Proteomic and Antibody Profiles Reveal Antigenic Composition and Signatures of Bacterial Ghost Vaccine of Brucella abortus A19.

Brucellosis is an important zoonotic disease that causes great economic losses. Vaccine immunisation is the main strategy for the prevention and control of brucellosis. Although live attenuated vaccines play important roles in the prevention of this disease, they also have several limitations, such as residual virulence and difficulty in the differentiation of immunisation and infection. We developed and evaluated a new bacterial ghost vaccine of Brucella abortus A19 by a new double inactivation method. The results showed that the bacterial ghost vaccine of Brucella represents a more safe and efficient vaccine for brucellosis. We further characterised the antigenic components and signatures of the vaccine candidate A19BG. Here, we utilised a mass spectrometry-based label-free relative quantitative proteomics approach to investigate the global proteomics changes in A19BGs compared to its parental A19. The proteomic analysis identified 2014 proteins, 1116 of which were differentially expressed compared with those in A19. The common immunological proteins of OMPs (Bcsp31, Omp25, Omp10, Omp19, Omp28, and Omp2a), HSPs (DnaK, GroS, and GroL), and SodC were enriched in the proteome of A19BG. By protein micro array-based antibody profiling, significant differences were observed between A19BG and A19 immune response, and a number of signature immunogenic proteins were identified. Two of these proteins, the BMEII0032 and BMEI0892 proteins were significantly different (P < 0.01) in distinguishing between A19 and A19BG immune sera and were identified as differential diagnostic antigens for the A19BG vaccine candidate. In conclusion, using comparative proteomics and antibody profiling, protein components and signature antigens were identified for the ghost vaccine candidate A19BG, which are valuable for further developing the vaccine and its monitoring assays.

Combined immunization with inactivated vaccine reduces the dose of live B. abortus A19 vaccine.

BACKGROUND: Brucella spp. is an important zoonotic pathogen responsible for brucellosis in humans and animals. Brucella abortus A19 strain is a widespread vaccine in China. However, it has a drawback of residual virulence in animals and humans. METHODS: In this study, the BALB/c mice were inoculated with either 100 µL PBS(control group, C group), 109 CFU/mL inactivated B. abortus A19 strain (I group), 105 CFU/mL (low-dose group, L group) 106 CFU/mL live B. abortus A19 strain (high-dose group, H group), or 105 CFU/mL live B. abortus A19 strain combined with 109 CFU/mL inactivated B. abortus A19 strain (LI group). Mice were challenged with B. abortus strain 2308 at 7 week post vaccination. Subsequently, the immune and protective efficacy of the vaccines were evaluated by measuring splenic bacterial burden, spleen weight, serum IgG, interferon-gamma (IFN-γ), interleukin-4 (IL-4) percentage of CD4 + and CD8 + T cells of mice via bacterial isolation, weighing, ELISA and flow cytometry, respectively. RESULTS: The splenic bacterial burden and spleen weight of the mice in group LI were mostly equivalent to the mice of group H. Moreover, Brucella-specific serum IgG, IFN-γ, IL-4, and the percentage of CD4+ and CD8+ T cells of the LI group mice were similar to those of the H group. In the subsequent challenge test, both vaccines conferred protective immunity to wild-type (WT) 2308 strain. In addition, the levels of IL-4 and IFN-γ, CD4+ and CD8+ T cells in these mice were similar to those of the mice in the H group. CONCLUSIONS: Combined immunization with low dose live vaccine and inactivated vaccine allowed to reduce the live B. abortus A19 vaccine, dose with an equivalent protection of the high-dose live vaccine.

High repetitive arginine in the anterior of PCV3 capsid protein is a severe obstacle for its expression in E. coli.

Porcine circovirus type 3 (PCV3) is a novel circovirus identified in sows with PDNS-like clinical signs and reproductive failure. The capsid protein (CAP) of PCV3 is expected to be an effective vaccine candidate. Here, we expressed the original capsid protein, truncated capsid protein without anterior highly repetitive arginine (ΔCAP) and their codon-optimized counterparts in E. coli. These results showed that lots of repeated arginine could severely lower the expression of PCV3 capsid protein in E. coli. At the same time, the recombined truncated PCV3 capsid protein forms typic virions. The efficient expression of capsid protein is expected to serve the development of PCV3 vaccines and other studies of PCV3 capsid protein.

Increased Expressions and Roles of CC Chemokine Ligand 21 and CC Chemokine Ligand 25 in Chronic Rhinosinusitis with Nasal Polyps.

