Setup of Aerosol Delivery System to Prevent Measles Virus Infection in Nonhuman Primate Model.

Measles virus is one of the most contagious airborne human viruses which keeps causing outbreaks in numerous countries over the world despite the existence of an efficient vaccine. Fusion inhibitory lipopeptides were shown to inhibit viral entry into target cells, and their adequate administration into the respiratory tract may provide a novel preventive approach against airborne infections. Aerosol delivery presents the best administration route to deliver such preventive compounds to the upper and lower respiratory tract. This approach offers a conceptually new strategy to protect the population at risk against infection by respiratory viruses, including measles. It is a noninvasive needle-free approach, which may be used when antiviral protection is required, without any medical assistance. In this chapter, we describe the nebulization approach of lipopeptide compounds in nonhuman primates and the subsequent measles virus challenge.

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.

Mouse Models of Henipavirus Infection.

The Nipah and Hendra viruses, belonging to henipavirus genus, are recently emerged zoonotic pathogens that cause severe and often fatal, neurologic, and/or respiratory diseases in both humans and various animals. As mice represent a small animal model convenient to study viral infections and provide a well-developed experimental toolbox for analysis in immunovirology, we describe in this chapter a few basic methods used in biosafety 4 level (BSL4) conditions to study henipavirus infection in mice.

SARS-CoV-2 awakens ancient retroviral genes and the expression of proinflammatory HERV-W envelope protein in COVID-19 patients.

Patients with COVID-19 may develop abnormal inflammatory response, followed in some cases by severe disease and long-lasting syndromes. We show here that in vitro exposure to SARS-CoV-2 activates the expression of the human endogenous retrovirus (HERV) HERV-W proinflammatory envelope protein (ENV) in peripheral blood mononuclear cells from a subset of healthy donors, in ACE2 receptor and infection-independent manner. Plasma and/or sera of 221 COVID-19 patients from different cohorts, infected with successive SARS-CoV-2 variants including the Omicron, had detectable HERV-W ENV, which correlated with ENV expression in T lymphocytes and peaked with the disease severity. HERV-W ENV was also found in postmortem tissues of lungs, heart, gastrointestinal tract, brain olfactory bulb, and nasal mucosa from COVID-19 patients. Altogether, these results demonstrate that SARS-CoV-2 could induce HERV-W envelope protein expression and suggest its involvement in the immunopathogenesis of certain COVID-19-associated syndromes and thereby its relevance in the development of personalized treatment of patients.

Interplay between RNA viruses and cGAS/STING axis in innate immunity.

While the function of cGAS/STING signalling axis in the innate immune response to DNA viruses is well deciphered, increasing evidence demonstrates its significant contribution in the control of RNA virus infections. After the first evidence of cGAS/STING antagonism by flaviviruses, STING activation has been detected following infection by various enveloped RNA viruses. It has been discovered that numerous viral families have implemented advanced strategies to antagonize STING pathway through their evolutionary path. This review summarizes the characterized cGAS/STING escape strategies to date, together with the proposed mechanisms of STING signalling activation perpetrated by RNA viruses and discusses possible therapeutic approaches. Further studies regarding the interaction between RNA viruses and cGAS/STING-mediated immunity could lead to major discoveries important for the understanding of immunopathogenesis and for the treatment of RNA viral infections.

Measles Virus-Induced Host Immunity and Mechanisms of Viral Evasion.

The immune system deploys a complex network of cells and signaling pathways to protect host integrity against exogenous threats, including measles virus (MeV). However, throughout its evolutionary path, MeV developed various mechanisms to disrupt and evade immune responses. Despite an available vaccine, MeV remains an important re-emerging pathogen with a continuous increase in prevalence worldwide during the last decade. Considerable knowledge has been accumulated regarding MeV interactions with the innate immune system through two antagonistic aspects: recognition of the virus by cellular sensors and viral ability to inhibit the induction of the interferon cascade. Indeed, while the host could use several innate adaptors to sense MeV infection, the virus is adapted to unsettle defenses by obstructing host cell signaling pathways. Recent works have highlighted a novel aspect of innate immune response directed against MeV unexpectedly involving DNA-related sensing through activation of the cGAS/STING axis, even in the absence of any viral DNA intermediate. In addition, while MeV infection most often causes a mild disease and triggers a lifelong immunity, its tropism for invariant T-cells and memory T and B-cells provokes the elimination of one primary shield and the pre-existing immunity against previously encountered pathogens, known as "immune amnesia".

