The potential of 3′,5′-cyclic diguanylic acid (c-di-GMP) as an effective vaccine adjuvant

Vaccine 28 (2010) 3080–3085

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Vaccine journal homepage: www.elsevier.com/locate/vaccine

Review

The potential of 3 ,5 -cyclic diguanylic acid (c-di-GMP) as an effective vaccine adjuvant Wangxue Chen a,b,∗ , Rhonda KuoLee a , Hongbin Yan c a

Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada Department of Biology, Brock University, St. Catharines, Ontario, Canada c Department of Chemistry, Brock University, St. Catharines, Ontario, Canada b

a r t i c l e

i n f o

Article history: Received 18 December 2009 Received in revised form 12 February 2010 Accepted 15 February 2010 Available online 1 March 2010 Keywords: Adjuvant c-di-GMP Vaccine Mucosal immunization

a b s t r a c t 3 , 5 -Cyclic diguanylic acid (c-di-GMP) is a bacterial intracellular signaling molecule that plays a crucial role in the regulation of bacterial motility, adhesion, cell-to-cell communication, exopolysaccharide synthesis, biofilm formation and virulence. The recent finding that c-di-GMP can act as a danger signal on eukaryotic cells has prompted the study of the immunostimulatory and immunomodulatory properties of c-di-GMP in an effort to determine whether c-di-GMP might be further developed as a potential vaccine adjuvant. In this review, we discussed the recent in vitro and in vivo studies of the immunostimulatory properties of c-di-GMP and the progress that has been made in the preclinical development of c-di-GMP as a potential vaccine adjuvant for systemic and mucosal vaccination. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3080 3 , 5 -Cyclic diguanylic acid (c-di-GMP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3081 Immunostimulatory properties of c-di-GMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3081 The promise of c-di-GMP as a vaccine adjuvant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3083 c-di-GMP as a mucosal adjuvant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3083 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3084 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3084 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3084

1. Introduction Despite significant medical advances and the improvement of human health, the control and eventual eradication of infectious diseases remain major challenges to public health in both developed and developing countries. To date, immunization with potent and safe vaccines remains the most efficient and cost-effective medical strategy in the prevention and control of infectious diseases. In this regard, vaccines prevent more than three million deaths each year with an estimated positive economic impact over a billion dollars per year [1]. The global vaccination program and

∗ Corresponding author at: Institute for Biological Sciences, National Research Council Canada, 100 Sussex Drive, Room 3100, Ottawa, ON K1A 0R6, Canada. Tel.: +1 613 991 0924; fax: +1 613 952 9092. E-mail address: [email protected] (W. Chen).

effort has successfully eradicated one infectious disease (smallpox) with another one (polio) expected soon [2]. Although substantial progress has been made in the prevention of many important infectious diseases (such as polio, measles, whooping cough, hepatitis A and B, etc.) by vaccination, infectious diseases still cause substantial morbidity and mortality and thus, there remains an urgent need for the development of new or improved vaccines [1]. This is particularly true for organisms that cause devastating diseases such as HIV, malaria and tuberculosis. In addition, the recent surge in emerging and re-emerging microbial pathogens and the mounting incidence of antimicrobial resistance are major concerns in the clinical management of infectious diseases, fueling the urgency for new and improved vaccines. A number of different strategies have been used in the development of vaccines. Vaccines made from live, attenuated microorganisms are usually very effective but the risk of reversion and limited self-replication makes this strategy less than ideal and

0264-410X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.02.081

W. Chen et al. / Vaccine 28 (2010) 3080–3085

Fig. 1. Chemical structures of c-di-GMP and its sulfur analogues (c-di-GMP-S1 and -S2).

