WO2005010034A1 - Soluble fragments of the sars-cov spike glycoprotein - Google Patents
Soluble fragments of the sars-cov spike glycoprotein Download PDFInfo
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- WO2005010034A1 WO2005010034A1 PCT/US2004/023345 US2004023345W WO2005010034A1 WO 2005010034 A1 WO2005010034 A1 WO 2005010034A1 US 2004023345 W US2004023345 W US 2004023345W WO 2005010034 A1 WO2005010034 A1 WO 2005010034A1
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- C—CHEMISTRY; METALLURGY
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- 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
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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- A—HUMAN NECESSITIES
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
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- 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
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- 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 generally to a spike polypeptide that is encoded by a coronavirus (herein SARS-CoN), which is etiologically linked to Severe Acute Respiratory Syndrome (SARS).
- SARS-CoN coronavirus
- the invention further relates to nucleic acids and polypeptides having amino acid sequences that correspond to fragments of spike protein of SARS-CoN, and conservative variants thereof.
- the invention also relates to use of these nucleic acids, polypeptides, variants, and fragments to produce antibodies that recognize the spike protein of SARS-CoN, and for the production of vaccines against SARS.
- Another aspect of the invention relates to spike protein fragments for inhibiting fusion of the SARS-CoN with animal cells.
- Severe acute respiratory syndrome is an infectious atypical pneumonia that has recently been recognized in patients in 32 countries and regions.
- the atypical pneumonia with unknown etiology was initially observed in Guangdongzhou, China. This observation was followed by reports from Hong Kong, Vietnam, Singapore, Canada and Beijing of severe febrile respiratory illness that spread to household members and health care workers.
- This disease was later designated "severe acute respiratory syndrome (SARS)" by the World Health Organization (WHO).
- WHO World Health Organization
- coronaviruses have been classified into coronaviruses. Coronaviruses have been grouped into three categories based on cross-reactivity of antibodies backed up by genetic data. Two previously known human viruses fell into different groups than SARS- CoN. The coronavirus that causes SARS does not fit into any of the previously known clusters. Rather, it forms a new group by itself. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. Most coronaviruses cause either a respiratory or an enteric disease, which is also transmitted by the faecal-oral route. The incubation period for SARS is usually 2 to 7 days.
- Infection is characterized by fever, non-productive cough, shortness of breath, and the presence of minimal auscultatory findings with consolidation on chest radiographs. Lymphopenia, leucopenia, thrombocytopenia, and elevated liver enzymes and creatinine kinase may also be present in most cases. Symptoms relating to the gastrointestinal tract were also noticed in SARS patients. Pathological studies of patients who died of SARS from Guangdong,
- the pathogenesis of this disorder remains to be determined.
- the mechanism of acute lung injury could involve direct damage by the virus to the alveolar wall by targeting either endothelial cells or epithelial cells.
- the virus could infect inflammatory cells with the injury mediated through cytokines, interleukins, or tumor necrosis factor-alpha.
- tissue damage in SARS is not directly related to viral infection in tissues but is a secondary effect of cytokines or other factors induced by viral infection proximal to but not within the lung tissue.
- the invention provides polypeptides; peptide fragments; viral fusion inhibitors; coupled proteins; immunopeptides; immune compositions; peptidomimetics; nucleic acid segments; expression cassettes; nucleic acid constructs; recombinant viruses; viral vaccines; peptide vaccines; microorganism vaccines; D ⁇ A vaccines; antibodies; aptamers; pharmaceutical compositions; methods to immunize an animal; a method to treat severe acute respiratory syndrome (SARS); methods to diagnose SARS; and kits.
- the invention provides polypeptides having an amino acid sequence corresponding to that of a polypeptide that is etiologically linked to SARS.
- the polypeptide is the spike protein from SARS-CoN that can inhibit SARS fusion with animal cells and or raise immune response in an animal, hi some embodiments, the polypeptide is a soluble form of the spike protein from SARS-CoN. In other embodiments, the polypeptide includes amino acids 17- 757 of the spike protein from SARS-CoN. In some embodiments, the polypeptide includes amino acids 762-1189 of the spike protein from SARS- CoN. In other embodiments, the polypeptide includes amino acids 17-757 of the spike protein from SARS-CoN. In some embodiments, the polypeptide includes amino acids 17-276 of the spike protein from SARS-CoN.
- the polypeptide includes amino acids 303-537 of the spike protein from SARS-CoN. hi some embodiments, the polypeptide includes amino acids 317-517 of the spike protein from SARS-CoN. In other embodiments, the polypeptide includes amino acids 272-537 of the spike protein from SARS-CoN. In some embodiments, the polypeptide includes amino acids 17-537 of the spike protein from SARS-CoN. In other embodiments, the polypeptide includes amino acids 17-1189 (relative to SEQ ID NO: 1) of the spike protein from SARS-CoN.
- the polypeptides of the invention can inhibit SARS-CoN fusion with animal cells.
- the nucleic acids and polypeptides of the invention can elicit an immune response when used to inoculate an animal.
- the nucleic acids and polypeptides of the invention elicit a cellular immune response when used to inoculate an animal.
- the nucleic acids and polypeptides of the invention elicit a humoral immune response when used to inoculate an animal.
- the animal can be a reptile.
- the animal is an avian.
- the animal is a mammal.
- the animal is a human.
- the invention provides peptide fragments of the spike protein from SARS-CoN. Preferably the peptide fragments are soluble in aqueous solution.
- a peptide fragment of the invention may lack one amino acid residue from the amino acid sequence of the full length spike protein from SARS-CoN.
- peptide fragments are at least three amino acids in length.
- peptide fragments are at least 10 amino acids in length.
- peptide fragments are at least 20 amino acids in length.
- peptide fragments are at least 30 amino acids in length, i some embodiments, peptide fragments are at least 40 amino acids in length.
- peptide fragments are at least 50 amino acids in length.
- peptide fragments are at least 60 amino acids in length.
- the peptide fragments may also be single amino acid unit additions to a fragment of a given length.
- peptide fragment may be 3, 4, 10, 11, 21, 22, 31, or 32 amino acids in length.
- the peptide fragments of the invention can inhibit SARS Co-N fusion with animal cells or elicit an immune response when used to inoculate an animal.
- Examples of peptides that can elicit an immune response after inoculation ofan animal include, for example, the D24 peptide having sequence DNQAPNYTQHTSSMRGC (SEQ ID NO:58) and the P540 peptide having sequence PSSKRFQPQQFGRDC (SEQ ID NO:59).
- the peptide fragments of the invention elicit a cellular immune response when used to inoculate an animal.
- the peptide fragments of the invention elicit a humoral immune response when used to inoculate an animal.
- the animal can be a reptile.
- the animal is an avian.
- the animal is a mammal.
- the animal is a human.
- the invention provides coupled proteins.
- the coupled proteins include a carrier protein that is coupled to a second polypeptide.
- the carrier protein is soluble.
- the carrier protein increases an immune response to the second polypeptide of the coupled protein when used to inoculate an animal.
- the carrier protein elicits a cellular immune response to the second polypeptide of the coupled protein when used to inoculate an animal.
- the carrier protein elicits a humoral immune response to the second polypeptide of the coupled protein when used to inoculate an animal.
- the second polypeptide can be a polypeptide or a peptide fragment of the invention, or a conservative variant thereof.
- the animal can be a reptile.
- the animal is an avian.
- the animal is a mammal.
- the animal is a human.
- the invention provides immunopeptides that include a polypeptide or peptide fragment of the invention, or a conservative variant thereof, that is coupled to an acetyl group, a picryl group, an arsanilic acid, or to a sulfanilic acid, hi some embodiments, the immunopeptide is coupled to an acetyl or a picryl group. In other embodiments, immunopeptide is coupled to arsanilic acid or sulfanilic acid. Preferably, the immunopeptide is soluble. Preferably, the immunopeptide elicits an immune response when used to inoculate an animal. In some embodiments, the immunopeptide elicits a humoral immune response when used to inoculate an animal.
- the immunopeptide elicits a cellular immune response when used to inoculate an animal.
- the animal can be a reptile.
- the animal is an avian.
- the animal is a mammal.
- the animal is a human.
- the invention provides peptidomimetics that are polypeptides or peptide fragments of the invention, and conservative variants thereof, in which a peptide bond has been replaced with a non-peptide bond, hi some embodiments, the peptidomimetic can inhibit SARS Co-N fusion with animal cells.
- the peptidomimetic elicits an immune response when used to inoculate an animal.
- the peptidomimetic can elicit a cellular immune response when used to inoculate an animal.
- the peptidomimetic elicits a humoral immune response when used to inoculate an animal.
- the animal can be a reptile.
- the animal is an avian.
- the animal is a mammal.
- the animal is a human.
- the invention provides compositions containing an adjuvant and a nucleic acid, polypeptide, a peptide fragment, or a peptidomimetic of the invention.
- the composition inhibits SARS-CoN fusion with animal cells.
- the composition elicits an immune response when used to inoculate an animal.
