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We investigated the adjuvant effect of CpG ODN alone or in combination with aluminum hydroxide on the immune response to the three main antigens presented in current acellular pertussis vaccines: pertussis toxoid, filamentous haemagglutinin and pertactin. The development of protection in mice was investigated for the intra-peritoneal and intra-nasal immunisation routes. The results showed that CpG ODN alone, or in combination with aluminum hydroxide, gave enhancement in anti-pertussis toxin, anti- filamentous haemagglutinin and especially anti-pertactin titers after mucosal immunisation. Higher macrophage NO levels indicating activation were found when the antigens were co-formulated with CpG ODN. Vaccines containing CpG ODN gave enhanced humoral and CMI responses with a shift toward Th-1 and increased protection against challenge infection with B.pertussis in mice.
Whole cell pertussis vaccines (WCVs) have now been largely replaced by less reactogenic acellular pertussis vaccines (ACVs) in many countries. ACVs have conferred protection against pertussis in clinical trials and have been associated with a reduced incidence of serious adverse events.1,2 Nevertheless, pertussis remains endemic worldwide and recently there has been a resurgence of reported cases in several regions of the world where the ACV vaccination coverage in young children is high.3-5 This suggests that the current vaccines may be failing to provide adequate protection.
Current ACVs containing detoxified antigens with aluminum adjuvant induce a predominantly Th-2 type response in humans.6,7 Although aluminum is efficient in inducing a strong antibody response, it is a weak adjuvant for the induction of Th1 type responses and cell mediated immunity (CMI).8,9 As CMI plays an essential role in the clearance of Bordetella pertussis it is necessary that vaccines should stimulate it. Therefore, improving this aspect of their performance may be the key to increasing the efficacy of the current ACVs.
Oligonucleotides containing immunostimulatory CpG motifs (CpG ODN) have been shown to be an effective mucosal and systemic adjuvant for vaccines against a variety of infectious diseases.10-12 CpG ODN favors a Th1 response to a wide variety of antigens and has broad effects on other arms of the immune response.12-15 CpG ODN interacts with toll-like receptor 9 (TLR9) which initiates a cascade of events resulting in secretion of Th1 type cytokines and chemokines and leading to maturation, differentiation and proliferation of B and T cells, macrophages, natural killer cells and monocytes which contribute to its adjuvant activity and also to stimulation of B cells to proliferate and secrete immunoglobulin.11,12 In several studies CpG ODN as adjuvant has been reported to shift the immune response to pertussis toxin (PT, one of the antigenic components of ACVs) substantially toward to Th1 as evidenced by the increase in IgG2a subclass titers in murine models.16-18
Since most ACVs contain multiple antigens, we examined the ability of CpG ODN /or CpG ODN in combination with aluminum hydroxide as adjuvant to improve the humoral and CMI responses to not only PT antigen, but also to the other two major antigen components presented in ACV, filamentous haemagglutinin (FHA) and pertactin (PRN), following either intraperitoneal (IP) or intra-nasal (IN) administration, and to relate this to protection in a mouse model.
Immune response to both CpC ODN or CpG ODN alone were assessed in pilot studies. There was no response observed for either of them (data not shown). Therefore, only CpC ODN alone was included in most of the later experiments as the negative control.
Mice intra-nasally immunised with CpC ODN alone as the negative control group showed no detectable antibody titers to PT, FHA or PRN. Mice receiving pertussis antigens alone showed very low or undetectable antibody response (data not shown). In the group of mice receiving pertussis antigens formulated with CpG ODN, increased anti-PRN IgA response was observed, but not for anti-PT and anti-FHA (data not shown). However, greatly increased serum and mucosal (lung supernatant) IgG antibody responses with approximately 10 to > 1000 fold increases in anti-PT, anti-FHA and anti-PRN titers respectively (p < 0.05) were seen after mucosal immunisation (Fig. 1A). Similar results were found after IP administration (data not shown). A 2 and 18-fold increase (p < 0.05) in antibody production to PT and FHA antigens respectively followed primary immunisation. Mice boosted with the same antigen formulations at 4 weeks after the primary immunisation, showed further 12, 65 and 5 fold increases in antibody production to PT, FHA and PRN respectively (p < 0.05) (data not shown).
