Synthesis of BLF1-containing trimethyl chitosan nanoparticles and evaluation of its immunogenicity and protection in syrian mice by oral and subcutaneous injections.

The bacterium Burkholderia pseudomallei is the cause of melioidosis infectious disease. In this bacterium, the BLF1 protein wide inhibits the synthesis of proteins in human cells. This disease is reported to cause a death rate of 40% in some parts of the world. Currently, no effective vaccine is available against this bacterial infection. In this study, therefore, a Nano vaccine was synthesized based on the trimethyl chitosan (TMC) polymer containing the BLF1 recombinant protein, and its immunogenicity and protection in Syrian mice were evaluated by oral and subcutaneous injections. The BLF1 recombinant protein expression was induced in Escherichia coli Bl21 (DE3) and purified by the affinity chromatography technique. Recombinant protein-containing nanoparticles (NPs) were then synthesized by the ionotropic gelation method. After oral and subcutaneous injections, antibody titration was assessed by the indirect ELISA assay. Finally, murine groups were challenged using the BLF1 toxin. The results indicated that the immune system showed more antibody titration in subcutaneous injection than in the oral form. However, the results were reversed in the challenge results, and the survival rate was more significant in the oral injection.

Oral Administration of Bacterial ß Cell Expansion Factor A (BefA) Alleviates Diabetes in Mice with Type 1 and Type 2 Diabetes.

Diabetes mellitus (DM) is a group of metabolic diseases, and there is an urgent need to develop new therapeutic DM oral drugs with fewer side effects and sound therapeutic efficacy. In this study, a ß cell expansion factor A (BefA) production strain of Escherichia coli (BL21-pet 28C-BefA) was constructed, and the antidiabetes effect of BefA was evaluated using type 1 DM (T1DM) and type 2 DM (T2DM) mice models. The T1DM mice results indicated that BefA significantly reduced blood glucose levels; exerted a protective effect on islet ß cell morphology; downregulated the expressions of TLR-4, p-NFκB/NFκB, and Bax/Bcl-2, and the secretion levels of IL-1ß and TNF-α; increased the expression of PDX-1 protein and insulin secretion in a concentration-dependent manner; and restored the disturbed microbial diversity to normal levels. Similarly with the T1DM mice, BefA obviously increased islet ß cells and reduced the inflammatory reaction and apoptosis in T2DM mice, as well as improved liver lipid metabolism by downregulating the expressions of CEBP-α, ACC, and Fasn; inhibited the synthesis of triglycerides; and induced Cpt-1, Hmgcs2, and Pparα in a concentration-dependent manner. In conclusion, BefA alleviates diabetes via increasing the number of islet ß cells, reducing the inflammatory reaction and apoptosis, improving liver lipid metabolism, and restoring microbial diversity to normal levels, which provides a new strategy for a DM oral drug.

Macrophage-Mediated Melanoma Reduction after HP-NAP Treatment in a Zebrafish Xenograft Model.

The Helicobacter pylori Neutrophil Activating Protein (HP-NAP) is endowed with immunomodulatory properties that make it a potential candidate for anticancer therapeutic applications. By activating cytotoxic Th1 responses, HP-NAP inhibits the growth of bladder cancer and enhances the anti-tumor activity of oncolytic viruses in the treatment of metastatic breast cancer and neuroendocrine tumors. The possibility that HP-NAP exerts its anti-tumor effect also by modulating the activity of innate immune cells has not yet been explored. Taking advantage of the zebrafish model, we examined the therapeutic efficacy of HP-NAP against metastatic human melanoma, limiting the observational window to 9 days post-fertilization, well before the maturation of the adaptive immunity. Human melanoma cells were xenotransplanted into zebrafish embryos and tracked in the presence or absence of HP-NAP. The behavior and phenotype of macrophages and the impact of their drug-induced depletion were analyzed exploiting macrophage-expressed transgenes. HP-NAP administration efficiently inhibited tumor growth and metastasis and this was accompanied by strong recruitment of macrophages with a pro-inflammatory profile at the tumor site. The depletion of macrophages almost completely abrogated the ability of HP-NAP to counteract tumor growth. Our findings highlight the pivotal role of activated macrophages in counteracting melanoma growth and support the notion that HP-NAP might become a new biological therapeutic agent for the treatment of metastatic melanomas.

