Chimeric Hepatitis B core virus-like particles harboring SARS-CoV2 epitope elicit a humoral immune response in mice.

BACKGROUND: Virus-like particles are an interesting vector platform for vaccine development. Particularly, Hepatitis B virus core antigen has been used as a promising VLP platform. It is highly expressed in different recombinant expression systems, such as E. coli, and self-assembled in vitro. It effectively improves the immunogenicity of foreign antigenic epitopes on its surface. Various foreign antigens from bacteria, viruses, and protozoa can be genetically inserted into such nanoparticles. The effective immunogenicity due to VLP vaccines has been reported. However, no research has been performed on the SARS-CoV2 vaccine within this unique platform through genetic engineering. Considering the high yield of target proteins, low cost of production, and feasibility of scaling up, E. coli is an outstanding expression platform to develop such vaccines. Therefore, in this investigation, we planned to study and develop a unique HBc VLP-based vaccine against SARS-Cov2 utilizing the E. coli expression system due to its importance. RESULTS: Insertion of the selected epitope was done into the major immunodominant region (MIR) of truncated (149 residues) hepatitis B core capsid protein. The chimeric protein was constructed in PET28a+ and expressed through the bacterial E. coli BL21 expression system. However, the protein was expressed in inclusion body forms and extracted following urea denaturation from the insoluble phase. Following the extraction, the vaccine protein was purified using Ni2 + iminodiacetic acid (IDA) affinity chromatography. SDS-PAGE and western blotting were used to confirm the protein expression. Regarding the denaturation step, the unavoidable refolding process was carried out, so that the chimeric VLP reassembled in native conformation. Based on the transmission electron microscopy (TEM) analysis, the HBC VLP was successfully assembled. Confirming the assembled chimeric VLP, we explored the immunogenic effectivity of the vaccine through mice immunization with two-dose vaccination with and without adjuvant. The utilization of adjuvant was suggested to assess the effect of adjuvant on improving the immune elicitation of chimeric VLP-based vaccine. Immunization analysis based on anti-spike specific IgG antibody showed a significant increase in antibody production in harvested serum from immunized mice with HBc-VLP harboring antigenic epitope compared to HBc-VLP- and PBS-injected mice. CONCLUSIONS: The results approved the successful production and the effectiveness of the vaccine in terms of humoral IgG antibody production. Therefore, this platform can be considered a promising strategy for developing safe and reasonable vaccines; however, more complementary immunological evaluations are needed.

Identification of faecal Escherichia coli isolates with similar patterns of virulence and antimicrobial resistance genes in dogs and their owners.

BACKGROUND: The presence of antimicrobial resistance and virulence genes in Escherichia coli allows them to survive and cause infections. The close contact between humans and pets can reinforce the risk of transmitting resistant and virulent bacteria between them. OBJECTIVES: This study aims to compare the patterns of the presence of tetracycline and streptomycin resistance genes, as well as important virulence genes in E. coli isolated from faeces of healthy dogs and their owners. METHODS: Polymerase chain reactions were performed for detection of antimicrobial resistance (tetA, tetB, tetC, tetD, strA and strB) and virulence (fimH, iss, sitA and malX) genes in 144 faecal E. coli isolates from 28 dog-owner pairs and 16 humans who did not keep any pets as controls. RESULTS: Among the investigated antimicrobial resistance and virulence genes, tetA (52.1%) and fimH (86.8%) genes had the highest prevalence. No statistically significant difference was found between the prevalence of antimicrobial resistance and virulence genes in isolates of dogs and their owners. In total, 46.4% of dog-owner pairs had the same patterns of presence or absence of six antimicrobial resistance genes, 50.0% had the same patterns of presence or absence of four virulence genes and 25.0% had the same patterns of presence or absence of all 10 tested genes. CONCLUSION: The presence of antimicrobial-resistant virulent E. coli in humans and pets may predispose them to infections that are hard to cure with conventional antibiotics. Notable frequency of dogs' and their owners' E. coli isolates with similar patterns of antimicrobial resistance and virulence genes may indicate the possibility of sharing virulent antimicrobial resistant E. coli between them.

