Development of broad‐spectrum human monoclonal antibodies for rabies post‐exposure prophylaxis
2016; Springer Nature; Volume: 8; Issue: 4 Linguagem: Inglês
10.15252/emmm.201505986
ISSN1757-4684
AutoresPaola De Benedictis, Andrea Minola, E. Rota Nodari, Roberta Aiello, Barbara Zecchin, Angela Salomoni, Mathilde Foglierini, Gloria Agatic, Fabrizia Vanzetta, Rachel Lavenir, Anthony Lepelletier, Emma M. Bentley, Robin A. Weiss, Giovanni Cattoli, Ilaria Capua, Federica Sallusto, Edward Wright, Antonio Lanzavecchia, Hervé Bourhy, Davide Corti,
Tópico(s)Viral Infections and Outbreaks Research
ResumoResearch Article18 March 2016Open Access Source DataTransparent process Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis Paola De Benedictis Paola De Benedictis FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Andrea Minola Andrea Minola Humabs BioMed SA, Bellinzona, Switzerland Search for more papers by this author Elena Rota Nodari Elena Rota Nodari FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Roberta Aiello Roberta Aiello FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Barbara Zecchin Barbara Zecchin FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Angela Salomoni Angela Salomoni FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Mathilde Foglierini Mathilde Foglierini Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Search for more papers by this author Gloria Agatic Gloria Agatic Humabs BioMed SA, Bellinzona, Switzerland Search for more papers by this author Fabrizia Vanzetta Fabrizia Vanzetta Humabs BioMed SA, Bellinzona, Switzerland Search for more papers by this author Rachel Lavenir Rachel Lavenir Institut Pasteur, Unit of Lyssavirus Dynamics and Host Adaptation, National Reference Centre for Rabies, World Health Organization Collaborating Centre for Reference and Research on Rabies, Paris Cedex 15, France Search for more papers by this author Anthony Lepelletier Anthony Lepelletier Institut Pasteur, Unit of Lyssavirus Dynamics and Host Adaptation, National Reference Centre for Rabies, World Health Organization Collaborating Centre for Reference and Research on Rabies, Paris Cedex 15, France Search for more papers by this author Emma Bentley Emma Bentley Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster, London, UK Search for more papers by this author Robin Weiss Robin Weiss Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Giovanni Cattoli Giovanni Cattoli FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Ilaria Capua Ilaria Capua FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Federica Sallusto Federica Sallusto Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Search for more papers by this author Edward Wright Edward Wright Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster, London, UK Search for more papers by this author Antonio Lanzavecchia Antonio Lanzavecchia Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Institute of Microbiology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Hervé Bourhy Hervé Bourhy Institut Pasteur, Unit of Lyssavirus Dynamics and Host Adaptation, National Reference Centre for Rabies, World Health Organization Collaborating Centre for Reference and Research on Rabies, Paris Cedex 15, France Search for more papers by this author Davide Corti Corresponding Author Davide Corti orcid.org/0000-0003-4046-7222 Humabs BioMed SA, Bellinzona, Switzerland Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Search for more papers by this author Paola De Benedictis Paola De Benedictis FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Andrea Minola Andrea Minola Humabs BioMed SA, Bellinzona, Switzerland Search for more papers by this author Elena Rota Nodari Elena Rota Nodari FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Roberta Aiello Roberta Aiello FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Barbara Zecchin Barbara Zecchin FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Angela Salomoni Angela Salomoni FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Mathilde Foglierini Mathilde Foglierini Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Search for more papers by this author Gloria Agatic Gloria Agatic Humabs BioMed SA, Bellinzona, Switzerland Search