WO2021155323A1 - Compositions and methods for preventing and treating coronavirus infection-sars-cov-2 vaccines - Google Patents

Compositions and methods for preventing and treating coronavirus infection-sars-cov-2 vaccines Download PDF

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WO2021155323A1
WO2021155323A1 PCT/US2021/015946 US2021015946W WO2021155323A1 WO 2021155323 A1 WO2021155323 A1 WO 2021155323A1 US 2021015946 W US2021015946 W US 2021015946W WO 2021155323 A1 WO2021155323 A1 WO 2021155323A1
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protein
seq
sars
nucleic acid
cov
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PCT/US2021/015946
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French (fr)
Inventor
Dan H. Barouch
Johannes Petrus Maria LANGEDIJK
Lucy RUTTEN
Mark Johannes Gerandus BAKKERS
Rinke Bos
Frank Wegmann
David Adrianus Theodorus Maria ZUIJDGEEST
An Vandebosch
Mathieu Claude Michel LE GARS
Jerald C. Sadoff
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Beth Israel Deaconess Medical Center, Inc.
Janssen Pharmaceuticals, Inc.
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Priority claimed from US202062969008P external-priority
Priority claimed from CA3101131A external-priority patent/CA3101131A1/en
Application filed by Beth Israel Deaconess Medical Center, Inc., Janssen Pharmaceuticals, Inc. filed Critical Beth Israel Deaconess Medical Center, Inc.
Priority to KR1020227030114A priority Critical patent/KR20220134622A/en
Priority to BR112022014808A priority patent/BR112022014808A2/en
Priority to EP21747489.9A priority patent/EP4097122A1/en
Publication of WO2021155323A1 publication Critical patent/WO2021155323A1/en

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Abstract

The invention relates to immunogenic compositions and vaccines containing a coronavirus (e.g., Wuhan coronavirus (2019-nCoV; also referred to as SARS-CoV-2)) protein or a polynucleotide encoding a coronavirus (e.g., Wuhan coronavirus (2019-nCoV; SARS-CoV-2)) protein and uses thereof. The invention also provides methods of treating and/or preventing a coronavirus (e.g., Wuhan coronavirus (2019-nCoV; SARS-CoV-2)) infection by administering an immunogenic composition or vaccine to a subject (e.g., a human). The invention also provides methods of detecting and/or monitoring a protective anti-coronavirus (e.g., Wuhan coronavirus (2019-nCoV; SARS-CoV-2)) antibody response (e.g., anti-coronavirus antibody response, e.g., anti-2019- nCoV antibody response, e.g., anti-Spike antibody response, e.g., anti-Spike neutralizing antibody response). The present invention relates to isolated nucleic and/or recombinant nucleic acid encoding a coronavirus S protein, in particular a SARS-CoV-2 S protein, and to the coronavirus S proteins, as well as to the use of the nucleic acids and/or proteins thereof in vaccines.

Description

COMPOSITIONS AND METHODS FOR PREVENTING AND TREATING CORONAVIRUS
INFECTION - SARS-COV-2 VACCINES
INTRODUCTION
The invention relates to the fields of virology and medicine. In particular, the invention relates to vaccines for the prevention of disease induced by SARS-CoV-2.
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No. 62/969,008, filed January 31, 2020; U.S. Provisional Application No. 62/994,630, filed March 25, 2020; U.S. Provisional Application No. 63/014,467, filed April 23, 2020; U.S. Provisional Application No. 63/025,782, filed May 15, 2020; U.S. Provisional Application No. 62/705,187, filed June 15, 2020; U.S. Provisional Application No. 62/705,308, filed June 21, 2020; U.S. Provisional Application No. 63/043,090, filed June 23, 2020; U.S Provisional Application No. 62/706,366, filed August 12, 2020; U.S. Provisional Application No. 63/066,147, filed August 14, 2020; U.S. Provisional Application No. 62/706,676, filed September 2, 2020; U.S. Provisional Application No. 62/706,937, filed September 18, 2020; U.S. Provisional Application No. 62/706,958, filed September 21, 2020; U.S. Provisional Application No. 63/198,089, filed September 28, 2020; U.S. Provisional Application No. 63/198,306, filed October 9, 2020; U.S. Provisional Application No. 63/112,900, filed on November 12, 2020; Canadian Patent Application No. 3,101,131, filed November 28, 2020; U.S. Provisional Application No. 63/121,482, filed December 4, 2020; U.S. Provisional Application No. 63/133,969, filed January 5, 2021; U.S. Provisional Application No. 63/135,182, filed January 8, 2021; U.S. Provisional Application No. 63/141,913, filed January 26, 2021; U.S. Provisional Application No. 63/142,977, filed January 28, 2021. Each disclosure is incorporated by reference in its entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on January 29, 2021, is named CRU6043WOPCT and is 1.36 MB in size.
STATEMENT AS TO FEDERALLY FUNDED RESEARCH
This invention was made with Government support under Agreement HHS0100201700018C, awarded by HHS. The Government has certain rights in the invention. BACKGROUND
SARS-CoV-2 is a coronavirus that was first discovered late 2019 in the Wuhan region in China. SARS-CoV-2 is a beta-coronavirus, like MERS-CoV and SARS-CoV, all of which have their origin in bats. There are currently several sequences available from several patients from the U.S., China and other countries, suggesting a likely single, recent emergence of this virus from an animal reservoir. The name of this disease caused by the virus is coronavirus disease 2019, abbreviated as COVID-19. Symptoms of COVID-19 range from mild symptoms to severe illness and death for confirmed COVID-19 cases.
As indicated above, SARS-CoV-2 has strong genetic similarity to bat coronaviruses, from which it likely originated, although an intermediate reservoir host such as a pangolin is thought to be involved. From a taxonomic perspective SARS-CoV-2 is classified as a strain of the severe acute respiratory syndrome (SARS)-related coronavirus species.
Coronaviruses are enveloped RNA viruses. The major surface protein is the large, trimeric spike glycoprotein (S) that mediates binding to host cell receptors as well as fusion of viral and host cell membranes. The S protein is composed of an N-terminal S1 subunit and a C-terminal S2 subunit, responsible for receptor binding and membrane fusion, respectively. Recent cryo-EM reconstructions of the CoV trimeric S structures of alpha-, beta-, and deltacoronaviruses revealed that the S1 subunit comprises two distinct domains: an N-terminal domain (S1 NTD) and a receptor-binding domain (S1 RBD). SARS-CoV-2 makes use of its S1 RBD to bind to human angiotensin-converting enzyme 2 (ACE2).
The rapid expansion of the COVID-19 pandemic has made the development of a SARS-CoV-2 vaccine a global health priority. Since the novel SARS-CoV-2 virus was first observed in humans in late 2019, over 8 million people have been infected and hundreds of thousands have died as a result of COVID-19. SARS-CoV-2, and coronaviruses more generally, lack effective treatment, leading to a large unmet medical need. In addition, there is currently no vaccine available to prevent coronavirus induced disease (COVID-19). The best way to prevent illness currently is to avoid being exposed to this virus. Since emerging infectious diseases, such as COVID-19 present a major threat to public health there is an urgent need for novel vaccines that can be used to prevent coronavirus induced respiratory disease.
Wuhan coronavirus (2019-nCoV; also referred to as SARS-CoV-2) is a coronavirus that is responsible for an unprecedented current epidemic in China. 2019-nCoV is known to cause respiratory symptoms and fever, which may result in death. The World Health Organization declared the 2019-nCoV outbreak a Public Health Emergency of International Concern on January 30, 2020 and has confirmed over 11,000 cases in 16 countries. While the rapid development of a safe and effective 2019-nCoV vaccine is a global health priority, very little is currently known about 2019-nCoV immunology and mechanisms of immune protection.
Accordingly, there is an unmet need in the field for 2019-nCoV therapies.
SUMMARY OF THE INVENTION
In the research that led to the present invention certain stabilized SARS-CoV-2 S proteins were constructed that were demonstrated to be useful as immunogens for inducing a protective immune response against SARS-CoV-2.
The invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a 2019-NCOV Spike (S) protein (also referred to as SARS-CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEC ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEC ID NO: 90).
In some embodiments, these are the only modifications made to the sequence of SEC ID NO: 29. In other embodiments, the isolated nucleic acid molecule encodes a 2019-NCOV Spike (S) protein that comprises the following further modification to the full-length amino acid sequence of SEC ID NO: 29: c. deletion of the signal sequence.
In some embodiments, the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (SEQ ID NO: 219) promoter, preferably the CMV immediate early promoter. In some embodiments, the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif. In specific embodiments, the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 219. In some embodiments, the CMV promotor consists of the nucleotide sequence of SEQ ID NO: 219. These nucleic acids typically form part of a vector.
In one aspect, the present invention thus relates to isolated and/or recombinant nucleic acids encoding a stabilized coronavirus S protein, in particular a SARS-CoV-2 S protein, said nucleic acids comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 211- 218, or fragments thereof. In a preferred embodiment, the present invention relates to an isolated and/or recombinant nucleic acid encoding a stabilized coronavirus S protein, in particular a SARS-CoV-2 S protein, said nucleic acid comprising a nucleotide of SEQ ID NO: 211, or fragments thereof.
The invention provides an isolated 2019-NCOV Spike (S) protein (also referred to as SARS- CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90).
In some embodiments, these are the only modifications made to the sequence of SEQ ID NO: 29. In other embodiments the isolated 2019-NCOV Spike (S) protein comprises the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
In another aspect the invention relates to isolated and/or recombinant coronavirus S proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 205-210, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof.
In a preferred embodiment, the invention relates to an isolated and/or recombinant coronavirus S proteins comprising an amino acid sequence of SEQ ID NO: 205, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof.
In yet another aspect, the invention relates to vectors comprising such nucleic acids. In certain embodiments, the vector is a recombinant human adenovirus of serotype 26.
In another aspect, the invention relates to compositions and vaccines comprising such nucleic acids, proteins and/or vectors.
In another aspect, the invention relates to methods for vaccinating a subject against COVID-19, the method comprising administering to the subject a vaccine or composition according to the invention.
In another aspect, the invention relates to an isolated host cell comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof.
In another aspect, the invention relates to methods for making a vaccine against COVID-19, comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and formulating the recombinant adenovirus in a pharmaceutically acceptable composition. The recombinant human adenovirus of this aspect may be any of the adenoviruses described herein.
In another aspect, the invention relates to an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises a nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof. The adenovirus may also be any of the adenoviruses as described in the embodiments above.
The invention also relates to a composition for use in prevention of molecularly confirmed, moderate to severe/critical COVID-19 in a subject in need thereof, comprising administering to the subject a composition or immunogenic composition of the invention as described herein, wherein the composition is administered at a dose of 5x1010 vp per dose in a one dose regimen (i.e. a single dose).
The present invention features optimized and/or non-naturally occurring coronavirus (e.g., 2019- nCoV) nucleic acid molecules and polypeptides for the generation of DNA or RNA vaccines, antibodies, and immunogenic compositions and their use in methods of preventing, reducing and/or treating a coronavirus (e.g., 2019-nCoV) infection in a subject (e.g., a mammalian subject (e.g., a human)).
One aspect of the invention features an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 1-84. In some embodiments, a) the polypeptide is capable of eliciting an immune response in a subject; or b) the polypeptide has at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to, or the polypeptide sequence of, any one of SEQ ID NOs: 1-84. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 56. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 51.
Another aspect features an isolated nucleic acid molecule comprising a nucleotide sequence having at least 85% sequence identity to all or a portion of any one of SEQ ID NOs: 93-181 , 190-195, and 199-204, or a complementary sequence thereof. In some embodiments, the nucleic acid molecule has at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to, or the nucleotide sequence of, any one of SEQ ID NOs: 93-181, 190-195, and 199-204. In some embodiments, the nucleic acid molecule, or a portion thereof, is capable of eliciting an immune response in a subject. In some embodiments, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 195. In some embodiments, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 143. In some embodiments, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 204. In some embodiments, the nucleic acid molecule has the nucleotide sequence of nucleotides 19-3837 of SEQ ID NO: 204. Another aspect features an isolated polypeptide comprising an amino acid sequence having at least 85% sequence identity to all or a portion of any one of SEQ ID NOs: 1-84. In some embodiments, said polypeptide has at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to, or the amino acid sequence of, any one of SEQ ID NOs: 1-84. In some embodiments, the polypeptide, or a portion thereof, is capable of eliciting an immune response in a subject. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 28. In some embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 51.
Another aspect features an isolated vector comprising one or more of the above nucleic acid molecules. In some embodiments, the vector is replication-defective (e.g., lacking an E1, E3, and/or E4 region). In some embodiments, the vector is a mammalian, bacterial, or viral vector.
In some embodiments, the vector is an expression vector. In some embodiments, the viral vector is a virus selected from the group consisting of a retrovirus, adenovirus, adeno- associated virus, parvovirus, coronavirus, negative strand RNA viruses, orthomyxovirus, rhabdovirus, paramyxovirus, positive strand RNA viruses, picornavirus, alphavirus, double stranded DNA viruses, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox, and canarypox. In some embodiments, the vector is an adenovirus. In some embodiments, the adenovirus is selected from the group consisting of Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52, Ad59, and Pan9. In some embodiments, the Ad52 is a rhesus Ad52 or the Ad59 is a rhesus Ad59. In some embodiments, the adenovirus is Ad26. In other embodiments, the adenovirus is an Ad26 vector that comprises a nucleic acid molecule with nucleotides 19-3837 of SEQ ID NO: 204 or all of the nucleotides of SEQ ID NO: 204. In another embodiment, the adenovirus is an Ad26 vector that comprises a nucleic acid molecule encoding a polypeptide with at least 85% or more (e.g., 90%, 95%, 99%, or 100%) sequence identity to the polypeptide of SEQ ID NOL 51.
Another aspect features an isolated antibody that specifically binds to any of the abovementioned polypeptides. In some embodiments, the antibody is generated by immunizing a mammal with the nucleic acid, the polypeptide, or the vector. In some embodiments, the mammal is a human, cow, goat, mouse, or rabbit. In some embodiments, the antibody is humanized. In some embodiments, the antibody is an IgG. In some embodiments, the antibody is a bis-Fab, Fv, Fab, Fab’-SH, F(ab’)2, a diabody, a linear antibody, or a scFV. Another aspect features a method of producing an anti-2019-Wuhan coronavirus (2019-nCoV) antibody, comprising administering an amount of the nucleic acid molecule, the polypeptide, and/or the vector to a subject sufficient to elicit the production of neutralizing anti-2019-nCoV antisera after administration to said subject.
Another aspect features an isolated anti-2019-nCoV antibody produced by any of the abovementioned methods. In some embodiments, the antibody binds to an epitope within any one of SEQ ID NOs: 1-84.
Another aspect features a composition comprising the nucleic acid molecule, the polypeptide, the vectors or the antibody. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the composition further comprises an adjuvant or an immunostimulatory agent.
Another aspect features an immunogenic composition comprising the nucleic acid molecule, the polypeptide, the vector, or the antibody. In some embodiments, the immunogenic composition is a vaccine. In some embodiments, the immunogenic composition is capable of treating or reducing the risk of a coronavirus infection (e.g., a 2019-nCoV infection) in a subject in need thereof. In some embodiments, the immunogenic composition elicits production of neutralizing anti-2019-nCoV antisera after administration to said subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human has an underlying health condition. In some embodiments, the underlying health condition is hypertension, diabetes, or cardiovascular disease.
Another aspect features a method of identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection comprising determining whether the subject has a protective level of an anti-coronavirus antibody (such as an anti-Spike antibody) in a sample from the subject, wherein preferably the protective level is: (i) a level that is at or above a titer of at least about 70, as determined using a pseudovirus neutralization assay; (ii) a level that is at or above a titer of at least about 25, as determined using a live virus neutralization assay; or (iii) a level that is at least 80% of a median level of an anti-coronavirus antibody in a cohort of convalescent humans, as determined by a pseudovirus neutralization assay or live virus neutralization assay. In some embodiments the protective level of an anti-coronavirus antibody is a level sufficient to prevent or reduce the development of severe disease. In some embodiments, the method further comprises administering an effective amount of the composition or the immunogenic composition to the subject having less than a protective level of the anti-coronavirus antibody. In some embodiments, the method further comprises identifying a subclass and/or an effector function of the anti-coronavirus antibody (e.g., the anti- Spike antibody). In some embodiments, the subclass is IgM, IgA, lgG1, lgG2, lgG3, or FcgR2A. In some embodiments, the effector function is antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), or antibody-dependent NK cell activation. In some embodiments, the sample is a bodily fluid from the subject, wherein preferably the bodily fluid is blood. In some embodiments, the coronavirus is 2019-nCoV.
Another aspect features a method of treating or reducing the risk of a coronavirus infection in a subject in need thereof, comprising administering a therapeutically effective amount of the composition or the immunogenic composition to said subject. In some embodiments, the method further comprises measuring an anti-coronavirus antibody (e.g., an anti-Spike antibody) level in the subject. In some embodiments, the anti-coronavirus antibody level in the subject is measured before and/or after the administration. In some embodiments, the anti-coronavirus antibody level in the subject is measured one or more times over about 1, 2, 3, 4, 5, or 6 days, 1, 2, 3, 4, 5, 6, or 7 weeks, 2, 3, 4, 5, or 6 months, 1, 2, 3, 4, or 5 years after administration. In some embodiments, the anti-coronavirus antibody level of the subject is below a protective level and wherein the method further comprises re-administering the composition or the immunogenic composition or administering a different anti-coronavirus composition to the subject. In some embodiments, the protective level is a level sufficient to reduce symptoms or duration of a coronavirus-mediated disease. In some embodiments, the protective level is a level sufficient to prevent or reduce the development of severe disease (e.g., which can be characterized by weight loss (e.g., a weight reduction of at least about 5% (e.g., at least about 7.5%, at least about 10%, at least about 12.5%, at least about 15%, at least about 20%, at least about 25% or more) relative to the subject’s initial weight pre-infection), the development of pneumonia and/or respiratory failure, and/or increased risk of death). In some embodiments, the protective level is: (i) a level that is at or above a titer of at least about 70, as determined using a pseudovirus neutralization assay; (ii) a level that is at or above a titer of at least about 25, as determined using a live virus neutralization assay; or (iii) a level that is at least 80% of a median level of an anti-coronavirus antibody in a cohort of convalescent humans, as determined by a pseudovirus neutralization assay or live virus neutralization assay. In some embodiments, the coronavirus is 2019-nCoV. In some embodiments, the method further includes measuring the coronavirus (e.g., 2019-nCoV) viral load in a sample from the subject. In some embodiments, the sample is a bronchoalveolar lavage (BAL) or a nasal swab (NS). In some embodiments, the sample is a bodily fluid (e.g., blood, e.g., whole blood or plasma) from the subject. In some embodiments, the sample is a tissue sample (e.g., a respiratory tract tissue sample) from the subject. In some embodiments, viral load is a detectible nucleic acid (e.g., subgenomic mRNA) level or a detectible protein (e.g., nucleocapsid protein (N)) level. In some embodiments, the detectible nucleic acid (e.g., subgenomic mRNA) is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, LAMP, microarray analysis, or hybridization (e.g., ISH (e.g., FISH)). In some embodiments, the detectible protein (e.g., nucleocapsid protein (N)) is determined by an immunoassay (e.g., an immunohistochemical (IHC) assay or a lateral flow immunoassay). In some embodiments, a detectable viral load indicates that the subject is susceptible to disease (e.g., a 2019-nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than at least about 3.5 logio sgmRNA copies/mL. In some embodiments, a viral load of greater than 3.85 logio sgmRNA copies/mL in BAL or 3.78 logio sgmRNA copies/mL in NS indicates that the subject is susceptible to disease (e.g., a 2019- nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than 3.85 logio sgmRNA copies/mL in BAL or 3.78 logio sgmRNA copies/mL in NS indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 2.0 logio sgmRNA copies/g of tissue indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 8.0 logio sgmRNA copies/g in lung tissue, about 7.0 logio sgmRNA copies/g in nares tissue, about 6.0 logio sgmRNA copies/g in trachea tissue, about 5.5 logio sgmRNA copies/g in heart tissue, or about 2.0 logio sgmRNA copies/g in Gl, spleen, liver, kidney, or brain tissue indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 3% SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to disease. In some embodiments, a viral load of greater than about 5% SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, coronavirus (e.g., 2019- nCoV) viral load is measured one or more times over about 1, 2, 3, 4, 5, or 6 days or 1 , 2, 3, 4,
5, 6, or 7 weeks post-infection. In some embodiments, a subject determined to be susceptible to disease (e.g., a 2019-nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease) is administered the composition or the immunogenic composition of the present disclosure alone or in combination with an additional therapeutic agent.
Another aspect features a method of reducing a coronavirus-mediated activity (e.g., 2019- nCoV-mediated activity) in a subject infected with a 2019-nCoV, comprising administering a therapeutically effective amount of the composition or the immunogenic composition to said subject. In some embodiments, the therapeutically effective amount is sufficient to produce a log serum anti-Spike antibody titer greater than 2 in a subject, as measured by an ELISA assay. In some embodiments, the therapeutically effective amount is between 15 pg and 300 pg of the composition or the immunogenic composition. In some embodiments, the activity is viral titer, viral spread, infection, or cell fusion. In some embodiments, the viral titer is decreased after administration of the composition or the immunogenic composition. In some embodiments, the viral titer is decreased by 25% or more. In some embodiments, the viral titer is decreased by 50% or more. In some embodiments, the viral titer is decreased by 75% or more. In some embodiments, the coronavirus is undetectable after said administration. In some embodiments, the administering occurs prior to exposure to the coronavirus. In some embodiments, the administering occurs at least 1 hour prior to exposure to said coronavirus. In some embodiments, the administering occurs at least 1 week, 1 month, or a year prior to exposure to said coronavirus. In some embodiments, the administering occurs post-exposure to the coronavirus. In some embodiments, the administering occurs at least 15 minutes post-exposure to said coronavirus. In some embodiments, the administering occurs at least 1 hour, 1 day, 1- week, post-exposure to said coronavirus. In some embodiments, the subject is administered at least one dose of the nucleic acid molecule, polypeptide, vector, composition, immunogenic composition, and antibody. In some embodiments, the subject is administered at least two doses. In some embodiments, the nucleic acid molecule, polypeptide, vector, composition, or immunogenic composition is administered to said subject as a prime, a boost, or as a prime boost. In some embodiments, the nucleic acid molecule, polypeptide, vector, composition, immunogenic composition, or antibody is administered intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivelly, intravesicularlly, mucosally, intraperi cardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in creams, or in lipid compositions. In some embodiments, the nucleic acid molecule, polypeptide, vector, composition, immunogenic composition, or antibody is administered intramuscularly. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the human has an underlying health condition. In some embodiments, the underlying health condition is hypertension, diabetes, or cardiovascular disease. In some embodiments, the method promotes an immune response in said subject. In some embodiments, the immune response is a humoral immune response. In some embodiments, the humoral immune response is an IgG response. Another aspect features a composition for use in treating or reducing the risk of a coronavirus infection, such as a 2019-nCoV infection, in a subject in need thereof, comprising a therapeutically effective amount of the composition or the immunogenic composition.
Another aspect features a composition for use in reducing a coronavirus-mediated activity (e.g., 2019-nCoV-mediated activity) in a subject infected with a 2019-nCoV, comprising a therapeutically effective amount of the composition or the immunogenic composition.
Another aspect features a method of manufacturing an immunogenic composition for treating or reducing the risk of a coronavirus (e.g., 2019-nCoV) infection in a subject in need thereof, said method comprising the steps of: (a) admixing at least one of the nucleic acid molecule, the polypeptide, the vector, the composition, and the antibody with a pharmaceutically acceptable carrier, excipient, or diluent to form the immunogenic composition; and (b) placing the immunogenic composition in a container.
Another aspect features a kit comprising: (a) a first container comprising at least one of the nucleic acid molecule, the polypeptide, the vector, the composition, the immunogenic composition, and the antibody; (b) instructions for use thereof; and optionally (c) a second container comprising a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the first container further comprises a pharmaceutically acceptable carrier, excipient, or diluent. The kit optionally includes an adjuvant and/or an immunostimulatory agent.
Another aspect features a kit comprising: one or more reagents for determining the presence of an anti-coronavirus antibody (such as an anti-Spike antibody) in a sample (e.g., a blood sample) from a subject and instructions for identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection. In some embodiments, the kit further comprises reagents for identifying a subclass and/or an effector function of the anti-coronavirus antibody. In some embodiments, the kit further comprises standards or samples for comparison.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings are included to illustrate embodiments of the invention and further an understanding of its implementations. The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise embodiments shown in the drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 is a diagram showing Spike protein immunogens. Annotated domains of 2019-nCoV Spike (SEQ ID NO: 1) including the S1 (SEQ ID NO: 4), S2 (amino acids 665-1191 of SEQ ID NO: 1), TM (SEQ ID NO: 86), and CT (SEQ ID NO: 85) domains. Full-length Spike (SEQ ID NO: 1), SdCT (SEQ ID NO: 2), S.Ecto (SEQ ID NO: 3), S1-foldon (SEQ ID NO: 9), RBD-foldon (SEQ ID NO: 10), and S.Ecto-PP-foldon (SEQ ID NO: 22) protein immunogens are labeled. White boxes indicate foldon domain and the double intersecting lines in S.Ecto-PP-foldon indicate the approximate position of two stabilizing mutations (proline substitutions corresponding to amino acids K969 and V970 of SEQ ID NO: 1).
FIG. 2 is a western blot showing the recognition of recombinant 2019-nCoV proteins by polyclonal anti-SARS antiserum. Cell lysates (left panel) and supernatants (right panel) from cells transfected with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), and SS-S.Ecto-dF-PP-foldon (lane 4) were probed using polyclonal anti-SARS antiserum. Numbered black lines to the left of each blot indicate approximate molecular weight in kDa and numbers at the top of each blot indicate lane number.
FIG. 3 is a graph showing the recognition of full-length Spike by antibodies produced in 2019-nCoV vaccinated mice. Serum was collected from mice 4-weeks post-vacci nation with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), SS-S1-foldon (lane 4), SS-RBD-foldon (lane 5), and SS-S.Ecto-dF-PP-foldon (lane 6) and used in an ELISA with full- length Spike (SEQ ID NO: 1). Gray bars represent mean ELISA titer.
FIG. 4 is a graph showing the recognition of S.dTM.PP by antibodies produced in 2019- nCoV vaccinated mice. Serum was collected from mice 4-weeks post-vaccination with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), SS-S1-foldon (lane 4), SS- RBD-foldon (lane 5), and SS-S.Ecto-dF-PP-foldon (lane 6) and used in an ELISA with full-length ectodomain S.Ecto-PP (SEQ ID NO: 19). Gray bars represent mean ELISA titer.
FIG. 5 is a graph showing the neutralizing activity of antibodies produced in 2019-nCoV vaccinated mice. Serum was collected from mice 4-weeks post-vaccination with DNA encoding SS-Spike (lane 1), SS-SdCT (lane 2), SS-S.Ecto (lane 3), SS-S1-foldon (lane 4), SS- RBD-foldon (lane 5), and SS-S.Ecto-dF-PP-foldon (lane 6) and used in an in vitro 2019-nCoV Spike pseudovirus neutralization assay. Gray bars represent mean IC50 titer.
FIGS. 6A-6E are graphs showing humoral immune responses in vaccinated rhesus macaques. Humoral immune responses were assessed following immunization by (FIG. 6A) binding antibody ELISA, (FIG. 6B) pseudovirus neutralization assays, and (FIG. 6C) live virus neutralization assays. (FIG. 6D) Comparison of pseudovirus neutralization titers in vaccinated macaques (all animals and SS-Spike / SS-SdCT groups), a cohort of 9 convalescent macaques, and a cohort of 27 convalescent humans from Boston who had recovered from 2019-nCoV infection. (FIG. 6E) S- and RBD-specific antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), and antibody-dependent NK cell activation (IFN-y secretion, CD107a degranulation, and MIR-1b expression) are shown. Radar plots show the distribution of antibody features across the vaccine groups. The size and color intensity of the wedges indicate the median of the feature for the corresponding group (blue depicts antibody functions, red depicts antibody isotype/subclass/FcyR binding). The principal component analysis (PCA) plot shows the multivariate antibody profiles across groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses shows the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution. Red bars reflect median responses. Dotted lines reflect assay limit of detection.
FIGS. 7A-7B are graphs showing cellular immune responses in vaccinated rhesus macaques. Cellular immune responses were assessed following immunization by (FIG. 7 A) IFN-g ELISPOT assays and (FIG. 7B) multiparameter intracellular cytokine staining assays in response to pooled S peptides. Red bars reflect mean responses.
FIGS. 8A-8D are graphs showing viral loads in 2019-nCoV challenged rhesus macaques.
Rhesus macaques were challenged by the intranasal and intratracheal route with 1.2x1012 VP (1.1x104 PFU) 2019-nCoV. (FIG. 8A) Logio sgmRNA copies/mL or copies/swab (limit 50 copies/mL) were assessed in bronchoalveolar lavage (BAL) and nasal swabs (NS) in sham controls at multiple timepoints following challenge. (FIG. 8B) Logio sgmRNA copies/mL in BAL and (FIG. 8C) logio sgmRNA copies/swab in NS in vaccinated animals. (FIG. 8D) Peak viral loads in BAL and NS following challenge. Red lines reflect median viral loads. P-values indicate two-sided Mann-Whitney tests. FIGS. 9A-9C are graphs showing immune correlates of protection. Correlations of (FIG.
