c-di-GMP is an effective immunomodulator and vaccine adjuvant against pneumococcal infection

Abstract

Cyclic diguanylate (c-di-GMP) is a unique bacterial intracellular signaling molecule capable of stimulating enhanced protective innate immunity against various bacterial infections. The effects of intranasal pretreatment with c-di-GMP, or intraperitoneal coadministration of c-di-GMP with the pneumolysin toxoid (PdB) or pneumococcal surface protein A (PspA) before pneumococcal challenge, were investigated in mice. We found that c-di-GMP had no significant direct short-term effect on the growth rate of Streptococcus pneumoniae either in vitro or in vivo. However, intranasal pretreatment of mice with c-di-GMP resulted in a significant decrease in bacterial load in lungs and blood after serotypes 2 and 3 challenge, and a significant decrease in lung titers after serotype 4 challenge. Potential cellular mediators of these enhanced protective responses were identified in lungs and draining lymph nodes. Intraperitoneal coadministration of c-di-GMP with PdB or PspA before challenge resulted in significantly higher antigen-specific antibody titers and increased survival of mice, compared to that obtained with alum adjuvant. These findings demonstrate that local or systemic c-di-GMP administration stimulates innate and adaptive immunity against invasive pneumococcal disease. We propose that c-di-GMP can be used as an effective broad spectrum immunomodulator and vaccine adjuvant to prevent infectious diseases.

Introduction

Streptococcus pneumoniae is the leading cause of bacterial pneumonia, meningitis, and otitis media in the United States [1]. In spite of the availability of antimicrobials, the capsular polysaccharide (PS) vaccine, and the 7-valent protein–PS conjugate vaccine, pneumococcal disease continues to be responsible for high morbidity and mortality worldwide, especially in groups at high risk [2]. Consequently, global efforts are focused on exploring alternative pneumococcal vaccine strategies to address the shortcomings of existing formulations, without compromising efficacy. One of these approaches involves the development of vaccines based on pneumococcal proteins that contribute to pathogenesis and are common to all serotypes. To date, the most promising vaccine candidates are the pneumolysin toxoid (PdB), pneumococcal surface protein A (PspA), pneumococcal surface protein C (PspC, also referred to as choline binding protein A) and the 37-kDa metal-binding lipoprotein PsaA (reviewed by [2]).

We have shown that c-di-GMP (3′,5′-cyclic diguanylic acid or cyclic diguanylate or cGpGp) initially identified in the bacterium Acetobacter xylinum is an intracellular signaling molecule [3], [4], [5]. It is present in multiple bacterial species but not in eukaryotes [6], [7], [8], [9], [10], [11], and is now recognized to control many key functions in bacteria, including survival, adherence, colonization, and biofilm formation [8], [9], [12], [13], [14], [15]. Recent studies indicate that it might also modulate host cellular responses [14], resulting in an enhanced control of infection [16]. Consistent with this notion, we recently demonstrated that c-di-GMP increases MIG/CXCL9 (a chemoattractant for activated T cells) suggesting possible antitumor activity [17], and inhibits basal and growth factor-induced proliferation of human colon carcinoma cells [18].

Recently, c-di-GMP was shown to modulate the immune system to prevent and fight various lethal bacterial infections [17]. Intranasal (i.n.) or subcutaneous administration of c-di-GMP before intratracheal challenge with Klebsiella pneumoniae also resulted in significantly increased survival and reduction in bacterial counts in lung and blood [19]. The response was characterized by enhanced accumulation of neutrophils, αβ T cells, and activated NK and αβ T lymphocytes, associated with earlier and more vigorous expression of chemokines and type-1 cytokines [19]. Moreover, lung macrophages recovered from Klebsiella-infected mice pretreated with c-di-GMP expressed greater quantities of inducible nitric oxide synthase (iNOS) and nitric oxide ex vivo than those isolated from mice pretreated with control cGMP [19]. These findings demonstrate that c-di-GMP delivered locally or systemically stimulates protective innate immunity in the lung, decrease bacterial burden and enhances protective responses against infection.

In this study, we investigated the ability of c-di-GMP to enhance resistance against systemic pneumococcal infection, using established mouse models. We provide additional direct evidence that c-di-GMP is immunostimulatory, can protect against infection, and acts as an effective vaccine adjuvant against systemic disease.