Virus replicon particle based Chikungunya virus neutralization assay using Gaussia luciferase as readout

Abstract

Background: Chikungunya virus (CHIKV) has been responsible for large epidemic outbreaks causing fever, headache, rash and severe arthralgia. So far, no specific treatment or vaccine is available. As nucleic acid amplification can only be used during the viremic phase of the disease, serological tests like neutralization assays are necessary for CHIKV diagnosis and for determination of the immune status of a patient. Furthermore, neutralization assays represent a useful tool to validate the efficacy of potential vaccines. As CHIKV is a BSL3 agent, neutralization assays with infectious virus need to be performed under BSL3 conditions. Our aim was to develop a neutralization assay based on non-infectious virus replicon particles (VRPs).

Methods: VRPs were produced by cotransfecting baby hamster kidney-21 cells with a CHIKV replicon expressing Gaussia luciferase (Gluc) and two helper RNAs expressing the CHIKV capsid protein or the remaining structural proteins, respectively. The resulting single round infectious particles were used in CHIKV neutralization assays using secreted Gluc as readout.

Results: Upon cotransfection of a CHIKV replicon expressing Gluc and the helper RNAs VRPs could be produced efficiently under optimized conditions at 32°C. Infection with VRPs could be measured via Gluc secreted into the supernatant. The successful use of VRPs in CHIKV neutralization assays was demonstrated using a CHIKV neutralizing monoclonal antibody or sera from CHIKV infected patients. Comparison of VRP based neutralization assays in 24- versus 96-well format using different amounts of VRPs revealed that in the 96-well format a high multiplicity of infection is favored, while in the 24-well format reliable results are also obtained using lower infection rates. Comparison of different readout times revealed that evaluation of the neutralization assay is already possible at the same day of infection.

Conclusions: A VRP based CHIKV neutralization assay using Gluc as readout represents a fast and useful method to determine CHIKV neutralizing antibodies without the need of using infectious CHIKV.

Figure 1

CHIKV VRP system. (A) Schematic presentation of the CHIKV replicon expressing Gluc marker and the CHIKV helper-C and helper-E RNAs. Lines represent non-translated regions and boxes represent translated regions, whereas white boxes indicate nonstructural proteins and gray boxes indicate structural proteins. The Gluc reporter is represented as black box. In the helper constructs 5’ terminal nucleotides of the nsP1 gene important for RNA replication were retained. Arrows indicate the position of the subgenomic promoter. The solid black circle at the 5’ end of each RNA represents the CAP structure; (A)_n indicates the poly(A) tail. (B) Kinetics of Gluc secretion after electroporation of CHIKV replicon expressing Gluc. In vitro-synthesized replicon RNA was electroporated into BHK cells and release of Gluc into the supernatant was measured at the indicated time points.

Figure 2

Optimum of VRP production. (A) In vitro-synthesized Gluc replicon RNA was coelectroporated with in vitro-synthesized helper-C and helper-E RNA and cells were either incubated at 32°C or 37°C. Supernatants were harvested at the indicated time points after electroporation and Gluc activity was measured in RLUs. (B) Same volumes of supernatants harvested at different time points from the different incubation temperatures in (A) were used to infect fresh BHK cells at 37°C. At 6 h post infection, Gluc activity was measured in the supernatant.

Figure 3

VRP based NT assay using a monoclonal antibody. Assays were performed in either 24- or 96-well format as indicated. Neutralizing monoclonal antibody directed against CHIKV E2 protein was serially diluted and preincubated with VRPs corresponding to an MOI of 5, 0.5 or 0.05, respectively. A monoclonal antibody against CHIKV capsid protein was used as control (Neg. mAb). Preincubated samples were used to infect the BHK cells in the 24- or 96-well plates and readout via Gluc secreted into the supernatant was performed at 6 or 24 h post infection as indicated. The bar labeled with VRP represents infection with the appropriate amount of VRPs not preincubated with monoclonal antibody. The bar labeled with Mock represents the background measured in untreated (non-infected) BHK cells. The % infectivity was normalized to VRP infection without monoclonal antibody incubation. Data represent means and standard deviation of experiments performed in triplicate.

Figure 4

VRP based NT assay using patient serum. Assays were performed in either 24- or 96-well format as indicated. Human serum from a CHIKV patient was serially diluted and preincubated with VRPs corresponding to an MOI of 5, 0.5 or 0.05, respectively. Preincubated samples were used to infect the BHK cells in the 24- or 96-well plates and readout via Gluc secreted into the supernatant was performed at 6 or 24 h post infection as indicated. The bar labeled with VRP represents infection with the appropriate amount of VRPs not preincubated with patient sera. The bar labeled with Mock represents the background measured in untreated (non-infected) BHK cells. Negative (Neg.) serum from a person not infected with CHIKV was used as control. The % infectivity was normalized to VRP infection without serum incubation. Data represent means and standard deviation of experiments performed in triplicate.

Figure 5

Comparison of plaque neutralization and VRP based NT assays. (A) CHIKV infected and non-infected (mock) BHK cells were used for indirect immunofluorescence analyses using the indicated monoclonal antibody or human sera. Nuclei were stained with DAPI. The bar represents 25 μm. (B) For the plaque neutralization assay, serial dilutions of monoclonal antibody D3.62 or three patient sera were preincubated with infectious CHIKV. NT assays were performed in 6-well format with readout at 48 h p.i. using crystal violet staining. The bar labeled with Virus represents a non-neutralized infectivity control, which was set 100%. The % infectivity was normalized to the non-neutralized virus infection. The gray dotted line indicates the 50% infectivity threshold. Data represent the means and ranges of duplicate infection experiments. (C) For the VRP based NT assay, serial dilutions of monoclonal antibody D3.62 or three patient sera were preincubated with VRPs. NT assays were performed in either 24-well plates (top panel) or 96-well plates (bottom panel) using VRPs at an MOI of 5. Readout was performed at 6 h p.i. via measurement of Gluc secreted into the supernatant. The bar labeled with VRP represents infection with the appropriate amount of VRPs not preincubated with patient sera/antibody. Negative (Neg.) serum from a person not infected with CHIKV was used as control. The % infectivity was normalized to VRP infection without serum/antibody incubation. The gray dotted line indicates the 50% infectivity threshold. Data represent average and standard deviation of experiments performed in triplicate.