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The novel coronavirus SARS-CoV-2 has caused significant global morbidity and mortality and continues to burden patients with persisting neurological dysfunction. COVID-19 survivors develop debilitating symptoms to include neuro-psychological dysfunction, termed “Long COVID”, which can cause significant reduction of quality of life. Despite vigorous model development, the possible cause of these symptoms and the underlying pathophysiology of this devastating disease remains elusive. Mouse adapted (MA10) SARS-CoV-2 is a novel mouse-based model of COVID-19 which simulates the clinical symptoms of respiratory distress associated with SARS-CoV-2 infection in mice. In this study, we evaluated the long-term effects of MA10 infection on brain pathology and neuroinflammation. 10-week and 1-year old female BALB/cAnNHsd mice were infected intranasally with 104plaque-forming units (PFU) and 103PFU of SARS-CoV-2 MA10, respectively, and the brain was examined 60 days post-infection (dpi). Immunohistochemical analysis showed a decrease in the neuronal nuclear protein NeuN and an increase in Iba-1 positive amoeboid microglia in the hippocampus after MA10 infection, indicating long-term neurological changes in a brain area which is critical for long-term memory consolidation and processing. Importantly, these changes were seen in 40-50% of infected mice, which correlates to prevalence of LC seen clinically. Our data shows for the first time that MA10 infection induces neuropathological outcomes several weeks after infection at similar rates of observed clinical prevalence of “Long COVID”. These observations strengthen the MA10 model as a viable model for study of the long-term effects of SARS-CoV-2 in humans. Establishing the viability of this model is a key step towards the rapid development of novel therapeutic strategies to ameliorate neuroinflammation and restore brain function in those suffering from the persistent cognitive dysfunction of “Long-COVID”.

A subset of individuals who recover from coronavirus disease 2019 (COVID-19) develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal tissue samples. The mouse-adapted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute to clinical recovery phases. At 15 to 120 days post-virus clearance, pulmonary histologic findings included subpleural lesions composed of collagen, proliferative fibroblasts, and chronic inflammation, including tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal up-regulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC., After recovery from acute SARS-CoV-2 infection, mice exhibit chronic lung disease similar to some humans, allowing for testing of therapeutics.

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.

COVID-19 survivors develop post-acute sequelae of SARS-CoV-2 (PASC), but the mechanistic basis of PASC-associated lung abnormalities suffers from a lack of longitudinal samples. Mouse-adapted SARS-CoV-2 MA10 produces an acute respiratory distress syndrome (ARDS) in mice similar to humans. To investigate PASC pathogenesis, studies of MA10-infected mice were extended from acute disease through clinical recovery. At 15-120 days post-virus clearance, histologic evaluation identified subpleural lesions containing collagen, proliferative fibroblasts, and chronic inflammation with tertiary lymphoid structures. Longitudinal spatial transcriptional profiling identified global reparative and fibrotic pathways dysregulated in diseased regions, similar to human COVID-19. Populations of alveolar intermediate cells, coupled with focal upregulation of pro-fibrotic markers, were identified in persistently diseased regions. Early intervention with antiviral EIDD-2801 reduced chronic disease, and early anti-fibrotic agent (nintedanib) intervention modified early disease severity. This murine model provides opportunities to identify pathways associated with persistent SARS-CoV-2 pulmonary disease and test countermeasures to ameliorate PASC.

The global COVID-19 pandemic has sparked intense interest in the rapid development of vaccines as well as animal models to evaluate vaccine candidates and to define immune correlates of protection. We recently reported a mouse-adapted SARS-CoV-2 virus strain (MA10) with the potential to infect wild-type laboratory mice, driving high levels of viral replication in respiratory tract tissues as well as severe clinical and respiratory symptoms, aspects of COVID-19 disease in humans that are important to capture in model systems. We evaluated the immunogenicity and protective efficacy of novel rhesus adenovirus serotype 52 (RhAd52) vaccines against MA10 challenge in mice. Baseline seroprevalence is lower for rhesus adenovirus vectors than for human or chimpanzee adenovirus vectors, making these vectors attractive candidates for vaccine development. We observed that RhAd52 vaccines elicited robust binding and neutralizing antibody titers, which inversely correlated with viral replication after challenge. These data support the development of RhAd52 vaccines and the use of the MA10 challenge virus to screen novel vaccine candidates and to study the immunologic mechanisms that underscore protection from SARS-CoV-2 challenge in wild-type mice.ImportanceWe have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibits a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative Ad vector for boosting. We sought to test these vaccines using a recently reported mouse-adapted SARS-CoV-2 (MA10) virus to i) evaluate the protective efficacy of RhAd52 vaccines and ii) further characterize this mouse-adapted challenge model and probe immune correlates of protection. We demonstrate RhAd52 vaccines elicit robust SARS-CoV-2-specific antibody responses and protect against clinical disease and viral replication in the lungs. Further, binding and neutralizing antibody titers correlated with protective efficacy. These data validate the MA10 mouse model as a useful tool to screen and study novel vaccine candidates, as well as the development of RhAd52 vaccines for COVID-19.