INTRODUCTION: Chronic rhinosinusitis (CRS) is a local inflammation of the nasal mucosa and sinus that persists for >12 weeks. As CC chemokine ligand (CCL) 19 expression is known to be elevated in CRS, and CCL 19, CCL21, and CCL25 share the same atypical chemokine receptor 4, so we focused on CCL21 and CCL25. OBJECTIVES: To investigate the expression of CCL21 and CCL25 in different types of CRS and their significance in CRS development. METHODS: A total of 116 patients participated in the study, and uncinate process mucosa or nasal polyp (NP) specimens were collected during surgery. Western blotting and immunohistochemistry were performed to detect the expression of CCL21 and CCL25, respectively, in the nasal mucosa. Immunofluorescence was used to determine their cellular localization in NPs, whereas macrophage culture was used to determine their relationships with macrophages. RESULTS: Immunohistochemistry revealed that the expressions of CCL21 and CCL25 were increased in NPs only. Western blotting revealed that these expressions were gradually increased in control, CRS without NP and CRS with NP groups and were positively correlated with disease severity. Furthermore, increased expressions of CCL21 and CCL25 in NPs were not related to eosinophil infiltration. Immunofluorescence results demonstrated colocalization of CCL25+ cells and CD68+ macrophages. CCL25 expression was increased in macrophage culture, especially in M1 macrophages, while CCL21 expression was not significantly associated with macrophages. CONCLUSIONS: CCL21 and CCL25 were significantly upregulated in NPs and positively correlated with disease severity. CCL25 upregulation was related to M1 macrophages.

Rapid and simple detection of Glaesserella parasuis in synovial fluid by recombinase polymerase amplification and lateral flow strip.

BACKGROUND: Glaesserella parasuis (G. parasuis) is an influential pathogen of the pig, which induces high morbidity and mortality in naive pig populations in the pig industry. Accurate and rapid detection of the agent is important for disease control. In this study, a simple recombinase polymerase amplification (RPA) with a Lateral flow (LF) strip (RPA-LF-GPS) was developed to detect G. parasuis. RESULTS: The RPA-LF-GPS can specifically detect G. parasuis a limit of 100 CFU from other common related pathogens causing arthritis in the pig. The RPA-LF-GPS assay can use boiled synovial fluid samples as a template with the same sensitivity as other DNA extraction methods. In the detection of clinic positive synovial fluid sample, RPA-LF-GPS is equally sensitive (98.1%) compared with that of PCR (90.4%) (P > 0.05). The whole procedure of the RPA-LF-GPS assay could be finished in 1 hour without professional equipment. CONCLUSIONS: RPA-LF-GPS assay is a rapid and simple method for point-of-care diagnostic testing for G. parasuis infection.

Development and application of a simple recombinase polymerase amplification assay for rapid point-of-care detection of feline herpesvirus type 1.

Feline herpesvirus type 1 (FHV-1) is a highly contagious pathogen of domestic cats and other members of the family Felidae. Point-of-care diagnosis of persistent infection in cats is essential for control of its spread. A recombinase polymerase amplification (RPA) assay (RPA-LFD-FHV) combined with a lateral flow dipstrip (LFD) was developed that uses human body heat for incubation. Sensitivity was evaluated by testing a serial dilution of a control plasmid, and specificity was evaluated by testing related viruses. Swab samples from cats with suspected infection were tested by RPA-LFD-FHV, and the results were compared to those obtained by PCR to evaluate its clinical performance. The RPA-FLD-FHV assay was carried out successfully within 20 min, using body heat for incubation. The RPA-FLD-FHV had a detection limit of 103 copies of the FHV-1 gD gene, which was lower than that of PCR, which was 104 copies. The assay could detect templates of FHV-1 but not those of other feline and canine viruses. Viruses in boiled samples could be efficiently detected by the RPA-FLD-FHV. Thirty-one out of the 80 samples were positive by the RPA-FLD-FHV assay, whereas only 27 were positive by PCR. DNA sequencing confirmed that the four samples that were positive by RPA-FLD-FHV but negative by PCR were indeed positive. These results indicate that RPA-FLD-FHV is applicable for clinical use. The RPA-FLD-FHV assay is a simple, rapid, and reliable method for point-of-care diagnosis of FHV-1 infection.

Intranasal Administration of Lentiviral miR-135a Regulates Mast Cell and Allergen-Induced Inflammation by Targeting GATA-3.

Mast cell (MC) degranulation is the foundation of the acute phase of allergic rhinitis (AR). Previously, downregulation of GATA binding protein 3 (GATA-3) was shown to suppress MC activation in an AR mouse model. Binding of microRNA-135a (miR-135a) to GATA-3 was also observed, and overexpression of this miRNA decreased GATA-3 mRNA and protein expression. However, the effects of miR-135a on MCs during AR are currently unknown. In the present study, we utilized a lentiviral (LV) vector to intranasally administer miR-135a to ovalbumin (OVA)-sensitized AR mice. Following miR-135a treatment, the total serum IgE concentration observed during AR was significantly reduced. In the nasal mucosa, the expression of T-box expressed in T cells (T-bet) was higher, whereas that of GATA-3 was lower in the AR mice following miRNA treatment. Notably, during AR, the ratio of type 1 T-helper cells (Th1) to type 2 (Th2) cells in the spleen is unbalanced, favoring Th2. However, administering miR-135a to the AR mice appeared to balance this ratio by increasing and decreasing the percentage of Th1 and Th2 cells, respectively. MiR-135a also appeared to strongly suppress the infiltration of eosinophils and MCs into the nasal mucosa, and it was specifically localized in the MCs, suggesting that its influence is modulated through regulation of GATA-3 in these cells. Additional work identifying the full therapeutic potential of miR-135a in the treatment of AR and diseases involving allergen-induced inflammation is warranted.