 (2003) 219e224. [3] A. Kadioglu, J.N. Weiser, J.C. Paton, P.W. Andrew, The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease, Nat. Rev. Microbiol. 6 (2008) 288e301. [4] J.A. Scott, The preventable burden of pneumococcal disease in the developing world, Vaccine 25 (2007) 2398e2405. [5] R. Novak, B. Henriques, E. Charpentier, S. Normark, E. Tuomanen, Emergence of vancomycin tolerance in Streptococcus pneumoniae, Nature 399 (1999) 590e593. [6] M. Jomaa, S. Terry, C. Hale, C. Jones, G. Dougan, J. Brown, Immunization with the iron uptake ABC transporter proteins PiaA and PiuA prevents respiratory infection with Streptococcus pneumoniae, Vaccine 24 (2006) 5133e5139. [7] E.D. Shapiro, A.T. Berg, R. Austrian, D. Schroeder, V. Parcells, A. Margolis, R.K. Adair, J.D. Clemens, The protective efficacy of polyvalent pneumococcal polysaccharide vaccine, N. Engl. J. Med. 325 (1991) 1453e1460. [8] D.E. Briles, S.K. Hollingshead, G.S. Naobors, J.C. Paton, A. BrooksWalter, The potential for using protein vaccines to protect against otitis media caused by streptococcus pneumoniae, Vaccine 19 (Suppl. 1) (2001) S87eS95. [9] K. Trzcin´ski, C.M. Thompson, A. Srivastava, A. Basset, R. Malley, M. Lipsitch, Protection against nasopharyngeal colonization by Streptococcus pneumoniae is mediated by antigen-specific CD4þT cells, Infect Immun. 76 (2008) 2678e2684. [10] C. Gianfaldoni, S. Censini, M. Hilleringmann, M. Moschioni, C. Facciotti, W. Pansegrau, V. Masignani, A. Covacci, R. Rappuoli, M.A. Barocchi, P. Ruggiero, Streptococcus pneumoniae pilus subunits protect mice against lethal challenge, Infect Immun. 75 (2007) 1059e1062. [11] B.G. Spratt, B.M. Greenwood, Prevention of pneumococcal disease by vaccination: does serotype replacement matter? Lancet 356 (2000) 1210e1211. [12] C. Giefing, A.L. Meinke, M. Hanner, T. Henics, M.D. Bui, D. Gelbmann, U. Lundberg, B.M. Senn, M. Schunn, A. Habel, B. Henriques-Normark, A. Ortqvist, M. Kalin, A. von Gabain, E. Nagy, Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies, J. Exp. Med. 205 (2008) 117e131. [13] J.E. Adamou, J.H. Heinrichs, A.L. Erwin, W. Walsh, T. Gayle, M. Dormitzer, R. Dagan, Y.A. Brewah, P. Barren, R. Lathigra, S. Langermann, S. Koenig, S. Johnson, Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis, Infect. Immun. 69 (2001) 949e958.
[14] J. Cao, D. Chen, W. Xu, T. Chen, S. Xu, J. Luo, Q. Zhao, B. Liu, D. Wang, X. Zhang, Y. Shan, Y. Yin, Enhanced protection against pneumococcal infection elicited by immunization with the combination of PspA, PspC, and ClpP, Vaccine 25 (2007) 4996e5005. [15] H.Y. Kwon, A.D. Ogunniyi, M.H. Choi, S.N. Pyo, D.K. Rhee, J.C. Paton, The ClpP protease of Streptococcus pneumoniae modulates virulence gene expression and protects against fatal pneumococcal challenge, Infect. Immun. 72 (2004) 5646e5653. [16] C. Morsczeck, T. Prokhorova, J. Sigh, M. Pfeiffer, M. Bille-Nielsen, J. Petersen, A. Boysen, T. Kofoed, N. Frimodt-Møller, P. NyborgNielsen, P. Schrotz-King, Streptococcus pneumoniae: proteomics of surface proteins for vaccine development, Clin. Microbiol. Infect. 14 (2008) 74e81. [17] A.M. Roche, S.J. King, J.N. Weiser, Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice, Infect. Immun. 75 (2007) 2469e2475. [18] J. Banchereau, R. Steinman, Dendritic cells and the control of immunity, Nature 392 (1998) 245e252. [19] M. Kane, Unsafe injections, Bull. WHO 76 (1998) 99e100. [20] A. Basset, C.M. Thompson, S.K. Hollingshead, D.E. Briles, E.W. Ades, M. Lipsitch, R. Malley, Antibody-independent, CD4þT-cell-dependent protection against pneumococcal colonization elicited by intranasal immunization with purified pneumococcal proteins, Infect. Immun. 75 (2007) 5460e5464. [21] R. Malley, S.C. Morse, L.C. Leite, A.P. Areas, P.L. Ho, F.S. Kubrusly, I.C. Almeida, P. Anderson, Multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear by killed nonencapsulated cells given intranasally with a nontoxic adjuvant, Infect. Immun. 72 (2004) 4290e4292. [22] P.A. Kozlowski, S.B. Williams, R.M. Lynch, T.P. Flanigan, R.R. Patterson, S. Cu-Uvin, M.R. Neutra, Differential induction of mucosal and systemic antibody responses in women after nasal, rectal, or vaginal immunization: influence of the menstrual cycle, J. Immunol. 