Vaccine-associated enhanced respiratory disease (VAERD) was previously observed in some preclinical models of severe acute respiratory syndrome (SARS) and MERS coronavirus vaccines. We used the SARS coronavirus 2 (SARS-CoV-2) mouse-adapted, passage 10, lethal challenge virus (MA10) mouse model of acute lung injury to evaluate the immune response and potential for immunopathology in animals vaccinated with research-grade mRNA-1273. Whole-inactivated virus or heat-denatured spike protein subunit vaccines with alum designed to elicit low-potency antibodies and Th2-skewed CD4+ T cells resulted in reduced viral titers and weight loss post challenge but more severe pathological changes in the lung compared to saline-immunized animals. In contrast, a protective dose of mRNA-1273 induced favorable humoral and cellular immune responses that protected from viral replication in the upper and lower respiratory tract upon challenge. A subprotective dose of mRNA-1273 reduced viral replication and limited histopathological manifestations compared to animals given saline. Overall, our findings demonstrate an immunological signature associated with antiviral protection without disease enhancement following vaccination with mRNA-1273., Graphical abstract, As vaccine-enhanced disease to respiratory viruses has been previously observed, a thorough safety evaluation of COVID-19 vaccines in preclinical animal models is essential. Here, DiPiazza and Leist et al. provide evidence for antiviral protection in the absence of lung disease following SARS-CoV-2 challenge in mice immunized with research-grade mRNA-1273.

There is a great need for the development of vaccines that induce potent and long-lasting protective immunity against SARS-CoV-2. Multimeric display of the antigen combined with potent adjuvant can enhance the potency and longevity of the antibody response. The receptor binding domain (RBD) of the spike protein is a primary target of neutralizing antibodies. Here, we developed a trimeric form of the RBD and show that it induces a potent neutralizing antibody response against live virus with diverse effector functions and provides protection against SARS-CoV-2 challenge in mice and rhesus macaques. The trimeric form induces higher neutralizing antibody titer compared to monomer with as low as 1μg antigen dose. In mice, adjuvanting the protein with a TLR7/8 agonist formulation alum-3M-052 induces 100-fold higher neutralizing antibody titer and superior protection from infection compared to alum. SARS-CoV-2 infection causes significant loss of innate cells and pathology in the lung, and vaccination protects from changes in innate cells and lung pathology. These results demonstrate RBD trimer protein as a suitable candidate for vaccine against SARS-CoV-2., Efficient vaccines for SARS-CoV-2 are needed. Here, the authors show that a trimeric form of the receptor-binding domain of SARS-CoV-2 spike adjuvanted with alum-3M-052 protects non-human primates from disease and inhibits infection.

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies., Graphical abstract, Hsieh et al. generate MERS-CoV spike stabilized stem (SS) antigens using a structure-guided approach. Mice immunized with MERS SS are protected against MERS-CoV challenge and used for isolation of cross-reactive monoclonal antibodies, including IgG22, which protects mice against MERS-CoV and SARS-CoV-2 challenges. Structures of Fab22-spike complexes reveal a conserved epitope.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2., Author summary Both mRNA-based and viral vector-based vaccines are currently being distributed to curtail the COVID-19 pandemic. Continued development of more varieties of SARS-CoV-2 vaccines will help battle the many variants of SARS-CoV-2. Here we have developed a virus-like particle (VLP) vaccine that combines the effectiveness of virus-based vaccines and safety of protein-based vaccines. Using the lumazine synthase nanoparticle protein as the structural scaffold and 120 copies of SARS-CoV-2 receptor-binding domain as the surface immunogen, this VLP vaccine induced high-titer neutralizing antibody responses in mice that lasted >2 months and potently inhibited SARS-CoV-2, SARS-CoV-1, and their variants. The VLP vaccine also protected mice from high-titer SARS-CoV-2 challenge. The novel VLP vaccine may contribute to the protection of the human population from SARS-CoV-2 and its variants.

A successful severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine must not only be safe and protective, but must also meet the demand on a global scale at a low cost. Using the current influenza virus vaccine production capacity to manufacture an egg-based inactivated Newcastle disease virus (NDV)/SARS-CoV-2 vaccine would meet that challenge. Here, we report pre-clinical evaluations of an inactivated NDV chimera stably expressing the membrane-anchored form of the spike (NDV-S) as a potent coronavirus disease 2019 (COVID-19) vaccine in mice and hamsters. The inactivated NDV-S vaccine was immunogenic, inducing strong binding and/or neutralizing antibodies in both animal models. More importantly, the inactivated NDV-S vaccine protected animals from SARS-CoV-2 infections. In the presence of an adjuvant, antigen-sparing could be achieved, which would further reduce the cost while maintaining the protective efficacy of the vaccine.

