A combination of two human monoclonal antibodies cures symptomatic rabies
2020; Springer Nature; Volume: 12; Issue: 11 Linguagem: Inglês
10.15252/emmm.202012628
ISSN1757-4684
AutoresGuilherme Dias de Melo, Florian Sonthonnax, Gabriel Lepousez, Grégory Jouvion, Andrea Minola, Fabrizia Zatta, Florence Larrous, Lauriane Kergoat, Camille Mazo, Carine Moigneu, Roberta Aiello, Angela Salomoni, Élise Brisebard, Paola De Benedictis, Davide Corti, Hervé Bourhy,
Tópico(s)Poxvirus research and outbreaks
ResumoArticle18 September 2020Open Access Transparent process A combination of two human monoclonal antibodies cures symptomatic rabies Guilherme Dias de Melo Guilherme Dias de Melo orcid.org/0000-0003-0747-7760 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Florian Sonthonnax Florian Sonthonnax orcid.org/0000-0003-4777-7274 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Sorbonne-Paris Cité, Cellule Pasteur, Université Paris-Diderot, Paris, France Search for more papers by this author Gabriel Lepousez Gabriel Lepousez Perception and Memory Unit, Institut Pasteur, Paris, France Search for more papers by this author Grégory Jouvion Grégory Jouvion Experimental Neuropathology Unit, Institut Pasteur, Paris, France INSERM, Pathophysiology of Pediatric Genetic Diseases, Sorbonne Université, Hôpital Armand-Trousseau, UF Génétique Moléculaire, Assistance Publique-Hôpitaux de Paris, Paris, France Search for more papers by this author Andrea Minola Andrea Minola Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland Search for more papers by this author Fabrizia Zatta Fabrizia Zatta Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland Search for more papers by this author Florence Larrous Florence Larrous Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Lauriane Kergoat Lauriane Kergoat Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Camille Mazo Camille Mazo Perception and Memory Unit, Institut Pasteur, Paris, France Search for more papers by this author Carine Moigneu Carine Moigneu Perception and Memory Unit, Institut Pasteur, Paris, France Search for more papers by this author Roberta Aiello Roberta Aiello Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy Search for more papers by this author Angela Salomoni Angela Salomoni Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy Search for more papers by this author Elise Brisebard Elise Brisebard Experimental Neuropathology Unit, Institut Pasteur, Paris, France Laboratoire d'Histopathologie, VetAgro-Sup, Université de Lyon, Lyon, France Search for more papers by this author Paola De Benedictis Paola De Benedictis orcid.org/0000-0001-6760-1933 Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy Search for more papers by this author Davide Corti Davide Corti orcid.org/0000-0003-4046-7222 Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland Search for more papers by this author Hervé Bourhy Corresponding Author Hervé Bourhy [email protected] orcid.org/0000-0002-2608-5589 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Guilherme Dias de Melo Guilherme Dias de Melo orcid.org/0000-0003-0747-7760 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Florian Sonthonnax Florian Sonthonnax orcid.org/0000-0003-4777-7274 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Sorbonne-Paris Cité, Cellule Pasteur, Université Paris-Diderot, Paris, France Search for more papers by this author Gabriel Lepousez Gabriel Lepousez Perception and Memory Unit, Institut Pasteur, Paris, France Search for more papers by this author Grégory Jouvion Grégory Jouvion Experimental Neuropathology Unit, Institut Pasteur, Paris, France INSERM, Pathophysiology of Pediatric Genetic Diseases, Sorbonne Université, Hôpital Armand-Trousseau, UF Génétique Moléculaire, Assistance Publique-Hôpitaux de Paris, Paris, France Search for more papers by this author Andrea Minola Andrea Minola Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland Search for more papers by this author Fabrizia Zatta Fabrizia Zatta Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland Search for more papers by this author Florence Larrous Florence