9A) pseudovirus NAb titers and (FIG. 9B) live NAb titers prior to challenge with log peak sgmRNA copies/ L in BAL or log peak sgmRNA copies/swab in nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests. (FIG. 9C) The heat map (top panel) shows the Spearman and Pearson correlations between antibody features and log peak sgmRNA copies/mL in BAL (*q<0.05, **q < 0.01, ***q<0.001, with q-values obtained by Benjamini- Hochberg correction for multiple testing). The bar graph (bottom left panel) shows the rank of the Pearson correlation of the most predictive combination or individual antibody features defined by recursive feature elimination for partial least square regression (PLSR) and random forest (RF) regression. The correlation heatmap (bottom right panel) represents pairwise Pearson correlations between features across all animals.
FIG. 10 is a graph showing correlation of pseudovirus and live virus NAb assays in vaccinated macaques. Red line reflects the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
FIG. 11 is a graph showing viral RNA following 2019-nCoV challenge in sham controls in BAL and nasal swabs. Red lines reflect median viral loads.
FIG. 12 is a graph showing viral RNA following 2019-nCoV challenge in vaccinated animals in BAL. Red lines reflect median viral loads.
FIG. 13 is a graph showing viral RNA following 2019-nCoV challenge in vaccinated animals in nasal swabs. Red lines reflect median viral loads.
FIG. 14 is a graph showing Peak viral RNA following 2019-nCoV challenge in vaccinated animals in BAL and nasal swabs. Red lines reflect median viral loads. P-values indicate two- sided Wilcoxon rank-sum tests.
FIG. 15 is a graph showing correlations of log ELISA titers prior to challenge with log sgmRNA in BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests. FIG. 16 is a graph showing correlations of log ELISPOT responses prior to challenge with log sgmRNA in BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
FIG. 17 is a graph showing correlations of log CD4+ ICS responses prior to challenge with log sgmRNA in BAL and nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
FIG. 18 is a graph showing correlations of log CD8+ ICS responses prior to challenge with log sgmRNA copies/mL in BAL and log sgmRNA copies/swab in nasal swabs following challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
FIG. 19 is a graph showing anamnestic ELISA responses following challenge. Responses on day 0 and day 14 following challenge are shown. Red lines reflect median responses.
FIG. 20 is a graph showing anamnestic pseudovirus NAb responses following challenge.
Responses on day 0 and day 14 following challenge are shown. Red lines reflect median responses.
FIG. 21 is a graph showing anamnestic live virus NAb responses following challenge.
Responses on day 0 and day 14 following challenge are shown. Red lines reflect median responses.
FIG. 22 is a graph showing anamnestic ELISPOT responses following challenge.
Responses on day 0 and day 14 following challenge are shown. Red lines reflect median responses.
FIGS. 23A-23C are graphs showing viral loads in 2019-nCoV challenged rhesus macaques. Rhesus macaques were inoculated by the intranasal and intratracheal route with 1.1x106 PFU (Group 1; N=3), 1.1x105 PFU (Group 2; N=3), or 1.1x104 PFU (Group 3; N=3) 2019-nCoV. (FIG. 23A) Log viral RNA copies/mL (limit 50 copies/mL) were assessed in bronchoalveolar lavage (BAL) at multiple timepoints following challenge. (FIG. 23B) Log™ viral RNA copies/swab and (FIG. 23C) log sgmRNA copies/swab (limit 50 copies/swab) were assessed in nasal swabs (NS) at multiple timepoints following challenge. Red horizontal bars reflect median viral loads.
FIG. 24A-24F are graphs showing immune responses in 2019-nCoV challenged rhesus macaques. Humoral immune responses were assessed following challenge by (FIG. 24A) binding antibody ELISA, (FIG. 24B) pseudovirus neutralization assays, (FIG. 24C) live virus neutralization assays, and (FIG. 24D) systems serology profiles including antibody subclasses and effector functions to receptor binding domain (RBD), soluble spike (S) ectodomain, and nucleocapsid (N) proteins on day 35. Antibody-dependent complement deposition (ADCD), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent neutrophil phagocytosis (ADNP), and antibody-dependent NK cell degranulation (NK CD107a) and cytokine secretion (NK Ml P1 b, NK IFNy) are shown. Cellular immune responses were also assessed following challenge by (FIG. 24E) IFN-g ELISPOT assays and (FIG. 24F) multiparameter intracellular cytokine staining assays in response to pooled S peptides. Red and horizontal bars reflect mean responses.
FIGS. 25A-25L is a set of micrographs showing that 2019-nCoV induces acute viral interstitial pneumonia. (FIGS. 25A-25F) H&E sections of fixed lung tissue from 2019-nCoV infected rhesus macaques 2 days following challenge showing (FIG. 25A) interstitial edema and regional lung consolidation, (FIG. 25B) intra-alveolar edema and infiltrates of neutrophils, (FIGS. 25C-25D) bronchiolar epithelial sloughing and necrosis, (FIG. 25E) bronchiolar epithelial syncytial cell formation, and (FIG. 25F) hyaline membranes within alveolar septa. (FIGS. 25G- 25H) IHC for SARS nucleocapsid showing virus infected cells within interstitial spaces including (FIG. 25G) a viral syncytial cell within the lumen and (FIG. 25H) virus infected alveolar lining cells. (FIG. 25I) Inflammatory infiltrate showing multiple cells containing 2019-nCoV RNA by RNASCOPE® in situ hybridization. (FIGS. 25J-25L) bronchial respiratory epithelium showing (FIG. 25J) inflammation within the submucosa and transmigration of inflammatory cells into the ciliated columnar respiratory epithelium of a bronchus, (FIG. 25K) 2019-nCoV RNA, and (FIG.
25 L) SARS nucleocapsid. Scale bars (FIG. 25A) = 200 microns; (25C, 25I, 25K-25L) = 100 micron; (FIG. 25G) = 50 micron; (FIGS. 25B, 25D-25F, 25J) = 20 microns, and (FIG. 25H) = 10 microns. H&E=hematoxylin and eosin; IHC=immunohistochemistry; RNAscope=2019-nCoV RNA staining. FIGS. 26A-26K is a set of micrographs showing that 2019-nCoV infects alveolar epithelial cells in rhesus macaques. Cyclic immunofluorescence (CyCIF) staining of fixed lung tissue from 2019-nCoV infected rhesus macaques 2 days following challenge. (FIG. 26A) Whole slide image of a lung with DAPI staining for cell nuclei, regions of nuclear consolidation (arrows), and foci of viral replication (box). (FIG. 26B) Higher magnification images of inset box showing (FIG. 26C) SARS nucleocapsid positive cells (green) and DAPI for cell nuclei (blue). (FIG. 26D) Bright-field IHC for SARS nucleocapsid from corresponding lung region depicted in (FIG. 26B and FIG. 26C). (FIGS. 26E-26K) SARS-N co-staining with CD206 (FIG. 26E and FIG. 26K), pan-cytokeratin (pan-CK) (FIG. 26G and FIG. 26H), CD68 (FIG. 26I), and lba-1(FIG. 26J) showing virally infected epithelial cells and macrophages near an infected epithelial cell. Scale bars (FIGS. 26F-26K) = 50 microns.
FIGS. 27A-27F are graphs showing viral loads following 2019-nCoV re-challenge in rhesus macaques. On day 35 following initial infection (see FIGS. 23A-23C and 24A-24F), rhesus macaques were re-challenged with 2019-nCoV by the intranasal and intratracheal route with 1.1x106 PFU (Group 1; N=3), 1.1x105 PFU (Group 2; N=3), or 1.1x104 PFU (Group 3; N=3). Three naive animals were included as a positive control in the re-challenge experiment. (FIG. 27A) Logio viral RNA copies/mL (limit 50 copies/mL) were assessed in bronchoalveolar lavage (BAL) at multiple timepoints following re-challenge. One of the naive animals could not be lavaged. (FIG. 27B) Comparison of viral RNA in BAL following primary challenge and re challenge. (FIG. 27C) Logio viral RNA copies/mL and (FIG. 27E) logio sgmRNA copies/swab (limit 50 copies/mL) were assessed in nasal swabs (NS) at multiple timepoints following re challenge. Comparison of (FIG. 27D) viral RNA and (FIG. 27F) sgmRNA in NS following primary challenge and re-challenge. Red horizontal bars reflect median viral loads. P-values reflect two-sided Mann- Whitney tests.
FIG. 28 is a series of graphs showing anamnestic immune responses following 2019- nCoV re-challenge in rhesus macaques. Virus-specific binding antibody ELISAs, pseudovirus neutralization assays, live virus neutralization assays, and IFN-g ELISPOT assays are depicted prior to and 7 days following 2019-nCoV re-challenge. Red lines reflect mean responses. P-values reflect two-sided Mann-Whitney tests.
FIG. 29 is a series of graphs showing plasma viral loads in 2019-nCoV challenged rhesus macaques. Groups 1-3 are described in FIG. 23. Logio viral RNA copies/mL (limit 50 copies/mL) were assessed in plasma at multiple timepoints following challenge. Red horizontal bars reflect median viral loads.
FIG. 30 is a series of graphs showing clinical scores of 2019-nCoV challenged rhesus macaques. Groups 1-3 are described in FIG. 23. Semi-quantitative clinical scoring of animals based on appearance, dyspnea, recumbency, appetite, and responsiveness respiratory distress at multiple timepoints following challenge. Red horizontal bars reflect median clinical scores.
FIG. 31 is a graph showing hematology in 2019-nCoV challenged rhesus macaques.
Neutrophil and lymphocyte counts (K/pL) are shown. Red horizontal bars reflect median values.
FIG. 32 is a graph showing tissue viral loads in 2019-nCoV challenged rhesus macaques.
Rhesus macaques were inoculated with 2019-nCoV and were necropsied on day 2 (N=2) and day 4 (N=2) following challenge. Logio viral RNA copies/g tissue (limit 200 copies/g) were assessed in multiple tissues. Red horizontal bars reflect median viral loads.
FIGS. 33A-33J are micrographs showing that 2019-nCoV replication induces polymorphonuclear and mononuclear inflammatory infiltrates associated with type 1 interferon responses. (FIG. 33A and FIG. 33F) Detection of 2019-nCoV RNA, (FIG. 33B and FIG. 33G) IHC for myeloperoxidase (MPO), (FIG. 33C and FIG. 33H) double staining for CD4 (brown) and macrophages (CD68 and CD163, red), (FIG. 33D and FIG. 33I) CD8 T lymphocytes, and (FIG. 33E and FIG. 33J) MX1 (type 1 interferon response gene) in (FIG. 33A- 33E) a 2019-nCoV infected rhesus macaque on day 2 following challenge and (FIG. 33F-33G) an uninfected rhesus macaque. Scale bars = 100 microns.
FIGS. 34A-34B is a graph showing quantitative analysis of inflammatory infiltrates in lung tissue. (FIG. 34A) Lung alveoli polymorphonuclear (PMN) cell infiltration and (FIG. 34B) extent of MX1 staining in 2019-nCoV infected versus uninfected rhesus macaques. P values reflect 2- sided Mann-Whitney tests.
FIGS. 35A-35J are micrographs showing inflammatory infiltrates within regions of lung consolidation in 2019-nCoV acute infection. (FIG. 35A) One lung section 2 days post infection, containing three regions of interest (ROI) showed tissue consolidation (ROI 1-3); scale bar = 5 mm. (FIG. 35B) Bronchus associated lymphoid tissue (BALT) and (FIG. 35C and FIG. 35D) bronchiolar epithelium within ROI 1 showing bronchiolar necrosis with (FIG. 35C) migration of CD3+ T lymphocytes into the bronchiole lumen and (FIG. 35D) peribronchiolar inflammation with numerous myeloperoxidase (MPO) expressing neutrophils and CD16+ macrophages; CD3 (gray), CD20 (blue), CD68 (yellow), HLADR (cyan), MPO (magenta), E- Cadherin (green); scale bar = 100 pm. (FIG. 35E and FIG. 35F) CD16 positivity (red) in consolidated tissue (DNA, gray) with (FIG. 35E, FIG. 35G, and FIG. 35I) low 2019-nCoV positivity in ROI 1 as compared to (FIG. 35F, FIG. 35H, and FIG. 35J) high 2019-nCoV positivity in ROI 3. SARS-N (green) and pan-CK (epithelium, red) staining in (FIG. 35G) ROI 1 versus (FIG. 35H) ROI 3. CD3 (gray), CD20 (red), and CD68 (green) positivity in ROI 1 (low SARS CoV2 positivity) (FIG. 35I) versus ROI 3 (high SARS CoV2 positivity) (FIG. 35J); scale bar = 0.5 mm.
FIG. 36 is a graph showing plaque assays of BAL and nasal swabs following 2019-nCoV primary challenge and re-challenge. Peak plaque-forming units (PFU) per ml for BAL or per swab for nasal swabs from days 1-7 following primary challenge or re-challenge are shown.
Red horizontal bars reflect median PFU titers.
FIG. 37 is a graph showing clinical scores of 2019-nCoV re-challenged rhesus macaques.
Groups 1-3 are described in FIG. 23. Semi-quantitative clinical scoring of animals based on appearance, dyspnea, recumbency, appetite, and responsiveness respiratory distress at multiple timepoints following challenge.
FIG. 38A is a diagram showing the construction of Ad26 vectors. Seven Ad26 vectors were produced expressing SARS-CoV-2 S protein variants: (i) tPA leader sequence with full- length S (tPA.S (SEQ ID NO: 1, with a tPA leader sequence fused to the N-terminus)), (ii) tPA leader sequence with full-length S with mutation of the furin cleavage site and two proline stabilizing mutations (tPA.S. PP (SEQ ID NO: 23, with a tPA leader sequence fused to the N- terminus)), (iii) wildtype leader sequence with native full-length S (S (SEQ ID NO: 29)), (iv) wildtype leader sequence with S with deletion of the cytoplasmic tail (S.dCT (SS-SdCT) (SEQ ID NO: 30)), (v) tandem tPA and wildtype leader sequences with full-length S (tPA.WT.S (SEQ ID NO: 29, with a tPA leader sequence fused to the N-terminus)), (vi) wildtype leader sequence with S with deletion of the transmembrane region and cytoplasmic tail, reflecting the soluble ectodomain, with mutation of the furin cleavage site, proline stabilizing mutations, and a foldon trimerization domain (S.dTM.PP (SS-S.Ecto-dF-PP-foldon) (SEQ ID NO: 56)), and (vii) wildtype leader sequence with full-length S with mutation of the furin cleavage site and proline stabilizing mutations (S.PP (SS-Spike-dF-PP) (SEQ ID NO: 51)). Red triangle depicts tPA leader sequence, black triangle depicts wildtype leader sequence, red X depicts furin cleavage site mutation, red vertical lines depict proline mutations, open square depicts foldon trimerization domain.
FIG. 38B is a western blot showing the recognition of SARS-CoV-2 S variatns by polyclonal anti-SARS antibody. Western blot analyses for expression from Ad26 vectors encoding tPA.S (lane 1), tPA.S.PP (lane 2), S (lane 3), S.dCT (SS-SdCT) (lane 4), tPA.WT.S (lane 5), S.dTM.PP (SS-S.Ecto-dF-PP-foldon) (lane 6), and S.PP (SS-Spike-dF-PP) (lane 7) in cell lysates using an anti-SARS polyclonal antibody.
FIGS. 39A-39D are graphs showing humoral immune responses in vaccinated rhesus macaques. Humoral immune responses were assessed at weeks 0, 2, and 4 by (FIG. 39A) RBD-specific binding antibody ELISA, (FIG. 39B) pseudovirus neutralization assays, and (FIG. 39C) live virus neutralization assays. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation. (FIG. 39D) S- and RBD-specific antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent monocyte cellular phagocytosis (ADCP), antibody- dependent complement deposition (ADCD), and antibody-dependent NK cell activation (ADNKA) at week 4 are shown as radar plots. The size and color intensity of the wedges indicate the median of the feature for the corresponding group (blue depicts antibody functions, red depicts antibody isotype/subclass/FcyR binding). The principal component analysis (PCA) plot shows the multivariate antibody profiles across groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses shows the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution.
FIGS. 40A-40B are graphs showing cellular immune responses in vaccinated rhesus macaques. Cellular immune responses were assessed at week 4 following immunization by (FIG. 40A) IFN-Y ELISPOT assays and (FIG. 40B) IFN-Y+CD4+ and IFN-Y+CD8+ T cell intracellular cytokine staining assays in response to pooled S peptides. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
FIGS. 41A-41D are graphs showing viral loads in rhesus macaques following SARS-CoV- 2 challenge. Rhesus macaques were challenged by the intranasal and intratracheal routes with 1.2x108 VP (1.1x104 PFU) SARS-CoV-2. (FIGS. 41A-41B) Log sgmRNA copies/mL (limit of quantification 50 copies/mL) were assessed in bronchoalveolar lavage (BAL) in sham controls and in vaccinated animals following challenge. (FIGS. 41C-41D) Log™ sgmRNA copies/swab (limit of quantification 50 copies/swab) were assessed in nasal swabs (NS) in sham controls and in vaccinated animals following challenge. One animal in the S.dTM.PP (SS- S.Ecto-dF-PP-foldon) group did not have peak BAL samples obtained following challenge. Red lines reflect median values.
FIG. 42 is a graph showing summary of peak viral loads following SARS-CoV-2 challenge.
Peak viral loads in BAL and NS following challenge. Peak viral loads occurred variably on day 1-4 following challenge. Red lines reflect median viral loads. P-values indicate two-sided Mann-Whitney tests (*P<0.05, **P<0.001, ***P<0.0001).
FIGS. 43A-43D are graphs showing antibody correlates of protection. Correlations of (FIG. 43A) binding ELISA titers, (FIG. 43B) pseudovirus NAb titers, and (FIG. 43C) live virus NAb titers at week 2 and week 4 with log peak sgmRNA copies/mL in BAL following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two- sided Spearman rank-correlation tests. (FIG. 43D) The heat map shows the differences in the means of z-scored features between completely protected (N=17) and partially protected and non-protected (N=22) animals. The two groups were compared by two-sided Mann-Whitney tests, and stars indicate the Benjamini-Hochberg corrected q-values (*q < 0.05). The dot plots show differences in the features that best discriminated completely protected and partially protected animals, including NAb titers, S-specific antibody-dependent NK cell activation (ADNKA), and antibody-dependent monocyte cellular phagocytosis (ADCP). P-values indicate two-sided Mann-Whitney tests. The bar plot shows the cross-validated area under the receiver operator characteristics curves using the features indicated on the x-axis in a logistic regression model. The top three 1 -feature and 2-feature models are shown. Error bars indicate the mean and standard deviation for 100 repetitions of 10-fold cross-validation.
FIGS. 44A-44B are graphs showing immune responses following SARS-CoV-2 challenge.
(FIG. 44A) Pseudovirus NAb titers prior to challenge and on day 14 following challenge and (FIG. 44B) IFN-Y+CD8+ and IFN-y+CD4+ T cell responses by intracellular cytokine staining assays in response to pooled spike (S1, S2), nucleocapsid (NCAP), and non-structural proteins (N6, N7a, N8) peptides on day 14 following challenge in sham controls and in Ad26-S.PP (Ad26 SS-Spike-dF-PP) vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
FIG. 45 is a pair of graphs showing correlation of pseudovirus NAb titers and ELISA or live virus NAb assays in vaccinated macaques. Red line reflects the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
FIG. 46 is a graph showing a comparison of pseudovirus NAb titers in vaccinated macaques and convalescent macaques and humans. Comparison of pseudovirus NAb in macaques vaccinated with Ad26-S.PP (Ad26 SS-Spike-dF-PP) with previously reported cohorts of convalescent macaques and convalescent humans who had recovered from SARS-CoV-2 infection.
FIG. 47 is a pair of graphs showing humoral immune responses in BAL in vaccinated rhesus macaques. S-specific IgG and IgA at week 4 in BAL by ELISA in sham controls and in Ad26-S.PP (Ad26 SS-Spike-dF-PP) vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
FIG. 48 is a series of graphs showing humoral immune responses in vaccinated rhesus macaques. S- and RBD-specific antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent monocyte cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent NK cell activation (ADNKA) are shown. Red bars reflect median responses.
FIG. 49 is a pair of graphs showing cellular immune responses in vaccinated rhesus macaques. IFN-y+, IL-2+, IL-4+, and IL-10+ CD4+ T cell intracellular cytokine staining assays in response to pooled S peptides in Ad26-S.dTM.PP (Ad26 SS-S.Ecto-dF-PP-foldon) and Ad26- S.PP (Ad26 SS-Spike-dF-PP) vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
FIG. 50 is a pair of graphs showing ELISA correlates of protection. Correlations of binding ELISA titers at week 2 and week 4 with log peak sgmRNA copies/swab in NS following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
FIG. 51 is a pair of graphs showing pseudovirus NAb correlates of protection.
Correlations of pseudovirus NAb titers at week 2 and week 4 with log peak sgmRNA copies/swab in NS following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
FIG. 52 is a pair of graphs showing live virus NAb correlates of protection. Correlations of live virus NAb titers at week 2 and week 4 with log peak sgmRNA copies/swab in NS following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests.
FIG. 53 is a series of graphs showing antibody correlates of protection. The dot plots show differences in the features between completely protected and partially protected animals. P-values indicate two-sided Mann-Whitney tests.
FIG. 54 is a series of graphs showing NAb titers following SARS-CoV-2 challenge.
Pseudovirus NAb titers prior to challenge and on day 14 following challenge in vaccinated animals. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation.
FIGS. 55A-55C are graphs showing clinical disease following SARS-CoV-2 infection in hamsters. Syrian golden hamsters (10-12 weeks old; male and female; N=20) were infected with 5x104 TCIDso (low dose; N=4) or 5x105 TCID50 (high dose; N=16) of SARS-CoV-2 by the intranasal route. (FIG. 55A) Median percent weight change following challenge. The numbers reflect the number of animals at each timepoint. In the high-dose group, 4 animals were necropsied on day 2, 4 animals were necropsied on day 4, 4 animals met euthanization criteria on day 6, and 2 animals met euthanization criteria on day 7. (FIG. 55B) Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 2 and day 4. (FIG. 55C) Tissue viral loads as measured by logio RNA copies per gram tissue (limit of quantification 100 copies/g) in the scheduled necropsies at day 2 and day 4 and in 2-5 of 6 animals that met euthanization criteria on days 6-7. Extended tissues were not harvested on day 6. FIGS. 56A-56L are a series of graphs showing pathologic features of high-dose SARS- CoV-2 infection in hamsters. (FIG. 56A) Necrosis and inflammation (arrow) in nasal turbinate, H&E (d2). (FIG. 56B) Bronchiolar epithelial necrosis with cellular debris and degenerative neutrophils in lumen (arrow) and transmigration of inflammatory cells in vessel wall (arrowhead), H&E (d2). (FIG. 56C) Interstitial pneumonia, hemorrhage, and consolidation of lung parenchyma, H&E (d2). (FIG. 56D) Nasal turbinate epithelium shows strong positivity for SARS-CoV-N by IHC (d2). (FIG. 56E) Bronchiolar epithelium and luminal cellular debris show strong positivity for SARS-CoV-N by IHC (d2). (FIG. 56F) Pneumocytes and alveolar septa show multifocal strong positivity for SARS-CoV-N by IHC (d2). (FIG. 56G) Diffuse vRNA staining by RNASCOPE® within pulmonary interstitium (arrow, interstitial pneumonia) and within bronchiolar epithelium (arrowhead; d2). (FIG. 56H) Diffuse vRNA staining by RNASCOPE® within pulmonary interstitium (d4). (FIG. 56I) lba-1 IHC (macrophages) within pulmonary interstitium (d7). (FIG. 56J) CD3+ T lymphocytes within pulmonary interstitium, CD3 IHC (d4). (FIG. 56K) MPO (neutrophil myeloperoxidase) IHC indicating presence of interstitial neutrophils (d7). (FIG. 56L) Interferon-inducible gene, MX1, IHC shows strong and diffuse positivity throughout the lung (d4). H&E, hematoxylin and eosin; IHC, immunohistochemistry; Iba1, ionized calcium binding adaptor protein 1. Representative sections are shown. Scale bars = 20 mGh (FIGS. 56B, 56D); 50 mGP (FIGS. 56A, 56E, 56F); 100 mGP (FIGS. 56C, 56G-56L).
FIGS. 57A-57F are a series of graphs showing longitudinal quantitative image analysis of viral replication and associated inflammation in lungs. (FIG. 57A) Percent lung area positive for anti-sense SARS-CoV-2 viral RNA (vRNA) by RNASCOPE® ISH. (FIG. 57B) Percentage of total cells positive for SARS-CoV-N protein (nuclear or cytoplasmic) by IHC.
(FIG. 57C) lba-1 positive cells per unit area by IHC. (FIG. 57D) CD3 positive cells per unit area. (FIG. 57E) MPO positive cells per unit area. (FIG. 57F) Percentage of MX1 positive lung tissue as a proportion of total lung area. ISH, in situ hybridization; IHC, immunohistochemistry; SARS- N, SARS-CoV nucleocapsid; MPO, myeloperoxidase; MX1, myxovirus protein 1 (a type 1 interferon inducible gene). Each dot represents one animal.
FIGS. 58A-580 are a series of graphs showing lung viral dynamics and ACE2 receptor expression patterns. Hamsters were necropsied before (SARS-CoV-2 Negative) or after high- dose SARS-CoV-2 challenge on day 2 (D2), day 4 (D4), day 7 (D7), and day 14 (D14) following challenge. Serial sections of lung tissue were stained for vRNA anti-sense RNASCOPE® (FIGS. 58A-58E), for vRNA sense RNASCOPE® (FIGS. 58F-58J), and ACE2 IHC (FIGS. 58K- 580). Anti-sense RNASCOPE® used a sense probe; sense RNASCOPE® used an anti-sense probe. IHC, immunohistochemistry. Representative sections are shown. Scale bars = 100 pm.
FIGS. 59A-59L are a series of graphs showing extrapulmonary pathology. (FIG. 59A) Anti- sense SARS-CoV-2 viral RNA (vRNA) in brainstem on day 2 following challenge. (FIG. 59B) Higher magnification showing cytoplasmic vRNA staining in neurons in the absence of inflammation and pathology. (FIG. 59C) Anti-sense SARS-CoV-2 vRNA staining in the lamina propria of small intestinal villus on day 2 following challenge. (FIG. 59D) Higher magnification showing cytoplasmic and nuclear vRNA staining in an individual mononuclear cell in the absence of inflammation and tissue pathology. (FIG. 59E) Anti-sense SARS-CoV-2 vRNA staining within the myocardium and along the epicardial surface of the heart on day 4 following challenge. (FIG. 59F) Higher magnification showing staining of inflammatory mononuclear cell infiltrates consistent with focal myocarditis. (FIG. 59G) Pulmonary vessel showing endothelialitis day 4 (d4) following challenge. (FIG. 59H) Pulmonary vessel showing CD3+ T lymphocyte staining by IHC adhered to endothelium and within vessel wall, d4 following challenge. (FIG. 59I) Pulmonary vessel showing lba-1+ staining by IHC of macrophages along endothelium and perivascularly, d4. (FIG. 59J) Pulmonary vessel showing minimal vascular staining for SARS-CoV-N by IHC, d4. (FIG. 59K) Heart from (FIGS. 59E, 59F) showing focal lymphocytic myocarditis as confirmed by CD3+ T lymphocyte staining (FIG. 59L) of cells by IHC, d4. Scale bars = 500 pm (FIGS. 59A, 59C, 59E); 100 pm (FIGS. 59B, 59D, 59F, 59G-59L).
FIGS. 60A-60F are a series of graphs showing SARS-CoV-2 in blood mononuclear cells and bone marrow. (FIG. 60A-60C) SARS-CoV-2 anti-sense vRNA staining within mononuclear cells within lung thrombus on day 2 following challenge. (FIG. 60D) Bone marrow from the nasal turbinate 4 days following challenge showing (FIG. 60E) hematopoetic cells (H&E) that show (FIG. 60F) positive staining for SARS-CoV-N IHC. vRNA, viral RNA; H&E, hematoxylin and eosin; IHC, immunohistochemistry. Scale bars = 500 pm (FIG. 60A); 200 pm (FIG. 60D); 100 pm (FIG. 60B, 60C, 60E, 60F).
FIGS. 61A-61F are graphs showing humoral immune responses in vaccinated hamsters.
(FIG. 61 A) SARS-CoV-2 spike (S) immunogens with (i) deletion of the transmembrane region and cytoplasmic tail reflecting the soluble ectodomain with a foldon trimerization domain (S.dTM.PP) or (ii) full-length S (S.PP), both with mutation of the furin cleavage site and two proline stabilizing mutations. Red X depicts furin cleavage site mutation, red vertical lines depict proline mutations, open square depicts foldon trimerization domain. S1 and S2 represent the first and second domain of the S protein, TM depicts the transmembrane region, and CT depicts the cytoplasmic domain. Hamsters were vaccinated with 1010 vp or 109 vp of Ad26-S.dTM.PP or Ad26-S.PP or sham controls (N=10/group). Humoral immune responses were assessed at weeks 0, 2, and 4 by (FIG. 61 B) RBD-specific binding antibody ELISA and (FIG. 61C) pseudovirus neutralization assays. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation. (FIG. 61 D) S- and RBD-specific IgG subclass, FcyR, and ADCD responses at week 4 are shown as radar plots. The size and color intensity of the wedges indicate the median of the feature for the corresponding group (antibody subclass, red; FcyR binding, blue; ADCD, green). (FIG. 61 E) Principal component analysis (PCA) plot showing the multivariate antibody profiles across vaccination groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses show the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution. (FIG. 61 F) The heat map shows the differences in the means of z-scored features between vaccine groups S.PP and S.dTM.PP. The two groups were compared by two-sided Mann-Whitney tests and stars indicate the Benjamini-Hochberg corrected q-values (*q < 0.05, ** q < 0.01, *** q < 0.001).