Significance The COVID-19 pandemic has already caused over 1 million deaths. Therefore, effective vaccine concepts are urgently needed. In search of such a concept, we have analyzed a measles virus-based vaccine candidate targeting SARS-CoV-2. Using this well-known, safe vaccine backbone, we demonstrate here induction of functional immune responses in both arms of adaptive immunity, yielding antiviral efficacy in vivo with the desired immune bias. Consequently, no immunopathologies became evident during challenge experiments. Moreover, the candidate still induces immunity against the measles, recognized as a looming second menace, when countries are forced to stop routine vaccination campaigns in the face of COVID-19. Thus, a bivalent measles-based COVID-19 vaccine could be the solution for two significant public health threats., The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread worldwide, with millions of cases and more than 1 million deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8+ and CD4+ T cell responses with S protein-specific killing activity were detected. Upon challenge using a mouse-adapted SARS-CoV-2, virus loads in vaccinated mice were significantly lower, while vaccinated Syrian hamsters revealed protection in a harsh challenge setup using an early-passage human patient isolate. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines.

Changing with the timesPandemic spread of a virus in naïe populations can select for mutations that alter pathogenesis, virulence, and/or transmissibility. The ancestral form of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that emerged from China has now been largely replaced by strains containing the mutation D614G (Asp614-to-Gly) in the viral spike protein. Houet al.compared the characteristics of the new variant against the ancestral form in a series of experiments in human cells and animal models. The variant is better at infecting upper-airway epithelial cells and replicates in greater numbers than the ancestral virus. Evidence indicates modest, if any, significant changes to virulence in animal models. Therefore, the virus appears to have evolved for greater transmissibility in humans rather than for greater pathogenicity. The mutation renders the new virus variant more susceptible to neutralizing antisera without altering the efficacy of vaccine candidates currently under development.Science, this issue p.1464

A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers ten-fold higher than the prefusion-stabilized spike despite a five-fold lower dose. Antibodies elicited by the RBD-nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-associated enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manufacture of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufacturing efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic., Highlights • Two-component nanoparticle platform enabled rapid generation of SARS-CoV-2 vaccines • The RBD-nanoparticle vaccines elicit potent neutralizing antibody responses • Nanoparticle vaccine-elicited antibodies target multiple non-overlapping epitopes • The lead nanoparticle vaccine candidate is being manufactured for clinical trials, Walls et al. describe a potential nanoparticle vaccine for COVID-19, made of a self-assembling protein nanoparticle displaying the SARS-CoV-2 receptor-binding domain in a highly immunogenic array reminiscent of the natural virus. Their nanoparticle vaccine candidate elicits a diverse, potent, and protective antibody response, including neutralizing antibody titers ten-fold higher than the prefusion-stabilized spike ectodomain trimer.

All known recently emerged human coronaviruses likely originated in bats. Here, we used a single experimental platform based on human lung-only mice (LoM) to demonstrate efficient in vivo replication of all recently emerged human coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2) and two highly relevant endogenous pre-pandemic SARS-like bat coronaviruses. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats harbor endogenous coronaviruses capable of direct transmission into humans. Further detailed analysis of pandemic SARS-CoV-2 in vivo infection of LoM human lung tissue showed predominant infection of human lung epithelial cells, including type II pneumocytes present in alveoli and ciliated airway cells. Acute SARS-CoV-2 infection was highly cytopathic and induced a robust and sustained Type I interferon and inflammatory cytokine/chemokine response. Finally, we evaluated a pre-exposure prophylaxis strategy for coronavirus infection. Our results show that prophylactic administration of EIDD-2801, an oral broad spectrum antiviral currently in phase II clinical trials for the treatment of COVID-19, dramatically prevented SARS-CoV-2 infection in vivo and thus has significant potential for the prevention and treatment of COVID-19.

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801—an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials—markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19. Human and bat coronaviruses replicate efficiently in immunodeficient mice implanted with human lung tissue, and treatment or prophylaxis using EIDD-2801 in this model suggests that this oral antiviral agent may be effective in preventing COVID-19.

The D614G substitution in the S protein is most prevalent SARS-CoV-2 strain circulating globally, but its effects in viral pathogenesis and transmission remain unclear. We engineered SARS-CoV-2 variants harboring the D614G substitution with or without nanoluciferase. The D614G variant replicates more efficiency in primary human proximal airway epithelial cells and is more fit than wildtype (WT) virus in competition studies. With similar morphology to the WT virion, the D614G virus is also more sensitive to SARS-CoV-2 neutralizing antibodies. Infection of human ACE2 transgenic mice and Syrian hamsters with the WT or D614G viruses produced similar titers in respiratory tissue and pulmonary disease. However, the D614G variant exhibited significantly faster droplet transmission between hamsters than the WT virus, early after infection. Our study demonstrated the SARS-CoV2 D614G substitution enhances infectivity, replication fitness, and early transmission.

Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus. Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type or a pre-fusion membrane anchored format. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, we report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs.Research in contextEvidence before this studyThe spike (S) protein of the SARS-CoV-2 is the major antigen that notably induces neutralizing antibodies to block viral entry. Many COVID-19 vaccines are under development, among them viral vectors expressing the S protein of SARS-CoV-2 exhibit many benefits. Viral vector vaccines have the potential of being used as both live or inactivated vaccines and they can induce Th1 and Th2-based immune responses following different immunization regimens. Additionally, viral vector vaccines can be handled under BSL-2 conditions and they grow to high titers in cell cultures or other species restricted-hosts. For a SARS-CoV-2 vaccine, several viral vectors are being tested, such as adenovirus, measles virus and Modified vaccinia Ankara.Added value of this studyThe NDV vector vaccine against SARS-CoV-2 described in this study has advantages similar to those of other viral vector vaccines. But the NDV vector can be amplified in embryonated chicken eggs, which allows for high yields and low costs per dose. Also, the NDV vector is not a human pathogen, therefore the delivery of the foreign antigen would not be compromised by any pre-existing immunity in humans. Finally, NDV has a very good safety record in humans, as it has been used in many oncolytic virus trials. This study provides an important option for a cost-effective SARS-CoV-2 vaccine.Implications of all the available evidenceThis study informs of the value of a viral vector vaccine against SARS-CoV-2. Specifically, for this NDV based SARS-CoV-2 vaccine, the existing egg-based influenza virus vaccine manufactures in the U.S. and worldwide would have the capacity to rapidly produce hundreds of millions of doses to mitigate the consequences of the ongoing COVID-19 pandemic.

Summary A severe acute respiratory syndrome coronavirus (SARS-CoV-2) vaccine is needed to control the global coronavirus infectious disease (COVID-19) public health crisis. Atomic-level structures directed the application of prefusion-stabilizing mutations that improved expression and immunogenicity of betacoronavirus spike proteins1. Using this established immunogen design, the release of SARS-CoV-2 sequences triggered immediate rapid manufacturing of an mRNA vaccine expressing the prefusion-stabilized SARS-CoV-2 spike trimer (mRNA-1273). Here, we show that mRNA-1273 induces both potent neutralizing antibody responses to wild-type (D614) and D614G mutant2 SARS-CoV-2 and CD8 T cell responses and protects against SARS-CoV-2 infection in lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in Phase 3 efficacy evaluation.

Coronaviruses are prone to emergence into new host species most recently evidenced by SARS-CoV-2, the causative agent of the COVID-19 pandemic. Small animal models that recapitulate SARS-CoV-2 disease are desperately needed to rapidly evaluate medical countermeasures (MCMs). SARS-CoV-2 cannot infect wildtype laboratory mice due to inefficient interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE2. We used reverse genetics to remodel the S and mACE2 binding interface resulting in a recombinant virus (SARS-CoV-2 MA) that could utilize mACE2 for entry. SARS-CoV-2 MA replicated in both the upper and lower airways of both young adult and aged BALB/c mice. Importantly, disease was more severe in aged mice, and showed more clinically relevant phenotypes than those seen in hACE2 transgenic mice. We then demonstrated the utility of this model through vaccine challenge studies in immune competent mice with native expression of mACE2. Lastly, we show that clinical candidate interferon (IFN) lambda-1a can potently inhibit SARS-CoV-2 replication in primary human airway epithelial cells in vitro, and both prophylactic and therapeutic administration diminished replication in mice. Our mouse-adapted SARS-CoV-2 model demonstrates age-related disease pathogenesis and supports the clinical use of IFN lambda-1a treatment in human COVID-19 infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 as the causative agent of the novel pandemic viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for safe, broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV), a monophosphoramidate prodrug of an adenosine analog, potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 µM). Weaker activity was observed in Vero E6 cells (EC50 = 1.65 µM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminished lung viral load and improved pulmonary function as compared to vehicle treated animals. These data provide evidence that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19. Funding: This project was funded in part by the National Institute of Allergy and Infectious Diseases, National 284 Institutes of Health, Department of Health and Human Service awards: 1U19AI142759 (Antiviral Drug 285 Discovery and Development Center awarded to M.R.D. and R.S.B); 5R01AI132178 awarded to T.P.S. 286 and R.S.B.; and 5R01AI108197 awarded to M.R.D. and R.S.B. D.R.M was funded by T32 AI007151 and 287 a Burroughs Wellcome Fund Postdoctoral Enrichment Program Award. The Marsico Lung Institute 288 Tissue Procurement and Cell Culture Core is supported by NIH grant DK065988 and Cystic Fibrosis 289 Foundation grant BOUCHE15RO. We also are grateful for support from the Dolly Parton COVID-19 290 Research Fund, the VUMC Office of Research, and the Elizabeth B. Lamb Center for Pediatric Research 291 at Vanderbilt University. Conflict of Interest: The authors affiliated with Gilead Sciences, Inc. are employees of the company and own company stock. The other authors have no conflict of interest to report. Ethical Approval: Human tracheobronchial epithelial cells provided by Dr. Scott Randell were obtained from airway specimens resected from patients undergoing surgery under University of North Carolina Institutional Review Board-approved protocols (#03-1396) by the Cystic Fibrosis Center Tissue Culture Core.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expres-sion in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-in-fected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings high-light the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host in-teractions in protective immunity, host susceptibility, and virus pathogenesis.