Nebulized fusion inhibitory peptide protects cynomolgus macaques from measles virus infection.

Measles is the most contagious airborne viral infection and the leading cause of child death among vaccine-preventable diseases. We show here that aerosolized lipopeptide fusion inhibitor, derived from heptad-repeat regions of the measles virus (MeV) fusion protein, blocks respiratory MeV infection in a non-human primate model, the cynomolgus macaque. We use a custom-designed mesh nebulizer to ensure efficient aerosol delivery of peptide to the respiratory tract and demonstrate the absence of adverse effects and lung pathology in macaques. The nebulized peptide efficiently prevents MeV infection, resulting in the complete absence of MeV RNA, MeV-infected cells, and MeV-specific humoral responses in treated animals. This strategy provides an additional means to fight against respiratory infection in non-vaccinated people, that can be readily translated to human trials. It presents a proof-of-concept for the aerosol delivery of fusion inhibitory peptides to protect against measles and other airborne viruses, including SARS-CoV-2, in case of high-risk exposure.

Hamster organotypic kidney culture model of early-stage SARS-CoV-2 infection highlights a two-step renal susceptibility.

Kidney pathology is frequently reported in patients hospitalized with COVID-19, the pandemic disease caused by the Severe acute respiratory coronavirus 2 (SARS-CoV-2). However, due to a lack of suitable study models, the events occurring in the kidney during the earliest stages of infection remain unknown. We have developed hamster organotypic kidney cultures (OKCs) to study the early stages of direct renal infection. OKCs maintained key renal structures in their native three-dimensional arrangement. SARS-CoV-2 productively replicated in hamster OKCs, initially targeting endothelial cells and later disseminating into proximal tubules. We observed a delayed interferon response, markers of necroptosis and pyroptosis, and an early repression of pro-inflammatory cytokines transcription followed by a strong later upregulation. While it remains an open question whether an active replication of SARS-CoV-2 takes place in the kidneys of COVID-19 patients with AKI, our model provides new insights into the kinetics of SARS-CoV-2 kidney infection and can serve as a powerful tool for studying kidney infection by other pathogens and testing the renal toxicity of drugs.

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Nebulized fusion inhibitory peptide protects cynomolgus macaques from measles virus infection.

Measles is the most contagious airborne viral infection and the leading cause of child death among vaccine-preventable diseases. We show here that aerosolized lipopeptide fusion inhibitors, derived from heptad-repeat regions of the measles virus (MeV) fusion protein, block respiratory MeV infection in a non-human primate model, the cynomolgus macaque. We used a custom-designed mesh nebulizer to ensure efficient aerosol delivery of peptides to the respiratory tract and demonstrated the absence of adverse effects and lung pathology in macaques. The nebulized peptide efficiently prevented MeV infection, resulting in the complete absence of MeV RNA, MeV-infected cells, and MeV-specific humoral responses in treated animals. This strategy provides an additional shield which complements vaccination to fight against respiratory infection, presenting a proof-of-concept for the aerosol delivery of fusion inhibitory peptides to protect against measles and other airborne viruses, including SARS-CoV-2, in case of high-risk exposure, that can be readily translated to human trials.

Fruit bats as natural reservoir of highly pathogenic henipaviruses: balance between antiviral defense and viral toleranceInteractions between Henipaviruses and their natural host, fruit bats.

Bats are the natural reservoir host for a number of zoonotic viruses, including Hendra and Nipah viruses of Henipavirus genus, which are highly pathogenic in humans and numerous other mammalian species. Despite being infected, bats present limited signs of disease but still retain the ability to transmit the infection to other susceptible hosts, presenting thus a permanent source of new viral outbreaks. Different mechanisms have evolved in fruit bats permitting them to efficiently control the Henipavirus infection. These mechanisms likely allow bats to establish an adequate equilibrium between viral tolerance and antiviral defense, enabling them thus to avoid both uncontrollable virus expansion as well as immunopathology linked to excessive antiviral responses.

Reprogrammed Pteropus Bat Stem Cells as A Model to Study Host-Pathogen Interaction during Henipavirus Infection.

