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WO2006071250A2 - Fragments solubles de la glycoproteine de spicule de cov-sras - Google Patents

Fragments solubles de la glycoproteine de spicule de cov-sras Download PDF

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WO2006071250A2
WO2006071250A2 PCT/US2005/011510 US2005011510W WO2006071250A2 WO 2006071250 A2 WO2006071250 A2 WO 2006071250A2 US 2005011510 W US2005011510 W US 2005011510W WO 2006071250 A2 WO2006071250 A2 WO 2006071250A2
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sars
antibody
cov
seq
polypeptide
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WO2006071250A3 (fr
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Bernard Moss
Himani Bisht
Linda S. Wyatt
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US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A61K39/1045—Moraxella
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to the treatment and prevent of severe acute respiratory syndrome (SARS) caused by the SARS-coronavirus (SARS-CoV).
  • SARS severe acute respiratory syndrome
  • SARS-CoV SARS-coronavirus
  • SARS Severe acute respiratory syndrome
  • the etiologic agent of SARS was identified as a coronavirus (CoV) and the sequence of the SARS virus genome established that it was a new member of the family. See Rota et al. (2003) Science 300, 1394-1399; Marra et al. (2003) Science 300, 1399-1404. Closely related coronaviruses were recovered from civet cats and other animals in southern China, although the source of human SARS infection remained uncertain. Other members of the CoV family can cause fatal diseases of livestock, poultry and laboratory rodents. Holmes, K. V. (2003) J. Clin. Invest. Ill, 1605-1609. The two previously identified human CoV, however, cause only mild upper respiratory infections. Id.
  • the invention provides SARS Coronavirus polypeptides, antibodies directed against those polypeptides and recombinant viruses that can express SARS Coronavirus polypeptides.
  • Administration of these SARS-related polypeptides, antibodies and recombinant viruses to animals is surprisingly effective for protecting those animals against SARS Coronavirus infection.
  • one aspect of the invention is an isolated polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
  • Another aspect of the invention is an isolated nucleic acid encoding a polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
  • such a nucleic acid can have SEQ ID NO:2 or 5.
  • Another aspect of the invention is an antibody that can bind to a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
  • Another aspect of the invention is a recombinant attenuated poxvirus comprising a genome with a nucleic acid insertion that encodes a SARS
  • Nucleic acid insertions that can be used in the recombinant attenuated poxvirus can, for example, have SEQ ID NO:2 or 5.
  • Many types of poxviruses are available for use.
  • the poxvirus is a modified MVA virus.
  • Another aspect of the invention is a recombinant attenuated baculo virus comprising a nucleic acid encoding a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
  • such a nucleic acid can have SEQ ID NO:2 or 5.
  • Another aspect of the invention is a DNA vaccine comprising a pharmaceutically acceptable carrier and a vector encoding a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:1, 3, 4, 6 or 7.
  • compositions comprising a carrier and an effective amount of SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6, 7, or a combination thereof.
  • the amount employed in the composition can be effective for generating antibody production in an animal.
  • compositions comprising a carrier and an effective amount of a recombinant attenuated poxvirus comprising a genome with a nucleic acid insertion that encodes a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:1, 3, 4, 6 or 7.
  • the amount employed in the composition can be effective for generating antibody production in an animal.
  • compositions comprising a carrier and an effective amount of antibody that can bind to a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
  • the amount employed in this composition can be effective to inhibit SARS Coronavirus replication in the animal.
  • Another aspect of the invention is a method for generating an immune response in an animal against a SARS Coronavirus S polypeptide comprising: administering to the animal an immunologically effective amount of any of the polypeptide or poxvirus compositions of the invention.
  • Another aspect of the invention is a method for inhibiting SARS Coronavirus infection in an animal comprising: administering to the animal an immunologically effective amount of any of the polypeptide, poxvirus or antibody compositions of the invention.
  • Another aspect of the invention is a method for treating SARS Coronavirus infection in an animal comprising: administering to the animal an effective amount of the composition of the invention.
  • an effective amount is effective to inhibit SARS Coronavirus replication in the animal.
  • Another aspect of the invention is a diagnostic kit for detection of a
  • SARS Coronavirus infection in a mammal comprising packaging material, an antibody that can bind to a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7, and instructions for detection of a SARS Coronavirus infection in a mammal.
  • Another aspect of the invention is a diagnostic kit for detection of a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7, and instructions for detection of a SARS Coronavirus infection in a mammal.
  • Another aspect of the invention is a diagnostic kit for detection of a
  • SARS Coronavirus infection in a mammal comprising packaging material, a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7, and instructions for detection of a SARS Coronavirus infection in a mammal.
  • FIG. IA-B provides a diagram of a recombinant Spike polypeptide expression cassette within a MVA viral vector and illustrates expression from this construct.
  • FIG. IA provides a diagram of selected portion of MVA/S.
  • the GFP and S open reading frames were inserted into a deletion site (del III) of the MVA genome.
  • the early/late mH5 and late PI l vaccinia virus promoters were used to regulate expression of S and GFP, respectively.
  • MVA/S-HA has an identical structure except for the presence of a short segment of DNA encoding the influenza virus HA tag at the C-terminus of the S open reading frame.
  • IB provides a Western-blot analysis of SARS-CoV S protein expressed by cells infected with MVA or MVA/S-HA. Uninfected HeLa cells were used as a control. Eighteen hours after infection, the cells were harvested and the cleared cell lysates were analyzed by SDS-PAGE. The electrophoretically separated proteins were transferred to a nitrocellulose membrane and detected with anti- HA mAb (lanes 1 , 2, and 3) or anti-SARS-CoV S polyclonal antibody (lanes 4, 5, and 6). The masses of marker proteins in kDa are shown on the left and the position of SARS-CoV S protein is indicated by an arrow on the right.
  • FIG. 2A-B illustrates that the SARS-CoV S protein is a glycoprotein.
  • FIG. IA shows that the molecular weight of the SARS-CoV S protein is sensitive to Endo H, which digests the N-linked high-mannose carbohydrate side chains of glycoproteins that are synthesized in the endoplasmic reticulum (ER), and PNGase F, which hydrolyzes all types of N-glycan chains.
  • HeLa cells were uninfected (lanes 1, 5) or infected with MVA (lanes 2, 6) or MVA/S-HA (lanes 3, 4, 7, 8). After 18 h, the cells were lysed, cleared by centrifugation, and incubated with anti-HA affinity matrix (Roche).
  • FIG. IB illustrates the kinetics of endo H sensitivity.
  • HeLa cells at 8 h after infection with MVA/S-HA were pulse-labeled with [ 35 S]methionine and
  • FIG. 3A-H illustrates the cellular localization of SARS-CoV S.
  • Unfixed and unpermeabilized CEF (FIG. 3A-F) that had been infected with MVA (FIG. 3A-B) 5 MVA/S (FIG. 3C-D) and MVA/S-HA (FIG. 3E-F) for 18 h were stained with anti-SARS-CoV mouse serum (FIG. 3A-D) or anti-HA mAb (FIG. 3E-F) followed by Alexa 594- conjugated-anti-mouse IgG and viewed by confocal microscopy.
  • CEF infected with MVA/S-HA (FIG. 3G-H) were fixed, permeabilized and stained with anti-HA mAb followed by Alexa 594- conjugated-anti-mouse IgG. Panels on the left and right show GFP and Alexa 594 fluorescence, respectively.
  • FIG. 4A-B illustrates the antibody responses after immunization with recombinant MVA/S by intranasal (IN) or intramuscular (IM) routes.
  • FIG. 4B shows the pre-challenge SARS-CoV neutralization titers of pooled serum were determined. The dilution of serum that completely prevented SARS-CoV cytopathic effect in 50% of the wells was calculated.
  • FIG. 5 illustrates that mice immunized with MVA/S, which expresses the SARS-CoV S polypeptide, were protected from subsequent challenge with live SARS-CoV.
  • Groups of 8 B ALB/c mice were mock vaccinated or vaccinated with MVA or MVA/S by the IN or IM routes at 0 time and 4 weeks and then challenged 4 weeks later with 10 4 TCID 50 of SARS-CoV administered by the IN route. Two days later the titers of SARS-CoV in the lungs and nasal turbinates of 4 mice in each group were determined. Virus titers are expressed as 1Og 10 TCID 5 o/g of tissue.
  • FIG. 6 provides amino acid and cDNA sequences (SEQ ID NO:4 and 5, respectively) for the SARS-CoV (Urbani strain) S ⁇ TM+CT polypeptide containing spike protein amino acids 14-1195.
  • FIG. 7A-C illustrates the construction, expression and characterization of SARS-CoV (Urbani strain) S ⁇ TM+CT polypeptide containing spike protein amino acids 14-1195.
  • FIG. 7A provides a schematic representation of pMelBacB-based baculovirus transfer vector. Abbreviations: P PH polyhedrin promoter; HBM, DNA encoding honeybee melittin signal sequence; nS, DNA segment encoding amino acids (aa) 14-762 of the SARS- CoV S protein; His 6 ; DNA encoding 6 histidine residues.
  • P PH polyhedrin promoter HBM, DNA encoding honeybee melittin signal sequence
  • nS DNA segment encoding amino acids (aa) 14-762 of the SARS- CoV S protein
  • His 6 DNA encoding 6 histidine residues.
  • FIG. 7B illustrates that the SARS CoV nS polypeptide is pure as analyzed by SDS polyacrylamide gel electrophoresis and Coomassie Blue staining (lane 1), silver staining (lane 2) and western blot analysis with anti-His mAb (lane 3) or anti-SARS CoV S polyclonal antibody (lane 4).
  • FIG. 7C shows that the SARS-CoV nS polypeptide is glycosylated. Purified nS protein was (+) or was not (-) treated with peptide N-glycosidase F and was analyzed by SDS polyacrylamide gel electrophoresis and western blotting with anti-His mAb and anti-SARS-CoV S polyclonal antibody. Molecular masses of marker proteins in kDa are shown on the left.