- the immune composition elicits a cellular immune response when used to inoculate an animal, hi other embodiments, the immune composition elicits a humoral immune response when used to inoculate an animal.
- the animal can be a reptile. hr some embodiments, the animal is an avian. In other embodiments, the animal is a mammal. In further embodiments, the animal is a human.
- the invention provides nucleic acid segments that encode polypeptides and peptide fragments of the invention, and conservative variants thereof.
- the invention provides expression cassettes having a promoter that is operably linked to a nucleic acid segment of the invention.
- the promoter is constitutive. In other embodiments, the promoter is inducible.
- the invention provides nucleic acid constructs that include a vector arid a nucleic acid segment of the invention.
- the nucleic acid construct can include an expression cassette of the invention.
- the vector can be a virus. In other embodiments, the vector is a plasmid. In further embodiments, the vector is an expression vector.
- the invention provides a recombinant virus that includes a viral vector and a nucleic acid segment of the invention.
- the viral vector is a herpes virus, hi other embodiments, the viral vector is a canarypox virus. In other embodiments, the viral vector is an adenovirus.
- the viral vector is a vaccinia virus.
- the invention provides a viral vaccine against SARS that includes a viral vector, a nucleic acid segment of the invention, and a pharmaceutical carrier, hi some embodiments, the viral vector is a herpes virus. In other embodiments, the viral vector is a canarypox virus. In other embodiments, the viral vector is an adenovirus. In further embodiments, the viral vector is a vaccinia virus.
- the pharmaceutical carrier is formulated for injection.
- the viral vaccine elicits an immune response when used to inoculate an animal, hi some embodiments, the viral vaccine elicits a cellular immune response when used to inoculate an animal.
- the viral vaccine elicits a humoral immune response when used to inoculate an animal.
- the animal can be a reptile.
- the animal is an avian.
- the animal is a mammal.
- the animal is a human.
- the invention provides a peptide vaccine against SARS that includes a peptidomimetic, polypeptide or a peptide fragment of the invention, or a conservative variant thereof, and a pharmaceutical carrier.
- the pharmaceutical carrier is formulated for injection.
- the peptide vaccine is formulated in unit dosage form.
- the peptide vaccine elicits an immune response when used to inoculate an animal.
- the peptide vaccine elicits a cellular immune response when used to inoculate an animal. In other embodiments, the peptide vaccine elicits a humoral immune response when used to inoculate an animal.
- the animal can be a reptile. In some embodiments, the animal is an avian. In other embodiments, the animal is a mammal. In further embodiments, the animal is a human.
- the invention provides a microorganism vaccine against SARS that includes a microorganism that expresses a polypeptide or a peptide fragment of the invention, or a conservative variant thereof, and a pharmaceutical carrier. Preferably, the microorganism is attenuated. In some embodiments, the microorganism is Salmonella.
- the microorganism is Listeria. hi further embodiments, the microorganism is Listeria monocytogenes.
- the pharmaceutical carrier is formulated for injection. In other embodiments, the pharmaceutical carrier is formulated for oral administration.
- the microorganism vaccine is formulated in unit dosage form.
- the microorganism vaccine elicits an immune response when used to inoculate an animal, hi some embodiments, the microorganism vaccine elicits a cellular immune response when used to inoculate an animal, hi other embodiments, the microorganism vaccine elicits a humoral immune response when used to inoculate an animal.
- the animal can be a reptile. In some embodiments, the animal is an avian.
- the animal is a mammal. In further embodiments, the animal is a human.
- the invention provides a DNA vaccine against SARS that includes a vector into which is inserted a nucleic acid segment of the invention, and a pharmaceutical carrier.
- the DNA vaccine may include an adjuvant.
- the DNA vaccine may include a myonecrotic agent.
- the myonecrotic agent can be bupivicaine.
- the myonecrotic agent is cardiotoxin.
- the vector can, for example, be a virus, hi other embodiments, the vector is a bacteriophage. In further embodiments, the vector is a plasmid.
- the vector containing the insert can be prepared in a eukaryotic cell.
- the vector containing the insert is prepared in a prokaryotic cell.
- the vector containing the insert can be prepared in a bacterium, h some embodiments, the pharmaceutical carrier is formulated for mucosal delivery. In other embodiments, the pharmaceutical carrier is formulated for injection.
- the DNA vaccine is formulated in unit dosage form.
- the DNA vaccine elicits an immune response when used to inoculate an animal. In some embodiments, the DNA vaccine elicits a humoral immune response when used to inoculate an animal. In other embodiments, the DNA vaccine elicits a cellular immune response when used to inoculate an animal. The animal can be a reptile.
- the animal is an avian. In other embodiments, the animal is a mammal. In further embodiments, the animal is a human.
- the invention provides an antibody that binds to a polypeptide or peptide fragment of the invention, or a conservative variant thereof. In some embodiments, the antibody is an antigen-binding antibody fragment. In other embodiments, the antibody is a polyclonal antibody, hi 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.
- the detectable tag can be a radiolabel. In some embodiments, the detectable tag is an affinity tag.
- the detectable tag is an enzyme. In further embodiments, the detectable tag is a fluorescent protein. In some preferred embodiments, the detectable tag is a fluorescent marker.
- the antibody may also be coupled to a toxin.
- the invention provides aptamers that bind to a polypeptide or peptide fragment of the invention, or a conservative variant thereof.
- the aptamer may be coupled to a detectable tag.
- the detectable tag is a radiolabel.
- the detectable tag is an affinity tag.
- the detectable tag is an enzyme.
- the detectable tag is a fluorescent protein. In some preferred embodiments, the detectable tag is a fluorescent marker.
- the aptamer may also be coupled to a toxin.
- the invention provides a pharmaceutical composition or a kit containing an antibody, S polypeptide or aptamer of the invention and a pharmaceutical carrier.
- the pharmaceutical composition is formulated for injection.
- FIG. 1 A illustrates an agarose gel electrophoresis of a DNA construct having an insert that encodes the spike protein of the invention.
- Lanes from left to right Lane 1 is a one kb DNA ladder (markers from bottom to top - 0.5, 1.0, 1.6, 2.0, 3.0, 4.0); Lane 2 shows the DNA construct digested with BamHI/Xbal, resulting in the distinctive vector band (upper band) and the DNA fragment that encodes the spike protein (lower band); Lane 3 shows the DNA construct digested with Hindlll which produced a smaller band and a larger band as expected due to the presence of a Hindlll site in the vector and within the DNA fragment encoding the spike protein.
- Lane 1 is a one kb DNA ladder (markers from bottom to top - 0.5, 1.0, 1.6, 2.0, 3.0, 4.0); Lane 2 shows the DNA construct digested with BamHI/Xbal, resulting in the distinctive vector band (upper band) and the DNA fragment that encodes the spike protein (lower band); Lane 3 shows the DNA construct digested with Hindlll which produced a smaller band and a larger band as expected due to the presence of
- IB provides a schematic diagram of a monomer of the full-length SARS-CoN S glycoprotein showing various soluble polypeptide fragments after removal of the signal sequence (residues 1-16, SEQ ID NO:60).
- the soluble fragments are spike protein fragments named "S” followed by numbers corresponding to the spike protein amino acids that constitute the termini of the fragment.
- S756 is a soluble spike protein fragment beginning at amino acid 17 (just after the signal sequence) and ending at amino acid 756.
- TM denotes the transmembrane segment and the arrow indicates a possible cleavage site within amino acid positions 758-761 (sequence RNTR).
- Fig. 2 illustrates a denaturing polyacrylamide gel electrophoresis (SDS- PAGE) of the expression of a peptide fragment of the spike protein from SARS- CoN in Escherichia coli.
- the peptide fragment corresponds to amino acids 17- 446 of SEQ ID NO: 1.
- the nucleic acid segment encoding amino acids 17-446 was cloned into a pRSET vector to create pRSET-S(l 7-446), which was expressed in BL21DE3 cells.
- the lanes contain the following polypeptides: M - molecular weight markers; lanes 1 and 2 - polypeptides of control E. coli containing the pRSET vector without the nucleic acid segment encoding amino acid residues 17-446 of SEQ ID NO: 1 and without isopropylthiogalactoside
- IPTG IPTG induction
- lane 3 polypeptides of control E. coli containing the pRSET vector without the nucleic acid segment encoding amino acid residues 17-446 of SEQ ID NO: 1 but with IPTG induction
- lane 4 analysis of E. coli containing the pRSET vector with a nucleic acid segment encoding amino acid residues 17- 446 of SEQ ID NO: 1 , and with IPTG induction.
- the arrow on the right side indicates the position of a peptide fragment corresponding to amino acid residues 17-446 of SEQ ID NO: 1 as expressed in E. coli.
- FIG. 3 illustrates a slot blot analysis of the expression of the indicated peptide fragments of the spike protein from SARS-CoN in mammalian cells.
- Nucleic acid segments coding for the peptide fragments were cloned into a pSecTag2B vector to express peptide fragments having the mouse k chain leader sequence at the N-terminus for secretion, and a c-Myc epitope plus a histidine tag at the C-terminus for detection and affinity purification.