169 (2002) 566e574. [23] F. Rharbaoui, B. Drabner, S. Borsutzky, U. Winckler, M. Morr, B. Ensoli, P.F. Mu¨hlradt, C.A. Guzma´n, The Mycoplasma-derived lipopeptide MALP-2 is a potent mucosal adjuvant, Eur. J. Immunol. 32 (2002) 2857e2865. [24] C.M. Soerensen, O.L. Nielsen, A. Willis, P.M. Heegaard, U. Holmskov, Purification, characterization and immunolocalization of porcine surfactant protein D, Immunology 114 (2005) 72e82. [25] L. Vernacchio, H. Bernstein, S. Pelton, C. Allen, K. MacDonald, J. Dunn, D.D. Duncan, G. Tsao, V. LaPosta, J. Eldridge, S. Laussucq, D.M. Ambrosino, D.C. Molrine, Effect of monophosphoryl lipid A (MPL) on T-helper cells when administered as an adjuvant with pneumocococcalCRM197 conjugate vaccine in healthy toddlers, Vaccine 20 (2002) 3658e3667. [26] J. McLay, E. Leonard, S. Petersen, D. Shapiro, N.S. Greenspan, J.R. Schreiber, Gamma 3 gene-disrupted mice selectively deficient in the dominant IgG subclass made to bacterial polysaccharides. II. Increased susceptibility to fatal pneumococcal sepsis due to absence of antipolysaccharide IgG3 is corrected by induction of anti-polysaccharide IgG1, J. Immunol. 168 (2002) 3437e3443. [27] C.J. Orihuela, G. Gao, M. McGee, J. Yu, K.P. Francis, E. Tuomanen, Organ-specific models of Streptococcus pneumoniae disease, Scand. J. Infect. Dis. 35 (2003) 647e652. [28] S. Hammerschmidt, S. Wolff, A. Hocke, S. Rosseau, E. Muller, M. Rohde, Illustration of pneumococcal polysaccharide capsule during adherence and invasion of epithelial cells, Infect. Immun. 73 (2005) 4653e4667. [29] B.A. Green, Y. Zhang, A.W. Masi, V. Barniak, M. Wetherell, R.P. Smith, M.S. Reddy, D. Zhu, PppA, a surface-exposed protein of Streptococcus pneumoniae, elicits cross-reactive antibodies that reduce colonization in a murine intranasal immunization and challenge model, Infect. Immun. 73 (2005) 981e989.
Original article
Mucosal immunization with purified ClpP could elicit protective efficacy against pneumococcal pneumonia and sepsis in mice Ju Cao a,b, Tingmei Chen a, Dairong Li a, Chun K. Wong b, Dapeng Chen a,b, Wenchun Xu a, Xuemei Zhang a, Christopher W.K. Lam b, Yibing Yin a,* a
Key Laboratory of Diagnostic Medicine designated by the Ministry of Education, Chongqing Medical University, #1 Yixueyuan Road, ChongQing 400016, PR China b Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong Received 2 June 2008; accepted 15 September 2008 Available online 1 October 2008
Abstract Caseinolytic protease (ClpP) has been found to be highly conserved among different strains of Streptococcus pneumoniae and intraperitoneal immunization with ClpP could elicit protection against invasive pneumococcal infections. In this study, mucosal immunization with ClpP antigen induced both systemic and mucosal antibodies, and in this way reduced lung colonization in an invasive pneumococcal pneumonia model and also protected mice against death in an intraperitoneal-sepsis model. Surface localization of ClpP was confirmed using flow cytometry analysis. Furthermore, characterization of human sera for anti-ClpP IgG antibody levels demonstrated that ClpP protein was immunogenic in healthy children and was expressed during disease based on the elevated antibody levels in infected individuals. Finally, we describe that in vitro functional anti-ClpP antibody could kill streptococcus pneumoniae by polymorphonuclear leukocytes in a complement-dependent assay. To our knowledge, this is the first study about the protective efficacy of mucosal immunization with ClpP as a promising pneumococcal protein antigen. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: ClpP; Vaccine; Mucosal immunization
1. Introduction Infections with Streptococcus pneumoniae are a major cause of human diseases [1]. The surface of the nasal mucosa is the major reservoir for pneumococcus, and at this site it resides primarily in a commensal relationship with its human host [2]. Under predisposing conditions, especially following infections with respiratory viruses and blockage of the estuation tube, the pneumococci colonizing the upper airways are able to cause symptomatic diseases [3]. Pneumococcal infections result in an estimated 1.5 million deaths per year worldwide [4]. The rapid emergence of multidrug-resistant pneumococcal strains throughout the world is leading to