Bats are natural hosts for numerous zoonotic viruses, including henipaviruses, which are highly pathogenic for humans, livestock, and other mammals but do not induce clinical disease in bats. Pteropus bats are identified as a reservoir of henipaviruses and the source of transmission of the infection to humans over the past 20 years. A better understanding of the molecular and cellular mechanisms allowing bats to control viral infections requires the development of relevant, stable, and permissive cellular experimental models. By applying a somatic reprogramming protocol to Pteropus bat primary cells, using a combination of ESRRB (Estrogen Related Receptor Beta), CDX2 (Caudal type Homeobox 2), and c-MYC (MYC proto-oncogene) transcription factors, we generated bat reprogrammed cells. These cells exhibit stem cell-like characteristics and neural stem cell molecular signature. In contrast to primary fibroblastic cells, these reprogrammed stem cells are highly permissive to henipaviruses and exhibit specific transcriptomic profiles with the particular expression of certain susceptibility factors such as interferon-stimulated genes (ISG), which may be related to viral infection. These Pteropus bat reprogrammed stem cells should represent an important experimental tool to decipher interactions during henipaviruses infection in Pteropus bats, facilitate isolation and production of bat-borne viruses, and to better understand the bat biology.

Nipah virus W protein harnesses nuclear 14-3-3 to inhibit NF-κB-induced proinflammatory response.

Nipah virus (NiV) is a highly pathogenic emerging bat-borne Henipavirus that has caused numerous outbreaks with public health concerns. It is able to inhibit the host innate immune response. Since the NF-κB pathway plays a crucial role in the innate antiviral response as a major transcriptional regulator of inflammation, we postulated its implication in the still poorly understood NiV immunopathogenesis. We report here that NiV inhibits the canonical NF-κB pathway via its nonstructural W protein. Translocation of the W protein into the nucleus causes nuclear accumulation of the cellular scaffold protein 14-3-3 in both African green monkey and human cells infected by NiV. Excess of 14-3-3 in the nucleus was associated with a reduction of NF-κB p65 subunit phosphorylation and of its nuclear accumulation. Importantly, W-S449A substitution impairs the binding of the W protein to 14-3-3 and the subsequent suppression of NF-κB signaling, thus restoring the production of proinflammatory cytokines. Our data suggest that the W protein increases the steady-state level of 14-3-3 in the nucleus and consequently enhances 14-3-3-mediated negative feedback on the NF-κB pathway. These findings provide a mechanistic model of W-mediated disruption of the host inflammatory response, which could contribute to the high severity of NiV infection.

Highly Potent Host-Specific Small-Molecule Inhibitor of Paramyxovirus and Pneumovirus Replication with High Resistance Barrier.

Multiple enveloped RNA viruses of the family Paramyxoviridae and Pneumoviridae, like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine. Therefore, there is an urgent need for treatment options to counteract these viral infections. The development of antiviral drugs in general stands as a huge challenge due to the rapid emergence of viral escape mutants. Here, we disclose the discovery of a small-molecule antiviral, compound 1 (ZHAWOC9045), active against several pneumo-/paramyxoviruses, including MeV, NiV, CDV, RSV, and parainfluenza virus type 5 (PIV-5). A series of mechanistic characterizations revealed that compound 1 targets a host factor which is indispensable for viral genome replication. Drug resistance profiling against a paramyxovirus model (CDV) demonstrated no detectable adaptation despite prolonged time of investigation, thereby mitigating the rapid emergence of escape variants. Furthermore, a thorough structure-activity relationship analysis of compound 1 led to the invention of 100-times-more potent-derivatives, e.g., compound 2 (ZHAWOC21026). Collectively, we present in this study an attractive host-directed pneumoviral/paramyxoviral replication inhibitor with potential therapeutic application. IMPORTANCE Measles virus, respiratory syncytial virus, canine distemper virus, and Nipah virus are some of the clinically significant RNA viruses that threaten substantial number of lives each year. Limited to no availability of treatment options for these viral infections makes it arduous to handle the outbreaks. This highlights the major importance of developing antivirals to fight not only ongoing infections but also potential future epidemics. Most of the discovered antivirals, in clinical trials currently, are virus targeted, which consequently poses the challenge of rapid emergence of escape variants. Here, we present compound 1 (ZHAWOC9045), discovered to target viral replication in a host-dependent manner, thereby exhibiting broad-spectrum activity against several members of the family Pneumo-/Paramyxoviridae. The inability of viruses to mutate against the inhibitor mitigated the critical issue of generation of escape variants. Importantly, compound 1 was successfully optimized to a highly potent variant, compound 2 (ZHAWOC21026), with a promising profile for pharmacological intervention.