  • FIG. 8A-H illustrates binding of antibodies from mice immunized with nS to full-length membrane-bound S.
  • HeLa cells were uninfected (FIG. 8 A-B), infected with non-recombinant MVA (FIG. 8C-D) or MVA expressing S (FIG. 8E-H) for 18 h.
  • the unpermeabilized cells were stained with pooled sera from mice immunized three times with nS and MPL + TDM (E-F) or nS and QS21 (FIG. 8A-D, G-H) followed by Alexa 594- conjugated-anti-mouse IgG and viewed by visible (FIG. 8A 5 C 5 E 5 G) or fluorescence (FIG. 8B 5 D 5 F 5 H) light microscopy.
  • FIG. 9A-B illustrates ELISA and neutralizing antibody responses to the nS (SEQ ID NO:6) polypeptide.
  • Groups of 7 BALB/c mice were immunized subcutaneously with 10 ⁇ g of purified nS and QS21 or MPL + TDM adjuvant at 4- week intervals (arrows) and challenged intranasally with 10 5 TCID 50 SARS- CoV on day 82 (arrow head).
  • Control mice were immunized at the same times with purified soluble vaccinia virus LlR protein.
  • FIG. 9 A shows end-point ELISA titers of pooled sera collected on days indicated were measured using nS as the capture antigen. The absorbance obtained with serum from mice immunized with LlR was subtracted.
  • FIG. 9B shows the dilution of serum that completely prevented cytopathic effects of SARS-CoV in 50% of wells containing Vero cells. Assays were performed on pooled serum collected on days 28 and 56 days and on individual mouse serum collected on day 78. Standard error bars are shown for the latter.
  • FIG. 10A-B illustrates that immunized mice are protected against SARS- CoV replication.
  • Groups of 7 BALB/c mice were immunized and challenged with SARS-CoV as described in the legend to FIG. 9.
  • Two days after the challenge the virus titers (mean 1Og 10 TCID 50 per g tissue with standard error) were measured in the lower (FIG. 10A) and upper (FIG. 10B) respiratory tract.
  • a full-length Spike (S) polypeptide of SARS-CoV 5 expressed by an attenuated poxvirus induces formation of neutralizing antibodies and protectively immunizes animals against a subsequent infection with SARS-CoV.
  • Antiserum collected from animals immunized with the attenuated poxvirus reduced SARS viral replication in infected animals.
  • a secreted, glycosylated S polypeptide including amino acids 14 to 762 of the SARS coronavirus (SARS-CoV) S protein provided complete protection of the upper and lower respiratory tract against SARS infection.
  • the invention provides immunological compositions of SARS-CoV polypeptides, and of live attenuated viruses that can express such SARS-CoV polypeptides.
  • the invention provides anti-S ARS-CoV S antibody compositions that are useful for passive immunization of animals that are infected, or may become infected, with SARS.
  • Attenuated recombinant virus refers to a virus that has been rendered less virulent than wild type, typically by deletion of specific genes or by serial passage in a non-natural host cell line or at cold temperatures.
  • Nucleic acid-based vaccines include both naked DNA and vectored DNA (within a viral capsid) where the nucleic acid encodes B-cell and T-cell epitopes and provides an immunoprotective response in the animal to which the vaccine has been administered.
  • Poxviruses are large, enveloped viruses with a genome of double- stranded DNA that is covalently closed at the ends. Poxviruses replicate entirely in the cytoplasm. They have been used as vaccines since the early 1980's (see, e.g., Panicali, D. et al. Construction of live vaccines by using genetically engineered pox viruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin, Proc. Natl. Acad. Sci. USA 80:5364- 5368, 1983).
  • "Viral load” is the amount of virus present in the blood of a patient. Viral load is also referred to as viral titer or viremia. Viral load can be measured using procedures available to one of skill in the art.
  • SARS-coronavirus has a nearly 30,000 nucleotides long RNA genome with eleven open reading frames that encode four major structural proteins consisting of nucleocapsid, spike (S), membrane and small envelope protein (Marra et al. (2003) Science 300, 1399-1404; Rota et al. (2003) Science 300, 1394-1399).
  • S nucleocapsid
  • S membrane and small envelope protein
  • the latter is a type-I transmembrane glycoprotein, which forms the characteristic corona of large protruding spikes on the virion surface and mediates binding to the host cell receptor and membrane fusion.
  • S was shown to be an important determinant of pathogenesis as well as the major target of protective immunity (8, 11).
  • SARS-CoV The S of SARS-CoV is quite divergent from those of other CoV, exhibiting only 20 to 27% overall amino acid identity (Rota et al. (2003) Science 300, 1394-1399). Recent studies have indicated that the SARS-CoV S polypeptide is expressed as a non-cleaved glycoprotein with an apparent mass of 180 to 200 kDa that interacts with a functional receptor identified as angiotensin-converting enzyme 2 (Li et al. (2003) Nature 426, 450-454; Xiao et al. (2003) Biochem. Biophys. Res. Commun. 312, 1159-1164.
  • S polypeptides are useful antigens for generating an immune response against SARS-CoV.
  • SARS-CoV Several different strains of SARS-CoV have been isolated and sequenced. Nucleic acid and amino acid sequences for different S polypeptides, and the nucleic acids that encode them can be found in the art, for example, in the NCBI database. See website at ncbi.nlm.nih.gov.
  • one amino acid sequence for the S polypeptide from the Urbani strain of SARS-CoV can be found in the NCBI database as accession number AAP 13441 (gi: 30027620). See website at ncbi.nlm.nih.gov. This Urbani S polypeptide sequence is provided below as follows (SEQ ID NO: 1).
  • a nucleotide sequence for this SARS-CoV Urbani S polypeptide can be found in the nucleotide sequence having accession number AY278741 (gi: 30027617), which provides the complete nucleotide sequence for the Urbani genome.
  • the S polypeptide sequence is encoded by nucleotides 21492 to 25259. This S nucleic acid sequence is provided below for easy reference (SEQ ID NO:2).
  • SARS-CoV S polypeptide NS-I strain
  • accession number AAR91586 gi: 40795747. See website at ncbi.nlm.nih.gov. This sequence for this SARS-CoV S polypeptide is provided below (SEQ ID NO:3). 1 MFIFLLFLTL TSGSDLDRCT TFDDVQAPNY TQHTSSMRGV
  • the invention provides antigenic fragments of SARS-CoV S polypeptides.
  • substantially full length SARS- CoV spike protein with native signal sequences as well as transmembrane and cytoplasmic regions are deleted from the S polypeptide ( ⁇ TM+CT).
  • ⁇ TM+CT SARS-CoV spike protein
  • a cDNA encoding amino acids 14 to 1195 of the SARS-CoV (Urbani Strain) S protein was used (see GenBank accession no. AY278741 , starting at nucleotide 21531) with a sequence for 6 histidine residues attached to its 3 'end.
  • GenBank accession no. AY278741 starting at nucleotide 21531
  • the sequences of the S( ⁇ TM+CT) polypeptide 14-1195AA, SEQ ID NO:4) and cDNA (SEQ ID NO:5) are shown in FIG. 6 and are provided below.
  • S( ⁇ TM+CT) spike polypeptide sequence (14-1195AA, SEQ ID NO:4) is as follows.
  • the S( ⁇ TM+CT) cDNA (SEQ ID NO:5) sequence is as follows. 1 AGTGACCTTG ACCGGTGCAC CACTTTTGAT GATGTTCAAG
  • S polypeptide encoding the N-teraiinal 14-762 amino acids is also highly antigenic and is provided for use in an immunogenic composition or vaccine.
  • This S polypeptide fragment was selected on the basis of hydrophilicity and secondary structure predictions using Kyte and Dolittle and Chou Fasman algorithms (McVactor 7.2) and also because it encompasses the receptor binding region as well as the region corresponding to Sl of other coronaviruses.
  • the sequence of this N-terminal 14-762 amino acid spike polypeptide is as follows (SEQ ID NO:6).
  • a C-terminal S polypeptide fragment is also provided for use in the immunogenic compositions and vaccines of the invention.
  • This C-terminal S polypeptide fragment includes amino acids 763-1195.
  • the sequence for this C- terminal 763-1195 amino acid SARS-CoV S polypeptide is as follows (SEQ ID NO:7).
  • the invention provides S polypeptides and antigenic fragments thereof that are useful for treating and preventing SARS infection.
  • peptide variants and derivatives of the S polypeptides and peptides are also useful in the practice of the invention.
  • Such peptide variants and derivatives can have one or more amino acid substitutions, deletions, insertions or other modifications so long as the S polypeptide variant or derivative can induce an immune response against an S polypeptide or against SARS-CoV.
  • Amino acid residues of the S polypeptides can be genetically encoded L- amino acids, naturally occurring non-genetically encoded L-amino acids, synthetic L-amino acids or D-enantiomers of any of the above.
  • the amino acid notations used herein for the twenty genetically encoded L-amino acids and common non-encoded amino acids are conventional and are as shown in Table 1.
  • S polypeptides that are within the scope of the invention can have one or more amino acids substituted with an amino acid of similar chemical and/or physical properties, so long as these variant or derivative S polypeptides retain the ability to induce an immune response in an animal against SARS-CoV.
  • amino acids that are substitutable for each other generally reside within similar classes or subclasses.
  • amino acids can be placed into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes may be further divided into subclasses.
  • Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids include amino acids having aromatic or apolar side chains.
  • Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids.
  • the definitions of the classes of amino acids as used herein are as follows:
  • Hydrophobic Amino Acid refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution.
  • genetically encoded hydrophobic amino acids include He, Leu and VaI.
  • non-genetically encoded hydrophobic amino acids include t- BuA.
  • Aromatic Amino Acid refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated ⁇ -electron system (aromatic group).
  • aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others.
  • Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan.
  • Non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, ⁇ -2-thienylalanine, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.
