Development Of Dromedary Antibody-Based Immunoassay For Detection Of Chikungunya Virus Infections

Abstract

Chikungunya virus (CHIKV) is an arthropod-borne togavirus belonging to the genus Alphavirus that is responsible for sporadic worldwide outbreaks of Chikungunya fever, an acute febrile illness often associated with severe polyarthralgia. In Kenya, Chikungunya virus is of great epidemiological concern, with the last outbreak in 2016 in North Eastern Kenya. CHIKV is transmitted to humans through the bite of an infected female Aedes aegypti or Aedes albopictus mosquito. Transmission of CHIKV is maintained in an enzootic (sylvatic) cycle. Reliable detection of CHIKV infections is key to controlling this re-emerging pathogen, for which no cure currently exists. Current diagnostic methods for CHIKV employ a combination of tests, particularly immunologic, serologic or virologic techniques. However, the independent scientific reviews on the validity and sensitivity of currently available commercial assays have been conflicting. This project aimed to develop, optimize and validate a camel antibody-based enzyme linked immunosorbent assay for detecting Chikungunya virus infections. Camels and llamas produce a unique set of immunoglobulin G (IgG) referred to as Heavy Chain Antibodies (HCAbs) that are homodimeric and devoid of the two light chains that typify the heterotetrameric structure of the conventional immunoglobulins. The HCAbs have remarkable thermal stability coupled with high affinity to the cognate antigens. The combination of these characteristics means that HCAbs have a high potential for applications in diagnostics and therapeutics. xviii To produce sufficient antigen for camel immunization, Chikungunya virus (strain Lamu 33) was propagated in confluent C6-36 E2 cells using Cytodex microcarrier system. Purified and inactivated CHIKV immunogen was used to inoculate two camels reared at the University of Nairobi farm in Kibwezi, Kenya. Both camels demonstrated strong immune responses enabling harvesting of anti-Chikungunya virus antibodies. Heavy Chain Antibodies were purified from camel serum and used to develop the immunoassay method. A total of 188 human sera samples were assayed using the developed dromedary-based enzyme linked immunosorbent assay to determine Chikungunya virus infections. The sensitivity of the dromedary HCAb IgG2 assay was 91.3% (95% CI: 0.831 - 0.994); while that for HCAb IgG3 assay was 95.7% (95% CI: 0.898 - 1.01). The specificity of HCAb IgG2 assay was 92.3% (95% CI: 0.879 - 0.967); while the specificity of HCAb IgG3 method was 90% (95% CI: 0.851 - 0.949). For HCAb IgG2 and IgG3 based assays, the positive predictive values were 79.2% and 75.8 % respectively; while the negative predictive values were 97% and 98.4% for HCAb IgG2 and IgG3 based assays respectively. There was no cross-reactivity of camel Anti-CHIKV HCAbs with the following viral antigens: Dengue virus, O’nyong’nyong virus, Sindbis virus, Rift valley fever virus, Yellow fever virus and Semliki Forest virus. The camel antibody based assay was found to be reliable assay with very good sensitivity and specificity, and can be deployed for detection of Chikungunya virus infections.