* Corresponding author. Tel./fax: þ86 23 6848 5658. E-mail address: [email protected] (Y. Yin). 1286-4579/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.micinf.2008.09.007
increased emphasis on the prevention of pneumococcal infections by vaccination [5]. The current 23-valent capsular polysaccharide vaccines classified as type 2 T-cell-independent antigens are poorly immunogenic in young children who suffer the highest rates of pneumococcal infections [7,8]. The conjugate vaccines with 7e11 serotypes could induce a memory immune response and are useful against invasive diseases caused by the vaccine-type strains [4]. However, there are shortcomings associated with the current capsular conjugate vaccines including high cost, serotype-specific protection, limited serotype coverage, and the likelihood of a concomitant increase in carriage and subsequent infection by nonvaccine serotypes [11]. At present, the new strategy tends to develop novel protein vaccines which may be highly immunogenic, T-cell dependent, antigenically conserved, and cross-protective among pneumococcal strains [3]. Protein antigens of S. pneumoniae
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have been evaluated for protective efficacy in animal models of pneumococcal infections. These include pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC), pneumococcal surface adhesion A (PsaA), pneumolysin (Ply), pneumococcal histidine triad (Pht) proteins, neuraminidases A and B, Pilus subunits, iron ABC transporters PiaA and PiuB, protein required for cell wall separation of group B streptococcus (PcsB) and serine/threonine protein kinase (StkP) [6,8,10,12,13]. Recent studies from several different research groups described a novel heat shock protein caseinolytic protease (ClpP) and immunization with ClpP has been suggested to possibly elicit protection against invasive pneumococcal infection [14e16]. Since the mucosal epithelium of the nasopharynx is the primary site of pneumococcal colonization and the natural route of infection with S. pneumoniae starts with colonization, which then progress to invasive diseases, mucosal immune responses may play a significant role [8]. Recent work from Richard Malley’s laboratory demonstrated that multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear could be achieved by killed nonencapsulated cells given intranasally with a nontoxic adjuvant [21], and they further showed that protection against nasopharyngeal colonization by S. pneumoniae is mediated by antigen-specific CD4þ T cells and intranasal immunization with a mixture of pneumococcal proteins protects against intranasal colonization in an antibody-independent, CD4þ T-cell-dependent manner [9,20]. However, mucosal vaccination with ClpP protein antigen against invasive pneumococcal infection has not been exploited. This study aimed to determine in detail whether or not the mucosal site would be efficient for induction of systemic and mucosal antibody responses after application of protein antigen ClpP. Protection elicited by intranasal immunization with ClpP was also evaluated in pneumonia and lethal sepsis models. Furthermore, in vitro opsonophagocytic killing activity induced by ClpP-specific antibody was investigated. 2. Materials and methods 2.1. Bacteria S. pneumoniae strain TIGR4 (ATCC BAA-334) was used for challenge experiments and the viability of bacterial stocks was analyzed prior to challenge. Escherichia coli DH5a (Invitrogen) was used as the host for routing plasmid cloning. Recombinant proteins were expressed in E. coli BL21(DE3) (Novagen). 2.2. Mice Six- to eight-week-old BALB/c mice were used for production of hyperimmune anti-ClpP sera. In pneumococcal pneumonia and sepsis model, we used 6- to 8-week-old CBA/N mice who have the Btk (XID) immune-response defect and fail to make natural antibodies to pneumococcal polysaccharides.
