Inoculation of raccoons with a wild-type-based recombinant canine distemper virus results in viremia, lymphopenia, fever, and widespread histological lesions.

Raccoons are naturally susceptible to canine distemper virus (CDV) infection and can be a potential source of spill-over events. CDV is a highly contagious morbillivirus that infects multiple species of carnivores and omnivores, resulting in severe and often fatal disease. Here, we used a recombinant CDV (rCDV) based on a full-genome sequence detected in a naturally infected raccoon to perform pathogenesis studies in raccoons. Five raccoons were inoculated intratracheally with a recombinant virus engineered to express a fluorescent reporter protein, and extensive virological, serological, histological, and immunohistochemical assessments were performed at different time points post inoculation. rCDV-infected white blood cells were detected as early as 4 days post inoculation (dpi). Raccoon necropsies at 6 and 8 dpi revealed replication in the lymphoid tissues, preceding spread into peripheral tissues observed during necropsies at 21 dpi. Whereas lymphocytes, and to a lesser extent myeloid cells, were the main target cells of CDV at early time points, CDV additionally targeted epithelia at 21 dpi. At this later time point, CDV-infected cells were observed throughout the host. We observed lymphopenia and lymphocyte depletion from lymphoid tissues after CDV infection, in the absence of detectable CDV neutralizing antibodies and an impaired ability to clear CDV, indicating that the animals were severely immunosuppressed. The use of a wild-type-based recombinant virus in a natural host species infection study allowed systematic and sensitive assessment of antigen detection by immunohistochemistry, enabling further comparative pathology studies of CDV infection in different species. IMPORTANCE Expansion of the human interface supports increased interactions between humans and peridomestic species like raccoons. Raccoons are highly susceptible to canine distemper virus (CDV) and are considered an important target species. Spill-over events are increasingly likely, potentially resulting in fatal CDV infections in domestic and free ranging carnivores. CDV also poses a threat for (non-human) primates, as massive outbreaks in macaque colonies were reported. CDV pathogenesis was studied by experimental inoculation of several species, but pathogenesis in raccoons was not properly studied. Recently, we generated a recombinant virus based on a full-genome sequence detected in a naturally infected raccoon. Here, we studied CDV pathogenesis in its natural host species and show that distemper completely overwhelms the immune system and spreads to virtually all tissues, including the central nervous system. Despite this, raccoons survived up to 21 d post inoculation with long-term shedding, supporting an important role of raccoons as host species for CDV.

Preclinical immunogenicity and protective efficacy of a SARS-CoV-2 RBD-based vaccine produced with the thermophilic filamentous fungal expression system Thermothelomyces heterothallica C1.

Introduction: The emergency use of vaccines has been the most efficient way to control the coronavirus disease 19 (COVID-19) pandemic. However, the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern has reduced the efficacy of currently used vaccines. The receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein is the main target for virus neutralizing (VN) antibodies. Methods: A SARS-CoV-2 RBD vaccine candidate was produced in the Thermothelomyces heterothallica (formerly, Myceliophthora thermophila) C1 protein expression system and coupled to a nanoparticle. Immunogenicity and efficacy of this vaccine candidate was tested using the Syrian golden hamster (Mesocricetus auratus) infection model. Results: One dose of 10-µg RBD vaccine based on SARS-CoV-2 Wuhan strain, coupled to a nanoparticle in combination with aluminum hydroxide as adjuvant, efficiently induced VN antibodies and reduced viral load and lung damage upon SARS-CoV-2 challenge infection. The VN antibodies neutralized SARS-CoV-2 variants of concern: D614G, Alpha, Beta, Gamma, and Delta. Discussion: Our results support the use of the Thermothelomyces heterothallica C1 protein expression system to produce recombinant vaccines against SARS-CoV-2 and other virus infections to help overcome limitations associated with the use of mammalian expression system.

SARS-CoV-2 Omicron variant causes mild pathology in the upper and lower respiratory tract of hamsters.