these vaccines are usually considered unsafe for use in humans [3]. Similar regulatory concerns plague recombinant protein and live vector vaccines. Vaccines based on killed, whole pathogen cells are somewhat effective but these could potentially contain toxic molecules such as lipopolysaccharides or be contaminated with live pathogens [4]. The safety of DNA vaccines have also come into question since there are concerns about insertion into the host genome and possible mutagenic and oncogenic potential as well as the potential to trigger pathogenic anti-DNA autoimmune antibody responses [5]. Subunit vaccines, on the other hand, do not have these safety concerns as they only contain purified antigens rather than whole organisms and are, therefore, not infectious. As such, they can be safely given to immunosuppressed people and are less likely to induce unfavorable immune reactions. However, these advantages are tempered by the fact that purified antigens are often less immunogenic and they require the use of strong adjuvants to increase immunogenicity. Adjuvants can be classified into one of two broad categories: they are either immunostimulatory molecules like CpG oligonucleotides (ODN), bacterial toxins and cytokines or they are delivery vehicles that have inherent immunostimulatory activity like liposomes, microparticles and emulsions. Licensing adjuvants for use in human vaccines has been a difficult undertaking, typically due to the safety profile of these substances [6]. There are very few adjuvants currently approved for human use. In fact, in the United States, alum is still the only adjuvant approved for human vaccination. In Europe and Canada combined, only three other adjuvants have been licensed for human use: MF59 (licensed in an influenza vaccine, Fluad), AS03 (licensed for the prepandemic H5N1 vaccine and is now being used in the current pandemic H1N1 influenza vaccine) and AS04 (approved for use in vaccines against hepatitis B and human papillomavirus) [7]. Both MF59 and AS03 are squalenebased oil-in-water emulsion adjuvants and AS04 is a combination of two adjuvants, alum and monophosphoryl lipid A [7]. Given the lack of licensed adjuvants, the search for new vaccine adjuvants is a high priority for vaccinologists. 2. 3 , 5 -Cyclic diguanylic acid (c-di-GMP) 3 , 5 -Cyclic diguanylic acid (Fig. 1 where X = Y = O) is an intracellular signaling molecule first identified in Gluconacetobacter xylinus (formerly Acetobacter xylinum) where it regulates cellulose production by modulating cellulose synthase activity [8]. Research has suggested that c-di-GMP-mediated signaling is widespread in bacterial species from Escherichia coli to Bacillus subtilis to Caulobacter crescentus [9–11]. However, it has not been found in higher eukaryotes [9], leading many to believe that c-di-GMP signaling is an exclusively bacterial characteristic. Its seemingly ubiquitous presence in bacteria would seem to suggest that c-di-GMP plays a role

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in one or more critical bacterial functions and in fact, an increasing body of research has revealed the importance of c-di-GMP as a bacterial second messenger (cf. [12–14]) in the regulation of many physiological processes important for bacterial survival (such as adhesion, cell-to-cell communication, exopolysaccharide synthesis, and motility [15–18]). The recent finding that c-di-GMP can act as a danger signal on eukaryotic cells [19] has prompted the study of the immunostimulatory and immunomodulatory properties of c-di-GMP in an effort to determine whether c-di-GMP might be further developed as a potential vaccine adjuvant. This review focuses on the recent studies of the immunostimulatory properties of c-di-GMP and the progress that has been made in the preclinical development of c-di-GMP as a potential vaccine adjuvant for systemic and mucosal vaccination (Table 1).