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All animal experiments were approved by the respective ethics committees of ChongQing Medical University. 2.3. Human sera samples and anti-ClpP immunoglobulin G (IgG) characterization Sera from 87 children not suffering from any infectious diseases at the time of sampling and sera from 12 children patients between 5 and 10 years of age with acute pneumococcal pneumonia were obtained at the Children’s Hospital of ChongQing Medical University in a study approved by the Ethical Committees of ChongQing Medical University and Children’s Hospital of ChongQing Medical University. These sera samples were analyzed for anti-ClpP IgG levels by ELISA using recombinant ClpP protein as coating antigens as described later. 2.4. Protein vaccines and adjuvants The cloning, expression, and purification of ClpP from TIGR4 pneumococci were done according to the procedures described previously [14]. Cholera toxin (CT) was obtained from Calbiochem Corporation. The recombinant ClpP protein and CT solutions for animal experiments did not contain any detectable LPS, as determined by the quantitative Limulus amoebocyte lysate assay (sensitivity limit 12 pg/ml; Biowhittaker Inc., MD, USA). 2.5. Production of hyperimmune mouse sera against ClpP in BALB/C mice Hyperimmune mouse sera specific for ClpP (anti-ClpP) were generated by intraperitoneal immunization of BALB/c mice with recombinant ClpP protein. Each mouse was primed with 10 mg of ClpP, emulsified in complete Freund’s adjuvant (CFA,1:1 ratio, v/v) on Day zero, and boosted with the same concentration of recombinant protein emulsified in incomplete Freund’s adjuvant (IFA,1:1 ratio, v/v) on Day 14 and on Day 28, each BALB/C mouse received the last dose of 10 mg 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. 2.6. Active immunization Six- to eight-week-old female CBA/N mice (12 per group) were immunized intranasally with recombinant ClpP twice a week for three consecutive weeks. The following conditions were used: CT, ClpP (5 mg), ClpP (5 mg) þ CT. The dose of antigen solution corresponded to a mixture of 25 ml of the ClpP and 5 ml (1 mg/ml) of CT. The intranasal immunization was carried out with the CBA/N mouse held in a supine position with the head down while 30 ml of the antigen solution was delivered slowly with a micropipette onto the nares so that the CBA/N mouse could sniff it in.
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2.7. Blood and saliva sampling for antibody measurements The CBA/N mice were bled from the retro-orbital sinus 15 days after the last immunization. Sera were then isolated and stored at 70 C. Saliva was collected from each CBA/N mouse by the insertion of absorbent sticks (Polyfiltronics) to the mouth. After 5minutes, the sticks were transferred to phosphate-buffered saline, pH 7.4 (PBS), containing 10.0 mg of protease inhibitor (Sigma) per ml to prevent the proteolysis of antibodies. The dissolved saliva was pooled for each group of CBA/N mice and was stored at 70 C. Anti-ClpP specific IgG and immunoglobulin A (IgA) from sera and saliva were determined by enzyme-linked immunosorbent assay (ELISA) as described below.
2.10. Pneumococci surface staining by flow cytometry The abilities of hyperimmune sera specific for ClpP from BALB/C mice to bind to the surface of live S. pneumoniae cells were determined using flow cytometry. TIGR4 from the early logarithmic growth phase were collected and washed twice in PBS. Bacterial suspensions were aliquoted in 300-ml samples, and primary serum antibodies were added for a final dilution of 1:100. The bacterial suspensions were incubated for 1 h at 4 C and washed twice before the addition of fluorescein isothiocyanate-conjugated goat antibodies to mouse (R & D systems) for 30 min at room temperature. After two additional washes, the bacteria were fixed with 0.25% (v/v) formaldehyde for 18 h at 4 C, and surface staining was detected by a flow cytometry (Beckman Coulter). 2.11. Challenge studies
2.8. Determination of ClpP-specific antibody titers in saliva and sera by ELISA Ninety-six-well ELISA plates were coated with recombinant ClpP (10 mg/ml, 100 ml per well in PBS) overnight at 4 C and blocked with 200 ml of 10% fetal calf serum (Sigma) in PBS for 3 h at room temperature. Individual sera or saliva from immunized CBA/N mice were tested in duplicate. Sera and saliva samples (100 ml) were added and serially diluted in PBS. After 1 h incubation at 37 C, plates were washed three times with 250 ml of PBS containing 0.05% Tween 20 (PBS-T) and titers of IgG and IgA were determined by addition of 100 ml of peroxidase conjugated goat anti-mouse IgG (Sigma) and peroxidase-conjugated goat anti-mouse IgA (Sigma), respectively. Following incubation for an additional hour at 37 C, the plates were washed six times and the color reaction for this ELISA was developed by adding 100 ml of tetramethylbenzidine (Sigma). The ELISA was allowed to react for 20 min and stopped with 50 ml 2% H2SO4. The plates were read spectrophotometrically at 405 nm and the endpoint titers were calculated for all samples. For human sera anti-ClpP IgG assays, antigen-specific antibodies in sera of children were analyzed by recombinant ClpP protein coated ELISA plates and peroxidase-conjugated goat anti-human IgG secondary antibodies (Sigma).
