A vaccine targeting antigen-presenting cells through CD40 induces protective immunity against Nipah disease.

Nipah virus (NiV) has been recently ranked by the World Health Organization as being among the top eight emerging pathogens likely to cause major epidemics, whereas no therapeutics or vaccines have yet been approved. We report a method to deliver immunogenic epitopes from NiV through the targeting of the CD40 receptor of antigen-presenting cells by fusing a selected humanized anti-CD40 monoclonal antibody to the Nipah glycoprotein with conserved NiV fusion and nucleocapsid peptides. In the African green monkey model, CD40.NiV induces specific immunoglobulin A (IgA) and IgG as well as cross-neutralizing responses against circulating NiV strains and Hendra virus and T cell responses. Challenge experiments using a NiV-B strain demonstrate the high protective efficacy of the vaccine, with all vaccinated animals surviving and showing no significant clinical signs or virus replication, suggesting that the CD40.NiV vaccine conferred sterilizing immunity. Overall, results obtained with the CD40.NiV vaccine are highly promising in terms of the breadth and efficacy against NiV.

Nipah Virus: An Overview of the Current Status of Diagnostics and Their Role in Preparedness in Endemic Countries.

Nipah virus (NiV) is a paramyxovirus responsible for a high mortality rate zoonosis. As a result, it has been included in the list of Blueprint priority pathogens. Bats are the main reservoirs of the virus, and different clinical courses have been described in humans. The Bangladesh strain (NiV-B) is often associated with severe respiratory disease, whereas the Malaysian strain (NiV-M) is often associated with severe encephalitis. An early diagnosis of NiV infection is crucial to limit the outbreak and to provide appropriate care to the patient. Due to high specificity and sensitivity, qRT-PCR is currently considered to be the optimum method in acute NiV infection assessment. Nasal swabs, cerebrospinal fluid, urine, and blood are used for RT-PCR testing. N gene represents the main target used in molecular assays. Different sensitivities have been observed depending on the platform used: real-time PCR showed a sensitivity of about 103 equivalent copies/reaction, SYBRGREEN technology's sensitivity was about 20 equivalent copies/reaction, and in multiple pathogen card arrays, the lowest limit of detection (LOD) was estimated to be 54 equivalent copies/reaction. An international standard for NiV is yet to be established, making it difficult to compare the sensitivity of the different methods. Serological assays are for the most part used in seroprevalence studies owing to their lower sensitivity in acute infection. Due to the high epidemic and pandemic potential of this virus, the diagnosis of NiV should be included in a more global One Health approach to improve surveillance and preparedness for the benefit of public health. Some steps need to be conducted in the diagnostic field in order to become more efficient in epidemic management, such as development of point-of-care (PoC) assays for the rapid diagnosis of NiV.

Hemostasis defects underlying the hemorrhagic syndrome caused by mammarenaviruses in a cynomolgus macaque model.

Viral hemorrhagic fevers (HF) are a group of acute febrile diseases with high mortality rates. Although hemostatic dysfunction appears to be a major determinant of the severity of the disease, it is still unclear what pathogenic mechanisms lead to it. In clinical studies it is found that arenaviruses, such as Lassa, Machupo, and Guanarito viruses cause HF that vary in symptoms and biological alterations. In this study we aimed to characterize the hemostatic dysfunction induced by arenaviral HF to determine its implication in the severity of the disease and to elucidate the origin of this syndrome. We found that lethal infection with Machupo, Guanarito, and Lassa viruses is associated with cutaneomucosal, cerebral, digestive, and pulmonary hemorrhages. The affected animals developed a severe alteration of the coagulation system, which was concomitant with acute hepatitis, minor deficit of hepatic factor synthesis, presence of a plasmatic inhibitor of coagulation, and dysfunction of the fibrinolytic system. Despite signs of increased vascular permeability, endothelial cell infection was not a determinant factor of the hemorrhagic syndrome. There were also alterations of the primary hemostasis during lethal infection, with moderate to severe thrombocytopenia and platelet dysfunction. Finally, we show that lethal infection is accompanied by a reduced hematopoietic potential of the bone marrow. This study provides an unprecedented characterization of the hemostasis defects induced by several highly pathogenic arenaviruses.