for more papers by this author Fabrizia Vanzetta Fabrizia Vanzetta Humabs BioMed SA, Bellinzona, Switzerland Search for more papers by this author Rachel Lavenir Rachel Lavenir Institut Pasteur, Unit of Lyssavirus Dynamics and Host Adaptation, National Reference Centre for Rabies, World Health Organization Collaborating Centre for Reference and Research on Rabies, Paris Cedex 15, France Search for more papers by this author Anthony Lepelletier Anthony Lepelletier Institut Pasteur, Unit of Lyssavirus Dynamics and Host Adaptation, National Reference Centre for Rabies, World Health Organization Collaborating Centre for Reference and Research on Rabies, Paris Cedex 15, France Search for more papers by this author Emma Bentley Emma Bentley Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster, London, UK Search for more papers by this author Robin Weiss Robin Weiss Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Giovanni Cattoli Giovanni Cattoli FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Ilaria Capua Ilaria Capua FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy Search for more papers by this author Federica Sallusto Federica Sallusto Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Search for more papers by this author Edward Wright Edward Wright Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster, London, UK Search for more papers by this author Antonio Lanzavecchia Antonio Lanzavecchia Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Institute of Microbiology, ETH Zurich, Zurich, Switzerland Search for more papers by this author Hervé Bourhy Hervé Bourhy Institut Pasteur, Unit of Lyssavirus Dynamics and Host Adaptation, National Reference Centre for Rabies, World Health Organization Collaborating Centre for Reference and Research on Rabies, Paris Cedex 15, France Search for more papers by this author Davide Corti Corresponding Author Davide Corti orcid.org/0000-0003-4046-7222 Humabs BioMed SA, Bellinzona, Switzerland Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland Search for more papers by this author Author Information Paola De Benedictis1,‡, Andrea Minola2,‡, Elena Rota Nodari1, Roberta Aiello1, Barbara Zecchin1, Angela Salomoni1, Mathilde Foglierini3, Gloria Agatic2, Fabrizia Vanzetta2, Rachel Lavenir4, Anthony Lepelletier4, Emma Bentley5, Robin Weiss6, Giovanni Cattoli1, Ilaria Capua1, Federica Sallusto3, Edward Wright5, Antonio Lanzavecchia3,7, Hervé Bourhy4 and Davide Corti 2,3 1FAO and National Reference Centre for Rabies, National Reference Centre and OIE Collaborating Centre for Diseases at the Animal-Human Interface, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padua, Italy 2Humabs BioMed SA, Bellinzona, Switzerland 3Institute for Research in Biomedicine, Università della Svizzera Italiana, Bellinzona, Switzerland 4Institut Pasteur, Unit of Lyssavirus Dynamics and Host Adaptation, National Reference Centre for Rabies, World Health Organization Collaborating Centre for Reference and Research on Rabies, Paris Cedex 15, France 5Viral Pseudotype Unit, Faculty of Science and Technology, University of Westminster, London, UK 6Division of Infection and Immunity, University College London, London, UK 7Institute of Microbiology, ETH Zurich, Zurich, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41 91 825 63 80; E-mail: [email protected] EMBO Mol Med (2016)8:407-421https://doi.org/10.15252/emmm.201505986 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20–RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. Synopsis Post-exposure prophylaxis of rabies virus infections relies on vaccination and local administration of expensive human or equine rabies immunoglobulins. Their limited supply, however, restricts access in endemic areas. Two novel human broadly neutralizing monoclonal antibodies offer hope. The replacement of rabies immunoglobulins (RIGs) with monoclonal antibody-based post-exposure prophylaxis (PEP) must be based on the identification of neutralizing monoclonal antibodies (mAbs) able to recognize G protein sequences of RABV from all lineages. According to WHO recommendations, the best approach to replace RIGs is to develop a cocktail of mAbs recognizing distinct antigenic sites to reduce the risk of PEP