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1.
Cell Rep ; 37(5): 109929, 2021 11 02.
Article in English | MEDLINE | ID: covidwho-1466097

ABSTRACT

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 ß-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.


Subject(s)
Antibodies, Viral/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Cell Line , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Cross Reactions , Drug Design , Epitope Mapping , Female , Immunoglobulin G/immunology , Male , Mice , Mice, Inbred BALB C , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Viral Vaccines/immunology
2.
J Virol ; 95(23): e0097421, 2021 11 09.
Article in English | MEDLINE | ID: covidwho-1410203

ABSTRACT

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. IMPORTANCE We have developed a series of SARS-CoV-2 vaccines using rhesus adenovirus serotype 52 (RhAd52) vectors, which exhibit a lower seroprevalence than human and chimpanzee vectors, supporting their development as novel vaccine vectors or as an alternative adenovirus (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 that 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.


Subject(s)
Adenovirus Vaccines/immunology , Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Pandemics/prevention & control , SARS-CoV-2/immunology , Adenoviridae Infections/immunology , Adenoviruses, Simian/immunology , Animals , Antibodies, Viral/immunology , Disease Models, Animal , Female , Humans , Immunogenicity, Vaccine , Macaca mulatta/virology , Mice , Mice, Inbred BALB C , SARS-CoV-2/pathogenicity , Vaccination
3.
PLoS Pathog ; 17(9): e1009897, 2021 09.
Article in English | MEDLINE | ID: covidwho-1398941

ABSTRACT

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.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine , Nanoparticles/therapeutic use , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Disease Models, Animal , Drug Design , Female , HEK293 Cells , Humans , Lung/virology , Mice , Mice, Inbred BALB C , Protein Domains/immunology
4.
Nat Commun ; 12(1): 3587, 2021 06 11.
Article in English | MEDLINE | ID: covidwho-1387350

ABSTRACT

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.


Subject(s)
Adjuvants, Immunologic/administration & dosage , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Heterocyclic Compounds, 3-Ring/administration & dosage , Stearic Acids/administration & dosage , Alum Compounds/administration & dosage , Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antibody Formation/immunology , COVID-19 Vaccines/administration & dosage , Disease Models, Animal , Heterocyclic Compounds, 3-Ring/immunology , Humans , Macaca mulatta , Mice , Protein Binding , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , Stearic Acids/immunology
5.
Immunity ; 54(8): 1869-1882.e6, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1293864

ABSTRACT

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.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Host-Pathogen Interactions/immunology , SARS-CoV-2/immunology , Vaccines, Synthetic/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Biopsy , COVID-19 Vaccines/administration & dosage , Disease Models, Animal , Humans , Immunoglobulin G , Immunohistochemistry , Mice , Outcome Assessment, Health Care , RNA, Messenger , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , Vaccines, Synthetic/administration & dosage
6.
mBio ; 12(2)2021 03 16.
Article in English | MEDLINE | ID: covidwho-1138305

ABSTRACT

The angiotensin-converting enzyme 2 (ACE2) receptor is a major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) host range determinant, and understanding SARS-CoV-2-ACE2 interactions will provide important insights into COVID-19 pathogenesis and animal model development. SARS-CoV-2 cannot infect mice due to incompatibility between its receptor binding domain and the murine ACE2 receptor. Through molecular modeling and empirical in vitro validation, we identified 5 key amino acid differences between murine and human ACE2 that mediate SARS-CoV-2 infection, generating a chimeric humanized murine ACE2. Additionally, we examined the ability of the humanized murine ACE2 receptor to permit infection by an additional preemergent group 2B coronavirus, WIV-1, providing evidence for the potential pan-virus capabilities of this chimeric receptor. Finally, we predicted the ability of these determinants to inform host range identification of preemergent coronaviruses by evaluating hot spot contacts between SARS-CoV-2 and additional potential host receptors. Our results identify residue determinants that mediate coronavirus receptor usage and host range for application in SARS-CoV-2 and emerging coronavirus animal model development.IMPORTANCE SARS-CoV-2 (the causative agent of COVID-19) is a major public health threat and one of two related coronaviruses that have caused epidemics in modern history. A method of screening potential infectible hosts for preemergent and future emergent coronaviruses would aid in mounting rapid response and intervention strategies during future emergence events. Here, we evaluated determinants of SARS-CoV-2 receptor interactions, identifying key changes that enable or prevent infection. The analysis detailed in this study will aid in the development of model systems to screen emergent coronaviruses as well as treatments to counteract infections.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Betacoronavirus/physiology , Virus Replication , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/genetics , Animals , Betacoronavirus/metabolism , Binding Sites , COVID-19/virology , Cell Line , Coronavirus Infections/virology , Host Specificity , Humans , Mice , Models, Molecular , Mutation , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism
7.
Nature ; 591(7850): 451-457, 2021 03.
Article in English | MEDLINE | ID: covidwho-1075231

ABSTRACT

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.


