| Abstract: | All lyssaviruses cause fatal encephalitis in mammals. There is sufficient antigenic variation within the genus to cause variable vaccine efficacy, but this variation is difficult to characterize quantitatively: sequence analysis cannot yet provide detailed antigenic information, and antigenic neutralization data have been refractory to high-resolution robust interpretation. Here, we address these issues by using state-of-the-art antigenic analyses to generate a high-resolution antigenic map of a global panel of 25 lyssaviruses. We compared the calculated antigenic distances with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from the glycoprotein amino acid sequence, there are in some cases substantial differences between genetic and antigenic distances, thus highlighting the risk of inferring antigenic relationships solely from sequence data at this time. These differences included epidemiologically important antigenic differences between vaccine strains and wild-type rabies viruses. Further, we quantitatively assessed the antigenic relationships measured by using rabbit, mouse, and human sera, validating the use of nonhuman experimental animals as a model for determining antigenic variation in humans. The use of passive immune globulin is a crucial component of rabies postexposure prophylaxis, and here we also show that it is possible to predict the reactivity of immune globulin against divergent lyssaviruses.Rabies remains a globally important zoonosis, despite being one of the oldest recognized infectious diseases (27, 55). The majority of rabies in terrestrial animals and humans is caused by classical rabies virus (RABV), a lyssavirus in the family Rhabdoviridae. Since the 1950s, many related lyssaviruses which are capable of causing clinical rabies have been identified. The majority of those viruses have been isolated from bats (Chiroptera), including four divergent viruses, which were isolated in separate geographic locations throughout Eurasia in the past 18 years (2, 29, 31). The Chiroptera, therefore, represent a global reservoir for lyssaviruses, creating the potential for “spillover” infection to terrestrial mammals, including humans. Occasionally transmission between members of a new host species will occur, with potential for a subsequent adaptation in that species (35). Phylogenetic evidence suggests that one or more host-switching events from bats into terrestrial mammals were originally responsible for the ongoing global epidemic of terrestrial RABV (6).Pre- or postexposure prophylaxis, using vaccination and passive immune globulin administration according to World Health Organization (WHO) guidelines, is currently the only effective way to prevent rabies after infection with a lyssavirus (1). The efficacy of both active and passive immunization is likely to be affected by antigenic differences between viruses. The lyssavirus trimeric glycoprotein is the primary surface antigen, the major target for neutralizing antibodies (8), and is involved in cell binding and entry (34, 36, 53). Antigenic sites on the glycoprotein have been described using monoclonal antibody escape mutants (8, 16, 47, 51). These studies have elucidated two major sites (sites II and III) and multiple minor sites. Although estimates of antigenic differences can be made using information regarding these known antigenic sites, protein structure, and amino acid properties, predictions of the relative importance of those sites and specific mutations within those sites cannot be quantitatively tested without a method to reliably measure antigenic effect.The use of serological cross neutralization data to measure antigenic difference is limited by the reliability of the serological test and, more importantly, by paradoxes, or irregularities in the data. These irregularities include higher heterologous than homologous titers and individual variations between sera raised against the same antigen (22, 52). Hence, serological data are considered to have low resolution, and they are often used only qualitatively. Despite these difficulties, studies have attempted to further quantify antigenic differences among lyssaviruses. Badrane et al. (5) showed a linear correlation between the glycoprotein amino acid identity and four cross neutralization titers. Other studies have demonstrated variable serological cross-reactivity between European bat lyssaviruses (EBLV) and RABVs (10, 11) and suggested that antigenic relationships between EBLV-1 and EBLV-2 may not be fully reflected in the genetic relationships (41). Recent investigations into the efficacy of biologics against the Eurasian lyssaviruses showed an array of relatedness between lyssavirus species, with, for example, a murine anti-Aravan virus (anti-ARAV) serum neutralizing Khujand virus (KHUV) and ARAV equally but an anti-KHUV serum being less effective at neutralizing ARAV than KHUV (22). Until recently, however, there were no established tools for the quantitative analysis of antigenic data.Here we resolve the issue of quantitative interpretation of antigenic data using antigenic cartography. Antigenic cartography is a theory and associated computational method that resolves the paradoxes in the interpretation of antigenic data and makes possible high-resolution quantitative analyses and visualizations of binding assay data (15, 20, 25, 44, 49, 52).Integrating antigenic data with direct sequencing data, here we quantify the antigenic and genetic variation among a global panel of lyssaviruses, including representatives from all lyssavirus species. Furthermore, we address two key issues in the development of antilyssavirus biologics: the appropriateness of animal models and the development of efficacious alternatives to human rabies immune globulins (HRIGs). |
