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WO2006071250A2 - Soluble fragments of the sars-cov spike glycoprotein - Google Patents

Soluble fragments of the sars-cov spike glycoprotein 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 (en
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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

The invention provides isolated SARS Coronavirus polypeptides, anti-SARS antibodies, recombinant poxviruses and compositions that are useful for treating and inhibiting SARS Coronavirus infection.

Description

Soluble Fragments of the SARS-CoV Spike Glycoprotein
This application claims benefit of the filing date of U.S. Provisional Application Ser. No. 60/558,995 filed April 5, 2004, which is hereby incorporated by reference in its entirety.
Government Funding
The invention described herein was developed with the support of the Department of Health and Human Services, National Institutes of Health. The United States Government has certain rights in the invention.
Field of the Invention
The invention relates to the treatment and prevent of severe acute respiratory syndrome (SARS) caused by the SARS-coronavirus (SARS-CoV).
Background of the Invention
Severe acute respiratory syndrome (SARS), an emerging infectious disease of humans, appeared in China in November 2002 and spread to thirty countries in early 2003. Before the epidemic ended, 8,098 probable cases of SARS and 774 associated deaths were reported to the World Health
Organization. See website at cdc.gov/mmwr/mguide sars.html. 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.
Although the 2002/2003 epidemic was eventually controlled by case isolation, the high morbidity and mortality, lack of specific treatment, and potential of re-emergence make it imperative to develop effective means to prevent or cure the disease should it reappear. Summary of the Invention
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.
Therefore, 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. For example, 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
Coronavirus polypeptide consisting essentially of SEQ ID NO:1, 3, 4, 6 or 7. 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. In one embodiment, 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. For example, 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.
Another aspect of the invention is a composition 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.
Another aspect of the invention is a composition 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.
Another aspect of the invention is a composition 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. For example, 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 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.
Description of the Figures
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. FIG. 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). The bound proteins were treated with endo H or PNGase F as indicated by plus signs and analyzed by SDS- PAGE and western blotting with anti-HA mAb. The positions of two glycosylated forms of S and a non-glycosylated (ngS) form are shown by arrows. FIG. IB illustrates the kinetics of endo H sensitivity. HeLa cells at 8 h after infection with MVA/S-HA were pulse-labeled with [35S]methionine and
[35S]cysteine for 10 min and then washed and chased for 0, 20, 40, 60 and 80 min in medium supplemented with unlabeled cysteine and methionine. Cells were lysed immediately after the pulse or chase and the S was captured with anti-HA affinity matrix (Roche), subjected to endo H digestion, resolved by SDS-PAGE and visualized by autoradiography. The masses of marker proteins in kDa are shown on the left.
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. 4A provides end-point ELISA titers of pooled serum (n=8), taken before (prebleed) or after immunizations, were determined using insect cell expressed Sl domain of the SARS-CoV S as the capture antigen. Sera from 2 mice were pooled after challenge and analyzed. Thin and thick arrows depict times of immunizations and challenge with SARS-CoV respectively. 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 104 TCID50 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 1Og10 TCID5o/g of tissue. Statistical comparison of MVA/S titers to unvaccinated controls was performed using a Mann Whitney U non-parametric analysis; *p = 0.02.
FIG. 6 provides amino acid and cDNA sequences (SEQ ID NO:4 and 5, respectively) for the SARS-CoV (Urbani strain) SΔTM+CT polypeptide containing spike protein amino acids 14-1195. FIG. 7A-C illustrates the construction, expression and characterization of
SARS-CoV nS glycoprotein, which include amino acids 14 to 762 (SEQ ID NO:6) of the SARS-CoV S polypeptide. FIG. 7A provides a schematic representation of pMelBacB-based baculovirus transfer vector. Abbreviations: PPH polyhedrin promoter; HBM, DNA encoding honeybee melittin signal sequence; nS, DNA segment encoding amino acids (aa) 14-762 of the SARS- CoV S protein; His6; 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. After fixation, 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. 8A5C5E5G) or fluorescence (FIG. 8B5D5F5H) 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 μg of purified nS and QS21 or MPL + TDM adjuvant at 4- week intervals (arrows) and challenged intranasally with 105 TCID50 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 1Og10TCID50 per g tissue with standard error) were measured in the lower (FIG. 10A) and upper (FIG. 10B) respiratory tract.
Detailed Description of the Invention As illustrated herein, a full-length Spike (S) polypeptide of SARS-CoV5 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. As also described herein, 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. Thus, the invention provides immunological compositions of SARS-CoV polypeptides, and of live attenuated viruses that can express such SARS-CoV polypeptides. In another embodiment, 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.
Definitions "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 Spike (S) Protein
SARS-coronavirus (SARS-CoV) 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). 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. In previously studied CoV, S was shown to be an important determinant of pathogenesis as well as the major target of protective immunity (8, 11). 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.
As described herein, S polypeptides are useful antigens for generating an immune response against 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. For example, 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).
1 MFIFLLFLTL TSGSDLDRCT TFDDVQAPNY TQHTSSMRGV
41 YYPDEIFRSD TLYLTQDLFL PFYSNVTGFH TINHTFGNPV
81 IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS
121 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT 161 FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY
201 QPiDvvRDLP SGFNTLKPIF KLPLGINITN FRΆILTAFSP 241 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ
281 NPLAELKCSV KSFEIDKGIY QTSNFRVVPS GDVVRFPNIT
321 NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF
361 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG 401 QTGVIADYNY KLPDDFMGCV LAWNTRNIDA TSTGNYNYKY
441 RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND
481 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI
521 KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ FGRDVSDFTD
561 SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD 601 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE
641 HVDTSYECDI PIGAGICASY HTVSLLRSTS QKSIVAYTMS
681 LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC
721 NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT
761 REVFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI 801 EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL
841 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF
881 AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL
921 TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN
961 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI 1001 RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH
1041 GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN
1081 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY
1121 DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN
1161 IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL 1201 GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE
1241 DDSEPVLKGV KLHYT
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).
