Cellular and molecular mechanisms of action of the novel adjuvant polyphosphazene

Adjuvants are critical components of modern vaccines. They are added to improve the host’s immune responses to the vaccine antigens. Understanding the mechanisms of action of adjuvants is critical in the rational design of vaccines. The novel adjuvant poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) has shown great potential as a vaccine adjuvant, but the mechanisms that mediate its adjuvant activity have not been investigated. Hence, the present investigations were undertaken to understand the molecular and cellular mechanisms of action of PCEP. First, we investigated in vivo the capacity of PCEP to induce innate immune responses at the site of injection. PCEP induced time-dependent changes in the gene expression of various “adjuvant core response genes” including cytokines, chemokines, innate immune receptors and interferon-induced genes. We also observed that PCEP enhanced production of various cytokines including pro-inflammatory cytokines and chemokines such as CCL2, CCL4, CCL12 and CXCL10 locally at the injection site but no systemic responses. Due to the potent chemotactic potential of local cytokines and chemokines produced post-injection of PCEP, we observed increased recruitment of various myeloid and lymphoid cells at the injection site. Neutrophils and macrophages were recruited in significantly higher numbers followed by monocytes and dendritic cells (DCs). In addition, there was increased recruitment of T and B lymphocytes at the injection site. Further, confocal studies revealed intracytoplasmic lysosomal localization of PCEP in recruited immune cells at the site of injection. Whole body in vivo imaging of mice injected intramuscularly with PCEP revealed localized distribution of PCEP post-injection in the muscle tissue. Approximately 70% of PCEP was cleared from the injection site within 24 h post-injection, but there was evidence of PCEP retention up to 12 weeks post-injection. Although we could not detect PCEP in the draining lymph nodes, we observed significant increase in neutrophil, macrophage, monocyte and DC numbers, with the latter cell population being most abundant. We observed that in vivo PCEP upregulates NLRP3 gene and pro-inflammatory cytokine expression at the injection site. Since caspase-1 is a critical component of NLRP3 inflammasome and known to plays an important role in the release of IL-1β and IL-18, we examined the role of caspase-1 in PCEP-mediated secretion of IL-1β and IL-18 by splenic DCs. Pre-treatment of splenic DCs with the caspase-inhibitor YVAD-fmk significantly inhibited IL-1β and IL-18 secretion in response to PCEP. Although PCEP was taken up by the DCs, it failed to induce DC maturation (expression of MHC class II and co-stimulatory molecules CD86 and CD40). In addition, PCEP did not induce direct activation of naïve T cells. However, when naïve B cells were directly activated, PCEP induced significant production of IgM and IL-6. Further, immunization of mice with OVA plus PCEP significantly increased the production of OVA-specific IFN-γ by CD4+ T cells and CD8+ T cells suggesting that PCEP can generate antigen-specific T cell responses. Taken together, these results suggest that adjuvant activity of PCEP depends on creating a strong immunocompetent environment at the site of injection by activating innate immune responses, which involves modulation of adjuvant core response genes, production of cytokines and chemokines, recruitment of various immune cells and presumably activation of inflammasomes. Together, all these mechanisms might contribute to the adjuvant activity of PCEP

Whole Genomic Characterization of <i>Streptococcus iniae</i> Isolates from Barramundi (<i>Lates calcarifer</i>) and Preliminary Evidence of Cross-Protective Immunization

Lates calcarifer, also known as Barramundi or Asian seabass, is a highly productive and fast-growing species that is well suited to large-scale aquaculture due to its attractive harvestable yields (premium fish). This fish has been envisioned as having the potential to be the “Salmon of Tropics”. Cultivating Lates calcarifer in aquaculture poses challenges, as the dense populations that make such aquaculture commercially viable facilitate the rapid spread of infectious diseases, which in turn significantly impact yield. Hence, the immunization of juveniles is necessary, and the development of new immunization agents enhances the efficiency of aquaculture and improves food security. In our study, we characterize seven novel strains of the bacterial pathogen Streptococcus iniae that were collected from commercial fish farms in Singapore and Australia. We find that the capsular operon in our strains is highly conserved and identify a number of major surface antigens previously described in Streptococcus. A genome analysis indicates that the present strains are closely related but form distinct strains within the S. iniae species. We then proceed to demonstrate that inoculation with the inactivated strain P3SAB cross-protects Lates calcarifer against S. iniae infections in vivo from a variety of strains found in both Singapore and Australia

Kinome Analysis to Define Mechanisms of Adjuvant Action: PCEP Induces Unique Signaling at the Injection Site and Lymph Nodes

Understanding the mechanism of action of adjuvants through systems biology enables rationale criteria for their selection, optimization, and application. As kinome analysis has proven valuable for defining responses to infectious agents and providing biomarkers of vaccine responsiveness, it is a logical candidate to define molecular responses to adjuvants. Signaling responses to the adjuvant poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP) were defined at the site of injection and draining lymph node at 24 h post-vaccination. Kinome analysis indicates that PCEP induces a proinflammatory environment at the injection site, including activation of interferon and IL-6 signaling events. This is supported by the elevated expression of proinflammatory genes (IFNγ, IL-6 and TNFα) and the recruitment of myeloid (neutrophils, macrophages, monocytes and dendritic cells) and lymphoid (CD4+, CD8+ and B) cells. Kinome analysis also indicates that PCEP’s mechanism of action is not limited to the injection site. Strong signaling responses to PCEP, but not alum, are observed at the draining lymph node where, in addition to proinflammatory signaling, PCEP activates responses associated with growth factor and erythropoietin stimulation. Coupled with the significant (p < 0.0001) recruitment of macrophages and dendritic cells to the lymph node by PCEP (but not alum) supports the systemic consequences of the adjuvant. Collectively, these results indicate that PCEP utilizes a complex, multi-faceted MOA and support the utility of kinome analysis to define cellular responses to adjuvants

Molecular characterization of MHC class I alpha 1 and 2 domains in Asian seabass (lates calcarifer)

The Asian seabass is of importance both as a farmed and wild animal. With the emergence of infectious diseases, there is a need to understand and characterize the immune system. In humans, the highly polymorphic MHC class I (MHC-I) molecules play an important role in antigen presentation for the adaptive immune system. In the present study, we characterized a single MHC-I gene in Asian seabass (Lates calcarifer) by amplifying and sequencing the MHC-I alpha 1 and alpha 2 domains, followed by multi-sequence alignment analyses. The results indicated that the Asian seabass MHC-I α1 and α2 domain sequences showed an overall similarity within Asian seabass and retained the majority of the conserved binding residues of human leukocyte antigen-A2 (HLA-A2). Phylogenetic tree analysis revealed that the sequences belonged to the U lineage. Mapping the conserved binding residue positions on human HLA-A2 and grass carp crystal structure showed a high degree of similarity. In conclusion, the availability of MHC-I α1 and α2 sequences enhances the quality of MHC class I genetic information in Asian seabass, providing new tools to analyze fish immune responses to pathogen infections, and will be applicable in the study of the phylogeny and the evolution of antigen-specific receptors.Singapore Food AgencyPublished versionThis research is supported by the Singapore Food Agency under the Singapore Food Story ("SFS") R&D Programme 1st Grant Call (Theme 1 "Sustainable Urban Food Production") (SFS_RND_SUFP_001_01)

Kinetics of Tear Fluid Proteins after Endothelial Keratoplasty and Predictive Factors for Recovery from Corneal Haze

10.3390/jcm9010063JOURNAL OF CLINICAL MEDICINE9