Genomic diversity of mpox virus in Paris area (France) during the 2022 outbreak.

In May 2022, several countries reported mpox cases from patients without history of traveling to endemic areas. France was one of the most affected European countries by this outbreak. In this study, the clinical characteristics of mpox cases in France were described, and the genetic diversity of the virus was studied. Patients diagnosed with mpox infection (quantitative polymerase chain reaction ct < 28) between May 21, and July 4, 2022 and between 16th August and 10th September 2022 were included to this study. Twelve amplicons corresponding to the most polymorphic regions of the mpox genome and covering ~30 000 nucleotides were generated and sequenced using the S5 XL Ion Torrent technology to evaluate the genetic diversity of mpox sequences. One hundred and forty-eight patients were diagnosed with mpox-infection. 95% were men, 5% transgender (M-to-F), 50% were taking human immunodeficiency virus (HIV) pre-exposure prophylaxis, and 25% were HIV seropositive. One hundred and sixty-two samples (some patients had two samples) were sequenced and compared to GenBank sequences. Overall, low genetic diversity of mpox sequences was found compared with pre-epidemic Western-African sequences, with 32 distinct mutational patterns. This study provides a first glance at the mutational landscape of early mpox 2022 circulating strains in Paris (France).

Rapid protection induced by a single-shot Lassa vaccine in male cynomolgus monkeys.

Lassa fever hits West African countries annually in the absence of licensed vaccine to limit the burden of this viral hemorrhagic fever. We previously developed MeV-NP, a single-shot vaccine protecting cynomolgus monkeys against divergent strains one month or more than a year before Lassa virus infection. Given the limited dissemination area during outbreaks and the risk of nosocomial transmission, a vaccine inducing rapid protection could be useful to protect exposed people during outbreaks in the absence of preventive vaccination. Here, we test whether the time to protection can be reduced after immunization by challenging measles virus pre-immune male cynomolgus monkeys sixteen or eight days after a single shot of MeV-NP. None of the immunized monkeys develop disease and they rapidly control viral replication. Animals immunized eight days before the challenge are the best controllers, producing a strong CD8 T-cell response against the viral glycoprotein. A group of animals was also vaccinated one hour after the challenge, but was not protected and succumbed to the disease as the control animals. This study demonstrates that MeV-NP can induce a rapid protective immune response against Lassa fever in the presence of MeV pre-existing immunity but can likely not be used as therapeutic vaccine.

A MOPEVAC multivalent vaccine induces sterile protection against New World arenaviruses in non-human primates.

Pathogenic New World arenaviruses (NWAs) cause haemorrhagic fevers and can have high mortality rates, as shown in outbreaks in South America. Neutralizing antibodies (Abs) are critical for protection from NWAs. Having shown that the MOPEVAC vaccine, based on a hyperattenuated arenavirus, induces neutralizing Abs against Lassa fever, we hypothesized that expression of NWA glycoproteins in this platform might protect against NWAs. Cynomolgus monkeys immunized with MOPEVACMAC, targeting Machupo virus, prevented the lethality of this virus and induced partially NWA cross-reactive neutralizing Abs. We then developed the pentavalent MOPEVACNEW vaccine, expressing glycoproteins from all pathogenic South American NWAs. Immunization of cynomolgus monkeys with MOPEVACNEW induced neutralizing Abs against five NWAs, strong innate followed by adaptive immune responses as detected by transcriptomics and provided sterile protection against Machupo virus and the genetically distant Guanarito virus. MOPEVACNEW may thus be efficient to protect against existing and potentially emerging NWAs.

Bombali Ebolavirus in Mops condylurus Bats (Molossidae), Mozambique.

We detected Bombali ebolavirus RNA in 3 free-tailed bats (Mops condylurus, Molossidae) in Mozambique. Sequencing of the large protein gene revealed 98% identity with viruses previously detected in Sierra Leone, Kenya, and Guinea. Our findings further support the suspected role of Mops condylurus bats in maintaining Bombali ebolavirus.

Natural History of Sudan ebolavirus to Support Medical Countermeasure Development.

