AbstractCholera is a severe diarrheal disease, mostly found in developing countries and caused by the etiological agent, Vibrio cholerae. Despite recent advances in health cares, cholera is still a big challenge.
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454 cholera cases with 1304 deaths was observed only in 2015. Different routine therapies like oral or injection serum or the present vaccines are not effective enough to prevent or treat cholera. Recombinant protein vaccines such as chimeric vaccines offer several advantages for cloning, expressing and purification of genes from different etiologic agents to test as a vaccine. Their ability in presenting multi antigens at the same time can play important roles in immunization. In this study, a chimeric construct consisted 2 immunogenic agents of Vibrio cholera, ompW & ctxB, was designed and after bioinformatics assessments, it expressed in E.coli. The accuracy of protein was confirmed by Western blotting.
Mice were immunized with 3 recombinant proteins (OmpW, CtxB, and chimeric protein). The antibody titers and specificity of the immune serums were analyzed by ELISA. The statistically significant difference was observed in antibody titer compared to the control group and in passive immunization, the infants of the chimeric group who received LD50 were survived.
Keywords: Vibrio cholerae, Recombinant protein, Immunization, OmpW, CtxB Introduction:Cholera, a severe lethal secretory diarrheal disease, is caused by the Vibrio cholerae, a gram-negative, motile, curved-rod bacterium(1). Although there are more than 200 serogroups of Vibrio cholerae, the epidemics of cholera are caused only by strains of serogroup O1 and O139(2, 3). These epidemics are most common in developing countries which suffer from the unsafe drinking water, limited health care, and poor sanitary system and if it remains untreated its fatality rate can be as high as 70%(4). Despite the fact that the disease is easily curable, it is still considered as one of the major problems in the field of health, with a rate of 3-5 million cases a year. Cholera is known to be the second leading cause of death in children under the age of 5 years(5, 6). One of the reasons for its sustainability is the absence of vaccines with long-term effects and protections(7).Bacterial action is possible by its attachment to the small intestine.
A large number of factors including lipopolysaccharide (LPS), OM porins (OMPs), TCP, accessory colonization factors (ACFs), the core oligosaccharide (core OS) lipid A and flagella are important in its colonization(8, 9).After proliferation, the release of heat-sensitive exotoxin stimulates the intestinal mucosa, disrupting sodium ion exchange in the intestinal mucosa cells and releasing large amounts of water and electrolyte into the intestine which will sharply reduce blood plasma levels and ultimately lead to death(10, 11).Cholera toxin (CT) is one of the key factors in creating these conditions. It is encoded by the ctxAB gene located in bacteriophage CTX?(12). CtxA is the active unit and CtxB is a pentameric binding unit which has no toxic properties and just binds cholera toxin to GM1 ganglioside receptors(13).Besides the importance of antitoxic immunity, antibacterial immunity also plays an important role in preventing cholera (14).
This immunity is against both lipopolysaccharide (LPS) (14, 15) and outer membrane (OM) proteins (OMPs) of V. cholerae (16, 17). The antibodies to V. cholerae OMPs which inhibit the intestinal colonization of Vibrio play an important role in protection (18).Outer membranes (OMs) in Gram-negative bacteria work as a barrier and there are channels within them which promote uptake of necessary nutrients for the cells. These protein channels are monomeric or trimeric barrels with 12-22 anti-parallel ?-strands.
There is also a noticeable number of smaller, monomeric ?-barrels which consist of 8 or 10 ?-strands(19). One of this small monomeric ?-barrel is OmpW, an 8-stranded ?-barrel, 22 kDa protein, comprising of a narrow, long, hydrophobic channel that acts as an ion channel. This hydrophobic channel causes distinction between OmpW and other OMP channels, which are usually hydrophilic(20). Various environmental conditions such as temperature, salinity, availability of food and oxygen are factors that influence the expression of OmpW in Vibrio cholerae(21).
As mentioned, this protein is effective in the attachment of pathogenic bacteria to the intestine and an immune response against them. Study on mutant Vibrio cholerae that lacked OmpW showed a 10-fold reduction in colonization of the bacteria in mice compared to those that contained OmpW(21). Studies have shown that OmpW is conserved in Vibrio cholerae strains, and this conservation and immunogenicity has made it very noticeable for producing a vaccine(22).Attenuated or inactivated pathogens are traditionally used as vaccines. But nowadays, recombinant protein vaccines, especially chimeric ones, offer several advantages for cloning, expressing and purification of genes from different etiologic agents to test as a vaccine.
The ability of chimeric construct in presenting multi antigens at the same time can play important roles in immunization (23, 24).Therefore, in this work, based on the immunogenic properties of these two proteins and the benefits of chimeric vaccines, a chimeric construct containing ompW & ctxB, was designed and expressed in E.coli and its immunization against Vibrio cholerae was evaluated in mice.