Development of IglC and GroEL recombinant vaccines for francisellosis in Nile tilapia, Oreochromis niloticus.

Warm-water piscine francisellosis is a granulomatous bacterial disease caused by Francisella orientalis (Fo). The disease has been detected in a wide range of fish species globally, causing mortalities as high as 90% and significant economic losses. Currently there are no commercially available vaccines and few treatment options exist. In the current study, two novel recombinant vaccines were prepared using diatom-expressed IglC or bacterial-expressed GroEL proteins. The vaccine antigens were emulsified with either nanoparticles or a commercially available oil-based adjuvant. Nile tilapia, Oreochromis niloticus, fingerlings were immunized intracoelomically with the recombinant IglC or GroEL vaccines, diatoms alone or phosphate buffer saline. Approximately 840-degree days post-vaccination, fish were challenged via immersion with 106 CFU/mL of wild-type Fo. Twenty-one days post challenge (dpc), the highest relative percent survival was recorded in the IglC-Montanide group (75%), compared to 53%, 50%, 22%, 19% and 16% in the IglC-nanoparticles, GroEL-Montanide, GroEL-nanoparticles, diatoms-Montanide and diatoms-nanoparticles groups, respectively. Protection correlated with significantly higher specific antibody responses in the IglC-Montanide group. Moreover, a significantly lower bacterial load was detected in spleen samples from the IglC-Montanide survivor tilapia compared to the other experimental groups. This is the first report of recombinant vaccines against piscine francisellosis in tilapia. The Fo vaccines described in our study may facilitate development of a safe, cost-effective and highly protective vaccine against francisellosis in farmed tilapia.

A Nanoparticle-Poly(I:C) Combination Adjuvant Enhances the Breadth of the Immune Response to Inactivated Influenza Virus Vaccine in Pigs.

Intranasal vaccination elicits secretory IgA (SIgA) antibodies in the airways, which is required for cross-protection against influenza. To enhance the breadth of immunity induced by a killed swine influenza virus antigen (KAg) or conserved T cell and B cell peptides, we adsorbed the antigens together with the TLR3 agonist poly(I:C) electrostatically onto cationic alpha-D-glucan nanoparticles (Nano-11) resulting in Nano-11-KAg-poly(I:C) and Nano-11-peptides-poly(I:C) vaccines. In vitro, increased TNF-α and IL-1ß cytokine mRNA expression was observed in Nano-11-KAg-poly(I:C)-treated porcine monocyte-derived dendritic cells. Nano-11-KAg-poly(I:C), but not Nano-11-peptides-poly(I:C), delivered intranasally in pigs induced high levels of cross-reactive virus-specific SIgA antibodies secretion in the nasal passage and lungs compared to a multivalent commercial influenza virus vaccine administered intramuscularly. The commercial and Nano-11-KAg-poly(I:C) vaccinations increased the frequency of IFNγ secreting T cells. The poly(I:C) adjuvanted Nano-11-based vaccines increased various cytokine mRNA expressions in lymph nodes compared to the commercial vaccine. In addition, Nano-11-KAg-poly(I:C) vaccine elicited high levels of virus neutralizing antibodies in bronchoalveolar lavage fluid. Microscopic lung lesions and challenge virus load were partially reduced in poly(I:C) adjuvanted Nano-11 and commercial influenza vaccinates. In conclusion, compared to our earlier study with Nano-11-KAg vaccine, addition of poly(I:C) to the formulation improved cross-protective antibody and cytokine response.

In vitro synergistic effects of three enzymes from Bacillus subtilis CH-1 on keratin decomposition.

Extracellular protease Vpr (Vpr), gamma-glutamyltranspeptidase (GGT; EC 2.3.2.2) and glyoxal/methylglyoxal reductase (YvgN; EC 1.1.1.21) are extracellular enzymes involved in feather degradation, which were identified by secretome analyses from an efficient feather-degrading strain Bacillus subtilis CH-1. The encoding sequences corresponding to the three secretory enzymes were cloned into vector pET22b for recombinant expression in Escherichia coli strain BL21 (DE3). Afterward, the proteins containing the C-terminal His-tag were purified using a Ni-IDA column. The optimal temperatures and pH values for protease activity of recombinant Vpr, GGT, and YvgN were identified as 45 °C/pH 7.0, 40 °C/pH 8.0, and 50 °C/pH 6.0 respectively when casein is the substrate. Furthermore, the synergistic effects of the three enzymes were studied using feather powder as substrate. Vpr was the core enzyme to hydrolyze keratin, while GGT and YvgN were coenzymes providing reducing activities for keratin decomposition. The keratinolytic activity was enhanced to about 1.4-folds when YvgN and Vpr applied together in comparison to Vpr alone. And the keratinolytic activity almost reached to 1.5-folds when all the three enzymes were combined to use. The study provides a novel perspective of the mechanism of keratin degradation by microorganisms, and thereby may also be of relevance for the design of an industrial process for enzymatic keratin degradation; however, additional experiments must be done to substantiate this conclusion.