21492 ATGTTTATT TTCTTATTAT TTCTTACTCT 21521 CACTAGTGGT AGTGACCTTG ACCGGTGCAC CACTTTTGAT
21561 GATGTTCAAG CTCCTAATTA CACTCAACAT ACTTCATCTA
21601 TGAGGGGGGT TTACTATCCT GATGAAATTT TTAGATCAGA
21641 CACTCTTTAT TTAACTCAGG ATTTATTTCT TCCATTTTAT
21681 TCTAATGTTA CAGGGTTTCA TACTATTAAT CATACGTTTG 21721 GCAACCCTGT CATACCTTTT AAGGATGGTA TTTATTTTGC
21761 TGCCACAGAG AAATCAAATG TTGTCCGTGG TTGGGTTTTT
21801 GGTTCTACCA TGAACAACAA GTCACAGTCG GTGATTATTA
21841 TTAACAATTC TACTAATGTT GTTATACGAG CATGTAACTT
21881 TGAATTGTGT GACAACCCTT TCTTTGCTGT TTCTAAACCC 21921 ATGGGTACAC AGACACATAC TATGATATTC GATAATGCAT
21961 TTAATTGCAC TTTCGAGTAC ATATCTGATG CCTTTTCGCT
22001 TGATGTTTCA GAAAAGTCAG GTAATTTTAA ACACTTACGA
22041 GAGTTTGTGT TTAAAAATAA AGATGGGTTT CTCTATGTTT 22081 ATAAGGGCTA TCAACCTATA GATGTAGTTC GTGATCTACC 22121 TTCTGGTTTT AACACTTTGA AACCTATTTT TAAGTTGCCT 22161 CTTGGTATTA ACATTACAAA TTTTAGAGCC ATTCTTACAG 22201 CCTTTTCACC TGCTCAAGAC ATTTGGGGCA CGTCAGCTGC 22241 AGCCTATTTT GTTGGCTATT TAAAGCCAAC TACATTTATG 22281 CTCAAGTATG ATGAAAATGG TACAATCACA GATGCTGTTG 22321 ATTGTTCTCA AAATCCACTT GCTGAACTCA AATGCTCTGT 22361 TAAGAGCTTT GAGATTGACA AAGGAATTTA CCAGACCTCT 22401 AATTTCAGGG TTGTTCCCTC AGGAGATGTT GTGAGATTCC 22441 CTAATATTAC AAACTTGTGT CCTTTTGGAG AGGTTTTTAA 22481 TGCTACTAAA TTCCCTTCTG TCTATGCATG GGAGAGAAAA 22521 AAAATTTCTA ATTGTGTTGC TGATTACTCT GTGCTCTACA 22561 ACTCAACATT TTTTTCAACC TTTAAGTGCT ATGGCGTTTC 22601 TGCCACTAAG TTGAATGATC TTTGCTTCTC CAATGTCTAT 22641 GCAGATTCTT TTGTAGTCAA GGGAGATGAT GTAAGACAAA 22681 TAGCGCCAGG ACAAACTGGT GTTATTGCTG ATTATAATTA 22721 TAAATTGCCA GATGATTTCA TGGGTTGTGT CCTTGCTTGG 22761 AATACTAGGA ACATTGATGC TACTTCAACT GGTAATTATA 22801 ATTATAAATA TAGGTATCTT AGACATGGCA AGCTTAGGCC 22841 CTTTGAGAGA GACATATCTA ATGTGCCTTT CTCCCCTGAT 22881 GGCAAACCTT GCACCCCACC TGCTCTTAAT TGTTATTGGC 22921 CATTAAATGA TTATGGTTTT TACACCACTA CTGGCATTGG 22961 CTACCAACCT TACAGAGTTG TAGTACTTTC TTTTGAACTT 23001 TTAAATGCAC CGGCCACGGT TTGTGGACCA AAATTATCCA 23041 CTGACCTTAT TAAGAACCAG TGTGTCAATT TTAATTTTAA 23081 TGGACTCACT GGTACTGGTG TGTTAACTCC TTCTTCAAAG 23121 AGATTTCAAC CATTTCAACA ATTTGGCCGT GATGTTTCTG 23161 ATTTCACTGA TTCCGTTCGA GATCCTAAAA CATCTGAAAT 23201 ATTAGACATT TCACCTTGCT CTTTTGGGGG TGTAAGTGTA 23241 ATTACACCTG GAACAAATGC TTCATCTGAA GTTGCTGTTC 23281 TATATCAAGA TGTTAACTGC ACTGATGTTT CTACAGCAAT 23321 TCATGCAGAT CAACTCACAC CAGCTTGGCG CATATATTCT 23361 ACTGGAAACA ATGTATTCCA GACTCAAGCA GGCTGTCTTA 23401 TAGGAGCTGA GCATGTCGAC ACTTCTTATG AGTGCGACAT 23441 TCCTATTGGA GCTGGCATTT GTGCTAGTTA CCATACAGTT 23481 TCTTTATTAC GTAGTACTAG CCAAAAATCT ATTGTGGCTT 23521 ATACTATGTC TTTAGGTGCT GATAGTTCAA TTGCTTACTC 23561 TAATAACACC ATTGCTATAC CTACTAACTT TTCAATTAGC 23601 ATTACTACAG AAGTAATGCC TGTTTCTATG GCTAAAACCT 23641 CCGTAGATTG TAATATGTAC ATCTGCGGAG ATTCTACTGA 23681 ATGTGCTAAT TTGCTTCTCC AATATGGTAG CTTTTGCACA 23721 CAACTAAATC GTGCACTCTC AGGTATTGCT GCTGAACAGG 23761 ATCGCAACAC ACGTGAAGTG TTCGCTCAAG TCAAACAAAT 23801 GTACAAAACC CCAACTTTGA AATATTTTGG TGGTTTTAAT 23841 TTTTCACAAA TATTACCTGA CCCTCTAAAG CCAACTAAGA 23881 GGTCTTTTAT TGAGGACTTG CTCTTTAATA AGGTGACACT 23921 CGCTGATGCT GGCTTCATGA AGCAATATGG CGAATGCCTA 23961 GGTGATATTA ATGCTAGAGA TCTCATTTGT GCGCAGAAGT 24001 TCAATGGACT TACAGTGTTG CCACCTCTGC TCACTGATGA 24041 TATGATTGCT GCCTACACTG CTGCTCTAGT TAGTGGTACT 24081 GCCACTGCTG GATGGACATT TGGTGCTGGC GCTGCTCTTC 24121 AAATACCTTT TGCTATGCAA ATGGCATATA GGTTCAATGG 24161 CATTGGAGTT ACCCAAAATG TTCTCTATGA GAACCAAAAA 24201 CAAATCGCCA ACCAATTTAA CAAGGCGATT AGTCAAATTC 24241 AAGAATCACT TACAACAACA TCAACTGCAT TGGGCAAGCT 24281 GCAAGACGTT GTTAACCAGA ATGCTCAAGC ATTAAACACA 24321 CTTGTTAAAC AACTTAGCTC TAATTTTGGT GCAATTTCAA 24361 GTGTGCTAAA TGATATCCTT TCGCGACTTG ATAAAGTCGA 24401 GGCGGAGGTA CAAATTGACA GGTTAATTAC AGGCAGACTT 24441 CAAAGCCTTC AAACCTATGT AACACAACAA CTAATCAGGG 24481 CTGCTGAAAT CAGGGCTTCT GCTAATCTTG CTGCTACTAA 24521 AATGTCTGAG TGTGTTCTTG GACAATCAAA AAGAGTTGAC 24561 TTTTGTGGAA AGGGCTACCA CCTTATGTCC TTCCCACAAG 24601 CAGCCCCGCA TGGTGTTGTC TTCCTACATG TCACGTATGT 24641 GCCATCCCAG GAGAGGAACT TCACCACAGC GCCAGCAATT 24681 TGTCATGAAG GCAAAGCATA CTTCCCTCGT GAAGGTGTTT 24721 TTGTGTTTAA TGGCACTTCT TGGTTTATTA CACAGAGGAA 24761 CTTCTTTTCT CCACAAATAA TTACTACAGA CAATACATTT 24801 GTCTCAGGAA ATTGTGATGT CGTTATTGGC ATCATTAACA 24841 ACACAGTTTA TGATCCTCTG CAACCTGAGC TCGACTCATT 24881 CAAAGAAGAG CTGGACAAGT ACTTCAAAAA TCATACATCA 24921 CCAGATGTTG ATCTTGGCGA CATTTCAGGC ATTAACGCTT 24961 CTGTCGTCAA CATTCAAAAA GAAATTGACC GCCTCAATGA 25001 GGTCGCTAAA AATTTAAATG AATCACTCAT TGACCTTCAA 25041 GAATTGGGAA AATATGAGCA ATATATTAAA TGGCCTTGGT 25081 ATGTTTGGCT CGGCTTCATT GCTGGACTAA TTGCCATCGT 25121 CATGGTTACA ATCTTGCTTT GTTGCATGAC TAGTTGTTGC 25161 AGTTGCCTCA AGGGTGCATG CTCTTGTGGT TCTTGCTGCA 25201 AGTTTGATGA GGATGACTCT GAGCCAGTTC TCAAGGGTGT 25241 CAAATTACAT TACACATAA
Another example of a SARS-CoV S polypeptide, NS-I strain, has 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
41 YYPDEIFRSD TLYLTQDLFL PFYSNVTGFH TINHTFGNPV
81 IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS
121 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT
161 FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY 201 QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP
241 AQDTWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ
281 NPLAELKCSV KSFEIDKGIY QTSNFRVVPS GDVVRFPNIT
321 NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF
361 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG 401 QTGVIADYNY KLPDDFMGCV LAWNTRNIDA TSTGNYNYKY
441 RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND
481 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI
521 KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ FGRDVSDFTD
561 SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD 601 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE
641 HVDTSYECDI PIGAGICASY HTVSLLRSTS QKSIVAYTMS
681 LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC
721 NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT 761 REVFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI
801 EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL
841 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF
881 AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL
921 TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN 961 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI iooi MASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH
1041 GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN
1081 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY
1121 DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN 1161 IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPWYVWL
1201 GFIAGLIAIV MVTILLCCMT SCCSCLKGAC SCGSCCKFDE
1241 DDSEPVLKGV KLHYT
In another embodiment, the invention provides antigenic fragments of SARS-CoV S polypeptides. In one embodiment, substantially full length SARS- CoV spike protein, with native signal sequences as well as transmembrane and cytoplasmic regions are deleted from the S polypeptide (ΔTM+CT). For the substantially full length S(ΔTM+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(ΔTM+CT) polypeptide (14-1195AA, SEQ ID NO:4) and cDNA (SEQ ID NO:5) are shown in FIG. 6 and are provided below.