Sudan ebolavirus (SUDV) is one of four members of the Ebolavirus genus known to cause Ebola Virus Disease (EVD) in humans, which is characterized by hemorrhagic fever and a high case fatality rate. While licensed therapeutics and vaccines are available in limited number to treat infections of Zaire ebolavirus, there are currently no effective licensed vaccines or therapeutics for SUDV. A well-characterized animal model of this disease is needed for the further development and testing of vaccines and therapeutics. In this study, twelve cynomolgus macaques (Macaca fascicularis) were challenged intramuscularly with 1000 PFUs of SUDV and were followed under continuous telemetric surveillance. Clinical observations, body weights, temperature, viremia, hematology, clinical chemistry, and coagulation were analyzed at timepoints throughout the study. Death from SUDV disease occurred between five and ten days after challenge at the point that each animal met the criteria for euthanasia. All animals were observed to exhibit clinical signs and lesions similar to those observed in human cases which included: viremia, fever, dehydration, reduced physical activity, macular skin rash, systemic inflammation, coagulopathy, lymphoid depletion, renal tubular necrosis, hepatocellular degeneration and necrosis. The results from this study will facilitate the future preclinical development and evaluation of vaccines and therapeutics for SUDV.

Pathogenesis of recent Lassa virus isolates from lineages II and VII in cynomolgus monkeys.

The area of Lassa virus (LASV) circulation is expanding, with the emergence of highly pathogenic new LASV lineages. Benin recently became an endemic country for LASV and has seen the emergence of a new LASV lineage (VII). The first two outbreaks in 2014 and 2016 showed a relatively high mortality rate compared to other outbreaks. We infected cynomolgus monkeys with two strains belonging to lineage II and lineage VII that were isolated from deceased patients during the 2016 outbreak in Benin. The lineage VII strain (L7) caused uniform mortality. Death was associated with uncontrolled viral replication, unbalanced inflammatory responses characterized by increased concentrations of pro- and anti-inflammatory mediators, and the absence of efficient immune responses, resembling the pathogenesis associated with the prototypic Josiah strain in monkeys. The lineage II strain (L2) showed apparently lower virulence than its counterpart, with a prolonged time to death and a lower mortality rate. Prolonged survival was associated with better control of viral replication, a moderate inflammatory response, and efficient T-cell responses. Transcriptomic analyses also highlighted important differences in the immune responses associated with the outcome. Both strains caused strong inflammation in several organs. Notably, meningitis and encephalitis were observed in the cerebral cortex and cerebellum in all monkeys, independently of the outcome. Due to their apparently high pathogenicity, emerging strains from lineage VII should be considered in preclinical vaccine testing. Lineage II would also be beneficial in pathogenesis studies to study the entire spectrum of Lassa fever severity.

Uncovering a conserved vulnerability site in SARS-CoV-2 by a human antibody.

An essential step for SARS-CoV-2 infection is the attachment to the host cell receptor by its Spike receptor-binding domain (RBD). Most of the existing RBD-targeting neutralizing antibodies block the receptor-binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non-RBM-targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a KD of 5.6 nM, neutralizes SARS-CoV-2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross-reactivity against SARS-CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted "ideal" vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20-binding but confers little or no resistance to neutralization. Finally, in vitro mode-of-action characterization and negative-stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS-CoV-2 Spike for the development of potential antiviral drugs.

A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection.

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1-6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7-17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 µg mL-1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak.

A single-shot Lassa vaccine induces long-term immunity and protects cynomolgus monkeys against heterologous strains.

A safe and protective Lassa virus vaccine is crucially needed in Western Africa to stem the recurrent outbreaks of Lassa virus infections in Nigeria and the emergence of Lassa virus in previously unaffected countries, such as Benin and Togo. Major challenges in developing a Lassa virus vaccine include the high diversity of circulating strains and their reemergence from 1 year to another. To address each of these challenges, we immunized cynomolgus monkeys with a measles virus vector expressing the Lassa virus glycoprotein and nucleoprotein of the prototypic Lassa virus strain Josiah (MeV-NP). To evaluate vaccine efficacy against heterologous strains of Lassa virus, we challenged the monkeys a month later with heterologous strains from lineage II or lineage VII, finding that the vaccine was protective against these strains. A second cohort of monkeys was challenged 1 year later with the homologous Josiah strain, finding that a single dose of MeV-NP was sufficient to protect all vaccinated monkeys. These studies demonstrate that MeV-NP can generate both long-lasting immune responses and responses that are able to protect against diverse strains of Lassa virus.