failure. RVC20 and RVC58 human mAbs were selected to recognize different antigenic sites and to broadly and potently neutralize multiple lineages of RABVs and non-RABV lyssavirus species. These two mAbs could be produced cost-effectively and in large quantities to be made available at affordable prices in low-income countries. Introduction Rabies virus (RABV) belongs to the family Rhabdoviridae, genus Lyssavirus, and causes acute encephalitis in mammals. Lyssaviruses are enveloped single-stranded (-) RNA viruses which have helical symmetry and display on the outer surface of the virion envelope the G protein, which is the target antigen of virus-neutralizing antibodies. RABV is the first of fourteen lyssavirus species identified to date (Dietzgen et al, 2011), with an additional yet unclassified putative species named Lleida bat lyssavirus (LLEBV) (Arechiga Ceballos et al, 2013). According to their viral genetic distances, two major phylogroups have been defined: Phylogroup I includes the species RABV, European bat lyssavirus type 1 (EBLV-1) and type 2 (EBLV-2), Duvenhage virus (DUVV), Australian bat lyssavirus (ABLV), Aravan virus (ARAV), Khujand virus (KHUV), Bokeloh bat lyssavirus (BBLV), and Irkut virus (IRKV); Phylogroup II includes Lagos bat virus (LBV), Mokola virus (MOKV), and Shimoni bat virus (SHIBV). The remaining viruses, West Caucasian bat virus (WCBV) and Ikoma lyssavirus, (IKOV) cannot be included in either of these phylogroups and have been temporarily assigned to putative phylogroups III and IV, respectively (Bourhy et al, 1992, 1993; Amengual et al, 1997; Hooper et al, 1997; Badrane et al, 2001; Kuzmin et al, 2010; Marston et al, 2012). It is currently thought that infection with all lyssavirus species culminates in viral encephalitis clinically indistinguishable from that caused by RABV and ultimately results in human and animal deaths. RABV is found almost ubiquitously worldwide in different animal reservoirs, with occasional spillover events to non-reservoir hosts, including humans. Although almost 100% fatal following the onset of symptoms, rabies can be controlled in the animal reservoirs through mass vaccination and prevented through the appropriate prophylactic treatment in humans exposed to the virus. Approximately 17 million people per year are treated after exposure to rabies, in most cases following a bite from an infected animal. Some 59,000 people are estimated to die each year, mainly in Africa, China, and India, and 50% of rabies cases worldwide occur in children (Fooks et al, 2014; Hampson et al, 2015). However, the true burden of rabies-related lyssaviruses in developing countries is unknown and largely under-diagnosed (Mallewa et al, 2007). In humans, rabies prevention is achieved by either pre- or post-exposure prophylaxis. If exposed to RABV, post-exposure prophylaxis (PEP) is recommended to prevent the advancement of infection and thus the clinical disease; however, it must be administered as early as possible. According to the World Health Organization (WHO) (World Health Organization. 2013), PEP includes the first-aid treatment of the wound and the administration of the rabies vaccine alone or in combination with rabies immunoglobulin (RIG) for category II or III exposures, respectively. In particular, patients with category III exposures should receive RIG administered into or around the wound site and four to five doses of vaccine. Two types of RIG are currently available for PEP: human or equine rabies immunoglobulin (HRIG and ERIG, respectively). The dose of HRIG recommended by the WHO is 20 IU/kg body weight (corresponding to 20 mg/kg); for ERIG and F(ab′)2 products, the recommended dose is 40 IU/kg body weight. Higher doses of RIGs have been shown to reduce vaccine efficacy (Atanasiu et al, 1956, 1961, 1967; Archer & Dierks, 1968; Sikes, 1969; Cabasso et al, 1971, 1974; Wiktor et al, 1971; Cabasso, 1974). HRIG is widely used in developed countries and considered safer than ERIG. The high cost of HRIG and its limited availability hamper its wide use in resource-limited countries, particularly in Africa (Dodet and Africa Rabies Bureau (AfroREB) 2009). Moreover, vaccine and HRIG or ERIG do not confer protection against infection