ANALYSIS OF IMMUNE RESPONSES AGAINST CANDIDATE VIRULENCE ANTIGEN/S DURING MICROBIAL INFECTION

Abstract

“Microbial infection” is the term used to describe an infection caused by any microbe which can cause harm to an individual’s body. Some major types of infections caused by microbes or micro-organisms are bacterial, fungal, and viral infections. The severity of microbial infections can range from asymptomatic, mild infection to life-threatening fatal forms. One of the major fungal infections, Candidiasis which is caused by an opportunistic fungus Candida, usually comes under the non-pathogenic and commensal category of microbes that produces oral thrush. However, in immunocompromised or patients suffering from underlying comorbidities, individuals undergoing ICU treatments, Candida shows interactions with epithelial cells, breaches the mucosal lining, can invade endothelial cells, and then finally enters the bloodstream. The invasion of Candida to blood stream and dissemination to tissues can cause severe damage to vital organs which includes kidney, lungs, brain and bones. Severe bloodstream Candida infections are termed as Systemic/Invasive/disseminated candidiasis. Candidemia is the most serious form of fungal infection that usually occurs in immunocompromised individuals. Moreover, invasive candidiasis can also affect the brain and heart that leads to meningitis and endocarditis respectively. Invasive Candida infections are attributable to high morbidity and mortality rates in immunocompromised individuals. Although various species of Candida have been identified, only 5 pathogenic species in particular C. albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis account for most invasive candidiasis cases. Current treatments available for systemic candidiasis is basically broad-spectrum antibiotic drugs, for example, fluconazole, amphotericin-B and echinocandins. Although liposomal Amphotericin-B is a good treatment option but it’s not feasible for low-income countries due to its high cost of medication. Further, the emergence of multi-drug resistant species C. auris, which is the leading cause of nosocomial infections, has become the global threat. Therefore, there is an emergent need for developing alternative treatment options. To develop vaccine candidates against Candidiasis, two putative Candida virulence antigens, Sap2 protein from C. parapsilosis and Enolase from C. albicans were selected, isolated, and expressed in prokaryotic expression system. Thereafter, these antigens were purified by Ni-NTA chromatography. The structural characterization of Sap2 and Enolase proteins was performed by CD and FTIR spectroscopy. This research study presents results on expression, purification, and structural characterization of two Candida virulence antigens (Sap2 and Enolase). Cell-mediated immunity has been displayed as a crucial component in mediating anti-Candida protective immune responses. However, very few studies examined the role of antibody mediated protection against Candida. The antibody effector mechanisms, such as neutralization, direct candidacidal activity, opsonization, biofilm inhibition and ADCC have been reported in literature. It has been hypothesized that polyclonal antibodies generated against Candida virulence antigens may have an important role against systemic candidiasis. However, the mechanism of antibody mediated protection has not been previously described elsewhere in the case of C. tropicalis biofilms. To test this hypothesis, immune sera containing polyclonal antibodies was collected from mice immunized with recombinant Sap2-parapsilosis protein. Sap-specific polyclonal antibodies were tested for their ability to suppress pre-formed C. tropicalis biofilms using an XTT-based in vitro experiment. This study presents novel results pertaining to C. tropicalis biofilm inhibition via Sap-2 specific polyclonal antibodies. Acute to chronic infections that persist are linked to viral infections like chikungunya. The Chikungunya virus (CHIKV), an alphavirus spread by mosquitoes that causes severe illnesses in humans, is the cause of chikungunya. Epidemic outbreaks of CHIKV have been reported in Africa, Asia, North and South America, and Europe. There are currently no approved vaccinations available for the treatment or prevention of CHIKV infection, despite the virus’s rising worldwide incidence. Among the potentially useful vaccine antigens against CHIKV is the envelope E2 protein. Thus, there was the usage of the E. Coli expression system for the cloning, production, and purification of CHIKV E2 proteins. In addition, a thorough refolding process was refined and carried out to produce the native conformation of the E2 proteins. Infrared spectroscopy using Fourier transform and circular dichroism were used to confirm the native structures of E2 proteins When compared to sham-immunized controls, E2-specific antibody titers were higher in mice following immunization with the distinct E2 proteins, suggesting a strong antibody mediated immune response. Out of the three E2 antigens employed in the investigation, the E2 full length protein was shown to be the most immune stimulating, upon examination of the antibody response specific to E2 protein produced in vaccinated mice. Additionally, compared to short versions of E2 protein, the E2 full length protein was more immunogenic due to the inclusion of immunogenic peptides in its N- and C-terminus end, that were not present in truncated forms of E2 protein, according to B- and T-cell epitope mapping data. This research study presents results on expression, purification, and E2 protein refolding and offers a detailed analysis of its potential B-cell and T-cell immunogenic epitopes, which can be used to create innovative multiepitope-based anti-CHIKV vaccination regimens. Overall, fungal, and viral infections are now a serious risk to immuno-compromised and hospitalized patients throughout the world. Therefore, this research study's primary objective was indeed the characterization of potential vaccine candidates against these microbial infections.