- the nucleic acid constructs were transformed into HEK293 and NeroE6 cells. Expression of the indicated peptide fragments was examined through use of slot blot analysis with an anti-c-Myc antibody.
- the numbers on the left and right indicate the amino acid residues included within the detected peptide fragments.
- the left column represents expression of the peptide fragments in HEK293 cells.
- the right column represents expression of the peptide fragments in NeroE6 cells.
- the upper half represents samples obtained from medium in which the cells were grown (secreted proteins), and the lower half represents samples obtained from cell lysate (intracellular portion).
- PC is a positive control, provided by the manufacturer of the plasmid that contains PSA with a c-Myc tag at the C- terminus.
- ⁇ C is a negative control that contains the full length spike protein from SARS-CoN that lacks a c-Myc epitope or histidine tag.
- FIG. 4 A illustrates a slot blot analysis of the expression of the indicated peptide fragments from the spike protein from SARS-CoN in human 293 or Monkey NeroE6 cells.
- Supematants of 293 and Nero E6 cells transfected with plasmids encoding S fragments (S276, S537, and S756) in the absence or presence of T7 polymerase expressed by recombinant vaccinia virus (NTF7.3) were transferred to nitrocellulose membranes and detected with anti-c-Myc epitope antibody.
- the numbers on the left and right indicate the amino acid residues included within the detected peptide fragments.
- PSA PC is a positive control that contains PSA with a c-Myc tag at the C-terminus.
- pCD ⁇ A-S ⁇ C is a negative control that contains the full length spike protein from SARS-CoN that lacks a c-Myc epitope or histidine tag.
- the lanes are as follows: (1) human 293 cells that were not infected with a NTF7.3 vaccinia virus, (2) human 293 cells that were infected with a NTF7.3 vaccinia virus, (3) monkey NeroE6 cells that were not infected with a NTF7.3 vaccinia virus, and (4) monkey NeroE6 cells that were infected with a NTF7.3 vaccinia virus.
- Fig. 4B Supematants from transfected cells as described above for Fig.
- Fig. 4A were incubated with ⁇ i- ⁇ TA agarose beads, washed, and subjected to Western blotting with the same anti-c-Myc epitope antibody as in Fig. 4A.
- Fig. 4C illustrates detection of S fragments by two rabbit polyclonal antibodies raised against peptides corresponding to sequences starting at residues 24 (D24, middle panel) and 540 (P540, right panel), respectively. The left panel shows for comparison Western blot where S537 and S756 were detected by the anti-c-Myc epitope antibody.
- Fig. 5 illustrates that the full-length membrane-associated S protein is expressed on the surface of cells, as shown by flow cytometry using the rabbit polyclonal antibody P540.
- a nucleic acid encoding the full-length S glycoprotein was used to transfect 293 cells, which were then infected with NTF7.3. Cells were collected and incubated with P540 polyclonal antibody plus anti-rabbit secondary antibody conjugated with FITC, washed, and subjected to flow cytometry analysis. The same plasmid used to express S but without the nucleic acids for S was used to transfect cells in a control experiment denoted as negative control ( ⁇ C); cells with nucleic acids encoding the full-length S glycoprotein are denoted as S.
- Fig. 6 A and 6B illustrate that substantially no cleavage of the S glycoprotein occurs naturally.
- FIG. 6A shows a Western blot of samples kept for three days at 4 °C before analysis to monitor the effect of nonspecific protease activity on the cleavage pattern.
- Fig. 6B shows blots with samples used immediately after preparation.
- Fig. 7A-C shows that cell fusion is mediated by the S glycoprotein.
- Fig.7A illustrates that there was no syncytium formation between 293T cells transfected with pSecTag2B-S and pCDNA3-ACE2-Ecto.
- Fig.7B illustrates syncytium formation between 293T cells transfected with ⁇ SecTag2B-S and ⁇ CDNA3-ACE2, respectively.
- FIG. 7C graphically illustrates cell fusion as measured by a reporter gene-based assay.
- S glycoprotein expressed in both pCDNA3 and pSecTag2B vectors can be detected in a /3-gal reporter gene-based cell-cell fusion assay.
- Fig. 8 A-C shows that the S glycoprotein receptor-binding domain (RBD) is localized between residues 272 and 537.
- Fig. 8A illustrates binding of two different S soluble fragments (S537 and S756) to 293 and Nero E6 cells.
- Fig. 8B illustrates binding of various S fragments to Nero E6 cells. The background OD 4 o 5 measured for the negative control was subtracted from the OD 405 values of each S fragment.
- OD 4 o 5 for each fragment was then presented as a percentage of the OD 4 o 5 for S537.
- Fig. 8C illustrates which S polypeptide fragments interact with purified soluble ACE2 as measured by ELISA.
- the negative control (NC) represents sample processed exactly the same way as the others except that the plasmid used for transfection did not encode any protein. Data shown here represent at least three independent experiments.
- OD o 5 for all samples is presented as percentages of the OD 4 o 5 for S537.
- Fig. 9 A-D illustrates that dimerization occurs between the N terminal fragments of the SARS-CoN S glycoprotein as demonstrated by co- immunoprecipitation and cross-linking.
- N-terminal fragments except the smallest fragment (S317-517) containing the receptor binding domain were coimmunoprecipiated with S756 by the P540 antibody.
- the P540 antibody is a rabbit polyclonal antibody that was developed against a peptide containing residues 540-555 of the S glycoprotein and it binds the S756 polypeptide but not the N-terminal fragments.
- plasmids encoding N-terminal fragments (denoted by the number of the ending amino acid residue or the number of the starting and ending residue) were used to transfect 293T cells alone (left six lanes) or in combination (right four lanes) with a S756-encoding plasmid.
- Fig. 9B shows that all N-terminal S fragments, except the smallest fragment (S317-517) that contained the receptor binding domain, were coimmunoprecipiated with S756 by the P540 antibody.
- the same medium samples used in Fig. 9 A were subjected first to immunoprecipitation with the P540 polyclonal antibody that recognizes only S756.
- Fig. 9C shows that a new band with a molecular weight corresponding to a dimer forms in the presence and absence of DTT.
- DTT was included in one of the coimmunoprecipitation experiment. DTT had no effect on either immunoprecipitation or coimmunoprecipitation of secreted S756 (left lanes) or S756+S276 (right lanes).
- Fig. 9D illustrates the size of the S polypeptide oligomers.
- the S537 fragment was cross-linked with BS 3 (Pierce, Rockford, IL) as described in the Examples and a Western blot was prepared after SDS-PAGE separation and the anti-c-Myc antibody was used for detection of the S537 monomer and its oligomers.
- BS 3 Pieris, Rockford, IL
- Fig. 10A illustrates dimerization of the N terminal fragment S537 as detected by size-exclusion chromatography. The elution profiles of S537 and S317-517 are shown with arrows and numbers indicating the position and molecular weight at which standard calibration proteins were eluted.
- Fig. 10B provides western blots of fractions collected for S537 and S317- 517 by using an anti-c-Myc epitope antibody.
- Fig. 11 A-B illustrates that the extreme N terminal domain is required for the S glycoprotein mediated cell-cell fusion.
- Fig. 10A illustrates dimerization of the N terminal fragment S537 as detected by size-exclusion chromatography. The elution profiles of S537 and S317-517 are shown with arrows and numbers indicating the position and molecular weight at which standard calibration proteins were eluted.
- Fig. 10B provides western blots of fractions collected for S537 and S317- 517 by using an anti
- FIG. 11 A provides a schematic representation of the S glycoprotein deletion mutants and a summary of the data from a cell-cell fusion assay where RBD denotes the approximate position of the receptor binding domain. The presence of signal due to fusion is denoted by a plus (+) and lack of measurable signal above background levels by a minus (-). Only wild type polypeptides with amino acids 17-1255 had fusion activity. Neither of the deletion mutants having amino acids 103-1255 (Dell) or 311- 1255 (Del2) had fusion activity.
- Fig. 1 IB shows the levels of expression of full length and deletion mutants of the S glycoprotein as measured by Western analysis. Equal amount of cell lysates were loaded for each sample and the rabbit polyclonal antibody P540 was used for detection.
- Fig. 1 IC illustrates that the full length S glycoprotein and the Dell and Del 2 deletion mutants are expressed on the cell surface as measured by flow cytometry. The level of surface expression was low although the negative control where the cells were transfected with an empty plasmid was clearly distinguishable to the left of the other three curves.
- Fig. 12A-B illustrates that dimeric SI binds more efficiently to the receptor ACE2 than monovalent fragments containing the receptor binding domain.
- Fig. 12A shows the relative levels of expression of different S fragments as detected by ELISA using 200 ⁇ l of culture supematants from cells transfected with S276, S319-518 and S537 constructs.
- Fig. 12B shows the level of binding by S fragments to ACE2 as measured by ELISA.