with all non-RABV lyssavirus species, and protection is thought to be inversely related to the genetic distance with the RABV vaccine strain (Brookes et al, 2005; Hanlon et al, 2005; Both et al, 2012). Thus, a search for a replacement to HRIG has been strongly encouraged by the WHO (World Health Organization, 2013). To this end, mouse and human monoclonal antibodies have been developed in the last decade, with two products in advanced clinical trials, namely CL184 (produced by Crucell, based on the combination of two antibodies called CR57 and CR4098, Bakker et al, 2005; Goudsmit et al, 2006) and RAB1 (produced by Mass Biologics and Serum Institute of India, based on a single monoclonal antibody, Sloan et al, 2007; Nagarajan et al, 2014). However, RABV isolates that are not neutralized by each of these monoclonal antibodies have been identified (Marissen et al, 2005; Kuzimina et al, 2013). These findings highlight the challenge to perform Phase 2 or 3 trials where the risk of monoclonal antibody-based PEP failures poses a serious ethical concerns. Indeed, for the lack of broad RABV coverage, the development of CL184 was recently halted, while RAB1 in still under Phase 2 or 3 development in India. Thus, in the selection and development of a safe and effective monoclonal antibody-based PEP of RABV infections, it is of paramount importance to identify neutralizing monoclonal antibodies that are able to recognize G protein sequences of RABV from all lineages. As previously described for other viral targets (Corti & Lanzavecchia, 2013), the combination of two antibodies that bind to different antigenic sites on the RABV G protein and are able to broadly neutralize both RABV and non-RABV lyssavirus isolates will significantly reduce the risk of PEP failure. Results Selection of rabies vaccinees and isolation of potent RABV-neutralizing antibodies In order to isolate broadly neutralizing antibodies against not only RABV isolates but also non-RABV lyssaviruses, sera from 90 RABV vaccinees were screened for the presence of high titers of antibodies that bind to the RABV (CVS-11 isolate) G protein by ELISA (Fig 1A) Of these, the 29 with the highest binding titers (ED50 > 50) were tested for their ability to neutralize a panel of 12 pseudotyped lyssaviruses representing RABV and non-RABV lyssaviruses isolates of phylogroup I, II, and III viruses (Fig 1B and Appendix Table S1). HRIG Berirab® was included as a reference. As expected, all samples neutralized, albeit with variable titers, the CVS-11 isolate (RABV). The neutralization profile of the other lyssavirus species varied considerably in all donors tested, but in a few cases, all species were neutralized. It was interesting to note that HRIG showed only modest activity against non-RABV phylogroup I species and no cross-reactivity with phylogroup II and III viruses. Figure 1. Selection of RABV vaccinees with broadly reactive neutralizing antibodies A panel of 90 sera were tested by ELISA for binding to RABV G protein. Shown are the 1/ED50 values. Sera samples selected for high binding titers (1/ED50 > 50) values were tested for the presence of neutralizing antibodies against a panel of 12 pseudotyped viruses. 1/ID50 values are shown. Black circles indicate HRIG (Berirab®), and colored circles indicate the four donors selected for the memory B-cell interrogation. Download figure Download PowerPoint Memory B cells from four vaccinees selected for the presence of serum antibodies capable of broadly neutralizing multiple lyssavirus species were immortalized with Epstein–Barr virus (EBV) and CpG, as previously described (Traggiai et al, 2004). Culture supernatants were then tested using a 384-well-based RABV (CVS-11) pseudotyped neutralization assay on BHK-21 cells. Five hundred human monoclonal antibodies were isolated for their ability to neutralize pseudotyped CVS-11 RABV. Twenty-one human monoclonal antibodies were selected for their high neutralizing potency against CVS-11 RABV pseudotyped virus, with IC90 (concentration of antibody neutralizing 90% of viral infectivity) ranging from 0.01 to 317 ng/ml (Appendix Table S2). These antibodies used different VH and VL genes, with a slight bias toward VH3 and VH4, carried heavy chain