Since its discovery in 2019, multiple variants of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been identified. This study investigates virus spread and associated pathology in the upper and lower respiratory tracts of Syrian golden hamsters at 4 days post intranasal SARS-CoV-2 Omicron infection, in comparison to infection with variants of concern (VOCs) Gamma and Delta as well as ancestral strain 614 G. Pathological changes in the upper and lower respiratory tract of VOC Omicron infected hamsters are milder than those caused by other investigated strains. VOC Omicron infection causes a mild rhinitis with little involvement of the olfactory epithelium and minimal lesions in the lung, with frequent sparing of the alveolar compartment. Similarly, viral antigen, RNA and infectious virus titers are lower in respiratory tissues of VOC Omicron infected hamsters. These findings demonstrate that the variant has a decreased pathogenicity for the upper and lower respiratory tract of hamsters.

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants of concern.

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays notable immune escape potential through mutations at key antigenic sites on the spike protein. Many of these mutations localize to the spike protein ACE2 receptor binding domain, annulling the neutralizing activity of therapeutic antibodies that were effective against other variants of concern (VOCs) earlier in the pandemic. Here, we identified a receptor-blocking human monoclonal antibody, 87G7, that retained potent in vitro neutralizing activity against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta, and Omicron (BA.1/BA.2) VOCs. Using cryo-electron microscopy and site-directed mutagenesis experiments, we showed that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protected mice and hamsters prophylactically against challenge with all current SARS-CoV-2 VOCs and showed therapeutic activity against SARS-CoV-2 challenge in both animal models. Our findings demonstrate that 87G7 holds promise as a prophylactic or therapeutic agent for COVID-19 that is more resilient to SARS-CoV-2 antigenic diversity.

Development of molecular clamp stabilized hemagglutinin vaccines for Influenza A viruses.

Influenza viruses cause a significant number of infections and deaths annually. In addition to seasonal infections, the risk of an influenza virus pandemic emerging is extremely high owing to the large reservoir of diverse influenza viruses found in animals and the co-circulation of many influenza subtypes which can reassort into novel strains. Development of a universal influenza vaccine has proven extremely challenging. In the absence of such a vaccine, rapid response technologies provide the best potential to counter a novel influenza outbreak. Here, we demonstrate that a modular trimerization domain known as the molecular clamp allows the efficient production and purification of conformationally stabilised prefusion hemagglutinin (HA) from a diverse range of influenza A subtypes. These clamp-stabilised HA proteins provided robust protection from homologous virus challenge in mouse and ferret models and some cross protection against heterologous virus challenge. This work provides a proof-of-concept for clamp-stabilised HA vaccines as a tool for rapid response vaccine development against future influenza A virus pandemics.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Susceptibility of rabbits to SARS-CoV-2.

Transmission of severe acute respiratory coronavirus-2 (SARS-CoV-2) between livestock and humans is a potential public health concern. We demonstrate the susceptibility of rabbits to SARS-CoV-2, which excrete infectious virus from the nose and throat upon experimental inoculation. Therefore, investigations on the presence of SARS-CoV-2 in farmed rabbits should be considered.

Immunogenicity and efficacy comparison of MDCK cell-based and egg-based live attenuated influenza vaccines of H5 and H7 subtypes in ferrets.