- the tagged ACE2 was bound to plates by an anti-C9 antibody that had been previously coated on the plates.
- the supematants from cell cultures where the cells were transfected with various S proteins were mixed and incubated in ELISA plates either with (hatched bars) or without (open bars) anti-c-Myc antibody. The highest level of expression or binding is assumed to be 100 %.
- Fig. 13A-B illustrates that the soluble S ectodomain is trimeric under the conditions of size-exclusion chromatography.
- purified Se was run on a gel filtration column that was calibrated by using proteins with known molecular weight. BSA in equal amount was included as an internal control.
- Fig. 13B different fractions were collected from the gel filtration column and analyzed by Western blot.
- Fig. 14A illustrates that a DNA vaccine of the invention can elicit very high titer anti-SARS-CoN sera in mice.
- Mice 1 A-5A were immunized with D ⁇ A encoding the S319-518 fragment that contains the spike protein receptor binding domain (RBD).
- Mice 1B-5B were immunized with RBD-encoding
- Fig. 14B illustrates that anti-sera from mice immunized with RBD- encoding D ⁇ A can prevent S-mediated cell fusion.
- PC denotes positive control where no serum was added.
- serum dilution factors 10 (designated 0.1), 100
- Fig. 15 illustrates that soluble S glycoprotein fragments inhibit S- mediated cell fusion. 10 ug/ml of various S fragments were incubated with ACE2-expressing cells first for 10 min at room temperature. The ACE2- expressing cells were then mixed with S expressing cells and the fusion assay was carried out as described in the Examples. The Y-axis is the OD 5 5 for each sample after the background noise was subtracted. Numbers of each construct represent the starting and ending residues of the respective polypeptide.
- SARS represents an important public health concern. Methods to diagnose and treat persons who are infected with SARS-CoN provide the opportunity to either prevent or confrol further spread of infection by SARS- CoN. These methods are especially important due to the ability of SARS-CoN to infect persons through an airborne route.
- the present invention provides nucleic acids that encode segments of the amino acid sequence of the spike protein of SARS-CoN.
- the present invention also provides polypeptides that , correspond in amino acid sequence to segments of the amino acid sequence of the spike protein of SARS-CoN.
- the invention also provides peptide fragments and conservative variants of the spike protein of SARS-CoN, in addition to coupled proteins and peptidomimetics that have portions which correspond in amino acid sequence to the spike protein.
- the spike protein is important because it is present on the outside of intact SARS-CoN. Thus, it presents a target that can be used to inhibit or eliminate an intact virus before the virus has an opportunity to infect a cell.
- the nucleic acids and polypeptides of the invention offer advantages over the full length spike protein because the nucleic acids are easy to produce and the polypeptides of the invention are produced in large amounts in soluble form.
- the polypeptides of the invention offer additional advantages over the native spike protein because they can be made to have increased resistant to degradation when administered to an animal.
- the polypeptides of the invention can also be formulated to increase their antigenicity to make them more efficient antigens to elicit an immune response when administered to an animal, such as a human.
- the invention provides nucleic acids and polypeptide antigens that may be used to formulate vaccines and immune compositions that can be used to immunize and treat persons who are infected with SARS-CoN.
- the invention provides antibodies that bind to the spike protein of SARS-CoN which may be used to diagnose, immunize, and treat persons infected with SARS-CoN.
- an "adjuvant” is generally defined as a substance that nonspecifically enhances the immune response to an antigen.
- adjuvants may be employed with the immunopeptides and immunofragopeptides of this invention.
- Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
- Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.
- Freund's Incomplete Adjuvant and Complete Adjuvant Difco Laboratories, Detroit, Mich.
- Merck Adjuvant 65 Merck and Company, Inc., Rahway, NJ.
- aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate
- salts of calcium, iron or zinc an insoluble suspension of acylated tyrosine
- Cytokines such as GM-CSF or interleukin- 2, -7, or -12, may also be used as adjuvants.
- An "animal” refers to an organism that can mount an immune response upon antigenic challenge. For example, reptiles, avians, and mammals are able to produce antibodies in response to an antigenic challenge. -Antibodies raised in non-human organisms are thought to be useful in diagnostic assays to reduce or eliminate cross-reactivity.
- An "aptamer” is a peptide, polypeptide or nucleic acid (RNA or DNA) that binds to a polypeptide or peptide fragment of the invention.
- a “carrier protein” refers to a polypeptide that can be coupled with a polypeptide or a peptide fragment of the invention to form a coupled protein.
- a carrier protein may be coupled to a polypeptide or peptide fragment in order to increase the solubility or the immunogenicity of the polypeptide or peptide fragment.
- a carrier protein may also be coupled to a polypeptide or peptide fragment to provide a tag which provides for separation or detection of the coupled protein.
- biotin may be used as a carrier protein that is coupled to a polypeptide or peptide fragment to create a coupled protein which can then be isolated through interaction with avidin, or detected through use of a fluorescently tagged avidin.
- a carrier protein that is bound by an antibody can be coupled to a polypeptide or peptide fragment to create a coupled protein that is bound by the antibody which binds to the carrier protein of the coupled protein.
- the invention encompasses isolated or substantially purified nucleic acids, peptides, polypeptides or proteins.
- an "isolated" nucleic acid, DNA or RNA molecule or an “isolated” polypeptide is a nucleic acid, DNA molecule, RNA molecule, or polypeptide that exists apart from its native environment and is therefore not a product of nature.
- nucleic acid, DNA molecule, RNA molecule or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell.
- a "purified" nucleic acid molecule, peptide, polypeptide or protein, or a fragment thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
- an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid ⁇ i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
- the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
- a protein, peptide or polypeptide that is substantially free of cellular material includes preparations of protein, peptide or polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating protein.
- culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
- polypeptide, peptide and protein are used interchangeably herein.
- a peptide or polypeptide "fragment" as used herein refers to a less than full length peptide, polypeptide or protein.
- a peptide or polypeptide fragment can have is at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids in length, or single unit lengths thereof.
- fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length.
- size of a peptide fragment There is no upper limit to the size of a peptide fragment.
- peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids or less than about 250 amino acids in length.
- the peptide fragment can elicit an immune response when used to inoculate an animal.
- a peptide fragment may be used to elicit an immune response by inoculating an animal with a peptide fragment in combination with an adjuvant, a peptide fragment that is coupled to an adjuvant, or a peptide fragment that is coupled to arsanilic acid, sulfanilic acid, an acetyl group, or a picryl group.
- a peptide fragment can include a non-amide bond, and can be a peptidomimetic.
- the term "soluble" as used herein refers to the ability of a polypeptide to be solvated in an aqueous solution.
- a soluble peptide can be mixed with an aqueous medium such that at least a detectable portion of the peptide is present in the aqueous medium.
- the peptide may be detected through use of common techniques, such as absorbance of light, fluorescence, the ability to bind / dyes, the ability to reduce silver ions, and the like.
- the term "specifically binds" refers to an antibody that binds to a single epitope, but which does not bind to more than one epitope. Accordingly, an antibody that specifically binds to a polypeptide will bind to an epitope that present on the polypeptide, but which is not present on other polypeptides.
- polypeptides Polypeptides. peptide fragments., coupled proteins., immunopeptides., and peptidomimetics of the invention
- the invention provides a polypeptide which has an amino acid sequence that corresponds to the amino acid sequence of the spike protein from the virus (SARS-CoN) that is etiologically linked to severe acute respiratory syndrome (SARS).
- SARS-CoN spike protein from the virus
- SARS-CoN severe acute respiratory syndrome
- a representative amino acid sequence is provided by SEQ ID NO: 1, whose sequence is provided below for easy reference.
- the invention also provides peptide fragments which have amino acid sequences that correspond to a fragment of the spike protein from the virus (SARS-CoN ) that is etiologically linked to severe acute respiratory syndrome (SARS).
- SARS-CoN spike protein from the virus
- SARS-CoN severe acute respiratory syndrome
- 25 be three or more amino acids in length, and produce an immune response when used to immunize an animal.
- the invention also provides coupled proteins having a carrier protein coupled to a polypeptide or peptide fragment of the invention.
- the carrier protein may be used to increase the solubility of the coupled protein.
- the carrier protein may also be used to increase the immunogenicity of the coupled protein to increase production of antibodies that bind to the polypeptide or peptide fragment of the invention.
- the carrier protein may also be used to provide for the separation or detection of a coupled protein. Accordingly, a coupled protein can be detected or isolated by interaction with other components that bind to the carrier protein portion of the coupled protein. For example, a coupled protein having avidin as a carrier protein can be detected or separated with biotin through use of known methods. Numerous carrier proteins may be used to create coupled proteins of the invention.
- carrier proteins examples include, keyhole limpet hemacyanin, bovine serum albumin, ovalbumin, mouse serum albumin, rabbit serum albumin, and the like.
- a carrier protein may be coupled to a polypeptide or peptide fragment of the invention by creation of a fusion protein through use of recombinant methods.