The S(ΔTM+CT) spike polypeptide sequence (14-1195AA, SEQ ID NO:4) is as follows.
14 SDLDRCT TFDDVQAPNY TQHTSSMRGV
41 YYPDEIFRSD TLYLTQDLFL PFYSNVTGFH TINHTFGNPV
81 IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS
121 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT 161 FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY
201 QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP
241 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ
281 NPLAELKCSV KSFEIDKGIY QTSNFRVVPS GDVVRFPNIT
321 NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF 361 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG
401 QTGVIADYNY KLPDDFMGCV LAWNTRNIDA TSTGNYNYKY
441 RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND
481 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI
521 KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ FGRDVSDFTD 561 SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD 601 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE 641 HVDTSYECDI PIGAGICASY HTVSLLRSTS QKSIVAYTMS 681 LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC 721 NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT 761 REVFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI 801 EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL
841 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF
881 AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL 921 TTTSTALGKL QDWNQNAQA LNTLVKQLSS NFGAISSVLN
961 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI
1001 RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH
1041 GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN
1081 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY 1121 DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN
1161 IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPHHHHH
1201 H
The S(ΔTM+CT) cDNA (SEQ ID NO:5) sequence is as follows. 1 AGTGACCTTG ACCGGTGCAC CACTTTTGAT GATGTTCAAG
41 CTCCTAATTA CACTCAACAT ACTTCATCTA TGAGGGGGGT
81 TTACTATCCT GATGAAATTT TTAGATCAGA CACTCTTTAT
121 TTAACTCAGG ATTTATTTCT TCCATTTTAT TCTAATGTTA
161 CAGGGTTTCA TACTATTAAT CATACGTTTG GCAACCCTGT 201 CATACCTTTT AAGGATGGTA TTTATTTTGC TGCCACAGAG
241 AAATCAAATG TTGTCCGTGG TTGGGTTTTT GGTTCTACCA
281 TGAACAACAA GTCACAGTCG GTGATTATTA TTAACAATTC
321 TACTAATGTT GTTATACGAG CATGTAACTT TGAATTGTGT
361 GACAACCCTT TCTTTGCTGT TTCTAAACCC ATGGGTACAC 401 AGACACATAC TATGATATTC GATAATGCAT TTAATTGCAC
441 TTTCGAGTAC ATATCTGATG CCTTTTCGCT TGATGTTTCA
481 GAAAAGTCAG GTAATTTTAA ACACTTACGA GAGTTTGTGT
521 TTAAAAATAA AGATGGGTTT CTCTATGTTT ATAAGGGCTA
561 TCAACCTATA GATGTAGTTC GTGATCTACC TTCTGGTTTT 601 AACACTTTGA AACCTATTTT TAAGTTGCCT CTTGGTATTA
641 ACATTACAAA TTTTAGAGCC ATTCTTACAG CCTTTTCACC
681 TGCTCAAGAC ATTTGGGGCA CGTCAGCTGC AGCCTATTTT
721 GTTGGCTATT TAAAGCCAAC TACATTTATG CTCAAGTATG
761 ATGAAAATGG TACAATCACA GATGCTGTTG ATTGTTCTCA 801 AAATCCACTT GCTGAACTCA AATGCTCTGT TAAGAGCTTT
841 GAGATTGACA AAGGAATTTA CCAGACCTCT AATTTCAGGG
881 TTGTTCCCTC AGGAGATGTT GTGAGATTCC CTAATATTAC
921 AAACTTGTGT CCTTTTGGAG AGGTTTTTAA TGCTACTAAA
961 TTCCCTTCTG TCTATGCATG GGAGAGAAAA AAAATTTCTA 1001 ATTGTGTTGC TGATTACTCT GTGCTCTACA ACTCAACATT
1041 TTTTTCAACC TTTAAGTGCT ATGGCGTTTC TGCCACTAAG
1081 TTGAATGATC TTTGCTTCTC CAATGTCTAT GCAGATTCTT
1121 TTGTAGTCAA GGGAGATGAT GTAAGACAAA TAGCGCCAGG
1161 ACAAACTGGT GTTATTGCTG ATTATAATTA TAAATTGCCA 1201 GATGATTTCA TGGGTTGTGT CCTTGCTTGG AATACTAGGA
1241 ACATTGATGC TACTTCAACT GGTAATTATA ATTATAAATA 1281 TAGGTATCTT AGACATGGCA AGCTTAGGCC CTTTGAGΆGA
1321 GACATATCTA ATGTGCCTTT CTCCCCTGAT GGCAAACCTT 1361 GCACCCCACC TGCTCTTAAT TGTTATTGGC CATTAAATGA 1401 TTATGGTTTT TACACCACTA CTGGCATTGG CTACCAACCT 1441 TACAGAGTTG TAGTACTTTC TTTTGAACTT TTAAATGCAC 1481 CGGCCACGGT TTGTGGACCA AAATTATCCA CTGACCTTAT 1521 TAAGAACCAG TGTGTCAATT TTAATTTTAA TGGACTCACT 1561 GGTACTGGTG TGTTAACTCC TTCTTCAAAG AGATTTCAAC 1601 CATTTCAACA ATTTGGCCGT GATGTTTCTG ATTTCACTGA 1641 TTCCGTTCGA GATCCTAAAA CATCTGAAAT ATTAGACATT 1681 TCACCTTGCT CTTTTGGGGG TGTAAGTGTA ATTACACCTG 1721 GAACAAATGC TTCATCTGAA GTTGCTGTTC TATATCAAGA
1761 TGTTAACTGC ACTGATGTTT CTACAGCAAT TCATGCAGAT 1801 CAACTCACAC CAGCTTGGCG CATATATTCT ACTGGAAACA 1841 ATGTATTCCA GACTCAAGCA GGCTGTCTTA TAGGAGCTGA
1881 GCATGTCGAC ACTTCTTATG AGTGCGACAT TCCTATTGGA 1921 GCTGGCATTT GTGCTAGTTA CCATACAGTT TCTTTATTAC 1961 GTAGTACTAG CCAAAAATCT ATTGTGGCTT ATACTATGTC 2001 TTTAGGTGCT GATAGTTCAA TTGCTTACTC TAATAACACC 2041 ATTGCTATAC CTACTAACTT TTCAATTAGC ATTACTACAG 2081 AAGTAATGCC TGTTTCTATG GCTAAAACCT CCGTAGATTG 2121 TAATATGTAC ATCTGCGGAG ATTCTACTGA ATGTGCTAAT 2161 TTGCTTCTCC AATATGGTAG CTTTTGCACA CAACTAAATC 2201 GTGCACTCTC AGGTATTGCT GCTGAACAGG ATCGCAACAC 2241 ACGTGAAGTG TTCGCTCAAG TCAAACAAAT GTACAAAACC 2281 CCAACTTTGA AATATTTTGG TGGTTTTAAT TTTTCACAAA 2321 TATTACCTGA CCCTCTAAAG CCAACTAAGA GGTCTTTTAT 2361 TGAGGACTTG CTCTTTAATA AGGTGACACT CGCTGATGCT 2401 GGCTTCATGA AGCAATATGG CGAATGCCTA GGTGATATTA 2441 ATGCTAGAGA TCTCATTTGT GCGCAGAAGT TCAATGGACT 2481 TACAGTGTTG CCACCTCTGC TCACTGATGA TATGATTGCT 2521 GCCTACACTG CTGCTCTAGT TAGTGGTACT GCCACTGCTG 2561 GATGGACATT TGGTGCTGGC GCTGCTCTTC AAATACCTTT 2601 TGCTATGCAA ATGGCATATA GGTTCAATGG CATTGGAGTT 2641 ACCCAAAATG TTCTCTATGA GAACCAAAAA CAAATCGCCA 2681 ACCAATTTAA CAAGGCGATT AGTCAAATTC AAGAATCACT 2721 TACAACAACA TCAACTGCAT TGGGCAAGCT GCAAGACGTT 2761 GTTAACCAGA ATGCTCAAGC ATTAAACACA CTTGTTAAAC 2801 AACTTAGCTC TAATTTTGGT GCAATTTCAA GTGTGCTAAA 2841 TGATATCCTT TCGCGACTTG ATAAAGTCGA GGCGGAGGTA 2881 CAAATTGACA GGTTAATTAC AGGCAGACTT CAAAGCCTTC 2921 AAACCTATGT AACACAACAA CTAATCAGGG CTGCTGAAAT 2961 CAGGGCTTCT GCTAATCTTG CTGCTACTAA AATGTCTGAG
3001 TGTGTTCTTG GACAATCAAA AAGAGTTGΆC TTTTGTGGAA 3041 AGGGCTACCA CCTTATGTCC TTCCCACAAG CAGCCCCGCA
3081 TGGTGTTGTC TTCCTACATG TCΆCGTATGT GCCATCCCAG
3121 GAGAGGAACT TCACCACAGC GCCAGCAATT TGTCATGAAG 3161 GCAAAGCATA CTTCCCTCGT GAAGGTGTTT TTGTGTTTAA 3201 TGGCACTTCT TGGTTTATTA CACAGAGGAA CTTCTTTTCT 3241 CCACAAATAA TTACTACAGA CAATACATTT GTCTCAGGAA 3281 ATTGTGATGT CGTTATTGGC ATCATTAACA ACACAGTTTA 3321 TGATCCTCTG CAACCTGAGC TCGACTCATT CAAAGAAGAG
3361 CTGGACAAGT ACTTCAAAAA TCATACATCA CCAGATGTTG
3401 ATCTTGGCGA CATTTCAGGC ATTAACGCTT CTGTCGTCAA
3441 CATTCAAAAA GAAATTGACC GCCTCAATGA GGTCGCTAAA 3481 AATTTAAATG AATCACTCAT TGACCTTCAA GAATTGGGAA