2.9. Western blot analysis Whole-cell lysates were prepared from S. pneumoniae strain TIGR4 by boiling cell suspensions containing SDSe PAGE sample buffer (Ambresco) for 5 min. After electrophoresis in 10% polyacrylamide gels, the proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes for immunoblotting with CBA/N hyperimmune mouse sera diluted 1:800. Detection of antigens was performed by an indirect antibody immunoassay using HRPlabeled anti-mouse IgG (Sigma) diluted 1:5000 in PBS-T and DAB staining.
In the sepsis model, intraperitoneal-challenge experiments were performed. The CBA/N mice were challenged 1 week after the last intranasal immunization with a challenge dose 1 105 CFU of the TIGR4. The challenged CBA/N mice were observed twice daily by an experienced person, and signs of sickness and dead CBA/N mice were recorded. CBA/N mice still alive after 21 days were considered to have survived the infection. In the pneumonia model, CBA/N mice were lightly anesthetized with 3% (v/v) halothane over oxygen by using a methacrylate box connected to Fluovac 240 (Anaesthetizing Systems). Following anesthetization, suspensions of 40 ml of PBS containing 1 106 bacteria were introduced into the nares of the CBA/N mice to induce aspiration pneumonia and the challenged CBA/N mice were euthanized on day 3 after challenge, and the lungs were removed and homogenized in PBS. Samples were serially diluted and plated on blood agar plates, and viable counts were determined after overnight incubation. 2.12. Passive protection studies CBA/N mice were injected in the tail vein with 150 ml of hyperimmune mouse sera specific for ClpP from BALB/C mice, and control CBA/N mice were injected with 150 ml of pooled preimmune sera from non-immunized BALB/C mice. CBA/N mice received a second dose of sera 24 h after intranasal infection as described above, under the same conditions as the first dose. 2.13. Antibody-dependent killing of S. pneumoniae by complement and human polymorphonuclear leukocytes (PMN) The ability of hyperimmune sera from BALB/c mice immunized with ClpP to opsonize pneumococcus for uptake and killing by human PMN was determined using an opsonophagocytosis assay as previously described [12,26]. TIGR4 were grown at 37 C overnight on blood agar and then inoculated into CþY medium. Bacteria were harvested in early
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stationary phase at 1500 g for 5 min, resuspended in sterile PBS, and diluted to 1 105 CFU/ml in RPMI 1640 medium (Invitrogen) with 10% FBS. PMN were isolated from 30 ml heparinized blood by Percoll gradient separation. Recovered PMNs were washed in HBSS without calcium chloride, magnesium chloride, magnesium sulfate, or phenol red (Life Technologies), and resuspended in RPMI 10% FBS to a concentration of 2 107cells/ml. The killing assays were performed by using BALB/c mice hyperimmune (HI) sera at a dilution of 1:10 or 1:100 and with their respective preimmune (PI) sera as control. A total of 100 ml of bacterial preparation (1 104 CFU), 50 ml of diluted sera, 200 ml of PMNs, and 40 ml of human sera obtained from an agammaglobulinemic patient as the complement source were combined (ratio of PMN to bacteria ¼ 400:1). The final volume was brought to 400 ml with RPMI 10% FBS and incubated at 37 C for 1 h with shaking. Aliquots (100 ml) from each tube were diluted in RPMI with 10% FBS, and 10 ml of each dilution was plated in duplicate on TSA II plates with 5% sheep blood. After overnight incubation at 37 C, CFU were counted and calculated for each original reaction tube, and data were expressed as the percentage of bacterial survival relative to the CFU at time 0.
