Development and application of monoclonal antibodies to Chikungunya virus proteins

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes arthritic disease in humans and is considered a serious health threat in regions where competent mosquito vectors are prevalent. The recent CHIKV epidemic (2004-2014) has resulted in an estimated 1.4-6.5 million cases, involving more than 40 countries. There are no commercially licensed human vaccines available against infection with this re-emerging alphavirus. Treatment of CHIKV rheumatic disease usually involves the use of analgesics and/or non-steroidal anti-inflammatory drugs, with relief often inadequate. Furthermore, the number of monoclonal antibodies (mAbs) specific for CHIKV is limited. This thesis describes the generation and characterisation of a suite of mAbs that are specific to the E2 or capsid protein (CP) of CHIKV. These mAbs were shown to be reactive in a range of assays including ELISA, Western blot, immunofluorescence and immunohistochemistry. Eleven CP-specific mAbs were capable of recognising isolates that represent the major genotypes of CHIKV, as well as several other alphaviruses. In contrast, five mAbs specific for the E2 glycoprotein were able to distinguish CHIKV from all other alphaviruses tested. Two of these E2-specific mAbs have the ability to provide complete protection against arthritis in a CHIKV mouse model when administered prior to infection. The anti-E2 mAbs have also been used successfully in a sensitive epitope-blocking ELISA to detect anti-CHIKV antibodies in clinical samples. Moreover, we have identified CHIKV in a serum sample from a patient using an antigen-capture ELISA. The recent re-emergence of CHIKV and its potential threat to human health has called for a better understanding of the virus for improved treatment, prevention and control measures. To do so, we have mapped the binding sites of the anti-CP mAbs with the help of N- and C-terminally truncated versions of the CHIKV CP. Two putative binding regions, residues 1 to 35 and 105-210 of CP, were identified. Competitive binding studies also revealed that five of the CP-specific mAbs recognised a series of overlapping epitopes that are likely to be the nuclear export signal of CHIKV CP. Furthermore, we have confirmed the presence of a smaller, truncated product of CP that may have structural and/or functional significance during viral replication. Evidence was also provided to show that the C-terminus of CP is required for antibody binding. Several ELISA-based diagnostic tests have been developed for the detection anti-CHIKV antibodies. However, international standardization and validation of these assay are currently limited. The use of human sera collected from infected or uninfected individuals is required for the validation of diagnostic assays, as well as to determine cut-off values. Numerous drawbacks associated with the routine usage of pooled reference sera from CHIKV-infected patients include obtaining sufficient quantities to meet demands, stringent safety procedures and ethical concerns to be taken into account, and the requirement of standardization for each batch of control material. Therefore, a recombinant, chimeric version of a selected anti-CP mAb was created as an alternative positive control in diagnostic kits. The chimeric 5.2H7 mAb was assessed in ELISA and Western blot, and was found to have specificity and sensitivity levels comparable to that of the original mAb. In addition, we have derived a novel method to specifically amplify the variable chain sequences of antibody genes that could be applied towards the generation of any other chimeric mAbs, while avoiding the notoriously troublesome aberrant heavy or light chain genes. Given the current shortage of widely-available reagents for CHIKV, these specific antibodies present not only useful tools for further investigations on the structure and function of the virus, but may also provide the basis for new diagnostics and treatment of the disease.