Summary Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the novel viral disease COVID-19. With no approved therapies, this pandemic illustrates the urgent need for broad-spectrum antiviral countermeasures against SARS-CoV-2 and future emerging CoVs. We report that remdesivir (RDV) potently inhibits SARS-CoV-2 replication in human lung cells and primary human airway epithelial cultures (EC50 = 0.01 μM). Weaker activity is observed in Vero E6 cells (EC50 = 1.65 μM) due to their low capacity to metabolize RDV. To rapidly evaluate in vivo efficacy, we engineered a chimeric SARS-CoV encoding the viral target of RDV, the RNA-dependent RNA polymerase, of SARS-CoV-2. In mice infected with chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared to vehicle treated animals. These data demonstrate that RDV is potently active against SARS-CoV-2 in vitro and in vivo, supporting its further clinical testing for treatment of COVID-19., Graphical Abstract, Highlights • Remdesivir binding of active site of polymerase is conserved across all human CoVs • Remdesivir inhibits SARS-CoV-2 in primary and continuous human lung cell cultures • Remdesivir potency depends on cell-type specific metabolism to its active form • Therapeutic remdesivir reduces viral loads and improves outcomes in mice, SARS-CoV-2 causes severe lung disease (COVID-19) in humans. Pruijssers et al. demonstrate that the antiviral drug remdesivir potently inhibits SARS-CoV-2 in human lung cell cultures. Therapeutic treatment of infected mice with remdesivir reduces viral loads and improves clinical outcomes, further supporting use of remdesivir for the treatment of COVID-19.

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2. Herein, we show that the ribonucleoside analog β-D-N4-hydroxycytidine (NHC, EIDD-1931) has broad spectrum antiviral activity against SARS-CoV 2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c Bat-CoVs, as well as increased potency against a coronavirus bearing resistance mutations to another nucleoside analog inhibitor. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC-prodrug (b-D-N4-hydroxycytidine-5’-isopropyl ester), improved pulmonary function, and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral but not host cell RNA, supporting a mechanism of lethal mutagenesis. The potency of NHC/EIDD-2801 against multiple coronaviruses, its therapeutic efficacy, and oral bioavailability in vivo, all highlight its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic coronaviruses.

Overall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans., Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with severe acute respiratory syndrome (SARS)-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two Middle East respiratory syndrome (MERS)-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate the infection of human gut cells but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that the addition of exogenous trypsin also rescues HKU5-CoV, a second bat group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate the emergence potential of bat CoVs and offers a means to recover previously unrecoverable zoonotic CoV strains. IMPORTANCE Overall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans.

A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant2 SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathology. mRNA-1273 is currently in a phase III trial to evaluate its efficacy.

Background Due to the lack of protective immunity of humans towards the newly emerged SARS-CoV-2, this virus has caused a massive pandemic across the world resulting in hundreds of thousands of deaths. Thus, a vaccine is urgently needed to contain the spread of the virus. Methods Here, we describe Newcastle disease virus (NDV) vector vaccines expressing the spike protein of SARS-CoV-2 in its wild type format or a membrane-anchored format lacking the polybasic cleavage site. All described NDV vector vaccines grow to high titers in embryonated chicken eggs. In a proof of principle mouse study, the immunogenicity and protective efficacy of these NDV-based vaccines were investigated. Findings We report that the NDV vector vaccines elicit high levels of antibodies that are neutralizing when the vaccine is given intramuscularly in mice. Importantly, these COVID-19 vaccine candidates protect mice from a mouse-adapted SARS-CoV-2 challenge with no detectable viral titer and viral antigen in the lungs. Interpretation The results suggested that the NDV vector expressing either the wild type S or membrane-anchored S without the polybasic cleavage site could be used as live vector vaccine against SARS-CoV-2. Funding This work is supported by an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS) contract, the Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract, philanthropic donations and NIH grants.