  • Apolar Amino Acid refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar.
  • Examples of genetically encoded apolar amino acids include glycine, proline and methionine. Examples of non-encoded apolar amino acids include Cha.
  • Aliphatic Amino Acid refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain.
  • genetically encoded aliphatic amino acids include Ala, Leu, VaI and He.
  • non-encoded aliphatic amino acids include NIe.
  • Hydrophilic Amino Acid refers to an amino acid having a side chain that is attracted by aqueous solution.
  • examples of genetically encoded hydrophilic amino acids include Ser and Lys.
  • examples of non-encoded hydrophilic amino acids include Cit and hCys.
  • Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
  • Basic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of greater than 7.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • genetically encoded basic amino acids include arginine, lysine and histidine.
  • non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.
  • Poly Amino Acid refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has a bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • genetically encoded polar amino acids include asparagine and glutamine.
  • non-genetically encoded polar amino acids include citrulline, N-acetyl lysine and methionine sulfoxide.
  • Cysteine-Like Amino Acid refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage.
  • cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group.
  • examples of genetically encoded cysteine-like amino acids include cysteine.
  • examples of non-genetically encoded cysteine-like amino acids include homocysteine and penicillamine.
  • cysteine has both an aromatic ring and a polar hydroxyl group.
  • cysteine has dual properties and can be included in both the aromatic and polar categories.
  • cysteine also has apolar character.
  • cysteine can be used to confer hydrophobicity to a peptide.
  • Certain commonly encountered amino acids that are not genetically encoded and that can be present, or substituted for an amino acid, in the peptides and peptide analogues include, but are not limited to, ⁇ -alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; ⁇ -aminoisobutyric acid (Aib); ⁇ - aminohexanoic acid (Aha); ⁇ -aminovaleric acid (Ava); methylglycine (MeGIy); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (MeIIe); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (
  • Table 2 is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues that may include the peptides and peptide analogues described herein.
  • Other amino acid residues that are useful for making the peptides and peptide analogues described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein.
  • Amino acids not specifically mentioned herein can be conveniently classified into the above-described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.
  • Polypeptides can have any amino acid substituted by any similarly classified amino acid to create a variant or derivative peptide, so long as the peptide variant or derivative retains the ability to induce an immune response in an animal.
  • the immune response is against SARS-CoV.
  • S polypeptides, derivatives and variants thereof can be assessed by procedures available to one of skill in the art.
  • the S polypeptide, derivative or variant thereof can be administered to the animal and, after a time period sufficient for production of antibodies, serum can be collected from the animal to ascertain whether the animal has produced circulating antibodies that are reactive with the S polypeptide, derivative or variant thereof.
  • S polypeptides, derivatives and variants thereof may also choose to test the S polypeptides, derivatives and variants thereof to ascertain whether they are useful for inhibiting SARS viral replication in an animal.
  • a rodent animal model has been developed in which SARS-CoV replicates but does not cause disease (Subbarao et al. (2004) J. Virol. 78, 3572-3577).
  • An S polypeptide, derivative or variant thereof can be administered to such a rodent animal, the animal can then be exposed to SARS-CoV and the respiratory tract or lungs of the animal can be monitored for SARS-CoV viral load.
  • the S polypeptide, derivative or variant thereof reduces the viral load relative to animals exposed to the SARS-CoV but not immunized with the S polypeptide, derivative or variant thereof, then the S polypeptide, derivative or variant is an effective immunogen that can be used to treat or protect an animal against SARS-CoV infections.
  • Attenuated recombinant viruses that express SARS-CoV specific epitopes are of use in immunological compositions of this invention. Attenuated viruses are modified from their wild type virulent form to a non-infective or weakened form when administered to humans.
  • the recombinant viruses that can be used are adenoviruses, adeno-associated viruses, retroviruses and poxviruses.
  • a recombinant, attenuated virus for use in an immunogenic composition or vaccine is a virus wherein the genome of the virus is defective with respect to a gene that is essential for the efficient production infectious virus.
  • the mutant virus acts as a vector for production of an immunogenic SARS-CoV S epitope or antigenic SARS-CoV S polypeptide by virtue of insertion of S polypeptide DNA into the genome of the virus.
  • Expression of the SARS-CoV S epitopes or antigens provokes or stimulates an immune response against S polypeptides and against SARS-CoV.
  • Attenuated viruses can be used.
  • examples of viral expression vectors include adenoviruses as described in M. Eloit et al., Construction of a Defective Adenovirus Vector Expressing the Pseudorabies Virus Glycoprotein gp50 and its Use as a Live Vaccine, J. Gen. Virol. 71(10):2425-2431 (Oct., 1990), adeno-associated viruses (see, e.g., Samulski et al., J. Virol. 61: 3096-3101 (1987); Samulski et al., J. Virol.
  • the viral vector may be derived from herpes simplex virus (HSV) in which, for example, the gene encoding glycoprotein H (gH) has been inactivated or deleted.
  • HSV herpes simplex virus
  • suitable viral vectors include retroviruses (see, e.g., Miller, Human Gene Ther. 1:5-14 (1990); Ausubel et al., Current Protocols in Molecular Biology).
  • Poxviruses can be used in the compositions of this invention.
  • attenuated poxviruses that are available for use as an immunological composition against SARS-CoV. These include attenuated vaccinia virus, cowpox virus and canarypox virus.
  • one technique for inserting foreign genes into live infectious poxvirus involves a recombination between pox DNA sequences flanking a foreign genetic element in a donor plasmid and homologous sequences present in the rescuing poxvirus as described in Piccini et al., Methods in Enzymology 153, 545-563 (1987).
  • the recombinant poxviruses are constructed in two steps using procedures like those for creating synthetic recombinants of poxviruses such as the vaccinia virus and avipox virus as described in U.S. Patent No. 4,769,330, U.S. Patent No.
  • a nucleic acid segment encoding an antigenic S polypeptide sequence such as an identified or known T-cell epitope, is selected to be inserted into the virus.
  • the nucleic acid segment to be inserted is generally operably ligated to a promoter.
  • the promoter-SARS-CoV segment is positioned in the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a DNA sequence flanking a region of pox DNA encoding a nonessential function.
  • the resulting plasmid construct is then amplified by growth in a host cell, for example, within E. coli cells.
  • a host cell for example, within E. coli cells.
  • the isolated vector or plasmid containing the SARS-CoV sequence to be inserted into the poxviral genome is transfected into animal cells (e.g. chick embryo fibroblasts), along with the poxvirus. Recombination between homologous pox DNA in the plasmid and the viral genome gives a poxvirus with a SARS-CoV insertion.
  • animal cells e.g. chick embryo fibroblasts
  • Recombination between homologous pox DNA in the plasmid and the viral genome gives a poxvirus with a SARS-CoV insertion.
  • one of skill in the art selects a nonessential region of the poxvirus genome to insert the foreign (SARS-CoV) DNA sequences. Attenuated recombinant pox viruses are often used as viral vectors
  • recombinant pox viruses include MVA, ALVAC, TROVAC, NYVAC, and vCP205 (ALVAC-MN 120TMG). These viruses have been deposited under the terms of the Budapest Treaty with the American Type Culture Collection (ATCC) 5 12301 Parklawn Drive, Rockville, Md., 20852, USA.
  • the NYVAC virus has been deposited under ATCC accession number VR-2559 on Mar. 6, 1997.
  • the vCP205 (ALVAC-MN 120TMG) virus has been deposited under ATCC accession number VR-2557 on Mar. 6, 1997.
  • the MVA virus has been deposited under ATCC accession number VR-1508 or VR-1566.
  • the TROVAC virus has been deposited under ATCC accession number VR- 2553 on Feb. 6, 1997, and the ALVAC virus has been deposited under ATCC accession number VR-2547 on Nov. 14, 1996.
  • NYVAC is a genetically engineered vaccinia virus strain generated by the specific deletion of eighteen open reading frames encoding gene products associated with virulence and host range.
  • NYVAC is highly attenuated by a number of criteria including: (a) decreased virulence after intracerebral inoculation in newborn mice, (b) inocuity in genetically (nuVnu 4 ) or chemically (cyclophoshamide) immunocompromised mice; (c) failure to cause disseminated infection in immunocompromised mice, (d) lack of significant induration and ulceration on rabbit skin; (e) rapid clearance from the site of inoculation; and (f) greatly reduced replication competency on a number of tissue culture cell lines including those of human origin.
  • TROVAC refers to an attenuated fowlpox that is a plaque-cloned isolate derived from the FP-I vaccine strain of fowlpoxvirus, which is licensed for vaccination of one-day old chicks.
  • ALVAC is an attenuated canarypox virus-based vector that was a plaque- cloned derivative of the canarypox vaccine, Kanapox (Taraglia et al., AIDS Res. Hum. Retroviruses 8:1445-47 (1992)).
  • Kanapox a plaque- cloned derivative of the canarypox vaccine
  • ALVAC has some general properties which are similar to the Kanapox.
  • ALVAC-based recombinant viruses expressing extrinsic immunogens have also been demonstrated to be efficacious as vaccine vectors.
  • This avipox vector is restricted to avian species for productive replication. In human cell cultures, canarypox virus replication is aborted early in the viral replication cycle prior to viral DNA synthesis.
  • NYVAC, ALVAC ad TROVAC have also been recognized as unique among poxviruses in the National Institutes of Health (U.S. Public Health Service), Recombinant DNA Advisory Committee, which issues guidelines for the physical containment of genetic material such as viruses and vectors. This Committee granted a reduction in physical containment level for NYVAC, ALVAC and TROVAC from BSL2 to BSLl.
  • MVA Modified Vaccinia virus Ankara
  • MVA Modified Vaccinia virus Ankara
  • MVA retains it original immunogenicity and its variola-protective effect and longer has any virulence and contagiousness for animals and humans.