Hamster organotypic modeling of SARS-CoV-2 lung and brainstem infection.

SARS-CoV-2 has caused a global pandemic of COVID-19 since its emergence in December 2019. The infection causes a severe acute respiratory syndrome and may also spread to central nervous system leading to neurological sequelae. We have developed and characterized two new organotypic cultures from hamster brainstem and lung tissues that offer a unique opportunity to study the early steps of viral infection and screening antivirals. These models are not dedicated to investigate how the virus reaches the brain. However, they allow validating the early tropism of the virus in the lungs and demonstrating that SARS-CoV-2 could infect the brainstem and the cerebellum, mainly by targeting granular neurons. Viral infection induces specific interferon and innate immune responses with patterns specific to each organ, along with cell death by apoptosis, necroptosis, and pyroptosis. Overall, our data illustrate the potential of rapid modeling of complex tissue-level interactions during infection by a newly emerged virus.

Identification of a Region in the Common Amino-terminal Domain of Hendra Virus P, V, and W Proteins Responsible for Phase Transition and Amyloid Formation.

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a ß-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200-310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

A Bioluminescent 3CLPro Activity Assay to Monitor SARS-CoV-2 Replication and Identify Inhibitors.

Our therapeutic arsenal against viruses is very limited and the current pandemic of SARS-CoV-2 highlights the critical need for effective antivirals against emerging coronaviruses. Cellular assays allowing a precise quantification of viral replication in high-throughput experimental settings are essential to the screening of chemical libraries and the selection of best antiviral chemical structures. To develop a reporting system for SARS-CoV-2 infection, we generated cell lines expressing a firefly luciferase maintained in an inactive form by a consensus cleavage site for the viral protease 3CLPro of coronaviruses, so that the luminescent biosensor is turned on upon 3CLPro expression or SARS-CoV-2 infection. This cellular assay was used to screen a metabolism-oriented library of 492 compounds to identify metabolic vulnerabilities of coronaviruses for developing innovative therapeutic strategies. In agreement with recent reports, inhibitors of pyrimidine biosynthesis were found to prevent SARS-CoV-2 replication. Among the top hits, we also identified the NADPH oxidase (NOX) inhibitor Setanaxib. The anti-SARS-CoV-2 activity of Setanaxib was further confirmed using ACE2-expressing human pulmonary cells Beas2B as well as human primary nasal epithelial cells. Altogether, these results validate our cell-based functional assay and the interest of screening libraries of different origins to identify inhibitors of SARS-CoV-2 for drug repurposing or development.

Rapid and Flexible Platform To Assess Anti-SARS-CoV-2 Antibody Neutralization and Spike Protein-Specific Antivirals.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is ongoing and has shown the community that flexible methods for rapidly identifying and screening candidate antivirals are needed. Assessing virus-neutralizing activity of human serum to monitor population immunity and response to infection and vaccination is key to pandemic control. We developed a virus neutralization platform strategy that relies only on bioinformatic and genetic information of the virus of interest. The platform uses viral envelope glycoprotein cDNAs to set up an assay that mimics multicycle infection but is safe and, therefore, amenable to biosafety level 2 (BSL2) conditions for viruses that require BSL3 facilities (e.g., SARS-CoV-1 and SARS-CoV-2). As a complement to this platform, we present a new cell-based immunofluorescent (CBI) assay that uses SARS-CoV-2 spike protein (S)-expressing cells to accurately measure the neutralization potential of human sera and is readily adaptable to variants of concern. These methods should be useful additions to the tools for assessing antiviral immunity, whether acquired via natural infection or vaccines. IMPORTANCE Assays for rapid biosafety level 2 (BSL2) evaluation of neutralizing properties of antibodies acquired via natural infection or through vaccination is urgently needed. Here, we propose a combinatorial approach in which sera are screened for SARS-CoV-2 spike protein (S) binding using a cell-based immunofluorescent (CBI) assay, and positive samples are further evaluated in a pseudotyped viral multicycle infection-mimicking protocol under BSL2 conditions.

Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain.

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.