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saliva. However, intranasal immunization with ClpP only or CT alone could not lead to systemic and mucosal anti-ClpP antibody responses. 3.2. Characterizing specific anti-ClpP antibodies in patients with pneumococcal pneumonia and healthy children In this part, 87 sera obtained from healthy children and 12 sera from pediatric patients with acute pneumococcal pneumonia were analyzed for anti-ClpP IgG levels. We detected a wide range of IgG titers from almost undetectable to high levels (Fig. 1). In the groups of healthy children, the ClpP-specific antibody levels showed a continuous increase, with peak median value in the 8e11 years old group. In sera from infants (<12 months old) and very young children (12e24 months old), the median ELISA values for ClpP were lower than those in older age groups. Further analysis of sera from the group of pediatric patients with acute pneumonia showed significantly (P < 0.05) higher ELISA activity than those from groups of healthy children, and the median value for anti-ClpP IgG ELISA unit in this group was about 1000. 3.3. Confirmation of surface localization of ClpP
2.14. Statistics Differences between the overall survival rates for groups of CBA/N mice were analyzed by Fisher’s exact test. A nonparametric test (ManneWhitney) was used to compare concentrations of anti-ClpP antibodies. Statistical comparisons of CFU levels were all carried out on log transformed data by the Wilcoxon tests. The limit statistical significance was a P value of 0.05. 3. Results 3.1. Anti-ClpP specific IgG and IgA in sera and saliva following immunization ELISA assays were performed in order to evaluate the ability of intranasal immunization with ClpP to induce antibody production in CBA/N mice. Results were shown in Table 1. It was evident from the results of this experiment that the nasal route, with CT as a mucosal adjuvant, was a way for the induction of serum IgG antibodies to purified ClpP, but IgG levels in saliva were not detectable in all groups. Notably, nasal immunization with ClpP in CT was clearly evident for induction of IgA antibodies to ClpP in sera, as well as in
Our previous work has shown that hyperimmune sera from BALB/C mice by immunization with the purified ClpP protein emulsified in Freund’s adjuvant could react with a single band of molecular mass 21 kDa in fraction of S. pneumoniae TIGR4 [14,15]. In this study, we demonstrated that antisera from CBA/N mice mucosally immunized with ClpP in CT also reacted with S. pneumoniae TIGR4 lysates and 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 (Fig. 2A). To further verify the surface location of ClpP, flow cytometry analysis was performed using anti-ClpP antisera obtained from immunized BALB/c mice. For bacteria stained with preimmune sera, geometric mean fluorescence intensity (GMFI) values were about 2.56; For bacteria stained with hyperimmune sera, GMFI values were about 4.86 (Fig. 2B). 3.4. Protection by ClpP in models of pneumococcal sepsis After analysis of anti-ClpP IgG, CBA/N mice underwent an intraperitoneal challenge with 1 105 CFU of S. pneumoniae strain TIGR4. Survival, observed up to 21 days after
Table 1 Serum and salivary antibody responses to mucosal immunization with ClpP Conditions
CT ClpP (5 mg) ClpP (5 mg) þ CT
IgG in serum
ND 1.8 (1.4e6.3) 2860 (2119e3890)
IgA Serum
Saliva
ND 0.1 (0.1e0.3) 120.1 (82.1e172.5)
ND ND 4.9 (3.4e10.9)
Median concentrations (ranges) of IgG and IgA antibodies to ClpP (kilounits per milliliter); ND, not detectable.
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challenge, was shown in Fig. 3. Partial protection was observed in CBA/N mice immunized with 5 mg of ClpP in CT (58.3%). This value was significantly higher than that for the mice treated with CT alone (P < 0.01). 3.5. Protection by ClpP in models of pneumococcal pneumonia
Fig. 1. ClpP protein antigen was immunologic in healthy children and antiClpP IgG could be elevated in children patients with acute pneumococcal pneumonia. Sera samples were analyzed for anti-ClpP IgG levels by ELISA using recombinant proteins as coating antigens. Each datum point represents an individual’s data. A horizontal line denotes the median anti-ClpP IgG titer for the group. Antibody titers are expressed as ELISA units (OD, 405 nm) at 1:1000 serum dilutions. m, months; y, years.
CBA/N mice were mucosally immunized with recombinant ClpP and challenged intranasally with TIGR4. Lung colonization of individual CBA/N mouse was shown at day 3 after challenge. ClpP in CT was capable of reducing the bacterial load in the lung about 100-fold relative to CBA/N mice immunized with either CT only or ClpP alone (P < 0.001, Table 2). In sera transfer experiments, anti-ClpP sera treatment was reproducibly as effective as intranasal immunization with ClpP in reducing lung infection by TIGR4 compared to control sera (P < 0.01) 3.6. Sera from ClpP-immunized BALB/c mice were efficiently opsonic for uptake and killing of pneumococcus by human PMN in a complement-dependent assay In this part, we used in vitro functional assay to measure the bactericidal activity of anti-ClpP hyperimmune sera. We were able to detect a dose-dependent opsonophagocytic killing activity for anti-ClpP sera (Fig. 4). Bacterial survival rate for the treatment of hyperimmune anti-ClpP sera diluted 1:10 was as low as 8%. For hyperimmune anti-ClpP sera diluted 1:100, there was an increase for bacterial survival rate which was about 40%. However, this bacterial survival rate was still much
Fig. 2. (A) Western immunoblot of pneumococcal lysates of TIGR4 by use of antisera from CBA/N mouse mucosally immunized with ClpP in CT. Lane 1 was developed with lysates from Staphylococcus aureus; lane 2 was developed with lysates from untransformed E. coli expression strain from which the recombinant ClpP protein was purified; lane 3 was developed with lysates from TIGR4 Streptococcus pneumoniae. Apparent molecular mass of ClpP in kilodaltons (KD) is indicated by arrow. (B) Confirmation of surface localization of ClpP. Flow cytometry analysis was performed using anti-ClpP hyperimmune sera obtained from immunized BALB/c mice. Shown are overlays of primary flow cytometry histograms demonstrating staining of TIGR4 with anti-ClpP hyperimmune (HI) or with their respective preimmune (PI) sera. Results are expressed as histograms of relative cell counts with fluorescence intensity. This figure is representative of three independent experiments with similar results.