Significance The emergence of new human and animal coronaviruses demand novel strategies that characterize the threat potential of newly discovered zoonotic strains. We synthetically recovered recombinant wild-type and derivative swine acute diarrhea syndrome coronaviruses (SADS-CoVs) that express indicator genes and characterized their growth, macromolecular biosynthesis, and replication efficiency in a variety of mammalian cell lines, including primary human cells. The data demonstrate that SADS-CoV has a broad host range and has inherent potential to disseminate between animal and human hosts, perhaps using swine as an intermediate species., Zoonotic coronaviruses represent an ongoing threat, yet the myriads of circulating animal viruses complicate the identification of higher-risk isolates that threaten human health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a newly discovered, highly pathogenic virus that likely evolved from closely related HKU2 bat coronaviruses, circulating in Rhinolophus spp. bats in China and elsewhere. As coronaviruses cause severe economic losses in the pork industry and swine are key intermediate hosts of human disease outbreaks, we synthetically resurrected a recombinant virus (rSADS-CoV) as well as a derivative encoding tomato red fluorescent protein (tRFP) in place of ORF3. rSADS-CoV replicated efficiently in a variety of continuous animal and primate cell lines, including human liver and rectal carcinoma cell lines. Of concern, rSADS-CoV also replicated efficiently in several different primary human lung cell types, as well as primary human intestinal cells. rSADS-CoV did not use human coronavirus ACE-2, DPP4, or CD13 receptors for docking and entry. Contemporary human donor sera neutralized the group I human coronavirus NL63, but not rSADS-CoV, suggesting limited human group I coronavirus cross protective herd immunity. Importantly, remdesivir, a broad-spectrum nucleoside analog that is effective against other group 1 and 2 coronaviruses, efficiently blocked rSADS-CoV replication in vitro. rSADS-CoV demonstrated little, if any, replicative capacity in either immune-competent or immunodeficient mice, indicating a critical need for improved animal models. Efficient growth in primary human lung and intestinal cells implicate SADS-CoV as a potential higher-risk emerging coronavirus pathogen that could negatively impact the global economy and human health.

Traditionally, the emergence of coronaviruses (CoVs) has been attributed to a gain in receptor binding in a new host. Our previous work with SARS-like viruses argued that bats already harbor CoVs with the ability to infect humans without adaptation. These results suggested that additional barriers limit the emergence of zoonotic CoV. In this work, we describe overcoming host restriction of two MERS-like bat CoVs using exogenous protease treatment. We found that the spike protein of PDF2180-CoV, a MERS-like virus found in a Ugandan bat, could mediate infection of Vero and human cells in the presence of exogenous trypsin. We subsequently show that the bat virus spike can mediate infection of human gut cells, but is unable to infect human lung cells. Using receptor-blocking antibodies, we show that infection with the PDF2180 spike does not require MERS-CoV receptor DPP4 and antibodies developed against the MERS spike receptor-binding domain and S2 portion are ineffective in neutralizing the PDF2180 chimera. Finally, we found that addition of exogenous trypsin also rescues replication of HKU5-CoV, a second MERS-like group 2c CoV. Together, these results indicate that proteolytic cleavage of the spike, not receptor binding, is the primary infection barrier for these two group 2c CoVs. Coupled with receptor binding, proteolytic activation offers a new parameter to evaluate emergence potential of CoVs and offer a means to recover previously unrecoverable zoonotic CoV strains.ImportanceOverall, our studies demonstrate that proteolytic cleavage is the primary barrier to infection for a subset of zoonotic coronaviruses. Moving forward, the results argue that both receptor binding and proteolytic cleavage of the spike are critical factors that must be considered for evaluating the emergence potential and risk posed by zoonotic coronaviruses. In addition, the findings also offer a novel means to recover previously uncultivable zoonotic coronavirus strains and argue that other tissues, including the digestive tract, could be a site for future coronavirus emergence events in humans.