3521 AATATGAGCA ATATATTAAA TGGCCTCATC ATCACCATCA 3561 CCATTGA
An 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).
14 SDLDRCT TFDDVQAPNY TQHTSSMRGV
41 YYPDEIFRSD TLYLTQDLFL PFYSNVTGFH TINHTFGNPV
81 IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS 121 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT
161 FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY
201 QPIDWRDLP SGFNTLKPIF KLPLG1NITN FRAILTAFSP
241 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ
281 NPLAELKCSV KSFEIDKGIY QTSNFRVVPS GDVVRFPNIT 321 NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF
361 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG
401 QTGVIADYNY KLPDDFMGCV LAWNTRNIDA TSTGNYNYKY
441 RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND
481 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI 521 KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ FGRDVSDFTD
561 SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD
601 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE
641 HVDTSYECDI PIGAGICASY HTVSLLRSTS QKSIVAYTMS
681 LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC 721 NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT
761 RE
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).
763 VFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI 801 EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL 841 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF
881 AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL
921 TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN
961 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI 1001 RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH
1041 GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN
1081 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY
1121 DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN
1161 IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPHHHHH 1201 H
Thus, the invention provides S polypeptides and antigenic fragments thereof that are useful for treating and preventing SARS infection. Moreover, 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.
Table 1
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
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. As known to one of skill in the art, 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. Examples of genetically encoded hydrophobic amino acids include He, Leu and VaI. Examples of 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 π-electron system (aromatic group). The aromatic group may be further substituted with 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. Commonly encountered non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, β-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. Examples of genetically encoded aliphatic amino acids include Ala, Leu, VaI and He. Examples of 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. Examples of genetically encoded basic amino acids include arginine, lysine and histidine. Examples of non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.
"Polar 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. Examples of genetically encoded polar amino acids include asparagine and glutamine. Examples of 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. Typically, 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.
As will be appreciated by those having skill in the art, the above classifications are not absolute. Several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category. For example, tyrosine has both an aromatic ring and a polar hydroxyl group. Thus, tyrosine has dual properties and can be included in both the aromatic and polar categories. Similarly, in addition to being able to form disulfide linkages, cysteine also has apolar character. Thus, while not strictly classified as a hydrophobic or apolar amino acid, in many instances 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, β-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; α-aminoisobutyric acid (Aib); ε- aminohexanoic acid (Aha); δ-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 (NIe); 2-naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4- fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1, ,2,3, A- tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p- aminophenylalanine (Phe(pNH2)); N-methyl valine (MeVaI); homocysteine (hCys) and homoserine (hSer). These amino acids also fall into the categories defined above.
The classifications of the above-described genetically encoded and non- encoded amino acids are summarized in Table 2. It is to be understood that 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.
Figure imgf000022_0001
Figure imgf000023_0001
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. Preferably, the immune response is against SARS-CoV.
The ability of S polypeptides, derivatives and variants thereof to generate an immune response in an animal can be assessed by procedures available to one of skill in the art. For example, 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.
One of skill in the art 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. If administration of 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 Encoding SARS-CoV Antigens
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. Among the recombinant viruses that can be used are adenoviruses, adeno-associated viruses, retroviruses and poxviruses. For example, 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.
A variety of 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. 63:3822-3828 (1989)), papillomavirus, Epstein Barr virus (EBV) and Rhino viruses (see, e.g., U.S. Patent No. 5,714,374). Human parainfluenza viruses are also reported to be useful, especially JS CP45 HPIV-3 strain. 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. Other 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. There are a variety of attenuated poxviruses that are available for use as an immunological composition against SARS-CoV. These include attenuated vaccinia virus, cowpox virus and canarypox virus.