Fig. 3. Protection by ClpP in model of pneumococcal sepsis. CBA/N mice (n ¼ 12) were intranasally immunized with recombinant ClpP and then intraperitoneally challenged with S. pneumoniae TIGR4. Control mice (n ¼ 12) were sham immunized with CT only. Mucosal immunization with ClpP significantly protected the CBA/N mice (P < 0.01) from an intraperitoneal lethal challenge with S. pneumoniae TIGR4.
J. Cao et al. / Microbes and Infection 10 (2008) 1536e1542 Table 2 Protection by ClpP in model of pneumococcal pneumonia Conditions
No. of mice
CFU/lung, mean log (standard error)
CT ClpP (5 mg) ClpP (5 mg) þ CT Preimmune sera Anti-ClpP sera
12 12 12 12 12
5.47 5.51 3.46 5.78 3.68
(0.34) (0.31) (0.20)* (0.29) (0.34)#
*
Value is significantly different from the value observed for CBA/N mice immunized with CT or ClpP alone (P < 0.001);# value is significantly different from the value observed for CBA/N mice treated with preimmune sera (P < 0.01).
lower than that for the treatment of preimmune sera which had a bacterial survival rate of about 91%. 4. Discussion Streptococcus pneumoniae is a major pathogen which enters the body through the respiratory mucosa and may cause serious infections [11]. Intranasal vaccination may offer an alternative approach to current strategies since it induces mucosal as well as systemic immune responses and systemic immunization does not induce mucosal immune responses [3,18]. Mucosal immunization would be a more effective route for immunizing young children, the elderly and the age groups most at risk of pneumococcal infections, as the mucosal immune system develops earlier in infants and lasts longer in the elderly than the systemic immune system [8,17].Mucosal delivery of pediatric vaccines has become an explicit goal of the NIH as well as of WHO from economical, logistical and safety standpoints [19] ClpP represents a unique family of serine proteases by itself and ClpP-mediated proteolysis copes with protein folding, repair, and degradation [15]. Accordingly, ClpP plays a complex and central role in the pathogenesis of pneumococcal infections. In this study, we could detect surface
Fig. 4. ClpP-immunized hyperimmune (HI) mouse sera at a dilution of 1:10 or 1:100 is opsonic for killing of pneumococcus by human PMN in the presence of complement, while their respective preimmune (PI) sera are much less opsonic. Data are expressed as the percentage of bacterial survival relative to the initial CFU at time 0. The average of three experiments is shown; error bars represent standard deviations.
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staining with BALB/C mouse hyperimmune sera specific for ClpP by FACS analysis and then demonstrated that mucosal immunization with ClpP could induce both systemic and mucosal antibodies when applied CT as a mucosal adjuvant. Besides that, intranasal immunization with ClpP protected CBA/N mice against death caused by S. pneumoniae TIGR4 in an intraperitoneal-sepsis model. This way could also reduce lung colonization in an invasive pneumococcal pneumonia model, and passive immunization with anti-ClpP sera demonstrated that this protection was antibody mediated. The induction of protective efficacy to pneumococci via the nasal route suggests that the nasopharyngeal mucosa possesses the necessary structures to make mucosal immunization a realistic alternative to the use of ClpP protein vaccines. Furthermore, we detected natural antibody responses against ClpP in healthy children and they were followed by the development of specific antibody levels as a function of age. Our results showed that anti-ClpP IgG titer was elevated in pediatric patients with acute pneumococcal pneumonia, so ClpP antigen is expressed during infection in the human host and may form part of the normal repertoire of protective antibodies induced by prior infection. Host defenses against S. pneumoniae depend largely on phagocytosis following opsonization by specific antibodies and complement [11]. Our sera transfer experiments suggested that protective efficacy of ClpP was antibody mediated. We further established in vitro assays to investigate opsonophagocytic killing activity of functional anti-ClpP antibody. In these assays, we used TIGR4 pneumococcus which is a virulent strain with less capsule [27]. At this stage, we still do not know whether or not functional anti-ClpP antibody could kill other encapsulated strains. However, previous studies have demonstrated that most of the polysaccharide capsule was shed on S. pneumoniae adhesion to