FIGS. 62A-62C are graphs showing the correlation of antibody titers and survival curves.
(FIG. 62A) Correlations of binding ELISA titers and pseudovirus NAb titers at week 2 and week 4. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests. (FIG. 62B) Survival curve for the vaccine study. P values indicate two-sided Fisher’s exact tests. N denotes number of animals in each group. (FIG. 62C) Combined analysis of the two hamster studies involving all animals that received the 5x105 TCID5o challenge dose and were followed longitudinally. P values indicate two-sided Fisher’s exact tests. N denotes number of animals in each group.
FIGS. 63A-63D are graphs showing clinical disease in hamsters following high-dose SARS-CoV-2 challenge. (FIG. 63A) Median percent weight change following challenge. (FIG. 63B) Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 4. (FIG. 63C) Maximal weight loss in the combined Ad26-S.dTM.PP (N=14), Ad26-S.PP (N=14), and sham control (N=7) groups, excluding the animals that were necropsied on day 4. P values indicate two-sided Mann-Whitney tests. (FIG. 63D) Quantification of percent lung area positive for anti-sense vRNA in tissue sections from Ad26-S.dTM.PP and Ad26-S.PP vaccinated hamsters as compared to control hamsters on day 4 following challenge. P values represent two-sided Mann- Whitney tests.
FIGS. 64A-64B are graphs showing antibody correlates of clinical protection. Correlations of (FIG. 64A) binding ELISA titers and (FIG. 64B) pseudovirus NAb titers at week 2 and week 4 with maximum percent weight loss following challenge. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
FIGS. 65A-65B are graphs showing tissue viral loads on day 4 and day 14. Tissue viral loads as measured by logio subgenomic RNA copies per gram tissue (limit of quantification 100 copies/g) on (FIG. 65A) day 4 (N=6 reflects both dose groups for each vaccine) and (FIG. 65B) day 14 (N=14 reflects both dose groups for each vaccine) following challenge. Red lines reflect median values. Each dot represents one animal.
FIGS. 66A-66D are graphs showing antibody correlates of protection. Correlations of (FIGS. 66A, 66C) binding ELISA titers and (FIGS. 66B, 66D) pseudovirus NAb titers at week 2 and week 4 with logio RNA copies per gram (FIGS. 66A, 66B) lung and (FIGS. 66C, 66D) nasal turbinate tissue in the animals that were necropsied on day 4. Red lines reflect the best linear fit relationship between these variables. P and R values reflect two-sided Spearman rank- correlation tests.
FIGS. 67A-67B are graphs showing antibody correlates of protection and anamnestic responses. (FIG. 67A) The heatmaps show the Spearman rank correlation between antibody features and weight loss (N=35), lung viral loads (N=12), and nasal turbinate viral loads (N=12). Significant correlations are indicated by stars after multiple testing correction using the Benjamini-Hochberg procedure (*q < 0.05, ** q < 0.01, *** q < 0.001). (FIG. 67B) ELISA and NAb responses in surviving hamsters on day 14 following SARS-CoV-2 challenge.
FIGS. 68A-68S is a series of graphs showing Ad26 vaccination protects against SARS- CoV-2 pathology. Histopathological H&E evaluation of (FIGS. 68A-68E, 68K-68N) sham controls and (FIGS. 68F-J, 680-R) Ad26-S.PP vaccinated hamsters shows in sham controls (FIG. 68A) severe consolidation of lung parenchyma and infiltrates of inflammatory cells, (FIG. 68B) bronchiolar epithelial syncytia and necrosis, (FIG. 68C) SARS-CoV-N positive (IHC) bronchiolar epithelial cells, (FIG. 68D) peribronchiolar CD3+ T lymphocyte infiltrates, and (FIG. 68E) peribronchiolar macrophage infiltrates (lba-1; IHC), and (FIGS. 68F-J) minimal to no corresponding pathology in Ad26-S.PP vaccinated animals. SARS-CoV-2 anti-sense RNASCOPE® ISH in (FIG. 68K), interstitial CD3+ T lymphocytes (FIG. 68L) MPO staining by IHC, and MX1 staining by IHC (FIG. 68N) in sham controls compared to similar regions in Ad26- S.PP vaccinated animals (FIGS. 680-68R) on day 4 following challenge. Quantification of percent lung area positive for anti-sense vRNA (FIG. 68S) in tissue sections from Ad26- S.dTM.PP and Ad26-S.PP vaccinated hamsters as compared to control hamsters 4 days following challenge. Representative sections are shown. P values represent two-sided Mann- Whitney tests. Scale bars = 20 mhi (FIGS. 68B, 68C, 68G, 68H, 681); 50 mhi (FIGS. 68A, 68D, 68E, 68J); 100 mGP (FIGS. 68F, 68K-68R).
FIG. 69: Schematic representation of several designs of SARS-CoV-2 S constructs (S1 light grey, S2 dark grey). In the bottom designs, the wildtype (wt) signal peptide was changed to the tPA signal peptide. Vertical line between S1 and S2 indicates the mutation in the furin cleavage site. Dotted line indicates the double proline mutations at position 986 and 987. Delta ERRS indicates deletion of the C-terminal residues that contain the endoplasmic reticulum retention signal.
FIG. 70: Detection of expression of SARS-CoV-2 S with several SARS-CoV-1 specific antibodies on the cell surface after transfection with several different S variants based on luminescence intensities using a cell-based ELISA. Data are represented as mean ± SEM.
FIG. 71 : Detection of expression of SARS-CoV-2 S with several SARS-CoV-1 specific antibodies and an antibody specific for a high-mannose patch on HIV gp120 (2G12), on the cell surface after transfection with several different S variants using flow cytometry. Data are represented as mean ± SEM.
FIG. 72: Serum SARS-CoV-2 Spike-specific antibody titers on day 19 by ELISA. ULOQ, upper limit of detection; LLOD, lower limit of detection. Statistical analysis: One-way ANOVA with Bonferroni multiple comparison correction; n.s., not significant (p>0.05).
FIG. 73: Effect of different signal peptides on protein expression. Detection of expression of SARS-CoV-2 S constructs with different signal peptides with a ACE2-antibody construct and SARS-CoV-1 specific antibody (CR3022) on the cell surface based on luminescence intensities using cell-based ELISA. Data are represented as mean ± SEM.
FIG. 74: Cloning the SARS-CoV-2 coding sequence in linearized target vector. Two DNA fragments coding parts of SARS-CoV-2 spike protein were assembled into their target vector.
FIG. 75: Introduction of expression cassette coding the SARS-CoV-2 sequence into the E1 region of the Ad26 vector.
FIGs. 76A-76B: S protein binding antibody titers as measured by ELISA. (FIG. 76A) week 2; (FIG. 76B) week 4. The median response per group is indicated with a horizontal line. The dotted line indicates the LLOD. Across-dose comparisons between Ad26NCOV030 (Ad26COVS1), Ad26NCOV006, and Ad26NCOV028 (data not shown) were performed with a t- test from ANOVA with Bonferroni correction for multiple comparisons. *: p<0.05. LLOD = lower limit of detection.
FIGs. 77A-77B: Neutralizing antibody titers as measured by wtVNA determining the cytopathic effect (CPE) of virus isolate Leidenl (L-001) on Vero E6 cells. (FIG. 77 A) week 2; (FIG. 77B) week 4. The median response per group is indicated with a horizontal line. The dotted line indicates the LLOD. Animals with a response at or below the LLOD are shown as open symbols. wtVNA = wild-type virus neutralization assay. Across-dose comparisons between Ad26NCOV030 (Ad26COVS1), Ad26NCOV006, and Ad26NCOV028 (data not shown) were performed with a z-test for Tobit ANOVA with Bonferroni correction for multiple comparisons. **: p<0.01.
FIG. 78: SARS-CoV-2 S Protein Specific Cellular Response in Mice; The sum of SFU from stimulation with peptide pools 1 and 2 is shown. IFN-y production by splenocytes was measured by ELISpot after 18 hours of peptide stimulation. The dotted line indicates the LLOD of 200 IFN- Y SFU/106 unstimulated splenocytes incubated with medium and DMSO. Animals with a response at or below the LLOD are shown as open symbols.
FIGs. 79A-79C: Cytokine concentration in cell supernatant was determined by a Multiplex ELISA based analysis, and ratios were calculated. (FIG. 79A) Ratio IFNY/IL-4; (FIG. 79B) Ratio IFNY/IL-5; (FIG. 79C) Ratio IFNY/IL-10. The median ratio per group is indicated with a horizontal line. Statistical difference between Ad26COVS1 (Ad26NCOV030) and adjuvanted S protein calculated by Mann-Whitney U-testwith 2-fold Bonferroni correction. ***: p<0.001.
FIGs. 80A-80C: S protein binding lgG1 and lgG2a titers were measured by ELISA. (FIG. 80A) lgG1; (FIG. 80B) lgG2a. The median response per group is indicated with a horizontal line. Dotted lines indicate the LLOD and ULOD of the assays. Animals with a response at or below the LLOD or at or above the ULOD are shown as open symbols. (FIG. 80C) The ratio of lgG2a/lgG1 titers was calculated. The median response per group is indicated with a horizontal line. The dotted line indicates an lgG2a/lgG1 ratio of 1. Animals with a response at or below the LLOD in the lgG2a ELISA are shown as open symbols. Statistical comparison of Ad26COVS1 (Ad26NCOV030) to the adjuvanted S protein analyzed by Mann-Whitney U-testwith a 2-fold Bonferroni correction. **: p<0.01.
FIGs. 81A-81B: (FIG. 81 A) S protein binding antibody titers as measured by ELISA. (FIG. 81 B) Neutralizing antibody titers as measured by wtVNA determining the cytopathic effect (CPE) of virus isolate Leidenl (L-001) on Vero E6 cells. The median response per group is indicated with a horizontal line. The lower dotted line indicates the LLOD of 1.699 logio for ELISA and 5.7 for wtVNA. Animals with a response at or below the LLOD are shown as open symbols. Across- dose comparisons were performed with a t-test from ANOVA (ELISA) and with a Cochran- Mantel-Haenszel test (wtVNA), with Bonferroni adjustment for multiple comparisons.
FIG. 82: IFN-y production by PBMC measured after 18 hours of peptide stimulation by ELISpot. The sum of SFU from stimulation with peptide pools 1 and 2 is shown. The dotted line indicates the LOB of 4.5 IFN-g SFU/106 PBMC, calculated as the 95th percentile of SFU from unstimulated cells incubated with medium and DMSO. Animals with a response at or below the LOB are shown as open symbols. Across-dose comparisons were performed with a t-test from ANOVA, with Bonferroni correction for multiple comparisons.
FIGs. 83A-83B: (FIG. 83A) S protein binding antibody titers as measured by ELISA.
Ad26. Empty sample N=18. (FIG. 83B) Neutralizing antibody titers as measured by wtVNA determining the cytopathic effect (CPE) of virus isolate Leidenl (L-001) on Vero E6 cells.
Ad26. Empty group shown as 2 pooled sera groups, from Group 7 (N=6) and Group 8 (N=6).
The median response per group is indicated with a horizontal line. The dotted line indicates the LLOD. Animals with a response at or below the LLOD are shown as open symbols. ELISA data comparisons were performed per dose between Ad26COVS1 (Ad26NCOV030) and
Ad26. Empty by Mann- Whitney U-test. No corrections for multiple comparisons were made. ***: p<0.001.
FIG. 84: Replication competent virus (TCID5o per gram lung) measured by plaque assay. The median viral load per group is indicated with a horizontal line. Animals with a response at or below the LLOD are shown as open symbols. Comparisons were performed between the Ad26. Empty group and the Ad26COVS1 (Ad26NCOV030) and Ad26NCOV006 groups by Mann-Whitney U-test. No corrections for multiple comparisons were made. **: p<0.01.
FIG. 85: Body weight as measured prior to challenge on Day relative to challenge [C]0 and 4 days post challenge on Day C4. The relative change in body weight is expressed as the body weight % relative to Day CO. The median change in body weight per group is indicated with a horizontal line. Comparisons were performed between the Ad26. Empty group and the Ad26COVS1 (Ad26NCOV030) and Ad26NCOV006 groups by t-test from ANOVA. No corrections for multiple comparisons were made. ***: p<0.001 ; **: p<0.01; *: p<0.05.
FIGs. 86A-86D: (FIG. 86A) S protein binding antibody titers as measured by ELISA. (FIG. 86B) Neutralizing antibody titers as measured by ppVNA. (FIG. 86C) Neutralizing antibody titers as measured by wtVNA using the Seattle Washington isolate, designed to express luciferase and GFP, incubated on Vero E6 cells. The titers were measured via Nano-Glo Luciferase Assay System. (FIG. 86D) IFN-y production of SARS-CoV-2 peptide stimulated PBMC as measured by ELISpot. The sum of SFU from stimulation with peptide pools 1 and 2 is shown. The dotted line indicates the LOB of 50 IFN-g SFU/106 PBMC. Animals with a response at or below the LOB are shown as open symbols. The median response per group is indicated with a horizontal line.
The dotted line indicates the LLOD of 25 for ELISA, 20 for ppVNA, and 4 for wtVNA. Animals with a response at or below the LLOD are shown as open symbols. ppVNA = pseudotyped virus neutralization assay; SFU = spot forming units; vp = virus particles; wtVNA = wild-type virus neutralization assay.
FIG. 87: Western blot analyses for expression from Ad26 vectors encoding tPA.S (lane 1), tPA.S.PP (lane 2), S (lane 3), S.dCT (lane 4), tPA.WT.S (lane 5), S.dTM.PP (lane 6), and S.PP (lane 7) in cell lysates using an anti-SARS polyclonal antibody. FIGs. 88A-88B: Humoral immune responses in vaccinated rhesus macaques. Humoral immune responses were assessed at weeks 0, 2, and 4 by (FIG. 88A) RBD-specific binding antibody ELISA, and (FIG. 88B) pseudovirus neutralization assays. Red (solid) bars reflect median responses.
FIGs. 89A-89B: Cellular immune responses in vaccinated rhesus macaques. Cellular immune responses were assessed at week 4 following immunization by (FIG. 89A) IFN-g ELISPOT assays and (FIG. 89B) IFN-y+CD4+ and IFN-y+CD8+ T cell intracellular cytokine staining assays in response to pooled S peptides. Red (solid) bars reflect median responses.
FIGs. 90A-90D: Viral loads in rhesus macaques following SARS-CoV-2 challenge. Rhesus macaques were challenged by the intranasal and intratracheal route with 1.2x108 VP (1.1x104 PFU) SARS-CoV-2. (FIG. 90A, FIG. 90B) Logio sgmRNA copies/ml (limit 50 copies/ml) were assessed in bronchoalveolar lavage (BAL) in sham controls and in vaccinated animals following challenge. (FIG. 90C, FIG. 90D) Logio sgmRNA copies/swab (limit 50 copies/swab) were assessed in nasal swabs (NS) in sham controls and in vaccinated animals following challenge. Red lines reflect median values.
FIG. 91 :_Summary of peak viral loads following SARS-CoV-2 challenge. Peak viral loads in BAL and NS following challenge. Peak viral loads occurred variably on day 1-4 following challenge. Red lines reflect median viral loads. P-values indicate two-sided Mann-Whitney tests (*P<0.05, **P<0.001, ***P<0.0001).
FIG. 92: Immune responses following SARS-CoV-2 challenge. Pseudovirus NAb titers prior to challenge and on day 14 following challenge. Red bars reflect median responses.
FIG. 93: Comparison of pseudovirus NAb titers in vaccinated macaques and convalescent macaques and humans. Comparison of pseudovirus NAb in macaques vaccinated with Ad26.S.PP with previously reported cohorts of convalescent macaques and convalescent humans who had recovered from SARS-CoV-2 infection.
FIG. 94: Humoral immune responses in BAL in vaccinated rhesus macaques. S-specific IgG and IgA at week 4 in BAL by ELISA in sham controls and in Ad26.S.PP vaccinated animals.
Red bars reflect median responses. FIG. 95: Cellular immune responses in vaccinated rhesus macaques. IFN-Y+, IL-2+, IL-4+, and IL-10+ CD4+ T cell intracellular cytokine staining assays in response to pooled S peptides in Ad26.S.dTM.PP and Ad26.S.PP vaccinated animals. Red bars reflect median responses.
FIGs. 96A-96C: (FIG. 96A) Median percent weight change following challenge. The numbers reflect the number of animals at each timepoint. In the high-dose group, 4 animals were necropsied on day 2, 4 animals were necropsied on day 4, 4 animals met euthanization criteria on day 6, and 2 animals met euthanization criteria on day 7. (FIG. 96B) Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 2 and day 4. (FIG. 96C) Tissue viral loads as measured by logio RNA copies per gram tissue (limit of quantification 100 copies/g) in the scheduled necropsies at day 2 and day 4 and in 2-5 of 6 animals that met euthanization criteria on days 6-7. Extended tissues were not harvested on day 6.
FIGs. 97A-97L: Pathologic features of high-dose SARS-CoV-2 infection in hamsters. (FIG. 97A) Necrosis and inflammation (arrow) in nasal turbinate, H&E (d2). (FIG. 97B) Bronchiolar epithelial necrosis with cellular debris and degenerative neutrophils in lumen (arrow) and transmigration of inflammatory cells in vessel wall (arrowhead), H&E (d2). (FIG. 97C) Interstitial pneumonia, hemorrhage, and consolidation of lung parenchyma, H&E (d2). (FIG. 97D) Nasal turbinate epithelium shows strong positivity for SARS-CoV-N by IHC (d2). (FIG. 97E)
Bronchiolar epithelium and luminal cellular debris show strong positivity for SARS-CoV-N by IHC (d2). (FIG. 97F) Pneumocytes and alveolar septa show multifocal strong positivity for SARS-CoV-N by IHC (d2). (FIG. 97G) Diffuse vRNA staining by RNAscope within pulmonary interstitium (arrow, interstitial pneumonia) and within bronchiolar epithelium (arrowhead; d2). (FIG. 97H) Diffuse vRNA staining by RNAscope within pulmonary interstitium (d4). (FIG. 97I) lba-1 IHC (macrophages) within pulmonary interstitium (d7). (FIG. 97J) CD3+ T lymphocytes within pulmonary interstitium, CD3 IHC (d4). (FIG. 97K) MPO (neutrophil myeloperoxidase) IHC indicating presence of interstitial neutrophils (d7). (FIG. 97L) Interferon-inducible gene, MX1,
IHC shows strong and diffuse positivity throughout the lung (d4). H&E, hematoxylin and eosin; IHC, immunohistochemistry; Iba1, ionized calcium binding adaptor protein 1. Representative sections are shown. Experiments were repeated at least 3 times with similar results. Scale bars = 20 pm (b, d); 50 pm (a, e, f); 100 pm (c, g-l). FIGs. 98A-98F: Humoral immune responses in vaccinated hamsters. (FIG. 98A) SARS-CoV-2 spike (S) immunogens with (i) deletion of the transmembrane region and cytoplasmic tail reflecting the soluble ectodomain with a foldon trimerization domain (S.dTM.PP) or (ii) full-length S (S.PP), both with mutation of the furin cleavage site and two proline stabilizing mutations. Red X depicts furin cleavage site mutation, red vertical lines depict proline mutations, open square depicts foldon trimerization domain. S1 and S2 represent the first and second domain of the S protein, TM depicts the transmembrane region, and CT depicts the cytoplasmic domain. Hamsters were vaccinated with 1010 vp or 109 vp of Ad26-S.dTM.PP or Ad26-S.PP or sham controls (N=10/group). Humoral immune responses were assessed at weeks 0, 2, and 4 by (FIG. 98B) RBD-specific binding antibody ELISA and (FIG. 98C) pseudovirus neutralization assays. Red bars reflect median responses. Dotted lines reflect assay limit of quantitation. (FIG. 98D) S- and RBD specific IgG subclass, FcyR, and ADCD responses at week 4 are shown as radar plots. The size and color intensity of the wedges indicate the median of the feature for the corresponding group (antibody subclass, red; FcyR binding, blue; ADCD, green). (FIG. 98E) Principal component analysis (PCA) plot showing the multivariate antibody profiles across vaccination groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses show the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution. (FIG. 98F) The heat map shows the differences in the means of z-scored features between vaccine groups S.PP and S.dTM.PP. The two groups were compared by two-sided Mann-Whitney tests and stars indicate the Benjamini-Hochberg corrected q-values (*q < 0.05, ** q < 0.01, *** q < 0.001).
FIGs. 99A-99C: Clinical disease in hamsters following high-dose SARS-CoV-2 challenge. (FIG. 99A) Median percent weight change following challenge. (FIG. 99B) Percent weight change following challenge in individual animals. Median weight loss is depicted in red. Asterisks indicate mortality. Grey lines indicate animals with scheduled necropsies on day 4. (FIG. 99C) Maximal weight loss in the combined Ad26-S.dTM.PP (N=14), Ad26-S.PP (N=14), and sham control (N=7) groups, excluding the animals that were necropsied on day 4. P values indicate two-sided Mann Whitney tests. N reflects all animals that were followed for weight loss and were not necropsied on day 4.
FIGs. 100A-100F: Longitudinal quantitative image analysis of viral replication and associated inflammation in lungs. (FIG. 100A) Percent lung area positive for anti-sense SARS-CoV-2 viral RNA (vRNA) by RNAscope ISH. (FIG. 100B) Percentage of total cells positive for SARS-CoV-N protein (nuclear or cytoplasmic) by IHC. (FIG. 100C) lba-1 positive cells per unit area by IHC. (FIG. 100D) CD3 positive cells per unit area. (FIG. 100E) MPO positive cells per unit area.
(FIG. 100F) Percentage of MX1 positive lung tissue as a proportion of total lung area. ISH, in situ hybridization; IHC, immunohistochemistry; SARS-N, SARS-CoV nucleocapsid; MPO, myeloperoxidase; MX1, myxovirus protein 1 (a type 1 interferon inducible gene). Each dot represents one animal.
FIG. 101: Participants were enrolled concurrently at Belgian and US sites. Participants were randomized in parallel in a 1:1:1 :1 :1 ratio to one of five vaccination groups to receive one or two IM injections of Ad26.COV2.S at two dose levels of either 5x1010 vp or 1x1011 vp, or placebo. For each cohort, in the absence of clinically significant findings 24 hours after the first vaccination was administered to five sentinel participants (one per dose level and one placebo), another ten participants were vaccinated across all groups. Safety data up to day 28 were then reviewed by an internal data review committee before the remaining participants were randomized.
FIGs. 102A-102C: Flow charts for cohort 1a (FIG. 102A), cohort 1b (FIG. 102B) and cohort 3 (FIG. 102C).
FIGs. 103A-103D: Humoral and cellular immune responses. (FIG. 103A) Log geometric mean titers (GMTs - as illustrated by the horizontal bars and the numbers below each timepoint) of serum SARS-CoV-2 binding antibodies, measured by ELISA (ELISA Units per mL [EU/mL]), at baseline and 29 days post vaccination, among all participants, according to schedule in cohort 1a and 3. Dotted lines indicate the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the assay, error bars indicate 95% confidence interval (Cl). For values below the LLOQ, LLOQ/2 values were plotted. (FIG. 103B) Log GMTs of serum SARS-CoV-2 neutralizing antibodies, measured by 50% microneutralization assay (ID50 Log GMT - as illustrated by the horizontal bars and the numbers below each timepoint), at baseline and 29 days post vaccination, among a subset of participants, according to schedule, in cohort 1a and 3. Dotted lines indicate the LLOQ and ULOQ of the assay, error bars indicate 95% Cl. For values below the LLOQ, LLOQ/2 values were plotted. (FIG. 103C) Expression of Th1 (IFNy and/or IL-2, not IL-4, IL-5 and IL-13), and Th2 (IL-4 and/or IL-5 and/or IL-13 and CD40L) cytokines by CD4 T cells was measured by intracellular cytokine staining (ICS). Median (as illustrated by the horizontal bars and the numbers below each timepoint) and individual ICS responses to SARS-CoV-2 S protein peptide pool in peripheral blood mononuclear cells, at baseline and 15 days post vaccination, among a subset of participants from cohort 1a and 3, according to schedule. Percent denotes the percentage of T cells positive for the Th1 or Th2 cytokines. Dotted line indicates the LLOQ. (FIG. 103D) Expression of IFNy and/or IL-2 cytokines by CD8 T cells was measured by ICS. Median (as illustrated by the horizontal bars and the numbers below each timepoint) and individual ICS responses to SARS-CoV-2 S protein peptide pool in peripheral blood mononuclear cells, at baseline and 15 days post vaccination, among a subset of participants from cohort 1a and 3, according to schedule. Percent denotes the percentage of CD8 T cells positive for IFNy and/or IL-2 cytokines. Dotted line indicates the LLOQ.
FIG. 104: Graphical representation of VNA responses against SARS-CoV-2 (geometric mean titers [GMTs] with corresponding 95% Cls) over time.
FIG. 105: depicts the effect of age on the neutralizing antibody response by Day 29.
FIG. 106: depicts the neutralizing antibody response and responder rates in the study cohorts.
FIG. 107: depicts reverse cumulative distribution curves for the 5c1010 vp vaccine group and the 1x1011 vp vaccine group.
FIG. 108: graphic representation of SARS-CoV-2 S protein binding antibody responses as measured by ELISA.
FIG. 109: depicts the effect of age on the binding antibody response at Day 29.
FIG. 110: graphic representation of SARS-CoV-2 S protein binding antibody responses as measured by ELISA.
FIG. 111: depicts reverse cumulative distribution curves for the 5c1010 vp vaccine group and the 1x1011 vp vaccine group. FIG. 112: shows that wtVNA titers highly correlated with ELISA titers at both Day 15 and Day 29, with Spearman Correlation coefficients of 0.734 and 0.72; respectively.
FIG. 113: shows the percentage of CD4+ T cells expressing IFNy and/or IL-2 (Th1), and not Th2 cytokines, and expressing IL-4 and/or IL-5/IL-13 and CD40L (Th2).
FIG. 114: shows the combined regimen profile.
FIG. 115: shows the descriptive statistics for CD8+ T cells producing IFNy and/or IL-2 in response to SARS-CoV-2 S peptide stimulation.
FIG. 116: shows the combined regimen profile.
FIG. 117A-117C: SARS-CoV-2-specific humoral immune responses to 1- and 2-dose Ad26.COV2.S vaccine regimes in adult rhesus macaques. (FIG. 117A) Spike (S) protein binding antibody levels were measured over time with a qualified ELISA for human samples, using a trimeric, soluble stabilized S protein produced in mammalian cells as coating antigen. Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The geometric mean titers (GMT) of binding antibody responses per group is indicated with the red line. The dotted lines indicate the lower limit of detection (LLOD) and lower limit of quantification (LLOQ). (FIG. 117B) S protein neutralizing antibody levels were measured over time with a qualified psVNA for human samples, using pseudotyped virus particles made from a modified Vesicular Stomatitis Virus (VSVAG) backbone and bear the S glycoprotein of SARS-CoV- 2. Neutralizing antibody responses are measured as the reciprocal of the sample dilution where 50% neutralization is achieved (IC50)· Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The GMT of neutralizing antibody responses per group is indicated with the red line. The dotted lines indicate the LLOD and LLOQ. (FIG. 117C) Correlation between S-specific binding antibody levels and neutralizing antibody titers per animal for all groups and timepoints except the sham control group and week 0 (baseline). The dotted lines indicate the LLOD for each assay.
FIG. 118A-118C: SARS-CoV-2-specific humoral and cellular immune responses after vaccination of aged rhesus macaques. (FIG. 118A) Spike (S) protein binding antibody levels were measured over time with a qualified ELISA for human samples, using a trimeric, soluble stabilized S protein produced in mammalian cells as coating antigen. Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The geometric mean titer (GMT) of binding antibody responses per group is indicated with the red line. The dotted lines indicate the lower limit of detection (LLOD) and lower limit of quantification (LLOQ). (FIG. 118B) SARS-Cov-2 neutralization antibody titers over time, as measured by wtVNA. Individual animal levels are depicted with grey points and paired measurements connected with grey lines. The GMT per group is indicated with the red line. The dotted line indicates the LLOD. (FIG. 118C) Correlation between S-specific binding antibody levels and neutralizing antibody titers per animal for all groups and timepoints except the sham control group and week 0. The dotted lines indicate the LLOD for each assay.