Zika virus (ZIKV) is a mosquito-borne positive sense RNA virus. Recently, ZIKV emerged into the Western hemisphere as a human health threat, with severe disease associated with developmental and neurological complications. The structural envelope protein of ZIKV and other neurotropic flaviviruses contains an extended CD-loop relative to non-neurotropic flaviviruses, and has been shown to augment ZIKV stability and pathogenesis. Here we show that shortening the CD-loop in ZIKV attenuates the virus in mice, by reducing the ability to invade and replicate in the central nervous system. The CD-loop mutation was genetically stable following infection in mice, though secondary site mutations arise adjacent to the CD-loop. Importantly, while shortening of the CD-loop attenuates the virus, the CD-loop mutant maintains antigenicity in immunocompetent mice, eliciting an antibody response that similarly neutralizes both the mutant and wildtype ZIKV. These findings suggest that the extended CD-loop in ZIKV is a determinant of neurotropism and may be a target in live-attenuated vaccine design, for not only ZIKV, but for other neurotropic flaviviruses., Author summary Zika virus (ZIKV) is a mosquito-transmitted virus that was recently introduced in Brazil and subsequently spread throughout the Americas. ZIKV is highly similar to the related dengue virus but causes unique disease outcomes including neurological disease in adults and fetal developmental complications. The ZIKV envelope protein coats the surface of the virus and allows entry into host cells. Here we investigate a portion of the ZIKV envelope protein, the CD-loop, which extends further than in dengue virus, and its role in ZIKV neurological disease. Our study finds that shortening the CD-loop reduces the ability of ZIKV to replicate in neuronal cells, that the longer CD-loop is a key factor for invasion of the central nervous system in mice, and that a deletion in the CD-loop is genetically stable with passage. Additionally, we show that infection with the CD-loop mutant induces a potent antibody response that can neutralize wildtype ZIKV, suggesting it may offer protection in mice. Shortening of the ZIKV CD-loop, and the CD-loop of other neurotropic flaviviruses, could contribute to development of rapid, effective, and safe vaccines.

Vimentin intermediate filament is involved in multiple steps of viral infection such as viral entry, trafficking and egress, as well as in various mechanisms of hyperinflammation such as the restraint of Treg cell functions and the activation of NLRP3 inflammasome. We evaluated a vimentin-binding small molecule compound ALD-R491 for its effects on cellular processes related to viral infection and for its efficacy in treating SARS-CoV2 infection in vitro and in vivo. In cultured cells, the compound could reduce endocytosis by 10%, endosomal trafficking by 40% and exosomal release by over 30%. In an infection system consisting of a lentiviral pseudotype bearing the SARS-CoV-2 spike protein and HEK293 cells over-expressing the human ACE2 receptor with multiplicity of infection (MOI) of 1, 10 and 100, the compound inhibited the infection up to a maximum of over 90%, with IC50 < 50 nM, CC50 > 10 μM, and SI > 200. After oral administration of ALD-R491 in rats, the plasma concentration of the compound reached the peak (Tmax) at around 5 h with a half-life (T1/2) of about 5 h. The compound was widely distributed and enriched in tissues in vivo in rats with a volume of distribution (Vd) of over 2,000 ml/kg. The lung and the lymph nodes were among the tissues with high drug exposures. In rats receiving oral gavage of the compound at 30 mg/kg, the drug exposure in the lung and the lymph nodes maintained at levels over 1 μM from 1 h to 6 h after the oral dosing. In the syngeneic mouse tumor CT26 model, ALD-R491 was found to activate regulatory T cells (Tregs) in vivo and enhance de novo generation of Tregs in lymph nodes of the mice. In the Mouse-Adapted SARS-CoV2 model, aged mice (11-12 months) were used to provide a harder test of recovery from infection that reflects the severeness of COVID-19 in old patients. For therapeutic treatment, the mice were orally administered with the compound 24 h after the SARS-CoV2 infection once per day on Day 1, Day 2 and Day 4. At 10 mg/kg, ALD-R491 significantly reduced the body weight loss of the mice (p Citation Format: Zhizhen Li, Jianping Wu, Baoshi Yuan, Kenneth Dinnon, III, Lian Mo, Fei Zhou, Yingying Dong, Kendra Gully, Ralph Baric, Rachel Graham, Ying Xu, Ruihuan Chen. Targeting vimentin impacts multiple cellular processes, blocks the spike protein-ACE2-mediated pseudoviral infection in vitro and reduces the symptom and lung injury in aged mice with mouse-adapted SARS-CoV-2 infection in vivo [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P35.

The SARS-CoV-2 pandemic has caused extreme human suffering and economic harm. We generated and characterized a new mouse-adapted SARS-CoV-2 virus that captures multiple aspects of severe COVID-19 disease in standard laboratory mice. This SARS-CoV-2 model exhibits the spectrum of morbidity and mortality of COVID-19 disease as well as aspects of host genetics, age, cellular tropisms, elevated Th1 cytokines, and loss of surfactant expression and pulmonary function linked to pathological features of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). This model can rapidly access existing mouse resources to elucidate the role of host genetics, underlying molecular mechanisms governing SARS-CoV-2 pathogenesis, and the protective or pathogenic immune responses related to disease severity. The model promises to provide a robust platform for studies of ALI and ARDS to evaluate vaccine and antiviral drug performance, including in the most vulnerable populations, i.e. the aged, using standard laboratory mice., Highlights - Serial in vivo evolution selected for a lethal mouse-adapted SARS-CoV-2 MA10 variant - SARS-CoV-2 MA10 shows a dose- and age-related increase in pathogenesis in BALB/c mice - Mice exhibit ALI, ARDS, and surfactant loss, key metrics in countermeasure performance - SARS-CoV-2 MA10 model enables access to immune reagents and genetically defined mice, Leist et al. present a mouse model for COVID-19 by serially passaging human SARS-CoV-2 in vivo to create an evolution selected lethal mouse-adapted virus variant, called MA10. MA10 shows a dose- and age-related increase in pathogenesis in standard laboratory mice and recapitulates key features of COVID-19 in humans.