Larrous Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Lauriane Kergoat Lauriane Kergoat Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Camille Mazo Camille Mazo Perception and Memory Unit, Institut Pasteur, Paris, France Search for more papers by this author Carine Moigneu Carine Moigneu Perception and Memory Unit, Institut Pasteur, Paris, France Search for more papers by this author Roberta Aiello Roberta Aiello Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy Search for more papers by this author Angela Salomoni Angela Salomoni Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy Search for more papers by this author Elise Brisebard Elise Brisebard Experimental Neuropathology Unit, Institut Pasteur, Paris, France Laboratoire d'Histopathologie, VetAgro-Sup, Université de Lyon, Lyon, France Search for more papers by this author Paola De Benedictis Paola De Benedictis orcid.org/0000-0001-6760-1933 Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy Search for more papers by this author Davide Corti Davide Corti orcid.org/0000-0003-4046-7222 Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland Search for more papers by this author Hervé Bourhy Corresponding Author Hervé Bourhy [email protected] orcid.org/0000-0002-2608-5589 Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France Search for more papers by this author Author Information Guilherme Dias de Melo1,‡, Florian Sonthonnax1,2,‡, Gabriel Lepousez3, Grégory Jouvion4,5, Andrea Minola6, Fabrizia Zatta6, Florence Larrous1, Lauriane Kergoat1, Camille Mazo3, Carine Moigneu3, Roberta Aiello7, Angela Salomoni7, Elise Brisebard4,8, Paola De Benedictis7, Davide Corti6 and Hervé Bourhy *,1 1Lyssavirus Epidemiology and Neuropathology Unit, Institut Pasteur, Paris, France 2Sorbonne-Paris Cité, Cellule Pasteur, Université Paris-Diderot, Paris, France 3Perception and Memory Unit, Institut Pasteur, Paris, France 4Experimental Neuropathology Unit, Institut Pasteur, Paris, France 5INSERM, Pathophysiology of Pediatric Genetic Diseases, Sorbonne Université, Hôpital Armand-Trousseau, UF Génétique Moléculaire, Assistance Publique-Hôpitaux de Paris, Paris, France 6Humabs BioMed SA, a subsidiary of Vir Biotechnology, Bellinzona, Switzerland 7Istituto Zooprofilattico Sperimentale delle Venezie, Padua, Italy 8Laboratoire d'Histopathologie, VetAgro-Sup, Université de Lyon, Lyon, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 45 68 87 85; E-mail: [email protected] EMBO Mol Med (2020)12:e12628https://doi.org/10.15252/emmm.202012628 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 Rabies is a neglected disease caused by a neurotropic Lyssavirus, transmitted to humans predominantly by the bite of infected dogs. Rabies is preventable with vaccines or proper post-exposure prophylaxis (PEP), but it still causes about 60,000 deaths every year. No cure exists after the onset of clinical signs, and the case-fatality rate approaches 100% even with advanced supportive care. Here, we report that a combination of two potent neutralizing human monoclonal antibodies directed against the viral envelope glycoprotein cures symptomatic rabid mice. Treatment efficacy requires the concomitant administration of antibodies in the periphery and in the central nervous system through intracerebroventricular infusion. After such treatment, recovered mice presented good clinical condition, viral loads were undetectable, and the brain inflammatory profile was almost normal. Our findings provide the unprecedented proof of concept of an antibody-based therapeutic approach for symptomatic rabies. Synopsis Rabies is an invariably fatal disease after the rabies virus has invaded the central nervous system and clinical signs have been manifested. This study brings the proof-of-concept that a cocktail of human monoclonal antibodies (mAbs) can cure symptomatic rabies. Intramuscular + intracerebroventricular administration of RVC20/RVC58 mAbs cures symptomatic rabies in a mouse model. Continuous administration of RVC20/RVC58mAbs directly in the central nervous system is crucial to treatment success. RVC20/RVC58 mAbs therapy promotes rabies virus clearance from infected brains