3. Immunostimulatory properties of c-di-GMP Several studies have now convincingly demonstrated that c-diGMP does indeed have strong immunostimulatory properties. In vitro experiments have shown that c-di-GMP stimulates human immature dendritic cell (DC) expression of MHC class II, costimulatory molecules CD80/CD86 and maturation marker CD83, increases their secretion of cytokines and chemokines interleukin (IL)-12, interferon (IFN)-␥, IL-8, monocyte chemotactic protein 1 (MCP1), IFN-␥ inducible protein 10 (IP-10), and regulated on activation normal T cell expressed and secreted (RANTES), and alters expression of chemokine receptors including CCR1, CCR7 and CXCR4 [20]. Also, c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity [20]. More importantly, the immunostimulatory properties of c-diGMP have also been demonstrated in vivo. Intraperitoneal (i.p.) or intranasal (i.n.) administration of mice with c-di-GMP induces recruitment of monocytes and granulocytes [20] and activates the host immune response [21,22]. In one study, the lungs and draining lymph nodes from mice intranasally treated either with c-di-GMP or phosphate buffered saline (PBS) were examined 24 or 48 h after treatment for differences in cell number or composition. Results showed that the draining lymph nodes of c-di-GMPtreated mice had significantly higher total cell numbers as well as higher percentages of CD44low cells and CD86 positive cells. In the lung, however, the picture was less clear with no difference in total numbers of monocytes or neutrophils or pulmonary DCs as determined by flow cytometry [21]. However, there was some indication that c-di-GMP did affect lung parenchymal cells in that the lungs from c-di-GMP-treated mice had a larger proportion of alveolar macrophages which were newly recruited (CD11chi MHCIIlow CD11b+ ). Also, DCs (CD11chi MHCIIhi ), although not significantly increased in number, expressed higher levels of CD40 and CD86 than PBS-treated control mice [21]. Work from our own laboratories has indicated that 24 h after a single i.n. administration of c-di-GMP, there is a significant increase in the number of pulmonary DCs with higher expression of CD40 and CD80 but not CD86 or MHCII [23]. The treatment also induced a rapid but transient recruitment of neutrophils and other inflammatory cells into the bronchoalveolar space [23] and increased levels of proinflammatory cytokines and chemokines IL-12p40, IL1␤, IL-6, keratinocyte derived chemokine (KC), MCP-1, macrophage inflammatory protein (MIP)-1␤, RANTES and tumor necrosis factor (TNF)-␣ in a dose-dependent manner [22]. A number of recent studies have shown that the innate immune response elicited by c-di-GMP is a potent immunomodulator for the treatment of bacterial infections. In this regard, studies of the effect of c-di-GMP on the course of bacterial infection have clearly shown a striking protective effect of c-di-GMP administration against a number of serious bacterial infections. Using a mouse

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Table 1 In vivo effects of c-di-GMP. Route of administration

Main findings

Reference

Immunostimulatory effects i.p. i.n. i.n. i.n. i.n.

Monocyte and granulocyte recruitment Neutrophil recruitment in BAL Increased proinflammatory cytokines in BAL Increased DC numbers and DC activation Increased newly recruited alveolar macrophages in lung

[20] [22,23] [23] [21,23] [21]

Immunomodulatory effects after bacterial infection Intramammary Decreased bacterial burden in mammary glands s.c. Decreased bacterial burdens in livers and spleens i.p. Decreased bacterial burdens in livers only i.n. Decreased bacterial burdens in lung and blood s.c. Improved survival i.n. More neutrophils, ␣␤T cells, total and activated NK cells Th1-biased increases in mRNA levels and cytokine levels

[20,24] [26] [26] [21,27] [21] [27]

Adjuvant properties s.c. s.c. i.m. s.c. i.p. i.p. i.n. i.n. i.n. i.n.

Balanced Th1/Th2 humoral response (IgG1 and IgG2a) Balanced Th1/Th2 cellular immune response (lymphocyte proliferation and IFN-␥, IL-2, IL-4 and TNF-␣ cytokine secretion) Balanced Th1/Th2 humoral response (IgG1 and IgG2a) Improved survival rates and decreased bacterial burdens after i.v. MRSA infection Humoral response predominantly IgG1 Longer median survival time and a higher survival rate Th1-biased response (more IgG2a in sera) Th1-biased response: cellular immune response (lymphocyte proliferation, IFN-␥, IL-2 cytokine secretion and cytotoxic T lymphocyte responses) Local and distal secretory IgA production Lower bacterial burdens in nasal cavities after S. pneumoniae infection