Regeneration of trachea graft with cartilage support, vascularization, and epithelization.

The repair and functional reconstruction of long-segment tracheal defects is always a great challenge in the clinic. Finding an ideal substitute for tracheal transplantation is the only way to solve this problem. The current study proposed a series of novel strategies for constructing a bionic living trachea substitute. For the issue of tubular cartilage support, cartilage sheet technique based on high-density culture of chondrocytes was adopted to avoid the inflammatory reaction triggered by the materials and thus formed mature cartilage-like tissue in autologous goat model. For the issue of epithelialization, the autologous transplantation of oral mucosal epithelium was used to realize mucosa coverage of the constructed trachea lumen. Finally, the flat trapezius fascia flap with double blood supply was separated by microsurgical techniques to achieve stable pre-vascularization of both the regenerated cartilage and the grafted epithelium simultaneously. By integrating the above strategies, the vascularized and epithelialized tracheal substitute with tubular cartilage support was successfully constructed in a goat model. The reconstructed trachea possessed a multiple layer structure of muscle-cartilage-fascia-mucosa comparable to the native trachea, and thus might realize stable survival and long-term airway function maintenance, providing a promising tracheal substitute for the repair and permanent functional reconstruction of long-segment tracheal defects. STATEMENT OF SIGNIFICANCE: The repair of long-segment tracheal defects is always a great challenge in the clinic. Finding an ideal substitute for tracheal transplantation is the only way to solve this problem. In the current study, by technical integration of cartilage regeneration, microsurgery, and oral mucosa transplantation, a complex tracheal substitute with satisfactory vascularization, epithelialization, and tubular cartilage support was successfully constructed in a goat autologous model. The reconstructed trachea substitute possessed a multiple layer structure of muscle-cartilage-fascia-mucosa exactly similar to native trachea, and thus might realize stable survival and long-term airway function maintenance. The current study provides feasible strategies and ideal tracheal substitutes for permanent functional reconstruction of long-segmental trachea defects.

Formulation of aluminum hydroxide adjuvant with TLR agonists poly(I:C) and CpG enhances the magnitude and avidity of the humoral immune response.

Subunit vaccines generally require adjuvants to achieve optimal immune responses. Toll-like receptor (TLR) agonists are promising immune potentiators, but rapid diffusion from the injection site reduces their local effective concentration and may cause systemic reactions. In this study, we investigated the potential of aluminum hydroxide adjuvant (AH) to adsorb the TLR3 agonist poly(I:C) and TLR9 agonist CpG and compared the effect of the combination adjuvant on the immune response with either the TLR agonists or AH alone in mice. Poly(I:C) and CpG readily adsorbed onto AH and this combination adjuvant induced a stronger IgG1 and IgG2a immune response with a significant increase of antibody avidity. The combination adjuvant enhanced antigen uptake and activation of dendritic cells in vitro. It induced an inflammatory response at the injection site similar to AH but without eosinophils which are typically observed with AH. A distinctive antigen-containing monocyte/macrophage population with an intermediate level of CD11c expression was identified in the draining lymph nodes after immunization with TLR agonists and the combination adjuvant. Injection of the combination adjuvant did not induce an increase of TNFα and CXCL10 in serum in contrast to the injection of soluble TLR agonists. These results indicate that this combination adjuvant is a promising formulation to solve some of the unmet needs of current vaccines.

Corn-derived alpha-D-glucan nanoparticles as adjuvant for intramuscular and intranasal immunization in pigs.