FIG. 119A-119C: SARS-CoV-2-specific cellular immune responses after vaccination of aged rhesus macaques. (FIG. 119A) Spike (S) protein-specific T cell responses as measured with an IFN-y/IL-4 Double-color ELISpot at indicated timepoints. The geometric mean titer (GMT) response per group is indicated with a horizontal line. Samples with background subtracted counts below or equal to 0 were set a 10 for visualization purposes, indicated by the dotted line. (FIG. 119B) Spike (S) protein-specific T cell responses as measured by intracellular cytokine staining at indicated timepoints. Frequency of CD4+CD69+ T cell expressing cytokines. The geometric mean titer (GMT) response per group is indicated with a horizontal line. The dotted line indicates the technical threshold. Open symbols denote samples at technical threshold.
(FIG. 119C) ratio of CD4+CD69+ T cells expressing Th1 (IFN-y or IL-2) or Th2 (IL-4, IL-5, IL-13) cytokines. The geometric mean titer (GMT) response per group is indicated with a horizontal line. Open symbols denote values were either cells expressing Th1, Th2 or any cytokine were at the technical threshold. The dotted horizontal line is set at a ratio of 1 for visualization purposes.
FIG. 120: Spike (S) protein-specific T cell responses as measured by intracellular cytokine staining at indicated timepoints. Frequency of CD8+CD69+ T cell expressing cytokines. The geometric mean titer (GMT) response per group is indicated with a horizontal line. The dotted line indicates the technical threshold. Open symbols denote samples at technical threshold.
FIGs. 121A-121D: Log geometric mean titers (GMTs - as illustrated by the horizontal bars and the numbers below each timepoint) of SARS-CoV-2 Spike protein binding antibodies in serum as measured by ELISA (ELISA Units per mL [EU/mL]), at baseline and at Day 29 post vaccination, among all participants, and Day 57 and Day 71 for those available for cohort 1a, according to schedule in cohort 1a (18-55 years old) (FIG. 121A) and cohort 3 (>65 years old) (FIG. 121 B). Dotted lines indicate the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) of the assay, error bars indicate 95% confidence interval (Cl). For values below the LLOQ, LLOQ/2 values were plotted.
Log GMTs of serum SARS-CoV-2 neutralizing antibodies (wtVNA), measured by 50% neutralization assay (IC50 Log GMT - as illustrated by the horizontal bars and the numbers below each timepoint), at baseline and at Day 29, Day 57 and Day 71 post vaccination, among a subset of participants, according to schedule, cohort 1a (18-55 years old) (FIG. 121C) and cohort 3 (>65 years old) (FIG. 121 D). Dotted lines indicate the LLOQ and ULOQ of the assay run with the current pre-dilution used for vaccine samples, error bars indicate 95% Cl. For values below the LLOQ, LLOQ/2 values were plotted.
FIGs. 122A-122F: Log GMTs of serum SARS-CoV-2 neutralizing antibodies (wtVNA), measured by 80% neutralization assay.
(IC80 Log GMT - as illustrated by the black dots and the numbers below each timepoint), at baseline and at Day 29, 57 and 71 post vaccination, among a subset of participants, according to schedule, cohort 1a (18-55 years old) and at day 1, 15 and 29 post vaccination cohort 3 (>65 years old).
FIG. 123: Correlation plot between Ad26 and SARS-CoV-2 neutralizing antibody titers: baseline Ad26 VNA baseline vs. wtVNA day 29, and Ad26 VNA at day 57 vs. wtVNA at day 71
FIGs. 124A-124C: Expression of Th1 (IFN-y and/or IL-2, and not IL-4, IL-5 and IL-13) (FIG. 124A), and Th2 (IL-4 and/or IL-5 and/or IL-13 and CD40L) (FIG. 124B) cytokines by CD4+ T cells was measured by intracellular cytokine staining (ICS). Median (as illustrated by the horizontal bars and the numbers below each timepoint) and individual ICS responses to a SARS-CoV-2 S protein peptide pool in peripheral blood mononuclear cells, at baseline and 15 days post vaccination, among a subset of participants from cohort 1a (18-55 years old) and cohort 3 (>65 years old), according to schedule, are given. The Y-axis denotes the percentage of T cells positive for the Th1 or Th2 cytokines. Dotted line indicates the LLOQ. Values below the LLOQ were plotted as LLOQ/2.
(FIG. 124C) Expression of IFN-g and/or IL-2 cytokines by CD8+ T cells was measured by ICS. Median (as illustrated by the horizontal bars and the numbers below each timepoint) and individual ICS responses to SARS-CoV-2 S protein peptide pool in peripheral blood mononuclear cells, at baseline and 15 days post vaccination, among a subset of participants from cohort 1a (18-55 years old) and cohort 3 (>65 years old), according to schedule, are given. The Y-axis denotes the percentage of CD8+ T cells positive for IFN-g and/or IL-2 cytokines. Dotted line indicates the LLOQ. Values below the LLOQ were plotted as LLOQ/2.
FIGs. 125A-125B: Relative productivity of Ad26NCOV030 (JNJ 78436735) and Ad26NCOV028 purified material in SPER.C6 and PER.C6 TetR cells.
A). SPER.C6 cells B) SPER.C6 TetR cells. Cells were transduced in shaker flasks with purified material of the Ad26 vector (70 or 300 VP/cell). Samples were taken at 0, 1, 2, 3, and 4 days post infection, and vector particle concentration was measured by VP-qPCR. Standard control (Ad26.ZEBOV) and low control (26ZIK001). VP, viral particles; qPCR, quantitative polymerase chain reaction; TetR, tetracycline repressor.
FIGs. 126A-126E: Titration of SARS-CoV-2 challenge dose and characterization of histopathology in Syrian hamsters. Syrian hamsters (N = 12 per group), were inoculated intra- nasally with 102, 1033, 1046or 1059TCID50 SARS-CoV-2 BetaCoV/Munich/BavPat1/2020, or mock-inoculated with Vero E6 cell-supernatant. Daily throat swabs were taken, and 2, 3, 4, and 7 days p.i., 3 hamsters per group were sacrificed and nose and lung tissue collected for virological analysis and histopathology. Replication competent viral load in (FIG. 126A) lung tissue, (FIG. 126B) nose tissue, and (FIG. 126C) throat swabs, was determined by TCID5o assay on Vero E6 cells. LLOD was calculated per animal per gram or milliliter of tissue, and animals with a response at or below the LLOD are shown as open symbols. (FIG. 126D) Lung tissue was analyzed and scored for presence and severity of alveolitis, alveolar damage, alveolar edema, alveolar hemorrhage, type II pneumocyte hyperplasia, bronchitis, bronchiolitis, peribronchial and perivascular cuffing. Sum of scores are presented as sum of LRT disease parameters (potential range: 0 - 24). (FIG. 126E) Nose tissue was analyzed and scored for severity of rhinitis on a scale from 0 to 3. Dotted lines indicate the minimal and maximal scores of histopathology. Median responses per group are indicated with horizontal lines, error bars in panel c indicate the range p.i. = post inoculation; LLOD = lower limit of detection; LRT = lower respiratory tract; N = number of animals; TCID50/g = 50% tissue culture infective dose per gram tissue; TCID50/ml = 50% tissue culture infective dose per milliliter sample; vp = virus particles.
FIGs. 127A-127C: SARS-CoV-2 neutralizing antibody response elicited by 1- and 2-dose Ad26.COV2.S vaccine regimes in Syrian hamsters. (FIG. 127A) Syrian hamsters were immunized with either 109 or 1010 VP (N=12 per dose level) of Ad26-based vaccines candidates, or with 1010 vp of an Ad26 vector without gene insert as control (Ad26. empty, N=6). Four weeks after immunization half the hamsters per group received a second immunization with the same Ad26-based vaccine candidate (N=6 per group). (FIG. 127B) SARS-CoV-2 neutralization titers were measured 4 weeks after dose 1 and (FIG. 127C) 4 weeks after dose 2 by wild-type VNA determining the inhibition of the cytopathic effect of SARS-CoV-2 on Vero E6 cells. The sera from Syrian hamsters immunized with Ad26. Empty were pooled into 2 groups for negative control samples. Median responses per group are indicated with horizontal lines. Dotted lines indicate the LLOD. Animals with a response at or below the LLOD are displayed as open symbols on the LLOD. CPE = cytopathic effect; LLOD = Lower Limit of Detection; p.i. = post inoculation; VNA = virus neutralization assay; VP = virus particles.
FIGs. 128A-128C: SARS-CoV-2 spike protein binding antibody response, and ratio with neutralizing antibody responses, elicited by 1- and 2-dose Ad26 vaccine regimes in Syrian hamsters. Syrian hamsters were immunized with either 109 or 1010 VP of Ad26-based vaccine candidates Ad26.S, Ad26.dTM.PP or Ad26.COV2.S, or with 1010 vp of an Ad26 vector without gene insert as control (Ad26. empty). Four weeks after immunization half the hamsters per group received a second immunization with the same Ad26-based vaccine candidate (N=6 per group). Two and 4 weeks after one immunization, and 1, 2 and 4 weeks after the second immunization (weeks 5, 6 and 8 respectively), blood samples were collected and serum isolated for serological analyses. (FIG. 128A) SARS-CoV-2 spike protein-specific antibody binding titers of hamsters receiving two immunizations (N=6 per group) were measured by ELISA. (FIG. 128B) Neutralization titers were measured 4 weeks after dose 1 (hamsters receiving one immunizations, N=6 per group) and (FIG. 128C) 4 weeks after dose 2 (hamsters receiving two immunizations, N=6 per group). The ratio of SARS-CoV-2 neutralizing antibodies over binding antibodies was calculated by dividing antibody titers as measured by wtVNA, by antibody titers as measured by ELISA. Animals with neutralization titers at or below the LLOD are displayed as open symbols. Median responses per group are indicated with horizontal lines. LLOD = Lower Limit of Detection; ELISA = Enzyme-linked Immunosorbent assay; wtVNA = wild-type virus neutralization assay; VP = virus particles
FIGs. 129A-129C: SARS-CoV-2 spike protein binding antibody response and neutralizing antibody response elicited by a 2-dose Ad26 vaccine regime in New Zealand White rabbits.
New Zealand white rabbits were intramuscularly immunized with a 2-dose regimen of 5x109 vp or 5x1010 vp Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or saline (FIG. 129A). Serum was sampled prior to immunization (day -3) and days 14, 21 , 35, 42, 56, 63, 70, 84 and at sacrifice (days 99- 101, depicted in the graph as day 100). (FIG. 129B) SARS-CoV-2 spike protein-specific antibody binding titers were measured by ELISA. (FIG. 129C) Neutralization titers were measured on days 14, 35, 56, 70 and at sacrifice. Median responses per group are indicated with horizontal lines, vertical lines denote group ranges. ELISA = Enzyme-linked Immunosorbent assay; wtVNA = wild-type virus neutralization assay; LLOQ = Lower Limit of Qualification; ULOQ = Upper Limit of Qualification; LLOD = Lower Limit of Detection; VP = virus particles.
FIGs. 130A-130F: Protection against SARS-CoV-2 viral replication in Syrian hamsters immunized with Ad26-based vaccine candidates. Syrian hamsters were intramuscularly immunized with a 1-dose regimen and a 2-dose regimen of Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or Ad26. empty (Ad26 vector not encoding any SARS-CoV-2 antigens).
Hamsters received an intranasal inoculation with 102 TCID50 SARS-CoV-2 strain BetaCoV/Munich/BavPat1/20204 weeks post-dose 1 (week 4) or 4 weeks post-dose 2 (week 8). (FIGs. 130A, 130B) Right lung tissue and (FIGs. 130C, 130D) right nasal turbinates were harvested at the end of the 4-day inoculation phase for viral load analysis. Replication competent virus was measured by TCID50 assay. (FIGs. 130E, 130F) Throat swab samples were taken daily after inoculation, and viral load area under the curve during the four-day follow up was calculated as TCIDso/ml x day. The median viral load per group is indicated with a horizontal line. LLOD was calculated per animal and animals with a response at or below the LLOD are shown as open symbols on the LLOD. Comparisons were performed between the Ad26.S, Ad26.dTM.PP and Ad26.COV2.S groups across dose level, with the Ad26. empty group by Mann-Whitney U-test. Statistical differences indicated by asterisks: *: p<0.05; **:p<0.01. LLOD = lower limit of detection; TCIDso/g = 50% tissue culture infective dose per gram tissue; TCID50/ml = 50% tissue culture infective dose per ml sample; VP = virus particles.
FIGs. 131A-131D: Protection against SARS-CoV-2 IHC and histopathology in lung tissue of Syrian hamsters immunized with Ad26-based vaccine candidates. Syrian hamsters were intramuscularly immunized with a 1-dose regimen and a 2-dose regimen of Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or Ad26. empty (Ad26 vector not encoding any SARS-CoV-2 antigens). The hamsters received intranasal inoculation with 102 TCID50 SARS-CoV-2 strain BetaCoV/Munich/BavPat1/20204 weeks post-dose 1 (week 4), or 4 weeks post-dose 2 (week 8). Left lung tissue was isolated 4 days after inoculation for analysis of immunohistochemical SARS-CoV-NP staining and histopathology. Due to a technical failure, tissues of only 2 out of 8 hamsters immunized with one dose of Ad26.Emtpy could be analyzed. (FIGs. 131 A and 131 B) Presence of SARS-CoV-2 NP was determined by immunohistochemical staining. (FIGs. 131C and 131 D) Lung tissue was scored for presence and severity of alveolitis, alveolar damage, alveolar edema, alveolar hemorrhage, type II pneumocyte hyperplasia, bronchitis, bronchiolitis, peribronchial and perivascular cuffing. Sum of scores are presented as sum of LRT disease parameters. Median scores per group are indicated with horizontal lines. Dotted lines indicate the LLOD. Comparisons were performed between the vaccine groups across dose level, with the Ad26. Empty group by Mann-Whitney U-test, except for panel c. Statistical differences indicated by asterisks: *:p<0.05; **:p<0.01. LLOD = lower limit of detection; VP = virus particles; NP = Nucleocapsid protein; IHC = Immunohistochemistry; LRT = Lower Respiratory Tract.
FIGs. 132A-132D: Protection against SARS-CoV-2 IHC and histopathology in nasal tissue of Syrian hamsters immunized with Ad26-based vaccine candidates. Syrian hamsters were intramuscularly immunized with a 1-dose regimen and a 2-dose regimen of Ad26.S, Ad26.dTM.PP, Ad26.COV2.S, or Ad26. empty (Ad26 vector not encoding any SARS-CoV-2 antigens). The hamsters received intranasal inoculation with 102 TCID50 SARS-CoV-2 strain BetaCoV/Munich/BavPat1/20204 weeks post-dose 1 (week 4), or 4 weeks post-dose 2 (week 8). Left nasal tissue was isolated 4 days after inoculation for analysis of immunohistochemical SARS-CoV-NP staining and histopathology. Due to a technical failure, tissues of only 5 out of 8 hamsters immunized with one dose of Ad26.Emtpy could be analyzed. (FIGs. 132A and 132B) Presence of SARS-CoV-NP was determined by immunohistochemical staining. (FIGs. 132C and 132D) Nasal tissue was scored for severity of rhinitis.
Median scores per group are indicated with horizontal lines. Dotted lines indicate the LLOD. Comparisons were performed between the vaccine groups across dose level, with the Ad26. Empty group by Mann-Whitney U-test. Statistical differences indicated by asterisks: *:p<0.05; **:p<0.01. LLOD = lower limit of detection; VP = virus particles; NP = Nucleocapsid protein; IHC = Immunohistochemistry.
FIGs. 133A-133D: Dose responsiveness of Ad26.COV2.S on immunogenicity and lung viral load in hamsters. Syrian hamsters were intramuscularly immunized with 107, 10s, 109 or 1010 VP of Ad26.COV2.S N=8 per group, or 1010 VP Ad26.lrr (an Ad26 vector not encoding any SARS- CoV-2 antigens, N=8). Four weeks after one immunization, SARS-CoV-2 Spike protein-specific antibody binding titers (FIG. 133A) and SARS-CoV-2 neutralizing antibodies (FIG. 133B) were determined. The median antibody responses per group is indicated with a horizontal line.
Dotted lines indicate the LLOD. Animals with a response at or below the LLOD were put on LLOD and are shown as open symbols. Hamsters received intranasal inoculation with 102 TCID5O SARS-CoV-2 strain BetaCoV/Munich/BavPat1/2020 4 weeks post immunization (week 4). Right lung tissue was isolated 4 days after inoculation for virological analysis and immunohistochemistry. (FIG. 133C) Lung viral load was determined by TCID50 assay on Vero E6 cells. The median viral load per group is indicated with a horizontal line. LLOD was calculated per animal, and animals with a response at or below the LLOD are shown as open symbols. (FIG. 133D) presence of SARS-CoV-2 NP was determined by immunohistochemical staining. Comparisons were performed between the Ad26.COV2.S dose level groups, with the Ad26.lrr group by Mann- Whitney U-test. Statistical differences indicated by asterisks: *:p<0.05; **:p<0.01 ; ***:p<0.001. Ad26.lrr = Ad26 vector not encoding any SARS-CoV-2 antigens; LLOD = lower limit of detection; N = number of animals; TCIDso/g = 50% tissue culture infective dose per gram tissue; VP = virus particles; NP = Nucleocapsid protein.
FIG. 134: No signs of VAERD in Ad26 immunized Syrian hamsters inoculated with SARS-CoV- 2 Four days after IN inoculation with 102 TCID50 SARS-CoV-2 (N = 8 per group), a) lung tissue was isolated and scored for presence and severity of alveolitis, alveolar damage, alveolar edema, alveolar hemorrhage, type II pneumocyte hyperplasia, bronchitis, bronchiolitis, peribronchial and perivascular cuffing. Sum of scores are presented as sum of LRT disease parameters b) Four days after inoculation, nose tissue was isolated and scored for severity of inflammation (rhinitis).
Horizontal lines denote a pathology score of 0, indicating no histopathology. Symbols in red denote samples from hamsters with breakthrough lung viral load (>102 TCID5o/g). Comparisons were performed between the Ad26.COV2.S dose level groups, with the Ad26.lrr group by Mann- Whitney U-test. Statistical differences indicated by asterisks: *:p<0.05; **:p<0.01 ; ***:p<0.001. Ad26.lrr = Ad26 vector not encoding any SARS-CoV-2 antigens; LRT = lower respiratory tract;
N = number of animals; VP = virus particles.
FIGs. 135A-135D: Binding and neutralizing antibodies correlate with protection. Protection per vaccine construct was defined as a viral load below 102 TCIDso/g in lung tissue, irrespective of vaccine regimen and dose level (see Fig 129A and B, and Fig 133C). Syrian hamsters were immunized once, or twice, with 109 or 1010 VP of Ad26.S or Ad26.dTM.PP (N=24 per construct), or 107, 108, 109, 1010 VP Ad26.CoV.S (N=56). Hamsters were inoculated with 102 TCID50 SARS- CoV-2, and four days later sacrificed for virological analysis of lung tissue. Prior to virus inoculation serum samples were analyzed for (FIG. 135A) antibody binding titers and (FIG. 135B) virus neutralizing antibodies. Median antibody responses per group is indicated with horizontal lines. Dotted lines indicate the LLOD. (FIG. 135C) Protection probability logistic regression models were built with Firth’s correction of binding and neutralizing antibody titers from pooled regimens and dose levels of Ad26.COV2.S. Dotted lines indicate the 95% confidence interval. LLOD = lower limit of detection; N = number of animals; TCIDso/g = 50% tissue culture infective dose per gram tissue; VP = virus particles.
FIGs. 136A-136C: Rapid induction of binding and neutralizing antibodies following Ad26.COV2.S vaccination. (FIG. 136A) S-specific binding antibodies by ELISA, (FIG. 136B) RBD-specific binding antibodies by ELISA, and (FIG. 136C) pseudovirus neutralizing antibodies (psVNA) on day 1 and day 8 in recipients of the high dose (HD) and low dose (LD) Ad26.COV2.S or placebo (PL). Red bars reflect geometric mean titers (GMT). P values reflect two-sided Mann-Whitney tests.
FIGs. 137A-137B: Kinetics and magnitude of binding and neutralizing antibodies following Ad26.COV2.S vaccination. (A) S- and RBD-specific binding antibodies by ELISA and (B) SARS-CoV-2 pseudovirus neutralizing antibody (psVNA) and Ad26 virus neutralizing antibody (Ad26 VNA) responses following Ad26.COV2.S vaccination. LD, low dose; HD, high dose; PL, placebo. Red bars reflect geometric mean titers (GMT). Dotted lines reflect lower limits of quantitation.
FIG. 138: Correlations of humoral immune responses. Correlations of logi0 S-specific ELISA titers, logio RBD-specific ELISA titers, and Iog10 neutralizing antibody (NAb) titers on day 29. P and R values reflect two-sided Spearman rank-correlation tests.
FIGs. 139A-139B: Antibody cross-reactivity following Ad26.COV2.S vaccination. Electrochemiluminescence assay (Meso Scale Discovery SARS-CoV-2 IgG Panel 2; K15369U- 2) assessing binding antibody responses to the S proteins from (FIG. 139A) SARS-CoV-2 and SARS-CoV-1 as well as (FIG. 139B) CoV-229E, CoV-HKLH, CoV-NL63, and CoV-OC43 following Ad26.COV2.S vaccination. LD, low dose; HD, high dose; PL, placebo. Red bars reflect geometric mean responses. Dotted lines reflect lower limits of quantitation. FIGs. 140A-140B: Kinetics and magnitude of cellular immune responses following Ad26.COV2.S vaccination. (FIG. 140A) IFN-g and IL-4 ELISPOT responses and (FIG. 140B) IFN-g central memory CD27+ CD45RA- CD4+ and CD8+ T cell responses by ICS assays following Ad26.COV2.S vaccination. ICS assays were performed in a subset of participants with sufficient peripheral blood mononuclear cells (PBMCs) on day 71/85. SFC, spot-forming cells; LD, low dose; HD, high dose; PL, placebo. Red bars reflect geometric mean responses. Dotted lines reflect lower limits of quantitation.
FIG. 141: Correlations of cellular and humoral immune responses. Correlations of logio ELISPOT responses with logio S-specific ELISA titers, logio RBD-specific ELISA titers, and Iog10 neutralizing antibody (NAb) titers on day 29. P and R values reflect two-sided Spearman rank-correlation tests.
FIG. 142: Cumulative Incidence of Molecularly Confirmed Moderate to Severe/Critical COVID- 19 Cases with Onset at Least 1 Day after Vaccination, Full Analysis Set.
FIG. 143: Cumulative Incidence of Molecularly Confirmed Moderate to Severe/Critical COVID- 19 Cases with Onset at Least 1 Day After Vaccination Until Day 29 by Serostatus; Full Analysis Set.
FIG. 144: Cumulative Incidence of Molecularly Confirmed Severe/Critical COVID-19 Cases with Onset at Least 1 Day after Vaccination, Full Analysis Set.
FIG. 145: Force of infection. Placebo COVID-19 incidence rate in different countries.
DETAILED DESCRIPTION
DEFINITIONS
As used herein, the term “about” means +/- 10% of the recited value.
The terms “adenovirus vector” and “adenoviral vector” are used interchangeably and refer to a genetically-engineered adenovirus that is designed to insert a polynucleotide of interest (e.g., a polynucleotide encoding a 2019-nCoV immunogen) into a eukaryotic cell, such that the polynucleotide is subsequently expressed. Examples of adenoviruses that can be used as a viral vector include those having, or derived from, the serotypes Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52), Ad59 (e.g., RhAd59), and Pan9 (also known as AdC68); these vectors can be derived from, for example, human, chimpanzee, or rhesus adenoviruses. In some embodiments, the adenovirus is Ad26.
The term “adjuvant” refers to a pharmacological or immunological agent that modifies the effect of other agents (e.g., vaccines) while having few if any direct effects when given by itself. They are often included in vaccines to enhance the recipient's immune response to a supplied antigen while keeping the injected foreign material at a minimum.
As used herein, by “administering” is meant a method of giving a dosage of a pharmaceutical composition (e.g., an immunogenic composition (e.g., a vaccine (e.g., a Wuhan coronavirus (2019-nCoV) vaccine))) to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions. The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated).
The terms “antibody” and “immunoglobulin (lg)” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity) and may also include certain antibody fragments. An antibody typically comprises both “light chains” and “heavy chains.” The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (K) and lambda (l), based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. The term “codon” as used herein refers to any group of three consecutive nucleotide bases in a given messenger RNA molecule, or coding strand of DNA, that specifies a particular amino acid or a starting or stopping signal for translation. The term codon also refers to base triplets in a DNA strand.
Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
The term “convalescent” as used herein refers to subjects who have recovered or are recovering from a coronavirus infection (e.g., 2019-nCoV). A “cohort of convalescent humans” refers to a group of humans that share common characteristics (e.g., sex, age, weight, medical history, race, ethnicity, or environment) and have recovered or are recovering from a coronavirus infection (e.g., 2019-nCoV). Preferably, a cohort of convalescent humans will share common characteristics with a subject having a risk of 2019-nCoV infection or suspected of being susceptible to a coronavirus infection. Preferably, samples from convalescent humans will be obtained at least 7 days after documented recovery (e.g., determined with a negative nasal swab).
The terms “ectodomain” and “extracellular domain” refer to the portion of a coronavirus Spike polypeptide that extends beyond the transmembrane domain into the extracellular space. The ectodomain mediates binding of a Spike polypeptide to one or more coronavirus receptors (e.g., ACE2). For instance, an ectodomain includes the S1 domain (e.g., SEQ ID NO: 4) and RBD (e.g., SEQ ID NO: 5) of a Spike polypeptide.
A “gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell. Examples of gene delivery vehicles are liposomes, biocompatible polymers, including natural polymers and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial viral envelopes; metal particles; and bacteria, or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art that have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
"Gene delivery," "gene transfer," and the like as used herein, are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, irrespective of the method used for the introduction. Such methods include a variety of techniques such as, for example, vector-mediated gene transfer (e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other techniques used for the introduction of polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are capable of mediating transfer of genes to mammalian cells.
By “gene product” is meant to include mRNAs or other nucleic acids (e.g., microRNAs) transcribed from a gene, as well as polypeptides translated from those mRNAs. In some embodiments, the gene product is from a virus (e.g., a 2019-nCoV) and may include, for example, any one or more of the viral proteins, or fragments thereof, described herein.
By “heterologous nucleic acid molecule” is meant a nucleotide sequence that may encode proteins derived or obtained from pathogenic organisms, such as viruses, which may be incorporated into a polynucleotide or vector. Heterologous nucleic acids may also encode synthetic or artificial proteins, such as immunogenic epitopes, constructed to induce immunity. An example of a heterologous nucleic acid molecule is one that encodes one or more immunogenic peptides or polypeptides derived from a coronavirus (e.g., 2019-nCoV). The heterologous nucleic acid molecule is one that is not normally associated with the other nucleic acid molecules found in the polynucleotide or vector into which the heterologous nucleic acid molecule is incorporated.
The term “host cell,” refers to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Host cells include cells within the body of a subject (e.g., a mammalian subject (e.g., a human)) into which an exogenous nucleic acid has been introduced.
By “immunogen” is meant any polypeptide that can induce an immune response in a subject upon administration. In some embodiments, the immunogen is encoded by a nucleic acid molecule that may be incorporated into, for example, a polynucleotide or vector, for subsequent expression of the immunogen (e.g., a gene product of interest, or fragment thereof (e.g., a polypeptide)).
The term “immunogenic composition” as used herein, is defined as material used to provoke an immune response and may confer immunity after administration of the immunogenic composition to a subject. The term “immunostimulatory agent” refers to substances (e.g., drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components. An immunostimulatory agent includes a cytokine (e.g., the granulocyte macrophage colony-stimulating factor) and interferon (e.g., IFN-a and/or IFN-y).
By “isolated” is meant separated, recovered, or purified from a component of its natural environment. For example, a nucleic acid molecule or polypeptide may be isolated from a component of its natural environment by 1% (2%, 3%, 4%, 5%, 6%, 7%, 8% 9% 10%, 20%,
30%, 40%, 50%, 60% 70%, 80%, or 90%) or more.
By “pharmaceutical composition” is meant any composition that contains a therapeutically or biologically active agent, such as an immunogenic composition or vaccine (e.g., a 2019-nCoV nucleic acid molecule, vector, and/or polypeptide), preferably including a nucleotide sequence encoding an antigenic gene product of interest, or fragment thereof, that is suitable for administration to a subject and that treats or prevents a disease (e.g., 2019-nCoV infection) or reduces or ameliorates one or more symptoms of the disease (e.g., 2019-nCoV viral titer, viral spread, infection, and/or cell fusion)). For the purposes of this invention, pharmaceutical compositions include vaccines, and pharmaceutical compositions suitable for delivering a therapeutic or biologically active agent can include, for example, tablets, gelcaps, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels, hydrogels, oral gels, pastes, eye drops, ointments, creams, plasters, drenches, delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. Any of these formulations can be prepared by well- known and accepted methods of art. See, for example, Remington: The Science and Practice of Pharmacy (21st ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is hereby incorporated by reference.
The terms “linked” or “links” or “link” as used herein are meant to refer to the covalent joining of two amino acid sequences or two nucleic acid sequences together through peptide or phosphodiester bonds, respectively, such joining can include any number of additional amino acid or nucleic acid sequences between the two amino acid sequences or nucleic acid sequences that are being joined.
“Nucleic acid molecule” or “polynucleotide,” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label.
A "nucleic acid vaccine" refers to a vaccine that includes a heterologous nucleic acid molecule under the control of a promoter for expression in a subject. The heterologous nucleic acid molecule can be incorporated into an expression vector, such as a plasmid. A “DNA vaccine” refers to a vaccine in which the nucleic acid is DNA. An “RNA vaccine” refers to a vaccine in which the nucleic acid is RNA (e.g., an mRNA).
