Detection and quantification of anti-rabies glycoprotein antibodies: current state and perspectives

Abstract

Rabies is an ancient fatal disease with no other available treatment than post-exposure vaccination, where the bite of infected animals, mainly dogs, is the leading cause of its transmission to human beings. In this context, global vaccination campaigns of companion animals, as well as wildlife reservoirs vaccination, are key factors to achieve the "Zero by 30" plan that pursues the eradication of dog-mediated human rabies by 2030. Rabies virus-neutralizing antibodies (VNAs) play an essential role in the disease protection, as it correlates with an adequate immune response and allows evaluating pre- or post-exposure prophylaxis efficacy. Hence, counting with reliable, accurate, and robust serological tests is of paramount importance. Currently, RFFIT and FAVN are the gold standard VNAs tests recommended by both the WHO and the OIE. Despite these methodologies are efficient and widely used, they present several drawbacks, as they are less easily to standardize and require the use of live rabies virus, containment facilities, and skilled professionals. Thus, in this review, we describe the state-of-the-art of alternative analytical methodologies currently available for rabies serology, with novel approaches based on pseudotyped recombinant viruses and emphasizing in the antigen binding methodologies that detect and quantify antibodies against the rabies glycoprotein. We discussed the wide range of assays that are interesting tools for a faster measurement of anti-rabies glycoprotein antibodies and, in some cases, less complex and more versatile than the gold standard methods. Finally, we discussed the key issues during the design and optimization steps of ELISA assays, highlighting the importance of validation and standardization procedures to improve rabies serology tests and, as a consequence, their results. KEY POINTS: • An exhaustive revision of rabies serology testing was made. • No rabies serology assay can be thought as better than others for all intents and purposes. • The validation procedure guarantees reliable and consistent results among the globe.

Keywords: Glycoprotein; Neutralizing antibodies; Rabies; Serology test.

Fig. 1

Rabies virus infection and the importance of pre-exposure prophylaxis (PREP) and post-exposure prophylaxis (PEP) in rabies control and prevention. Both PREP and PEP are mainly based on vaccination of individuals in order to induce an immune response where antibodies play a central role for the control of the infection. Besides, human rabies immunoglobulins (HRIGs) are frequently administered in PEP protocols

Fig. 2

Gold standard serum neutralization tests. A RFFIT. Previously diluted serum samples are mixed with a fix amount of RABV (CVS 11 strain) in 8-well chambers (1). Following the neutralization period, a suspension of BHK cells is added and incubated for 20–24 h (2). After a wash and fix step (3), FITC conjugated anti-RABV Abs are employed as the detection system (4). Results are assessed using a fluorescence microscope (5). The presence of fluorescence in the cells correlates with non-neutralized RABV (6). The ED₅₀ neutralization titer is defined as the dilution at which 50 % of the observed microscopic field contains one or more infected cells (7). B FAVN. Serially diluted serum samples are mixed with a fixed amount of RABV (CVS 11 strain) in 96-well microplates (1). Following the neutralization period, a suspension of BHK cells is added and incubated for 48 h (2). After a wash and fix step (3), FITC-conjugated anti-RABV Abs are employed as the detection system (4). Results are assessed using a fluorescence microscope (5). The well is considered negative if no signal is observed. By the contrary, if one or more fluorescent cells were observed, the well is considered as positive (6). Thereafter, the ED₅₀ neutralization titer is calculated (7)

Fig. 3

Schematic representation of a lentivirus pseudotyped assay, employing GFP as reporter gene. First, packaging cells are co-transfected with the lentiviral structural plasmids and with the transfer vector containing the reporter gene. A RABV glycoprotein expressing plasmid is used to pseudotype the recombinant viruses (1). Forty-eight hours post transfection, culture supernatants containing the lentivirus are harvested and titrated (2). Thereafter, a known amount of lentiviral particles are pre-incubated with diluted serum samples (3). Later, the mix is added to a target cell culture and incubated for 48 h (4). Finally, the GFP signal is measured by flow cytometry (5)

Fig. 4

A schematic representation of bELISA assay. First, the antigen is immobilized in 96-well plates (1). Second, Abs present in the serum sample recognizes an epitope of the target antigen (2). Third, a biotin-conjugated Ab interacts with the Ag not blocked by the Abs of the test sample (3). Fourth, a streptavidin-peroxidase conjugate interacts with the biotin of the conjugated Ab (4). The peroxidase reacts with a substrate to produce a detectable signal (5). Here, the higher the signal, the lesser the Ab amount in the test serum samples