Nitric oxide (NO) production in the macrophage cultures was used as a marker for macrophage activation.9 Macrophages from mice immunised (IP) with pertussis antigens with/or without aluminum hydroxide adjuvant only produced moderate NO. The NO concentrations in macrophages from mice immunised with antigens plus CpG ODN were approximately 3 fold higher than in the antigen alone and aluminum hydroxide groups (p < 0.05) (Fig. 1B). A 4.5 fold increase in NO production was observed in macrophage cultures from mice immunised with antigens plus CpG ODN in combination with aluminum hydroxide as the adjuvant in comparison with the antigen alone or antigen plus aluminum hydroxide groups. Moreover, spleen cell supernatants from mice that received antigen plus CpG ODN in combination with aluminum hydroxide had significantly higher levels (5 folds, p < 0.05) of type 1 cytokine, IFN-γ production than the antigen alone group or antigen plus aluminum hydroxide groups (Fig. 1C). The number of IFN-γ secreting cells were examined by ELISPOT experiments which showed 69% higher cells in the antigen plus CpG ODN in combination with aluminum hydroxide group than the antigen alone group (data not shown). These results indicated that apart from PT, CpG ODN also has an adjuvant effect on the other main pertussis antigens presented in ACVs, FHA and PRN, and was able to generate a memory response as well as Th1 Type cellular responses. Most importantly, the adjuvant effect of CpG ODN /or CpG ODN in combination of aluminum hydroxide could produce macrophage activation.
The adjuvant effect of CpG ODN on protection by ACV against B. pertussis infection was investigated in an aerosol challenge model. Mice were loaded with approximately 104 bacteria per lung on the aerosol challenge day which increased to approximately 107 CFU/lung in the control group on day 7. The group immunised IP with antigens alone in comparison with the CpC ODN control group showed an approximately 4 log reduction in CFU (p < 0.05, Fig. 2A). The groups receiving antigens plus either CpG ODN or aluminum hydroxide showed similar results, with a further 1.0 log reduction in CFU (p < 0.05). The numbers of viable bacteria in the lungs of mice that received antigens plus CpG ODN in combination with aluminum hydroxide as the adjuvant were below the detection limit.
After the mucosal immunisation there was no protection observed in the antigens alone group (Fig. 2B). When CpG ODN was used as an adjuvant there was an approximately 4 log reduction in lung CFU compared with the controls (p < 0.05) and this was similar to the CFU counts observed in the antigen plus aluminum hydroxide group. Viable bacteria were below the detection limit in the group immunised with antigens plus CpG ODN in combination with aluminum hydroxide.