in vivo and restoration of normal clinical condition in infected mice. The paper explained Problem Rabies is an almost invariably fatal disease. Despite being an ancient illness, rabies is still nowadays considered a neglected disease, being responsible of 60,000 estimated deaths each year, mainly related to young people coming from remote areas from developing countries. Rabies can be prevented by using vaccination and passive serotherapy, but currently no treatment is able to effectively cure rabies after the onset of the neurological symptoms. Results Here, we established a protocol to treat symptomatic rabies in mice using a cocktail of two potent neutralizing human monoclonal antibodies (mAbs RVC20/RVC58). The efficacy of this treatment is linked to the concomitant administration of these antibodies locally at the site of the infection and directly into the central nervous system. This therapy could lead to survival of infected mice, restoring their clinical condition and clearing rabies virus from their brain, with higher survival rates found in mice receiving the mAbs cocktail in early time points after the onset of the neurological symptoms. Impact Currently, despite some descriptions of patients surviving clinical rabies, there is no effective and reproducible therapy. Rabies can be prevented in rabies-exposed patients by the timely administration of a post-exposure prophylaxis (PEP) combining rabies vaccine and immunoglobulins. However, this PEP is not always accessible to target populations, and therefore, there is an urgent need for an affordable treatment against this infection to cure rabies-infected patients. The mAbs RVC20/RVC58 cocktail represents this unprecedented possibility to develop an effective treatment of brain infection by rabies virus, increasing survival rate and repairing neurological symptoms. Such a therapy would require a combined implementation of rapid and early diagnosis of rabies in infected patients as early administration of mAbs appears to be instrumental in the success of this therapeutic approach. Introduction Rabies is a lethal acute encephalomyelitis caused by a neurotropic Lyssavirus mainly transmitted to humans by the bite of domestic dogs (WHO, 2018). Rabies is an ancient illness (Tarantola, 2017), but it is still considered one of the most neglected diseases, especially in developing countries. The first vaccine against this infection was developed more than 130 years ago by Louis Pasteur (Bourhy et al, 2010), and today, rabies is fully preventable with proper post-exposure prophylaxis (PEP), with an estimation that 15–29 million patients exposed to rabies receive the PEP annually (WHO, 2018). However, after the onset of clinical symptoms, which correlates with the presence of the virus in the central nervous system (CNS), rabies is nearly 100% fatal in infected patients, even with advanced supportive care (Dacheux et al, 2011; Jackson, 2018; Ugolini & Hemachudha, 2018; WHO, 2018). The mortality due to rabies is estimated to be about 60,000 deaths each year, mostly in Asia and Africa, and among them, 50% are children under 15 years of age (Hampson et al, 2015; WHO, 2018; Cantaert et al, 2019). Several attempts have been performed to treat symptomatic rabies (Dacheux et al, 2011; Smith et al, 2019). In 2004, an infected patient from Wisconsin (USA) survived after a therapeutic approach that was named Milwaukee protocol (Willoughby et al, 2005). Since then, changes have been made in this protocol to arrive at its current version, which includes therapeutic coma, ketamine infusion, amantadine, and the management of cerebral vasospasm (Zeiler & Jackson, 2016). Nevertheless, its effectiveness is questionable since at least 31 documented failures have been reported (Zeiler & Jackson, 2016; Jackson, 2018). In the quest for a novel therapeutic possibility, we have previously reported the selection of two human monoclonal antibodies (mAbs), RVC20 and RVC58, that were able to bind to two distinct antigenic sites on the RABV glycoprotein protein (sites I and III), to potently neutralize RABV isolates of all lineages, and of all phylogroup