  • NYVAC and ALVAC viruses expression of recombinant polypeptides by MVA occurs during an abortive infection of human cells, thus providing a safe, yet effective, delivery system for antigenic S polypeptides.
  • Vaccinia virus vectors including the highly attenuated modified vaccinia virus Ankara (MVA) strain, have been used to express and characterize glycoproteins of numerous pathogens and some of those are being evaluated as candidate prophylactic and therapeutic vaccines (Moss, B. (1996) Proc. Natl. Acad. Sci. USA 93, 11341-11348). MVA accumulated multiple deletions and other mutations during more than 500 passages in chicken embryo fibroblasts (CEF) resulting in a severe host range restriction in most mammalian cells.
  • CEF chicken embryo fibroblasts
  • MVA expresses viral and recombinant proteins in non-permissive as well as in permissive cells. MVA is highly attenuated due to its replication defect in mammalian cells and no adverse effects were reported even when high doses of MVA were given to immune deficient non-human primates or severe combined immunodeficiency disease mice.
  • nucleic acids encoding antigenic SARS-CoV S polypeptides can be inserted into viral genomes such as those of the poxviruses described herein, to generate a recombinant virus that can express the SARS-CoV s polypeptide after administration to an animal (e.g. a human).
  • the recombinant virus is introduced into an animal (e.g. a human) by standard methods for administering immunogenic compositions or for vaccination with live vaccines.
  • a composition containing live recombinant virus can be administered at, for example, about 10 4 - 10 8 organisms/dose, or 10 6 to 10 10 pfu per dose.
  • NYVAC, ALVAC or MVA recombinant poxviruses can be administered by an intramuscular route using a dosage of about 10 7 to 10 9 pfu per inoculation, for a patient of about 100 to 200 pounds.
  • compositions containing such recombinant viruses can be delivered in a physiologically compatible solution such and phosphate buffered saline in a volume of about 0.05 to about 1.5 ml. Such dosages can be administered once or several times in a continuous or intermittent fashion, using a regimen that is readily determined by one of ordinary skill in the field.
  • an immunological or vaccine composition of the invention may contain DNA encoding one or more of the SARS S polypeptides described herein, such that the polypeptide is generated in situ.
  • the DNA may thus be "naked,” as described, for example, in Ulmer et al., Science 259:1745-1749 (1993), and reviewed by Cohen, Science 259:1691-1692 (1993).
  • the DNA may be present within any of a variety of delivery systems known to those of ordinary skill in the art, including nucleic acid expression systems, bacteria and viral expression systems. Appropriate nucleic acid expression systems contain the necessary DNA sequences for expression in the patient (such as a suitable promoter and terminating signal).
  • Bacterial delivery systems involve the administration of a bacterium (such as Bacillus- Calmette-Guerrin) that expresses an immunogenic portion of the polypeptide on its cell surface.
  • any of the conventional vectors used for expression in eukaryotic cells may be used directly introducing DNA into tissue.
  • Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, for example, SV40 vectors, pMSG, PAV009/A+, pMAMneo-5, baculovirus pDSVE, and other vectors that permit expression of proteins under the direction of promoters such as the SV40 early promoter, SV40 later promoter, metallothionein promoter, human cytomegalovirus promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedron promoter, or other promoters effective for expression in eukaryotic cells.
  • promoters such as the SV40 early promoter, SV40 later promoter, metallothionein promoter, human cytomegalovirus promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, poly
  • Therapeutic quantities of plasmid DNA can be produced, for example, by expansion in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and the cells are grown to saturation in shaker flasks or bioreactors using procedures available in the art. Plasmid DNA can be purified using available bioseparation techniques such as solid phase anion-exchange resins. If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
  • Preferred plasmid DNA can be prepared for administration using a variety of formulations.
  • the simplest is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS).
  • PBS sterile phosphate-buffer saline
  • This formulation known as "naked DNA”
  • IM intramuscular
  • ID intradermal
  • Cationic lipids can be used in the formulation, for example, as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S.
  • PINC protective, interactive, non-condensing compounds
  • the invention provides a preparation of antibodies that can bind to a SARS-CoV, or a SARS CoV S polypeptide, derivative or variant thereof.
  • the antibody can be directed against an SARS-CoV S polypeptide comprising any one of SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, or a combination thereof.
  • the antibody preparations are useful for treating and preventing SARS-CoV infection in an animal.
  • the invention provides an antibody that binds to a polypeptide or peptide fragment of the invention, or a variant thereof.
  • the antibody is an antigen-binding antibody fragment.
  • the antibody is a polyclonal antibody.
  • the antibody is a single-chain antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is a humanized antibody.
  • the antibody may be coupled to a detectable tag.
  • the detectable tag can be a radiolabel.
  • the detectable tag is an affinity tag.
  • the detectable tag is an enzyme.
  • the detectable tag is a fluorescent protein.
  • the detectable tag is a fluorescent molecule.
  • the antibody may also be coupled to a toxin.
  • immunoglobulins All antibody molecules belong to a family of plasma proteins called immunoglobulins, whose basic building block, the immunoglobulin fold or domain, is used in various forms in many molecules of the immune system and other biological recognition systems.
  • a typical immunoglobulin has four polypeptide chains, containing an antigen binding region known as a variable region and a non- varying region known as the constant region.
  • Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.
  • VH variable domain
  • VL variable domain at one end
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al., J. MoI. Biol. 186, 651-66, 1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4596 (1985).
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG- 1, IgG-2, IgG-3 and IgG-4; IgA-I and IgA-2.
  • the heavy chains constant domains that correspond to the different classes of immunoglobulins are called alpha ( ⁇ ), delta ( ⁇ ), epsilon ( ⁇ ), gamma ( ⁇ ) and mu ( ⁇ ), respectively.
  • the light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda ( ⁇ ), based on the amino sequences of their constant domain.
  • K kappa
  • ⁇ lambda
  • variable in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies.
  • the variable domains are for binding and determine the specificity of each particular antibody for its particular antigen.
  • variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • variable domains The more highly conserved portions of variable domains are called the framework (FR).
  • the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies.
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector function, such as participation of the antibody in antibody-dependent cellular toxicity.
  • an antibody that is contemplated for use in the present invention thus can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody that includes the variable domain complementarity determining regions (CDR), and the like forms, all of which fall under the broad term "antibody,” as used herein.
  • the present invention contemplates the use of any specificity of an antibody, polyclonal or monoclonal, and is not limited to antibodies that recognize and irnrnunoreact with a specific epitope. In some embodiments, however, the antibodies of the invention may react with selected epitopes within various domains of the SARS-CoV S protein.
  • antibody fragment refers to a portion of a full-length antibody, generally the antigen binding or variable region.
  • antibody fragments include Fab, Fab 1 , F(ab') 2 and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments, which are capable of cross- linking antigen, and a residual other fragment (which is termed pFc').
  • Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • “functional fragment” with respect to antibodies refers to Fv, F(ab) and F(ab') 2 fragments.
  • Antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
  • Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule.
  • a Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.
  • Fab' is the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule. Fab 1 fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • (Fab') 2 is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction.
  • F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds.
  • Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody.
  • a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single chain antibody (“SCA”), defined as a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
  • SCA Single chain antibody
  • Single chain antibodies are also referred to as "single-chain Fv” or "sFv” antibody fragments.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to a small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy chain variable domain
  • VL light chain variable domain
  • VH-VL polypeptide chain
  • polyclonal antibodies The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green, et al., Production of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1 (1992), which are hereby incorporated by reference. The preparation of monoclonal antibodies likewise is conventional.
  • the monoclonal antibodies for use with the present invention may also be isolated from antibody libraries using the techniques described in Clackson et al. Nature 352: 624-628 (1991), as well as in Marks et al., J. MoI Biol. 222: 581-597 (1991).
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan, et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al., Purification of Immunoglobulin G (IgG), in: Methods in Molecular Biology, Vol. 10, pages 79- 104 (Humana Press (1992).
  • Selected Lymphocyte Antibody Method Another method for generating antibodies involves a Selected Lymphocyte Antibody Method (SLAM).
  • SLAM Selected Lymphocyte Antibody Method
  • the methodology principally involves the growth of antibody forming cells, the physical selection of specifically selected antibody forming cells, the isolation of the genes encoding the antibody and the subsequent cloning and expression of those genes.
  • an animal is immunized with a source of specific antigen.
  • the animal can be a rabbit, mouse, rat, or any other convenient animal.
  • This immunization may consist of purified protein, in either native or recombinant form, peptides, DNA encoding the protein of interest or cells expressing the protein of interest.
  • blood, spleen or other tissues are harvested from the animal. Lymphocytes are isolated from the blood and cultured under specific conditions to generate antibody-forming cells, with antibody being secreted into the culture medium.
  • Another method involves humanizing a monoclonal antibody by recombinant means to generate antibodies containing human specific and recognizable sequences. See, for review, Holmes, et al., J. Immunol., 158:2192- 2201 (1997) and Vaswani, et al., Annals Allergy, Asthma & Immunol., 81:105- 115 (1998).
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations that typically include different antibodies directed against different determinants
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the antibody is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567); Morrison et al. Proc. Natl. Acad Sci. 81, 6851-6855 (1984).
  • chimeric antibodies immunoglobulins in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived
  • Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5 S Fab monovalent fragments.
  • a thiol reducing agent optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages
  • an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.
  • Fv fragments comprise an association of V H and V L chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde.
  • the Fv fragments comprise V H and V L chains connected by a peptide linker.
  • sFv single-chain antigen binding proteins
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick, et al., Methods: a Companion to Methods in Enzymologv, Vol. 2, page 106 (1991).
  • the invention further contemplates human and humanized forms of non- human (e.g. murine) antibodies.
  • humanized antibodies can be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
  • CDR complementary determining region
  • humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • humanized antibodies can comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the Fv regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • mutant antibody refers to an amino acid sequence variant of an antibody.
  • one or more of the amino acid residues in the mutant antibody is different from what is present in the reference antibody.