- a carrier protein may also be coupled to a polypeptide or peptide fragment of the invention through use of chemical linking methods, or through use of a chemical linker. Such coupling methods are known in the art and have been described. Harlow et al.,
- the invention provides immunopeptides having a polypeptide or a peptide fragment of the invention coupled to arsanilic acid, sulfanilic acid, an acetyl group, or a picryl group. Methods to couple such groups to peptides are known and have been reported. Weigle, J. Exp. Med.. 116:913-928 (1962); Weigle, J. Exp. Med.. 122:1049-1062 (1965); Weigle, J. Exp. Med.. 121:289- 308 (1965).
- polypeptides and peptide fragments of the invention may be in glycosylated form, or in unglycosylated form.
- a polypeptide or peptide fragment of the invention may be soluble or insoluble in aqueous solution.
- the polypeptides and peptide fragments of the invention may be conservative variants.
- a conservative variant is a polypeptide or peptide fragment derived from a full-length polypeptide, such as that exemplified by SEQ ID NO: 1, by deletion (so-called truncation), addition, or subtraction of one or more amino acids to the N-terminal and/or C-terminal end of the full-length polypeptide; deletion, addition or subtraction of one or more amino acids at one or more sites in the full-length polypeptide.
- Such variants may result from, for example, genetic polymorphism or from human manipulation. Methods for such manipulations are generally known in the art.
- amino acid sequence variants of SEQ ID NO: 1 can be prepared by mutagenesis of DNA encoding the polypeptide.
- Conservative substitutions such as exchanging one amino acid with another having similar properties, are preferred. For example, substitution of a hydrophobic amino acid for another, or substitution of a hydrophilic amino acid for another.
- Routine screening assays can be used to determine if a substituted polypeptide or peptide fragment derived from SEQ ID NO: 1 produces an immune response when administered to a mammal. Examples of such screening assays are well known in the art and include enzyme linked immunosorbant assays, radioimmuno assays, chromium release assays, and the like. Such assays have been described. Harlow et al., Antibodies: A Laboratory Manual, page 319 (Cold Spring Harbor Pub. 1988).
- the invention provides peptidomimetics of the polypeptides and peptide fragments of the invention.
- a peptidomimetic describes a peptide analog, such as those commonly used in the pharmaceutical industry as non-peptide drugs, with properties analogous to those of the template peptide. (Fauchere, J., Adv. Drag Res.. 15: 29 (1986) and Evans et al., J. Med. Chem.. 30:1229 (1987)).
- Advantages of peptide mimetics over natural polypeptide embodiments may include more economical production, greater chemical stability, altered specificity and enhanced pharmacological properties such as half-life, absorption, potency and efficacy.
- polypeptides, peptide fragments, coupled proteins, and peptidomimetics of the invention can be modified for in vivo use by the addition, at the amino-terminus and/or the carboxyl-terminus, of a blocking agent to decrease degradation in vivo. This can be useful in those situations in which the polypeptide termini tend to be degraded by proteases prior to cellular uptake.
- blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the polypeptide, peptide fragment, coupled protein, and peptidomimetic to be administered.
- polypeptides and peptide fragments that are amino-terminally and carboxyl-terminally blocked. The ability of a polypeptide or peptide fragment of the invention to produce an immune response may be tested through numerous art recognized methods.
- polypeptides and peptide fragments of the invention may be used within screening assays to identify or isolate antibodies that bind to the polypeptides or peptide fragments of the invention, or the spike protein from SARS-CoN.
- the polypeptides or peptide fragments may be used in phage display assays to isolate antibodies that bind to the polypeptides or peptide fragments.
- the polypeptides or peptide fragments of the invention may be bound to a solid support to which antibodies are contacted such that antibodies wliich bind to the polypeptides or peptide fragments become immobilized on the solid support.
- polypeptides and peptide fragments of the invention may be used to isolate antibodies according to many other methods known in the art.
- Expression systems that may be used for small or large scale production of the, coupled proteins, polypeptides or peptide fragments of the invention include, but are not limited to, cells or microorganisms that are transformed with a recombinant nucleic acid construct that contains a nucleic acid segment of the invention.
- recombinant nucleic acid constructs may include bacteriophage DNA, plasmid DNA, cosmid DNA, or viral expression vectors.
- cells and microorganisms that may be transformed include bacteria (for example, E. coli or B.
- subtilis subtilis
- yeast for example, Saccharomyces and Pichia
- insect cell systems for example, baculovirus
- plant cell systems or mammalian cell systems (for example, COS, CHO, BHK, 293, NERO, HeLa, MDCK, W138, and ⁇ IH 3T3 cells).
- mammalian cell systems for example, COS, CHO, BHK, 293, NERO, HeLa, MDCK, W138, and ⁇ IH 3T3 cells.
- Also useful as host cells are primary or secondary cells obtained directly from a mammal that are transfected with a plasmid vector or infected with a viral vector.
- suitable expression vectors include, without limitation, plasmids and viral vectors such as herpes viruses, retroviruses, vaccinia viruses, attenuated vaccinia viruses, canary pox viruses, adenoviruses, adeno-associated viruses, lentivirases and herpes viruses, among others. Synthetic methods may also be used to produce polypeptides and peptide fragments of the invention. Such methods are known and have been reported. Merrifield, Science. 85:2149 (1963).
- nucleic acid segments, expression cassettes, and nucleic acid constructs of the invention The present invention provides isolated nucleic acid segments that encode the polypeptides, peptide fragments, and coupled proteins of the invention.
- the nucleic acid segments of the invention also include segments that encode for the same amino acids due to the degeneracy of the genetic code.
- the amino acid threonine is encoded by ACU, ACC, ACA and ACG and is therefore degenerate. It is intended that the invention includes all variations of the polynucleotide segments that encode for the same amino acids. Such mutations are known in the art (Watson et al, Molecular Biology of the Gene, Benjamin Cummings 1987).
- Mutations also include alteration of a nucleic acid segment to encode for conservative amino acid changes, for example, the substitution of leucine for isoleucine and so forth. Such mutations are also known in the art.
- the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
- the nucleic acid segments of the invention may be contained within a vector.
- a vector may include, but is not limited to, any plasmid, phagemid, F- factor, virus, cosmid, or phage in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable.
- the vector can also transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extra-chromosomally (e.g. autonomous replicating plasmid with an origin of replication).
- the nucleic acid segment in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in vitro or in a host cell, such as a eukaryotic cell, or a microbe, e.g. bacteria.
- the vector may be a shuttle vector that functions in multiple hosts.
- the vector may also be a cloning vector that typically contains one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion.
- a cloning vector may also contain a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Examples of marker genes are tetracycline resistance or ampicillin resistance. Many cloning vectors are commercially available (Stratagene, New England Biolabs, Clonetech). The nucleic acid segments of the invention may also be inserted into an expression vector.
- an expression vector typically contains prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance gene to provide for the amplification and selection of the expression vector in a bacterial host; regulatory elements that control initiation of transcription such as a promoter; and DNA elements that control the processing of transcripts such as introns, or a transcription termination / polyadenylation sequence.
- a vector into which a nucleic acid segment is to be inserted is treated with one or more restriction enzymes (restriction endonuclease) to produce a linearized vector having a blunt end, a "sticky" end with a 5' or a 3' overhang, or any combination of the above.
- the vector may also be treated with a restriction enzyme and subsequently treated with another modifying enzyme, such as a polymerase, an exonuclease, a phosphatase or a kinase, to create a linearized vector that has characteristics useful for ligation of a nucleic acid segment into the vector.
- the nucleic acid segment that is to be inserted into the vector is treated with one or more restriction enzymes to create a linearized segment having a blunt end, a "sticky" end with a 5' or a 3' overhang, or any combination of the above.
- the nucleic acid segment may also be treated with a restriction enzyme and subsequently treated with another DNA modifying enzyme.
- DNA modifying enzymes include, but are not limited to, polymerase, exonuclease, phosphatase or a kinase, to create a nucleic acid segment that has characteristics useful for ligation of a nucleic acid segment into the vector.
- the treated vector and nucleic acid segment are then ligated together to form a construct containing a nucleic acid segment according to methods available in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). Briefly, the treated nucleic acid fragment and the treated vector are combined in the presence of a suitable buffer and ligase. The mixture is then incubated under appropriate conditions to allow the ligase to ligate the nucleic acid fragment into the vector.
- the invention also provides an expression cassette which contains a nucleic acid sequence capable of directing expression of a particular nucleic acid segment of the invention, such as SEQ ID NO: 2, either in vitro or in a host cell.
- a nucleic acid segment of the invention may be inserted into the expression cassette such that an anti-sense message is produced.
- the expression cassette is an isolatable unit such that the expression cassette may be in linear form and functional for in vitro transcription and translation assays.
- the materials and procedures to conduct these assays are commercially available from Promega Corp. (Madison, Wisconsin).
- an in vitro transcript may be produced by placing a nucleic acid sequence under the control of a T7 promoter and then using T7 RNA polymerase to produce an in vitro transcript. This transcript may then be translated in vitro through use of a rabbit reticulocyte lysate.