During a pandemic, the availability of specific pathogen free chicken eggs is a major bottleneck for up-scaling response to the demand for influenza vaccine. This has led us to explore the use of Madin-Darby Canine Kidney (MDCK) cells for the manufacture of live attenuated influenza vaccine (LAIV) that provides production flexibility and speed. The present study reports the comparison of the immunogenicity and efficacy of two MDCK-based LAIVs against two egg-based LAIVs prepared from the same pandemic potential strains of H5 and H7 subtypes after a single dose of the vaccine followed by a challenge with a homologous wild type strain. The vaccine strains have been generated by classical method of reassortment using the A/Leningrad/134/17/57 master donor strain. Additionally, a prime-boost regimen of the MDCK-based vaccine followed by a challenge with a homologous wild type strain for H5 and H7 immunized ferrets and also a heterologous wild type strain for the H5 immunized animals was studied. No difference in the hemagglutination inhibition and virus neutralization antibody titers against the homologous virus was observed following a single dose of either egg-based or MDCK-based H5 and H7 LAIV vaccine. A second dose of MDCK-based vaccine significantly boosted antibody titers in the vaccinated animals. Both a single dose or two doses of LAIV provided complete protection from lower respiratory tract infection and resulted in a significant reduction in the virus titers recovered from the throat, nasal turbinates and lungs after challenge with the homologous wild type strain. Protection from a challenge with a heterologous strain of H5 was also observed after two doses of the MDCK-based LAIVs. This data strongly supports the use of MDCK as a substrate for the manufacture of LAIV which ensures reliable quality, safety, production flexibility, speed and breadth of protection, features that are highly critical during a pandemic.

Influenza-induced thrombocytopenia is dependent on the subtype and sialoglycan receptor and increases with virus pathogenicity.

Thrombocytopenia is a common complication of influenza virus infection, and its severity predicts the clinical outcome of critically ill patients. The underlying cause(s) remain incompletely understood. In this study, in patients with an influenza A/H1N1 virus infection, viral load and platelet count correlated inversely during the acute infection phase. We confirmed this finding in a ferret model of influenza virus infection. In these animals, platelet count decreased with the degree of virus pathogenicity varying from 0% in animals infected with the influenza A/H3N2 virus, to 22% in those with the pandemic influenza A/H1N1 virus, up to 62% in animals with a highly pathogenic A/H5N1 virus infection. This thrombocytopenia is associated with virus-containing platelets that circulate in the blood. Uptake of influenza virus particles by platelets requires binding to sialoglycans and results in the removal of sialic acids by the virus neuraminidase, a trigger for hepatic clearance of platelets. We propose the clearance of influenza virus by platelets as a paradigm. These insights clarify the pathophysiology of influenza virus infection and show how severe respiratory infections, including COVID-19, may propagate thrombocytopenia and/or thromboembolic complications.

Particulate multivalent presentation of the receptor binding domain induces protective immune responses against MERS-CoV.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a WHO priority pathogen for which vaccines are urgently needed. Using an immune-focusing approach, we created self-assembling particles multivalently displaying critical regions of the MERS-CoV spike protein ─fusion peptide, heptad repeat 2, and receptor binding domain (RBD) ─ and tested their immunogenicity and protective capacity in rabbits. Using a "plug-and-display" SpyTag/SpyCatcher system, we coupled RBD to lumazine synthase (LS) particles producing multimeric RBD-presenting particles (RBD-LS). RBD-LS vaccination induced antibody responses of high magnitude and quality (avidity, MERS-CoV neutralizing capacity, and mucosal immunity) with cross-clade neutralization. The antibody responses were associated with blocking viral replication and upper and lower respiratory tract protection against MERS-CoV infection in rabbits. This arrayed multivalent presentation of the viral RBD using the antigen-SpyTag/LS-SpyCatcher is a promising MERS-CoV vaccine candidate and this platform may be applied for the rapid development of vaccines against other emerging viruses such as SARS-CoV-2.

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model.

The current pandemic coronavirus, severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), was recently identified in patients with an acute respiratory syndrome, coronavirus disease 2019 (COVID-19). To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or Middle East respiratory syndrome (MERS)-CoV and compared the pathology and virology with historical reports of SARS-CoV infections. In SARS-CoV-2-infected macaques, virus was excreted from nose and throat in the absence of clinical signs and detected in type I and II pneumocytes in foci of diffuse alveolar damage and in ciliated epithelial cells of nasal, bronchial, and bronchiolar mucosae. In SARS-CoV infection, lung lesions were typically more severe, whereas they were milder in MERS-CoV infection, where virus was detected mainly in type II pneumocytes. These data show that SARS-CoV-2 causes COVID-19-like disease in macaques and provides a new model to test preventive and therapeutic strategies.

