A Chikungunya Virus Multiepitope Recombinant Protein Expressed from the Binary System Insect Cell/Recombinant Baculovirus Is Useful for Laboratorial Diagnosis of Chikungunya

Abstract

Chikungunya virus (CHIKV) is an arbovirus currently distributed worldwide, causing a disease that shares clinical signs and symptoms with other illnesses, such as dengue and Zika and leading to a challenging clinical differential diagnosis. In Brazil, CHIKV emerged in 2014 with the simultaneous introduction of both Asian and East/Central/South African (ECSA) genotypes. Laboratorial diagnosis of CHIKV is mainly performed by molecular and serological assays, with the latter more widely used. Although many commercial kits are available, their costs are still high for many underdeveloped and developing countries where the virus circulates. Here we described the development and evaluation of a multi-epitope recombinant protein-based IgG-ELISA (MULTREC IgG-ELISA) test for the specific detection of anti-CHIKV antibodies in clinical samples, as an alternative approach for laboratorial diagnosis. The MULTREC IgG-ELISA showed 86.36% of sensitivity and 100% of specificity, and no cross-reactivity with other exanthematic diseases was observed. The recombinant protein was expressed from the binary system insect cell/baculovirus using the crystal-forming baculoviral protein polyhedrin as a carrier of the target recombinant protein to facilitate recovery. The crystals were at least 10 times smaller in size and had an amorphous shape when compared to the polyhedrin wild-type crystal. The assay uses a multi-epitope antigen, representing two replicates of 18 amino acid sequences from the E2 region and a sequence of 17 amino acids from the nsP3 region of CHIKV. The recombinant protein was highly expressed, easy to purify and has demonstrated its usefulness in confirming chikungunya exposure, indeed showing a good potential tool for epidemiological surveillance.

Keywords: E2; IgG-ELISA; baculovirus expression; chikungunya; recombinant protein.

Figure 1

Schematic representation and light microscope of recombinants 6xHis-polh and 6xHis-R1-polh. (A) Schematic representation of modified polh gene cloned into pFastBac1 to generate 6xHis-polh and CHIKV multi-epitope gene (R1) fused at 5′ region of polh (6xHis-R1-polh); (B) Sf9 cells at 72 h.p.i. with the respective second passage virus stock (m.o.i. of five): mock-infected cells, cells infected with vAc-6xHis-polh, vAc-polh-R1-6xHis, and AcMNPV. The insets show details of infected cells with the amorphous crystal that accumulate in nucleus of the infected cells.

Figure 2

Scanning electron microscopy (SEM) of the purified recombinant OBs. (A) Native AcMNPV OB; (B) 6xHis-polh OBs; and (C) 6xHis-R1-polh OBs (Scale bar = 1 or 10 µm).

Figure 3

Protein expression analyses of the recombinant 6xHis-R1-polh in insect cells infected with different recombinant baculoviruses, including vAc-6xHis-polh, vAc-6xHis-R1-polh, and AcMNPV/occ- (without polh expression). (A) SDS-PAGE of insect cells extracts infected with different recombinant baculoviruses; (B,C) Membranes immune-stained with (B) anti-6xHis (Promega), (C) anti-CHIKV-positive patient’s serum as primary antibodies, incubated with mouse and human anti-IgG, respectively, and conjugated to alkaline phosphatase enzyme (Invitrogen). The proteins’ reacting bands were detected using the substrate NBT/BCIP (Promega). Black arrowheads point the specified bands.

Figure 4

IgG antibody response in chikungunya patients and control groups (n = 495) to the chikungunya recombinant polypeptide as determined by the MULTREC IgG-ELISA. Absorbance values obtained using the MULTREC IgG-ELISA on a panel (n = 495) of chikungunya cases and controls. (-) represents the mean value for each group and (---) dashed lines, the cut-off value for the test (0.3503) as determined at a wavelength of 450 nm and a reference value between 620 nm and 650 nm.