- the expression cassette can be incorporated into a vector allowing for replication and amplification of the expression cassette within a host cell or also in vitro transcription and translation of a nucleic acid segment.
- Such an expression cassette may contain one or a plurality of restriction sites allowing for placement of the nucleic acid segment under the regulation of a regulatory sequence.
- the expression cassette can also contain a termination signal operably linked to the nucleic acid segment as well as regulatory sequences required for proper translation of the nucleic acid segment.
- the expression cassette containing the nucleic acid segment may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
- the expression cassette may also be oonnee which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Expression of the nucleic acid segment in the expression cassette may be under the control of a constitutive promoter or an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
- the expression cassette may include in the 5 '-3' direction of transcription, a transcriptional and translational initiation region, a nucleic acid segment and a transcriptional and translational termination region functional in vivo and /or in vitro.
- the termination region may be native with the transcriptional initiation region, may be native with the nucleic acid segment, or may be derived from another source.
- the regulatory sequence can be a polynucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing or stability, or translation of the associated coding sequence.
- Regulatory sequences can include, but are not limited to, enhancers, promoters, repressor binding sites, translation leader sequences, introns, and polyadenylation signal sequences. They may include natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.
- regulatory sequences are not limited to promoters, some useful regulatory sequences include constitutive promoters, inducible promoters, regulated promoters, tissue-specific promoters, viral promoters and synthetic promoters.
- a promoter is a nucleotide sequence which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
- a promoter includes a minimal promoter, consisting only of all basal elements needed for transcription initiation, such as a TATA-box and/or initiator that is a short DNA sequence comprised of a TATA- box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
- a promoter may be derived entirely from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments.
- a promoter may contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
- the invention also provides a construct containing a vector and an expression cassette.
- the vector may be selected from, but not limited to, any vector previously described. Into this vector may be inserted an expression cassette through methods known in the art and previously described (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)).
- the regulatory sequences of the expression cassette may be derived from a source other than the vector into which the expression cassette is inserted.
- a construct containing a vector and an expression cassette is formed upon insertion of a nucleic acid segment of the invention into a vector that itself contains regulatory sequences.
- an expression cassette is formed upon insertion of the nucleic acid segment into the vector.
- Vectors containing regulatory sequences are available commercially and methods for their use are known in the art (Clonetech, Promega, Stratagene).
- Immune compositions and vaccines of the invention provides immune compositions and vaccines that can be used to produce an immune response against the virus that is etiologically linked to severe acute respiratory syndrome when administered to an animal.
- the immune response may be a humoral immune response or a cellular immune response.
- An immune composition of the invention can include an adjuvant and a nucleic acid, polypeptide, peptide fragment, a peptidomimetic, a coupled protein, an immunopeptide of the invention, or any combination thereof.
- An immune composition can contain an adjuvant that is not chemically linked to a polypeptide, peptide fragment, a peptidomimetic, a coupled protein, or an immunopeptide of the invention.
- An immune composition can contain an adjuvant that is chemically linked to a polypeptide, peptide fragment, a peptidomimetic, a coupled protein, or an immunopeptide of the invention.
- An immune composition of the invention can also include a pharmaceutically acceptable diluent or carrier.
- An immune composition may be manufactured conventionally.
- a nucleic acid, polypeptide, peptide fragment, peptidomimetic, coupled protein, immunopeptide, or any combination thereof that is contained in the composition may be combined with a pharmaceutically acceptable diluent or carrier.
- Examples of pharmaceutically acceptable diluent or carriers include water or a saline solution, such as phosphate-buffered saline (PBS), hi general, the pharmaceutically acceptable diluent or carrier is selected on the basis of the mode and route of administration and of standard pharmaceutical practices.
- PBS phosphate-buffered saline
- compositions may contain adjuvants as disclosed herein and as known in the art.
- Aluminum compounds may be used as adjuvants. Such aluminum compounds include, aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and the like.
- the nucleic acid, polypeptide, peptide fragment, peptidomimetic, coupled protein, immunopeptide, or any combination thereof may be absorbed or precipitated on an aluminum compound according to standard methods.
- adjuvants include polyphosphazene (WO 95/2415), DC-chol (3-beta-[N-(N', N'-dimethylaminomethane) carbamoyl) cholesterol] (U.S. Pat. No. 5,283,185 and WO 96/14831), QS-21 (WO 88/9336) and RIBI from ImmunoChem (Hamilton, Montana), hnmunostimulatory oligonucleotides containing unmethylated CpG dinucleotides (“CpG”) are known in the art as being adjuvants when administered by both systemic and mucosal routes (WO 96/02555, EP 468520, Davis et al., J.
- CpG when formulated into immune compositions or vaccines, is generally administered in free solution together with free antigen (WO 96/02555; McCluskie and Davis, Immunol., 161:4463 (1998)) or covalently conjugated to an antigen (PCT Publication No. WO 98/16247), or formulated with a carrier such as aluminum hydroxide. (Brazolot-Millan et al., Proc.Natl.Acad.Sci., 95:15553 (1998)).
- the invention also provides vaccines that include a nucleic acid, polypeptide, a peptide fragment, a peptidomimetic, a coupled protein, an immunopeptide of the invention, a nucleic or any combination thereof.
- Such vaccines can be formulated as described herein or as known in the vaccine arts.
- a viral vaccine may be created that expresses a polypeptide, a peptide fragment, or a coupled protein of the invention according to methods known in the art.
- viral vectors that may be used include, adenoviruses, herpes viruses, vaccinia viruses, canarypox viruses, and the like.
- Vaccines can also be formulated as a liposome. Such formulations are known to those skilled in the art.
- the invention also provides nucleic acid based vaccines that express a polypeptide, a peptide fragment, or a coupled protein of the invention.
- a nucleic acid vaccine can express a polypeptide having SEQ ID NO: 1, 13, 14, 15, 20-59, 61-63 or a fragment of SEQ ID NO: 1. Inoculation ofan animal with a nucleic acid construct that encodes a polypeptide, a peptide fragment, or a coupled protein of the invention may lead to a humoral and cell- mediated immune response to the encoded antigen.
- nucleic acid vaccines provide for eliciting strong cytotoxic T-lymphocyte (CTL) responses. These responses occur because the nucleic acid-encoded polypeptides are synthesized in the cytosol of transfected cells.
- CTL cytotoxic T-lymphocyte
- nucleic acid constructs that are produced in bacteria are rich in unmethylated CpG nucleotides that are recognized as foreign by macrophages. Thus, they elicit an innate immune response that enhances adaptive immunity.
- nucleic acid vaccines are effective even when administered without adjuvants.
- Direct injection ofan expression cassette into living host cells transforms a number of the cells and causes them to express the introduced nucleic acid and thereby express a gene product.
- the transfected cells may display fragments of the expressed antigens on their cell surfaces together with major histocompatibility class I (MHC I) or class II (MHC II) complexes.
- MHC I major histocompatibility class I
- MHC II class II complexes.
- Nucleic acid constructs can be introduced into cells more efficiently by inducing muscle degeneration prior to the injection of the nucleic acid construct into an animal, including a human (Vitadello et. al., Hum. Gene.
- Nucleic acid constructs can be administered in a pharmaceutically acceptable carrier.
- Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to a human or other mammalian subject, e.g., physiological saline.
- a therapeutically effective amount is an amount of the nucleic acid construct that is capable of producing an immune response (e.g., an enhanced T-cell response or antibody production) in a treated animal.
- the dosage for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drags being administered concurrently. Dosages will vary, but a preferred dosage for administration of a nucleic acid construct is from approximately 10 6 to 10 12 copies of the nucleic acid constract. This does can be repeatedly administered, as needed. Numerous routes of administration may be used to administer nucleic acid constructs.
- nucleic acid based vaccines can also be administered through use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages.
- PLGA poly-lacto-co-glycolide
- the nucleic acid construct is encapsulated in these microparticles, which are taken up by macrophages and gradually biodegraded within the cell, thereby releasing the nucleic acid construct. Once released, the nucleic acid is expressed within the cell.
- liposomes can be prepared by standard methods.
- the nucleic acid constructs can be incorporated alone into these delivery vehicles or co- incorporated with tissue-specific antibodies.
- a molecular conjugate can be prepared that is composed of a nucleic acid constract attached to poly-L-lysine by electrostatic or covalent forces.
- lymphoid tissue specific targeting can be achieved by the use of lymphoid tissue-specific transcriptional regulatory elements (TRE) such as a B lymphocyte, T lymphocyte, or dendritic cell specific TRE. Lymphoid tissue specific TRE are known (Thompson et al., Mol. Cell. Biol, 12:1043 (1992); Todd et al., J. Exp. Med., 177:1663 (1993); Penix et al., J. Exp. Med..178:1483 (1993)).
- TRE lymphoid tissue-specific transcriptional regulatory elements
- the invention also provides microbe based vaccines.