Adjuvant potential of positively charged corn-derived nanoparticles (Nano-11) was earlier revealed in mice. We evaluated its adjuvant role to electrostatically adsorbed inactivated/killed swine influenza virus antigen (KAg) (Nano-11 + KAg) in pigs. Nano-11 facilitated the uptake of KAg by antigen presenting cells and induced secretion of proinflammatory cytokines. In pigs vaccinated by an intranasal mist containing Nano-11 + KAg, expression of T-helper 1 and T-helper 2 transcription factors and secretion of cross-reactive influenza antigen-specific mucosal IgA in the nasal cavity were observed. The enhanced frequencies of IFN-γ positive T-helper and cytotoxic T-cells in Nano-11 + KAg-vaccinates after heterologous virus challenge were also observed. Clinically, slightly reduced influenza signs and pneumonic lesions, with mild reduction in virus load in the respiratory tract of vaccinates were observed. In pigs immunized with Nano-11 adsorbed ovalbumin administered by intramuscular (IM) route, enhanced IgG1 and IgG2 antibodies were detected in serum. Thus, Nano-11 vaccine delivery system confers adjuvant effect in pigs.

Porous decellularized trachea scaffold prepared by a laser micropore technique.

Rapid development of tissue engineering technology provides new methods for tracheal cartilage regeneration. However, the current lack of an ideal scaffold makes engineering of trachea cartilage tissue into a three-dimensional (3-D) tubular structure a great challenge. Although a decellularized trachea matrix (DTM) has become a recognized scaffold for trachea cartilage regeneration, it is difficult for cells to detach from or penetrate the matrix because of its non-porous structure. To tackle these problems, a laser micropore technique (LMT) was applied in the current study to enhance trachea sample porosity, and facilitate decellularizing treatment and cell ingrowth. Furthermore, after optimizing LMT and decellularizing treatment parameters, LMT-treated DTM (LDTM) retained its natural tubular structure with only minor extracellular matrix damage. Moreover, compared with DTM, the current study showed that LDTM significantly improved the adherence rate of cells with perfect cell biocompatibility. Moreover, the optimal implantation cell density for chondrogenesis with LDTM was determined to be 1 × 108 cells/ml. Collectively, the results suggest that the novel LDTM is an ideal scaffold for trachea tissue engineering.

Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs.

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly (p < 0.05) reduced in CNPs-KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, an increased frequency of T helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells were observed. In summary, chitosan SwIAV nanovaccine delivered by IN route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in a reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate IN human influenza vaccines.

Validation of RUNX1 as a potential target for treating circadian clock-induced obesity through preventing migration of group 3 innate lymphoid cells into intestine.

The impact of unrhythmic circadian clock on obesity has started to be increasingly appreciated nowadays. Recently it was discovered that interaction between intestinal microbiota and unrhythmic circadian clock plays a key role in such a process. It involves relaying signals from microbiota through dendritic cells to group 3 innate lymphoid cells in the intestine and in the end impacting some of the key transcription factors of circadian clock. Breaking such a signal relay may prove to be an effective way reducing unrhythmic circadian clock-induced obesity. Here, we propose a hypothesis and design experiments to prove that suppressing one of the transcription factors, RUNX1, plays a key role in the homing of ILC3 cells to intestine. Such suppression is in response to a retinoic acid-RARα binding initiated pathway and results in the upregulation of gut-homing chemokine receptor CCR9 and downregulation of lymphoid tissue-homing receptor CCR7, which can then guide ILC3 cells to intestine. Therapies that can specifically sustain Runx1 expression in ILC3 cells may assist preventing the ever-escalating obesity problem in modern society.

Alpha-D-glucan nanoparticulate adjuvant induces a transient inflammatory response at the injection site and targets antigen to migratory dendritic cells.

Biodegradable nanoparticles with functionalized surfaces are attractive candidates as vaccine adjuvants. Nano-11 are cationic dendrimer-like α-D-glucan nanoparticles with a diameter of 70-80 nm. Mice injected with antigen formulated with Nano-11 developed antibody titers that were similar or greater than antigen with aluminum adjuvant. Utilizing an in vivo imaging system, Nano-11 was shown to remain at the injection site after administration and cleared gradually over the course of 3 weeks. Injection of Nano-11 induced a transient inflammatory response characterized by recruitment of a mixed population of inflammatory cells, predominantly monocytes and macrophages with relatively few neutrophils. Recruited Mac-2+macrophages efficiently phagocytized the majority of Nano-11 at the injection site. Fluorescently labeled Nano-11 was present in cells in the draining lymph nodes 1 day after injection, with the majority contained in migratory dendritic cells. Injection of ovalbumin adsorbed to Nano-11 resulted in an increase of ovalbumin-containing cells in draining lymph nodes. Nano-11 delivered more antigen to antigen-presenting cells on a per cell basis and demonstrated more specific targeting to highly immunopotentiating migratory dendritic cells compared with soluble or aluminum hydroxide adsorbed ovalbumin. These results support the efficacy of Nano-11 and its potential use as a next generation vaccine adjuvant.

