In silico design and validation of a Plasmodium falciparum multi-epitope diagnostic antigen (PfMEDA1) for the diagnosis of falciparum malaria

Abstract

BACKGROUND: Despite efforts to curb the debilitating effects of malaria, incidence is still on the rise. Rapid diagnosis and effective treatment remain key strategies for reducing morbidity and mortality. The limitations of common antigens used for rapid diagnosis necessitate the search for alternatives. Studies carried out by Azcárate and colleagues have identified immunogenic proteins expressed in both clinical and subclinical malaria, which can therefore serve as diagnostic targets. This study is aimed at designing and validating a multi-epitope antigen to detect Plasmodium falciparum. METHODS: The amino acid sequences of five proteins previously reported to be immunogenic in infected malaria cases were retrieved from the Uniprot database, and linear B-cell epitopes were predicted and joined using GSGSG linkers to produce a chimeric protein (PfMEDA1). The physicochemical properties of the protein, as well as the secondary and tertiary structures, were predicted. The gene sequence of the protein after reverse transcription and codon optimization was synthesized, cloned into a pET30a(+) vector, expressed, and purified from E coli. Antibody reactivity against PfMEDA1 was assessed using an indirect ELISA with human serum samples from infected (n = 223) and non-infected (n = 81) malaria cases collected from hospitals in Fako Division of the South West Region of Cameroon. RESULTS: The in silico-designed protein was predicted to be soluble, stable in varying climatic conditions, and immunogenic. There was a significant difference (P < 0.0001) observed between the mean OD in infected and non-infected samples, with a diagnostic sensitivity of 89.69% (95 CI: 84.93 to 93.35), diagnostic specificity of 75.31(95% CI: 64.47 to 84.22) and a Cohen kappa value of 0.64 implying slight substantial agreement with the PCR serving as the reference method. CONCLUSION: Despite limitations of sample size and the need for field validation in settings of varied endemicity to establish its true diagnostic potential, PfMEDA1, in the current study, has shown potential as a diagnostic antigen. CLINICAL TRIAL: Not applicable.