Evaluation of the indirect impact of the 10-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine in a cluster-randomised trial.

BACKGROUND: In the nation-wide double-blind cluster-randomised Finnish Invasive Pneumococcal disease trial (FinIP, ClinicalTrials.gov NCT00861380, NCT00839254), we assessed the indirect impact of the 10-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV10) against five pneumococcal disease syndromes. METHODS: Children 6 weeks to 18 months received PHiD-CV10 in 48 clusters or hepatitis B/A-vaccine as control in 24 clusters according to infant 3+1/2+1 or catch-up schedules in years 2009-2011. Outcome data were collected from national health registers and included laboratory-confirmed and clinically suspected invasive pneumococcal disease (IPD), hospital-diagnosed pneumonia, tympanostomy tube placements (TTP) and outpatient antimicrobial prescriptions. Incidence rates in the unvaccinated population in years 2010-2015 were compared between PHiD-CV10 and control clusters in age groups <5 and ≥5 years (5-7 years for TTP and outpatient antimicrobial prescriptions), and in infants <3 months. PHiD-CV10 was introduced into the Finnish National Vaccination Programme (PCV-NVP) for 3-month-old infants without catch-up in 9/2010. RESULTS: From 2/2009 to 10/2010, 45398 children were enrolled. Vaccination coverage varied from 29 to 61% in PHiD-CV10 clusters. We detected no clear differences in the incidence rates between the unvaccinated cohorts of the treatment arms, except in single years. For example, the rates of vaccine-type IPD, non-laboratory-confirmed IPD and empyema were lower in PHiD-CV10 clusters compared to control clusters in 2012, 2015 and 2011, respectively, in the age-group ≥5 years. CONCLUSIONS: This is the first report from a clinical trial evaluating the indirect impact of a PCV against clinical outcomes in an unvaccinated population. We did not observe consistent indirect effects in the PHiD-CV10 clusters compared to the control clusters. We consider that the sub-optimal trial vaccination coverage did not allow the development of detectable indirect effects and that the supervening PCV-NVP significantly diminished the differences in PHiD-CV10 vaccination coverage between the treatment arms.

Toxicological Evaluation of a Probiotic-Based Delivery System for P8 Protein as an Anti-Colorectal Cancer Drug.

PURPOSE: This study aimed to toxicological evaluate a probiotics-based delivery system for p8 protein as an anti-colorectal cancer drug. INTRODUCTION: Lactic acid bacteria (LAB) have been widely ingested for many years and are regarded as very safe. Recently, a Pediococcus pentosaceus SL4 (PP) strain that secretes the probiotic-derived anti-cancer protein P8 (PP-P8) has been developed as an anti-colorectal cancer (CRC) biologic by Cell Biotech. We initially identified a Lactobacillus rhamnosus (LR)-derived anti-cancer protein, P8, that suppresses CRC growth. We also showed that P8 penetrates specifically into CRC cells (DLD-1 cells) through endocytosis. We then confirmed the efficacy of PP-P8, showing that oral administration of this agent significantly decreased tumor mass (~42%) relative to controls in a mouse CRC xenograft model. In terms of molecular mechanism, PP-P8 induces cell-cycle arrest in G2 phase through down-regulation of Cyclin B1 and Cdk1. In this study, we performed in vivo toxicology profiling to obtain evidence that PP-P8 is safe, with the goal of receiving approval for an investigational new drug application (IND). METHODS: Based on gene therapy guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea, the potential undesirable effects of PP-P8 had to be investigated in intact small rodent or marmoset models prior to first-in-human (FIH) administration. The estimated doses of PP-P8 for FIH are 1.0×1010 - 1.0×1011 CFU/person (60 kg). Therefore, to perform toxicological investigations in non-clinical animal models, we orally administered PP-P8 at doses of 3.375 × 1011, 6.75 × 1011, and 13.5×1011 CFU/kg/day; thus the maximum dose was 800-8000-fold higher than the estimated dose for FIH. RESULTS: In our animal models, we observed no adverse effects of PP-P8 on clinicopathologic findings, relative organ weight, or tissue pathology. In addition, we observed no inflammation or ulceration during pathological necropsy. CONCLUSION: These non-clinical toxicology studies could be used to furnish valuable data for the safety certification of PP-P8.