Differences in innate IFNγ and IL-17 responses to Bordetella pertussis between BALB/c and C57BL/6 mice: role of γδT cells, NK cells, and dendritic cells.

Cell-mediated immune responses characterized by the secretion of IFNγ and IL-17 play an important role in the immune response to Bordetella pertussis (B. pertussis). We investigated innate sources of IFNγ and IL-17 upon stimulation of spleen cells from BALB/c (B/c) and C57BL/6 (B6) mice with heat-killed B. pertussis (hkBp). Spleen cells from B/c mice secreted less IFNγ and more IL-17 than those from B6 mice. Innate IFNγ was produced predominantly by NK cells in B/c mice and by CD8 T cells and NK cells in B6 mice. Innate IL-17 was produced primarily by γδT cells in both mouse strains. The secretion of IFNγ was abrogated by anti-IL-12, and the production of IL-17 was abolished by anti-IL-1ß- and anti-IL23-neutralizing antibodies. B/c dendritic cells (DCs) stimulated with hkBp secreted significantly more IL-1ß and less IL-12 than B6 DCs. Differences in JNK phosphorylation in DCs suggest that this pathway plays a role in the differences between B/c and B6 strains. Mixed cultures of DCs and γδT cells from B/c and B6 showed that cytokines from DCs as well as γδT cell-intrinsic factors contributed to the robust innate IL-17 response in B/c strain. Stimulation of γδT cells with IL-1ß and IL-23 was sufficient for IL-17 secretion whereas IL-12 inhibited the secretion of IL-17. A larger fraction of γδT cells were γδT-17 cells in B/c mice than B6 mice. Our data indicate important roles for genetically determined factors in the innate IFNγ and IL-17 responses to B. pertussis.

Preclinical safety study of a recombinant Streptococcus pyogenes vaccine formulated with aluminum adjuvant.

A recombinant vaccine composed of a fusion protein formulated with aluminum hydroxide adjuvant is under development for protection against diseases caused by Streptococcus pyogenes. The safety and local reactogenicity of the vaccine was assessed by a comprehensive series of clinical, pathologic and immunologic tests in preclinical experiments. Outbred mice received three intramuscular injections of 1/5th of the human dose (0.1 ml) and rabbits received two injections of the full human dose. Control groups received adjuvant or protein antigen. The vaccine did not cause clinical evidence of systemic toxicity in mice or rabbits. There was a transient increase of peripheral blood neutrophils after the third vaccination of mice. In addition, the concentration of acute phase proteins serum amyloid A and haptoglobin was significantly increased 1 day after injection of the vaccine in mice. There was mild transient swelling and erythema of the injection site in both mice and rabbits. Treatment-related pathology was limited to inflammation at the injection site and accumulation of adjuvant-containing macrophages in the draining lymph nodes. In conclusion, the absence of clinical toxicity in two animal species suggest that the vaccine is safe for use in a phase I human clinical trial. Copyright © 2016 John Wiley & Sons, Ltd.

Dendrimer-like alpha-d-glucan nanoparticles activate dendritic cells and are effective vaccine adjuvants.

The use of nanoparticles for delivery of vaccine antigens and as vaccine adjuvants is appealing because their size allows efficient uptake by dendritic cells and their biological properties can be tailored to the desired function. Here, we report the effect of chemically modified phytoglycogen, a dendrimer-like α-d-glucan nanoparticle, on dendritic cells in vitro, and the utility of this type of nanoparticle as a vaccine adjuvant in vivo. The modified phytoglycogen nanoparticle, termed Nano-11, has a positive surface charge which enabled electrostatic adsorption of negatively charged protein antigens. The Nano-11-antigen complexes were efficiently phagocytized by dendritic cells. Nano-11 induced increased expression of costimulatory molecules and the secretion of IL-1ß and IL-12p40 by dendritic cells. Intramuscular injection of Nano-11-antigen formulations induced a significantly enhanced immune response to two different protein antigens. Examination of the injection site revealed numerous monocytes and relatively few neutrophils at one day after injection. The inflammation had nearly completely disappeared by 2 weeks after injection. These studies indicate that Nano-11 is an effective vaccine delivery vehicle that significantly enhances the immune response. This type of plant based nanoparticle is considered highly cost-effective compared with fully synthetic nanoparticles and appears to have an excellent safety profile making them an attractive adjuvant candidate for prophylactic vaccines.

