Development and qualification of a fast, high-throughput and robust imaging-based neutralization assay for respiratory syncytial virus

Abstract

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.

Introduction

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection among infants and young children (Nair et al., 2010; Meng et al., 2014). RSV can also cause repeated infections throughout life, especially among the elderly and immunocompromised patients (Falsey et al., 2005; Glezen et al., 1986; Hall et al., 1991). At present, there is no vaccine licensed for RSV to protect children, the elderly or immunocompromised individuals (Rudraraju et al., 2013). The challenges for an effective RSV vaccine include (1) gaps in understanding of immunological mechanism of protection in different target population; (2) lack of consensus regarding clinical endpoints; (3) transient protection by natural immunity and repeated infection (Rudraraju et al., 2013; Mazur et al., 2018; Ruckwardt et al., 2019). Despite these obstacles, recent advances in understanding of the structural biology of RSV Fusion (F) protein provided a new target for preventive vaccines and monoclonal antibodies (mAbs) against RSV (McLellan et al., 2010; Cohen, 2013; Ngwuta et al., 2015). Palivizumab (Synagis), a humanized Immunoglobulin G1 (IgG1) was approved by US Food and Drug Administration (FDA) in 1998 to confer RSV prophylaxis in selected high-risk infants (Boivin et al., 2008). Due to its high cost and multiple dosing logistics (5 doses to cover a RSV season), this drug is not always accessible in lower resource settings. A second generation mAb candidates with improved pharmacokinetics (PK), pharmacodynamics (PD) and/or lower cost are needed. The development of an effective RSV vaccine or a better therapeutic antibody is still an important unmet medical need and an area of active research.

Neutralizing antibody titer measurement is a critical component of vaccine immunogenicity study. The traditional method for testing neutralizing antibodies against RSV is the plaque reduction neutralization test (PRNT), which is laborious, highly variable, and has a slow-turnaround time (8 days). Tremendous efforts have been taken to simplify or shorten plaque assay procedures (McKimm-Breschkin, 2004; Kim et al., 2017; Wen et al., 2019). A rapid, simple and accurate immunoplaque assay was developed using antibodies and diaminobenzidine (DAB) to visualize the plaques at two days post infection (Kim et al., 2017). Other studies have developed microneutralization assays, which measure enzymatic activity as the endpoint, usually at three days post infection (Ellis et al., 1995; Cheng et al., 2002). A quantitative polymerase chain reaction (qPCR) assay was developed to detect RSV neutralizing antibody as early as 24 h post infection (Varada et al., 2013). This approach is not widely used because it detects viral nucleic acid as the endpoint, which does not differentiate replicating virus from non-replicating virus. In addition, the process of nucleic acid extraction and purification is labor-intensive and expensive. RSV reporter viruses harboring a reporter gene such as luciferase or green fluorescence protein (GFP) were also evaluated in RSV neutralization assay (van Remmerden et al., 2012; Shambaugh et al., 2017; Fuentes et al., 2013; Phan et al., 2014) with attractive advantages such as simple and fast. However, it relies on a strain specific RSV reporter virus, which needs substantial time and resources to generate. In addition, to justify the use of a RSV reporter virus in vaccine clinical assay, a bridging study using wild type RSV and the RSV reporter virus may be needed to show the correlation of neutralization titers. Therefore, a simple and general neutralization assay platform which is easy to adapt to different serotypes of RSV is needed.

We recently developed a virus reduction neutralization test (VRNT) for Dengue virus (Whiteman et al., 2018), which uses an imaging cytometer to automatically count the number of virus infected cells at one day post infection in a 96-well plate format. In this study, we have described the development and qualification of a similar VRNT for RSV. Compared to PRNT, RSV VRNT is faster, higher throughput, robust and more precise, while retaining good correlation with PRNT titers. This method can test serum neutralization titers against multiple RSV strains and could also be applied to other viruses.