- these vaccines relate to microbes that have been transformed with a nucleic acid construct that provides for the expression of a polypeptide, a peptide fragment, or a coupled protein of the invention.
- Listeria monocytogenes may be used as a vector to elicit T-cell immunity. This is because it infects antigen- presenting cells and also because infection originates at the mucosa. Lieberman and Frankel, Vaccine. 20:2007-10 (2002).
- Listeria may be transformed with a nucleic acid constract that provides for the expression of a polypeptide, a peptide fragment, or a coupled protein that elicits an immune response against the spike protein from the coronaviras that causes severe acute respiratory syndrome.
- Highly attenuated forms of Listeria may be constructed according to methods reported in the art. Lieberman and Frankel, Vaccine, 20:2007 (2002).
- Salmonella may also be used as a vector to elicit a cytotoxic T lymphocyte (CTL) response against the coronavirus that causes severe acute respiratory syndrome. Pasetti et al., Infect hnmun., 70:4009 (2002).
- CTL cytotoxic T lymphocyte
- an immune composition or vaccine may be admimstered by any conventional route used in the field of vaccines.
- an immune composition or vaccine can be admimstered 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, peptidomimetic, coupled protein, immunopeptide, 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.
- Antibodies and aptamers of the invention provides antibodies that bind to an amino acid sequence as set forth in SEQ ID NO: 1, 13, 14, 15, 20-59, 60, 61, 62, 63 or a fragment of SEQ ID NO: 1, or conservative variants thereof. Such antibodies are useful for the diagnosis, immunization against, and treatment of severe acute respiratory syndrome (SARS). In some embodiments, the antibody binds to a peptide having SEQ ID NO:58 or 59.
- Antibodies that bind to the P540 peptide (SEQ ID NO:59) are highly effective, and can detect spike polypeptides even after extensive dilution. For example, a P540 antibody preparation diluted 1:10,000 could still detect spike polypeptides.
- Antibodies can be prepared using an intact polypeptide or peptide fragment of interest as the immunizing antigen.
- the polypeptide or fragment used to immunize an animal can be derived from translated cDNA or chemical synthesis.
- a polypeptide or peptide fragment can be coupled to a carrier protein, if desired.
- carrier proteins wliich are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- a coupled protein can be used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
- polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or peptide fragment to which the antibodies were raised is bound.
- a matrix to which the polypeptide or peptide fragment to which the antibodies were raised is bound.
- monoclonal antibodies Colligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference
- an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.
- An antibody suitable for binding to a polypeptide or peptide fragment is specific for at least one portion of a region of the polypeptide.
- one of skill in the art can use a peptide fragment to generate appropriate antibodies of the invention.
- Antibodies of the invention include polyclonal antibodies, monoclonal antibodies, and fragments of polyclonal and monoclonal antibodies.
- polypeptide or peptide fragment is injected into an animal host, preferably according to a predetermined schedule incorporating one or more booster immunizations, and the animal is bled periodically.
- Polyclonal antibodies specific for the polypeptide or peptide fragment may then be purified from such antisera by, for example, affinity chromatography using the polypeptide or peptide fragment coupled to a suitable solid support.
- affinity chromatography using the polypeptide or peptide fragment coupled to a suitable solid support.
- the preparation of monoclonal antibodies likewise is conventional (Kohler & Milstein, Nature, 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. 1988)), which are hereby incorporated by reference.
- monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
- Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques.
- isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (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)).
- Methods of in vitro and in vivo multiplication of monoclonal antibodies is well-known to those skilled in the art.
- Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally replenished by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
- suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
- a mammalian serum such as fetal calf serum or trace elements
- growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages.
- Production in vitro provides relatively pure antibody preparations and allows scale-up to yield large amounts of the desired antibodies.
- Large scale hybridoma cultivation can be carried out by homogenous suspension culture in an air reactor, in a continuous stirrer reactor, or immobilized or entrapped cell culture.
- Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., osyngeneic mice, to cause growth of antibody-producing tumors.
- the animals are primed with a hydrocarbon, especially oils such as pristine tetramethylpentadecane prior to injection.
- the desired monoclonal antibody is recovered from the body fluid of the animal.
- Antibodies can also be prepared through use of phage display techniques.
- an organism is immunized with an antigen, such as a polypeptide or peptide fragment of the invention. Lymphocytes are isolated from the spleen of the immunized organism.
- RNA is isolated from the splenocytes and mRNA contained within the total RNA is reverse transcribed into complementary deoxyribonucleic acid (cDNA).
- cDNA encoding the variable regions of the light and heavy chains of the immunoglobulin is amplified by polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- the light and heavy chain amplification products may be linked by splice overlap extension PCR to generate a complete sequence and ligated into a suitable vector.
- E. coli are then transformed with the vector encoding the scFV, and are infected with helper phage, to produce phage particles that display the antibody on their surface.
- the heavy chain amplification product can be fused with a nucleic acid sequence encoding a phage coat protein, and the light chain amplification product can be cloned into a suitable vector.
- E. coli expressing the heavy chain fused to a phage coat protein are transformed with the vector encoding the light chain amplification product.
- the disulphide linkage between the light and heavy chains are established in the periplasm of E. coli.
- the result of this procedure is to produce an antibody library with up to 10 9 clones.
- the size of the library can be increased to 10 phage by later addition of the immune responses of additional immunized organisms that may be from the same or different hosts.
- Antibodies that recognize a specific antigen can be selected through panning. Briefly, an entire antibody library can be exposed to an immobilized antigen against which antibodies are desired. Phage that do not express an antibody that binds to the antigen are washed away. Phage that express the desired antibodies are immobilized on the antigen. These phage are then eluted and again amplified in E. coli. This process can be repeated to enrich the population of phage that express antibodies that specifically bind to the antigen. After phage are isolated that express an antibody that binds to an antigen, a vector containing the coding sequences for the antibody can be isolated from the phage particles and the coding sequences can be recloned into a suitable vector to produce an antibody in soluble form.
- a human phage library can be used to select for antibodies, such as monoclonal antibodies, that bind to the spike protein from SARS-CoV.
- splenocytes may be isolated from a human that is infected, or not infected, with SARS-CoV and used to create a human phage library according to methods as described above and known in the art. These methods may be used to obtain human monoclonal antibodies that bind to the spike protein of SARS-CoV. Phage display methods to isolate antigens and antibodies are known in the art and have been described (Gram et al., Proc. Natl. Acad.
- An antibody of the invention may be derived from a "humanized" monoclonal antibody.
- Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts.
- the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
- General techniques for cloning murine immunoglobulin variable domains are described (Orlandi et al, Proc. Nat'l Acad. Sci. USA. 86:3833 (1989) which is hereby incorporated in its entirety by reference).
- antibodies of the present invention may be derived from a human monoclonal antibody.
- Such antibodies are obtained from fransgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
- elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci.
- the fransgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
- Methods for obtaining human antibodies from fransgenic mice are described (Green et al., Nature Genet., 7:13 (1994); Lonberg et al., Nature, • 368:856 (1994); and Taylor et al., Int.
- Antibody fragments of the 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 by 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 3.5S Fab' monovalent fragments.
- Fv fragments comprise, an association of VH and V L chains. This association may be noncovalent (Inbar et al, Proc. Nat'l Acad. Sci. USA, 69:2659 (1972)).
- the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (Sandhu, Crit. Rev. Biotech.. 12:437 (1992)).
- the Fv fragments comprise VH and V L chains connected by a peptide linker.
- These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L 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 (Whitlow et al, Methods: A Companion to Methods in Enzymology, Vol. 2, page 97 (1991); Bird et al, Science.
- 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 (Larrick et al, Methods: A Companion to Methods in Enzymology, Vol.
- An antibody of the invention may be coupled to a toxin.
- Such antibodies may be used to treat animals, including humans, that are infected with the virus that is etiologically linked to severe acute respiratory syndrome.
- an antibody that binds to the spike protein of the coronaviras that is etiologically linked to severe acute respiratory syndrome may be coupled to a tetanus toxin and administered to an animal suffering from infection by the aforementioned virus.
- the toxin-coupled antibody is thought to bind to a portion of a spike protein presented on an infected cell, and then kill the infected cell.
- An antibody of the invention may be coupled to a detectable tag.
- Such antibodies may be used within diagnostic assays to determine if an animal, such as a human, is infected with SARS-CoV.
- detectable tags include, fluorescent proteins (i.e., green fluorescent protein, red fluorescent protein, yellow fluorescent protein), fluorescent markers (i.e., fluorescein isothiocyanate, rhodamine, texas red), radiolabels (i.e., H, P, I), enzymes (i.e., ⁇ - galactosidase, horseradish peroxidase, /3-glucuronidase, alkaline phosphatase), or an affinity tag (i.e., avidin, biotin, sfreptavidin).
- fluorescent proteins i.e., green fluorescent protein, red fluorescent protein, yellow fluorescent protein
- fluorescent markers i.e., fluorescein isothiocyanate, rhodamine, texas red
- radiolabels i.e., H, P, I
- the invention also provides aptamers to the polypeptides and peptide fragments of the invention.