A nucleic acid is “operably linked” when it is placed into a structural or functional relationship with another nucleic acid sequence. For example, one segment of DNA may be operably linked to another segment of DNA if they are positioned relative to one another on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding sequence so as to facilitate transcription of the coding sequence; a ribosome binding site that is positioned relative to a coding sequence so as to facilitate translation; or a pre-sequence or secretory leader that is positioned relative to a coding sequence so as to facilitate expression of a pre-protein (e.g., a pre-protein that participates in the secretion of the encoded polypeptide). In other examples, the operably linked nucleic acid sequences are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.
By “optimized” is meant an immunogenic polypeptide that is not a naturally-occurring peptide, polypeptide, or protein, such as a non-naturally occurring viral polypeptide (e.g., a Spike polypeptide). Optimized viral polypeptide sequences are initially generated by modifying the amino acid sequence of one or more naturally-occurring viral gene products (e.g., peptides, polypeptides, and proteins) to increase the breadth, intensity, depth, or longevity of the antiviral immune response (e.g., cellular or humoral immune responses) generated upon immunization (e.g., when incorporated into a composition, e.g., vaccine) of a subject (e.g., a human). Thus, the optimized viral polypeptide may correspond to a “parent” viral gene sequence; alternatively, the optimized viral polypeptide may not correspond to a specific “parent” viral gene sequence but may correspond to analogous sequences from various strains or quasi-species of a virus. Modifications to the viral gene sequence that can be included in an optimized viral polypeptide include amino acid additions, substitutions, and deletions. In one embodiment, the optimized polypeptide is a Spike polypeptide, which has been further altered to include a leader/signal sequence (e.g., a Spike signal sequence or a tPA signal sequence) for maximal protein expression, a factor Xa site, a foldon trimerization domain (see, e.g., SEQ ID NO: 87), and/or linker or spacer (e.g., SEQ ID NOs: 88 or 89) sequences. An optimized polypeptide may, but need not, also include a cleavage site mutation(s) (e.g., a furin cleavage site mutation (e.g., SEQ ID NO:91)). Methods of generating an optimized viral polypeptide are described in, e.g., Fisher et al. “Polyvalent Vaccine for Optimal Coverage of Potential T-Cell Epitopes in Global HIV-1 Variants,” Nat. Med. 13(1): 100-106 (2007) and International Patent Application Publication WO 2007/024941 , herein incorporated by reference. Once the optimized viral polypeptide sequence is generated, the corresponding polypeptide can be produced or administered by standard techniques (e.g., recombinant viral vectors, such as the adenoviral vectors disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, herein incorporated by reference) and optionally assembled to form a stabilized polypeptide trimer.
The terms “optimized codon” and “codon optimized” as used herein refer to a codon sequence that has been modified to match codon frequencies in a target (e.g., a subject) or host organism, but that does not alter the amino acid sequence of the original translated protein.
By “pharmaceutically acceptable diluent, excipient, carrier, or adjuvant” is meant a diluent, excipient, carrier, or adjuvant that is physiologically acceptable to the subject while retaining the therapeutic properties of the pharmaceutical composition with which it is administered. One exemplary pharmaceutically acceptable carrier is physiological saline. Other physiologically acceptable diluents, excipients, carriers, or adjuvants and their formulations are known to one skilled in the art (see, e.g., U.S. Pub. No. 2012/0076812).
By “portion” or “fragment” is meant a part of a whole. A portion may comprise at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the entire length of a polynucleotide or polypeptide sequence region. For polynucleotides, for example, a portion may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 or more contiguous nucleotides of a reference polynucleotide molecule. For polypeptides, for example, a portion may include at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 125, 150, 175,
200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 600 or more continuous amino acids of a reference polypeptide molecule.
In some instances, a fragment of a nucleic acid molecule may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more consecutive nucleotides of the polynucleotide SS-Spike (SEQ ID NO: 121). In some instances, a fragment of a nucleic acid molecule may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more consecutive nucleotides of the polynucleotide SS-Spike-dF-PP (SEQ ID NOs: 143 and 204). In some instances, a fragment of a nucleic acid molecule may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,
1200, 1300, 1400, 1500, or more consecutive nucleotides of the polynucleotide SS-SdCT (SEQ ID NO: 122). In some instances, a fragment of a nucleic acid molecule may include at least 20,
30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, or more consecutive nucleotides of the polynucleotide SS-S.Ecto (SEQ ID NO: 123). In some instances, a fragment of a nucleic acid molecule may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, or more consecutive nucleotides of the polynucleotide SS-S1-foldon (SEQ ID NO: 129). In some instances, a fragment of a nucleic acid molecule may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, or more consecutive nucleotides of the polynucleotide SS-RBD-foldon (SEQ ID NO: 130). In some instances, a fragment of a nucleic acid molecule may include at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or more consecutive nucleotides of the polynucleotide SS-S.Ecto-dF-foldon (SEQ ID NO: 136 or 193). In some instances, a fragment of a nucleic acid molecule may include at least 5, 6, 7, 8, 9, 10, 20, 30,
40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, 1500, 1600, 1700, 1800, 1900, 2000, or more consecutive nucleotides of the polynucleotide SS-S.Ecto-PP-foldon (SEQ ID NO: 142). In some instances, a fragment of a nucleic acid molecule may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800,
1900, 2000, or more consecutive nucleotides of the polynucleotide SS-S.Ecto-dF-PP-foldon (SEQ ID NO: 195).
In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100,
125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, or more consecutive amino acids of polypeptide SS-Spike (SEQ ID NO: 29). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of polypeptide SS-SdCT (SEQ ID NO: 30). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of polypeptide SS-Spike-dF-PP (SEQ ID NO: 51). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or more consecutive amino acids of polypeptide SS-S.Ecto (SEQ ID NO: 31). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200,
225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or more consecutive amino acids of polypeptide SS-S1-foldon (SEQ ID NO: 37). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more consecutive amino acids of polypeptide SS-RBD-foldon (SEQ ID NO: 38). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, or more consecutive amino acids of polypeptide SS-S.Ecto-dF-foldon (SEQ ID NO: 44). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or more consecutive amino acids of polypeptide S.Ecto-PP-foldon (SEQ ID NO: 50). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or more consecutive amino acids of polypeptide SS-S.Ecto-dF-PP-foldon (SEQ ID NO: 56). In some instances, a fragment of a polypeptide may include at least 20, 25, 50, 75, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, or more consecutive amino acids of polypeptide prM-Env with JEV Stem/TM (SEQ ID NO: 27).
In some instances, administration of a fragment of a polynucleotide (e.g., SEQ ID NOs: 93-181, 190-195, and 199-204) and/or a polypeptide (e.g., SEQ ID NOs: 1-84, e.g., SEQ ID NO: 51) to a subject may illicit an immune response in the subject.
A “promoter” is a nucleic acid sequence enabling the initiation of the transcription of a gene sequence in a messenger RNA, such transcription being initiated with the binding of an RNA polymerase on or nearby the promoter.
By “promotes an immune response” is meant eliciting a humoral response (e.g., the production of antibodies) or a cellular response (e.g., the activation of T cells, macrophages, neutrophils, and/or natural killer cells) directed against, for example, one or more infective agents (e.g., a virus (e.g., a 2019-nCoV)) or protein targets in a subject to which the pharmaceutical composition (e.g., an immunogenic composition or vaccine) has been administered.
The term "2019-nCoV-mediated disease," used interchangeably with "Coronavirus disease 2019 (COVID-19)" herein, as well as grammatical variants thereof, refers to any pathology or sequelae known in the art to be caused by (alone or in association with other mediators), exacerbated by, or associated with 2019-nCoV (SARS-CoV-2) infection or exposure in the subject having the disease. Disease can be acute (e.g., fever) or chronic (e.g., chronic fatigue), mild (e.g., hair loss) or severe (e.g., organ failure), and early-onset (e.g., 2-14 days post infection) or late-onset (e.g., 2 weeks post-infection). Non-limiting examples of severe disease include pneumonia, acute respiratory distress syndrome (ARDS), acute respiratory failure, pulmonary edema, organ failure, or death. Non-limiting examples of symptoms include weight loss, fever, cough, difficulty breathing, fatigue, headache, loss of taste or smell, hair loss, rash, sore throat, nausea, and diarrhea. Symptoms can be mild or severe (e.g., weight loss of greater than about 5% within a week and high fever) and temporary or permanent.
A “protective level” refers to an amount or level of a marker (e.g., an antibody, a cell (e.g., an immune cell, e.g., a T cell, a B cell, an NK cell, or a neutrophil)) that is indicative of partial or complete protection from coronavirus infection or disease. An amount or level of a marker that is above the protective level indicates protection from coronavirus infection (e.g., a 2019-nCoV infection) or disease (e.g., a 2019-nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). An amount or level of a marker that is below the protective level indicates susceptibility to coronavirus infection or disease (e.g., a 2019-nCoV-mediated disease, e.g., COVID-19, e.g., severe clinical disease). The marker may be a single measure (e.g., neutralizing antibody level) or the marker may be a combination of multiple measures (e.g., neutralizing antibody level and RBD-specific lgG2 level). In some instances, the protective level is an anti-coronavirus antibody titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, an anti-coronavirus antibody titer of at least about 25 as measured using the live virus neutralization assay described herein, or an anti-coronavirus antibody titer that is above a level of at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. In some instances, the protective level is an anti- coronavirus antibody titer of at least about 100 as measured using the pseudovirus neutralization assay described herein.
As used herein, the term “sample” is a composition that is obtained or derived from a subject that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. A sample may be solid tissue as from a fresh, frozen, and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid. The sample may also be primary or cultured cells or cell lines. The sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, wax, nutrients, antibiotics, or the like.
By “sequence identity” or “sequence similarity” is meant that the identity or similarity, respectively, between two or more amino acid sequences, or two or more nucleotide sequences, is expressed in terms of the identity or similarity between the sequences.
Sequence identity can be measured in terms of “percentage (%) identity,” in which a higher percentage indicates greater identity shared between the sequences. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similarity shared between the sequences. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Sequence identity/similarity can be determined across all or a defined portion of the two or more sequences compared.
By “signal peptide” is meant a short peptide (e.g., 5-30 amino acids in length, such as 17 amino acids in length, e.g., SEQ ID NO: 92) at the N-terminus of a polypeptide that directs a polypeptide towards the secretory pathway (e.g., the extracellular space). The signal peptide is typically cleaved during secretion of the polypeptide. The signal sequence may direct the polypeptide to an intracellular compartment or organelle, e.g., the Golgi apparatus. A signal sequence may be identified by homology, or biological activity, to a peptide with the known function of targeting a polypeptide to a particular region of the cell. One of ordinary skill in the art can identify a signal peptide by using readily available software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Ws. 53705, BLAST, or PILEUP/PRETTYBOX programs). A signal peptide can be one that is, for example, substantially identical to the amino acid sequence of SEC ID NO: 92.
As used herein, the phrase “specifically binds” refers to a binding reaction which is determinative of the presence of an antigen in a heterogeneous population of proteins and other biological molecules that is recognized, e.g., by an antibody or antigen-binding fragment thereof, with particularity. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (e.g., between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD of greater than 100 nM (e.g., greater than 500 nm, 1 mM, 100 mM, 500 pM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein or carbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
As used herein, the term “stabilized polypeptide trimer” or “stabilized trimer” refers, but is not limited to, an oligomer that includes a protein and/or polypeptide sequence that increases the stability (e.g., via the presence of one or more oligomerization domains) of the trimeric structure (e.g., reduces dissociation of a trimer into monomeric units). The stabilized polypeptide trimer, for example, may be a homotrimer. An “oligomerization domain” refers, but is not limited to, a polypeptide sequence that can be used to increase the stability of an oligomeric envelope protein such as, e.g., to increase the stability of a Spike trimer. Oligomerization domains can be used to increase the stability of homooligomeric polypeptides as well as heterooligomeric polypeptides. Oligomerization domains are well known in the art and include “trimerization domains.” A trimerization domain refers to an oligomerization domain that stabilizes trimeric polypeptides (e.g., trimers consisting of one or more of the Spike polypeptides). Examples of trimerization domains include, but are not limited to, the T4-fibritin “foldon” trimerization domain; the coiled-coil trimerization domain derived from GCN4 (Yang et al. (2002) J. Virol. 76:4634); and the catalytic subunit of E. coli aspartate transcarbamoylase as a trimer tag (Chen et al. (2004) J. Virol. 78:4508).
A “subject” is a vertebrate, such as a mammal (e.g., a primate and a human, in particular a human with underlying health conditions (e.g., hypertension, diabetes, or cardiovascular disease)). Mammals also include, but are not limited to, farm animals (such as cows), sport animals (e.g., horses), pets (such as cats, and dogs), mice, rats, bats, civets, and raccoon dogs. A subject to be treated according to the methods described herein (e.g., a subject in need of protection from a 2019-nCoV infection or having a 2019-nCoV infection may be one who has been diagnosed by a medical practitioner as having such a need or infection. Diagnosis may be performed by any suitable means. A subject in whom the development of an infection is being prevented may or may not have received such a diagnosis. One skilled in the art will understand that a subject to be treated according to the present invention may have been subjected to standard tests or may have been identified, without examination, as one with a suspected infection or at high risk of infection due to the presence of one or more risk factors (e.g., exposure to a 2019-nCoV, for example, due to travel to an area where 2019-nCoV infection is prevalent). Additionally, humans with underlying health conditions (e.g., hypertension, diabetes, or cardiovascular disease) are identified as subjects at high risk of infection with a coronavirus (e.g., 2019-nCoV). The methods of treating a human subject with a composition are, therefore, particularly useful in treating, reducing, and/or preventing a 2019- nCoV infection in humans with underlying health conditions.
As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of an exogenous nucleic acid molecule (e.g., DNA, such as an expression vector) into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection, and the like.
As used herein, and as well understood in the art, “treatment” is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms (e.g., fever, joint pain, rash, conjunctivitis, muscle pain, headache, retro-orbital pain, edema, lymphadenopathy, malaise, asthenia, sore throat, cough, nausea, vomiting, diarrhea, and hematospermia) or conditions (Zammarchi et al. , J. Clin. Virol. 63:32-5, 2015; Waddell et al., PLoS One 11(5): e0156376, 2016); diminishment of the extent of disease, disorder, or condition; stabilization (e.g., not worsening) of a state of disease, disorder, or condition; prevention of spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Palliating” a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or the time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. A treatment can include one or more therapeutic agents, such as one or more of the compositions described herein and/or one or more additional therapeutic agents. Additional therapeutic agents can include agents that stimulate (e.g., interferons) or inhibit (e.g., an anti-inflammatory agent, such as corticosteroids, e.g., dexamethasone) the immune response. A treatment can include one or more therapeutic interventions, such as surgery or prone positioning.
The term “vaccine” as used herein, is defined as material used to provoke an immune response and that confers immunity for a period of time after administration of the vaccine to a subject.
By “vector” is meant a DNA construct that includes one or more polynucleotides, or fragments thereof, such as from a viral species, such as a 2019-nCoV species. The vector can be used to infect cells of a subject, which results in the translation of the polynucleotides of the vector into a protein product. One type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “recombinant vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may, at times, be used interchangeably as the plasmid is the most commonly used form of vector. Other vectors include, e.g., viral vectors, such as adenoviral vectors (e.g., an Ad26 vector), in particular, those described herein.
The term “virus,” as used herein, is defined as an infectious agent that is unable to grow or reproduce outside a host cell and that infects mammals (e.g., humans).
A “viral vector” is defined as a recombinantly produced virus or viral; particle that comprises a polynucleotide to be delivered into a host cell. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors (e.g., see PCT publication no. WO 2006/002203), alphavirus vectors and the like.
In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus (Ad (e.g., Ad26)) or adeno-associated virus (AAV), a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene. Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes (WO 95/27071). Ads are easy to grow and do not require integration into the host cell genome. Recombinant Ad derived vectors, particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed (WO 95/00655 and WO 95/11984). Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo. To optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the clones to eliminate extra, potential inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression, either at the level of transcription or translation.
Other features and advantages will be apparent from the following Detailed Description, the drawings, and the claims.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a 2019-NCOV Spike (S) protein (also referred to as SARS-CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90).
SEQ ID NO: 29 provides the amino acid sequence of the full-length 2019-NCOV Spike (S) protein; see also NCBI Reference Sequence: YP_009724390.1. The stabilising mutations are from the original amino acid in this sequence to proline. The original amino acid at position 986 is lysine (lys, K) and at position 987 is valine (val, V) as shown in SEQ ID NO: 29.
The furin cleavage site within the 2019-NCOV Spike (S) protein comprises, or has, the amino acid sequence RARR (SEQ ID NO: 90). Suitable mutations may comprise mutation to SRAG (SEQ ID NO: 225) or GGSG (SEQ ID NO: 91). The SRAG mutation is achieved by introducing a R682S and a R685G mutation into the amino acid sequence. The GGSG mutation is achieved by introducing a R682G, a R683G, a A684S and a R685G mutation into the amino acid sequence.
In some embodiments, these are the only modifications made to the sequence of SEQ ID NO: 29. Thus, a preferred nucleic acid molecule encodes a full-length 2019-NCOV Spike (S) protein with the stabilising mutations and mutations to the furin cleavage site as the only modifications. In other embodiments, the isolated nucleic acid molecule encodes a 2019-NCOV Spike (S) protein that comprises the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
In some embodiments, the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter, preferably the CMV immediate early promoter. In some embodiments, the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif. In specific embodiments, the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 219. In some embodiments, the CMV promotor consists of the nucleotide sequence of SEQ ID NO: 219. These nucleic acids typically form part of a vector. Vectors are described in further detail herein.
The invention also provides an isolated 2019-NCOV Spike (S) protein (also referred to as SARS-CoV-2 S protein herein) comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90).
In some embodiments, these are the only modifications made to the sequence of SEQ ID NO: 29. In other embodiments the isolated 2019-NCOV Spike (S) protein comprises the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
The term ‘recombinant’ for a nucleic acid, protein and/or adenovirus, as used herein implicates that it has been modified by the hand of man, e.g. in case of an adenovector it has altered terminal ends actively cloned therein and/or it comprises a heterologous gene, i.e. it is not a naturally occurring wild type adenovirus.
Nucleotide sequences herein are provided from 5’ to 3’ direction, as custom in the art.
The Coronavirus family contains the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. All of these genera contain pathogenic viruses that can infect a wide variety of animals, including birds, cats, dogs, cows, bats, and humans. These viruses cause a range of diseases including enteric and respiratory diseases. The host range is primarily determined by the viral spike protein (S protein), which mediates entry of the virus into host cells. Coronaviruses that can infect humans are found both in the genus Alphacoronavirus and the genus Betacoronavirus. Known coronaviruses that cause respiratory disease in humans are members of the genus Betacoronavirus. These include SARS-CoV-1, SARS-CoV-2 and MERS, OC43 and HKU1.
As described above, SARS-CoV-2 can cause severe respiratory disease in humans. A safe and effective SARS-CoV-2 vaccine may be required to end the COVID-19 pandemic.
The SARS CoV-2 viral spike (S) protein binds to angiotensin-converting enzyme 2 (ACE2), which is the entry receptor utilized by SARS-CoV-2. ACE2 is a type I transmembrane metallocarboxypeptidase with homology to ACE, an enzyme long-known to be a key player in the Renin-Angiotensin system (RAS) and a target for the treatment of hypertension. It is expressed in, inter alia, vascular endothelial cells, the renal tubular epithelium, and in Leydig cells in the testes. PCR analysis revealed that ACE-2 is also expressed in the lung, kidney, and gastrointestinal tract, tissues shown to harbor SARS-CoV-2. The spike (S) protein of coronaviruses is a major surface protein and target for neutralizing antibodies in infected patients (Lester et al., Access Microbiology 2019; 1) and is therefore considered a potential protective antigen for vaccine design. In the research that led to the present invention, several antigen constructs based on the S protein of the SARS-CoV-2 virus were designed. It was surprisingly found that the nucleic acid of the invention (i.e. SEQ ID NO: 211) was superior in immunogenicity when expressed and that adenovectors containing this nucleic acid could be manufactured in high yields. An Ad26 vector containing a nucleic acid encoding the SARS CoV- 2 S protein of SEQ ID NO: 205 induced robust neutralizing antibody responses and provided complete protection in bronchoalveolar lavage and or near-complete protection in nasal swabs following SARS-CoV-2 challenge. In addition, as shown in the Examples, it showed a robust single-shot vaccine protection against SARS-CoV-2 in nonhuman primates.
In one aspect, the present invention thus provides isolated and/or recombinant nucleic acids encoding a stabilized coronavirus S protein, in particular a SARS-CoV-2 S protein, said nucleic acids comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 211- 218, or fragments thereof.
In a preferred embodiment, the present invention provides an isolated and/or recombinant nucleic acid encoding a stabilized coronavirus S protein, in particular a SARS-CoV-2 S protein, said nucleic acid comprising, or consisting of, a nucleotide sequence of SEQ ID NO: 211, or fragments thereof.
The invention also provides isolated and/or recombinant coronavirus S proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 205-210, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof. In a preferred embodiment, the invention provides an isolated and/or recombinant coronavirus S protein comprising an amino acid sequence of SEQ ID NO: 205, or fragments thereof, as well as to nucleic acids encoding such coronavirus S proteins, or fragments thereof. The S protein may or may not comprise the signal peptide (or leader sequence). The signal peptide may comprise the amino acids 1-13 of SEQ ID NO: 205. In certain embodiments, the coronavirus S protein consists of an amino acid sequence of SEQ ID NO: 205. In certain embodiments, the coronavirus S protein consists of an amino acid sequence of SEQ ID NO: 205 without the signal peptide. It is understood by a skilled person that numerous different nucleic acids can encode the same polypeptide or protein as a result of the degeneracy of the genetic code. It is also understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleic acids, to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Therefore, unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
The invention thus also provides nucleic acids encoding a coronavirus S protein, in particular a SARS-CoV-2 S protein, of SEQ ID NO: 205, or a fragment thereof. In certain embodiments, the nucleic acid is codon optimized for expression in human cells.
The term “fragment” as used herein refers to a protein or (poly)peptide that has an amino- terminal and/or carboxy-terminal and/or internal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence of a SARS-CoV-2 S protein, for example, the full-length sequence of a SARS-CoV-2 S protein. It will be appreciated that for inducing an immune response and in general for vaccination purposes, a protein does not need to be full length nor have all its wild type functions, and fragments of the protein (i.e. without signal peptide) are equally useful.
A fragment according to the invention is an immunologically active fragment, and typically comprises at least 15 amino acids, or at least 30 amino acids, of the SARS-CoV-2 S protein. In certain embodiments, it comprises at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550 amino acids, of the SARS-CoV-2 S protein.
The person skilled in the art will also appreciate that changes can be made to a protein, e.g., by amino acid substitutions, deletions, additions, etc., e.g., using routine molecular biology procedures. Generally, conservative amino acid substitutions may be applied without loss of function or immunogenicity of a polypeptide. This can easily be checked according to routine procedures well known to the skilled person.
The present invention further provides a vector comprising a nucleic acid sequence according to the invention.
In certain embodiments of the invention, the vector is an adenovirus vector, such as a recombinant human adenoviral vector. An adenovirus according to the invention belongs to the family of the Adenoviridae, and preferably is one that belongs to the genus Mastadenovirus. It can be a human adenovirus, but also an adenovirus that infects other species, including but not limited to a bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), a canine adenovirus (e.g., CAdV2), a porcine adenovirus (e.g., PAdV3 or 5), or a simian adenovirus (which includes a monkey adenovirus and an ape adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or a rhesus monkey adenovirus (RhAd). In the invention, a human adenovirus is meant if referred to as Ad without indication of species, e.g., the brief notation “Ad26” means the same as HAdV26, which is human adenovirus serotype 26. Also as used herein, the notation “rAd” means recombinant adenovirus, e.g., “rAd26” refers to recombinant human adenovirus 26.
Most advanced studies have been performed using human adenoviruses, and human adenoviruses are preferred according to certain aspects of the invention. In certain preferred embodiments, a recombinant adenovirus according to the invention is based upon a human adenovirus. In preferred embodiments, the recombinant adenovirus is based upon a human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the invention, an adenovirus is a human adenovirus of serotype 26. Advantages of these serotypes include a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and experience with use in human subjects in clinical trials. Simian adenoviruses generally also have a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population, and a significant amount of work has been reported using chimpanzee adenovirus vectors (e.g., US6083716; WO 2005/071093; WO 2010/086189; WO 2010/085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al, 2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346: 394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see also review by Bangari and Mittal, 2006, Vaccine 24: 849-62; and review by Lasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other embodiments, the recombinant adenovirus according to the invention is based upon a simian adenovirus, e.g. a chimpanzee adenovirus. In certain embodiments, the recombinant adenovirus is based upon simian adenovirus type 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 or SA7P. In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as ChAdOx 1 (see, e.g., WO 2012/172277), or ChAdOx 2 (see, e.g., WO 2018/215766). In certain embodiments, the recombinant adenovirus is based upon a chimpanzee adenovirus such as BZ28 (see, e.g., WO 2019/086466). In certain embodiments, the recombinant adenovirus is based upon a gorilla adenovirus such as BLY6 (see, e.g., WO 2019/086456), or BZ1 (see, e.g., WO 2019/086466). In a preferred embodiment of the invention, the adenoviral vectors comprise capsid proteins from rare serotypes, e.g. including Ad26. In the typical embodiment, the vector is an rAd26 virus. An “adenovirus capsid protein” refers to a protein on the capsid of an adenovirus (e.g., Ad26, Ad35, rAd48, rAd5HVR48 vectors) that is involved in determining the serotype and/or tropism of a particular adenovirus. Adenoviral capsid proteins typically include the fiber, penton and/or hexon proteins. As used herein a “capsid protein” for a particular adenovirus, such as an “Ad26 capsid protein” can be, for example, a chimeric capsid protein that includes at least a part of an Ad26 capsid protein. In certain embodiments, the capsid protein is an entire capsid protein of Ad26. In certain embodiments, the hexon, penton, and fiber are of Ad26.
One of ordinary skill in the art will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenovirus vector. Thus, a chimeric adenovirus that combines desirable properties from different serotypes can be produced. Thus, in some embodiments, a chimeric adenovirus of the invention could combine the absence of pre-existing immunity of a first serotype with characteristics such as temperature stability, assembly, anchoring, production yield, redirected or improved infection, stability of the DNA in the target cell, and the like. See for example WO 2006/040330 for chimeric adenovirus Ad5HVR48, that includes an Ad5 backbone having partial capsids from Ad48, and also e.g. WO 2019/086461 for chimeric adenoviruses Ad26HVRPtr1, Ad26HVRPtr12, and Ad26HVRPtr13, that include an Ad26 virus backbone having partial capsid proteins of Ptr1, Ptr12, and Ptr13, respectively)
In certain preferred embodiments the recombinant adenovirus vector useful in the invention is derived mainly or entirely from Ad26 (i.e. , the vector is rAd26). In some embodiments, the adenovirus is replication deficient, e.g., because it contains a deletion in the E1 region of the genome. For adenoviruses being derived from non-group C adenovirus, such as Ad26 or Ad35, it is typical to exchange the E4-orf6 coding sequence of the adenovirus with the E4-orf6 of an adenovirus of human subgroup C such as Ad5. This allows propagation of such adenoviruses in well-known complementing cell lines that express the E1 genes of Ad5, such as for example 293 cells, PER.C6 cells, and the like (see, e.g., Havenga, et al. , 2006, J Gen Virol 87: 2135-43; WO 03/104467). However, such adenoviruses will not be capable of replicating in non complementing cells that do not express the E1 genes of Ad5.
The preparation of recombinant adenoviral vectors is well known in the art. Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81 (9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO: 1 of WO 2007/104792. Examples of vectors useful for the invention for instance include those described in WO2012/082918, the disclosure of which is incorporated herein by reference in its entirety.
Typically, a vector useful in the invention is produced using a nucleic acid comprising the entire recombinant adenoviral genome (e.g., a plasmid, cosmid, or baculovirus vector). Thus, the invention also provides isolated nucleic acid molecules that encode the adenoviral vectors of the invention. The nucleic acid molecules of the invention can be in the form of RNA or in the form of DNA obtained by cloning or produced synthetically. The DNA can be double-stranded or single-stranded.
The adenovirus vectors useful in the invention are typically replication deficient. In these embodiments, the virus is rendered replication deficient by deletion or inactivation of regions critical to replication of the virus, such as the E1 region. The regions can be substantially deleted or inactivated by, for example, inserting a gene of interest, such as a gene encoding a synthetic SARS CoV2 S protein (usually linked to a promoter), or a gene encoding an SARS CoV2 S antigenic polypeptide (usually linked to a promoter) within the region. In some embodiments, the vectors of the invention can contain deletions in other regions, such as the E2, E3 or E4 regions, or insertions of heterologous genes linked to a promoter within one or more of these regions. For E2- and/or E4-mutated adenoviruses, generally E2- and/or E4- complementing cell lines are used to generate recombinant adenoviruses. Mutations in the E3 region of the adenovirus need not be complemented by the cell line, since E3 is not required for replication.