  • Such mutant antibodies necessarily have less than 100% sequence identity or similarity with the reference amino acid sequence.
  • mutant antibodies have at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody.
  • mutant antibodies have at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody.
  • the antibodies of the invention are isolated antibodies.
  • An isolated antibody is one that has been identified and separated and/or recovered from the environment in which it was produced.
  • the isolated antibodies of the invention are substantially free of at least some contaminant components of the environment in which they were produced. Contaminant components of its production environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include cells, enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • isolated antibody also includes antibodies within recombinant cells because at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
  • the antibodies of the invention can be purified by any available procedure.
  • the antibodies can be affinity purified by binding an antibody preparation to a solid support to which the antigen used to raise the antibodies is bound. After washing off contaminants, the antibody can be eluted by known procedures.
  • Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (see for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference) .
  • the antibody will be purified as measurable by at least three different methods: 1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight; 2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.
  • the invention provides a method to immunize an animal against severe acute respiratory syndrome.
  • the method involves administering to an animal a therapeutically effective amount of a SARS-CoV S polypeptide having, for example, SEQ ID NO: 1, 3, 4, 6, 7 or a fragment of SEQ ID NO: 1, or a conservative variant thereof.
  • the method involves administering to an animal a therapeutically effective amount of an antibody that binds to a SARS-CoV S polypeptide, for example, a polypeptide having SEQ ID NO: 1, 3, 4, 6, 7 or a fragment thereof, or a conservative variant thereof.
  • the method involves administering to an animal an effective amount of a live recombinant virus that encodes and can express a SARS-CoV S polypeptide, for example, one having SEQ ID NO:1, 3, 4, 6, 7 or a fragment thereof, or a variant thereof.
  • the animal may be a mammal, such as a human. Methods to administer vaccines and immune compositions have been described herein and are available in the art. An animal may also be treated for infection by SARS-CoV through passive immunization according to the invention.
  • antibodies that bind to an amino acid sequence such as SEQ ID NO: 1, 3, 4, 6, 7 or a fragment of SEQ ID NO: 1, or a conservative variant thereof may be administered to an animal, such as a human, that is infected with SARS-CoV.
  • Such administration may be suitable in a variety of situations, for example, where a patient is immunocompromised and is unable to mount an effective immune response against SARS-CoV, or to a vaccine or immune composition.
  • the invention provides a method to diagnose severe acute respiratory syndrome in an animal that involves contacting a biological sample obtained from the animal, such as tissue samples, blood, mucus, or saliva, with an antibody that binds to an amino acid sequence as set forth in SEQ ID NO: 1, 3, 4, 6, 7 or a fragment of SEQ ID NO: 1, and determining if the antibody binds to the biological sample.
  • Diagnostic assays that utilize antibodies to detect the presence of an antigen in a biological sample are available in the art. Briefly, an antibody of the invention may be immobilized on a surface. A biological sample can then be contacted with the immobilized antibody such that an antigen contained in the sample is bound by the antibody to form an antibody-antigen complex. The sample may then be optionally washed to remove unbound materials.
  • a second antibody of the invention that is coupled to a detectable tag such as an enzyme, fluorophore or radiolabel
  • a detectable tag such as an enzyme, fluorophore or radiolabel
  • the detectable tag can then be detected to determine if an antigen was present in the biological sample.
  • a biological sample can be immobilized on a surface.
  • An antibody of the invention that is coupled to a detectable tag is then contacted with the immobilized biological sample and any unbound material is washed away. The presence of the detectable tag is then detected to determine whether the biological sample contained an antigen.
  • assays are available in the art and include, enzyme-linked immunosorbant assays, sandwich assays, radioimmuno assays, and the like.
  • Nucleic acid based methods may also be used to diagnose severe acute respiratory syndrome.
  • polymerase chain reaction PCR
  • a biological sample such as a tissue sample, blood, mucus, or saliva, is obtained from an animal.
  • the nucleic acids within the sample are then extracted using common methods, such as organic extraction.
  • the extracted nucleic acids are then mixed with forward and reverse primers that anneal to nucleic acids that encode SARS proteins, polymerase, nucleotides, and typically a buffer that includes components that allow the polymerase to extend the forward and reverse primers using the SARS nucleic acid as a template.
  • nucleic acid hybridization techniques such as Northern and Southern blotting, may also be used to detect the presence of SARS nucleic acids in a biological sample.
  • a SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, recombinant virus encoding a S polypeptide or an anti-S polypeptide antibody can be formulated as a pharmaceutical composition.
  • a pharmaceutical composition of the invention includes a SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, recombinant virus encoding a S polypeptide or an anti-S polypeptide antibody in combination with a pharmaceutically acceptable carrier.
  • the compositions of the invention can be immune (or immunogenic) compositions or vaccines.
  • compositions can contain any S polypeptide or fragment thereof, for example, an S polypeptide having any one of SEQ ID NO:1, 3, 4, 6, 7 or a combination thereof.
  • the invention also provides pharmaceutical compositions containing an antibody that binds to an S polypeptide, for example, any of SEQ ID NO: 1, 3, 4, 6, 7 or a combination thereof, and a pharmaceutically acceptable carrier.
  • the antibody binds to a peptide having SEQ ID NO:4 or 6.
  • Antibodies that bind to the polypeptide including amino acids 14 to 762 of the SARS coronavirus (SARS-CoV) spike protein (SEQ ID NO:6) are highly effective, and can inhibit viral replication in vivo.
  • the compositions can include a live recombinant virus that can express a SARS-CoV S polypeptide.
  • a substantially full-length Spike (S) polypeptide of SARS-CoV having SEQ ID NO:1 which was encoded within and expressed by a recombinant MVA, induces formation of neutralizing antibodies.
  • the invention provides compositions of live recombinant viruses that encode and express SARS-CoV antigens.
  • the compositions may contain an adjuvant.
  • adjuvants examples include, for example, a combination of monophosphoryl lipid A (e.g. 3-de-O- acylated monophosphoryl lipid A (3D-MPL)), and a saponin derivative such as combination of QS21 and 3D-MPL as described in WO 94/00153.
  • monophosphoryl lipid A can be combined with an aluminum salt to form an adjuvant for use in the compositions of the invention.
  • MPL adjuvants are available from Corixa Corporation (Seattle, Wash.; see U.S. Pat. Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated
  • oligonucleotides are available and are described, for example, in WO 96/02555 and WO 99/33488.
  • Immunostimulatory DNA sequences are also described, for example, by Sato et al, Science 273:352, 1996.
  • the adjuvant that can be used is a saponin, preferably QS21 (Aquila Biopharmaceuticals Inc., Framingham, Mass.), which may be used alone or in combination with other adjuvants.
  • a combination of a monophosphoryl lipid A and saponin derivative can be employed, as described above.
  • a less reactogenic composition is used where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • the excellent results were obtained with a combination of QS21 and an S polypeptide, which provided the highest antibody response as well as complete protection of the upper and lower respiratory tract.
  • Other formulations of the invention comprise an oil-in- water emulsion and tocopherol.
  • a particularly potent adjuvant formulation involving QS21, 3D- MPL and tocopherol in an oil-in- water emulsion is described in WO 95/17210.
  • Additional adjuvants that may be employed include Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from Montanide ISA 720 (Seppic, France), SAF (Chiron, Calif, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of
  • an immune composition or vaccine may be administered by any conventional route used in the field of vaccines.
  • an immune composition or vaccine can be administered orally or by intravenous infusion, or injected subcutaneously, intramuscularly, intraperitoneally, intrarectally, intravaginally, intranasally, intragastrically, intratracheally, or intrapulmonarily.
  • the choice of the administration route depends on a number of parameters such as the nature of the active principle; the identity of the polypeptide, peptide fragment, immunopeptide, recombinant virus, DNA vaccine; or the adjuvant that is combined with the aforementioned molecules.
  • an immune composition may take place in a single dose or in a dose repeated once or several times over a certain period.
  • the appropriate dosage varies according to various parameters. Such parameters include the individual treated (adult or child), the immune composition or antigen itself, the mode and frequency of administration, the presence or absence of adjuvant and, if present, the type of adjuvant and the desired effect (e.g. protection or treatment), as will be determined by persons skilled in the art.
  • compositions of the invention maybe prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, and liposomes and other slow-release formulations, such as shaped polymeric gels.
  • Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, nonaqueous vehicles (which may include edible oils), or preservatives.
  • An oral dosage form may be formulated such that the SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, live recombinant virus or anti-S polypeptide antibody is released into the intestine after passing through the stomach.
  • Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non- aqueous vehicles (which may include edible oils), or preservatives.
  • a SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, or anti-S polypeptide antibody can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, pre-filled syringes, small volume infusion containers or multi-dose containers with an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, or anti-S polypeptide antibody may be in powder form, obtained by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile saline, before use.
  • a suitable vehicle e.g., sterile saline
  • An antibody can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative.
  • the pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles.
  • the antibody compositions may also contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions suitable for rectal administration can be prepared as unit dose suppositories.
  • Suitable carriers include saline solution and other materials commonly used in the art.
  • a SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, or anti-S polypeptide antibody can be conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray.
  • Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • a SARS- CoV S polypeptide, S polypeptide derivative, S polypeptide variant, or anti-S polypeptide antibody may take the form of a dry powder composition, for example, a powder mix of a modulator and a suitable powder base such as lactose or starch.
  • the powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, e.g., gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
  • a SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, or anti-S polypeptide antibody may be administered via a liquid spray, such as via a plastic bottle atomizer.
  • compositions of the invention may also contain other ingredients such as flavorings, colorings, anti-microbial agents, anti- inflammatory agents or preservatives. It will be appreciated that the amount of a SARS-CoV S polypeptide, S polypeptide derivative, S polypeptide variant, live recombinant virus or anti-S polypeptide antibody required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the severity of the infection being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage.