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live-attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2’O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the SARS-CoV mouse model, a NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2’O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2’O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live-attenuated coronavirus vaccines.SignificanceEmergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations like the aged and immune-compromised. This study illustrates that despite success in young models, the NSP16 mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the NSP16 mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for NSP16 attenuation as a major component of future live-attenuated coronavirus vaccines.

With an ongoing threat posed by circulating zoonotic strains, new strategies are required to prepare for the next emergent coronavirus (CoV). Previously, groups had targeted conserved coronavirus proteins as a strategy to generate live attenuated vaccine strains against current and future CoVs. With this in mind, we explored whether manipulation of CoV NSP16, a conserved 2′O methyltransferase (MTase), could provide a broad attenuation platform against future emergent strains. Using the severe acute respiratory syndrome-CoV mouse model, an NSP16 mutant vaccine was evaluated for protection from heterologous challenge, efficacy in the aging host, and potential for reversion to pathogenesis. Despite some success, concerns for virulence in the aged and potential for reversion makes targeting NSP16 alone an untenable approach. However, combining a 2′O MTase mutation with a previously described CoV fidelity mutant produced a vaccine strain capable of protection from heterologous virus challenge, efficacy in aged mice, and no evidence for reversion. Together, the results indicate that targeting the CoV 2′O MTase in parallel with other conserved attenuating mutations may provide a platform strategy for rapidly generating live attenuated coronavirus vaccines. IMPORTANCE Emergent coronaviruses remain a significant threat to global public health and rapid response vaccine platforms are needed to stem future outbreaks. However, failure of many previous CoV vaccine formulations has clearly highlighted the need to test efficacy under different conditions and especially in vulnerable populations such as the aged and immunocompromised. This study illustrates that despite success in young models, the 2′O methyltransferase mutant carries too much risk for pathogenesis and reversion in vulnerable models to be used as a stand-alone vaccine strategy. Importantly, the 2′O methyltransferase mutation can be paired with other attenuating approaches to provide robust protection from heterologous challenge and in vulnerable populations. Coupled with increased safety and reduced pathogenesis, the study highlights the potential for 2′O methyltransferase attenuation as a major component of future live attenuated coronavirus vaccines.

Coronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2′O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2′O-MTase function across CoVs., Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2′O-methyltransferase (2′O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2′O-MTase activity had only a marginal impact on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures and in vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2′O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting. IMPORTANCE Coronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2′O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2′O-MTase function across CoVs.

Coronaviruses encode a mix of highly conserved and novel genes as well as genetic elements necessary for infection and pathogenesis, raising the possibility for common targets for attenuation and therapeutic design. In this study, we focus on the highly conserved nonstructural protein (NSP) 16, a viral 2’O methyl-transferase (MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of MERS NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2’O MTase activity had only marginal impact on propagation and replication in Vero cells, the MERS dNSP16 mutant demonstrated significant attenuation relative to control both in primary human airway cultures andin vivo.Further examination indicated the MERS dNSP16 mutant had a type I IFN based attenuation and was partially restored in the absence of IFIT molecules. Importantly, the robust attenuation permitted use of MERS dNSP16 as a live attenuated vaccine platform protecting from challenge with a mouse adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2’O MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.SignificanceCoronavirus emergence in both human and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of SARS-CoV, MERS-CoV, PEDV and swine delta coronavirus in the 21stcentury. These studies describe an approach that effectively targets the highly conserved 2’O methyl-transferase activity of coronaviruses for attenuation. With clear understanding of the IFN/IFIT based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-coronavirus functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2’O MTase function across coronaviruses.

With severe disease manifestations including microcephaly, congenital malformation, and Guillain-Barre syndrome, Zika virus (ZIKV) remains a persistent global public health threat. Despite antigenic similarities with dengue viruses, structural studies have suggested the extended CD-loop and hydrogen-bonding interaction network within the ZIKV envelope protein contribute to stability differences between the viral families. This enhanced stability may lead to the augmented infection, disease manifestation, and persistence in body fluids seen following ZIKV infection. To examine the role of these motifs in infection, we generated a series of ZIKV recombinant viruses that disrupted the hydrogen-bonding network (350A, 351A, and 350A/351A) or the CD-loop extension (Δ346). Our results demonstrate a key role for the ZIKV extended CD-loop in cell-type-dependent replication, virion stability, and in vivo pathogenesis. Importantly, the Δ346 mutant maintains similar antigenicity to wild-type virus, opening the possibility for its use as a live-attenuated vaccine platform for ZIKV and other clinically relevant flaviviruses.