model of mastitis, Karaolis and co-workers showed that, despite no direct bactericidal activity, c-di-GMP co-administered with S. aureus directly into the mammary glands significantly decreases bacterial burdens [24]. Previous work by the same group had shown that c-di-GMP inhibits biofilm formation of the same S. aureus strain as well as its adherence to HeLa cells [25]. To rule out the possibility that the c-di-GMP-mediated protection is solely due to its role in the inhibition of biofilm formation, subsequent work showed that pretreatment with c-di-GMP 12 and 6 h before intramammary infection with S. aureus also results in a 1.5 log and a 3.8 log reduction in bacterial burdens, respectively [20], suggesting that the anti-bacterial effect associated with c-di-GMP treatment is most likely due to the immunomodulating, rather than bactericidal, properties of c-di-GMP. More recently, Hu et al. [26] also showed that a pretreatment regimen of c-di-GMP administered alone subcutaneously three times at 2-week intervals followed by intravenous (i.v.) methicillin-resistant S. aureus (MRSA) challenge 7 days after the last administration decreased bacterial burdens in both the liver and the spleen and also improved the 12-day survival rate after challenge [26]. To a lesser degree, i.p. injection of c-di-GMP 24 h before i.v. infection with MRSA results in some decrease in bacterial burdens in the liver but not in the spleen. Similarly, subcutaneous treatment of mice with c-di-GMP 48 and 24 h before intratracheal challenge with Klebsiella pneumoniae resulted in significantly improved survival rates over saline treatment [27]. The immunomodulatory effects of c-di-GMP are not limited to systemic administration. There also appears to be substantial immunomodulatory effects when c-di-GMP is delivered intranasally. Two separate studies in mouse models of bacterial pneumonia indicate that i.n. treatment with c-di-GMP has protective efficacy against respiratory pathogens despite no direct bactericidal activity in vitro. c-di-GMP pretreatment is able to significantly reduce local bacterial burdens and decrease dissemination from the lungs [21,27]. In an S. pneumoniae mouse model of lung infection, i.n. pretreatment of mice with c-di-GMP 48 and 24 h before infection led to significant decreases in bacterial burdens 24 h post-infection in mice challenged with Type 2 (both lung

[29,26] [29] [20] [26] [21] [21] [39,23] [39] [39,23] [23]

and blood bacterial burdens) or Type 4 (lung bacterial burdens) S. pneumoniae [21]. In mice challenged with bioluminescent Type 3 S. pneumoniae, i.n. c-di-GMP pretreatment showed no effect on bacterial burdens at early time points but did result in lower lung bacterial burdens at 42 and 48 h post challenge. Similarly, when cdi-GMP was administered intranasally to mice before, at the time of, and after intratracheal challenge with K. pneumoniae, results showed that co-administration of c-di-GMP with K. pneumoniae plus treatment 6-h post-infection did not significantly affect survival rates while i.n. pretreatment 48 and 24 h before infection significantly improved survival rates. In the latter case, the bacterial burdens were lowered by 5-fold in the lung and ∼3 log in the blood on day 2 post-infection as compared to untreated mice [27]. Although the above studies convincingly demonstrate the immunomodulatory effect of c-di-GMP in the prevention of systemic and mucosal infection with various bacterial pathogens, the mechanism responsible for the immunomodulatory properties of c-di-GMP remains unknown. In an effort to begin dissecting this, Karaolis et al. [27] characterized the host immune response after c-di-GMP administration and K. pneumoniae challenge. Mice pretreated with c-di-GMP had significantly more neutrophils and ␣␤T cells in the lung than controls (mice pretreated with cGMP) at day 2 post-infection. c-di-GMP pretreatment also seemed to increase total NK cells as well as NK cells expressing CD69 in response to K. pneumoniae challenge. Moreover, when lung macrophages from mice infected with K. pneumoniae were cultured ex vivo, both spontaneous nitric oxide (NO) production as well as inducible nitric oxide synthase (iNOS) mRNA expression were significantly higher in c-di-GMP-pretreated mice. c-di-GMP stimulation of the innate immune response was also accompanied by increased mRNA levels and cytokine levels for IL-12p40, IP-10 and IFN-␥, in lungs of mice pretreated with c-di-GMP followed by infection with K. pneumoniae [27], indicating that in addition to stimulating an innate immune response, c-di-GMP pretreatment also induces a Th1-biased cytokine response pattern. Unfortunately, these studies failed to establish whether the observed Th1-biased immune response plays an important role in host defense against K. pneu-