Subject(s)
COVID-19/drug therapy , COVID-19/prevention & control , Cytidine/analogs & derivatives , Hydroxylamines/administration & dosage , Hydroxylamines/therapeutic use , Administration, Oral , Alveolar Epithelial Cells/immunology , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , COVID-19/immunology , Chemoprevention , Chiroptera/virology , Clinical Trials, Phase II as Topic , Clinical Trials, Phase III as Topic , Cytidine/administration & dosage , Cytidine/therapeutic use , Cytokines/immunology , Epithelial Cells/virology , Female , Heterografts , Humans , Immunity, Innate , Interferon Type I/immunology , Lung/immunology , Lung/pathology , Lung/virology , Lung Transplantation , Male , Mice , Post-Exposure Prophylaxis , Pre-Exposure Prophylaxis , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Virus Replication
9.
Vaccines (Basel) ; 8(4)2020 Dec 17.
Article in English | MEDLINE | ID: covidwho-979800

ABSTRACT

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.

10.
Proc Natl Acad Sci U S A ; 117(51): 32657-32666, 2020 12 22.
Article in English | MEDLINE | ID: covidwho-951832

ABSTRACT

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.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Measles virus/immunology , SARS-CoV-2/immunology , Th1 Cells/immunology , Animals , Antibodies, Viral/immunology , COVID-19/epidemiology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Humans , Measles Vaccine/genetics , Measles Vaccine/immunology , Measles virus/genetics , Mice , Pandemics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/administration & dosage , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes/immunology
11.
EBioMedicine ; 62: 103132, 2020 Dec.
Article in English | MEDLINE | ID: covidwho-938895

ABSTRACT

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.


Subject(s)
COVID-19 Vaccines , COVID-19 , Gene Expression Regulation, Viral/immunology , Newcastle disease virus , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , COVID-19/genetics , COVID-19/immunology , COVID-19/prevention & control , COVID-19 Vaccines/genetics , COVID-19 Vaccines/immunology , Chlorocebus aethiops , Female , Mice , Mice, Inbred BALB C , Newcastle disease virus/genetics , Newcastle disease virus/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vaccines, Live, Unattenuated/genetics , Vaccines, Live, Unattenuated/immunology , Vero Cells
12.
Science ; 370(6523): 1464-1468, 2020 12 18.
Article in English | MEDLINE | ID: covidwho-922513

ABSTRACT

The spike aspartic acid-614 to glycine (D614G) substitution is prevalent in global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains, but its effects on viral pathogenesis and transmissibility remain unclear. We engineered a SARS-CoV-2 variant containing this substitution. The variant exhibits more efficient infection, replication, and competitive fitness in primary human airway epithelial cells but maintains similar morphology and in vitro neutralization properties, compared with the ancestral wild-type virus. Infection of human angiotensin-converting enzyme 2 (ACE2) transgenic mice and Syrian hamsters with both viruses resulted in similar viral titers in respiratory tissues and pulmonary disease. However, the D614G variant transmits significantly faster and displayed increased competitive fitness than the wild-type virus in hamsters. These data show that the D614G substitution enhances SARS-CoV-2 infectivity, competitive fitness, and transmission in primary human cells and animal models.


Subject(s)
COVID-19/transmission , COVID-19/virology , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/genetics , Animals , Asparagine/genetics , Cricetinae , Genetic Fitness/genetics , Glycine/genetics , Humans , Mesocricetus , Mice , Mice, Transgenic , Respiratory Mucosa/virology , Virulence/genetics , Virus Replication/genetics
13.
Cell ; 183(5): 1367-1382.e17, 2020 11 25.
Article in English | MEDLINE | ID: covidwho-893667

ABSTRACT

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 10-fold higher than the prefusion-stabilized spike despite a 5-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.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Nanoparticles/chemistry , Protein Domains/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Vaccination , Adolescent , Adult , Aged , Animals , COVID-19/virology , Chlorocebus aethiops , Cohort Studies , Epitopes/immunology , Female , HEK293 Cells , Humans , Macaca nemestrina , Male , Mice, Inbred BALB C , Middle Aged , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells , Young Adult
14.
Proc Natl Acad Sci U S A ; 117(43): 26915-26925, 2020 10 27.
Article in English | MEDLINE | ID: covidwho-851432

ABSTRACT

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.