Techniques for inserting SARS-CoV S polypeptides into the recombinant virus are available. For example, 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). In some embodiments, 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. 4,722,848, U.S. Patent No. 4,603,112, U.S. Patent No. 5,110,587 and U.S. Patent No. 5,174,993, the disclosures of which are incorporated herein by reference. Thus, for example, 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. Thus, 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. In general, 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 in the compositions of the invention. A review of this technology is found in U.S. Patent No. 5,863,542, which is incorporated by reference herein. Representative examples of 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 (nuVnu4) 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)). 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. Nevertheless, when engineered to express immunogens, authentic expression and processing are observed in vitro in mammalian cells and inoculation into numerous mammalian species induces antibody and cellular immune responses to the extrinsic immunogen and provides protection against challenge with the cognate pathogen.
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.
Another attenuated poxvirus for use in the invention is the Modified Vaccinia virus Ankara (MVA), which acquired defects in its replication ability in humans, as well as most mammalian cells, following over 500 serial passages in chicken fibroblasts (see, e.g., Mayr et al., Infection 3: 6-14 (1975); Carrol, M and Moss, B., Virology 238: 198-211 (1997)). MVA retains it original immunogenicity and its variola-protective effect and longer has any virulence and contagiousness for animals and humans. As for the 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.
Because the restriction occurs at a late stage of virus assembly, 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.
Hence, 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 104 - 108 organisms/dose, or 106 to 1010 pfu per dose. For example, NYVAC, ALVAC or MVA recombinant poxviruses can be administered by an intramuscular route using a dosage of about 107 to 109 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.
Nucleic Acid- Based Immunological Composition
Alternatively, 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). In such compositions, 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.
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). This formulation, known as "naked DNA," is particularly suitable for intramuscular (IM) or intradermal (ID) administrations. To maximize the immunotherapeutic effects of plasmid DNA vaccines, alternate methods for formulating purified plasmid DNA may be desirable. A variety of methods has been described, and such methods are available to one of skill in the art. 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. Patent No. 5,279,833; WO 91/06309; and Feigner et al., Proc. Nat'l Acad. Sci. USA 84: 7413 (1987). In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) can also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion or trafficking to specific organs or cell types. The uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into the cells.
Antibody Preparations
In another embodiment, the invention provides a preparation of antibodies that can bind to a SARS-CoV, or a SARS CoV S polypeptide, derivative or variant thereof. For example, 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. In some embodiments, 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. In some embodiments, the antibody is an antigen-binding antibody fragment. In other embodiments, the antibody is a polyclonal antibody. In further embodiments, the antibody is a single-chain antibody. In other embodiments, the antibody is a monoclonal antibody. In some preferred embodiments, the antibody is a humanized antibody. The antibody may be coupled to a detectable tag. For example, the detectable tag can be a radiolabel. In some embodiments, the detectable tag is an affinity tag. In other embodiments, the detectable tag is an enzyme. In further embodiments, the detectable tag is a fluorescent protein. In some embodiments, the detectable tag is a fluorescent molecule. The antibody may also be coupled to a toxin.
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. 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). Depending on the amino acid sequences of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. 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 (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino sequences of their constant domain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.
The term "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. However, the 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.
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 β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-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.
The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of antibody fragments include Fab, Fab1, 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. As used herein, "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:
(1) 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.
(2) 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. Fab1 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.
(3) (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. (4) 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 (VH -VL 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 VH -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even 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. (5) 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. Such single chain antibodies are also referred to as "single-chain Fv" or "sFv" antibody fragments. Generally, 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. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, N. Y., pp. 269- 315 (1994).
The term "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). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161, and Hollinger et al, Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993).
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. See, for example, Kohler & Milstein, Nature, 256:495 (1975); Coligan, et al., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. (1988)), which are hereby incorporated by reference. Methods of in vitro and in vivo manipulation of monoclonal antibodies are also available to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or they may be made by recombinant methods, for example, as described in U.S. Patent No. 4,816,567. 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).
Another method for generating antibodies involves a Selected Lymphocyte Antibody Method (SLAM). The SLAM technology permits the generation, isolation and manipulation of monoclonal antibodies without the process of hybridoma generation. 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.
More specifically, 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. After a suitable period, during which antibodies can be detected in the serum of the animal (usually weeks to months), 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. These cells are detected by any of several means (complement mediated lysis of antigen-bearing cells, fluorescence detection or other) and then isolated using micromanipulation technology. The individual antibody forming cells are then processed for eventual single cell PCR to obtain the expressed Heavy and Light chain genes that encode the specific antibody. Once obtained and sequenced, these genes are cloned into an appropriate expression vector and recombinant, monoclonal antibody produced in a heterologous cell system. These antibodies are then purified via standard methodologies such as the use of protein A affinity columns. These types of methods are further described in Babcook, et al., Proc. Natl. Acad. Sci. (USA) 93: 7843-7848 (1996); U.S. Patent No. 5,627,052; and PCT WO 92/02551 by Schrader.
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).
The term "monoclonal antibody" as used herein 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
(epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In additional to their specificity, 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).
Methods of making antibody fragments are also known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, (1988), incorporated herein by reference). 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. For example, 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. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, in U.S. Patents No. 4,036,945 and No. 4,331,647, and references contained therein. These patents are hereby incorporated in their entireties by reference.
Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of VH and VL 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. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow, et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97 (1991); Bird, et al, Science 242:423-426 (1988); Ladner, et al, US Patent No. 4,946,778; and Pack, et al., Bio/Technology 11:1271-77 (1993).
Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). 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. Such 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. For the most part, 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.
In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, 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. In general, 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. For further details, see: Jones et al., Nature 321, 522-525
(1986); Reichmann et al., Nature 332, 323-329 (1988); Presta, Curr. Op. Struct. Biol. 2, 593-596 (1992); Holmes, et al., J. Immunol., 158:2192-2201 (1997) and Vaswani, et al., Annals Allergy, Asthma & Immunol, 81:105-115 (1998); U.S. Patent Nos. 4,816,567 and 6,331,415; PCT/GB84/00094; PCT/US 86/02269; PCT/US89/00077; PCT/US88/02514; and WO91/09967, each of which is incorporated herein by reference in its entirety.
The invention also provides methods of mutating antibodies to optimize their affinity, selectivity, binding strength or other desirable property. A mutant antibody refers to an amino acid sequence variant of an antibody. In general, 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. In general, 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. Preferably, 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. In general, 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. The term "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.
If desired, the antibodies of the invention can be purified by any available procedure. For example, 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) .
In some embodiments, 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.
Methods to immunize, treat, and diagnose an animal against SARS
The invention provides a method to immunize an animal against severe acute respiratory syndrome. In one embodiment, 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. In another embodiment, 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. In another embodiment, 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. For example, 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, can then be contacted with the antibody-antigen complex such that the enzyme, fluorophore or radiolabel is immobilized on the surface. The detectable tag can then be detected to determine if an antigen was present in the biological sample. In another example, 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. Examples of such 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. In one example, polymerase chain reaction (PCR) may be used to diagnose SARS-CoV infection. Briefly, 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. The presence of amplified DNA between the forward and reverse primers is then detected to determine if the sample contained SARS originated nucleic acid. 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.
Compositions
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.
Thus the 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. In some embodiments, 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. In other embodiments, the compositions can include a live recombinant virus that can express a SARS-CoV S polypeptide. Thus, as described herein 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. An immunogenic composition of this recombinant MVA-SARS-CoV S poxvirus protectively immunized mice against a subsequent infection with SARS-CoV. Hence, the invention provides compositions of live recombinant viruses that encode and express SARS-CoV antigens. In some embodiments, the compositions may contain an adjuvant.