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moniae infection as seen in this model or whether it is merely a “bystander” immune response. 4. The promise of c-di-GMP as a vaccine adjuvant The ability of c-di-GMP to stimulate and modulate the host innate immune response suggests that c-di-GMP (and its analogs) can be a potential vaccine adjuvant, a concept which was first formalized in a patent by Karaolis [28]. To evaluate this possibility, Ebensen et al. [29] co-administered c-di-GMP subcutaneously with model antigen ␤-galactosidase (␤-Gal) using a standard immunization protocol. Stronger antigen-specific systemic humoral (IgG1 and IgG2a) and cellular immune responses (lymphocyte proliferation and IFN-␥, IL-2, IL-4 and TNF-␣ cytokine secretion) were induced after co-administration with c-di-GMP as compared to antigen alone immunization [29]. Also, work from Karaolis et al. [20] demonstrated that intramuscular vaccination of mice with a mixture of S. aureus clumping factor A (ClfA) and c-di-GMP induced significantly higher anti-ClfA antibodies in the serum. As with ␤Gal, vaccination with c-di-GMP and ClfA led to significantly higher antigen-specific total IgG as well as both IgG1 and IgG2a subtypes [20]. Taken together, the presence of IgG1 and IgG2a subclasses in sera and the cytokine profile in restimulated spleen cells show that c-di-GMP-adjuvanted vaccines induce a balanced Th1 and Th2 immune response, making c-di-GMP a good adjuvant candidate for vaccine development. With these encouraging results, researchers proceeded to evaluate the adjuvant potential of c-di-GMP in a vaccination/challenge mouse model of systemic infection. Mice were immunized three times at 2-week intervals with one of two MRSA antigens, ClfA or staphylococcal enterotoxin C (SEC), mixed with either alum or c-di-GMP. One week after the last immunization, mice were intravenously challenged with a lethal dose of MRSA. Mice immunized with c-di-GMP-adjuvanted vaccine showed better survival rates compared to mice immunized with c-di-GMP alone or shamimmunized mice. Those mice also showed significantly reduced bacterial burdens in the spleens and livers 3 days post challenge [26], to a level comparable to the burdens in mice immunized with alum-adjuvanted vaccines. Also, for each of the two MRSA antigens, only the c-di-GMP-adjuvanted vaccines induced significant levels of various specific IgG subtypes. Surprisingly, alum-adjuvanted vaccines did not induce strong, specific anti-SEC or anti-ClfA antibodies in the sera. The potential for the use of c-di-GMP as a vaccine adjuvant was also demonstrated in a mouse model of i.p. pneumococcal infection. In this case, mice were intraperitoneally vaccinated with either S. pneumoniae pneumolysin toxoid (PdB) or pneumococcal surface protein A (PspA) adjuvanted with either c-di-GMP or alum. A predominantly IgG1 response was elicited as determined by antigen-specific antibody responses but again pneumococcal antigen adjuvanted with c-di-GMP resulted in stronger specific antibody response than antigen adjuvanted with alum. Furthermore, mice immunized with PdB + c-di-GMP showed a significantly longer median survival time (>504 h) and a better survival rate than control mice vaccinated with c-di-GMP alone (∼60 h). Similar data were observed in mice immunized with PspA + c-di-GMP although in this case the difference failed to reach statistical significance [21]. This may be due to the fact that c-di-GMP alone seemed to have some protective efficacy (4/15 mice immunized with c-di-GMP alone survived). More encouragingly, PdB + c-di-GMP vaccinated mice survived significantly longer than the positive control mice immunized with PdB + alum vaccine. Interestingly, results from this work also mirrored those from the MRSA challenge study in that antigen adjuvanted with c-di-GMP elicited higher levels of specific antibodies and better protective immunity than antigen adjuvanted with alum.