Construction and potential application of bacterial superoxide dismutase expressed in Bacillus subtilis against mycotoxins.

Oxidative stress, which could be evoked by numerous inducements including mycotoxins like deoxynivalenol (DON), cause severe damages to organisms. Antioxidants are promising protectants against oxidative stress that could be applied in pharmaceutical, cosmetic, and food and feed industries. In this study, a thermostable and acidophilic superoxide dismutase (AaSOD) was used to develop an antioxidant product that can potentially protect organisms from oxidative stress related damages. The enzyme was successfully expressed as an extracelluar protein in Bacillus subtilis with a high yield. To obtain a feasible protocol for industrial production of AaSOD, the fermentation mediums that are commonly used for culturing B. subtilis were screened, the feasibility of expressing AaSOD without antibiotic as selection pressure was confirmed, and the effect of using lactose as an inducer instead of isopropyl-ß-d-thiogalactoside (IPTG) was investigated. Batch fermentation was conducted to validate the optimized conditions for AaSOD production, and 6530 U mL-1 of SOD activity was obtained in the fermentation broth. The dry powder product of AaSOD with an activity of 22202 U g-1 was prepared by spray-drying and was administrated on zebrafish to test its function as a protectant against DON, and thus gained a significant redress of the reactive oxygen species (ROS) accumulation induced by DON. Taken together, this study provides a feasible protocol to prepare the AaSOD-based antioxidant product that is potentially applied in livestock industry.

An Albumin-Binding Domain Peptide Confers Enhanced Immunoprotection Against Viral Myocarditis by CVB3 VP1 Vaccine.

Coxsackievirus B3 (CVB3)-induced viral myocarditis is a common clinical cardiovascular disease without effective available vaccine. In this study, we tried to potentiate the immunoprotection efficacy of our previous CVB3-specific VP1 protein vaccine by introducing a streptococcal protein G-derived, draining lymph nodes (dLNs)-targeting albumin-binding domain (ABD) peptide. We found that compared with the original VP1 vaccine, ABD-fused VP1 (ABD-VP1) vaccine gained the new ability to efficiently bind murine albumin both in vitro and in vivo, possessed a much longer serum half-life in serum and exhibited more abundance in the dLNs after immunization. Accordingly, ABD-VP1 immunization not only significantly facilitated the enrichment and maturation of dendritic cells (DCs), induced higher percentages of IFN-γ+ CD8 + cells in the dLNs, but also robustly promoted VP1-induced T cell proliferation and cytotoxic T lymphocyte (CTL) responses in the spleens. More importantly, ABD-VP1 also elicited higher percentages of protective CD44hi CD62Lhi memory T cells in dLNs and spleens. Consequently, obvious protective effect against viral myocarditis was conferred by ABD-VP1 vaccine compared to the VP1 vaccine, reflected by the less body weight loss, improved cardiac function, alleviated cardiac histomorphological changes and an increased 28-day survival rate. Our results indicated that the ABD might be a promising immune-enhancing regime for vaccine design and development.

Intranasal Vaccination With Recombinant Antigen-FLIPr Fusion Protein Alone Induces Long-Lasting Systemic Antibody Responses and Broad T Cell Responses.

