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

Abstract

There is a compelling need for more effective vaccine adjuvants to augment induction of antigen specific adaptive immune responses. Recent reports suggested the bacterial second messenger bis-(3′–5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) acts as an innate immune system modulator. We recently incorporated a Vibrio cholerae diguanylate cyclase (DGC) into an adenovirus (Ad) vaccine, fostering production of c-di-GMP as well as pro-inflammatory responses in mice. Here, we recombined a more potent DGC, VCA0848, into a non-replicating adenovirus serotype 5 (AdVCA0848) that produces elevated amounts of c-di-GMP when expressed in mammalian cells in vivo. This novel platform further improved induction of type I interferon β (IFN-β) and activation of innate and adaptive immune cells early after administration into mice as compared to control vectors. Co-administration of the extracellular protein ovalbumin (OVA) and the AdVCA0848 adjuvant significantly improved OVA-specific T cell responses as detected by IFN-γ and IL-2 ELISPOT, while also improving OVA-specific humoral B cell adaptive responses. Additionally, we found that co-administration of AdVCA0848 with another Ad5 vector expressing the HIV-1 derived antigen Gag (AdGag) or the Clostridium difficile-derived Toxin B (AdToxB), resulted in significant inhibitory effects on the induction of Gag and ToxB-specific adaptive immune responses. As a proof of principle, these data confirm that in vivo synthesis of c-di-GMP stimulates strong innate immune responses that correlate with enhanced adaptive immune responses to concomitantly administered extracellular antigen, which can be utilized as an adjuvant to heighten effective immune responses for protein-based vaccine platforms against microbial infections and cancers.

The Promise and Challenges of Cyclic Dinucleotides as Molecular Adjuvants for Vaccine Development

“…[ 97 , 114 , 150 , 151 ]. Others have attempted to synthesize c-di-GMP in vivo by transducing a diguanylate cyclase gene into the nonreplicating adenovirus serotype 5 (Ad5) vector, which produced enhanced amounts of c-di-GMP when expressed in mammalian cells in vivo [ 152 , 153 ]. Furthermore, cGAMP has been incorporated into viral particles such as lentivirus and herpesvirus virions [ 154 ].…”

“…Furthermore, cGAMP has been incorporated into viral particles such as lentivirus and herpesvirus virions [ 154 ]. The proof of concept of these approaches has been demonstrated in animal models with extracellular protein Ova, HIV-1 Gag peptides, or the C. difficile toxin B [ 152 , 153 ]. Quintana and colleagues have explored the feasibility to engineer a live Lactococcus lactis vector containing a single plasmid with both cdaA and tscf genes under different promoters, respectively, to simultaneously synthesize the adjuvant c-di-AMP as well as a heterologous vaccine antigen of interests in order to develop a simple and economical system for vaccine development [ 155 ].…”

Au naturale: use of biologically derived cyclic di-nucleotides for cancer immunotherapy

“…As the preponderance of efforts to activate STING has relied on injection of natural CDNs or other small molecule STING agonists, either free or incorporated into delivery systems like nanoparticles, there is little research exploring harnessing the power of CON synthases to activate STING ( figure 4 c ). We have shown that the gene encoding two DGCs from the bacterial pathogen V. cholerae delivered into eukaryotic cells using an adenovirus type 5 delivery vector can synthesize high concentrations of intracellular c-di-GMP that robustly activates STING both in vitro in cell culture and in vivo in mouse models [ 118 , 119 ]. Such activation enhances both innate and adaptive immune responses.…”

Cyclic dinucleotide (c-di-GMP, c-di-AMP, and cGAMP) signalings have come of age to be inhibited by small molecules

“…169 In a more recent study, they combined VCA0848 (more potent dgc) with nonreplicating adenovirus serotype 5 (AdVCA0848) for an improved stimulation of innate immunity in mice. 170 On the other hand, the Gursel group used the arginine peptide (positively charged) to complex with c-di-GMP (negatively charged) for delivery into mouse cells with enhanced immunostimulation of c-di-GMP being observed. 168 In addition, Hayakawa loaded c-di-GMP on liposomes (YSK05 lipid), facilitating the delivery of c-di-GMP directly into the cytosol.…”