Lack of Middle East Respiratory Syndrome Coronavirus Transmission in Rabbits.

Middle East respiratory syndrome coronavirus (MERS-CoV) transmission from dromedaries to humans has resulted in major outbreaks in the Middle East. Although some other livestock animal species have been shown to be susceptible to MERS-CoV, it is not fully understood why the spread of the virus in these animal species has not been observed in the field. In this study, we used rabbits to further characterize the transmission potential of MERS-CoV. In line with the presence of MERS-CoV receptor in the rabbit nasal epithelium, high levels of viral RNA were shed from the nose following virus inoculation. However, unlike MERS-CoV-infected dromedaries, these rabbits did not develop clinical manifestations including nasal discharge and did shed only limited amounts of infectious virus from the nose. Consistently, no transmission by contact or airborne routes was observed in rabbits. Our data indicate that despite relatively high viral RNA levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission.

Influenza A/H3N2 virus infection in immunocompromised ferrets and emergence of antiviral resistance.

Influenza viruses can cause severe life threatening infections in high-risk patients, including young children, the elderly and patients with compromised immunity due to underlying medical conditions or immunosuppressive treatment. The impaired immunity of these patients causes prolonged virus infection and combined with antiviral treatment facilitates the emergence of viruses with resistance mutations. The diverse nature of their immune status makes them a challenging group to study the impact of influenza virus infection and the efficacy of antiviral therapy. Immunocompromised ferrets may represent a suitable animal model to assess influenza virus infection and antiviral treatment strategies in immunocompromised hosts. Here, ferrets were given a daily oral solution of mycophenolate mofetil, tacrolimus and prednisolone sodium phosphate to suppress their immune system. Groups of immunocompromised and immunocompetent ferrets were inoculated with an A/H3N2 influenza virus and were subsequently treated with Oseltamivir or left untreated. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed on the throat and nose specimens to study virus replication during the course of infection. All immunocompromised ferrets had prolonged presence of viral RNA and a higher total amount of virus shedding compared to the immunocompetent ferrets. Although Oseltamivir reduced the total amount of virus shedding from the nose and throat of treated ferrets, it also resulted in the emergence of the neuraminidase R292K resistance substitution in all these animals, as determined by mutation specific RT-PCR and next-generation sequencing. No additional mutations that could be associated with the emergence of the R292K resistance mutation were detected. The immunocompromised ferret model can be used to study A/H3N2 virus shedding and is a promising model to study new antiviral strategies and the emergence of antiviral resistance in immunocompromised hosts.

Analysis of Mouse Brain Transcriptome After Experimental Duvenhage Virus Infection Shows Activation of Innate Immune Response and Pyroptotic Cell Death Pathway.

Rabies is an important neglected disease, characterized by invariably fatal encephalitis. Several studies focus on understanding the pathogenic mechanisms of the prototype lyssavirus rabies virus (RABV) infection, and little is known about the pathogenesis of rabies caused by other lyssaviruses. We sought to characterize the host response to Duvenhage virus infection and compare it with responses observed during RABV infection by gene expression profiling of brains of mice with the respective infections. We found in both infections differentially expressed genes leading to increased expression of type I interferons (IFNs), chemokines, and proinflammatory cytokines. In addition several genes of the IFN signaling pathway are up-regulated, indicating a strong antiviral response and activation of the negative feedback mechanism to limit type I IFN responses. Furthermore we provide evidence that in the absence of significant neuronal apoptotic death, cell death of neurons is mediated via the pyroptotic pathway in both infections. Taken together, we have identified several genes and/or pathways for both infections that could be used to explore novel approaches for intervention strategies against rabies.