Subject(s)
Alphacoronavirus/physiology , Coronavirus Infections/virology , Disease Susceptibility/virology , Virus Replication , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Alphacoronavirus/genetics , Alphacoronavirus/growth & development , Animals , Cells, Cultured , Chlorocebus aethiops , Coronavirus Infections/transmission , Gene Expression , Host Specificity , Humans , Luminescent Proteins/genetics , Mice , Vero Cells , Virus Replication/drug effects
15.
bioRxiv ; 2020 Sep 29.
Article in English | MEDLINE | ID: covidwho-835242

ABSTRACT

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.

16.
Cell ; 183(4): 1070-1085.e12, 2020 11 12.
Article in English | MEDLINE | ID: covidwho-785288

ABSTRACT

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.


Subject(s)
Acute Lung Injury/pathology , Betacoronavirus/pathogenicity , Coronavirus Infections/pathology , Pneumonia, Viral/pathology , Animals , Betacoronavirus/isolation & purification , Betacoronavirus/physiology , COVID-19 , Cell Line , Chemokines/blood , Coronavirus Infections/mortality , Coronavirus Infections/virology , Cytokines/blood , Disease Models, Animal , Female , Humans , Lung/pathology , Lung/physiology , Lung/virology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Pandemics , Pneumonia, Viral/mortality , Pneumonia, Viral/virology , Respiratory Distress Syndrome/pathology , SARS-CoV-2 , Severity of Illness Index , Survival Rate
17.
Nature ; 586(7830): 560-566, 2020 10.
Article in English | MEDLINE | ID: covidwho-733515

ABSTRACT

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.


Subject(s)
Betacoronavirus , Coronavirus Infections/drug therapy , Coronavirus Infections/prevention & control , Disease Models, Animal , Interferons/pharmacology , Interferons/therapeutic use , Interleukins/pharmacology , Interleukins/therapeutic use , Pandemics/prevention & control , Pneumonia, Viral/drug therapy , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Aging/immunology , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/drug effects , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Female , Forkhead Transcription Factors/genetics , Humans , Interferon-alpha/administration & dosage , Interferon-alpha/pharmacology , Interferon-alpha/therapeutic use , Interferons/administration & dosage , Interleukins/administration & dosage , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Models, Molecular , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/genetics , Pneumonia, Viral/immunology , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2
18.
Nature ; 586(7830): 567-571, 2020 10.
Article in English | MEDLINE | ID: covidwho-703377

ABSTRACT

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.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Viral Vaccines/immunology , Animals , Antibodies, Neutralizing/immunology , Betacoronavirus/genetics , CD8-Positive T-Lymphocytes/immunology , COVID-19 , COVID-19 Vaccines , Clinical Trials, Phase III as Topic , Coronavirus Infections/genetics , Coronavirus Infections/virology , Female , Lung/immunology , Lung/virology , Mice , Mutation , Nose/immunology , Nose/virology , Pneumonia, Viral/virology , RNA, Messenger/genetics , RNA, Viral/genetics , SARS-CoV-2 , Th1 Cells/immunology , Toll-Like Receptor 4/agonists , Toll-Like Receptor 4/immunology , Viral Vaccines/chemistry , Viral Vaccines/genetics
19.
Cell Rep ; 32(3): 107940, 2020 07 21.
Article in English | MEDLINE | ID: covidwho-635658

ABSTRACT

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) because of 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 the chimeric virus, therapeutic RDV administration diminishes lung viral load and improves pulmonary function compared with 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.

20.
Cell ; 182(2): 429-446.e14, 2020 07 23.
Article in English | MEDLINE | ID: covidwho-381993

ABSTRACT

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) expression 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-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight 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 interactions in protective immunity, host susceptibility, and virus pathogenesis.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/pathology , Coronavirus Infections/virology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Respiratory System/virology , Reverse Genetics/methods , Aged , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Betacoronavirus/immunology , Betacoronavirus/pathogenicity , COVID-19 , Cell Line , Cells, Cultured , Chlorocebus aethiops , Coronavirus Infections/immunology , Coronavirus Infections/therapy , Cystic Fibrosis/pathology , DNA, Recombinant , Female , Furin/metabolism , Humans , Immunization, Passive , Lung/metabolism , Lung/pathology , Lung/virology , Male , Middle Aged , Nasal Mucosa/metabolism , Nasal Mucosa/pathology , Nasal Mucosa/virology , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Respiratory System/pathology , SARS-CoV-2 , Serine Endopeptidases/metabolism , Vero Cells , Virulence , Virus Replication
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