In certain embodiments, the recombinant human adenovirus has a deletion in the E1 region, a deletion in the E3 region, or a deletion in both the E1 and the E3 region of the adenoviral genome. Thus, in certain embodiments, an adenoviral vector according to the invention is deficient in at least one essential gene function of the E1 region, e.g. the E1a region and/or the E1b region, of the adenoviral genome that is required for viral replication. In certain embodiments, an adenoviral vector according to the invention is deficient in at least part of the non-essential E3 region. In certain embodiments, the vector is deficient in at least one essential gene function of the E1 region and at least part of the non-essential E3 region. The adenoviral vector can be "multiply deficient," meaning that the adenoviral vector is deficient in one or more essential gene functions in each of two or more regions of the adenoviral genome. For example, the aforementioned E1 -deficient or E1-, E3-deficient adenoviral vectors can be further deficient in at least one essential gene of the E4 region and/or at least one essential gene of the E2 region (e.g., the E2A region and/or E2B region). In certain embodiments, the vector is a recombinant human adenovirus of serotype 26 (rAd26 vectors). This serotype generally has a low seroprevalence and/or low pre-existing neutralizing antibody titers in the human population. Preparation of rAd26 vectors is described, for example, in WO 2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO 2007/104792.
In preferred embodiments, the adenovirus is replication deficient, e.g. because it contains a deletion in the E1 region of the genome. As known to the skilled person, in case of deletions of essential regions from the adenovirus genome, the functions encoded by these regions have to be provided in trans, preferably by the producer cell, i.e. when parts or whole of E1, E2 and/or E4 regions are deleted from the adenovirus, these have to be present in the producer cell, for instance integrated in the genome thereof, or in the form of so-called helper adenovirus or helper plasmids. The adenovirus may also have a deletion in the E3 region, which is dispensable for replication, and hence such a deletion does not have to be complemented.
In a preferred embodiment, the recombinant, replication-incompetent human adenovirus type 26 (Ad26) vector is constructed to encode the SARS-CoV-2 Spike (S) protein, stabilized in its prefusion conformation. Preferably, the adenovirus comprises the nucleic acid of SEQ ID NO: 205.
A packaging cell line is typically used to produce sufficient amounts of adenovirus vectors for use in the invention. A packaging cell is a cell that comprises those genes that have been deleted or inactivated in a replication deficient vector, thus allowing the virus to replicate in the cell. Suitable packaging cell lines for adenoviruses with a deletion in the E1 region include, for example, PER.C6, 911, 293, and E1 A549.
In a preferred embodiment of the invention, the vector is an adenovirus vector, and more preferably a rAd26 vector, most preferably a rAd26 vector with at least a deletion in the E1 region of the adenoviral genome, e.g. such as that described in Abbink, J Virol, 2007. 81(9): p. 4654-63, which is incorporated herein by reference. Typically, the nucleic acid sequence encoding the synthetic SARS CoV-2 S antigens is cloned into the E1 and/or the E3 region of the adenoviral genome.
In certain embodiments, the nucleic acid encoding the coronavirus S protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif. This allows for the cost-effective, large-scale manufacturing of adenoviral particles comprising the SARS CoV-2 S protein insert. Without intending to be limited by theory, it is believed that the SARS CoV-2 S protein leads to lower levels of adenoviral particle production. The addition of the TetO motif to the CMV promoter allows for higher levels of adenoviral particle production. As used herein, a “promoter” is a nucleic acid sequence enabling the initiation of the transcription of a gene sequence in a messenger RNA, such transcription being initiated with the binding of an RNA polymerase on or nearby the promoter.
As defined above, in certain embodiments, the promoter is a cytomegalovirus promoter comprising at least one tetracycline operator (TetO) motif. The TetO motif can be referred to a “regulatory sequence” or “regulatory element,” which as used herein refers to a segment of nucleic acid, typically, but not limited to DNA, that modulates the transcription of the nucleic acid sequence to which it is operatively linked, and, thus, acts as a transcriptional modulator. A regulatory sequence often comprises nucleic acid sequences that are transcription binding domains that are recognized by the nucleic acid-binding domains of transcriptional proteins and/or transcription factors, enhancers, or repressors, etc. For example, it is possible to operably couple a repressor sequence to the promoter, which repressor sequence can be bound by a repressor protein that can decrease or prevent the expression of the transgene in a production cell line that expresses the repressor protein. This can improve genetic stability and/or expression levels of the nucleic acid molecule upon passaging and/or when this is produced at high quantities in the production cell line. Such systems have been described in the art. A regulatory sequence can include one or more tetracycline operator (TetO) motifs/sequences, such that expression is inhibited in the presence of the tetracycline repressor protein (TetR). In the absence of tetracycline, the TetR protein is able to bind to the TetO sites and to repress transcription of a transgene (e.g., SARS CoV-2 S antigen) operably linked to the TetO motifs/sequences. In certain embodiments, the nucleic acid encoding the SARS-CoV-2 S protein, when present in the adenoviral vector, is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif, such that the expression of the SARS CoV-2 S protein is inhibited in recombinant adenoviruses that are produced in the producer cell line in which the TetR protein is expressed. Expression will not be inhibited when the recombinant adenoviral vector is introduced into a subject or into cells that do not express the TetR protein. The invention, however, is not limited to use of a cytomegalovirus promoter comprising at least one tetracycline operator (TetO) motif.
As used herein, the term “repressor” refers to molecules (e.g., proteins) having the capability to inhibit, interfere, retard, and/or repress the production of a heterologous protein product of a recombinant expression vector (e.g., an adenoviral vector). The repressor can inhibit expression by interfering with a binding site at an appropriate location along the expression vector, such as in an expression cassette (e.g., a TetR can bind the TetO motif in the CMV promoter). Repression of vector transgene expression during vector propagation can prevent transgene instability and can increase yields of vectors having the transgene during production. A nucleic acid is “operably linked” when it is placed into a structural or functional relationship with another nucleic acid sequence. For example, one segment of DNA can be operably linked to another segment of DNA if they are positioned relative to one another on the same contiguous DNA molecule and have a structural or functional relationship, such as a promoter or enhancer that is positioned relative to a coding sequence so as to facilitate transcription of the coding sequence; a ribosome binding site that is positioned relative to a coding sequence so as to facilitate translation; or a pre-sequence or secretory leader that is positioned relative to a coding sequence so as to facilitate expression of a pre-protein (e.g., a pre-protein that participates in the secretion of the encoded polypeptide). In other examples, the operably linked nucleic acid sequences are not contiguous, but are positioned in such a way that they have a functional relationship with each other as nucleic acids or as proteins that are expressed by them. Enhancers, for example, do not have to be contiguous. Linking may be accomplished by ligation at convenient restriction sites or by using synthetic oligonucleotide adaptors or linkers.
In certain embodiments, the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 219, preferably the CMV promotor consists of SEQ ID NO: 219.
In a preferred embodiment, the adenoviral vector is produced in PER.C6-tetracycline repressor (TetR) cells.
The invention further provides compositions comprising a nucleic acid, a protein, and/or vector according to the invention. For administering to humans, the invention may employ pharmaceutical compositions comprising the nucleic acid, a protein, and/or vector and a pharmaceutically acceptable carrier or excipient. In the present context, the term “Pharmaceutically acceptable” means that the carrier or excipient, at the dosages and concentrations employed, will not cause any unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The purified nucleic acid, a protein, and/or vector preferably is formulated and administered as a sterile solution although it is also possible to utilize lyophilized preparations. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solutions are then lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, preferably in the range of pH 5.0 to 7.5. The nucleic acid, a protein, and/or vector typically is in a solution having a suitable pharmaceutically acceptable buffer, and the solution may also contain a salt. Optionally stabilizing agent may be present, such as albumin. In certain embodiments, detergent is added. In certain embodiments, nucleic acid, a protein, and/or vector may be formulated into an injectable preparation. These formulations contain effective amounts of nucleic acid, a protein, and/or vector, are either sterile liquid solutions, liquid suspensions or lyophilized versions and optionally contain stabilizers or excipients.
For instance, adenovirus may be stored in the buffer that is also used for the Adenovirus World Standard (Hoganson et al. , Development of a stable adenoviral vector formulation, Bioprocessing March 2002, p. 43-48): 20 mM Tris pH 8, 25 mM NaCI, 2.5% glycerol. Another useful formulation buffer suitable for administration to humans is 20 mM Tris, 2 mM MgCI2, 25 mM NaCI, sucrose 10% w/v, polysorbate-800.02% w/v. Obviously, many other buffers can be used, and several examples of suitable formulations for the storage and for pharmaceutical administration of purified (adeno)virus preparations can for instance be found in European Patent No. 0853660, US patent 6,225,289 and in international patent applications WO 99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763, WO 03/078592, WO 03/061708.
In certain embodiments, the adenovirus composition is a sterile suspension for intramuscular injection, containing the following inactive ingredients:
• Citric acid monohydrate
• Trisodium citrate dihydrate
• Ethanol
• 2-hydroxypropyl^-cyclodextrin (HBCD)
• Polysorbate 80
• Sodium chloride
• Sodium hydroxide
• Water for injections and optionally hydrochloric acid, and wherein the product contains no preservatives.”
In a preferred embodiment, the initial shelf life of the adenoviral vector vaccine is 24 months when stored frozen at the recommended storage condition of -25°C to -15°C (-13°F to 5°F), and within these 24 months, 3 months when stored at 2°C to 8°C (36°F to 46°F).ln certain embodiments, a composition according to the invention comprises a(n) (adeno) vector according to the invention in combination with a further active component. Such further active components may comprise one or more SARS-CoV-2 protein antigens, e.g., a SARS-CoV-2 protein according to the invention, or any other SARS-CoV-2 protein antigen, or additional vectors comprising nucleic acid encoding similar or alternative SARS-CoV-2 antigens. Such vectors again may be non-adenoviral or adenoviral, of which the latter can be of any serotype.
In certain embodiments a composition comprising the adenovirus further comprises one or more adjuvants. Adjuvants are known in the art to further increase the immune response to an applied antigenic determinant, and pharmaceutical compositions comprising adenovirus and suitable adjuvants are for instance disclosed in WO 2007/110409, incorporated by reference herein. The terms “adjuvant” and “immune stimulant” are used interchangeably and are defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance an immune response to the adenovirus vectors of the invention. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see, e.g., US 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO 2005/002620); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG- motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like. It is also possible to use vector- encoded adjuvant, e.g. by using heterologous nucleic acid that encodes a fusion of the oligomerization domain of C4-binding protein (C4bp) to the antigen of interest (e.g., Solabomi et al, 2008, Infect Immun 76: 3817-23). In certain embodiments the compositions of the invention comprise aluminum as an adjuvant, e.g., in the form of aluminum hydroxide, aluminum phosphate, aluminum potassium phosphate, or combinations thereof, in concentrations of 0.05 - 5 mg, e.g. from 0.075-1.0 mg, of aluminum content per dose.
In other embodiments, the compositions do not comprise adjuvants.
The present invention further provides vaccines against COVID-19 comprising a nucleic acid, a protein, and/or vector according to the invention. The term “vaccine” refers to an agent or composition containing an active component effective to induce a therapeutic degree of immunity in a subject against a certain pathogen or disease. According to the present invention, the vaccine may comprise an effective amount of a recombinant adenovirus of serotype 26 that encodes a SARS CoV-2 S protein, in particular a SARS CoV-2 protein that comprises the amino acid sequence of SEQ ID NO: 1 , or an antigenic fragment thereof, which results in an immune response, preferably a protective immune response, against the S protein of SARS CoV-2. The vaccine of the invention may be used in a method of preventing serious lower respiratory tract disease leading to hospitalization and the decrease the frequency of complications such as pneumonia and bronchiolitis due to SARS-CoV-2 infection and replication in a subject. The vaccine may also be used in so-called Postexposure prophylaxis (PEP), i.e. for preventing illness after potential or documented exposure to the coronavirus and/or for reducing the risk of secondary spread of infection. The “vaccine” according to the invention typically includes a pharmaceutically acceptable diluent, carrier or excipient. It may or may not comprise further active ingredients.
In certain embodiments it may be a combination vaccine that further comprises other components that induce an immune response, e.g., against other proteins of SARS.CoV2 and/or against other infectious agents.
In certain embodiments, the vaccine is a combination vaccine comprising a vector according to the invention and a SARS CoV-2 S protein and optionally an adjuvant, wherein the vector and SARS CoV-2 protein are for concurrent administration. The SARS CoV-2 protein may be a protein as described herein or any other suitable SARS CoV-2 S protein that is known in the art. According to the invention, the vector and protein are preferably for concurrent administration (i.e. administered concurrently). “Concurrent administration or co-administration,” in the context of the administration of the vector and protein to a subject, refers to the use of the vector and protein in combination, wherein said vector and protein are administered to the subject within a period of 24 hours.
In certain embodiments, the vector and protein are co-formulated, for example, with a pharmaceutically acceptable buffer, carrier, excipient and/or adjuvant, in a single composition for administration, for example admixed, and administered to a subject together at the same time. In other embodiments, the vector and protein are formulated, for example, with a pharmaceutically acceptable buffer, carrier, excipient and/or adjuvant, in separate compositions, and are administered to a subject in separate compositions within 24 hours, such as within 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 2 hours, or within 1 hour or less, e.g. in the same arm or in two different arms of the subject at about the same time.
The invention provides methods for active immunization to prevent coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in individuals 18 years of age and older. The invention provides methods for active immunization to prevent coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in individuals 16 years of age and older.
The invention provides methods for active immunization to prevent coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in individuals 12 years of age and older.
The invention provides methods for active immunization to prevent coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in individuals 2 months of age and older.
The invention also provides methods for active immunization to prevent coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants.
The SARS CoV-2 variants preferably are selected from the group consisting of the UK variant (SARS-CoV-2 lin. B.1.1.7), South Africa (SARS-CoV-2 501Y.V2) and Denmark variant (Cluster 5).
The SARS CoV-2 variants may comprise one or more mutations selected from the group consisting of del69-70, del145, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, L242H, R246I, K417N, E484K, N501Y, Y435F, 11692V, M1229I and A701V.
The SARS CoV-2 variants may have the E484K mutation and one or more mutations selected from the group consisting of del69-70, del145, A570D, D614G, P681H, T716I, S982A, D1118H, D80A, D215G, L242H, R246I, K417N, E484K, N501Y, Y435F, 11692V, M1229I and A701V. Examples of such SARS CoV-2 variants are B.1.351, B.1.1.7, P.1 and CAL20C.
The invention also provides a method for inducing SARS-CoV-2 binding antibodies in a subject in need thereof, as measured, e.g., by ELISA, comprising administering to the subject a composition or vaccine as described herein.
The invention also provides a method for inducing SARS-CoV-2 neutralizing antibodies in a subject in need thereof, as measured, e.g., by VNA, comprising administering to the subject a composition or vaccine as described herein.
The invention also provides a method for inducing a SARS-CoV-2 specific T cell response in a subject in need thereof, as assessed e.g. by flow cytometry after SARS-CoV-2 S protein peptide stimulation of peripheral blood mononuclear cells (PBMCs) and intracellular staining, comprising administering to the subject a composition or vaccine as described herein.
The invention also provides a method for reducing infection and/or replication of SARS-CoV-2 or variants thereof in, e.g., the nasal tract and lungs of, a subject, comprising administering to the subject a composition or vaccine as described herein. This will reduce adverse effects resulting from SARS-CoV-2 infection in a subject, and thus contribute to protection of the subject against such adverse effects. In certain embodiments, adverse effects of SARS-COV-2 infection may be essentially prevented, i.e. reduced to such low levels that they are not clinically relevant. The recombinant adenovirus may be in the form of a vaccine according to the invention, including the embodiments described above. The administration of further active components may for instance be done by separate administration or by administering combination products of the vaccines of the invention.
The invention also provides a method for prevention of molecularly confirmed, moderate to severe/critical COVID-19, comprising administering to the subject a composition or vaccine as described herein, when given as a one or two dose vaccine. In certain embodiment, the invention provides a method for prevention of molecularly confirmed, moderate to severe/critical COVID-19 as compared to placebo, in SARS-CoV-2 seronegative adults, comprising administering to the subject a composition or vaccine as described herein when given as a one or two dose vaccine.
The invention also provides a method for prevention of molecularly confirmed, severe/critical COVID-19, comprising administering to the subject a composition or vaccine as described herein, when given as a one or two dose vaccine. In certain embodiment, the invention provides a method for prevention of molecularly confirmed, severe/critical COVID-19 as compared to placebo, in SARS-CoV-2 seronegative adults, comprising administering to the subject a composition or vaccine as described herein when given as a one or two dose vaccine. According to the invention, moderate COVID-19 is defined as: a SARS-CoV-2 positive RT-PCR or molecular test result from any available respiratory tract sample or other sample, molecularly confirmed at central laboratory, AND at any time during the observation period until signs and symptoms disappear:
Any 1 of the following new or worsening signs or symptoms:
Respiratory rate ³20 breaths/minute;
Abnormal saturation of oxygen (Sp02) but still >93% on room air at sea level;
Clinical or radiologic evidence of pneumonia;
Radiologic evidence of DVT;
Shortness of breath or difficulty breathing;
OR
Any 2 of the following new or worsening signs or symptoms:
Fever (³38.0°C or ³100.4°F); Heart rate ³90 beats/minute;
Shaking chills or rigors;
New or changing olfactory or taste disorders;
Sore throat;
Malaise;
Headache;
Cough;
Muscle pain (myalgia);
Gastrointestinal symptoms;
Red or bruised looking feet or toes.
According to the invention, severe/critical COVID-19 is defined as: a SARS-CoV-2 positive RT- PCR or molecular test result from any available respiratory tract sample or other sample, molecularly confirmed at central laboratory; AND
Clinical signs at rest indicative of severe systemic illness (respiratory rate ³30 breaths/minute, heart rate ³125 beats/minute, Sp02 £93% on room air at sea level, or Pa02/Fi02 <300 mmHg); Respiratory failure (defined as needing high-flow oxygen, non-invasive ventilation, mechanical ventilation, or ECMO [extracorporeal membrane oxygenation])
Evidence of shock (defined as systolic blood pressure <90 mmHg, diastolic blood pressure <60 mmHg, or requiring vasopressors);
Significant acute renal, hepatic, or neurologic dysfunction;
Admission to the ICU;
Death.
In certain embodiments, severe COVID-19 is as defined by FDA guidance.
According to the invention, mild COVID-19 is defined as: A SARS-CoV-2 positive RT-PCR or molecular test result from any available respiratory tract sample or other sample, molecularly confirmed at central laboratory, AND one of the following symptoms: fever (³38.0°C or ³100.4°F) sore throat, headache, muscle pain (myalgia), gastrointestinal symptoms, cough, chest congestion, runny nose, wheezing, skin rash eye irritation or discharge, chills, new or changing olfactory or taste disorders, red or bruised looking feet or toes, shaking chills or rigors, malaise (loss of appetite, generally unwell, fatigue, physical weakness.
A case is considered mild when it meets the above case definition but not the moderate to severe/critical definition.
Asymptomatic or undetected SARS CoV-2 infection is defined as: participant does not fulfill the criteria for suspected COVID-19 based on signs and symptoms; AND has a SARS-CoV-2 positive RT-PCR or molecular test result from any available respiratory tract sample (eg, nasal swab sample, sputum sample, throat swab sample, saliva sample) or other sample;
OR develops a positive serology (non-S protein) test (serological conversion).
The invention also provides a method for reducing SARS-CoV-2 Viral Load as Assessed by Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) in Participants with Molecularly Confirmed, Moderate to Severe/Critical COVID-19.
The invention also provides a method for preventing or reducing the occurrence of pneumonia linked to any molecularly confirmed COVID-19 when compared to placebo.
The invention also provides a method for reducing sympoms caused by SARS CoV-2 infection. The invention also provides a method for preventing or reducing the occurrence of hospitalization linked to any molecularly confirmed COVID-19 when compared to placebo.
The invention also provides a method for preventing or reducing the occurrence of acute respiratory distress syndrome linked to any molecularly confirmed COVID-19 when compared to placebo.
The invention also provides a method for preventing or reducing the occurrence of sepsis linked to any molecularly confirmed COVID-19 when compared to placebo. The invention also provides a method for preventing or reducing the occurrence of septic shock linked to any molecularly confirmed COVID-19 when compared to placebo.
The invention also provides a method for preventing or decreasing the mortality linked to any molecularly confirmed COVID-19.
In a preferred embodiment, the methods involve adminstraton of the composition or vaccine as a single dose of 0.5 ml_ comprising 5 x 1010 vp of the vaccine according to the invention.
In certain embodiments, the effects of the vaccine (i.e. the induction of SARS-CoV-2 binding and/or neutralizing antibodies and/or the induction of a SARS-CoV-2 specific T cell response) occur already at 8 days after administration of the vaccine.
In certain embodiments the effects of the vaccine occur at least 14 days after the 1st dose of study vaccine depending on the regimen. In certain embodiments the effects of the vaccine occur at least 28 days after the 1st dose of study vaccine depending on the regimen.
In preferred embodiments, the invention provides a method for preventing molecularly confirmed moderate to severe COVID-19.
The compositions or vaccines according to the invention preferably have a vaccine efficacy of at least 50, 55, 60, 65 or 70% against molecularly confirmed moderate to severe COVID-19. Preferably, the compositions or vaccines according to the invention have a vaccine efficacy of at least 60, preferably at least 65 % against molecularly confirmed moderate to severe COVID-19 with onset at least 14 days after vaccination.
According to the invention, the compositions or vaccines are effective against COVID-10 caused by SARS CoV-2, as well as to at least some of the circulating SARS-CoV-2 variants that have been associated with rapidly increasing case numbers and have particular prevalence in the UK (B1.1.7/501 Y.V1), South Africa 212 (501Y.V2) and Brazil (B1.1.28/501. V3).
According to the invention it has been shown that a single dose of the vaccine was efficacious in the prevention of moderate to severe/critical COVID-19 with a vaccine efficacy (VE) of 66% both post Day14 and post Day 28 post vaccination.
Vaccine efficacy against moderate/severe disease after Day 28 is 71 % in US, 66% in Brazil and 57% in South Africa, where most of the strains (90%) were of the variant 501 Y.V2 (South Africa variant). High vaccine efficacy (85% overall) was noted against severe/critical COVID-19 with over 90% efficacy in 18-59 year old. This finding was consistent across countries and regions (North and South America, South Africa), including South Africa where almost all cases were infected with the new variant of SARS-CoV-2
The onset of efficacy was estimated at day 14, with efficacy increasing through day 56, especially against severe disease consistent with the finding of neutralizing antibody titers detected from day 14 onwards, which continued to increase up to day 56 with no indication of waning up to day 85.
The compositions or vaccines according to the invention may be administered to a subject, e.g., a human subject. The total dose of the adenovirus provided to a subject during one administration is generally between 1x107 viral particles (vp) and 1x1012 vp, preferably between 1x108 vp and 1x1011 vp, for instance between 3x108 and 5x1010 vp, for instance between 109 and 3x1010 vp.
In a preferred embodiment the vaccine of the invention is administered to a human subject at a dose of 1.25 x 1010, 2.5 x 1010, 5x1010 or 1x1011 vp per dose in a one dose or two dose regimen wherein the doses are administered about 1, 2, or 3 months apart.
In certain embodiments, the vaccine of the invention is administered to a human subject at a dose of 1x1011 vp per dose in a one dose regimen followed by a second vaccination at 6, 12, or 24 months with same dose.
In a preferred embodiment, the vaccine of the invention is administered to a human subject at a dose of 5x1010 vp per dose in a one dose regimen.
In another preferred embodiment, the vaccine of the invention is administered to a human subject at a dose of 1x1011 vp per dose in a one dose regimen.
In another preferred embodiment, the vaccine is administered to a human subject in a two dose regimen comprising a first administration of a dose of 5 x 1010 vp per dose and a second dose of 5 x 1010 vp per dose administered about 2 months (8 weeks or 56 days) apart.
In certain embodiments, the vaccine is administered to a human subject at a dose of 5 x 1010 vp per dose in a 2-dose regimen administered about 2 months (8 weeks) apart, followed by a further vaccination at 8 months, 14 months, and 26 months (that is, 6 months, 12 months, or 24 months after completion of the two dose regimen) with the same dose.
In a preferred embodiment, the composition is administered at a dose of 5x1010 vp per dose in a one dose regimen.
Administration of adenovirus compositions can be performed using standard routes of administration. Non-limiting embodiments include parenteral administration, such as by injection, e.g., intramuscular, intradermal, etc., or subcutaneous, transcutaneous, or mucosal administration, e.g., intranasal, oral, and the like. However, it is particularly preferred according to the present invention to administer the vaccine intramuscularly. The advantage of intramuscular administration is that it is simple and well-established and does not carry the safety concerns for intranasal application in infants younger than 6 months. In one embodiment a composition is administered by intramuscular injection, e.g. into the deltoid muscle of the arm, or vastus lateralis muscle of the thigh.
A subject as used herein preferably is a mammal, for instance a rodent, e.g. a mouse, a cotton rat, or a non-human-primate, or a human. Preferably, the subject is a human subject. The subject can be of any age, e.g., from about 1 month to 100 years old, e.g., from about 2 months to about 80 years old, from about 6 months of age to about 3 years old, from about 3 years to about 18 years old, from about 12 years to about 18 years old, from about 18 years to about 55 years old, from about 50 years to about 75 years old, etc. In certain preferred embodiments, the subject is a human from 2 years of age. In other preferred embodiments, the human subject is a human from 18 years of age, preferably a human from 60 years of age, or a human from 65 years of age.
In certain embodiments, the composition or vaccine is administered to the subject more than once, e.g. once a year. In certain embodiments, the method of vaccination consists of a single administration of the composition or vaccine to the subject. It is also possible to provide one or more second (or booster) administrations of the vaccine of the invention. If a second vaccination is performed, typically, such a second vaccination will be administered to the same subject at a moment between one week and one year, preferably between two weeks and four months, after administering the composition to the subject for the first time (which is in such cases sometimes referred to as ‘priming vaccination’)
The invention further provides isolated host cells comprising a recombinant human adenovirus of serotype 26 comprising nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof.
A host cell (sometimes also referred to in the art and herein as ‘packaging cell’ or ‘complementing cell’ or ‘producer cell’) that can be used can be any host cell wherein a desired adenovirus can be propagated. A host cell line is typically used to produce sufficient amounts of adenovirus vectors of the invention. A host cell is a cell that comprises those genes that have been deleted or inactivated in a replication-defective vector, thus allowing the virus to replicate in the cell. Suitable cell lines include, for example, PER.C6, 911, 293, and E1 A549.
In certain embodiments, the host cell further comprises a nucleotide sequence encoding a tetracycline repressor (TetR) protein. The nucleotide sequence encoding the TetR protein can, for example, be integrated in the genome of the host cell. By way of an example, the nucleotide sequence encoding the TetR protein can be integrated in chromosome 1. The host cell line can, for example, be a PER.C6 cell.
In a preferred embodiment, the host cell is a PER.C6 cell comprising a nucleotide sequence encoding a tetracycline repressor (TetR) protein. Such “PER.C6 TetR” cells are PER.C6 cells (human retina cells immortalized by E1) to which a nucleotide encoding TetR has been introduced, as described in PCT/EP2018/053201 (incorporated herein by reference). In such embodiments, preferably the adenovirus comprises a nucleic acid encoding a SARS-CoV-2 S protein or fragment thereof which is operably linked to a promoter comprising one or more Tet operator (TetO) motifs. In preferred embodiments, the promoter is a CMV promoter comprising one or more TetO motifs. Preferably the promoter is a CMV promoter comprising two TetO motifs. Preferably the promoter is the CMV promoter and comprises a nucleotide sequence of SEQ ID NO: 219. In some embodiments, the promotor consists of the nucleotide sequence of SEQ ID NO: 219.
The invention further provides methods for making a vaccine against SARS Coronavirus virus (SARS-COV-2), comprising providing a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS-COV-2 S protein or fragment thereof as described herein, propagating said recombinant adenovirus in a culture of host cells, isolating and purifying the recombinant adenovirus, and bringing the recombinant adenovirus in a pharmaceutically acceptable composition. In certain embodiments, provided herein are methods of producing an adenoviral particle comprising a SARS-CoV-2 antigen. The methods comprise (a) contacting a host cell of the invention with an adenoviral vector of the invention and (b) growing the host cell under conditions wherein the adenoviral particle comprising the SARS- CoV-2 antigen is produced. Recombinant adenovirus can be prepared and propagated in host cells, according to well-known methods, which entail cell culture of the host cells that are infected with the adenovirus. The cell culture can be any type of cell culture, including adherent cell culture, e.g. cells attached to the surface of a culture vessel or to microcarriers, as well as suspension culture.
Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. Nowadays, continuous processes based on perfusion principles are becoming more common and are also suitable (see, e.g., WO 2010/060719, and WO 2011/098592, both incorporated by reference herein, which describe suitable methods for obtaining and purifying large amounts of recombinant adenoviruses).
The invention further provides an isolated recombinant nucleic acid that forms the genome of a recombinant human adenovirus of serotype 26 that comprises nucleic acid encoding a SARS- CoV2 S protein or fragment thereof.