A simple formulation is urgently needed for mucosal vaccine development. We employed formyl peptide receptor-like 1 inhibitory protein (FLIPr), an FcγR antagonist secreted by Staphylococcus aureus, as a vector to target ovalbumin (OVA) to dendritic cells (DCs) via intranasal administration. Our results demonstrate that intranasal administration of recombinant OVA-FLIPr fusion protein (rOVA-FLIPr) alone efficiently delivers OVA to DCs in nasal lymphoid tissue. Subsequently, OVA-specific IgG and IgA antibodies in the circulatory system and IgA antibodies in mucosal tissue were detected. Importantly, activation of OVA-specific CD4+ and CD8+ T cells and induction of a broad-spectrum cytokine secretion profile were detected after intranasal administration of rOVA-FLIPr alone in immunocompetent C57BL/6 mice. Furthermore, we employed immunodeficient AG129 mice as a Zika virus infection model and demonstrated that intranasal administration of recombinant Zika virus envelope protein domain III-FLIPr fusion protein induced protective immune responses against the Zika virus. These results suggest that antigen-FLIPr fusion protein alone via intranasal administration can be applied to mucosal vaccine development.

Streptococcus equi-derived extracellular vesicles as a vaccine candidate against Streptococcus equi infection.

Streptococcus equi subspecies equi is a pathogenic bacterium that causes strangles, a highly contagious respiratory infection in horses and other equines. The limitations of current vaccines against S. equi infection warrants the development of an affordable, safe, and effective vaccine. Because gram-positive extracellular vesicles (EVs) transport various immunogenic antigens, they are attractive vaccine candidates. Here, we purified the EVs of S. equi ATCC 39506 and evaluated them as a vaccine candidate against S. equi infection in mice. As an initial step, comparative proteomic analysis was performed to characterize the functional features of the EVs. Reverse vaccinology and knowledge-based annotations were then used to screen potential vaccine candidates (PVCs) for S. equi ATCC 39506. Finally, 32 PVCs were found to be enriched in the EV fraction, suggesting the usefulness of this fraction as a vaccine. Importantly, a significantly higher survival rate after S. equi infection was detected in mice immunized with S. equi-derived EVs via the intraperitoneal route than in mice immunized with heat-killed bacteria. Of note, immunoprecipitation-mass spectrometry results validated various immunogenic antigens within the EV proteome. In conclusion, our results suggest that S. equi-derived EVs can serve as a vaccine candidate against S. equi infection.

Oral Tolerance Induced by Heat Shock Protein 65-Producing Lactococcus lactis Mitigates Inflammation in Leishmania braziliensis Infection.

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.

Anticancer activity of Helicobacter pylori ribosomal protein (HPRP) with iRGD in treatment of colon cancer.

PURPOSE: As the conventional therapeutic approaches were not completely successful in the treatment of colon cancer, there is still a need for finding the most efficient therapeutic agents. Here we investigated the anticancer activity of HPRP-A1 that was derived from the N-terminal region of Helicobacter pylori ribosomal protein L1 (RpL1) alone or in combination with tumor-homing peptide iRGD and 5-Fluorouracil (5FU) on colon cancer cell lines (CT26 and HT29) and isograft models of colon cancer. METHOD: We assessed the tumor growth inhibitory activity of HPRP-A1 with or without iRGD and 5FU on colon cancer in vitro and in vivo. In the in vitro part, we investigate the effect of HPRP-A1 alone and in combination with iRGD/5FU. RESULTS: Our results demonstrated that co-administration of HPRP-A1 with iRGD increased the apoptosis, while these two peptides in combination with 5FU increased the intracellular level of p53 that upregulate the pro-apoptotic gene BAX and downregulate the anti-apoptotic gene BCL2. HPRP-A1 blocks the cell cycle progression in G0/G1. Co-administration of two peptides significantly reduced the size and weight of the tumors, while the group that received 5FU in combination with the peptides increased the necrotic and decrease the fibrotic area significantly in the tumor tissues, which also disrupt the oxidant/antioxidant balance. CONCLUSIONS: Our results indicated that HPRP-A1 could be considered an effective agent toward colon cancer in vitro and in vivo with the ability to enhance the effects of conventional chemotherapy agent 5FU.

