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1.
Cell ; 185(13): 2279-2291.e17, 2022 06 23.
Article in English | MEDLINE | ID: covidwho-1866951

ABSTRACT

The isolation of CCoV-HuPn-2018 from a child respiratory swab indicates that more coronaviruses are spilling over to humans than previously appreciated. We determined the structures of the CCoV-HuPn-2018 spike glycoprotein trimer in two distinct conformational states and showed that its domain 0 recognizes sialosides. We identified that the CCoV-HuPn-2018 spike binds canine, feline, and porcine aminopeptidase N (APN) orthologs, which serve as entry receptors, and determined the structure of the receptor-binding B domain in complex with canine APN. The introduction of an oligosaccharide at position N739 of human APN renders cells susceptible to CCoV-HuPn-2018 spike-mediated entry, suggesting that single-nucleotide polymorphisms might account for viral detection in some individuals. Human polyclonal plasma antibodies elicited by HCoV-229E infection and a porcine coronavirus monoclonal antibody inhibit CCoV-HuPn-2018 spike-mediated entry, underscoring the cross-neutralizing activity among ɑ-coronaviruses. These data pave the way for vaccine and therapeutic development targeting this zoonotic pathogen representing the eighth human-infecting coronavirus.


Subject(s)
Coronavirus 229E, Human , Coronavirus Infections , Coronavirus , Animals , CD13 Antigens/chemistry , CD13 Antigens/metabolism , Cats , Cell Line , Coronavirus/metabolism , Coronavirus 229E, Human/metabolism , Dogs , Humans , Receptors, Virus/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Swine
2.
NPJ Vaccines ; 7(1): 55, 2022 May 23.
Article in English | MEDLINE | ID: covidwho-1860374

ABSTRACT

Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice-alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)-for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

3.
Science ; 374(6575): 1621-1626, 2021 Dec 24.
Article in English | MEDLINE | ID: covidwho-1506414

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission leads to the emergence of variants, including the B.1.617.2 (Delta) variant of concern that is causing a new wave of infections and has become globally dominant. We show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing their immune evasion. Mutations in the B.1.617.1 (Kappa) and Delta spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including remodeling of the Delta amino-terminal domain. The angiotensin-converting enzyme 2 binding affinities of the Kappa and Delta receptor binding domains are comparable to the Wuhan-Hu-1 isolate, whereas B.1.617.2+ (Delta+) exhibits markedly reduced affinity.


Subject(s)
COVID-19 Vaccines/immunology , Immune Evasion , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , /immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibodies, Viral/metabolism , Antigens, Viral/chemistry , Antigens, Viral/immunology , Cryoelectron Microscopy , Humans , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Domains , Protein Folding , Receptors, Coronavirus/metabolism , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
4.
Cell ; 184(21): 5432-5447.e16, 2021 10 14.
Article in English | MEDLINE | ID: covidwho-1454060

ABSTRACT

Understanding vaccine-elicited protection against SARS-CoV-2 variants and other sarbecoviruses is key for guiding public health policies. We show that a clinical stage multivalent SARS-CoV-2 spike receptor-binding domain nanoparticle (RBD-NP) vaccine protects mice from SARS-CoV-2 challenge after a single immunization, indicating a potential dose-sparing strategy. We benchmarked serum neutralizing activity elicited by RBD-NPs in non-human primates against a lead prefusion-stabilized SARS-CoV-2 spike (HexaPro) using a panel of circulating mutants. Polyclonal antibodies elicited by both vaccines are similarly resilient to many RBD residue substitutions tested, although mutations at and surrounding position 484 have negative consequences for neutralization. Mosaic and cocktail nanoparticle immunogens displaying multiple sarbecovirus RBDs elicit broad neutralizing activity in mice and protect mice against SARS-CoV challenge even in the absence of SARS-CoV RBD in the vaccine. This study provides proof of principle that multivalent sarbecovirus RBD-NPs induce heterotypic protection and motivates advancing such broadly protective sarbecovirus vaccines to the clinic.

5.
Nature ; 594(7862): 253-258, 2021 06.
Article in English | MEDLINE | ID: covidwho-1192479

ABSTRACT

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).


Subject(s)
Adjuvants, Immunologic , Antibodies, Neutralizing/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Vaccines, Subunit/immunology , Alum Compounds , Animals , Antibodies, Viral/immunology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , COVID-19/virology , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Disease Models, Animal , Immunity, Cellular , Immunity, Humoral , Macaca mulatta/immunology , Male , Oligodeoxyribonucleotides , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Squalene
6.
Nature ; 591(7850): 482-487, 2021 03.
Article in English | MEDLINE | ID: covidwho-1049967

ABSTRACT

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.


Subject(s)
Antibodies, Viral/analysis , Biosensing Techniques/methods , Hepatitis B virus/immunology , SARS-CoV-2/chemistry , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/analysis , Troponin I/analysis , Antibodies, Viral/immunology , Biosensing Techniques/standards , Botulinum Toxins/analysis , Coronavirus Nucleocapsid Proteins/immunology , Immunoglobulin G/analysis , Immunoglobulin G/immunology , Limit of Detection , Luminescence , Phosphoproteins/immunology , Proto-Oncogene Proteins c-bcl-2/analysis , Receptor, ErbB-2/analysis , Sensitivity and Specificity , Viral Matrix Proteins/immunology
7.
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
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