I non-RABV isolates, and that presented a protective role when used as early PEP in hamsters as well (De Benedictis et al, 2016; Hellert et al, 2020). Here, we show that a combination of the RVC20 and RVC58 monoclonal antibodies can effectively cure already symptomatic mice (late infection) when concomitantly administered both directly in the central nervous system, through intracerebroventricular infusion, and in the periphery, at the site of the infection. After such treatment, mice that survived the infection presented good clinical condition, the viral load was absent, and the inflammatory profile in the brain was close to that of uninfected animals. Altogether, our findings provide proof of concept that a targeted administration of human monoclonal antibodies represents a possibility with an unprecedented breadth and potency for the development of a low-risk product to treat rabies. Results and Discussion Peripheral immunotherapy is not efficient in curing rabies To investigate whether the mAbs RVC20 and RVC58 display therapeutic activity against a lethal RABV infection in vivo, we set up a model of infection in Balb/c mice using a field RABV strain. In this model, the virus was detected from 4 days post-infection (dpi) in the spinal cord and at 5 dpi in the brain (Fig 1A). Weight loss and diminished motor performance are noticed from 7 dpi, while typical clinical signs (ruffled fur, lethargy, ataxia, paralysis) are detected from 8 dpi onwards (Fig 1B and C). Death occurred in all animals at 10–13 dpi (Fig 2A). In this condition, a single intramuscular (IM) injection of the 1:1 combination of RVC20/RVC58 (2 + 2 mg/kg) provided only modest survival effects when administered 2 or 4 dpi (Fig EV1), and no protective effect was observed when administered later. The blood half-life of the RVC20/RVC58 cocktail was determined as 6.16 days (Appendix Fig S1). At a higher dose (20 + 20 mg/kg), RVC20/RVC58 protected most animals from morbidity and mortality when administered 2 or 4 dpi, but displayed a limited success when treated 6 dpi (1/5). Intriguingly, some delayed deaths occurred in three treated animals (35, 55, and 68 dpi; Fig EV1B and C), which denoted the need to include late treatments to ensure the complete clearance of the remaining virus in the periphery, possibly avoiding a delayed wave of viral spread toward the CNS. Of note, we found similar results in CVS-11-infected golden Syrian hamsters treated just before the onset of clinical signs (Fig EV2). Altogether, these data support a dose-dependent effect of the mAbs cocktail and lead to the hypothesis that a strictly peripheral immunotherapy is not efficient in advanced rabies infection. Figure 1. Therapeutic efficacy of intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibody cocktail in Tha-RABV-infected mice A. Rabies virus infection kinetics in the central nervous system of infected mice. Tha-RABV load (copies/μg of RNA) detected in the thoracolumbar spinal cord, in the brainstem plus cerebellum, and in the cerebral cortex of infected mice according to different time points post-infection (n = 4 per time point). nd: not detected. Horizontal lines indicate the median. B. Follow-up of the clinical signs of mice under different treatments. Heat maps were established based on a progressive 0–7 clinical score scale (0: no apparent changes; 1: ruffled fur; 2: slow movement, hind limb ataxia; 3: apathy; 4: monoplegia; 5: hind limb paralysis, tremors; 6: paralysis, conjunctivitis/keratitis, urine staining of the haircoat of the perineum; 7: death). Each line represents one animal throughout time. C. Body weight progression of mice under different treatments. Mice were weighed on a daily basis during the treatment administration and then twice a week up to 100 days post-infection. All the mice were equipped with iPRECIO pumps weighing ca. 3.3 g. Each line represents one animal throughout time. D, E. Mouse behavioral testing. The tested animals were non-infected (n = 3 mice with iPRECIO pump + n = 4 age-related mice without iPRECIO pump), infected non-treated (n = 5), infected and treated at 7 dpi (n = 5, mice #9 to #13), and