Transmission of Human Respiratory Syncytial Virus in the Immunocompromised Ferret Model.

Human respiratory syncytial virus (HRSV) causes substantial morbidity and mortality in vulnerable patients, such as the very young, the elderly, and immunocompromised individuals of any age. Nosocomial transmission of HRSV remains a serious challenge in hospital settings, with intervention strategies largely limited to infection control measures, including isolation of cases, high standards of hand hygiene, cohort nursing, and use of personal protective equipment. No vaccines against HRSV are currently available, and treatment options are largely supportive care and expensive monoclonal antibody or antiviral therapy. The limitations of current animal models for HRSV infection impede the development of new preventive and therapeutic agents, and the assessment of their potential for limiting HRSV transmission, in particular in nosocomial settings. Here, we demonstrate the efficient transmission of HRSV from immunocompromised ferrets to both immunocompromised and immunocompetent contact ferrets, with pathological findings reproducing HRSV pathology in humans. The immunocompromised ferret-HRSV model represents a novel tool for the evaluation of intervention strategies against nosocomial transmission of HRSV.

Needle-free delivery of measles virus vaccine to the lower respiratory tract of non-human primates elicits optimal immunity and protection.

Needle-free measles virus vaccination by aerosol inhalation has many potential benefits. The current standard route of vaccination is subcutaneous injection, whereas measles virus is an airborne pathogen. However, the target cells that support replication of live-attenuated measles virus vaccines in the respiratory tract are largely unknown. The aims of this study were to assess the in vivo tropism of live-attenuated measles virus and determine whether respiratory measles virus vaccination should target the upper or lower respiratory tract. Four groups of twelve cynomolgus macaques were immunized with 104 TCID50 of recombinant measles virus vaccine strain Edmonston-Zagreb expressing enhanced green fluorescent protein. The vaccine virus was grown in MRC-5 cells and formulated with identical stabilizers and excipients as used in the commercial MVEZ vaccine produced by the Serum Institute of India. Animals were immunized by hypodermic injection, intra-tracheal inoculation, intra-nasal instillation, or aerosol inhalation. In each group six animals were euthanized at early time points post-vaccination, whereas the other six were followed for 14 months to assess immunogenicity and protection from challenge infection with wild-type measles virus. At early time-points, enhanced green fluorescent protein-positive measles virus-infected cells were detected locally in the muscle, nasal tissues, lungs, and draining lymph nodes. Systemic vaccine virus replication and viremia were virtually absent. Infected macrophages, dendritic cells and tissue-resident lymphocytes predominated. Exclusive delivery of vaccine virus to the lower respiratory tract resulted in highest immunogenicity and protection. This study sheds light on the tropism of a live-attenuated measles virus vaccine and identifies the alveolar spaces as the optimal site for respiratory delivery of measles virus vaccine.

Pigeon paramyxovirus type 1 from a fatal human case induces pneumonia in experimentally infected cynomolgus macaques (Macaca fascicularis).

Although avian paramyxovirus type 1 is known to cause mild transient conjunctivitis in human beings, there are two recent reports of fatal respiratory disease in immunocompromised human patients infected with the pigeon lineage of the virus (PPMV-1). In order to evaluate the potential of PPMV-1 to cause respiratory tract disease, we inoculated a PPMV-1 isolate (hPPMV-1/Netherlands/579/2003) from an immunocompromised human patient into three healthy cynomolgus macaques (Macaca fascicularis) and examined them by clinical, virological, and pathological assays. In all three macaques, PPMV-1 replication was restricted to the respiratory tract and caused pulmonary consolidation affecting up to 30% of the lung surface. Both alveolar and bronchiolar epithelial cells expressed viral antigen, which co-localized with areas of diffuse alveolar damage. The results of this study demonstrate that PPMV-1 is a primary respiratory pathogen in cynomolgus macaques, and support the conclusion that PPMV-1 may cause fatal respiratory disease in immunocompromised human patients.

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo.

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.