  • Kits The invention provides a kit which contains packaging material and a
  • kits may also contain a syringe to allow for injection of the polypeptide contained within the kit into an animal, such as a human.
  • the invention provides a kit that may contain packaging material, and an antibody that binds to a SARS-CoV S polypeptide, for example, an S polypeptide having SEQ ID NO: 1, 3, 4, 6, 7 or a fragment of SEQ ID NO: 1, or a conservative variant thereof that is formulated for administration to an animal, such as a human.
  • a kit may optionally contain a syringe to allow for injection of the antibody contained within the kit into an animal, such as a human.
  • the invention also provides a kit which contains packaging material and DNA vaccine having a DNA molecule or expression vector encoding a polypeptide with an amino acid sequence as set forth in SEQ ID NO: 1, 3, 4, 6, 7, or a fragment of SEQ ID NO: 1, or a conservative variant thereof.
  • the kit may also contain a device for administering the DNA vaccine (e.g. a syringe or gene gun) to allow for administration of the vaccine contained within the kit into an animal, such as a human.
  • the invention also provides a kit which contains packaging material and immunogenic composition or a vaccine composition that includes a polypeptide with an amino acid sequence as set forth in SEQ ID NO: 1, 3, 4, 6, 7, or a fragment of SEQ ID NO: 1, or a conservative variant thereof.
  • the kit may also contain a device for administering the composition or vaccine (e.g. a syringe) to allow for administration of the vaccine contained within the kit into an animal, such as a human.
  • the invention also provides a kit for detecting SARS-CoV infection, which contains packaging material and a polypeptide with an amino acid sequence as set forth in SEQ ID NO: 1, 3, 4, 6, 7 or a fragment of SEQ ID NO: 1, or a conservative variant thereof.
  • the polypeptide(s) can be immobilized onto a solid support.
  • Such a kit may be used for detection of antibodies directed against the SARS-CoV in the serum of infected animals or humans.
  • the kit can also contain a means for detecting binding of such antibodies to the S polypeptide(s).
  • the invention also provides a kit for detecting SARS-CoV infection, which contains packaging material and an antibody that can bind a SARS-CoV S polypeptide as forth in SEQ ID NO: 1, 3, 4, 6, 7 or a fragment of SEQ ID NO: 1, or a conservative variant thereof.
  • the antibodies can be immobilized onto a solid support.
  • Such a kit may be used for detection of SARS viruses or SARS S polypeptides in the serum of infected animals or humans.
  • the kit can also contain a means for detecting binding of such S polypeptide(s) by the antibodies.
  • EXAMPLE 1 Recombinant MVA Encoding SARS-CoV Spike Polypeptides Effectively Immunizes Animals against SARS-CoV Infection
  • This Example shows that a full-length Spike (S) polypeptide of SARS- CoV, expressed by MVA, induces formation of neutralizing antibodies.
  • S Spike
  • Such an immunogenic composition of this recombinant MVA-SARS-CoV S poxvirus protectively immunizes mice against a subsequent infection with SARS-CoV.
  • CEF Primary chicken embryo fibroblast cells
  • MEM minimum essential medium
  • FBS fetal bovine serum
  • the entire S gene was PCR amplified with or without an influenza virus hemagglutinin (HA) epitope tag and inserted into theXm ⁇ l site of the pLW44 transfer vector (provided by L. Wyatt) bringing it under the control of the early/late modified vaccinia virus H5 early late promoter (Wyatt et al. (1996) Vaccine 14, 1451-1458) and adjacent to the gene encoding enhanced green fluorescent protein (GFP) regulated by the vaccinia virus PI l late promoter.
  • HA hemagglutinin
  • MVAs were made by transfecting transfer plasmids into CEF that were infected with 0.05 plaque forming units (PFU) of MVA per cell. Florescent plaques were cloned by six successive rounds of plaque isolation, propagated in CEF, and purified by sedimentation through a sucrose cushion as described by Earl et al. (1998) in Current Protocols in Molecular Biology, eds. Ausubel et al. (Greene Publishing Associates & Wiley Interscience, New York), Vol. 2, pp. 16.17.1- 16.17.19. Titers of MVA/S and MVA/S-HA were determined by staining plaques with anti-vaccinia virus rabbit and anti-HA mouse antibodies, respectively.
  • PFU plaque forming units
  • Proteins were transferred to a nitrocellulose membrane, blocked with 5% skimmed milk in phosphate buffered saline (PBS), and incubated for 1 h at room temperature with anti-HA mouse mAb (Covance) or anti-S ARS-Co V S rabbit polyclonal antibody (IMG-541, Imgenex) diluted 1:1000 or 1:500 in blocking buffer, respectively.
  • the membrane was washed in PBS containing Tween-20 (0.1%) and incubated for 1 h with horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibody (Calbiochem) diluted 1 :2000.
  • the membrane was washed and proteins were visualized with the Super Signal chemiluminescence substrate (Pierce).
  • HeLa cells were mock infected or infected with 5 PFU per cell of MVA or MVA/S-HA and 18 h later were incubated for 30 min in Dulbecco's Modified Eagle's medium lacking methionine and cysteine, labeled with 100 ⁇ Ci of [ 35 S]methionine and [ 35 S] cysteine per ml of medium for 10 min, washed and chased with medium supplemented with 2 mM methionine and 2 mM cysteine. At each time, cells were harvested, lysed in ice-cold RIPA buffer, and clarified lysates were incubated with 20 ⁇ l of anti-HA affinity matrix overnight at 4°C as above. Washed agarose beads were treated with endo H and the samples were resolved by SDS-PAGE and detected by autoradiography.
  • CEF or HeLa cells on coverslips were infected with 5 PFU per cell of MVA, MVA/S or MVA/S-HA, incubated for 18 h, and either left unfixed and unpermeabilized or fixed with cold 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 2.5% digitonin in PBS for 5 min on ice.
  • the coverslips were washed and incubated with anti- SARS mouse serum kindly provided by Larry Anderson (CDC, Atlanta) or anti- HA mouse mAb for 1 h at room temperature, washed again, and incubated with Alexa 594-conjugated-anti-mouse IgG (Molecular Probes) diluted in PBS containing 10% FBS for 30 min at room temperature. Coverslips were mounted in 20% glycerol and examined with an inverted confocal microscope.
  • Enzyme-linked Immunosorbent Assay ELISA.
  • a 96-well plate was coated overnight at 4 0 C with 50 ng per well of soluble recombinant protein containing the S 1 domain of SARS-CoV S made in insect cells, blocked with 5% skimmed milk in PBS containing 0.2% Tween-20 for 1 h at 37°C and incubated with two-fold dilutions of serum from unimmunized or immunized mice for 1 h at 37°C.
  • the plate was incubated for 1 h with horse radish peroxidase-conjugated secondary anti-mouse antibody (Roche) diluted in blocking buffer, washed again and incubated with substrate solution (3,3'-5,5'- tetramethylbenzidine, Roche).
  • substrate solution (3,3'-5,5'- tetramethylbenzidine, Roche). The difference in absorbance at 370 and 492 nm was determined, readings from wells lacking antigen were subtracted and end point titers were calculated when the absorbance difference was ⁇ 0.1.
  • Neutralization Assay Neutralizing antibody was determined by the inhibition of cytopathic effects mediated by SARS-CoV on Vero cell monolayers as described by Subbarao et al. (2004) J. Virol. 78, 3572-3577. The dilution of serum that completely prevented cytopathic effect in 50% of the wells was calculated (Reed, L. J. & Muench, H. (1938) Am. J. Hyg. 27, 493-497).
  • mice Animal Challenge Experiments. Groups of 8 BALB/c mice were inoculated intranasally (IN) or intramuscularly (IM) with 10 7 PFU of MVA or MVA/S at 0 and 4 weeks. Four weeks after the second immunization, animals were challenged IN with 10 4 tissue culture infectious doses 50 (TCID 50 ) of SARS- CoV as described (14). Two days later the lungs and nasal turbinates of 4 animals in each group were removed and the SARS-CoV titers were determined as described by Subbarao et al. (2004) J. Virol. 78, 3572-3577.
  • TCID 50 tissue culture infectious doses 50
  • mice received MVA/S or MVA IM at 0 and 4 weeks. Three weeks after the last immunization, sera were collected and pooled. Undiluted or diluted MVA/S or MVA serum in a total volume of 0.4 ml was injected intraperitoneally (IP) to 2 to 4 na ⁇ ve mice. Mice were bled the following day to determine their levels of SARS-CoV specific neutralizing antibody and then each was challenged with 10 5 TCID 50 of SARS-CoV and analyzed as above.
  • IP intraperitoneally
  • a cDNA clone containing the entire open reading frame encoding SARS-CoV S was modified by introducing silent mutations that eliminated two poxvirus transcription termination signals and was placed under the control of an early/late vaccinia virus promoter (mH5) and inserted by homologous recombination into the site of an existing deletion (del HI) within the MVA genome to produce MVA/S (FIG. IA).
  • MVA/S- HA was also constructed with a 9-amino acid HA epitope tag coding sequence at the end of the S open reading frame.
  • the gene encoding GFP regulated by a vaccinia virus promoter was co-inserted into the MVA genome in order to facilitate the screening and isolation of recombinant viruses by repeated plaque purifications. Both viruses replicated well in CEF and the SARS-CoV S insert was genetically stable as assayed by plaque immunostaining with S- specif ⁇ c antibodies.
  • some S was trapped near the top of the gel, presumably due to aggregates or oligomers that were not dissociated by treatment with SDS and reducing agent at 100 0 C.
  • the SARS-CoV S has 23 potential N-linked glycosylation sites (Rota et al. (2003) Science 300, 1394-1399), the presence of which could contribute to the mass of the protein determined by SDS-PAGE.
  • S expressed in HeLa cells was treated with PNGase F, which hydrolyzes all types of N-glycan chains.
  • PNGase F treatment converted the 200-kDa doublet to a single sharp band of approximately 160 kDa (FIG. 2A), which was still greater than the 135 kDa estimated from the gene sequence.
