Whole Genomic Characterization of Streptococcus iniae Isolates from Barramundi (Lates calcarifer) 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.

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.

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.

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

Endothelial keratoplasty (EK) is less invasive with faster recovery as compared to conventional penetrating keratoplasty, however, it relies on the clarity of the host corneal stroma. Corneal transplantation involves the induction of immune tolerance for allogeneic tissues as well as the corneal wound healing process, in which coordinated interactions between cytokines and growth factors are critical. In this study, we profiled the expression of 51 soluble factors in the tear fluid over the course of EK and have provided evidence of dynamic changes in cytokine expression in the ipsilateral and contralateral eyes. Cluster analyses classified the cytokine expression kinetics into five groups. Group 1 proteins included TGF-b1, IL-1b, and innate proinflammatory cytokines, which bilaterally increased after surgery, despite the use of topical corticosteroid in the transplanted eyes. Local corticosteroids suppressed cytokines involved in adaptive immunity in the transplanted eyes but not in the contralateral eyes. We found tear protein expression at baseline and one week post-surgery to be a potential predictive biomarker of delayed recovery after EK in terms of the corneal haze and visual acuity. Furthermore, Group 1 tear proteins were most associated with persistent corneal haze pre-surgery as well as visual acuity at one month-post transplant.

The adjuvant PCEP induces recruitment of myeloid and lymphoid cells at the injection site and draining lymph node.

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. Previously, we had reported the potential of PCEP to induce cytokines and chemokines at the site of injection. Hence, we hypothesized that PCEP creates strong immuno-competent environment leading to recruitment of immune cells at the injection site. Intramuscular injection of mice with PCEP induced significant recruitment of neutrophils, macrophages, monocytes, dendritic cells (DCs), and lymphocytes at the site of injection as well as in the draining lymph nodes. Flow cytometric analysis showed that the majority of the recruited immune cells took up and/or were associated with PCEP at the injection site, with lymphocytes taking up PCEP in lesser quantity. Further, confocal analysis revealed intracytoplasmic lysosomal localization of PCEP in recruited immune cells. These observations suggest that recruitment of distinct immune cells to the site of injection site may be an important mechanism by which PCEP potentiates immune responses to antigens.

Vaccine Potentiation by Combination Adjuvants.

Adjuvants are crucial components of vaccines. They significantly improve vaccine efficacy by modulating, enhancing, or extending the immune response and at the same time reducing the amount of antigen needed. In contrast to previously licensed adjuvants, current successful adjuvant formulations often consist of several molecules, that when combined, act synergistically by activating a variety of immune mechanisms. These "combination adjuvants" are already registered with several vaccines, both in humans and animals, and novel combination adjuvants are in the pipeline. With improved knowledge of the type of immune responses needed to successfully induce disease protection by vaccination, combination adjuvants are particularly suited to not only enhance, but also direct the immune responses desired to be either Th1-, Th2- or Th17-biased. Indeed, in view of the variety of disease and population targets for vaccine development, a panel of adjuvants will be needed to address different disease targets and populations. Here, we will review well-known and new combination adjuvants already licensed or currently in development-including ISCOMs, liposomes, Adjuvant Systems Montanides, and triple adjuvant combinations-and summarize their performance in preclinical and clinical trials. Several of these combination adjuvants are promising having promoted improved and balanced immune responses.

Caspase-1 Dependent IL-1ß Secretion and Antigen-Specific T-Cell Activation by the Novel Adjuvant, PCEP.

The potent adjuvant activity of the novel adjuvant, poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP), with various antigens has been reported previously. However, very little is known about its mechanisms of action. We have recently reported that intramuscular injection of PCEP induces NLRP3, an inflammasome receptor gene, and inflammatory cytokines, including IL-1ß and IL-18, in mouse muscle tissue. Caspase-1 is required for the processing of pro-forms of IL-1ß and IL-18 into mature forms and is a critical constituent of the NLRP3 inflammasome. Hence, in the present study, we investigated the role of caspase-1 in the secretion of IL-1ß and IL-18 in PCEP-stimulated splenic dendritic cells (DCs). Caspase inhibitor YVAD-fmk-treated splenic DCs showed significantly reduced IL-1ß and IL-18 secretion in response to PCEP stimulation. Further, PCEP had no effect on the expression of MHC class II or co-stimulatory molecules, CD86 and CD40, suggesting that PCEP does not induce DC maturation. However, PCEP directly activated B-cells to induce significant production of IgM. In addition, PCEP+ovalbumin (OVA) immunized mice showed significantly increased production of antigen-specific IFN-γ by CD4⁺ and CD8⁺ T-cells. We conclude that PCEP activates innate immunity, leading to increased antigen-specific T-cell responses.

Mechanisms of action of adjuvants.

Adjuvants are used in many vaccines, but their mechanisms of action are not fully understood. Studies from the past decade on adjuvant mechanisms are slowly revealing the secrets of adjuvant activity. In this review, we have summarized the recent progress in our understanding of the mechanisms of action of adjuvants. Adjuvants may act by a combination of various mechanisms including formation of depot, induction of cytokines and chemokines, recruitment of immune cells, enhancement of antigen uptake and presentation, and promoting antigen transport to draining lymph nodes. It appears that adjuvants activate innate immune responses to create a local immuno-competent environment at the injection site. Depending on the type of innate responses activated, adjuvants can alter the quality and quantity of adaptive immune responses. Understanding the mechanisms of action of adjuvants will provide critical information on how innate immunity influences the development of adaptive immunity, help in rational design of vaccines against various diseases, and can inform on adjuvant safety.

Activation of adjuvant core response genes by the novel adjuvant PCEP.

Adjuvants are critical components of many vaccines but their mechanisms of action are often poorly understood. Understanding the mechanisms of adjuvant activity is critical in defining how innate immunity influenced adaptive immunity. We investigated the capacity of a novel adjuvant, poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP), to induce innate immune responses at the site of injection. PCEP induced time-dependent changes in the gene expression of many "adjuvant core response genes" including cytokines, chemokines, innate immune receptors, interferon-induced genes, adhesion molecules and antigen-presentation genes. In addition, PCEP triggered local production of cytokines and the chemokine CCL-2 as indicated by ELISA. Interestingly, PCEP up-regulated the gene expression of the inflammasome receptor, Nlrp3, and induced the production of pro-inflammatory cytokines IL-1ß, and IL-18 at the site of injection. Secretion of these cytokines is predominantly a result of activation of the inflammasome, a multi-protein complex that activates caspase-1, leading to the processing and secretion of proinflammatory cytokines. These results suggest that PCEP may modulate antigen-specific immune responses by strongly activating early innate immune responses and promoting a strong immuno-stimulatory environment at the site of injection.