Facile quantitative diagnostic testing for neutralizing antibodies against Chikungunya virus

Abstract

Background: Viral neutralization (NT) assays can be used to determine the immune status of patients or assess the potency of candidate vaccines or therapeutic monoclonal antibodies (mAbs). Focus reduction neutralization test (FRNT) is a conventional neutralization test (cVNT) with superior specificity for measurement of neutralizing antibodies against a specific virus. Unfortunately, the application of FRNT to the chikungunya virus (CHIKV) involves a highly pathogenic bio-agent requiring biosafety level 3 (BSL3) facilities, which inevitably imposes high costs and limits accessibility. In this study, we evaluated a safe surrogate virus neutralization test (sVNT) that uses novel CHIKV replicon particles (VRPs) expressing eGFP and luciferase (Luc) to enable the rapid detection and quantification of neutralizing activity in clinical human serum samples.

Methods: This unmatched case-control validation study used serum samples from laboratory-confirmed cases of CHIKV (n = 19), dengue virus (DENV; n = 9), Japanese encephalitis virus (JEV; n = 5), and normal individuals (n = 20). We evaluated the effectiveness of sVNT, based on mosquito cell-derived CHIK VRPs (mos-CHIK VRPs), in detecting (eGFP) and quantifying (Luc) neutralizing activity, considering specificity, sensitivity, and reproducibility. We conducted correlation analysis between the proposed rapid method (20 h) versus FRNT assay (72 h). We also investigated the correlation between sVNT and FRNT in NT titrations in terms of Pearson's correlation coefficient (r) and sigmoidal curve fitting.

Results: In NT screening assays, sVNT-eGFP screening achieved sensitivity and specificity of 100%. In quantitative neutralization assays, we observed a Pearson's correlation coefficient of 0.83 for NT50 values between sVNT-Luc and FRNT.

Conclusions: Facile VRP-based sVNT within 24 h proved highly reliable in the identification and quantification of neutralizing activity against CHIKV in clinical serum samples.

Keywords: Chikungunya virus; Diagnostics; Neutralizing antibodies; Surrogate virus neutralization test; Surveillance; Vaccine; Virus-like replicon particle.

Fig. 1

Qualitative NT evaluation. (A) A schematic diagram of VRP production is illustrated. A recombinant baculovirus was designed to carry two DNA cassettes: one containing the CHIKV replicon with dual reporters—enhanced green fluorescent protein (eGFP) and luciferase (Luc)—under the control of a cytomegalovirus (CMV) promoter, and another encoding the CHIKV structural proteins (sP) driven by a mosquito-specific hr1pag1 promoter. Upon transduction, the recombinant baculovirus introduces these DNA cassettes into mosquito cells, enabling the expression of all necessary components for VRP packaging. CHIK VRP contains a defective genome with dual reporters (eGFP and Luc) under the control of subgenomic RNA promoter, where nsP refers to nonstructural proteins, T2A refers to Thosea asigna virus 2 A self-cleaving peptides, and UTR indicates the untranslated region of the CHIKV genome; (B) Reproducibility assessment of mos-CHIK VRP-based sVNT-eGFP screening results obtained by three operators (1-3) on 52 serum samples from CHIKV patients (n =19), DENV patients (n =8), JEV patients (n =5), and 20 normal individuals (N). Sample numbers (corresponding eGFP intensities) are indicated beneath each well in the left panel; (C) Statistical analysis comparing eGFP intensity data from (Fig. 1B) between CHIKV patients and unrelated groups. The significance levels are (P < 0.0001), with a significance level set at p < 0.0001 and (_****) denoting extreme significance; (D) Overall performance of sVNT-eGFP screening

Fig. 2

Quantitative NT evaluation results (A) Titration curves of neutralizing antibodies for the sera of 19 CHIKV patients. CHK265 mAb serves as a positive control. Data represent means ± standard deviation (triplicate in 1 experiment). (B) Mean neutralizing titers from the sera of 19 CHIKV patients, as determined by mos-CHIK VRP-based sVNT50 (sVNT-Luc) and FRNT50; (C) Performance of sVNT-Luc based on the correlation between sVNT50 and FRNT50 values for the sera of 19 CHIKV patients

Fig. 3

Flowchart showing the application of mos-CHIK VRP-based sVNT as a tool for the rapid measurement of CHIKV NT activity. After pre-incubating VRPs with test serum for 1 h, Vero cells are infected with the pre-incubated VRPs for 1 h and washed. After incubation at 28 °C for 20 h, the cells are subjected to either sVNT-eGFP to qualify NT activity by inhibiting eGFP expression or sVNT-luc to quantify NT activity by inhibiting luciferase activity