  • FIG. 2A shows that S contains additional post-translational modifications.
  • endo H which digests the N- linked high-mannose carbohydrate side chains of glycoproteins that are synthesized in the endoplasmic reticulum (ER), but not after conversion to a more complex form in the medial Golgi apparatus. Only a subpopulation of S was digested, since both the original size protein and a faster migrating one were detected (FIG. 2A). The latter had a slightly higher mass than the PNGase F- treated protein, consistent with N-acetylglucosamine residues remaining after hydrolysis by endo H.
  • mice inoculated IN with SARS-CoV exhibit no overt signs of disease but have elevated virus titers in the respiratory tract that peak within 2 days and are cleared by 7 days (Subbarao et al., (2004) J. Virol. 78, 3572-3577).
  • the present study employed three control and two experimental groups. The controls were mice that were uninoculated or that had received the MVA vector IM or IN. When these mice were challenged with 10 4 TCID 50 of SARS-CoV, approximately 10 5 TCID50 of SARS-CoV per g of lung was recovered on day 2 (FIG. 5).
  • MVA can induce both humoral and cell mediated immune responses.
  • sera were pooled that were obtained from mice that had been immunized IM with 10 7 PFU of MVA/S or MVA on day 0 and 28 and bled three weeks later.
  • the ELISA titer to S was about 1 :25,000 and the mean neutralizing titer was 1 :284.
  • Undiluted or diluted serum (0.4 ml) was administered IP to na ⁇ ve mice to evaluate the protective role of antibody.
  • hyperimmune SARS-CoV serum was administered to two mice (Subbarao et al., (2004) J. Virol.
  • mice received an intranasal challenge of 10 5 TCID 50 of SARS-CoV, and two days later, their nasal turbinates and lungs were removed to measure the virus titers.
  • administration of undiluted MVAJS serum reduced the lung titers by 10 5 1 compared to recipients of MVA control serum.
  • SARS-CoV S polypeptide can be expressed in a
  • the secretory pathway of a cell has an important quality control function and the trafficking of a protein from the ER to the plasma membrane is a sign of proper folding.
  • the N-linked oligosaccharide pathway is frequently used for tracking protein movement. Addition of N-linked oligosaccharides occurs in the
  • the S open reading frame of SARS-CoV was expressed by recombinant MVA as a protein of approximately 200 kDa, which was reduced to 160 kDa by a glycosidase specific for N-linked carbohydrates. Trafficking of S to the medial Golgi apparatus was indicated by acquisition of endo H resistance by a subpopulation of molecules within 40 min after pulse labeling.
  • the staining of the surface of unpermeabilized cells infected with MVA/S by S-specific antibody provided direct evidence for insertion into the plasma membrane. Furthermore, the inability of antibody to a C-terminal epitope tag to stain cells unless they were permeabilized indicated that S has a type 1 topology in the membrane.
  • the MVA/S construct was then tested to determine whether it would elicit neutralizing antibodies.
  • IM intramuscular
  • IN intranasal
  • the ability of intramuscular (IM) or intranasal (IN) inoculation of a recombinant MVA to prevent upper and lower respiratory infections has previously been observed using a rodent model of parainfluenza virus 3 (Wyatt, L. S., Shors, S. T., Murphy, B. R. & Moss, B. (1996) Vaccine 14, 1451-1458).
  • Mice immunized with MVA/S by IN or IM routes developed antibodies that bound to the S 1 domain of S and neutralized SARS-CoV in vitro.
  • mice immunized IM or IN exhibited little or no replication of SARS CoV in the upper and lower respiratory tracts following an IN inoculation.
  • Control mice vaccinated with the MVA vector by IN or IM routes were unprotected, indicating that the effect was specific for the expressed S protein and was not due enhanced non-specific immunity.
  • mice that were immunized with MVAJS prior to challenge with SARS-CoV were immunized with MVAJS prior to challenge with SARS-CoV, as has been found after immunization with a vaccinia virus vector expressing S from feline infectious peritonitis virus and challenge with the corresponding virus (Vennema et al. (1990) J. Virol. 64, 1407-1409).
  • the latter effect is thought to be due to S antibody-dependent enhanced infection of macrophages. See Corapi et al. (1992) J. Virol. 66, 6695-6705; Olsen et al. (1992) J. Virol. 66, 956-965.
  • the present study provides encouraging results for the development of SARS-CoV vaccines based on the highly attenuated MVA vector expressing S.
  • This Example illustrates expression of a secreted, glycosylated polypeptide including amino acids 14 to 762 of the SARS coronavirus (SARS- CoV) spike protein and a polyhistidine tag in recombinant baculovirus-infected insect cells.
  • SARS-CoV (Urbani strain) S protein (GenBank accession no. AY278741) with 6 histidine residues appended to the C-terminus was inserted into the BamHI and EcoRI sites of the baculovirus transfer vector pMelBacB (Invitrogen) so that the honeybee melittin signal peptide was in frame with the S protein.
  • the plasmid and linearized Autographa californica multiple nuclear polyhedrosis virus DNA were transfected into SF9 and a recombinant baculovirus was clonally purified following the Bac-N-Blue system protocol (Invitrogen).
  • the recombinant baculoviruses were constructed to express the substantially full length SARS-CoV spike protein, or N- or C- terminal fragments of the SARS-CoV spike protein (nS or cS).
  • native signal sequences as well as transmembrane and cytoplasmic regions ( ⁇ TM+CT) were deleted.
  • ⁇ TM+CT full length S( ⁇ TM+CT) polypeptide
  • a cDNA encoding amino acids 14 to 1195 of the SARS-CoV (Urbani Strain) S protein was used (see GenBank accession no. AY278741, starting at nucleotide 21531) with a sequence for 6 histidine residues attached to its 3 'end.
  • the sequences of the S( ⁇ TM+CT) polypeptide 14-1195AA, SEQ ID NO:4) and cDNA (SEQ ID NO:5) are shown in FIG. 6 and are provided hereinabove.
  • This S( ⁇ TM+CT) cDNA was cloned into the BamEI and EcoRl sites of the baculovirus transfer vector pMelBacB (Invitrogen) in frame with the honeybee melittin signal peptide under a strong polyhedrin promoter. N- (nS) and C- (cS) terminal fragments encoding gene sequences were cloned in a similar way.
  • a spike polypeptide encoding the N-terminal 14-762 amino acids was selected on the basis of hydrophilicity and secondary structure predictions using Kyte and Dolittle and Chou Fasman algorithms (McVactor 7.2) and also because it encompasses the receptor binding region as well as the region corresponding to Sl of other coronaviruses.
  • the sequence of this N-terminal 14-762 amino acid spike polypeptide is as follows (SEQ ID NO:6). 14 SDLDRCT TFDDVQAPNY TQHTSSMRGV
  • the C-terminal fragment employed consisted of the remaining 763-1195 amino acid residues of the spike protein.
  • the sequence for this C-terminal 763- 1195 amino acid spike polypeptide is as follows (SEQ ID NO:7).
  • Recombinant plasmids encoding the spike polypeptides and the linearized Autographa californica multiple nuclear polyhedrosis virus DNA were transfected into Sf9 insect cells.
  • Recombinant baculoviruses were purified following the Bac-N-Blue system protocol (Invitrogen). The expression was checked by western blotting that showed -110 kDa band of nS, -200 kDa band of S( ⁇ TM+CT) and -50 kDa band of cS.
  • nS has been purified further on large scale with a yield of 1 Omg/1 of culture supernatant.
  • nS protein Expression and purification of recombinant nS protein.
  • High Five cells were infected with recombinant baculo virus at a multiplicity of infection of 10 for 120 h.
  • the culture supernatant was concentrated five fold with a Millipore Labscale transverse flow filter system and was clarified by centrifugation in a Sorvall H6000A rotor at 3000 rpm for 30 min at 4 °C.
  • the supernatant was dialyzed against phosphate pH 7.4 buffered saline (PBS) and then incubated with a 50% (wt/vol) slurry of nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) for 3-4 h at 4 0 C.
  • Ni-NTA nickel-nitrilotriacetic acid
  • the mixture was loaded into a column that was washed with 10 bed-volumes of wash buffer (50 mM phosphate pH 8 buffer/300 mM NaCl/10 mM imidazole/1 mM phenyl methyl sulfonyl fluoride), 10 bed- volumes of wash buffer containing 25 mM imidazole, 2 bed- volumes of wash buffer containing 40 mM imidazole, and 3 bed-volumes of wash buffer containing 200 mM imidazole.
  • the pooled 200 mM imidazole eluate containing nS was dialyzed against PBS and concentrated using a Millipore Amicon ultra filter.
  • Protein samples were analyzed on a 4-12% bis-Tris polyacrylamide gel (Invitrogen) and stained with GelCode Blue stain reagent (Pierce) and with Silver Stain Plus kit (BioRad). Where indicated, N-glycosidase F treatment was carried out as described in Bisht et al. (2004) Proc. Natl. Acad. Sci. USA 101, 6641-6646.
  • mice were intranasally challenged with 10 5 TCID 50 of SARS-CoV in 50 ⁇ l. Two days later, their lungs and nasal turbinates were removed and SARS-CoV titers were determined as described in Subbarao et al. (2004) J Virol. 78, 3572-3577. A non-parametric Mann- Whitney U test was used for statistical analysis.
  • baculovirus/insect cell system was used to express an N-terminal fragment of S (nS) as a secreted glycosylated protein that could be readily purified under native conditions.
  • the N-terminal 762 amino acids of the S protein was selected on the basis of hydrophilicity and secondary structure predictions using Kyte and Dolittle and Chou Fasman algorithms (Mc Vector 7.2) and because it includes the region corresponding to Sl of other coronaviruses.
  • a transfer vector was constructed in which the polyhedrin promoter regulates expression of an nS protein comprised of amino acids 14 to 762 of S preceded by the honeybee melittin signal peptide and followed by six histidines (FIG. 7A).
