Evaluation of the Immunogenicity of Recombinant Espb, Espc Proteins from Mycobacterium Tuberculosis and the Fusion Espc/Espb Protein in BALB/C Mice.

Background: Two newly identified proteins, EspB and EspC are involved in the pathogenesis of Mycobacterium tuberculosis. The objective of the present study was to evaluate the immunogenicity of recombinant EspC, EspB, and EspC/EspB fusion proteins in mice. Methods: BALB/c mice were immunized subcutaneously with recombinant EspC, EspB, and fusion EspC/EspB proteins, three times with along with Quil-A as an adjuvant. The cellular and humoral immune responses were evaluated by quantifying IFN-γ, IL-4, IgG, IgG1, and IgG2a antibodies against the antigens. Results: The results showed that the mice immunized with recombinant EspC, EspB, and EspC/EspB proteins did not produce IL-4, whereas IFN-γ was secreted in response to all three proteins. EspC/EspB group produced significant amounts of IFN-γ in response to stimulation with all the three recombinant proteins (P<0.001). In mice immunized with EspC, high levels of IFN-γ were detected in response to EspC/EspB, and EspC (P<0.0001); while mice immunized with EspB produced lower levels of IFN-γ in response to EspC/EspB, and EspB (P<0.05).Mice immunized with recombinant EspC, EspB, and EspC/EspB proteins exhibited significantly high levels of IgG and IgG2a/IgG1 ratio (P< 0.001). Moreover, high levels of IgG and IgG2a were detected in the sera of mice immunized with EspC/EspB fusion protein. Conclusions: All the three recombinant proteins induced Th1-type immune responses in mice against EspB and EspC; however, EspC/EspB protein is more desirable due to the presence of epitopes from both EspC and EspB proteins and the production of immune responses against both.

A Mixed Model Approach for Estimating the Optimal Food Fortification of Vitamin D: Experiment Based on Mashhad Cohort Study in Iran.

BACKGROUND: Vitamin D deficiency is a prevalent problem in worldwide healthcare related to several system disorders. Food fortification as a solution is associated with several challenges including insufficient coverage of the entire population, required degree of fortification, the vehicles used for fortification and potential toxicity. This study aimed to determine the optimal amount of vitamin D for fortification without surpassing the upper intake level (UL) of intake at the 95th percentile of the Iranian population and compare two methods of food fortification. METHODS: This study is aimed to develop a model of two different fortifying approaches related to an available dataset called MASHAD cohort study. The dataset comprised demographic and nutritional data of 9704 Iranian individuals living in the Greater Mashhad region. The first approach was a computational method necessary to implement a range of eight foods and calculate the optimal approach. In the second case, we used the European formula method called ILSI. RESULTS: To find the appropriate value for fortification, we calculated the consumption of 400 IU and 1000 IU supplements of vitamin D. Three micrograms per 100 g in each food was the optimal output. We also used Flynn and Rasmussen's formula on our data. Using these methods, we found that 2.1 micrograms per 100 kcal provides the best result. Hence, using the two different approaches, the results appear to be consistent and promising. CONCLUSION: One interesting finding was that supplement consumption did not greatly affect the impact of fortification. This observation may support the hypothesis to determine the amount of fortification, and we can ignore the study population's supplement consumption.

Cloning, Expression and Purification of Espc, Espb and Espc/Espb Proteins of Mycobacterium tuberculosis ESX-1 Secretion System.

BACKGROUND: It is estimated that one third of the world's population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of Tuberculosis (TB). The BCG vaccine is widely used to fight against TB; however, many question its ability to provide complete protection from Mtb. Recently, the "Region of Difference 1" (RD1) set of genes were shown to be involved in the pathogenesis of Mtb. Downstream of RD1 transcription region, two proteins are encoded, known as EspB and EspC, which were found to contribute to Mtb virulence.In this study these two proteins are targeted as potential vaccine candidates against TB. METHODS: The EspB and EspC Mtb genes were codon-optimized for expression and synthesis in Escherichia coli (E. coli). The amplicons were cloned into a pET21a expression vector and transformed into E. coli BL21(DE3). The expression and purity of the expressed proteins (i.e. rEspC, rEspB and rEspC/EspB) were confirmed by SDS-PAGE and Western blotting. Moreover, BALB/c mice were immunized against Mtb using the recombinant proteins. Finally, the mice sera were analyzed via Western blotting. RESULTS: EspC, EspB, and EspC/EspB fusion genes were cloned and expressed in E. coli. Both SDS-PAGE and Western blots confirmed the presence and successful purification of the desired proteins. Moreover, antisera produced against the purified recombinant proteins reacted with Mtb proteins. CONCLUSION: rEspC, rEspB, and rEspC/EspB could be expressed and purified using an E. coli expression system. The recombinant proteins induced the production of antibodies in BALB/c mice that reacted with Mtb proteins.