complementarity-determining region 3 (H-CDR3) of different lengths (11–21), and had a variable load of somatic mutations (Appendix Table S2). HRIG and three other human monoclonal antibodies in clinical development (CR57, CR4098, and RAB1) were used as a reference. As expected, all antibodies bound to the CVS-11 RABV G protein by ELISA. In order to understand whether the cognate epitope is conformational or not, RABV G protein was run on a SDS–PAGE gel under reducing or non-reducing conditions and probed by Western blot with all the isolated human monoclonal antibodies. With a few exceptions (RVB143, RVC44, and RVC68), all antibodies did not bind to RABV G protein under reducing conditions, thus suggesting that the epitopes recognized, in most cases, are conformational (Appendix Table S2). Antibody competition studies: determination of antigenic sites on RABV G protein Competition studies were then performed to determine the spatial proximity of each of the conformational epitopes recognized by the selected neutralizing monoclonal antibodies. The two reference antibodies CR57 and CR4098 were previously shown to recognize G protein antigenic sites I and III, respectively (Marissen et al, 2005; de Kruif et al, 2007), and were therefore used in this assay as probes to map the specificity of the other antibodies. Results shown in Fig 2 were used to cluster the 21 tested antibodies into 6 groups. Figure 2. RABV G protein antigenic site mapping using cross-competition ELISA-based binding studiesMonoclonal antibody cross-competition matrix performed by ELISA on the 21 isolated antibodies and two reference antibodies of known epitope specificity (CR57 and CR4098). The percentage of binding inhibition of the biotinylated antibodies (upper row) by the unlabeled antibodies listed in the left column is shown. Results are classified using color shading codes with values ≥ 80% in orange, < 80% and ≥ 50% in yellow, < −100% in light blue, and no shading for values < 50%. Download figure Download PowerPoint RVA125, RVC3, RVC20, and RVD74 antibodies were assigned to the antigenic site I group, according to the competition with CR57 and their reciprocal competitions. Interestingly, the binding of antigenic site I antibodies to G protein enhanced the binding by several non-antigenic site I antibodies. RVA122, RVA144, RVB492, RVC4, RVC69, RVC38, and RVC58 were assigned to the antigenic site III group, according to the competition with CR4098 and their reciprocal competitions. RVC58 showed only a partial competition with CR4098 (64%) and with antibodies that bind to sites different from site III, or I, suggesting that the RVC58 antibody recognizes a yet undefined epitope that only partially overlaps with the one recognized by CR4098. The binding of RVB181, RVC56, RVB185, RVC21, RVB161, and RVC111 was blocked by antigenic site III antibodies, but reciprocally, these antibodies did not block binding of several other antigenic site III antibodies, such as CR4098, RVC4, and RVC69. In interpreting competition results, it should be taken into account that when two epitopes overlap, or even when the areas covered by the arms of the two antibodies overlap, competition should be almost complete and mutually cross-competitive. Thus, only marked mutual cross-competition should be taken as unequivocal evidence of overlapping epitopes, since weak or one-way inhibition may simply reflect a decreased affinity due to steric or allosteric effects. Thus, the latter results suggest that these antibodies form a third cluster that recognizes a distinct, hereafter dubbed III.2, antigenic site. Three additional sites were further defined and named A, B, and C. Site A is defined by the unique antibody RVB686, whose binding compromises the binding of the majority of the labeled antibodies in the panel, but reciprocally the binding of the labeled RBV686 is not blocked by any antibody in the panel. These results might suggest that RVB686 binding induces an allosteric effect on the G protein that compromises the binding of most other antibodies. Site B is defined by antibody RVC44, whose binding is not blocked by any other antibody in the panel. Similarly, site C is defined by antibodies RVB143 and RVC68, which also recognize a