  • a baculovirus expressing nS was derived by recombination in insect cells.
  • the yield of secreted and affinity purified nS was approximately 10 mg/1 of culture supernatant, and a single major band of ⁇ 110 kDa was seen by SDS-polyacrylamide gel electrophoresis after staining with Coomassie Blue (FIG. 7B, lane 1) or silver nitrate (FIG. 7B, lane 2).
  • Coomassie Blue FIG. 7B, lane 1
  • FIG. 7B, lane 2 Upon western blotting, the same 110-kDa band was recognized by antibodies to the polyhistidine tag and SARS-CoV S protein (FIG. 7B, lanes 3 and 4).
  • QS21 adjuvant was injected subcutaneously into BALB/c mice on days 0, 28, and 56.
  • Control mice were immunized with adjuvant and a secreted form of the vaccinia virus membrane protein LlR that was also produced in the baculo virus system and purified by affinity chromatography (Fogg et al., (2004) J. Virol. 78, 10230-10237).
  • affinity chromatography Frogg et al., (2004) J. Virol. 78, 10230-10237.
  • sera from the mice were tested for antibodies that recognize S protein expressed on the surface of cells by recombinant modified vaccinia virus Ankara (MVA/S) (Bisht et al. (2004) Proc. Natl. Acad. Sci. USA 101, 6641-6646.
  • mice immunized with nS in QS21 or MPL + TDM adjuvant stained the surface of cells infected with MVA/S but did not detectably stain uninfected cells or cells infected with non-recombinant MVA (FIG. 8).
  • serum from control mice that were immunized with the vaccinia virus LlR protein stained cells infected with non-recombinant and MVA/S equally (not shown).
  • the relative binding activity of pooled serum from mice immunized with nS and QS21 or MPL + TDL adjuvant were analyzed using nS as the capture antigen. Antibody was detected after the primary inoculation of nS with QS21 and the reciprocal ELISA titer was boosted to 1 :409,600 after two more inoculations (FIG. 9A). With MPL + TDM adjuvant, the antibody response to nS was detected only after boosting but subsequently reached approximately 25% of the level achieved with QS21. The IgG2a/IgGl ratio is an indicator of ThI help.
  • mice immunized with QS21 and MPL + TDM were 0.25 and 0.03 respectively, suggesting a greater ThI response with the former adjuvant.
  • a determining effect of adjuvant on helper T cell responses has been noted (Cribbs et al. (2003) hit. Immunol. 15, 505-514; Santos et al. (2002) Vaccine 21, 30-43).
  • Virus was detected in the nasal turbinates of 4 of 7 test mice immunized with nS and the MPL + TDM adjuvant whereas the titers were uniformly below detection in the turbinates of mice immunized with nS and QS21.
  • the failure of the nS antibody response to be boosted after challenge (FIG. 9A) was also consistent with the absence of virus replication.
  • the protein vaccine described herein induced higher neutralizing antibody and complete protection against an intranasal SARS-CoV challenge than that achieved by inoculation of mice with live SARS-CoV (Subbarao et al. (2004) J. Virol. 78, 3572-3577), MVA expressing the full length S (Bisht et al., Proc. Natl. Acad. Sci. USA 101, 6641-6646 (2004)), or DNA expressing full length S or S lacking the transmembrane and cytoplasmic domains (Yang et al., Nature 428, 561-564 (2004)). The better protection achieved in this study is correlated with the higher antibody response.
  • nS with either QS21 or MPL + TDM was effective, the former adjuvant induced higher binding and neutralizing antibody and better protection of the upper respiratory tract.
  • Vaccination with QS21 also induced a more balanced helper T-cell response than MPL + TDM as indicated by the higher IgG2a/IgGl ratio.
  • Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J.
  • Drosten, C Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S.,
  • Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450-454. Lu, L., Manopo, L, Leung, B. P., Chng, H. H., Ling, A. E., Chee, L. L., Ooi, E. E., Chan, S. W., Kwang, J., 2004. Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus. J. Clin. Microbiol. 42, 1570-1576. Marra, M. A., Jones, S. J., Astell, C. R., Holt, R. A., Brooks-Wilson, A.,
  • a DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 428, 561-564. Zhou, T., Wang, H., Luo, D., Rowe, T., Wang, Z., Hogan, R. J., Qiu, S., Bunzel, R. J., Huang, G., Mishra, V., Voss, T. G., Kimberly, R., Luo, M., 2004.
  • a reference to "a host cell” includes a plurality (for example, a culture or population) of such host cells, and so forth.
  • a host cell includes a plurality (for example, a culture or population) of such host cells, and so forth.
  • the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein.
  • the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

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Abstract

L'invention concerne des polypeptides isolés du coronavirus SRAS, des anticorps anti-SARS, des poxviroses recombinées et des compositions qui sont utiles pour le traitement et l'inhibition d'une infection au coronavirus SRAS.
PCT/US2005/011510 2004-04-05 2005-04-05 Fragments solubles de la glycoproteine de spicule de cov-sras Ceased WO2006071250A2 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008155316A1 (fr) * 2007-06-19 2008-12-24 Glaxosmithkline Biologicals S.A. Compositions immunogènes associées à la protéine s du coronavirus associé au sras
WO2010063685A1 (fr) * 2008-12-02 2010-06-10 Glaxosmithkline Biologicals S.A. Vaccin
CN104292339A (zh) * 2013-07-18 2015-01-21 特菲(天津)生物医药科技有限公司 含sars病毒rbd抗原的重组蛋白及展示rbd蛋白的杆状病毒
CN111856027A (zh) * 2020-04-16 2020-10-30 中国科学院苏州纳米技术与纳米仿生研究所 适用于无明显症状者排查的新冠病毒抗体检测试剂盒
WO2021195694A1 (fr) * 2020-03-31 2021-10-07 Sementis Limited Vaccin à base de vecteur de poxvirus atténué pour la protection contre la covid-19
EP3928789A1 (fr) * 2020-06-24 2021-12-29 Consejo Superior de Investigaciones Científicas (CSIC) Vaccin à base de mva contre le covid-19 exprimant les antigènes du sars-cov-2
WO2021260065A1 (fr) * 2020-06-24 2021-12-30 Consejo Superior De Investigaciones Científicas (Csic) Vaccin à base de mva contre la covid-19 exprimant des antigènes de sras-cov-2
US11951174B2 (en) 2021-01-20 2024-04-09 Singh Biotechnology, Llc Therapeutics directed against coronavirus
EP4153228A4 (fr) * 2020-05-17 2024-07-10 City of Hope Vaccins contre le coronavirus à base de virus de la vaccine ankara modifiée synthétique (smva)

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CA2522379C (fr) * 2003-04-10 2012-10-23 Chiron Corporation Le coronavirus du syndrome respiratoire aigu grave
WO2004091524A2 (fr) * 2003-04-14 2004-10-28 Acambis Inc. Vaccins contre des virus des voies respiratoires
US8080642B2 (en) * 2003-05-16 2011-12-20 Vical Incorporated Severe acute respiratory syndrome DNA compositions and methods of use
TW200510450A (en) * 2003-07-21 2005-03-16 Nat Inst Health Soluble fragments of the SARS-CoV spike glycoprotein
WO2005027963A2 (fr) * 2003-09-15 2005-03-31 The United States Of America As Represented By Thesecretary Of Health And Human Services, Nih Procedes et compositions permettant de generer une reponse immunitaire protectrice contre sars-cov

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008155316A1 (fr) * 2007-06-19 2008-12-24 Glaxosmithkline Biologicals S.A. Compositions immunogènes associées à la protéine s du coronavirus associé au sras
WO2009085025A3 (fr) * 2007-06-19 2009-10-29 Glaxosmithkline Biologicals S.A. Vaccin
JP2011506267A (ja) * 2007-06-19 2011-03-03 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム Ｄｂｍｓベースのシステムにおいてパラメータ化ｓｐａｒｑｌクエリを使用するｓｐａｒｑｌクエリプロセシングのためのシステムおよび方法
WO2010063685A1 (fr) * 2008-12-02 2010-06-10 Glaxosmithkline Biologicals S.A. Vaccin
CN102316896A (zh) * 2008-12-02 2012-01-11 葛兰素史密丝克莱恩生物有限公司 疫苗
JP2012510449A (ja) * 2008-12-02 2012-05-10 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム ワクチン
CN104292339A (zh) * 2013-07-18 2015-01-21 特菲(天津)生物医药科技有限公司 含sars病毒rbd抗原的重组蛋白及展示rbd蛋白的杆状病毒
WO2021195694A1 (fr) * 2020-03-31 2021-10-07 Sementis Limited Vaccin à base de vecteur de poxvirus atténué pour la protection contre la covid-19
CN111856027A (zh) * 2020-04-16 2020-10-30 中国科学院苏州纳米技术与纳米仿生研究所 适用于无明显症状者排查的新冠病毒抗体检测试剂盒
CN111856027B (zh) * 2020-04-16 2022-05-10 中国科学院苏州纳米技术与纳米仿生研究所 适用于无明显症状者排查的新冠病毒抗体检测试剂盒
EP4153228A4 (fr) * 2020-05-17 2024-07-10 City of Hope Vaccins contre le coronavirus à base de virus de la vaccine ankara modifiée synthétique (smva)
EP3928789A1 (fr) * 2020-06-24 2021-12-29 Consejo Superior de Investigaciones Científicas (CSIC) Vaccin à base de mva contre le covid-19 exprimant les antigènes du sars-cov-2
WO2021260065A1 (fr) * 2020-06-24 2021-12-30 Consejo Superior De Investigaciones Científicas (Csic) Vaccin à base de mva contre la covid-19 exprimant des antigènes de sras-cov-2
US11951174B2 (en) 2021-01-20 2024-04-09 Singh Biotechnology, Llc Therapeutics directed against coronavirus

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