Wuhan coronavirus (2019-nCoV) polypeptides can be used to elicit protective and therapeutic immune responses (e.g., humoral responses or cellular responses) against a coronavirus infection (e.g., 2019-nCoV infection) when administered to a subject (e.g., a human subject) infected with or exposed to a coronavirus (e.g., 2019-nCoV). The compositions that can be prepared for administration to a subject include a 2019-nCoV protein (e.g., Spike (S) protein or a portion thereof (e.g., a polypeptide with the sequence of any one of SEQ ID NOs: 1-84, e.g., SEQ ID NO: 51, or a polypeptide with at least 85% (e.g., at least 90%, 95%, 99%, or more) sequence identity to a polypeptide with the sequence of any one of SEQ ID NOs: 1-84)) or a vector (e.g., an expression vector, such as a plasmid, or a viral vector, such as an adenovirus (e.g., Ad26), poxvirus, adeno-associated virus, retroviral, or other viral vector, or naked or encapsulated DNA) containing a nucleic acid sequence that encodes the 2019-nCoV protein (e.g., a nucleic acid molecule with the sequence of any one of SEQ ID NOs: 93-181, 190-195, and 199-204; or a nucleic acid molecule with at least 85% (e.g., at least 90%, 95%, 99%, or more) sequence identity to a nucleic acid molecule with the sequence of any one of SEQ ID NOs: 93-181, 190-195, and 199-204).
The generation of DNA vaccines expressing a 2019-nCoV Spike (S) protein are described. The 2019-nCoV DNA vaccines can be generated by incorporating a polynucleotide (e.g., SEQ ID NOs: 93-181, 190-195, and 199-204 or a variant thereof with up to 85% or more sequence identity thereto) encoding S or a portion thereof (e.g., SEQ ID NOs: 1-84, e.g., SEQ ID NO: 51, or a variant thereof with up to 85% or more sequence identity thereto) into a mammalian expression vector (e.g., pcDNA3.1+; Invitrogen, CA, USA) to generate a vaccine.
Generation of recombinant viral vectors (e.g., Ad26 viral vectors) expressing 2019-nCoV Spike (S) protein is also described. 2019-nCoV viral vectors can be generated by incorporating a polynucleotide (e.g., SEQ ID NOs: 93-181, 190-195, and 199-204 or a variant thereof with up to 85% or more sequence identity thereto) encoding S or a portion thereof (e.g., SEQ ID NOs: 1- 84, or a variant thereof with up to 85% or more sequence identity thereto, e.g., SEQ ID NO: 51) into a viral vector (e.g., an Ad26 viral vector).
Anti-coronavirus antibodies (e.g., anti-2019-nCoV antibodies, e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies) present in a sample from a subject (e.g., a human subject) can be used to detect and/or monitor a protective antibody response. The anti-coronavirus antibodies (e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies) may be measured in a short timeframe (e.g., between 1 day post-administration and 8-weeks post administration) or a longer timeframe (e.g., between 2 month post-administration and 15 years post-administration) after administration of a therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein).
The nucleic acid molecules, polypeptides, vectors, vaccines, compositions, antibodies, and methods treating and preventing a 2019-nCoV infection are described herein. I. COMPOSITIONS AND METHODS
Nucleic Acid Molecules
The nucleic acid molecules (e.g., SEQ ID NOs: 93-181, 190-195, and 199-204 or a variant thereof with up to 85% or more sequence identity thereto) were designed based on the Wuhan coronavirus (2019-nCoV). The nucleic acid molecules encode regions of the 2019-nCoV Spike (S) protein, for example, the full-length (SEQ ID NO: 121), Spike with a deletion of the cytoplasmic region (SEQ ID NO: 94), the ectodomain (SEQ ID NO: 95), S1 (SEQ ID NO: 96), and the receptor binding domain (SEQ ID NO: 97). The invention also features additional modifications to the abovementioned regions of S, including deletion of or inclusion of signal sequences (e.g., SEQ ID NO: 189), stabilizing mutations (e.g., proline substitutions corresponding to amino acids K969 and V970 of SEQ ID NO: 1), mutations to a furin cleavage site (e.g., SEQ ID NO: 188), introduction of a trimerization domain (e.g., a foldon trimerization domain, e.g., SEQ ID NO: 184), introduction of linker or spacer sequences (e.g., SEQ ID NOs: 185 and 186), and combinations thereof. The nucleic acid molecules have been optimized relative to the wild-type 2019-nCoV Spike nucleotide sequence for improved expression in host cells (e.g., mammalian (e.g., human) host cells). Optimization can include the addition of a leader sequence, restriction site, and/or a Kozak sequence.
The nucleic acid molecules have a nucleotide sequence with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to, all or a portion of any one of SEQ ID NOs: 93-181, 190-195, and 199-204, or a complementary sequence thereof. For example, a nucleic acid molecule can have the nucleotide sequence of SEQ ID NO: 195. Alternatively, an isolated nucleic acid molecule has a nucleotide sequence that encodes a 2019-nCoV polypeptide with at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to an amino acid sequence of any one of SEQ ID NOs: 1-84. For example, an isolated nucleic acid molecule can have a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 56.
The nucleic acid molecules may be further optimized, such as by codon optimization, for expression in a targeted mammalian subject (e.g., human or a non-human animal for vaccine production).
The nucleic acid molecules may also be inserted into expression vectors, such as a plasmid, or a viral vector, such as an adenovirus, poxvirus, adeno-associated virus, retroviral, or other viral vector, or prepared as naked or encapsulated DNA and incorporated into compositions. Polypeptides
The polypeptides are coronavirus polypeptides (e.g., 2019-nCoV polypeptides) corresponding to, for example, regions of the 2019-nCoV Spike (S) protein (SEQ ID NOs: 1-84), for example, the full-length (SEQ ID NO: 29), Spike with a deletion of the cytoplasmic region (SEQ ID NO: 2), the ectodomain (SEQ ID NO: 3), S1 (SEQ ID NO: 4), the receptor binding domain (SEQ ID NO: 5) and variants having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to, all or a portion of any one of SEQ ID NOs: 1-84. The polypeptides may include at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, or 1800 or more continuous or non-continuous amino acids of any one of SEQ ID NOs: 1-84. Polypeptides may also include a deletion of or an inclusion of a signal sequence (e.g., SEQ ID NO: 92), stabilizing mutations (e.g., proline substitutions corresponding to amino acids K969 and V970 of SEQ ID NO: 1), mutations to a furin cleavage site (e.g., SEQ ID NO:
91), introduction of a trimerization domain (e.g., a foldon trimerization domain, e.g., SEQ ID NO: 87), introduction of linker or spacer sequences (e.g., SEQ ID NOs: 88 and 89), and combinations thereof. For example, a polypeptide can have the amino acid sequence of SEQ ID NO: 28. The polypeptides may also be isolated from other components (e.g., components with which the polypeptides are natively associated) and incorporated into compositions. Vectors
The invention also features recombinant vectors (e.g., an Ad26 viral vector) including any one or more of the polynucleotides described above. The vectors can be used to deliver a nucleic acid expressing an immunogen (e.g., one of more of SEQ ID NOs: 1-84 or variants thereof, having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto), and include mammalian, viral, and bacterial expression vectors. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 104 or 204) expressing an immunogen with the amino acid sequence of SEQ ID NO: 51. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 195) expressing an immunogen with the amino acid sequence of SEQ ID NO: 56. The mammalian, viral, and bacterial vectors can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 93-181, 190-195, and 199-204 or variants thereof, having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto, and complements thereof. The vectors may be, for example, plasmids, artificial chromosomes (e.g. BAG, PAC, YAC), and virus or phage vectors, and may optionally include a promoter, enhancer, or regulator for the expression of the polynucleotide. The vectors may also contain one or more selectable marker genes, for example an ampicillin, neomycin, and/or kanamycin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example, to produce DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell, e.g., for the production of protein encoded by the vector. The vectors may also be adapted to be used in vivo, for example in a method of DNA vaccination, RNA vaccination, or gene therapy.
Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, mammalian promoters include the metallothionein promoter, which can be induced in response to heavy metals, such as cadmium, and the b-actin promoter. A viral promoter, which can be obtained from the genome of a virus, such as, for example, polyoma virus, fowlpox virus, adenovirus (A), bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus, hepatitis-B virus, and Simian Virus 40 (SV40), and human papillomavirus (HPV), may also be used. These promoters are well known and readily available in the art.
A preferred promoter element is the CMV immediate early promoter. In some embodiments, the expression plasmid is pcDNA3.1+ (Invitrogen, CA, USA). In some embodiments, the expression vector is a viral vector, such as a vector derived from adenovirus or poxvirus.
Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into the genome of a cell (e.g., a eukaryotic or prokaryotic cell). Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes are typically incorporated into the genome of a target cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents to induce gene integration. Examples of viral vectors that can be used to deliver a nucleic acid expressing an immunogen (e.g., one of more of SEQ ID NOs: 1-84 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto) include a retrovirus, adenovirus (e.g., Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48,
Ad49, Ad50, Ad52 (e.g., a RhAd52), Ad59 (e.g., a RhAd59), and Pan9 (also known as AdC68)), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses, such as picornavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox and canarypox). Other viruses useful for delivering polynucleotides encoding immunogens (e.g., polypeptides) include Norwalk virus, togavirus, coronavirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology , Third Edition, B. N. Fields, et al. , Eds., Lippincott- Raven Publishers, Philadelphia, 1996). For example, the vector can be Ad26. These adenovirus vectors can be derived from, for example, human, chimpanzee, or rhesus adenoviruses. Other examples include murine leukemia viruses, murine sarcoma viruses, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia virus, Mason Pfizer monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other examples of vectors are described, for example, in McVey et al., (U.S. Patent. No. 5,801,030); incorporated herein in its entirety by reference. The nucleic acid material (e.g., including a nucleic acid molecule) of the viral vector may be encapsulated, e.g., in a lipid membrane or by structural proteins (e.g., capsid proteins), that may include one or more viral polypeptides (e.g., a glycoprotein). The viral vector can be used to infect cells of a subject, which, in turn, promotes the translation of the heterologous gene(s) of the viral vector into the immunogens. For example, a viral vector can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 93-181, 190- 195, and 199-204 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto, and complements thereof. Adenoviral vectors disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, each incorporated by reference herein, are particularly useful as vectors. These adenoviral vectors can encode and/or deliver one or more of the immunogens (e.g., 2019-nCoV polypeptides) to treat a subject having a pathological condition associated with a viral infection (e.g., a 2019-nCoV infection). In some embodiments, one or more recombinant adenovirus vectors can be administered to the subject in order to express more than one type of immunogen (e.g., 2019-nCoV polypeptide). In some embodiments, a recombinant adenovirus vector can be modified to change the hexon HVR domains (e.g., replace one or more HVRs with those of a different serotype). Besides adenoviral vectors, other viral vectors and techniques are known in the art that can be used to facilitate delivery and/or expression of one or more of the immunogens in a subject (e.g., a human). These viruses include poxviruses (e.g., vaccinia virus and modified vaccinia virus Ankara (MVA); see, e.g., U.S. Patent Nos. 4,603,112 and 5,762,938, each incorporated by reference herein), herpesviruses, togaviruses (e.g., Venezuelan Equine Encephalitis virus; see, e.g., U.S. Patent No. 5,643,576, incorporated by reference herein), picornaviruses (e.g., poliovirus; see, e.g., U.S. Patent No. 5,639,649, incorporated by reference herein), baculoviruses, and others described by Wattanapitayakul and Bauer ( Biomed . Pharmacother. 54:487 (2000), incorporated by reference herein).
Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide into the host genome, although such recombination is not preferred. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.
Vectors capable of driving expression in insect cells (for example baculovirus vectors), in human cells, in yeast or in bacteria may be employed in order to produce quantities of the 2019-nCoV protein encoded by the polynucleotides of the present invention, for example, for use as subunit vaccines or in immunoassays.
Antibodies
Anti-2019-nCoV antibodies are capable of specifically binding to a 2019-nCoV polypeptide and are capable of inhibiting a 2019-nCoV-mediated activity (e.g., viral spread, infection, and or cell fusion) in a subject (e.g., a human). The result of such binding may be, for example, a reduction in viral titer (e.g., viral load), by about 1% (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or more, after administration of an antibody to a subject infected with 2019-nCoV. The anti-2019-nCoV antibodies may selectively bind to an epitope comprising all, or a portion of, the Env region of the 2019-nCoV polyprotein. In particular, the anti-2019-nCoV antibodies may selectively bind to an epitope comprising all, or a portion of, any one of SEQ ID NOs: 1-84. For Example, antibodies may bind to an epitope comprising all, or a portion of, SEQ ID NO: 28. The antibodies can therefore be used to prevent or treat a 2019-nCoV infection.
The specific binding of an antibody or antibody fragment to a 2019-nCoV polyprotein can be determined by any of a variety of established methods. The affinity can be represented quantitatively by various measurements, including the concentration of antibody needed to achieve half-maximal inhibition of viral spread (e.g., viral titer) in vitro (IC50) and the equilibrium constant (KD) of the anti body-2019-nCoV polyprotein complex dissociation. The equilibrium constant, KD, that describes the interaction of 2019-nCoV polyprotein with an antibody is the chemical equilibrium constant for the dissociation reaction of a 2019-nCoV polyprotein-antibody complex into solvent-separated 2019-nCoV polyprotein and antibody molecules that do not interact with one another.
Antibodies are those that specifically bind to a 2019-nCoV polyprotein (e.g., the Env region of 2019-nCoV) with a KD value of less than 1 mM (e.g., 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM). In certain cases, antibodies are those that specifically bind to a 2019-nCoV polyprotein with a KD value of less than 1 nM (e.g., 990 pM, 980 pM, 970 pM, 960 pM, 950 pM, 940 pM, 930 pM, 920 pM, 910 pM, 900 pM, 890 pM, 880 pM, 870 pM, 860 pM, 850 pM, 840 pM,
830 pM, 820 pM, 810 pM, 800 pM, 790 pM, 780 pM, 770 pM, 760 pM, 750 pM, 740 pM, 730 pM,
720 pM, 710 pM, 700 pM, 690 pM, 680 pM, 670 pM, 660 pM, 650 pM, 640 pM, 630 pM, 620 pM,
610 pM, 600 pM, 590 pM, 580 pM, 570 pM, 560 pM, 550 pM, 540 pM, 530 pM, 520 pM, 510 pM,
500 pM, 490 pM, 480 pM, 470 pM, 460 pM, 450 pM, 440 pM, 430 pM, 420 pM, 410 pM, 400 pM,
390 pM, 380 pM, 370 pM, 360 pM, 350 pM, 340 pM, 330 pM, 320 pM, 310 pM, 300 pM, 290 pM,
280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM,
170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, 10 pM, 5 pM, or 1 pM).
Antibodies can also be characterized by a variety of in vitro binding assays. Examples of experiments that can be used to determine the KD or IC50 of a 2019-nCoV antibody include, e.g., surface plasmon resonance, isothermal titration calorimetry, fluorescence anisotropy, and ELISA-based assays, among others. ELISA represents a particularly useful method for analyzing antibody activity, as such assays typically require minimal concentrations of antibodies. A common signal that is analyzed in a typical ELISA assay is luminescence, which is typically the result of the activity of a peroxidase conjugated to a secondary antibody that specifically binds a primary antibody (e.g., a 2019-nCoV antibody). Antibodies are capable of binding 2019-nCoV and epitopes derived thereof, such as epitopes containing one or more of residues of any one of SEQ ID NOs: 1-84, as well as isolated peptides derived from 2019-nCoV that structurally pre-organize various residues in a manner that may simulate the conformation of these amino acids in the native protein. For instance, antibodies may bind peptides containing the amino acid sequence of any one of SEQ ID NOs: 1-84, or a peptide containing between about 10 and about 30 continuous or discontinuous amino acids of any one of SEQ ID NOs: 1-84. In a direct ELISA experiment, this binding can be quantified, e.g., by analyzing the luminescence that occurs upon incubation of an HRP substrate (e.g., 2,2’-azino-di-3- ethylbenzthiazoline sulfonate) with an antigen-antibody complex bound to an HRP-conjugated secondary antibody.
Antibodies include those that are generated by immunizing a host (e.g., a mammalian host, such as a human) with the polypeptides of SEQ ID NOs: SEQ ID NOs: 1-84. The antibodies can be prepared recombinantly and, if necessary, humanized, for subsequent administration to a human recipient if the host in which the anti-2019-nCoV antibodies are generated is not a human.
Compositions
Compositions include DNA or RNA vectors containing a heterologous nucleic acid molecule encoding an antigenic or therapeutic gene product, or fragment thereof, from a 2019-nCoV (e.g., all or a portion of the nucleic acid molecule of any one of SEQ ID NOs: 93-181, 190-195, and 199-204, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of SEQ ID NOs: 93-181, 190-195, and 199-204, and complements thereof). For example, a composition can contain a DNA vector containing the nucleic acid molecule of SEQ ID NO: 143 or SEQ ID NO: 204. For example, a composition can contain a DNA vector containing the nucleic acid molecule of SEQ ID NO: 195. Additional compositions include an immunogenic polypeptide, or fragment thereof, from a 2019-nCoV polyprotein (e.g., all or a portion of the polypeptide of SEQ ID NO: 1-84, or a variant thereof having at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NOs: 1-84). For example, a composition can include an immunogenic polypeptide with the amino acid sequence of SEQ ID NO: 51. For example, a composition can include an immunogenic polypeptide with the amino acid sequence of SEQ ID NO: 28. The compositions may also include a 2019-nCoV antibody (e.g., an anti-Spike antibody) capable of binding 2019-nCoV and epitopes derived thereof, such as epitopes containing one or more of residues of any one of SEQ ID NOs: 1-84. For example, a composition can include an antibody capable of binding epitopes containing one or more of residues of SEQ ID NO: 28. The antibody may be generated by immunization of a host with a polypeptide of any one of SEQ ID NOs: 1-84. For example, an antibody may be generated by immunization of a host with a polypeptide having the amino acid sequence of SEQ ID NO: 28.
Optionally, the compositions can be formulated, for example, for administration via a viral vector (e.g., an adenovirus vector or a poxvirus vector). Recombinant adenoviruses offer several significant advantages for use as vectors for the expression of, for example, one or more of the immunogens (e.g., 2019-nCoV polypeptides). The viruses can be prepared to high titer, can infect non-replicating cells, and can confer high-efficiency transduction of target cells ex vivo following contact with a target cell population. Furthermore, adenoviruses do not integrate their DNA into the host genome. Thus, their use as expression vectors has a reduced risk of inducing spontaneous proliferative disorders. In animal models, adenoviral vectors have generally been found to mediate high-level expression for approximately one week. The duration of transgene expression (expression of a nucleic acid molecule) can be prolonged by using cell or tissue-specific promoters. Other improvements in the molecular engineering of the adenovirus vector itself have produced more sustained transgene expression and less inflammation. This is seen with so-called “second generation” vectors harboring specific mutations in additional early adenoviral genes and “gutless” vectors in which virtually all the viral genes are deleted utilizing a Cre-Lox strategy (Engelhardt et al. , Proc. Natl. Acad. Sci. USA 91:6196 (1994) and Kochanek et al., Proc. Natl. Acad. Sci. USA 93:5731 (1996), each herein incorporated by reference).
Therapeutic formulations of the compositions are prepared for administration to a subject (e.g., a human) using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Therapeutic formulations of the compositions are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Acceptable carriers, include saline, or buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™, or PEG.
Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations can contain a pharmaceutically acceptable preservative. The preservative concentration may range from about 0.1 to about 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts, such as benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben. Optionally, the formulations can include a pharmaceutically acceptable surfactant at a concentration of about 0.005 to about 0.02%.
Optionally, the compositions may be formulated to include for co-administration, or sequential administration with, an adjuvant and/or an immunostimulatory agent, (e.g., a protein), such as receptor molecules, nucleic acids, immunogenic proteins, pharmaceuticals, chemotherapy agents, and accessory cytokines. For example, interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), lipid A, phospholipase A2, endotoxins, staphylococcal enterotoxin B, Type I interferon, Type II interferon, transforming growth factor-b (TGF-b), lymphotoxin migration inhibition factor, granulocyte-macrophage colony-stimulating factor (CSF), monocyte-macrophage CSF, granulocyte CSF, vascular epithelial growth factor (VEGF), angiogenin, transforming growth factor (TGF-a), heat shock proteins (HSPs), carbohydrate moieties of blood groups, Rh factors, fibroblast growth factors, nucleotides, DNA, RNA, mRNA, MART, MAGE, BAGE, mutant p53, tyrosinase, AZT, angiostatin, endostatin, or a combination thereof, may be included in formulations of, or for co-administration with, the compositions.
The pharmaceutical compositions can be administered in a therapeutically effective amount that provides an immunogenic and/or protective effect against an infective agent (e.g., a 2019- nCoV). In some embodiments, a composition comprising a nucleic acid molecule, polypeptide, vector, and/or antibodies may be formulated for administration at a dose of at least 1-1 ,000 pg (e.g., at least 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, or 300 pg or more). The dose may be in a volume of 0.2 ml_ to 1.0 ml_ or up to 1 L (e.g., if prepared as an infusion). In some embodiments, a composition comprising a nucleic acid molecule, vector, and/or vaccine is administered at a dose of 50 pg.
The compositions utilized in the methods described herein can be formulated, for example, for administration intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, by gavage, in cremes, or in lipid compositions.
Pharmaceutical compositions according to the invention described herein may be formulated to release the composition immediately upon administration (e.g., targeted delivery) or at any predetermined time period after administration using controlled or extended release formulations. Administration of the pharmaceutical composition in controlled or extended release formulations is useful where the composition, either alone or in combination, has (i) a narrow therapeutic index (e.g., the difference between the plasma concentration leading to harmful side effects or toxic reactions and the plasma concentration leading to a therapeutic effect is small; generally, the therapeutic index, Tl, is defined as the ratio of median lethal dose (LD5O) to median effective dose (ED5o)); (N) a narrow absorption window at the site of release (e.g., the gastro-intestinal tract); or (iii) a short biological half-life, so that frequent dosing during a day is required in order to sustain a therapeutic level.
Many strategies can be pursued to obtain controlled or extended release in which the rate of release outweighs the rate of metabolism of the pharmaceutical composition. For example, controlled release can be obtained by the appropriate selection of formulation parameters and ingredients, including, e.g., appropriate controlled release compositions and coatings. Suitable formulations are known to those of skill in the art. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, nanoparticles, patches, and liposomes.
The compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation may be administered in powder form or combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11 , more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of an immunogenic composition (e.g., a vaccine or an anti-2019-nCoV antibody) and, if desired, one or more immunomodulatory agents, such as in a sealed package of tablets or capsules, or in a suitable dry powder inhaler (DPI) capable of administering one or more doses.
Methods of Treatment Using Compositions
The pharmaceutical compositions (e.g., immunogenic compositions and anti-2019-nCoV antibodies) can be used to treat a subject (e.g., a human) at risk of exposure (e.g., due to travel to a region where coronavirus (e.g., 2019-nCoV) infection is prevalent) to a coronavirus (e.g.,
2019-nCoV), a subject susceptible to a coronavirus (e.g., 2019-nCoV) infection, or to treat a subject having a coronavirus (e.g., 2019-nCoV) infection. In particular, the compositions can be used to treat (pre- or post-exposure) infection by a 2019-nCoV. In some embodiments, the treatment can induce a protective level of anti-coronavirus antibodies (e.g., anti-2019-nCoV antibodies, e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies). In some embodiments, the protective level is a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is above a level of at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. In some embodiments, treatment with a composition may reduce a 2019-nCoV-mediated activity in a subject, such as viral titer, viral spread, infection, and or cell fusion. In some embodiments, 2019-nCoV-mediated activity is viral load in the respiratory tract (e.g., the upper respiratory tract and/or the lower respiratory tract). In some embodiments, 2019-nCoV-mediated activity is viral load in the lung, nares, and/or trachea. In some embodiments, the 2019-nCoV viral load is decreased by about 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, 99.99%, or more). In some embodiments, 2019-nCoV titer in a treated subject infected with 2019-nCoV is decreased by at least about 1% or more (e.g., 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 95%, 99%, 99.9%, 99.99%, or more) after administration of a composition (e.g., vaccine) to the subject.
The compositions (e.g., any of the compositions described herein) can be used to induce an immune response (e.g., a humoral and/or cellular immune response) in a subject (e.g., a human subject). The immune response induced may be different (e.g., different in the specificity, robustness, or durability) depending on the composition or combination of compositions administered. For example, a composition can induce an antibody response with different antibody types (e.g., different proportions of IgM, IgA, lgG1, lgG2, lgG3, or FcgR2A.1) or different functional characteristics (e.g., ability to induce antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody- dependent monocyte cellular phagocytosis (ADCP), or antibody-dependent NK cell activation (IFN-g secretion, CD107a degranulation, and MIR-1b expression)). Compositions described herein (e.g., SS-Spike and SS-SdCT) may induce an ADCD response that can be monitored (e.g., to assess therapeutic efficacy). Compositions described herein (e.g., SS-RBD-foldon and SS-S.Ecto-dF-PP-foldon) may induce an antibody-dependent NK cell activation response that can be monitored (e.g., to assess therapeutic efficacy). Compositions may also induce cellular responses with different characteristics (e.g., Th1, Th2, or Th17 responses). Compositions described herein (e.g., SS-Spike, SS-SdCT, and SS-S.Ecto-dF-PP-foldon) may induce an S- specific CD4+ or CD8+ T cell response that can be monitored (e.g., to assess therapeutic efficacy).
The vectors (e.g., mammalian, bacterial, or viral (e.g., Ad26) derived expression vectors) can be used to deliver a nucleic acid expressing an immunogen (e.g., one of more of SEQ ID NOs: 1- 84 or variants thereof, having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto) to a subject in a method of preventing and/or treating a 2019-nCoV infection. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NOs: 104 or 204) expressing an immunogen with the amino acid sequence of SEQ ID NO: 51. For example, a vector can be used to deliver a nucleic acid (e.g., a nucleic acid containing the nucleotide sequence of SEQ ID NO: 195) expressing an immunogen with the amino acid sequence of SEQ ID NO: 56. The vectors (e.g., mammalian, bacterial, or viral derived expression vectors) can be genetically modified to contain one or more nucleic acid sequences set forth in SEQ ID NOs: 93-181, 190- 195, and 199-204 or variants thereof having at least 85-99% sequence identity thereto, for example at least greater than 90% sequence identity thereto, and complements thereof. In particular, adenoviral vectors (e.g., vectors derived from Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (RhAd52), Ad59 (RhAd59), and Pan9 (also known as AdC68)) disclosed in International Patent Application Publications WO 2006/040330 and WO 2007/104792, each incorporated by reference herein, are particularly useful as vectors in methods of delivering an immunogen to a subject. For example, the vector can be Ad26. Other examples of vectors are described, for example, in McVey et al. , (U.S. Patent. No. 5,801,030); incorporated herein, in its entirety, by reference.
Useful gene therapy methods for the delivery of immunogens to a subject in need thereof include those described in PCT publication no. WO 2006/060641, U.S. Patent No. US 7,179,903, and PCT publication no. WO 2001/036620, which described the use of, for example, an adenovirus vector (e.g., vectors derived from Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (RhAd52), Ad59 (RhAd59), and Pan9 (also known as AdC68)) for therapeutic protein delivery.
One or more of any of the compositions (e.g., pharmaceutical compositions (e.g., immunogenic compositions and anti-2019-nCoV antibodies)) described herein can be used in a treatment.
The treatment can include one or more additional therapeutic agents (e.g., proinflammatory (e.g., interferons) or anti-inflammatory agents (e.g., corticosteroids, e.g., dexamethasone)) and/or one or more therapeutic interventions (e.g., surgery and prone positioning). The therapeutic agents and/or interventions can be administered sequentially (e.g., administration of one or more of any of the compositions described herein before disease or at an early stage of disease (e.g., within a week of symptom onset), then administration of an additional therapeutic agent (e.g., an anti-inflammatory agent (e.g., a corticosteroid, e.g., dexamethasone) at a later stage of disease (e.g., after a week of symptom onset))) or simultaneously (e.g., administration of one or more of any of the compositions described herein and/or one or more additional therapeutic agents). Additional therapeutic agents can include corticosteroids (e.g., glucocorticoids (e.g., dexamethasone, prednisone, and hydrocortisone)), interferons (e.g., interferon beta), deoxycholic acid, colony stimulating factors (e.g., G-CSF and GM-CSF), and non-steroidal anti-inflammatory drugs (e.g., aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as sulindac, indomethacin, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib). Other agents that can be administered in combination with the compositions described herein include remdesivir, chloroquine, hydroxychloroquine, baricitinib, lopinavir/ritonavir, umifenovir, favipiravir, tocilizumab, and ribavirin.
Administration
The pharmaceutical compositions can be administered to a subject (e.g., a human) pre- or post exposure to an infective agent (e.g., a coronavirus, such as 2019-nCoV) to treat, prevent, ameliorate, inhibit the progression of, or reduce the severity of one or more symptoms of infection (e.g., a coronavirus infection, such as a 2019-nCoV infection). For example, the compositions can be administered to a subject having a 2019-nCoV infection. Examples of symptoms of diseases caused by a viral infection, such as 2019-nCoV, that can be treated using the compositions include, for example, fever, pneumonia, respiratory failure, weight loss, joint pain, rash, conjunctivitis, muscle pain, headache, retro-orbital pain, edema, lymphadenopathy, malaise, asthenia, sore throat, cough, nausea, vomiting, diarrhea, and hematospermia. These symptoms, and their resolution during treatment, may be measured by, for example, a physician during a physical examination or by other tests and methods known in the art. A pharmaceutical composition described herein can be administered to a subject (e.g., a human) pre- or post-exposure to an infective agent (e.g., a coronavirus, such as 2019-nCoV) to reduce or prevent the risk of mortality caused by the agent.