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5. c-di-GMP as a mucosal adjuvant The above studies have used c-di-GMP as a systemic adjuvant. While the results are quite encouraging, the possibility of using c-di-GMP as a mucosal adjuvant is an even more exciting prospect since human mucosal surfaces (such as respiratory, gastrointestinal (GI) and urogenital tracts) are the major portals of entry and sites of diseases caused by microbial pathogens [30,31]. Thus, development of adjuvants/vaccines that elicit effective and sustained mucosal immune responses to prevent the attachment, invasion and replication of the pathogen would be a significant advancement in the prevention and treatment of many socially and economically important infectious diseases. Most of the currently approved human vaccines are administered systemically, and they generally fail to elicit effective mucosal immunity [3,31,32]. Hence, there are ongoing world-wide efforts in developing mucosal adjuvants and vaccine delivery systems [3,30,31]. An effective mucosal vaccine must reach and breach the epithelial barrier. However, the mucosal epithelium is composed of a thin layer of cells sealed at their apical membranes by tight junctions, which is further protected by mucus and secretory IgA. The thick glycocalyx on the epithelial cell wall also contains hydrolytic enzymes that degrade most pathogens and macromolecules [33]. Orally delivered vaccines have the additional challenges of surviving the harsh gastric and intestinal environments while being present in high enough concentrations so that they are not too diluted in the intralumenal fluid of the gut [3]. This has prompted extensive research for developing mucosal adjuvants and nonreplicating delivery systems such as detoxified cholera toxin (CT) and E. coli heat labile toxin (LT), CpG-OGN, and various types of microparticulates [34–37]. Although there remain many unresolved issues related to the final clinical application of these experimental mucosal adjuvants [31,34–38], the relative success in early clinical trials of CpG-ODN as a mucosal adjuvant demonstrates the feasibility of development of effective mucosal adjuvants with acceptable side effects. The first direct evidence for the potential application of cdi-GMP as a mucosal adjuvant came from Ebensen et al. who demonstrated that i.n. co-administration of c-di-GMP with ␤-Gal or ovalbumin (OVA) induces efficient antigen-specific secretory IgA production in the lung and vagina as well as cytotoxic T lymphocyte (CTL) responses [39]. When ␤-Gal was co-administered intranasally with c-di-GMP three times at 2-week intervals, ␤Gal specific serum IgG antibody titers were significantly higher in ␤-Gal + c-di-GMP mice than in mice vaccinated with antigen alone. More importantly, ␤-Gal specific IgA titers in the lung and vaginal lavages were significantly higher in mice immunized with c-di-GMP-adjuvanted ␤-Gal [39]. In addition to strong humoral immune responses at mucosal sites, ␤-Gal specific cellular immune responses were induced in spleens from mice vaccinated with ␤-Gal + c-di-GMP as assessed by lymphocyte proliferation. Also, i.n. immunization with OVA + c-di-GMP resulted in an in vivo CTL response (approximately 28% versus 5% specific lysis by spleens from mice immunized with OVA only) [39]. In contrast to their earlier work with systemic immunization, which leads to a balanced Th1 and Th2 host immune response, i.n. immunization with ␤-Gal + c-di-GMP seems to skew the immune response toward a predominantly Th1 type as evidenced by higher serum levels of IgG2a and high IFN-␥ and IL-2 secretion by splenocytes from mice immunized with ␤-Gal + c-di-GMP [39]. Recent work in our laboratories further demonstrated, for the first time, that the mucosal immune response induced with cdi-GMP-adjuvanted vaccine does indeed translate into protective immunity against bacterial infection [23]. We showed that i.n. immunization of mice with c-di-GMP-adjuvant pneumococcal surface adhesion A (PsaA) induces specific IgA in both the local