infected and treated at 8 dpi (n = 5, mice #24 to #28). (D) Open-field test performed at 7 dpi (prodromal phase) and after the remission of clinical signs for the survivors (80 dpi). (E) Rotarod motor performance at 7 dpi (prodromal phase), 8 dpi (symptomatic phase), and after the remission of clinical signs for the survivors (20, 30, and 60 dpi). Horizontal lines indicate the median. Kruskal–Wallis followed by Dunn' multiple comparisons test (7 and 8 days post-infection) and Mann–Whitney test (20, 30, and 60 days post-infection). The rotarod performance presented a negative correlation (Spearman's r = −0.768) with the clinical score at 8 dpi. *P < 0.05, **P < 0.01, and ***P < 0.001. Treatment description = NI: non-infected (n = 5); NT: infected, non-treated (n = 5); infected, treated at 6 dpi (2 + 2 mg/kg) (n = 4); infected, treated at 7 dpi (2 + 2 mg/kg) (n = 9); infected, treated at 8 dpi (2 + 2 mg/kg) (n = 15); infected, treated at 8 dpi (10 + 10 mg/kg) (n = 4). Exact P values are shown in Appendix Table S2. Download figure Download PowerPoint Figure 2. Intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibody cocktail cures symptomatic Tha-RABV-infected mice A. Cumulative Kaplan–Meier survival curves of mice treated at different time points post-infection. Log-rank (Mantel–Cox) test to compare treated groups with the infected, non-treated group. **P < 0.01, ***P < 0.001. Treatment description = infected, non-treated (n = 5); infected, treated at 6 dpi (2 + 2 mg/kg) (n = 4); infected, treated at 7 dpi (2 + 2 mg/kg) (n = 9); infected, treated at 8 dpi (2 + 2 mg/kg) (n = 15); infected, treated at 8 dpi (10 + 10 mg/kg) (n = 4). B. Cumulative Kaplan–Meier survival curves of all the treated animals that presented with clinical signs (n = 31, all treatments combined). Log-rank (Mantel–Cox) test to compare treated groups with the infected, non-treated group. ****P < 0.0001. C. Virus-neutralizing antibodies detected in the serum of surviving mice from different experimental groups. D. Residual human monoclonal antibodies in the serum of representative surviving mice at 100 dpi (LOD: limit of detection = 0.02 μg/ml). E. Viral load in the brain of mice from different experimental groups. The samples were either collected at the time of death, or at 100 dpi for the survivors. F–N. Cytokines and innate immune mediators' profile in the brain of mice from different experimental groups: (F) IFN-β1, (G) ISG15, (H) Mx1, (I) IFN-γ, (J) CXCL10, (K) CCL5, (L) IL-6, (M) TNF-α, and (N) IL-1β, detected in one brain hemisphere of mice from different experimental groups. The expression of the genes of interest was normalized to the GAPDH housekeeping gene. Data information: Horizontal lines indicate the median. The orange crosshatched areas correspond to the 95% CI of the median from the infected and non-treated mice, and the blue crosshatched areas correspond to the 95% CI of the median from the non-infected mice. nd: not detected.Treatment description = ni: non-infected (n = 5); nt: infected, non-treated (n = 5); 6 (2 + 2): infected, treated at 6 dpi (2 + 2 mg/kg) (n = 4); 7 (2 + 2): infected, treated at 7 dpi (2 + 2 mg/kg) (n = 9); 8 (2 + 2): infected, treated at 8 dpi (2 + 2 mg/kg) (n = 15); 8 (10 + 10): infected, treated at 8 dpi (10 + 10 mg/kg) (n = 4). Exact P values are shown in Appendix Table S2. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Efficacy of intramuscular administration of the RVC20 and RVC58 monoclonal antibody cocktail in Tha-RABV-infected mice A–C. Kaplan–Meier survival curve of non-treated mice (A), or treated at different time points with a single intramuscular injection of 1:1 combination of RVC20 and RVC58 human monoclonal antibodies at the dose of 2 + 2 mg/kg (B) and 20 + 20 mg/kg (C) (n = 5 per treatment). Of note, the survivor treated at 6 dpi presented with persistent monoplegia as sequelae of the infection. Statistical analysis was performed using log-rank (Mantel–Cox) test with α = 0.05. *P < 0.05, **P < 0.01. ni: non-infected; nt: infected, non-treated. Exact P values are shown in Appendix Table S2. D–H. Brain viral load (D) detected in the brain, virus-neutralizing antibodies (e), and human antibodies (F) detected in the serum of mice from different experimental groups, and the relative brain expression of CCL5 (G) and CXCL10 (H). Data information: Horizontal lines indicate the median. The expression of the genes of interest was normalized to the GAPDH housekeeping gene. The samples were either collected at the time of death, or at 100 dpi for the survivors. ni: non-infected; nt: infected, non-treated; nd: not detected (n = 5 per group). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Efficacy of intramuscular administration of the RVC20 and RVC58 monoclonal antibody cocktail in CVS-11-infected golden Syrian hamsters A. Cumulative Kaplan–Meier survival curves of hamsters under different treatments (n = 12 per treatment). Log-rank (Mantel–Cox) test to compare treated groups with the infected, non-treated group. ****P < 0.0001. B–D. Follow-up of the clinical signs of hamsters under different treatments, based on a progressive 0–5 clinical score scale. E–G. Viral load in the cerebrum (E), cerebellum and medulla oblongata (F), and spinal cord (G) of hamsters from different experimental groups. The samples were either collected at the time of death, or at 120 and 340 dpi for the survivors. Data information: Horizontal lines indicate the median. The orange crosshatched areas correspond to the 95% CI of the median from the infected and non-treated hamsters. nt: infected, non-treated; nd: not detected (n = 12 per group). Download figure Download PowerPoint Combined CNS and peripheral immunotherapy cures symptomatic rabies We then further tested the therapeutic potential of RVC20/RVC58 on advanced phases of rabies virus infection, by combining IM injections with intracerebroventricular (ICV) administration in mice equipped with automated microinfusion pumps (Fig EV3A–C). The therapeutic protocol consisted of one IM injection of the RVC20/RVC58 cocktail (20 + 20 mg/kg) concomitantly with a continuous ICV infusion (2 + 2 mg/kg/day) during 20 days, starting at 6 dpi (presymptomatic phase, RABV already in the CNS; Fig 1A), at 7 dpi (prodromal phase, no clinical signs detected, but motor performance already impacted; Fig 1D and E), or at 8 dpi (symptomatic phase). At these time points, some cytokines and innate immune mediators' expression were already impacted (Appendix Fig S2 and Appendix Table S1). A second IM injection (20 + 20 mg/kg) two days after the end of the ICV infusion was administered to all treated animals. Click here to expand this figure. Figure EV3. Intracerebroventricular (ICV) drug delivery in mice, and the therapeutic efficacy of intramuscular and ICV administration of the RVC20-LALA and RVC58-LALA antibody cocktail against rabies A. Stereotaxic implantation of brain infusion kit connected to iPRECIO pump in mice to deliver the monoclonal antibody cocktail in the right lateral ventricle. The stereotaxic coordinates, taken bregma as reference, were −0.5 mm anteroposterior, +1.0 mm mediolateral (arrow), and −2.4 mm dorsoventral. B, C. Non-infected mice received fluorescent antibodies (goat IgG anti-chicken IgY conjugated to Alexa Fluor® 647; Invitrogen A-21449) by ICV, 10 μg/day during 3 days (1, n = 1), by intraperitoneal injection, 10 μg/mouse, 6 h before imaging (2, n = 1), and non-injected control (3, n = 1). Mice were euthanized, and the brains were extracted and imaged using the IVIS Spectrum (PerkinElmer); the fluorescence in the brain was detected only in the animal receiving the antibodies by ICV, with a diffusion spectrum from the site of injection throughout the brain (B; top panels: dorsal view of the whole brain; bottom panels: sagittal views). The total fluorescence was quantified in the whole brain (C). D. Neutralization of Tha-RABV by RVC20 and RVC58 antibodies, wild type (WT), or LALA, alone or in 1:1 combination. Data are expressed as IC50 (ng/ml). Horizontal lines indicate mean ± SD for two independent experiments. E–G. Therapeutic efficacy of intramuscular and intracerebroventricular administration of the RVC20-LALA and RVC58-LALA antibody cocktail against rabies. (E) Kaplan–Meier survival curves