Development of a recombinant fusion protein vaccine formulation to protect against Streptococcus pyogenes.

Diseases resulting from infection by group A streptococcus (GAS) are an increasing burden on global health. A novel vaccine was developed targeting infection by Streptococcus pyogenes. The vaccine incorporates a recombinant fusion protein antigen (SpeAB) which was engineered by combining inactive mutant forms of streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin B (SpeB) from S. pyogenes. A rational, scientific approach to vaccine development was utilized to determine optimal formulation conditions with aluminum adjuvants. Investigations of the pH stability profile of SpeAB concluded the antigen was most stable near pH 8. Incorporation of the stabilizers sucrose and mannitol significantly enhanced the stability of the antigen. Vaccines were formulated in which most of the SpeAB was adsorbed to the adjuvant or remained in solution. A SpeAB vaccine formulation, stabilized with sucrose, in which the antigen remains adsorbed to the aluminum adjuvant retained the greatest potency as determined by evaluation of neutralizing antibody responses in mice. This vaccine has great potential to provide a safe and effective method for prevention of GAS disease.

Control of antigen-binding to aluminum adjuvants and the immune response with a novel phosphonate linker.

The strongest mechanism for adsorption of antigens to aluminum adjuvants is ligand exchange, which involves the replacement of a surface hydroxyl on the adjuvant by a terminal phosphate group of the antigen. A novel phosphonate linker was developed that allows the addition of phosphonate (C-PO3) groups to proteins under controlled and chemically mild conditions. Increasing the number of linkers per protein molecule progressively increased the adsorption strength to aluminum hydroxide adjuvant (AH) as measured by elution in serum. The effect of phosphonate conjugation on the antibody response was determined with hen egg lysozyme (HEL), a protein that has the same charge as AH at neutral pH and does not adsorb to AH. The phosphonylated form of HEL (HEL-P) adsorbed to AH, indicating that the ligand exchange interaction could overcome the electrostatic repulsion. Mice injected with HEL-P/AH had a higher antibody titer to HEL than mice injected with HEL/AH, especially at lower antigen doses, suggesting that adsorption of antigen has a dose-sparing effect. Conjugation of CRM197, an antigen that adsorbs electrostatically to AH, with phosphonate linkers did not enhance the antibody response, indicating that adsorption by either electrostatic or ligand exchange to AH is sufficient to enhance the antibody response.

Kinetics of the inflammatory response following intramuscular injection of aluminum adjuvant.

Aluminum-containing adjuvants are widely used in human and veterinary vaccines, but their mechanism of action is not well understood. Recent evidence suggests an important role for inflammation in the immune response to aluminum-adjuvanted vaccines. To better understand this process, vaccines with aluminum adjuvant were injected into naïve or previously immunized mice and the injection sites were characterized for the corresponding primary and secondary inflammatory response at different time points after immunization. Inflammatory cells appeared at the injection site between 2h and 6h after vaccination, dominated by neutrophils at first, followed by macrophages, and later eosinophils and MHCII(+) cells. The number of cells at the injection site increased over time, except neutrophils, which decreased in number after day 2. There was extensive phagocytosis of aluminum adjuvant particles by macrophages. In secondary immunized mice, a faster and more robust recruitment of eosinophils, macrophages, and antigen presenting cells was observed at the injection site. The enhanced recruitment of inflammatory cells in previously immunized mice coincided with increased expression of relevant chemokines at the injection site. Since neutrophils accumulated first in response to aluminum-adjuvanted vaccines, their role was evaluated by depleting them prior to vaccination. Neutrophil depletion transiently reduced the recruitment of macrophages but it did not change the recruitment of eosinophils and MHCII(+) cells or the quality and magnitude of the antibody response.