Early control of viral load by favipiravir promotes survival to Ebola virus challenge and prevents cytokine storm in non-human primates.

Ebola virus has been responsible for two major epidemics over the last several years and there has been a strong effort to find potential treatments that can improve the disease outcome. Antiviral favipiravir was thus tested on non-human primates infected with Ebola virus. Half of the treated animals survived the Ebola virus challenge, whereas the infection was fully lethal for the untreated ones. Moreover, the treated animals that did not survive died later than the controls. We evaluated the hematological, virological, biochemical, and immunological parameters of the animals and performed proteomic analysis at various timepoints of the disease. The viral load strongly correlated with dysregulation of the biological functions involved in pathogenesis, notably the inflammatory response, hemostatic functions, and response to stress. Thus, the management of viral replication in Ebola virus disease is of crucial importance in preventing the immunopathogenic disorders and septic-like shock syndrome generally observed in Ebola virus-infected patients.

Systemic viral spreading and defective host responses are associated with fatal Lassa fever in macaques.

Lassa virus (LASV) is endemic in West Africa and induces a viral hemorrhagic fever (VHF) with up to 30% lethality among clinical cases. The mechanisms involved in control of Lassa fever or, in contrast, the ensuing catastrophic illness and death are poorly understood. We used the cynomolgus monkey model to reproduce the human disease with asymptomatic to mild or fatal disease. After initial replication at the inoculation site, LASV reached the secondary lymphoid organs. LASV did not spread further in nonfatal disease and was rapidly controlled by balanced innate and T-cell responses. Systemic viral dissemination occurred during severe disease. Massive replication, a cytokine/chemokine storm, defective T-cell responses, and multiorgan failure were observed. Clinical, biological, immunological, and transcriptomic parameters resembled those observed during septic-shock syndrome, suggesting that similar pathogenesis is induced during Lassa fever. The outcome appears to be determined early, as differentially expressed genes in PBMCs were associated with fatal and non-fatal Lassa fever outcome very early after infection. These results provide a full characterization and important insights into Lassa fever pathogenesis and could help to develop early diagnostic tools.

Longitudinal antibody and T cell responses in Ebola virus disease survivors and contacts: an observational cohort study.

BACKGROUND: The 2013-16 Ebola virus disease epidemic in west Africa caused international alarm due to its rapid and extensive spread resulting in a significant death toll and social unrest within the affected region. The large number of cases provided an opportunity to study the long-term kinetics of Zaire ebolavirus-specific immune response of survivors in addition to known contacts of those infected with the virus. METHODS: In this observational cohort study, we worked with leaders of Ebola virus disease survivor associations in two regions of Guinea, Guéckédou and Coyah, to recruit survivors of Ebola virus disease, contacts from households of individuals known to have had Ebola virus disease, and individuals who were not knowingly associated with infected individuals or had not had Ebola virus disease symptoms to serve as negative controls. We did Zaire ebolavirus glycoprotein-specific T cell analysis on peripheral blood mononuclear cells (PBMCs) on location in Guinea and transported plasma and PBMCs back to Europe for antibody quantification by ELISA, functional neutralising antibody analysis using live Zaire ebolavirus, and T cell phenotype studies. We report on the longitudinal cellular and humoral response among Ebola virus disease survivors and highlight potentially paucisymptomatic infection. FINDINGS: We recruited 117 survivors of Ebola virus disease, 66 contacts, and 23 negative controls. The mean neutralising antibody titre among the Ebola virus disease survivors 3-14 months after infection was 1/174 (95% CI 1/136-1/223). Individual results varied greatly from 1/10 to more than 1/1000 but were on average ten times greater than that induced after 1 month by single dose Ebola virus vaccines. Following reactivation with glycoprotein peptide, the mean T cell responses among 116 Ebola virus disease survivors as measured by ELISpot was 305 spot-forming units (95% CI 257-353). The dominant CD8+ polyfunctional T cell phenotype, as measured among 53 Ebola virus disease survivors, was interferon γ+, tumour necrosis factor+, interleukin-2-, and the mean response was 0·046% of total CD8+ T cells (95% CI 0·021-0·071). Additionally, both neutralising antibody and T cell responses were detected in six (9%) of 66 Ebola virus disease contacts. We also noted that four (3%) of 117 individuals with Ebola virus disease infections did not have circulating Ebola virus-specific antibodies 3 months after infection. INTERPRETATION: The continuous high titre of neutralising antibodies and increased T cell response might support the concept of long-term protective immunity in survivors. The existence of antibody and T cell responses in contacts of individuals with Ebola virus disease adds further evidence to the existence of sub-clinical Ebola virus infection. FUNDING: US Food & Drug Administration, Horizon 2020 EU EVIDENT, Wellcome, UK Department for International Development. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Long-lasting severe immune dysfunction in Ebola virus disease survivors.