Examples of adjuvants that can be used in the compositions of the invention 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. In other embodiments, 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) can also be used in the compositions of the invention. Such 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. In some embodiments, 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. For example, a combination of a monophosphoryl lipid A and saponin derivative can be employed, as described above. In other embodiments, a less reactogenic composition is used where the QS21 is quenched with cholesterol, as described in WO 96/33739. As described herein, 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
SmithKline Beecham, Rixensart, Belgium), Detox (Ribi ImmunoChem Research Inc., Hamilton, Mont.), RC-529 (Ribi ImmunoChem Research Inc., Hamilton, Mont.) and Aminoalkyl glucosaminide 4-phosphates (AGPs).
An immune composition or vaccine may be administered by any conventional route used in the field of vaccines. For example, 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.
Administration of 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.
The pharmaceutical 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. Such formulations are described in U.S. Patent No. 6,306,434 and in the references contained therein. 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. Alternatively, 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.
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.
Pharmaceutical 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. For administration by inhalation, 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. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, 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. For intra-nasal administration, 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.
Pharmaceutical 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
SARS-CoV S polypeptide, for example, an S polypeptide having any one of 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 syringe to allow for injection of the polypeptide contained within the kit into an animal, such as a human. In another embodiment, 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. Such 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.
The invention is further illustrated by the following non-limiting Examples.
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. Such an immunogenic composition of this recombinant MVA-SARS-CoV S poxvirus protectively immunizes mice against a subsequent infection with SARS-CoV.
Materials and Methods
Viruses and Cells. Primary chicken embryo fibroblast cells (CEF) prepared from 10-day old embryos were grown in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and used to propagate and titer MVA and recombinant MVA strains.
Spike Nucleic Acid Isolation and Recombinant Virus Construction. The 3,768-nucleotide open reading frame encoding the SARS-CoV S of the Urbani strain was copied and amplified from SARS-CoV virion RNA by RT- PCR, and cloned and sequenced. A clone was identified that exactly matched the published sequence (Gene Bank accession number AY278741). Two poxvirus transcription termination motifs (TTTTTNT) in S were altered using the QuickChange Multi Site-Directed Mutagenesis kit (Invitrogen). After mutagenesis, the entire S gene was PCR amplified with or without an influenza virus hemagglutinin (HA) epitope tag and inserted into theXmαl 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. The correct sequence of the entire S DNA insert was confirmed and recombinant 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.
Western Blotting. CEF and HeLa cells were infected with 5 PFU of recombinant MVA for 18 h. Infected cells were lysed in ice-cold RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate] supplemented with protease inhibitor cocktail (Sigma). Lysates were kept on ice for 10 min, centrifuged and resolved by SDS polyacrylamide gel electrophoresis (PAGE) on a bis-Tris 4 -12% polyacrylamide gel. 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).
Endoglycosidase (endo) H and Peptide N-Glycosidase (PNGase) F Treatments. Cleared cell lysates were incubated with 20 μl of anti-HA affinity matrix (Roche) overnight at 4°C. The agarose beads were washed and incubated with endo H and PNGase F (New England Biolabs) according to manufacturer's instructions and the proteins were analyzed by western blotting using peroxidase-conjugated anti-HA mouse mAb (Roche).
Pulse-Chase Analysis. 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 μCi of [35S]methionine and [35S] 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 μl 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.
Confocal Microscopy. 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 40C 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. After extensive washes, 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). 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).
Animal Challenge Experiments. Groups of 8 BALB/c mice were inoculated intranasally (IN) or intramuscularly (IM) with 107 PFU of MVA or MVA/S at 0 and 4 weeks. Four weeks after the second immunization, animals were challenged IN with 104 tissue culture infectious doses50 (TCID50) 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.
To obtain serum for passive protection studies, two groups of 8 BALB/c 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 naϊve mice. Mice were bled the following day to determine their levels of SARS-CoV specific neutralizing antibody and then each was challenged with 105 TCID50 of SARS-CoV and analyzed as above.
Results Characterization of SARS-CoV S Expressed by Recombinant MVA.
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). A second recombinant virus, 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. In each case, 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- specifϊc antibodies.
A protein doublet with an estimated mass of approximately 200 kDa, significantly higher than the value of 135 kDa for the unmodified protein predicted from the nucleotide sequence, was detected by SDS-PAGE of lysates of cells infected with MVAJS and MVA/S-HA and Western blotting with polyclonal antibody to S or a mAb to HA (FIG. IB; data for MVA/S not shown). In addition, 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 1000C.
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. To evaluate this possibility, 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. However, differences of this magnitude between the theoretical mass and the mass estimated by SDS- PAGE are commonly found, and this discrepancy does not necessarily indicate that S contains additional post-translational modifications.
Further experiments were carried out using 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. To determine the kinetics of acquisition of endo H resistance, cells infected with MVA/S-HA were pulse labeled for 10 min with [35S]methionine and [35S]cysteine and then chased in medium containing unlabeled amino acids. At each time point, the epitope tagged S protein was isolated using an HA mAb affinity matrix; one portion was analyzed directly by SDS-PAGE and autoradiography and an equal portion was first digested with endo H. Immediately after the pulse, a sharp 200-kDa band was detected that became more diffuse during the chase and was resolved as a doublet by 60 min in the absence of endo H treatment (FIG. 2B). The pulse-labeled S was completely digested to a 160-kDa species by endo H (FIG. 2B). A faint endo H- resistant band appeared by 40 min of chase (seen as a diffuse band in this particular experiment) indicating that a small fraction of S had become resistant to digestion (FIG. 2B). Even after 80 min, however, there was still considerable endo H-sensitive S.
Cellular Localization of S. The glycosylation and partial resistance to endo H was consistent with trafficking of the SARS-CoV S through the ER to the Golgi compartment. To determine whether S was expressed at the cell surface, unpermeabilized CEF that had been infected with MVA/S were stained with antibody to S followed by Alexa 594-conjugated-anti-mouse IgG. Whereas the fluorescence due to co-expressed GFP was present throughout the cytoplasm, the labeling of S was restricted to the cell surface (FIG. 3, row 2). Moreover no labeling occurred in cells infected with the MVA vector (FIG. 3, row 1) or uninfected cells (not shown). Experiments were also carried out with cells infected with MVA/S-HA except that antibody to the epitope tag was used. The absence of staining of unpermeabilized cells (FIG. 3, row 3) was consistent with the S protein having a type 1 topology with the tagged C-terminus in the cytoplasm. After permeabilization of the plasma membrane with digitonin, both plasma membrane and juxtanuclear staining were evident (FIG. 3, row 4). Similar patterns were found when infected HeLa cells were examined by confocal microscopy (not shown).
Immunogenicity of MVA/S in mice. Mice were inoculated IN or IM with 107 PFU of MVA/S at 0 time and again at 4 weeks. Antibody was determined by an endpoint ELISA using a recombinant protein consisting of the S 1 domain of SARS-CoV made in insect cells and purified by affinity chromatography. Antibody was detected at 4 weeks and peaked at 6 weeks (FIG. 4A). Similar titers were obtained after either route of inoculation. The titers began to decline with time and were not boosted at two weeks after the SARS- CoV challenge described in the next section (FIG. 4A).
The ability of sera to neutralize SARS CoV infectivity for VERO cells was determined as described by Subbarao et al., (2004) J. Virol. 78, 3572-3577. Neutralizing antibody was detected after the second immunization by either the IN or IM route (FIG. 4B).