of mice under different treatments (n = 5 per treatment). Log-rank (Mantel–Cox) test: *P < 0.05, **P < 0.01. (F–G) Follow-up of the clinical signs (F) and body weight (G) of mice under different treatments. Heat maps were established based on a progressive 0–7 clinical score scale (0: no apparent changes; 1: ruffled fur; 2: slow movement, hind limb ataxia; 3: apathy; 4: monoplegia; 5: hind limb paralysis, tremors; 6: paralysis, conjunctivitis/keratitis, urine staining of the haircoat of the perineum; 7: death). Each line represents one animal throughout time. The infected, non-treated group is also displayed in Fig 2. Download figure Download PowerPoint IM + ICV treatment was 100% efficient in resolving the clinical signs and controlling the infection when started at 6 dpi (Figs 1 and 2). When started at 7 dpi, the treatment was able to promote survival and to ameliorate the clinical condition in 55.6% (5/9) of the infected animals, and when started at 8 dpi, the treatment was efficient in curing 33.3% (5/15) of the infected animals. Higher ICV doses of RVC20/RVC58 (10 + 10 mg/kg/day) did not increase survival rate (Figs 1 and 2). Remarkably, in the group of animals treated at 8 dpi, three animals which died during the ICV administration (16, 22, and 23 dpi) and another one that died after the end of the treatment (38 dpi) presented low viral load in their brains (Fig 2E), indicating that viral clearance by the RVC20/RVC58 cocktail had already started (Hunter et al, 2010). The causa mortis might be related to bad overall clinical condition associated with sequelae of the infection, such as brain damage or uncontrolled brain inflammation; intensive care in these cases could be suitable. Overall, combining all the treated animals, the IM + ICV treatment was efficient in curing 45.2% (14/31) of mice that presented with clinical signs (Fig 2B). Surviving animals were monitored until 100 dpi and did not develop further signs of disease, except two mice presenting permanent monoplegia despite an overall good condition and normal behavior (i.e., alert and active). Food intake and body weight became normal in the surviving mice after clinical phase (Fig 1C); spontaneous locomotor activity, exploratory behavior, and anxiety-related behavior, as monitored in the open-field test, were similar between controls and survivors (Fig 1D). Virus-neutralizing antibodies and human monoclonal antibodies were detected in the serum of surviving mice at 100 dpi (Fig 2C and D). No virus was detected in the brains of the surviving mice (except two animals with low but detectable viral load, Fig 2E), there was a long-lasting expression of antiviral mediators, and the inflammatory mediator profile in their brains was close to the one exhibited in the brain of non-infected mice (Figs 2F–N and EV4). Histological analysis of the brain of surviving mice revealed only rare signs of residual neuro-inflammation: perivascular cuffing (2/7), minimal meningitis (3/7), and reactive microglial cells (more marked in 2/7) (Fig 3A–R). Finally, we cannot attest that virus clearance occurred without neuronal loss, especially due to long-lasting deficit in the fine motor coordination of surviving mice, even if clinically healthy (Fig 1E). Click here to expand this figure. Figure EV4. Principal component analysis (PCA) of immune mediators in the brain of Tha-RABV-infected mice treated with intramuscular and intracerebroventricular administration of the RVC20 and RVC58 monoclonal antibody cocktail A. Variable correlation plot showing the correlation of the gene expression of nine immune mediators in the brain of mice. The two-first principal components explained 63.65% of samples variability. IFN-γ, CXCL10, and the type I IFN-stimulated genes ISG15 and Mx1 contributed to PC1 (principal component 1; 66.7%), whereas IL-1β and TNF-α loaded positively on PC2 (principal component 2; 76.1%). B, C. PCA plots. Each symbol represents one animal, colored according to the experimental groups. Non-infected mice clustered together with survivors and even with some treated mice that di
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