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In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens.

ABSTRACT: Background:L7/L12 is a protective antigen conserved in main Brucella pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all Brucella pathogens. Materials and Methods:The purpose of the current study was to in silico design a L7/L12-SOmp2b fusion protein and in vitro production of the chimera. Two possible fusion forms, L7/L12-SOmp2b and SOmp2b-L7/L12, were subjected to in silico modeling and analysis. Cloning and expression of the fusion protein has been done in the pET28a vector and Escherichia coli Bl21 (DE3), respectively. Results:Analysis and validation of the fusion proteins three-dimensional models showed that both models are in the range of native proteins. However, L7/L12-SOmp2b structure was more valid than the SOmp2b-L7/L12 model and subjected to in vitro production. The major histocompatibility complex II (MHC-II) epitope mapping using Immune Epitope DataBase indicated that the model contained good MHC-II binders. The L7/L12-Omp2b coding sequence was cloned in pET28a vector. The fusion was successfully expressed in E. coli BL21 by induction with isopropyl-?-d-thiogalactopyranoside. The rL7/L12-SOmp2b was purified with Ni-NTA column. The yield of the purified rL7/L12-SOmp2b was estimated by Bradford method to be 240 ?g/ml of the culture. Western blot analysis revealed a specific reactivity with purified rL7/L12-SOmp2b produced in E. coli cells and showed the expression in the prokaryotic system. Conclusions:Our data indicates that L7/L12-SOmp2b fusion protein has a potential to induce both B- and T-cell-mediated immune responses and it can be evaluated as a new subunit vaccine candidate against brucellosis.

SUBMITTER: Golshani M

PROVIDER: S-EPMC5840964 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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<i>In silico</i> Design, and <i>In vitro</i> Expression of a Fusion Protein Encoding <i>Brucella abortus</i> L7/L12 and SOmp2b Antigens.

Golshani Maryam M Ghasemian Melina M Gheibi Nematollah N Bouzari Saeid S

Advanced biomedical research 20180216

<h4>Background</h4>L7/L12 is a protective antigen conserved in main <i>Brucella</i> pathogens and is considered as potential vaccine candidate. Outer membrane protein 2b is an immunogen conserved in all <i>Brucella</i> pathogens.<h4>Materials and methods</h4>The purpose of the current study was to <i>in silico</i> design a L7/L12-SOmp2b fusion protein and <i>in vitro</i> production of the chimera. Two possible fusion forms, L7/L12-SOmp2b and SOmp2b-L7/L12, were subjected to <i>in silico</i> mode ...[more]

PMID: 29531919

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Project description:BACKGROUND: We generated novel, effective candidate vaccine against Brucella abortus based on recombinant influenza viruses expressing the Brucella ribosomal protein L7/L12 or outer membrane protein (Omp)-16 from the NS1 open reading frame. The main purpose of this work was to evaluate the safety, immunogenicity and protectiveness of vaccine candidate in laboratory animals. METHODS AND RESULTS: Four recombinant influenza A viral constructs of the subtypes ?5N1 or H1N1 expressing the Brucella proteins L7/L12 or Omp16 were obtained by a reverse genetics method: Flu-NS1-124-L7/L12-H5N1, Flu-NS1-124-Omp16-H5N1, Flu-NS1-124-L7/L12-H1N1 and Flu-NS1-124-Omp16-H1N1. Despite of substantial modification of NS1 gene, all constructs replicated well and were retain their Brucella inserts over five passages in embryonated chicken eggs (CE). Administration of the mono- or bivalent vaccine formulation via prime-boost intranasal (i.n.), conjunctival (c.) or subcutaneous (s.c.) immunization was safe in mice; no deaths, body weight loss or pathomorphological changes were observed over 56 days. Moreover, guinea pigs vaccinated i.n. with vaccine vectors did not shed the vaccine viruses through their upper respiratory tract after the prime and booster vaccination. These findings confirmed the replication-deficient phenotype of viral vectors. The highest antibody response to Brucella antigen was obtained with constructs expressing L7/L12 (ELISA, GMT 242.5-735.0); whereas the highest T-cell immune response- with construct expressing Omp16 (ELISPOT, 337?±?52-651?±?45 spots/4×105cells), which was comparable (P?>?0.05) to the response induced by the commercial vaccine B. abortus 19. Interestingly, c. immunization appeared to be optimal for eliciting T-cell immune response. In guinea pigs, the highest protective efficacy after challenge with B. abortus 544 was achieved with Omp16 expressing constructs in both monovalent or bivalent vaccine formulations; protective efficacy was comparable to those induced by a commercial live B. abortus 19 vaccine. CONCLUSION: Thus, influenza vectors expressing Brucella protective antigens can be developed as novel influenza vectored vaccine against B. abortus infection.

Project description:Brucella, a genus of bacteria that causes brucellosis, infects and threatens domestic animals, and humans in endemic areas. Presently, some live attenuated vaccines of Brucella are used to immunize livestock; however, these vaccines are pathogenic to humans, can provoke abortion when administered to pregnant livestock, and induce antibodies in vaccinated livestock that affect the diagnosis of field infection. It is, therefore, very important for improving the safety and immune protection effects of Brucella vaccine. Currently, recombinant protein-based subunit vaccines are considered promising safe and effective alternatives against brucellosis. Here, we separately expressed the recombinant Omp10-Omp28-L7/L12 proteins of Brucella using eukaryotic and prokaryotic expression systems, which were then used as immunogens for evaluating their immune responses. Taishan Pinus massoniana pollen polysaccharides (TPPPS), an already verified natural adjuvant, was utilized to evaluate the immune conditioning effect on the recombinant proteins. Antibody levels, spleen lymphocyte proliferation, percentages of CD4+ and CD8+ T cells, and cytokine secretion in mice were examined after three successive immunizations. The protective effects against Brucella challenge were also evaluated in mice, and used a live vaccine as a positive control. The results indicated that the immune responses of the recombinant Omp10-Omp28-L7/L12 protein groups were significantly higher than those of the PBS control group. The recombinant Omp10-Omp28-L7/L12 protein expressed in Pichia pastoris (P. pastoris) exhibited a slightly higher expression level and immunogenicity than that expressed in Escherichia coli (E. coli), and the Omp10-Omp28-L7/L12 (P. pastoris) + TPPPS group provided the most pronounced immune effect. The protective results showed that the recombinant Omp10-Omp28-L7/L12 proteins expressed in the two expression systems had significantly better protective effects against Brucella melitensis challenge compared with the negative control, and the addition of TPPPS adjuvant could significantly improve the protective effects of subunit vaccines. However, we also noticed that all of the evaluated subunit vaccines induced less protection than the B. melitensis M5 live vaccine. These results indicate that the combination of recombinant Omp10-Omp28-L7/L12 antigen and TPPPS adjuvant shows potential as an effective brucellosis subunit vaccine, and P. pastoris is a preferred expression system to prepare this recombinant subunit antigen.

Dataset Information

In silico Design, and In vitro Expression of a Fusion Protein Encoding Brucella abortus L7/L12 and SOmp2b Antigens.

Publications

<i>In silico</i> Design, and <i>In vitro</i> Expression of a Fusion Protein Encoding <i>Brucella abortus</i> L7/L12 and SOmp2b Antigens.

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