Development of recombinant protiens as diagnostic intermediates for chikungunya infection

Abstract

INTRODUCTION: Chikungunya (CHIK) is an important disease with explosive outbreaks occurring in Sri Lanka. Confirmation of CHIK outbreaks is important for clinicians for proper clinical management of patients and epidemiological studies. There is a need to develop a laboratory diagnostic assay which can be discriminated CHIK from other diseases showing similar symptoms, produced at low cost and easily standardized for use in field settings. At present Enzyme-Linked Immunosorbent Assay (ELISA) is widely used for laboratory diagnosis of CHIK infection because of its rapidity, cost effectiveness and ability to standardize easily for field settings. These currently available ELISAs depend on whole viral lysate antigens which cause biohazard risk, high initial production cost and cross reactivity with other organisms of the same genus/family. A diagnostic intermediate using a single recombinant protein antigen to detect both Immuno globulin (Ig) M and G antibodies of CHIK is important to overcome problems associated with whole viral lysate antigen in ELISA. Overall objective of this study was to assist for clinical management of patients and confirmation of CHIK outbreaks through developing rapid laboratory diagnostic assays. Methodology: Surface proteins of Ribonucleic Acid (RNA) viruses are the targets of neutralizing antibodies. Envelope (E) region of the Chikungunya Virus (CHIKV) is the most immunodominant region of the virus. These protein antigens were prepared using the E region of the Virus. Synthetic genes of CHIK named Envelope 1 (El) and Envelope 2 (E2) were custom designed and chemically synthesized and resulted proteins were expressed in both bacteria (Escherichia coli} and yeast (Pichia pastoris) vector expression systems. Resulted recombinant proteins were purified using a single affinity chromatographic step using Ni- Nitrilotriacetic Acid (NTA) columns and evaluated to be used as a diagnostic intermediate using panels of well characterized serum samples. A total of 55 serum samples confirmed as positives and 186 confirmed as negatives for CHIK IgM antibodies were used to evaluate the antigens using novel IgM ELISA. A total of 78 serum samples confirmed as positives and 227 samples confirmed as negatives for CHIK IgG antibodies were used to evaluate the antigens using novel IgG ELISA. These samples were tested by Heamagglutination Inhibition (HAI) test, IgM Antibody capture (MAC) ELISA and indirect IgG ELISA. RESULTS: The recombinant proteins were purified using Ni-NTA columns under the denature conditions. For the detection of anti-CHIK IgM antibodies, the El recombinant protein expressed in E. coli showed 5% (3/55) sensitivity and 99% (184/186) specificity while the E2 recombinant protein expressed in E. coli showed 65 % (36/55) sensitivity and 70% (131/186) specificity when compared with MAC ELISA using total viral lysate as the antigen. The El recombinant protein expressed in P. pastoris showed 15% (8/55) sensitivity and 97% (181/186) specificity and the E2 recombinant protein expressed in P. pastoris showed 49% (27/55) sensitivity and 78% (146/186) specificity compared with MAC ELISA using total viral lysate as the antigen for detection of IgM antibodies. For the detection of anti-CHIK IgG antibodies, the El recombinant protein expressed in E. coll showed 60 % (47/78) sensitivity and 64% (94/148) specificity while the E2 recombinant protein expressed hi E. coli showed 83 % (65/78) sensitivity and 86% (195/227) specificity compared with the HAI test and indirect IgG ELISA using purified CHIKV. The El recombinant protein expressed in P. pastoris showed 86% (67/78) sensitivity and 61% (90/ 148) specificity and the E2 recombinant protein expressed in P. pastoris showed 76% (59/78) sensitivity and 81% (183/227) specificity compared with the HAI test and indirect IgG ELISA using purified CHIKV for the detection of IgG antibodies. Discussion: When considering El and E2 recombinant antigens, E2 recombinant proteins have showed better performance in identifying both anti-CHIK IgM and IgG antibodies in ELISAs, Here CHIK E2 protein is positioned in a way that it buries much of its partner El in the virus structure giving a CHIK El a less chance of exposure to the human immune system explaining the lower sensitivity and specificity towards detecting anti-CHIK antibodies. The E2 recombinant protein antigens expressed in bacterial expression system have performed better than the E2 recombinant antigens expressed in yeast system. In bacterial expression system the expressed recombinant proteins must have aggregated in cytoplasm in such a way, when denatured, most of the immuno dominant epitopes became linearized and exposed to the outside. CONCLUSION: Recombinant CHIK-E2 protein antigen expressed in E. coli showed higher specificity and sensitivity in detection of both IgM and IgG anti-CHIK antibodies. This type of study on development of diagnostic intermediates have a significant effect for rapid confirmation of outbreaks and take necessary steps to limit the spread of such outbreaks from one geographical area to another. Further, confirmation of disease outbreaks will allow the physicians to provide the necessary treatments rapidly and thereby avoid loss of working hours and socio-economical impact on individuals and government.