the epithelial cell [28]. Therefore, shedding of the polysaccharide capsule may reveal cell wall-associated proteins that are otherwise masked so that functional antibodies would interact with these protein antigens [29]. To our knowledge, this is the first report that in vitro functional anti-ClpP antibodies could kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent assay, which hints that anti-ClpP sera may have therapeutic utility, particularly in immunocompromised patients. Based on the results presented in this study, mucosal immunization with ClpP appears therefore as a novel, noninvasive vaccine approach that could protect mice against invasive pneumococcal pneumonia and sepsis. The findings of the present study should stimulate attempts to extend the development of ClpP as a promising pneumococcal protein vaccine component. However, it is also apparent that elicitation of protection by mucosal immunization with ClpP is dependent on CT, and CT is not an acceptable adjuvant for humans due to toxicity [21]. There are other potential mucosal adjuvants including mycoplasma-associated lipoprotein (MALP-2), porcine lung surfactant (PLS), Bordetella pertussis monophosphoryl lipid A (MPLA) and cholera toxin binding B subunit (CTB) which has been given safely by the intranasal route to humans and shown to be effective as an adjuvant by
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this route [22e25]. Therefore, our next goal is to identify a mucosal adjuvant that could be used safely and economically to develop ClpP-based pneumococcal protein vaccines for human use. Acknowledgment This work was supported by grants from National Natural Science Foundation grants of China (Nos. 30400376, 30471838, 30371275). References [1] H.K. Parsons, S.C. Metcalf, K. Tomlin, R.C. Read, D.H. Dockrell, Invasive pneumococcal disease and the potential for prevention by vaccination in the United Kingdom, J. Infect. 54 (2007) 435e438. [2] J. Casal, D. Tarrago´, Immunity to Streptococcus pneumoniae: Factors affecting production and efficacy, Curr. Opin. Infect. Dis. 16 (2003) 219e224. [3] A. Kadioglu, J.N. Weiser, J.C. Paton, P.W. Andrew, The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease, Nat. Rev. Microbiol. 6 (2008) 288e301. [4] J.A. Scott, The preventable burden of pneumococcal disease in the developing world, Vaccine 25 (2007) 2398e2405. [5] R. Novak, B. Henriques, E. Charpentier, S. Normark, E. Tuomanen, Emergence of vancomycin tolerance in Streptococcus pneumoniae, Nature 399 (1999) 590e593. [6] M. Jomaa, S. Terry, C. Hale, C. Jones, G. Dougan, J. Brown, Immunization with the iron uptake ABC transporter proteins PiaA and PiuA prevents respiratory infection with Streptococcus pneumoniae, Vaccine 24 (2006) 5133e5139. [7] E.D. Shapiro, A.T. Berg, R. Austrian, D. Schroeder, V. Parcells, A. Margolis, R.K. Adair, J.D. Clemens, The protective efficacy of polyvalent pneumococcal polysaccharide vaccine, N. Engl. J. Med. 325 (1991) 1453e1460. [8] D.E. Briles, S.K. Hollingshead, G.S. Naobors, J.C. Paton, A. BrooksWalter, The potential for using protein vaccines to protect against otitis media caused by streptococcus pneumoniae, Vaccine 19 (Suppl. 1) (2001) S87eS95. [9] K. Trzcin´ski, C.M. Thompson, A. Srivastava, A. Basset, R. Malley, M. Lipsitch, Protection against nasopharyngeal colonization by Streptococcus pneumoniae is mediated by antigen-specific CD4þT cells, Infect Immun. 76 (2008) 2678e2684. [10] C. Gianfaldoni, S. Censini, M. Hilleringmann, M. Moschioni, C. Facciotti, W. Pansegrau, V. Masignani, A. Covacci, R. Rappuoli, M.A. Barocchi, P. Ruggiero, Streptococcus pneumoniae pilus subunits protect mice against lethal challenge, Infect Immun. 75 (2007) 1059e1062. [11] B.G. Spratt, B.M. Greenwood, Prevention of pneumococcal disease by vaccination: does serotype replacement matter? Lancet 356 (2000) 1210e1211. [12] C. Giefing, A.L. Meinke, M. Hanner, T. Henics, M.D. Bui, D. Gelbmann, U. Lundberg, B.M. Senn, M. Schunn, A. Habel, B. Henriques-Normark, A. Ortqvist, M. Kalin, A. von Gabain, E. Nagy, Discovery of a novel class of highly conserved vaccine antigens using genomic scale antigenic fingerprinting of pneumococcus with human antibodies, J. Exp. Med. 205 (2008) 117e131. [13] J.E. Adamou, J.H. Heinrichs, A.L. Erwin, W. Walsh, T. Gayle, M. Dormitzer, R. Dagan, Y.A. Brewah, P. Barren, R. Lathigra, S. Langermann, S. Koenig, S. Johnson, Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis, Infect. Immun. 69 (2001) 949e958.
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