A novel fusion protein candidate for the serodiagnosis of Mycoplasma agalactiae infection.

BACKGROUND: The aim of current study was to construct, express, purify and immunogenicity evaluate of a novel recombinant fusion protein including Pyruvate dehydrogenase beta subunit (PDHB) and high antigenic region of lipoprotein P80 of Mycoplasma agalactiae. Using bioinformatics tools, antigenicity and physiochemical properties of fused protein were assessed. MATERIAL AND METHODS: The recombinant fusion protein of GST-PDHB-P80 were expressed in pGEX4T-1 and purified then verified by Western blot assay. The purified protein was successfully used for immunization of mice. 30 female BALB/c mice were divided into three groups (10 mice per each group) injected with GST-PDHB-P80, inactivated bacteria vaccine and PBS as negative control, separately. RESULTS: Western blot analysis confirmed the interaction between the immunized mice serum and the blotted recombinant protein GST-PDHB-P80, demonstrating the immunogenicity of this protein. Moreover, the sera of vaccinated mice with inactivated bacteria vaccine, containing whole cell proteins, detected the recombinant protein GST-PDHB-P80 confirming the antigenicity of PDHB-P80. Negative control displayed no reactivity with GST-PDHB-P80. CONCLUSION: We proposed a novel designed chimeric protein of Mycoplasma agalactiae as a potential marker for serodiagnostic assays but still further field research is required.

A novel chimeric recombinant protein PDHB-P80 of Mycoplasma agalactiae as a potential diagnostic tool.

The aim of this study was to construct, expression of a novel recombinant chimeric protein consisting of Pyruvate dehydrogenase beta subunit (PDHB) and high antigenic region of integral membrane lipoprotein P80 of Mycoplasma agalactiae as a potential diagnostic tool. The full-length sequence of pdhb and a portion of antigenic regions of P80 were selected and analyzed by CLC main workbench 5.5 software. Several linkers and three dimensional structure of PDHB-P80 were compared to the native PDHB and analyzed to select a proper one for expression. The fusion gene sequence was optimized and synthesized in pMAT cloning vector. The synthetic pMAT-pdhb-p80 was digested using Bam HI and Sal I restriction enzymes and ligated into pMAL-p5X expression vector. The pMAL-pdhb-p80 construct was transfected into E.coli BL21 strain cells and expressed protein were purified using amylose resin. and the purified protein was analyzed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. In silico analysis demonstrated that fusion proteins using IgG4 middle hinge (CPSCP) with TM-score of 0.99 showed the higher similarity between three dimensional structure of PDHB before and after fusion with high antigenic region of P80. Successful cloning verified by PCR colony, double digestion and sequence analysis. Besides, SDS-PAGE analysis and Western blotting indicated and confirmed the expression of intact recombinant chimeric protein MBP-PDHB-P80 along with some truncated forms of the recombinant protein. it could be concluded that the fusion construct has a potential for serodiagnostic assay in future studies.

In vitro histological investigation of interactions between rat decellularized large intestine scaffold and human adipose derived mesenchymal stem cells.

The aim of this study was to investigate the interactions between rat intestine decellularized scaffold and human adipose derived mesenchymal stem cells. Rat large intestine was dissected in fragments and decellularized by physicochemical methods. The scaffolds were loaded by human adipose derived mesenchymal stem cells expressing green fluorescent protein. Microscopic sections were prepared from the scaffolds after two weeks of culture with stem cells and studied by histological methods. The interactions of scaffolds with MSCs were also studied by electron microscopy. Histological and electron microscopy studies revealed human mesenchymal stem cell adhesion, migration, division and maintenance during the 14 days of culture in vitro. According to the results, scaffolds prepared from rat intestine matrix could be a suitable scaffold for studying in vitro cell behaviors such as division, migration and attachment. These various behaviors of cultured cells might be due to inductive effects of the extracellular matrix derived scaffold. However, more investigations are required to discover the exact effects of this scaffold and its interactions with mesenchymal stem cells.