unique and distinct site as compared to all the other antibodies. Identification of broad-spectrum lyssavirus-neutralizing antibodies Twelve of the 21 antibodies were selected for testing based on their neutralizing potency and recognition of distinct sites on the RABV G protein. In addition, CR57, CR4098, RAB1, and Berirab® (HRIG) were included for testing against a large panel of lyssaviruses using pseudotyped (N = 22) and infectious viruses (N = 16) covering RABV, LBV, MOKV, DUVV, EBLV-1, EBLV-2, ABLV, IRKV, KHUV, ARAV, SHIBV, BBLV, IKOV, and WCBV species (Fig 3A and B) (all viruses that neutralized with an IC50 or IC90 < 10,000 ng/ml were scored as positive). Figure 3. Neutralization of lyssaviruses by human monoclonal antibodiesA selection of 12 human monoclonal antibodies, three reference antibodies (CR57, CR4098, and RAB1) and the polyclonal human immunoglobulins (HRIG, Berirab®) were tested for their neutralizing activity against 13 different lyssavirus species using pseudotyped viruses or live viruses. Complete viral strain designations are shown in Appendix Table S4. Results of neutralization assays using 22 pseudotyped viruses expressed as inhibitory concentration 90 (IC90). Results of neutralization assays using 15 live viruses expressed as inhibitory concentration 50 (IC50). Summary of the percentage of non-RABV lyssavirus isolates and phylogroup I non-RABV lyssavirus isolates neutralized with IC50 (for viruses) or IC90 (for pseudotyped viruses) below 10,000 ng/ml for RVC20, RVC58, CR57, CR4098, and RAB1 monoclonal antibodies, HRIG or a combination of RVC20 with RVC58 or CR57 with CR4098. Color coding indicates the potency, with IC90 (for pseudotyped viruses) or IC50 (for viruses) < 100 ng/ml in red shading, 100 ng/ml < IC50 < 1,000 ng/ml in orange shading, and IC50 ≥ 1,000 ng/ml in yellow shading. IC50 > 10,000 ng/ml were scored as negative. *HRIG was scored as negative when IC50 or IC90 was > 100,000 ng/ml; **RAB1 was tested against 20 pseudotyped viruses and 9 viruses. Download figure Download PowerPoint Among the antigenic site I antibodies tested in the pseudotyped neutralization assay (Wright et al, 2008, 2009), RVC20 showed the largest breadth of reactivity being able to neutralize all phylogroup I viruses tested as well as SHIBV from phylogroup II and IKOV from putative phylogroup IV (Fig 3A). As a comparison, the antigenic site I antibody CR57 did not neutralize EBLV-1, SHIBV, and IKOV isolates. When tested on infectious viruses (Cliquet et al, 1998; Warrell et al, 2008), RVC20 broadly neutralized most of the RABV, DUVV, EBLV-1, EBLV-2, ABLV, and BBLV isolates tested as well as the phylogroup II MOKV (Fig 3B). In the same analysis, CR57 did not neutralize EBLV-1 and MOKV isolates. Among the antigenic site III antibodies tested in the pseudotyped neutralization assay, RVC58 potently neutralized all phylogroup I viruses with an IC90 of < 10 ng/ml. As a comparison, the antigenic site III antibodies CR4098 and RAB1 were less broad and potent and were unable to neutralize most of the non-RABV tested. When tested on infectious viruses, RVC58 potently neutralized all phylogroup I viruses tested. In the same analysis, CR4098 and RAB1 showed a limited breadth of neutralization. Of note, antibody RVC68 neutralized all phylogroups I and II pseudotyped viruses tested (only WCBV was not neutralized), although with IC90 values 10- to 100-fold higher than compared to RVC20 and RVC58 (Fig 3A). When tested on infectious viruses, antibody RVC68 was, however, not able to effectively (i.e. IC50 < 10,000 ng/ml) neutralize all phylogroups I and II isolates tested (Fig 3B). Limiting the analysis of antibody breath to non-RABV lyssaviruses, RVC58 (antigenic site III) was able to neutralize 68% of all non-RABV lyssaviruses tested and, remarkably, all the phylogroup I non-RABV lyssaviruses tested (Fig 3C). In comparison, antibody CR4098 and RAB1 neutralized only 19 and 18% of the non-RABV lyssaviruses and 24 and 31% of phylogroup I non-RABV lyssaviruses, respectively. Further analysis showed that RVC20 (antigenic site I) was able to neutralize 74 and 95% of the non-RABV lyssaviruses and phylogroup I non-RABV lyssaviruses, res
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