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bronchoalveolar space and distal mucosal sites (feces) as well as serum IgG1 and IgG2a responses. As was found by Ebensen et al. [39], results from our study showed that IgG2a levels were higher than IgG1 levels, suggesting a predominant Th1 response. Also, PsaA-specific antibodies both in serum and in fecal and bronchoalveolar lavage fluid were somewhat higher in mice immunized with PsaA + c-di-GMP than the control group immunized with PsaA + CT. More importantly, when these mice were intranasally challenged with S. pneumoniae, mice immunized with PsaA + c-diGMP harbored significantly less S. pneumoniae in their nasal cavities than did mice immunized with c-di-GMP alone, CT alone or saline. In fact, both immunization with PsaA + c-di-GMP and PsaA + CT had similar protective effects against nasopharyngeal colonization with S. pneumoniae [23]. This finding was very encouraging since CT is considered the “gold standard” of mucosal adjuvanticity and is the most potent experimental mucosal adjuvant; however, its considerable toxicity precludes its direct application in human vaccination. The potent immunostimulatory properties of c-di-GMP have provoked studies to evaluate its potential as a vaccine adjuvant and the results from these preliminary studies have demonstrated its potential as a mucosal adjuvant. In addition, there is emerging evidence that other structurally related cyclic dinucleotides, 3 , 5 -cyclic di-inosinic acid (c-di-IMP) and di-adenylic acid (cdi-AMP) [40,41], also exhibit strong mucosal adjuvant properties [42,43]. However, the structural requirements for the mucosal adjuvanticity of these cyclic dinucleotides remain largely uncharacterized. For example, the optimal structures/modifications of c-di-GMP for its use as a mucosal adjuvant are not known. Indeed, the magnitude of immunostimulation seen after c-di-GMP administration may in fact result in excessive tissue inflammation which is detrimental to the host. With this in mind, we have successfully replaced the non-bridging oxygen at the internucleotide linkages with either one (c-di-GMP-S1) or two sulfur atoms (c-di-GMP-S2) (Fig. 1). Both these sulfur analogs, when administered intranasally, recruit inflammatory cells including neutrophils into the lungs and induce the same pattern of proinflammatory cytokines and chemokines as unmodified c-di-GMP does but at lower levels [22]. As such, these sulfur analogues may be able to induce effective immune responses without the excessive tissue inflammation associated with strong immunostimulation and be superior to c-di-GMP as mucosal adjuvants. More work is needed in order to establish the structure–adjuvanticity relationship. Another fundamental question yet to be investigated is the mechanism by which c-di-GMP stimulates the host immune response. The first clues may have come to light in a very recent study by McWhirter et al. [44] who suggest that c-di-GMP is detected in the cytoplasm of mammalian cells and then triggers a transcriptional response similar to what occurs after stimulation with cytosolic DNA [44]. Results from their cell culture work indicate that c-di-GMP induces type I IFN and coregulates genes via induction of TBK1, IRF3, nuclear factor ␬B and MAP kinases. These data were corroborated by in vivo experiments using IRF3/7 doubledeficient mice. Whereas c-di-GMP treatment elicited Type 1 IFN in wild-type B6 mice, IRF3/7 double-deficient mice produced very little Type 1 IFN. In fact, while a single immunization with human serum albumin (HSA) + c-di-GMP elicited HSA-specific antibodies in B6 mice, this response was virtually undetectable in IRF3/7 double knockout mice [44]. McWhirter et al. postulated that since the transcriptional responses after c-di-GMP and cytosolic DNA are similar, this may add value to the use of c-di-GMP as a small molecule adjuvant. Since c-di-GMP is nonself and non-DNA, it is able to induce similar responses as DNA without the risk of autoimmune attack or mutagenic potential associated with DNA vaccines [44].

