The Expression of Chikungunya Virus Envelope 2 Glycoprotein Variants in Nicotiana benthamiana for the Development of a Diagnostic Reagent

Naude, Jason Christopher Delville

The Expression of Chikungunya Virus Envelope 2 Glycoprotein Variants in Nicotiana benthamiana for the Development of a Diagnostic Reagent

Master Thesis

2020

Abstract

Chikungunya fever is a non-fatal but highly debilitating disease that affects primates, birds and humans. The causative agent is the chikungunya virus (CHIKV), an arbovirus of the Alphavirus genus. CHIKV is responsible for the largest epidemic recorded for an Alphavirus, infecting an estimated 1.4 to 6 million patients worldwide. Furthermore, it has been recognised by the United States army as a potential biological weapon used for bioterrorism owing to the potential for infection via aerosol. CHIKV is primarily transmitted by infected Aedes aegypti mosquitos and is currently distributed in Africa, parts of Asia and South, Central and North America. As a result of the virus genetically adapting to infect the Aedes albopictus mosquito, its recent and rapid spread to non-endemic regions has occasioned increasing anxiety as well. Infection in humans presents as a sudden onset of fever, rash and severe arthralgia that persists for years. At present, there is no fast and effective diagnostic test to distinguish CHIKV from other similar viruses. This is a problem because viral infection displays the same symptoms as that of dengue, Zika, Ebola and yellow fevers while prognosis, patient care, and persistent symptoms of these viruses are very different. Usually, during the development of a diagnostic reagent, Biosafety Level 3 (BSL3) containment is required for purifying antigens from live viruses. These lab diagnostic tests are expensive to perform and, in regions facing a CHIKV epidemic, are inefficient due to their long waiting periods. This results in patients going undiagnosed or misdiagnosed and/or falling outside the window of prophylactic treatment. As such, a cheap and rapid diagnostic reagent to detect the presence of CHIKV antibodies would be most advantageous. In this study, two recombinant variants of the CHIKV E2 glycoprotein were expressed in Nicotiana benthamiana plants to assess their viability for use in a diagnostic reagent for CHIKV infection. Two versions of a tobacco sp. codon-optimised, 6xhis-tagged CHIKV E2 envelope glycoprotein gene were synthesised and cloned into the plant expression vector, pTRAkc-ERH. The E2 glycoprotein is a desirable protein candidate used for a diagnostic reagent as it is a major target for neutralizing antibody production against CHIKV during early infection. One variant contained a ~52 kDa full length E2 glycoprotein (CHIKV E2-HIS) while the other contained a ~49 kDa truncated E2 glycoprotein lacking its transmembrane domain (CHIKV E2ΔTM-HIS). Following this, an expression time trial was performed whereby the recombinant proteins were expressed in N. benthamiana plants via Agrobacterium-mediated small-scale 6 syringe-infiltration at different optical densities, OD600 = 1.0 and 0.5. To improve expression, both genes were co-infiltrated and co-expressed with a human chaperone proteins calreticulin (CRT) or calnexin (CNX), or a plant silencing-suppressor protein NSs. Expression of the recombinant protein variants alone showed low to undetectable levels of expression in plant leaves across 7 days post infiltration (dpi) for both ODs tested. CHIKV E2ΔTM-HIS yielded the highest levels of all combinations tested at an OD600 = 1.0 when co-expressed with CRT and harvested at 3 dpi. These parameters were used for subsequent scaling up and production of E2 using vacuum infiltration. Attempts at purifying CHIKV E2ΔTM-HIS proteins using Ni-NTA affinity chromatography and further investigation into the exposure of the 6xHis-tag on the native conformation of CHIKV E2ΔTM-HIS indicated that the 6xHis-tag was insufficiently exposed on E2 and thus inaccessible to facilitate purification by Ni-NTA affinity chromatography. Further attempts at purifying recombinant CHIKV E2ΔTM-HIS proteins by pH purification were also unsuccessful as large amounts of plant-protein contaminants present in all samples prevented adequate separation from CHIKV E2ΔTM-HIS. A different approach utilising ammonium sulphate precipitation facilitated separation of recombinant CHIKV E2ΔTM-HIS from some of the contaminating plant proteins in the 30 - 60% ammonium sulphate fraction; however, large amounts of recombinant CRT were co-purified with E2 in this fraction. Although expression of a candidate diagnostic reagent in plants for detecting CHIKV antibodies in the form of E2 glycoprotein was achieved, further research needs to be done to optimise a purification strategy for CHIKV E2ΔTM-HIS proteins.

Keywords

Molecular and Cell Biology

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