Protection of Mice Immunized with MVA/S. Previous studies demonstrated that 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 104TCID50 of SARS-CoV, approximately 105 TCID50 of SARS-CoV per g of lung was recovered on day 2 (FIG. 5). By contrast, the titers of SARS-CoV from the lungs of mice immunized with MVA/S either IM or IN were reduced by about 104 to levels that were barely above the limit of detection (FIG. 5). About 103 TCID50 per g of SARS- CoV were recovered from the nasal turbinates of control mice, but this too was significantly reduced in the immunized animals (FIG. 5). The severe reduction in SARS-CoV replication may explain the absence of an amnestic ELISA antibody response to S following challenge (FIG. 4A). Neutralizing titers to SARS CoV were not measured after challenge.
Passive Protection Mediated by Serum from MVA/S Immunized Mice. MVA can induce both humoral and cell mediated immune responses. To determine a role for antibody, sera were pooled that were obtained from mice that had been immunized IM with 107 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 naϊve mice to evaluate the protective role of antibody. As a positive control, hyperimmune SARS-CoV serum was administered to two mice (Subbarao et al., (2004) J. Virol. 78, 3572-3577). On the next day, the mice received an intranasal challenge of 105 TCID50 of SARS-CoV, and two days later, their nasal turbinates and lungs were removed to measure the virus titers. As shown in Table 3, administration of undiluted MVAJS serum reduced the lung titers by 105 1 compared to recipients of MVA control serum. These data indicate that antibodies to SARS CoV S polypeptide conferred the observed protection. Protection was observed despite a neutralization titer of only 1 :35 in recipient mice. Replication of SARS-CoV increased as the quantity of passively transferred serum decreased, but significant reductions in lung virus titers still occurred at sera dilutions of 1:4, 1:16 and 1:64. The absence of detectable neutralizing antibody in mice receiving these dilutions of passively transferred serum probably reflects a low sensitivity of the in vitro neutralization assay as indicated by the fact that the ELISA titers to S were more than 100-fold higher than the neutralization titers (FIG. 4). The recovery of SARS-CoV from the nasal turbinates was also reduced, but to a relatively lesser extent than from the lungs.
Table 3
Figure imgf000054_0001
*The indicated dilutions of antibody in 400 μl were administered to recipient mice by intraperitioneal injection. fMice were challenged with 105 TdD50 SARA-CoV intranasally (IN).
*Virus titers are expressed as logio TCIDso/g tissue.
§P values comparing titers with those seen in mice that received undiluted MVA control antibody in a Mann Whitney U non-parametic analysis.
¥Small sample size affected statistical significance.
10 Virus not detected; the lower limit of detection of infectious virus in a 5% w/v suspension of nasal turbinates was 1.8 logio TCIDso/g tissue and in a 10% w/v suspension of lung homogenate the detection limit was 1.5 logio TCID5o/g tissue.
As illustrated above the SARS-CoV S polypeptide can be expressed in a
15 native conformation and can induce antibodies that neutralize SARS-CoV.
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
20 ER and the conversion of the high mannose form to complex endo H-resistant N- linked chains occurs upon transport from the cis to the medial Golgi compartment. 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.
S 1 and S2 cleavage products were not detected, as found for group 2 but not group 1 CoV S proteins (Gallagher, T. M. & Buchmeier, M. J. (2001) Virology 279, 371-374). Xiao and co-workers ((2003) Biochem. Biophys. Res. Commun. 312, 1159-1164) expressed full length S by transfection and detected low amounts of several smaller than full-length S fragments, which they suggested might include specific cleavage products, though no evidence to support this was presented. The characterization studies provided herein strongly suggested that MVA expressed a properly folded form of S.
The MVA/S construct was then tested to determine whether it would elicit neutralizing antibodies. 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. Furthermore, 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.
Previous studies showed that IP inoculation of hyperimmune serum from mice inoculated twice with SARS CoV provided protection against SARS-CoV in the lower respiratory tract and to a lesser extent in the upper respiratory tract (Subbarao et al. (2004) J. Virol. 78, 3572-3577). Protection with serum from MVA/S-immunized animals was demonstrated in the present study. Because serum from animals inoculated with the MVA vector had no effect, the protection was likely due to S-specific antibodies. These results indicated that the S of SARS CoV, like that of other CoV, is an important target of neutralizing antibodies both in vitro and in vivo.
No enhanced virus replication or obvious disease was found in mice that 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.
Thus, the present study provides encouraging results for the development of SARS-CoV vaccines based on the highly attenuated MVA vector expressing S.
EXAMPLE 2: Immunogenic Spike Polypeptides Protect Against SARS
Infection
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. Mice that received the affinity-purified protein with either a saponin (QS21) or a Ribi (MPL + TDM) adjuvant subcutaneously and were challenged intranasally with SARS-CoV, produced neutralizing antibodies and protection against SARS-CoV intranasal infection. The best results were obtained with QS21 and protein, which provided the highest antibody as well as complete protection of the upper and lower respiratory tract.
Materials and Methods Vector construction. A cDNA encoding amino acids 14 to 762 of the
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).
In particular, 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). In all cases, native signal sequences as well as transmembrane and cytoplasmic regions (ΔTM+CT) were deleted. For the full length S(ΔTM+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(ΔTM+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(ΔTM+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
41 YYPDEIFRSD TLYLTQDLFL PFYSNVTGFH TINHTFGNPV
81 IPFKDGIYFA ATEKSNVVRG WVFGSTMNNK SQSVIIINNS
121 TNVVIRACNF ELCDNPFFAV SKPMGTQTHT MIFDNAFNCT
161 FEYISDAFSL DVSEKSGNFK HLREFVFKNK DGFLYVYKGY 201 QPIDVVRDLP SGFNTLKPIF KLPLGINITN FRAILTAFSP
241 AQDIWGTSAA AYFVGYLKPT TFMLKYDENG TITDAVDCSQ
281 NPLAELKCSV KSFEIDKGIY QTSNFRVVPS GDVVRFPNIT
321 NLCPFGEVFN ATKFPSVYAW ERKKISNCVA DYSVLYNSTF
361 FSTFKCYGVS ATKLNDLCFS NVYADSFVVK GDDVRQIAPG 401 QTGVIADYNY KLPDDFMGCV LAWNTRNIDA TSTGNYNYKY
441 RYLRHGKLRP FERDISNVPF SPDGKPCTPP ALNCYWPLND
481 YGFYTTTGIG YQPYRVVVLS FELLNAPATV CGPKLSTDLI 521 KNQCVNFNFN GLTGTGVLTP SSKRFQPFQQ FGRDVSDFTD 561 SVRDPKTSEI LDISPCSFGG VSVITPGTNA SSEVAVLYQD
601 VNCTDVSTAI HADQLTPAWR IYSTGNNVFQ TQAGCLIGAE
641 HVDTSYECDI PIGAGICASY HTVSLLRSTS QKSIVAYTMS
681 LGADSSIAYS NNTIAIPTNF SISITTEVMP VSMAKTSVDC 721 NMYICGDSTE CANLLLQYGS FCTQLNRALS GIAAEQDRNT
761 RE
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).
763 VFAQVKQM YKTPTLKYFG GFNFSQILPD PLKPTKRSFI
801 EDLLFNKVTL ADAGFMKQYG ECLGDINARD LICAQKFNGL
841 TVLPPLLTDD MIAAYTAALV SGTATAGWTF GAGAALQIPF
881 AMQMAYRFNG IGVTQNVLYE NQKQIANQFN KAISQIQESL 921 TTTSTALGKL QDVVNQNAQA LNTLVKQLSS NFGAISSVLN
961 DILSRLDKVE AEVQIDRLIT GRLQSLQTYV TQQLIRAAEI
1001 RASANLAATK MSECVLGQSK RVDFCGKGYH LMSFPQAAPH
1041 GVVFLHVTYV PSQERNFTTA PAICHEGKAY FPREGVFVFN
1081 GTSWFITQRN FFSPQIITTD NTFVSGNCDV VIGIINNTVY 1121 DPLQPELDSF KEELDKYFKN HTSPDVDLGD ISGINASVVN
1161 IQKEIDRLNE VAKNLNESLI DLQELGKYEQ YIKWPHHHHH
1201 H
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(ΔTM+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.