The preferred method of administration can vary depending on various factors (e.g., the components of the composition being administered, and the severity of the condition being treated). Formulations suitable for oral or nasal administration may consist of liquid solutions, such as an effective amount of the composition dissolved in a diluent (e.g., water, saline, or PEG-400), capsules, sachets, tablets, or gels, each containing a predetermined amount of the chimeric Ad5 vector composition. The pharmaceutical composition may also be an aerosol formulation for inhalation, for example, to the bronchial passageways. Aerosol formulations may be mixed with pressurized, pharmaceutically acceptable propellants (e.g., dichlorodifluoromethane, propane, or nitrogen). In particular, administration by inhalation can be accomplished by using, for example, an aerosol containing sorbitan trioleate or oleic acid, for example, together with trichlorofluoromethane, dichlorofluoromethane, dichlorotetrafluoroethane, or any other biologically compatible propellant gas.
Immunogenicity of the composition may be significantly improved if it is co-administered with an immunostimulatory agent and/or adjuvant. Suitable adjuvants well-known to those skilled in the art include, for example, aluminum phosphate, aluminum hydroxide, QS21, Quil A (and derivatives and components thereof), calcium phosphate, calcium hydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of an amino acid, muramyl dipeptides, polyphosphazene, lipoproteins, ISCOM matrix, DC-Chol, DDA, cytokines, and other adjuvants and derivatives thereof.
The compositions may be administered to provide pre-exposure prophylaxis or after a subject has been diagnosed as having a viral infection (e.g., 2019-nCoV infection) or a subject exposed to an infective agent, such as a virus (e.g., a coronavirus infection, such as a 2019-nCoV). The composition may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40,
45, 50, 55, or 60 minutes, 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6 months pre-exposure to a 2019-nCoV, or may be administered to the subject 15- 30 minutes or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48, or 72 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, 3, 4, 6, or 9 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years or post-exposure to a coronavirus (e.g., 2019-nCoV).
When treating viral infection (e.g., a 2019-nCoV infection), the compositions may be administered to the subject either before the occurrence of symptoms or a definitive diagnosis or after diagnosis or symptoms become evident. For example, the composition may be administered, for example, immediately after diagnosis or the clinical recognition of symptoms or 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days after diagnosis or detection of symptoms.
One or more doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) of an immunogenic composition or anti-2019-nCoV antibody-containing composition may be administered to a subject in need thereof. In some embodiments, a subject is administered at least one dose. In some embodiments, a subject is administered at least two doses. In some embodiments, doses are administered on the same day. In some embodiments, doses are administered on different days. In some embodiments, an immunogenic composition is administered to a subject in need thereof as a prime, a boost, or as a prime-boost. In some embodiments, the boost is administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21 , 22, or 23 months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years after the prime of a prime-boost regimen. In other embodiments, multiple boost doses are administered, in which each boost does is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 months, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years apart.
One or more doses of any of the compositions described herein (e.g., any of the immunogenic compositions described herein) may be administered with one or more additional therapeutic agents either sequentially or simultaneously.
Dosages
The dose of the compositions or the number of treatments using the compositions may be increased or decreased based on the severity of, occurrence of, or progression of, the disease in the subject (e.g., based on the severity of one or more symptoms of, e.g., viral infection).
The pharmaceutical compositions can be administered in a therapeutically effective amount that provides an immunogenic and/or protective effect against an infective agent (e.g., a 2019- nCoV). In some embodiments, a composition comprising a nucleic acid molecule, polypeptide, vector, and/or antibodies may be administered in a dose of at least 1 pg to 100 mg (e.g., at least 10 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 125 pg, 150 pg, 175 pg, 200 pg, 225 pg, 250 pg, 275 pg, 300 pg, 325 pg, 350 pg, 375 pg, 400 pg, 425 pg, 450 pg, 475 pg, 500 pg, 525 pg, 550 pg, 575 pg, 600 pg, 625 pg, 650 pg, 675 pg, 700 pg, 725 pg, 750 pg,
775 pg, 800 pg, 825 pg, 850 pg, 875 pg, 900 pg, 925 pg, 950 pg, 975 pg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6mg, 7mg, 8mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg,
18 mg, 19 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or more). In some embodiments, a composition comprising a nucleic acid molecule, vector, and/or antibody is administered at a dose of about 50 pg (e.g., a dose between about 25 pg and about 75 pg).
In some embodiments, a composition comprising a nucleic acid molecule, vector, and/or antibody is administered at a dose of about 5 mg (e.g., a dose of about 1 mg to about 10 mg). In some instances, administration of an effective amount of a composition (e.g., an immunogen, such as SEQ ID NOs: 1-84) induces a protective level (e.g., above a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, above a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is above a level of at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein) of anti-coronavirus antibodies (e.g., anti-2019-nCoV antibodies, e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies). In some instances, the protective level is a titer of at least about 70 (e.g., at least about 80, at least about 100, or at least about 120) as measured using the pseudovirus neutralization assay described herein. In some instances, the protective level is a titer of at least about 100, as measured using the pseudovirus neutralization assay described herein. In some instances, administration of an effective amount of a composition results in a protective level of anti-coronavirus antibodies (e.g., anti-2019-nCoV antibodies, e.g., anti-Spike antibodies, e.g., anti-Spike neutralizing antibodies) that are maintained for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 years or more.
In some instances, administration of an effective amount of a composition (e.g., an immunogen, such as SEQ ID NOs: 1-84) reduces 2019-nCoV serum viral loads determined from a subject having a 2019-nCoV infection by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to viral loads determined from the patient prior to administration of an effective amount of a composition. In some instances, administration of an effective amount of a composition reduces serum viral loads to an undetectable level compared to viral loads determined from the patient prior to administration of an effective amount of a composition. In some instances, administration of an effective amount of a composition results in a reduced and/or undetectable serum viral load that may be maintained for at least about 1,
2, 3, 4, 5, 6, 7 days; 1, 2, 3, 4, weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months; or 1 year or more.
The dosage administered depends on the subject to be treated (e.g., the age, body weight, capacity of the immune system, and general health of the subject being treated), the form of administration (e.g., as a solid or liquid), the manner of administration (e.g., by injection, inhalation, or dry powder propellant), and the cells targeted (e.g., epithelial cells, such as blood vessel epithelial cells, nasal epithelial cells, or pulmonary epithelial cells). The composition is preferably administered in an amount that provides a sufficient level of the antigenic or therapeutic gene product, or fragment thereof (e.g., a level of an antigenic gene product that elicits an immune response without undue adverse physiological effects in the host caused by the antigenic gene product).
The method of delivery, for example of a DNA or RNA vaccine, may also determine the dose amount. In some cases, dosage administered by injections by intravenous (i.v.) or intramuscular (i.m.) route may require variable amounts of a DNA or RNA vaccine, for example from 10 pg-1 mg. However, administration using a gene gun may require a dose of a DNA or RNA vaccine between 0.2 pg and 20 pg (e.g., 0.2, 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pg). In some instances, the use of a gene gun to deliver a dose of a DNA or RNA vaccine may require only ng quantities of DNA or RNA, for example between 10 ng and 200 ng (e.g., 10, 12, 13, 14, 15, 16, 17, 18, 19, 20.30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, or 200 ng).
In other embodiments wherein the delivery vector is a virus (e.g., an Ad26 virus), the subject can be administered at least about 1x103 viral particles (VP)/dose or between 1x101 and 1x1020 VP/dose (e.g., 1x101, 1x102, 1x103, 1x104, 1x105, 1x106, 1x107, 1x108, 1x109, 1x1010, 1x1011, 1x1012, 1x1013, 1x1014, 1x1015, 1x1016, 1x1017, 1x1018, 1x1019, or 1x1020 VP/dose). For example, the subject can be administered about 1x106 to about 1x1014 VP/dose (e.g., about 1x107, about 1x108, about 1x109, about 1x1010, about 1x1011, about 1x1012, about 1x1013, about 1x1014, or about 1x1015 VP/dose). For example, the subject can be administered about 1x1011, about 1x1012, about 1x1013, or about 1x1014 VP/dose.
In addition, single or multiple administrations of the compositions of the present invention may be given (pre- or post-exposure and/or pre- or post-diagnosis) to a subject (e.g., one administration or administration two or more times). For example, subjects who are particularly susceptible to, for example, viral infection (e.g., a 2019-nCoV infection) may require multiple treatments to establish and/or maintain protection against the virus. Levels of induced immunity provided by the pharmaceutical compositions described herein can be monitored by, for example, measuring amounts of neutralizing secretory and serum antibodies. The dosages may then be adjusted or repeated as necessary to trigger the desired level of immune response. For example, the immune response triggered by a single administration (prime) of a composition may not sufficiently potent and/or persistent to provide effective protection. Accordingly, in some embodiments, repeated administration (boost), such that a prime boost regimen is established, can significantly enhance humoral and cellular responses to the antigen of the composition.
Alternatively, the efficacy of treatment can be determined by monitoring the level of the antigenic or therapeutic gene product, or fragment thereof, expressed in a subject (e.g., a human) following administration of the compositions. For example, the blood or lymph of a subject can be tested for antigenic or therapeutic gene product, or fragment thereof, using, for example, standard assays known in the art.
In some instances, efficacy of treatment can be determined by monitoring a change in the serum viral load from a sample from the subject obtained prior to and after administration of an effective amount of a composition (e.g., an immunogen, such as any one of SEQ ID NOs: 1-84). A reduction in serum viral load of at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to viral load determined from the subject prior to administration of an effective amount of a composition may indicate that the subject is receiving benefit from the treatment. If a viral load does not decrease by at least about 10%, 20%, 30%, or more after administration of a composition, the dosage of the composition to be administered may be increased. For example, by increasing the pg or mg amount of a DNA vaccine (e.g., a DNA vaccine containing one or more of SEQ ID NOs: 93-181, 190-195, and 199-204) administered to the subject or by increasing the number of viral particles (VP) of an adenovirus vector-based vaccine (e.g., an adenovirus vector-based vaccine containing one or more of SEQ ID NOs: 93- 181, 190-195, and 199-204).
A single dose of a composition may achieve protection, pre-exposure or pre-diagnosis. In addition, a single dose administered post-exposure or post-diagnosis can function as a treatment according to the present invention.
A single dose of a composition can also be used to achieve therapy in subjects being treated for an infection (e.g., a coronavirus infection, such as a 2019-nCoV infection). Multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more doses) can also be administered, in necessary, to these subjects.
Methods of Diagnosing and Predicting Susceptibility to Coronavirus Infection
Diagnostic Methods
Provided herein are methods for identifying, diagnosing, and/or predicting the susceptibility of a subject to a coronavirus infection. The method includes measuring the level or amount of an anti-coronavirus antibody (e.g., an anti-Spike antibody) in a sample (e.g., a whole blood sample, e.g., a serum or plasma sample) from the subject. In some embodiments, the coronavirus is 2019-nCoV. In some embodiments, the anti-coronavirus antibody (e.g., an anti-Spike antibody) is a neutralizing antibody. In some embodiments, the subject is determined to be susceptible to the coronavirus infection if the anti-coronavirus antibody (e.g., an anti-Spike antibody) amount or level is below a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or below 80% of a median level of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., 2019-nCoV)) as determined by a pseudovirus neutralization assay or live virus neutralization assay) and determined to not be susceptible to the coronavirus infection if the anti-coronavirus antibody (e.g., an anti-Spike antibody) level is above a protective level. In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike neutralizing antibody titer) of at least about 70 (e.g., about 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 110, 115, 120, 125, 130, 140, 150, 175, 200, 225,
250, 275, 300, 325, 350 or more) as determined in a pseudovirus neutralization assay. In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike neutralizing antibody titer) of at least about 83 as determined in a pseudovirus neutralization assay. In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike neutralizing antibody titer) of at least about 25 (e.g., about 25, 26, 27, 28, 29, 30, 31 ,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,
61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 110, 115,
120, 125, 130, 140, 150, 175, 200 or more) as determined in a live virus neutralization assay. In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike neutralizing antibody titer) of at least about 35 as determined in a live virus neutralization assay In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti- Spike neutralizing antibody titer) that is at least about 60% (e.g., about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about
71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about
79%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about
88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
96%, about 97%, about 98%, about 99%, about 100%, about 110%, about 120%) of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike neutralizing antibody titer) that is at least about 80% of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. A subject determined to be susceptible to the coronavirus infection (a subject with an anti-coronavirus antibody (e.g., an anti-Spike antibody) amount or level is below a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or below 80% of a median level of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., 2019-nCoV)) as determined by a pseudovirus neutralization assay or live virus neutralization assay)) can be administered a therapy (e.g., administered any of the compositions described herein), such as an effective amount of one or more of the pharmaceutical compositions (e.g., immunogenic compositions and anti-2019-nCoV antibodies) described herein. A subject may be re-administered a therapy until the subject is determined to not be susceptible to the coronavirus infection (e.g., until the subject has an anti-coronavirus antibody (e.g., an anti-Spike antibody) level is above a protective level (e.g., a level above a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, above a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is at a level that is at least 80% of a median level (and preferably at or above a median level) of an anti-coronavirus antibody of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., 2019-nCoV)) as determined by a pseudovirus neutralization assay or live virus neutralization assay)). The method may also involve determining whether the anti-Spike antibody is an RBD-specific antibody. The method may also involve determining whether the anti-Spike antibody is an S1-specific antibody. The method may also involve determining whether the anti-Spike antibody is an S2-specific antibody. The method may also involve identifying the subclass (e.g., IgM, IgA, lgG1, lgG2, lgG3, or FcgR2A.1) and/or effector function (e.g., antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), or antibody-dependent NK cell activation (IFN-y secretion, CD107a degranulation, and MIR-1b expression)) of the anti-coronavirus antibody. The method may further include administering one or more of the pharmaceutical compositions (e.g., immunogenic compositions and anti-2019-nCoV antibodies) described herein to a subject determined to be in need of further therapy.
The method may include measuring the coronavirus (e.g., 2019-nCoV) viral load in a sample from the subject. In some embodiments, the sample is a bronchoalveolar lavage (BAL) or a nasal swab (NS). In some embodiments, the sample is a bodily fluid (e.g., blood, e.g., whole blood or plasma) from the subject. In some embodiments, the sample is a tissue sample (e.g., a respiratory tract tissue sample) from the subject. In some embodiments, viral load is a detectible nucleic acid (e.g., subgenomic mRNA) level or a detectible protein (e.g., nucleocapsid protein (N)) level. In some embodiments, the detectible nucleic acid (e.g., subgenomic mRNA) is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, LAMP, microarray analysis, or hybridization (e.g., ISH (e.g., FISH)). In some embodiments, the detectible protein (e.g., nucleocapsid protein (N)) is determined by an immunoassay (e.g., an immunohistochemical (IHC) assay or a lateral flow immunoassay). In some embodiments, a detectable viral load indicates that the subject is susceptible to disease (e.g., a 2019-nCoV- mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than at least about 3.5 logio sgmRNA copies/mL (e.g., about 3.75 logio sgmRNA copies/mL, about 3.8 logio sgmRNA copies/mL, about 3.9 logio sgmRNA copies/mL, about 4.0 logio sgmRNA copies/mL, about 4.25 logio sgmRNA copies/mL, about 4.5 logio sgmRNA copies/mL, about 4.75 logio sgmRNA copies/mL, about 5.0 logio sgmRNA copies/mL, about 5.5 logio sgmRNA copies/mL, about 6.0 logio sgmRNA copies/mL, about 6.5 logio sgmRNA copies/mL, about 7.0 logio sgmRNA copies/mL, about 7.5 logio sgmRNA copies/mL, about 8.0 logio sgmRNA copies/mL, about 8.5 logio sgmRNA copies/mL, about 9 logio sgmRNA copies/mL, about 10 logio sgmRNA copies/mL, about 11 logio sgmRNA copies/mL, about 12 logio sgmRNA copies/mL, about 13 logio sgmRNA copies/mL or more). In some embodiments, a viral load of greater than 3.85 logio sgmRNA copies/mL in BAL or 3.78 logio sgmRNA copies/mL in NS indicates that the subject is susceptible to disease (e.g., a 2019-nCoV- mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than 3.85 logio sgmRNA copies/mL in BAL or 3.78 logio sgmRNA copies/mL in NS indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 2.0 logio sgmRNA copies/g (e.g., about 2.0 logio sgmRNA copies/g, about 2.5 logio sgmRNA copies/g, about 3.0 logio sgmRNA copies/g, about 3.5 logio sgmRNA copies/g, about 4.0 logio sgmRNA copies/g, about 4.25 logio sgmRNA copies/g, about 4.5 logio sgmRNA copies/g, about 4.75 logio sgmRNA copies/g, about 5.0 logio sgmRNA copies/g, about 5.5 logio sgmRNA copies/g, about 6.0 logio sgmRNA copies/g, about 6.5 logio sgmRNA copies/g, about 7.0 logio sgmRNA copies/g, about 7.5 logio sgmRNA copies/g, about 8.0 logio sgmRNA copies/g, about 8.5 logio sgmRNA copies/g, about 9 logio sgmRNA copies/g, about 10 logio sgmRNA copies/g, about 11 logio sgmRNA copies/g, about 12 logio sgmRNA copies/g, about 13 logio sgmRNA copies/g or more) of tissue indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 8.0 logio sgmRNA copies/g in lung tissue, about 7.0 logio sgmRNA copies/g in nares tissue, about 6.0 logio sgmRNA copies/g in trachea tissue, about 5.5 logio sgmRNA copies/g in heart tissue, or about 2.0 log sgmRNA copies/g in Gl, spleen, liver, kidney, or brain tissue indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 3% (e.g., about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to disease (e.g., a 2019-nCoV-mediated disease, e.g., COVID-19, e.g., severe COVID-19 disease). In some embodiments, a viral load of greater than about 5% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, a viral load of greater than about 5% (e.g., about 5%, about 6%, about 7%, about 8%, about 9%, about 10%) SARS-CoV-2 vRNA staining by ISH indicates that the subject is susceptible to severe COVID-19 disease. In some embodiments, coronavirus (e.g., 2019- nCoV) viral load is measured one or more times over about 1, 2, 3, 4, 5, or 6 days or 1 , 2, 3, 4,
5, 6, or 7 weeks post-infection.
Monitoring Responsiveness
Provided herein are methods for monitoring an anti-coronavirus immune response of a subject to a therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein or known in the art) for treating or reducing the risk of a coronavirus infection. The method includes measuring the level or amount of an anti-coronavirus antibody (e.g., an anti-Spike antibody) in the subject. In some embodiments, the coronavirus is 2019-nCoV. In some embodiments, the anti-coronavirus antibody (e.g., an anti-Spike antibody) is a neutralizing antibody. The anti-coronavirus antibody (e.g., an anti-Spike antibody, e.g., an anti-Spike neutralizing antibody) may be measured in a short timeframe (e.g., in order to measure the robustness of the antibody response) or a longer timeframe (e.g., in order to measure the durability of the antibody response) after administration of a therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein). In some embodiments, the anti-coronavirus antibody (e.g., an anti-Spike antibody) is measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days, about 5, 6, 7, 8, 9, 10, 11, or 12 weeks, about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 months, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 years after the subject is administered the therapeutic composition (e.g., any of the compositions or immunogenic compositions described herein).
The subject is determined to be responsive to the therapeutic composition if the anti-coronavirus antibody (e.g., an anti-Spike antibody) detected in the subject (e.g., in the subject’s blood) is above a protective level (e.g., above a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, above a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is at a level that is at least 80% of a median level (and preferably at or above a median level) of an anti-coronavirus antibody of a cohort of convalescent humans (e.g., a group of humans who have recovered or are recovering from a coronavirus infection (e.g., 2019-nCoV)) as determined by a pseudovirus neutralization assay or live virus neutralization assay). Alternatively, the subject is determined to be non-responsive to the therapeutic composition if the anti-coronavirus antibody (e.g., an anti-Spike antibody) detected in the subject is below a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or is at a level that is below 80% of a median level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay). A protective level of an anti-coronavirus antibody (e.g., an anti-Spike neutralizing antibody) corresponds to a titer of at least about 70 (e.g., about 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 110, 115, 120, 125, 130, 140, 150, 175, 200, 225, 250, 275, 300, 325, 350 or more) as determined in a pseudovirus neutralization assay (e.g., the pseudovirus neutralization assay described herein). In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike neutralizing antibody titer) of at least about 25 (e.g., about 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,
105, 110, 115, 120, 125, 130, 140, 150, 175, 200 or more) as determined in a live virus neutralization assay(e.g., the pseudovirus neutralization assay described herein). In some embodiments, the protective level is an anti-coronavirus antibody titer (e.g., an anti-Spike neutralizing antibody titer) that is at least about 60% (e.g., about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 110%, about 120%) of a median or mean level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay as described herein. If, over time, an anti-coronavirus antibody (e.g., an anti-Spike antibody) titer in the subject (e.g., in the blood of a subject) falls below or fails to reach a protective level (e.g., below a titer of at least about 70 as measured using the pseudovirus neutralization assay described herein, below a titer of at least about 25 as measured using the live virus neutralization assay described herein, or below 80% of a median level of a cohort of convalescent humans as determined by a pseudovirus neutralization assay or live virus neutralization assay described herein), the subject may be administered or may be re-administered a coronavirus vaccine composition (e.g., one or more of the therapeutic or immunogenic compositions described herein) alone or in combination with an additional therapeutic agent, such as one or more of the additional therapeutic agents described herein. Administration of a composition of the present disclosure to a subject in need thereof can be performed one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times) over one or more days (e.g., 1, 2, 3, 4, 5, 6, or 7 days), weeks (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 weeks), months (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months), or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years), or over the life of the subject, as needed to maintain a protective level of an anti-coronavirus antibody in the subject, thereby protecting the subject against coronavirus infection (e.g., infection by 2019-nCoV).
The method may include measuring the coronavirus (e.g., 2019-nCoV) viral load in a sample from the subject. In some embodiments, the coronavirus is 2019-nCoV. In some embodiments, the viral load is measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42, or 48 hours or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days post-infection. In some embodiments, the sample is a bronchoalveolar lavage (BAL) or a nasal swab (NS). In some embodiments, the sample is a bodily fluid (e.g., blood, e.g., whole blood or plasma) from the subject. In some embodiments, the sample is a tissue sample (e.g., a respiratory tract tissue sample) from the subject. In some embodiments, viral load is a detectible nucleic acid (e.g., subgenomic mRNA) level or a detectible protein (e.g., nucleocapsid protein (N)) level. In some embodiments, the detectible nucleic acid (e.g., subgenomic mRNA) is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, LAMP, microarray analysis, or hybridization (e.g., ISH (e.g.,
FISH)). In some embodiments, the detectible protein (e.g., nucleocapsid protein (N)) is determined by an immunoassay (e.g., an immunohistochemical (IHC) assay or a lateral flow immunoassay). The subject is determined to be responsive to the therapeutic composition if the viral load is below a pre-assigned level. In some embodiments, the pre-assigned level is less than about 3.5 logio sgmRNA copies/mL BAL or NS or less than about 5.0 logio sgmRNA copies/g of tissue (e.g., lung, nares, trachea, heart, Gl, spleen, liver, kidney, or brain tissue). In some embodiments, the subject is determined to be responsive to the therapeutic composition if the viral load decreases in the subject.
If the subject is not determined to be responsive as determined by viral load, then the subject may be administered or may be re-administered a coronavirus vaccine composition (e.g., one or more of the therapeutic or immunogenic compositions described herein) alone or in combination with an additional therapeutic agent, such as one or more of the additional therapeutic agents described herein.
Administration of a composition of the present disclosure to a subject in need thereof can be performed one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times) over one or more days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, or 28 days) as needed to reduce the viral load.
Other Embodiments
All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations following, in general, the principles and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.
The present invention further provides:
Embodiment 1 is an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a 2019-NCOV Spike (S) protein comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90).
Embodiment 2 is the isolated nucleic acid molecule of embodiment 1 comprising a nucleotide sequence of SEQ ID NO: 211. Embodiment 3 is the isolated nucleic acid molecule of embodiment 1 or 2 comprising a nucleotide sequence that encodes a 2019-NCOV Spike (S) protein comprising an amino acid sequence of SEQ ID NO: 205.
Embodiment 4 is the isolated nucleic acid molecule of embodiment 1 comprising a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 51.
Embodiment 5 is the isolated nucleic acid molecule of embodiment 1 or 4 comprising a nucleotide sequence that encodes a polypeptide having at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 51.
Embodiment 6 is the isolated nucleic acid molecule of embodiment 1 comprising a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 54.
Embodiment 7 is the isolated nucleic acid molecule of embodiment 1 comprising a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 56.
Embodiment 8 is the isolated nucleic acid molecule of embodiment 1 comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 121, or a complementary sequence thereof.
Embodiment 9 is the isolated nucleic acid molecule of embodiment 4 or 5 comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 143, or a complementary sequence thereof.
Embodiment 10 is the isolated nucleic acid molecule of embodiment 6 or 7 comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 146, or a complementary sequence thereof. Embodiment 11 is the isolated nucleic acid molecule of embodiment 6 or 7 comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 148, or a complementary sequence thereof.
Embodiment 12 is the isolated nucleic acid molecule of embodiment 1 that encodes a 2019- NCOV Spike (S) protein comprising the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
Embodiment 13 is the isolated nucleic acid molecule of embodiment 1 or 12 comprising a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 23.
Embodiment 14 is the isolated nucleic acid molecule of embodiment 1, 12 or 13 comprising a nucleotide sequence that encodes a polypeptide having at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 23.
Embodiment 15 is the isolated nucleic acid molecule of embodiment 1 or 12 comprising a nucleotide sequence that encodes a polypeptide having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 26.
Embodiment 16 is the isolated nucleic acid molecule of embodiment 1, 12 or 13 comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 115, or a complementary sequence thereof.
Embodiment 17 is the isolated nucleic acid molecule of embodiment 15 comprising a nucleotide sequence having at least 85% sequence identity to SEQ ID NO: 118, or a complementary sequence thereof.
Embodiment 18 is the isolated nucleic acid molecule of any preceding embodiment, wherein the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter, preferably the CMV immediate early promoter. Embodiment 19 is the isolated nucleic acid molecule of any preceding embodiment, wherein the nucleic acid encoding the 2019-NCOV Spike (S) protein is operably linked to a cytomegalovirus (CMV) promoter comprising at least one tetracycline operator (TetO) motif.
Embodiment 20 is the isolated nucleic acid molecule according to embodiment 19, wherein the CMV promoter comprising at least one TetO motif comprises a nucleotide sequence of SEQ ID NO: 219.
Embodiment 21 is the isolated nucleic acid molecule according to embodiment 19 or 20, wherein the CMV promotor consists of the nucleotide sequence of SEQ ID NO: 219.
Embodiment 22 is an isolated 2019-NCOV Spike (S) protein comprising the following modifications to the full-length amino acid sequence of SEQ ID NO: 29: a. stabilising mutations to proline at amino acids 986 and 987; and b. mutations to the furin cleavage site (SEQ ID NO: 90.
Embodiment 23 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 comprising an amino acid sequence of SEQ ID NO: 205.
Embodiment 24 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 51.
Embodiment 25 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 or 23 comprising an amino acid sequence having at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 51.
Embodiment 26 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 54.
Embodiment 27 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 56. Embodiment 28 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 comprising the following further modification to the full-length amino acid sequence of SEQ ID NO: 29: c. deletion of the signal sequence.
Embodiment 29 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 or 28 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 23.
Embodiment 30 is the isolated 2019-NCOV Spike (S) protein of embodiment 22, 28 or 29 comprising an amino acid sequence having at least 99% sequence identity to an amino acid sequence of SEQ ID NO: 23.
Embodiment 31 is the isolated 2019-NCOV Spike (S) protein of embodiment 22 or 28 comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence of SEQ ID NO: 26.
Embodiment 32 is an isolated vector comprising one or more of the nucleic acid molecules of any one of embodiments 1-21.
Embodiment 33 is the vector of embodiment 32, wherein the vector is replication-defective.
Embodiment 34 is the vector of embodiment 32, wherein the vector is a mammalian, bacterial, or viral vector.
Embodiment 35 is the vector of embodiment 32, wherein the vector is an expression vector.
Embodiment 36 is the vector of embodiment 32, wherein the viral vector is a virus selected from the group consisting of a retrovirus, adenovirus, adeno-associated virus, parvovirus, coronavirus, negative strand RNA viruses, orthomyxovirus, rhabdovirus, paramyxovirus, positive strand RNA viruses, picornavirus, alphavirus, double stranded DNA viruses, herpesvirus, Epstein-Barr virus, cytomegalovirus, fowlpox, and canarypox.
Embodiment 37 is the vector of embodiment 36, wherein the vector is an adenovirus. Embodiment 38 is the vector of embodiment 37, wherein the adenovirus is selected from the group consisting of Ad26, Ad52, Ad59, Ad2, Ad5, Ad11, Ad12, Ad24, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, and Pan9, in particular Ad26.
Embodiment 39 is a composition comprising the nucleic acid molecule of any one of embodiments 1-21 , the polypeptide of any one of embodiments 22-31 or the vector of any one of embodiments 32-38.
Embodiment 40 is the composition of embodi