6. Conclusion There is a largely unmet requirement for safe and effective vaccine adjuvants. In fact, only a few adjuvants have been approved for use in humans and as such the development of novel adjuvants and immunostimulatory agents to enhance the innate immunity and vaccine efficacies is a high priority. The fortuitous discovery of c-di-GMP and its ability to stimulate the host immune response has jumpstarted research to investigate its potential adjuvanticity. The initial evidence suggesting the possibility of using c-di-GMP as a mucosal adjuvant is particularly exciting since mucosal immunization poses its own set of challenges. Nevertheless, another group of small synthetic molecules, CpG-ODNs, have generated a great deal of excitement as mucosal vaccine adjuvants and a number of vaccines containing CpG-ODN are currently in clinical trials [45]. c-di-GMP may represent another candidate with equal promise as a vaccine adjuvant. It has been less than 5 years since the immunostimulatory properties of c-di-GMP were first observed. During the past 5 years, few laboratories have examined the potential for c-di-GMP as a vaccine adjuvant. However, with the promising data that have come out from these studies, interest in this bacterial signaling molecule has quickly grown. Over the next few years, more data is needed to support the protective efficacy of c-di-GMP in its capacity as a potential vaccine adjuvant and both c-di-GMP immunogenicity and adjuvanticity must be evaluated in other species. In addition, understanding the mechanism underlying c-di-GMP stimulation of the host response is an important step towards the successful application of c-di-GMP as a vaccine adjuvant. Also, although some preliminary data indicate that there is no lethal cytotoxicity in normal rat kidney cells or human neuroblastoma cells as well as no adverse toxigenic or carcinogenic effects in vitro [19,26], the in vivo safety profile for c-di-GMP must be assessed and there is some concern that its potent immunostimulatory properties may in fact lead to excessive tissue inflammation. To this end, phosphorothioate analogues of c-di-GMP may be useful in providing c-di-GMP analogues that may be superior to c-di-GMP as adjuvants since they will likely have fewer side effects due to excessive tissue inflammation. A great deal of research is still needed before c-di-GMP could be included as a vaccine adjuvant in human clinical trials but initial research has highlighted the tremendous potential for c-di-GMP to be used as a vaccine adjuvant. Acknowledgements The c-di-GMP research in our laboratories was partially funded by Natural Sciences and Engineering Research Council (NSERC) of Canada (H. Yan) and by National Research Council Canada (A-base) (W. Chen). References [1] Rappuoli R, Ulmer J. Vaccines research and development: poised for rapid growth. Curr Opin Biotechnol 2007;18(6):521–2. [2] Rey M, Girard MP. The global eradication of poliomyelitis: progress and problems. Comp Immunol Microbiol Infect Dis 2008;31(2–3):317–25. [3] Neutra MR, Kozlowski PA. Mucosal vaccines: the promise and the challenge. Nat Rev Immunol 2006;6(2):148–58. [4] Ryan EJ, Daly LM, Mills KH. Immunomodulators and delivery systems for vaccination by mucosal routes. Trends Biotechnol 2001;19(8):293–304. [5] Schalk JA, Mooi FR, Berbers GA, van Aerts LA, Ovelgonne H, Kimman TG. Preclinical and clinical safety studies on DNA vaccines. Hum Vaccin 2006;2(2):45–53. [6] Levine MM, Sztein MB. Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol 2004;5(5):460–4. [7] Tritto E, Mosca F, De Gregorio E. Mechanism of action of licensed vaccine adjuvants. Vaccine 2009;27(25–26):3331–4. [8] Ross P, Weinhouse H, Aloni Y, Michaeli D, Weinberger-Ohana P, Mayer R, et al. Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 1987;325(6101):279–81.

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