Both macrophages and neutrophil leukocytes have been shown capable of killing B. pertussis in vitro. NO and reactive oxygen intermediates are potentially important mediators involved in this killing role and clearance of B. pertussis from mice involves activated macrophages.19,20 The NO concentrations in macrophages from mice immunised with antigens plus CpG ODN were approximately three times higher than in the antigen alone group. Our results indicate that CpG ODN in combination with the antigens was effective in activating macrophages and this was correlated with increased protection to challenge. Moreover, the enhanced NO production is apparently not due to direct innate immune stimulation of macrophages since CpG ODN alone does not induce significant NO production. It is not clear if an antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism or opsonization effects were involved wherein high levels of anti-pertussis antibodies enhance NO synthase activity. Further studies to delineate the underlying mechanisms would be needed. CpG ODN in combination with aluminum hydroxide as adjuvant synergized the pertussis immunity toward to a stronger Th1 type response than CpG ODN used alone, as evidenced by the significantly higher levels of both NO and IFN-γ found in macrophages or in the spleen supernatants respectively from mice immunised with antigens plus both CpG ODN and aluminum hydroxide as adjuvants in comparison with the single adjuvant group. This is in agreement with the previous findings by Sugai et al.16 where the immune response with CpG ODN as an adjuvant was shifted substantially toward Th1. Co-administration of CpG ODN with aluminum hydroxide and antigen has been reported to stimulate strongly both Th1 and Th2 type immune responses in mice and other species.21,22 This similar synergistic adjuvant effect was recently observed in a clinical trial in humans with Anthrax Vaccine Adsorbed (AVA) plus 1mg of CpG ODN.23 The exact mechanism by which aluminum enhances the CpG ODN induced immune-stimulatory effects is not clear. It has been attributed to depot formation by the aluminum at the site of injection, which also may activate local cells and both factors might enhance the CpG ODN-mediated effects.22,24,25
Since pertussis is a respiratory tract disease, in the present study the ability of CpG ODN to act as a mucosal adjuvant for immunisation against pertussis was evaluated. Much higher antigen specific IgG antibodies were found in both serum samples and supernatant of lung homogenate when CpG ODN was used as the adjuvant. Greater stimulation of production of antibody to PRN than of antibodies to PT and FHA was observed. Enhanced production of IgA antibodies against PT was also seen in supernatant of lung homogenate when CpG ODN and aluminum were used in combination as adjuvant (data not shown). A moderate increase in IgG antibodies to PT, FHA and PRN was observed in the combined adjuvant group in comparison to the adjuvant alone group. Chemical or physical association of CpG with the antigen has been shown to be important for the ability of the CpG to enhance immunogenicity and the optimal formulation of aluminum adjuvanted vaccines containing CpG is likely to require both the antigen and the CpG to be fully bound to the aluminum hydroxide since this would optimize the co-presentation of both antigen and CpG.26,27 Taken together, the results indicate that CpG can also be used as a powerful mucosal adjuvant for ACV. Our results are in agreement with previous studies where the intranasal delivery of CpG as an adjuvant resulted in strong systemic and mucosal immune responses to co-administered antigens including hepatitis B surface antigen,28 β-galactosidase,29 and whole killed influenza virus.30
Our results are in support of the findings from other studies where a significant feature of CpG is its unique ability to stimulate both antibody and cell-mediated responses in immunised animals.10-15 The ability to induce potent Th1-type immune responses is important because with many pathogens, including B. pertussis, both CMI and antibody contribute to protection.31-34 Here we have shown that enhanced humoral as well as cellular responses are correlated with protection against B.pertussis when CpG ODN is used as an adjuvant and moreover the protection is enhanced further when CpG ODN and aluminum hydroxide in combination is used as adjuvant. The synergistic effect in protection against aerosol challenge with live B.pertussis was observed in mice via both IP and IN route of administration which indicates that CpG ODN or CpG ODN in combination with aluminum hydroxide combination could generate both mucosal and systemic protective immune responses to acellular pertussis antigens. Taking together our and others findings, it indicates that CpG ODN is able to shift the immune response of acellular pertussis antigens toward either a stronger Th-1 type or a more balanced response and hence enhance the protective activity of ACV against the challenge. Since the Th1-biasing and adjuvanting effects of CpG ODN are TLR9-mediated, further work would be needed to study the role of the CpG ODN receptor on the observed effects using TLR9 knockout mice. Furthermore, in the present study, the persistence of protection produced by the CpG ODN was not addressed. It would be interesting to investigate the long-term protective effects of using CpG ODN as adjuvant for ACVs.
Since CMI response plays an essential role in the clearance of B.pertussis, the improvement of CMI response may be the key to increasing the efficacy of the current ACVs. In this study we have provided the evidence that CpG ODN in combination with the antigens was effective in activating macrophages and this was correlated with increased protection to B. pertussis challenge. Since pertussis is a respiratory tract disease, we also showed that CpG ODN can be a powerful mucosal adjuvant for pertussis antigens. Furthermore, all acellular pertussis vaccines on the market contain multiple aP antigens except one. Evaluation of adjuvant effect of CpG ODN to each of the antigen is important since CpG ODN may show different adjuvant effect on different antigen, for example, in the present study CpG ODN showed greater enhanced response to anti-PRN than to PT and FHA.