Selenium-containing protein from selenium-enriched Spirulina platensis antagonizes oxygen glucose deprivation-induced neurotoxicity by inhibiting ROS-mediated oxidative damage through regulating MPTP opening.

CONTEXT: Selenium-containing protein from selenium-enriched Spirulina platensis (Se-SP) (syn. Arthrospira platensis [Microcoleaceae]) showed novel antioxidant activity. However, the protective effect of Se-SP against oxygen glucose deprivation (OGD)-induced neural apoptosis has not been reported yet. OBJECTIVE: To verify whether Se-SP can inhibit OGD-induced neural apoptosis and explore the underlying mechanism. MATERIALS AND METHODS: Primary hippocampal neurons were separated from Sprague-Dawley (SD) rats. 95% N2 + 5% CO2 were employed to establish OGD model. Neurons were treated with 5 and 10 µg/mL Se-SP under OGD condition for 6 h. Neurons without treatment were the control group. Neural viability and apoptosis were detected by MTT, immunofluorescence and western blotting methods. RESULTS: Se-SP significantly improved neuronal viability (from 57.2% to 94.5%) and inhibited apoptosis in OGD-treated primary neurons (from 45.6% to 6.3%), followed by improved neuronal morphology and caspases activation. Se-SP co-treatment also effectively suppressed OGD-induced DNA damage by inhibiting ROS accumulation in neurons (from 225.6% to 106.3%). Additionally, mitochondrial dysfunction was also markedly improved by Se-SP co-treatment via balancing Bcl-2 family expression. Moreover, inhibition of mitochondrial permeability transition pore (MPTP) by CsA (an MPTP inhibitor) dramatically attenuated OGD-induced ROS generation (from 100% to 56.2%), oxidative damage, mitochondrial membrane potential (MPP) loss (from 7.5% to 44.3%), and eventually reversed the neuronal toxicity and apoptosis (from 57.4% to 79.6%). DISCUSSION AND CONCLUSIONS: Se-SP showed enhanced potential to inhibit OGD-induced neurotoxicity and apoptosis by inhibiting ROS-mediated oxidative damage through regulating MPTP opening, indicating that selenium-containing protein showed broad application in the chemoprevention and chemotherapy against human ischaemic brain injury.

Dual vaccination against IL-4 and IL-13 protects against chronic allergic asthma in mice.

Allergic asthma is characterized by elevated levels of IgE antibodies, type 2 cytokines such as interleukin-4 (IL-4) and IL-13, airway hyperresponsiveness (AHR), mucus hypersecretion and eosinophilia. Approved therapeutic monoclonal antibodies targeting IgE or IL-4/IL-13 reduce asthma symptoms but require costly lifelong administrations. Here, we develop conjugate vaccines against mouse IL-4 and IL-13, and demonstrate their prophylactic and therapeutic efficacy in reducing IgE levels, AHR, eosinophilia and mucus production in mouse models of asthma analyzed up to 15 weeks after initial vaccination. More importantly, we also test similar vaccines specific for human IL-4/IL-13 in mice expressing human IL-4/IL-13 and the related receptor, IL-4Rα, to find efficient neutralization of both cytokines and reduced IgE levels for at least 11 weeks post-vaccination. Our results imply that dual IL-4/IL-13 vaccination may represent a cost-effective, long-term therapeutic strategy for the treatment of allergic asthma as demonstrated in mouse models, although additional studies are warranted to assess its safety and feasibility.

Evaluation of different antigenic preparations against necrotic enteritis in broiler birds using a novel Clostridium perfringens type G strain.