Long-term follow up studies from Ebola virus disease (EVD) survivors (EBOV_S) are lacking. Here, we evaluate immune and gene expression profiles in 35 Guinean EBOV_S from the last West African outbreak, a median of 23 months (IQR [18-25]) after discharge from treatment center. Compared with healthy donors, EBOV_S exhibit increases of blood markers of inflammation, intestinal tissue damage, T cell and B cell activation and a depletion of circulating dendritic cells. All survivors have EBOV-specific IgG antibodies and robust and polyfunctional EBOV-specific memory T-cell responses. Deep sequencing of the genes expressed in blood reveals an enrichment in 'inflammation' and 'antiviral' pathways. Integrated analyses identify specific immune markers associated with the persistence of clinical symptoms. This study identifies a set of biological and genetic markers that could be used to define a signature of "chronic Ebola virus disease (CEVD)".

Modeling Favipiravir Antiviral Efficacy Against Emerging Viruses: From Animal Studies to Clinical Trials.

In 2014, our research network was involved in the evaluation of favipiravir, an anti-influenza polymerase inhibitor, against Ebola virus. In this review, we discuss how mathematical modeling was used, first to propose a relevant dosing regimen in humans, and then to optimize its antiviral efficacy in a nonhuman primate (NHP) model. The data collected in NHPs were finally used to develop a model of Ebola pathogenesis integrating the interactions among the virus, the innate and adaptive immune response, and the action of favipiravir. We conclude the review of this work by discussing how these results are of relevance for future human studies in the context of Ebola virus, but also for other emerging viral diseases for which no therapeutics are available.

Ribavirin does not potentiate favipiravir antiviral activity against Ebola virus in non-human primates.

BACKGROUND: In spite of recurrent and dramatic outbreaks, there are no therapeutics approved against Ebola virus disease. Favipiravir, a RNA polymerase inhibitor active against several RNA viruses, recently demonstrated significant but not complete protection in a non-human primate model of Ebola virus disease. In this study, we assessed the benefit of the combination of favipiravir and ribavirin, another broad spectrum antiviral agent, in the same model. METHODS: 15 female cynomolgus macaques were challenged intramuscularly with 1,000 FFU of Ebola virus Gabon 2001 strain and followed for 21 days. All animals received favipiravir 180 mg/kg twice a day (BID), either as monotherapy (n = 5) or in combination with ribavirin (n = 10). Ribavirin was given either at the dose 10 mg/kg BID (n = 5) or 5 mg/kg BID (n = 5). Favipiravir and ribavirin were initiated two and one days before viral challenge respectively and treatment were continued for 14 days. Treatment effects on viral and hematological markers were assessed using a mathematical model. Survival rate of 0% and 20% were obtained in macaques receiving favipiravir plus ribavirin 10 and 5 mg/kg BID, respectively, compared to 40% in the favipiravir monotherapy group (P = 0.061 when comparing monotherapy and bitherapy, log rank). Viral dynamic modeling analysis did not identify an association between plasma concentrations of ribavirin and viral load levels. Using a model of erythropoiesis, plasma concentrations of ribavirin were strongly associated with a hemoglobin drop (p = 0.0015). CONCLUSION: Ribavirin plus favipiravir did not extend survival rates and did not lower viral replication rate compared to favipiravir monotherapy in this animal model. Patients receiving this combination in other indications, such as Lassa fever, should be closely monitored to prevent potential toxicity associated with anemia.