In summary, our results suggest that the use of CpG ODN in ACVs could emphasize the Th1 type response and improve protection. If confirmed by clinical trials, the performance of ACVs could be greatly improved by using CpG ODN/ or CpG ODN in combination with aluminum and achieve more effective control of pertussis.
All experiments were performed using female NIH strain mice (3–4 weeks), obtained from Harlan, UK and maintained in pathogen-free conditions. All experiments were regulated under the Animal (Scientific Procedures Act) 1986.
Purified detoxified PT (PTd), FHA and PRN, as well as native pertussis antigens used for coating ELISA plates were kindly donated by GlaxoSmithKline (GSK), Rixensart, Belgium. A mouse-specific synthetic CpG oligodeoxynucleotides (CpG ODN 1826, TCC ATG ACG TTC CTG ACG TT) in Tris-EDTA buffer (pH 8.0) and an inactive negative control (CpC ODN 1982, TCC AGG ACT TCT CTC AGG TT) of same size were obtained from Loeb Research Institute, Canada. Aluminum hydroxide gel (Alhydrogel) was from Superfos Biosector, Vedbaek, Denmark. Heat-killed B. pertussis 18.323 (HKC) was prepared by growing cultures on charcoal agar plates for 18–24h and re-suspending in 1% casein in saline. The suspension (1 × 109/ml) was then heat killed at 80°C for 30 min.
For intra-peritoneal (IP) immunisation, mice were immunised with 0.5 ml of control CpC ODN alone (30 μg/dose), a mixture of antigens alone, (PT at 5 μg, FHA at 5 μg and PRN at 2.5 μg per dose), antigen mixture in combination with aluminum hydroxide (1.3 mg per dose) or CpG ODN (30 μg per dose) and antigen mixture in combination with aluminum hydroxide (0.26 mg per dose) and CpG ODN (30 μg per dose). These dosages were chosen to maximise CMI responses rather than minimise antigen load. For intra-nasal (IN) immunisation, antigen concentrations were increased to PT 6.7 μg/dose, FHA 6.7 μg/dose and PRN 3.35 μg/dose. A total volume of 40 μl of immunogens were applied as droplets into each nostril. For both immunisation routes, groups of five mice were administered indicated amounts of B. pertussis antigens and adjuvants followed by a booster at four weeks with terminal bleeds and sampling for macrophage and spleen at two weeks post boost.
This was done as described in Xing et al.31
Groups of five mice were terminally bled at two weeks post boost and sera and supernatants from lung/tracheal homogenates from individual mice were assayed for IgG antibodies to B. pertussis antigens PT, FHA and PRN using ELISA. The ELISA units in the sera samples to each antigen were calculated against the First International Reference Mouse Serum for Pertussis (NIBSC, code 97/642).
Aerosol infection was performed as described in Xing et al.35 The lower limit of sensitivity of the counting method was 100 CFU/per lung. This does not represent an absolute detection limit but is the lowest reliably quantifiable value, taking into account the physical and biological nature of the process as well as the statistical dilution model.
A student T-test was used to compare the difference between groups. A p-value ≤ 0.05 was considered as statistically significant.
Grateful acknowledgments are due to Dr. Heather L. Davis, Loeb Health Research Institute at the Ottawa Hospital, Canada for provision of CpG ODN samples used in this study and helpful scientific discussion. Authors would also like to thank Dr. A.M. Krieg, Department of Internal Medicine, University of Iowa College of Medicine for supporting this study.
No potential conflicts of interest were disclosed.
Previously published online: www.landesbioscience.com/journals/vaccines/article/22755