OBJECTIVE: Keeping in view, the constraints faced by the Indian broiler industry with lack of a suitable vaccine against Necrotic Enteritis (NE), a study has been proposed to explore the prevalence and detail characterization of C. perfringens type G in NE suspected broiler chicken in the process of suitable vaccine development. METHODS: Intestinal scrapings/faecal contents of NE suspected broiler chickens were screened to establish the prevalence of C.perfringens type G in broiler birds. A most pathogenic, highly resistant type G isolate of C. perfringens, bearing both tpeL and gapC gene was selected for preparation of three different vaccine formulations, and to evaluate their immunogenic potential in broiler birds. RESULTS: Screening of clinical samples of NE suspected broiler birds revealed C. perfringens type G, bearing gapC gene in 51.22% samples, of which 47.62% revealed tpeL gene. Seven of the tpeLpos type G isolates were comparatively more pathogenic for mice, of which, one exhibited multidrug resistance towards ciprofloxacin, norfloxacin, tetracycline and levofloxacin. The sonicated supernatant (SS) prepared from the selected tpeL and gapC positive isolate could maintain a significantly higher protective IgG response than toxoid and bacterin preparation from the 21st to 28thday of age in immunized birds. CONCLUSION: The additional TpeL toxin in C. perfringens type G has been proved to be an additional key biological factor in the pathogenesis of NE in broiler chickens. Considering the release of more immunogenic proteins, the SS proved to be a better immunogenic preparation against NE with a multiple immunization dose.

Suppression of ß-catenin signaling in colon carcinoma cells by a bacterial protein.

Colorectal cancer is one of the leading causes of cancer-related death worldwide. The adenomatous polyposis coli (APC) gene is mutated in hereditary colorectal tumors and in more than 80% of sporadic colorectal tumors. APC mutations impair ß-catenin degradation, leading to its permanent stabilization and increased transcription of cancer-driving target genes. In colon cancer, impairment of ß-catenin degradation leads to its cytoplasmic accumulation, nuclear translocation, and subsequent activation of tumor cell proliferation. Suppressing ß-catenin signaling in cancer cells therefore appears to be a promising strategy for new anticancer strategies. Recently, we discovered a novel Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA), that affects both invertebrate and vertebrate hosts. It promotes bacterial survival and proliferation in invertebrate predators but has unknown biological role(s) in mammalian hosts. Here, we report that MakA can cause lethality of tumor cells via induction of apoptosis. Interestingly, MakA exhibited potent cytotoxic activity, in particular against several tested cancer cell lines, while appearing less toxic toward nontransformed cells. MakA bound to the tumor cell surface became internalized into the endolysosomal compartment and induced leakage of endolysosomal membranes, causing cytosolic release of cathepsins and activation of proapoptotic proteins. In addition, MakA altered ß-catenin integrity in colon cancer cells, partly through a caspase- and proteasome-dependent mechanism. Importantly, MakA inhibited ß-catenin-mediated tumor cell proliferation. Remarkably, intratumor injection of MakA significantly reduced tumor development in a colon cancer murine solid tumor model. These data identify MakA as a novel candidate to be considered in new strategies for development of therapeutic agents against colon cancer.

Characterization and Specification of a Trivalent Protein-Based Pneumococcal Vaccine Formulation Using an Adjuvant-Free Nanogel Nasal Delivery System.

We previously developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan. Here, we generated three pneumococcal surface protein A (PspA) fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel and mixed the three resulting monovalent formulations into a trivalent nanogel-PspA formulation. First, to characterize the nanogel-PspA formulations, we used native polyacrylamide gel electrophoresis (PAGE) to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, we adopted two methods-a densitometric method based on lithium dodecyl sulfate (LDS)-PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)-to determine the PspA content in the nanogel formulations. Third, treatment of nanogel-PspA formulations by adding methyl-ß-cyclodextrin released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. However, when nanogel-PspA formulations were heat-treated at 80 °C for 16 h, CD spectroscopy showed that each PspA was released in a denatured form. Fourth, we confirmed that the nanogel-PspA formulations were internalized into nasal mucosa effectively and that each PspA was gradually released from the nanogel in epithelial cells in mice. Fifth, LDS-PAGE densitometry and ELISA both indicated that the amount of trivalent PspA was dramatically decreased in the heat-treated nanogel compared with that before heating. When mice were immunized nasally using the heat-treated formulation, the immunologic activity of each PspA was dramatically reduced compared with that of the untreated formulation; in both cases, the immunologic activity correlated well with the content of each PspA as determined by LDS-PAGE densitometry and ELISA. Finally, we confirmed that the trivalent nanogel-PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent PspA nasal vaccine formulation.