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 0C. 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.
Immunological assays. Western blotting was carried out using standard procedures and an anti-His mouse mAb (Qiagen) or anti-S ARS-CoV S rabbit polyclonal antibody (IMG-541, Imgenex, San Diego) diluted 1:1000 and 1 :500 in blocking buffer respectively. ELISA and confocal microscopy were carried out as described in Bisht et al. (2004) Proc. Natl. Acad. Sci. USA 101, 6641- 6646. Immunization protocol and SARS-CoV challenge. Groups of seven female 6- week BALB/c mice were injected subcutaneously with 10 μg of nS protein or with an unrelated vaccinia virus protein LlR on days 0, 28 and 56. Approximately four weeks after the third immunization, mice were intranasally challenged with 105 TCID50 of SARS-CoV in 50 μl. 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.
Results A 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). 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). Treatment with peptide N-glycosidase F reduced the mobility of the protein to ~85 kDa, demonstrating that the higher than expected apparent mass was due to N- glycosylation (FIG. 7C). To analyze immunogenicity, nS protein mixed with MPL + TDM or
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). As an initial evaluation of immunogenicity, 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. Because the endoplasmic reticulum acts as a filter for misfolded proteins, S present on the cell surface is likely to be correctly folded. Although SARS-CoV-infected cells could be used for the same purpose, considerably higher containment levels would be required. Uninfected HeLa cells or HeLa cells infected with non-recombinant MVA or MVA/S were fixed and stained with pooled mouse serum followed by Alexa 594-conjugated-anti-mouse IgG and analyzed by confocal microscopy. The serum obtained from 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). In contrast, 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). These data indicated that the antibodies produced by nS were able to bind to the membrane-associated form of full length S.
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. The specific IgG2a/IgGl titers from 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). For comparative purposes, we also determined the IgG2a/IgGl ratio of serum previously obtained from mice immunized with MVAJS. Although the overall IgG titers were lower in mice immunized with MVA/S (Bisht et al. (2004) Proc. Natl. Acad. Sci. USA 101, 6641-6646) than with the nS protein, the IgG2a/IgGl ratios were higher with values of 2 and 4 for pooled sera of mice immunized intranasally and intramuscularly, respectively. The high titer of nS-binding antibody and its recognition of full-length membrane-bound S encouraged us to evaluate the ability of the immune sera to neutralize the infectivity of SARS-CoV. Significant neutralizing activity was observed after the second inoculation of nS with either adjuvant (FIG. 9B). However, the mean neutralizing titer of 1 : 1269 achieved with QS21 was 4.6-fold higher than that obtained with MPL + TDM. Thus there was good correspondence between the relative binding and neutralizing activities of sera obtained with QS21 and MPL + TDM adjuvants.
Subbarao et al. (J. Virol. 78, 3572-3577 (2004)) demonstrated that SARS-CoV replicates in the respiratory tract of BALB/C mice and that replication was reduced following passive administration of neutralizing antibody. In this model, peak titers were reached within 1 to 2 days depending on the dose and clearing occurred by 7 days. Two days after the intranasal administration of 105 TCID50 of SARS-CoV, 10s TCID50 of virus per g of lung was recovered in control mice immunized with the vaccinia virus LlR protein in either adjuvant (FIG. 10A). By contrast, there was at least a 106-fold reduction in viral load in the lungs of mice immunized with nS regardless of the adjuvant (FIG. 4A). The difference was highly significant (p=0.0017) as determined using the Mann- Whitney non-parametric statistical method. Indeed, virus was detected in only one mouse out of seven in each of the test groups. The virus titers in the nasal turbinates showed a 103-fold reduction relative to controls when nS was administered with MPL + TDM adjuvant and >104-fold reduction when nS was given with QS21 (FIG. 10B). The effect of vaccination with either adjuvant was highly significant when compared with controls (p=0.0017) determined as above. 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 better protection obtained with the QS21 adjuvant was also statistically significant (p=0.0250), using the Mann- Whitney non-parametric statistical method corrected for ties, consistent with the higher binding and neutralizing antibody titers. The failure of the nS antibody response to be boosted after challenge (FIG. 9A) was also consistent with the absence of virus replication.
Thus, a recombinant polypeptide containing amino acids 14 to 762 of the SARS-CoV S protein that was administered with adjuvant induced neutralizing antibody and protectively immunized mice against upper and lower respiratory infections with SARS-CoV. Although the ability of a protein vaccine to protectively immunize against SARS-CoV has not previously been reported, recent studies have shown that the protein segment used herein contains the angiotensin-converting enzyme 2 receptor-binding region (Babcock et al. (2004) J. Virol. 78, 4552-4560; Wong et al. (2004) J. Biol. Chem. 279, 3197-3201; Xiao et al. (2003) Biophys. Res. Commun. 312, 1159-1164).
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. Although 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. However, we attribute the greater protection with QS21 adjuvant to the higher overall antibody response since MVA/S induced a considerably higher IgG2a/IgGl ratio but was less protective than nS with QS21.
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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications. The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality (for example, a culture or population) of such host cells, and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may 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.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The invention has been described broadly and genetically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

WHAT IS CLAIMED:
1. An isolated polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
2. An isolated nucleic acid encoding a polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
3. The nucleic acid of claim 2, wherein the nucleic acid comprises SEQ ID NO:2 or 5.
4. An antibody that can bind to a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
5. 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.
6. The recombinant attenuated poxvirus of claim 5, wherein the nucleic acid insertion comprises SEQ ID NO:2 or 5.
7. The recombinant attenuated poxvirus of claim 5, wherein the poxvirus is a modified MVA virus.
8. A recombinant attenuated baculo virus comprising a nucleic acid encoding a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6 or 7.
9. The recombinant attenuated baculovirus of claim 5, wherein the nucleic acid comprises SEQ ID NO:2 or 5.
10. A DNA vaccine comprising a pharmaceutically acceptable carrier and a vector encoding a SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:l, 3, 4, 6 or 7.
11. A composition comprising a carrier and an effective amount of SARS Coronavirus polypeptide consisting essentially of SEQ ID NO:4, 6, 7, or a combination thereof.
12. The composition of claim 11 , wherein the amount is effective for generating antibody production in an animal.
13. A composition 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.
14. The composition of claim 13, wherein the amount is effective for generating antibody production in an animal.
15. A composition comprising a carrier and an effective amount of antibody that can bind to a SARS Coronavirus polypeptide consisting essentially ofSEQ ID NO:4, 6 or 7.
16. The composition of claim 15, wherein the amount is effective to inhibit SARS Coronavirus replication in the animal.
17. 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 the composition of any one of claims 11 or 13.
18. A method for inhibiting SARS Coronavirus infection in an animal comprising: administering to the animal an immunologically effective amount of the composition of any one of claims 11, 13 or 15.
19. A method for treating SARS Coronavirus infection in an animal comprising: administering to the animal an effective amount of the composition of claim 15.
20. The method of claim 19, wherein the amount is effective to inhibit SARS Coronavirus replication in the animal.
21. 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.
22. 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.
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