Stimulation of Innate Immunity byIn VivoCyclic di-GMP Synthesis Using Adenovirus

Abstract

The bacterial second messenger cyclic di-GMP (c-di-GMP) stimulates inflammation by initiating innate immune cell recruitment and triggering the release of proinflammatory cytokines and chemokines. These properties make c-di-GMP a promising candidate for use as a vaccine adjuvant, and numerous studies have demonstrated that administration of purified c-di-GMP with different antigens increases protection against infection in animal models. Here, we have developed a novel approach to produce c-di-GMP inside host cells as an adjuvant to exploit a host-pathogen interaction and initiate an innate immune response. We have demonstrated that c-di-GMP can be synthesized in vivo by transducing a diguanylate cyclase (DGC) gene into mammalian cells using an adenovirus serotype 5 (Ad5) vector. Expression of DGC led to the production of c-di-GMP in vitro and in vivo, and this was able to alter proinflammatory gene expression in murine tissues and increase the secretion of numerous cytokines and chemokines when administered to animals. Furthermore, coexpression of DGC modestly increased T-cell responses to a Clostridium difficile antigen expressed from an adenovirus vaccine, although no significant differences in antibody titers were observed. This adenovirus c-di-GMP delivery system offers a novel method to administer c-di-GMP as an adjuvant to stimulate innate immunity during vaccination.

In Vivo Synthesis of Cyclic-di-GMP Using a Recombinant Adenovirus Preferentially Improves Adaptive Immune Responses against Extracellular Antigens

“…We previously demonstrated the feasibility of in vitro and in vivo production of c-di-GMP in mammalian cells by using Ad5 vectors to transduce DGCs (30). Our prior unpublished studies suggested that use of an alternative DGC, VCA0848, which has greater enzymatic activities, might generate a significantly elevated amount of c-di-GMP in vivo .…”

“…AdNull and AdGag were constructed as previously described (31, 32). AdVCA0848 was constructed similarly to AdVCA0956 as previously described (30). Briefly, the V. cholerae gene VCA0848 gene (GeneBank sequence: CP007635.1) was sub-cloned into pShuttle-CMV as previously described (33).…”

“…Primers used for AdToxB construction: forward: 5′-GCTACTACGAGGACGGCCTG-3′ and reverse: 5′-CTCATCGATGATCAGCTTGCC-3′. The C-terminal region of the new synthetic gene did not contain the enzymatic domain and recombination and viral propagation were carried out as previously described (30, 33, 37). Constructs were confirmed to be replication-competent adenovirus (RCA) negative using RCA PCR and direct sequencing methods as previously described (32, 38).…”

“…injected (retro-orbitally) with AdNull (n=3), AdVCA0956 (n=4), or AdVCA0848 (n=4) in 200 μl of a phosphate-buffered saline solution (PBS, pH 7. 4) containing 2×10 11 viral particles (vps)/mouse; or not injected (naïves) (n=3) as previously described (30). The same viral dose was also used for additional experiments in which mice were injected with AdVCA0848, AdVCA0848 mut , or not injected (naïves).…”

“…Transduction of VCA0956 and subsequent production of c-di-GMP stimulated several innate immune responses, including significant induction of IFN-β and other cytokines as well as the expression of several IFN-responsive genes. When co-administered with an adenovirus-based vector carrying the Clostridium difficile -derived Toxin A (TA) antigen, the Ad5 vaccine expressing VCA0956 also enhanced, although moderately, the induction of adaptive immune responses against the TA antigen in mice (30). …”