Protective response against Acinetobacter baumannii with ferric iron receptors HemTR-BauA in a murine sepsis model.

Aim: Iron uptake and metabolism pathways are promising targets in vaccine development as an alternative strategy for antibiotics. Methods & methods: HemTR, a putative heme receptor of Acinetobacter baumannii, was expressed and its protectivity against A. baumannii was determined singly or in combination with the siderophore receptor, BauA, in mice. Results: High level of IgG was elicited. There was a delay in mice mortality with reduced bacterial loads in internal organs in the sublethal challenge. Protection was better in the HemTR-BauA group in both lethal and sublethal challenges. Passive transfer of anti-HemTR and anti-BauA partially protected mice against A. baumannii infection. Conclusion: HemTR in combination with other iron receptors could contribute to the development of protective vaccines against A. baumannii.

Immunological characterization of chimeras of high specificity antigens from Mycobacterium tuberculosis H37Rv.

Tuberculosis remains a serious global health problem. BCG is the only prophylactic TB vaccine and it shows variable protective efficacy. Chimeric protein subunit vaccines hold great potential as stand-alone vaccines or heterologous BCG prime boosters. We have designed a protein chimera, PP31, by combining Mtb ESAT-6 family antigen Rv1198 and MoCo biosynthesis family antigen Rv3111. Further, PP31 was extended by addition of latency antigen Rv1813c to yield PP43. Immunization of BALB/c mice with PP31 or PP43 with FIA adjuvant elicited strong humoral immune response. Restimulation of splenocytes of the immunized mice lead to significant proliferation of lymphocytes, secretion of cytokines IFN-γ, TNF, IL-2 of the Th1 class, IL-17A of the Th17 class, and IL-6. PP31 and PP43 also induced intracellular cytokine expression (IFN-γ, TNF, and IL-2) from both CD4+-CD44high and CD8+-CD44high T-cells. Antigen-specific IFN-γ+/IL-2+ double positive CD4+ T-cells were significantly higher in case of PP43 than PP31-immunized mice and control group. PP43 showed protection equivalent to heat-inactivated BCG in response to challenge of the immunized mice with Mtb H37Ra. Based on its immunogenicity and protective efficacy, PP43 appears to be a potential candidate for further development as a subunit vaccine against TB.

Construction of a Vibrio anguillarum flagellin B mutant and analysis of its immuno-stimulation effects on Macrobrachium rosenbergii.

Vibrio anguillarum is a globally distributed aquatic pathogen, and its flagellin B (FlaB) protein can evoke innate immune responses in hosts. In order to explore the role of FlaB in V. anguillarum infection, we constructed a FlaB-deficient mutant using overlapping PCR and two-step homologous recombination, and gene sequencing confirmed successful knockout of the FlaB gene. Scanning electron microscopy showed no significant differences in the morphological structure of the flagellum between wild-type and FlaB-deficient strains. The mutant was subsequently injected into the freshwater prawn (Macrobrachium rosenbergii) to explore its pathogenicity in the host, and expression of myeloid differentiation factor 88, prophenoloxidase, catalase, superoxide dismutase and glutathione peroxidase was investigated by real-time PCR. The results showed that deletion of FlaB had little effect on V. anguillarum-induced expression of these immune-related genes (p > 0.05). In general, the FlaB mutant displayed similar flagella morphology and immune characteristics to the wild-type strain, hence we speculated that knockout of FlaB might promote the expression and function of other flagellin proteins. Furthermore, this study provides a rapid and simple method for obtaining stable mutants of V. anguillarum free from foreign plasmid DNA.