ROTADIAL: The first nanobody-based immunoassay to detect Group A Rotavirus

Highlights

• Group A Rotavirus is the pathogen most frequently associated with severe cases of diarrhea in children worldwide.

• ROTADIAL is a rapid nanobody-based ELISA assay able to identify Group A Rotavirus in feces from pediatric patients.

• ROTADIAL recognized all human Group A Rotavirus strains tested.

• ROTADIAL showed excellent diagnostic and analytical performance.

• ROTADIAL is systematically used by the Argentine Laboratory-based Surveillance Network for Viral Gastroenteritis since 2017.

Abstract

ROTADIAL is a rapid nanobody (Nb)-based ELISA assay able to identify Rotavirus group A (RVA) in feces from pediatric patients. The assay is based on a sandwich of two patented llama-derived Nbs directed to the inner capsid viral protein VP6 from RVA. Nbs are directed to conformational epitopes of VP6 and recognized all human RVA strains tested, representing ideal reagents for their use in immunodiagnostic tests for RVA detection. All the steps are carried out at room temperature, bringing results in less than two hours. This assay, named ROTADIAL, was validated with a reference panel of feces from pediatric patients from Argentina. The overall sensitivity and specificity of the ROTADIAL test, when compared to a commercial test, was 100 % (100/100) and 99 % (99/100) respectively. ROTADIAL presented optimal analytical performance, being capable of detecting RVA regardless of the presence of other common human enteric infectious agents and is the first RVA-diagnostic assay developed using Nbs, worldwide.

Introduction

Diarrhea is the second most important cause of childhood death worldwide and Rotavirus group A (RVA) is the pathogen most frequently associated with severe cases (Burnett et al., 2017; Lanata et al., 2013). Before the vaccine era, RVA infected nearly every child by the age of 3–5 years (World Health Organization, 2013). This virus was associated with one-third of the estimated 578,000 deaths from childhood gastroenteritis, more than two million hospitalizations, and 25 million outpatient clinic visits among children <5 years of age each year (Parashar et al., 2015). Because of the tremendous impact of this viral disease, the prevention of RVA is a priority for global health agencies. Several virus-attenuated vaccines were licensed in the last years (Burnett et al., 2020; Soares-Weiser et al., 2019). Vaccines showed high efficacy against severe RVA gastroenteritis in Europe and the Americas, even conferring good protection against disease caused by RVA strains different from the ones included in the vaccines (Burnett et al., 2020; Burnett et al., 2017).

Diagnostic assays are critical to assess the impact of RVA vaccine implementation and to monitor viral circulation among children. Typically, RVA is detected mainly by assays directed to viral structural protein 6 (VP6), as it is highly conserved (around 87–92 %) among mammalian RVA strains, very immunogenic and abundant (Desselberger, 2014; Mathieu et al., 2001; Tang et al., 1997). The VP6 is the only protein present in the middle layer of Rotavirus and has 397 amino acids, representing a molecular mass of approximately 45 kDa (Pesavento et al., 2006). Also, it is used to classify these viruses in tentative 10 species, from A to J (Lestari et al., 2020). These characteristics make VP6 preferable to be used as a comprehensive diagnostic component on enzyme-linked immunosorbent assays (ELISA), which are considered the most suitable methods based on antigen detection in stool material for RVA diagnosis in acute diarrhea cases (Masoudi et al., 2020; Soltan et al., 2020; Who, 2009). These assays should be reliable, easy to perform, capable of detecting all RVA strains, and economically affordable for all levels of healthcare (Memon et al., 2017). Acute diarrhea cases related to RVA are still a major concern for pediatricians considering their impact on dehydration and electrolyte imbalance. Thus, to ensure an optimal point-of-care test, these assays must be also simple and fast so that children can be properly and timely treated. This way, the unnecessary use of antibiotics can be avoided and potential outbreaks can be prevented.

We have developed nanobodies (Nb) to RVA VP6 protein (Garaicoechea et al., 2008). A Nb or VHH antibody is the single antigen-binding domain derived from heavy chain-antibodies present in all types of camelids. These Nbs are produced through recombination and employed as research tools or for diagnostic and therapeutic applications. They are robust, monomeric, and easy to tailor into more complex entities to meet the requirements of their application (Muyldermans, 2020). We have shown that these Nbs are broadly reactive reagents that can be engineered for their use in immunodiagnostic tests for RVA detection (Garaicoechea et al., 2015; Maffey et al., 2016; Vega et al., 2013). This study aimed to develop a sensitive and specific antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) to detect RVA in human feces. In this study, we developed a rapid Nb-based ELISA assay able to identify RVA in feces from pediatric patients, named ROTADIAL, that was subject to validation and interlaboratory proficiency testing. This assay is based on a sandwich of two different patented llama-derived Nbs directed to the inner capsid viral protein VP6 (Garaicoechea et al., 2008) that is performed at room temperature with optimal analytical performance. Also, it represents the first RVA-diagnostic assay developed using Nbs worldwide.