- Aptamers of the invention can be peptide or nucleic acid aptamers.
- Peptide aptamers are peptides that bind to a polypeptide or peptide fragment of the invention with affinities that are often comparable to those for monoclonal antibody-antigen complexes.
- nucleic acid aptamers are nucleic acids that bind to a polypeptide or peptide fragment of the invention with strong affinities, for example, affinities that are often comparable to those for monoclonal antibody-antigen complexes.
- nucleic acid aptamers can be isolated through use of a library of random oligonucleotide sequences. The library is screened to ascertain which oligonucleotide binds to the S polypeptides and peptide fragments of the invention. The bound oligonucleotides are eluted from the immobilized polypeptides or peptide fragments and are then amplified by PCR.
- Peptide aptamers can be isolated by mRNA display of a library that contains a promoter, a start codon, a nucleic acid sequence that encodes random peptides.
- the DNA library also includes a nucleic acid segment that codes for a histidine tag.
- This library is transcribed using a suitable polymerase, such as T7 RNA polymerase, after which a puromycin-containing poly A linker is ligated onto the 3' end of the newly formed mRNAs.
- a suitable polymerase such as T7 RNA polymerase
- the nascent peptides form covalent bonds to the puromycin of the linker to form an mRNA-peptide fusion molecule.
- the mRNA-peptide fusion molecules are then purified through use of Ni-NTA agarose and oligo-dT-cellulose. The mRNA portion of the fusion molecule is then reverse franscribed.
- the double-stranded DNA/RNA-peptide fusion molecules are then incubated with a polypeptide or peptide fragment of the invention and unbound fusion molecules are washed away.
- the bound fusion molecules are eluted from the immobilized polypeptides or peptide fragments and are then amplified by PCR. This process may be repeated to select for aptamers having high affinity for the polypeptides and peptide fragments of the invention.
- the sequence of the nucleic acid coding for the aptamers can then be determined and cloned into a suitable vector. Methods for the preparation of peptide aptamers have been described (Wilson et al, Proc. Natl. Acad. Sci., 98:3750 (2001)). Accordingly, the invention provides aptamers that recognize the polypeptides and peptide fragments of the invention.
- compositions of the invention provides pharmaceutical compositions containing an antibody that binds to an amino acid sequence as set forth in SEQ ID NO: 1, 13, 14, 15, 20-59, 60, 61, 62, 63 or a fragment of SEQ ID NO: 1, or a conservative variant thereof, and a pharmaceutically acceptable carrier.
- the antibody binds to a peptide having SEQ ID NO:58 or 59.
- Antibodies that bind to the P540 peptide (SEQ ID NO:59) are highly effective, and can detect spike polypeptides even after extensive dilution. For example, a P540 antibody preparation at dilution 1 : 10,000 could still detect spike ' polypeptides.
- compositions of the invention may be 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.
- An oral dosage form may be formulated such that the 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, nonaqueous vehicles (which may include edible oils), or preservatives.
- An antibody can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and maybe presented in unit dosage form in ampules, 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, and may 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.
- an 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.
- an 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.
- an 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, or preservatives.
- a pharmaceutical composition may be formulated as a single unit dosage form.
- the invention provides a method to immunize an animal against severe acute respiratory syndrome.
- the method relates to administering a therapeutically effective amount ofan antibody that binds to an amino acid sequence as set forth in SEQ ID NO: 1, 13, 14, 15, 20-59, 60, 61, 62, 63 or a fragment of SEQ ID NO: 1, or a conservative variant thereof to an animal; administering an effective amount of an immune composition to an animal; administering an effective amount of a viral vaccine to an animal; or administering an effective amount of a nucleic acid vaccine to an animal.
- the animal may be a mammal, such as a human.
- 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 as set forth in SEQ ID NO: 1, 13, 14, 15, 20-55, 60, 61, 62, 63 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 situations where a patient is immune compromised 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, 13, 14, 15, 20-59, 60, 61, 62, 33 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 well known 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 or radiolabel, can then be contacted with the antibody-antigen complex such that the enzyme 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.
- 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.
- PCR polymerase chain reaction
- 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.
- Kits The invention provides a kit which contains packaging material and an antibody that binds to an amino acid sequence as set forth in SEQ ID NO: 1, 13, 14, 15, 45, 46, or 47, 58, 59, 61, 62, 63 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 antibody 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 an amino acid sequence as set forth in SEQ ID NO: 1, 13, 14, 15, 20-59, 60, 61, 62, 63 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.
- the antibody binds to an amino acid sequence set forth in SEQ ID NO:59.
- the antibody binds to an amino acid sequence as set forth in SEQ ID NO: 58.
- 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, 13, 14, 15, 45, 46, or 47, 58, 59, 61, 62, 63 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 vaccine composition that includes a polypeptide with an amino acid sequence as set forth in SEQ ID NO: 1, 13, 14, 15, 45, 46, or 47, 58, 59, 61, 62, 63 or a fragment of SEQ ID NO: 1, or a conservative variant thereof.
- the kit may also contain a device for administering the 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, wliich contains packaging material and a polypeptide with an amino acid sequence as set forth in SEQ ID NO: 1, 13, 14, 15, 45, 46, or 47, 58, 59, 61, 62, 63 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).
- Example 1 Cloning of the spike protein The nucleic acid sequence encoding the full length spike protein was obtained through use of overlapping polymerase chain reaction (PCR).
- Overlapping clones containing fragments of the spike protein were obtained from the British Columbia Cancer Agency (Vancouver, British Columbia). The following primers were used during the PCR reactions to amplify the nucleic acid sequence encoding the full-length spike protein of SARS-CoV: Clone 1 : Forward primer: 5'- A GTC GGA TCC GGT AGG CTT ATC ATT AGA G - 3' (SEQ ID NO: 3); Reverse primer: 5'- CCA TCA GGG GAG AAA GGC AC-3 (SEQ ID NO: 4).
- Clone 2 Forward primer: 5'- GTG CCT TTC TCC CCT GAT GG-3' (SEQ ID NO: 5); Reverse primer: 5'- GAA GAG CAG CGC CAG CAC C-3 ' (SEQ ID NO: 6).
- Clone 3 Forward primer: 5 '- GGT GCT GGC GCT GCT CTT C-3' (SEQ ID NO: 7); Reverse primer: 5'- A CTG TCT AGA GTT CGT TTA TGT GTA ATG-3 (SEQ ID NO: 8).
- the nucleic acid segment that resulted from overlapping PCR between the nucleic acid segments generated with the above pairs of primers contain amino acid residues from number 1 to number 1255 of the spike protein of the virus (SARS-CoV) that is etiologically linked to severe acute respiratory syndrome.
- the underlined primer sequences represent restriction enzyme cutting sites for BamHI and Xbal that were used to clone the amplified fragment into pCDNA3(+) (Invitrogen, Carlsbad, California).
- the full length spike protein gene has been cloned as shown in Fig. 1.
- Fig. 1 shows a gel for the nucleic acid segment encoding the full length spike protein inserted into the pCDNA3.1(+) vector that has been digested with the restriction enzymes (Lane 2: BamHI and Xbal; Lane 3: Hindlll).
- Example 2 Generation of amino-terminal (SI) and carboxyl-terminal (S2) fragments of the full length spike protein
- Computer analysis identified a potential functional separation site between the amino-terminus (SI) and the carboxyl-terminus (S2) of the spike protein.
- the separation site between SI and S2 is between positions between 758 and 761 ( 758 RNTR 761 ) relative to SEQ ID NO: 1.
- PCR was used to create nucleic acids that code for the amino-terminal fragment (SI), and the carboxyl- terminal fragment (S2) of the spike protein.
- SI forward primer 5'-AGTC GGA TCC GAC CGG TGC ACC ACT TTT G-3' (SEQ ID NO: 9)
- S 1 Reverse primer 5'-AGTC GGG CCC CTG TTC AGC AGC AAT ACC-3' (SEQ ID NO: 10
- SEQ ID NO: 10 were used to prepare a nucleic acid segment coding for amino acid residues 17-757 of the spike protein.
- Two restriction sites, BamHI and Apal, underlined in the two primers were used to clone the nucleic acid segment coding for the amino-terminal fragment of the spike protein (SI) gene into the pSecTag2B plasmid for expression.
- the following pair of primers S2 Forward: 5'-ACTG GGATCC GAA GTG TTC GCT CAA GTC-3' (SEQ ID NO: 11), and S2 Reverse: 5'-ACTG TCTAGA TTG CTC ATA TTT TCC C-3 ' (SEQ ID NO: 12), were used within a PCR reaction to prepare a nucleic acid segment coding for amino acid residues 762-1189 of the spike protein.
- Two restriction sites, BamHI and Xbal, underlined in the two primers were used to clone the nucleic acid segment coding for the carboxyl-terminal fragment of the spike protein (S2) gene into pCDNA3.1 (+) plasmid for expression.
- S2 spike protein
- the following pair of primers was used for PCR amplification: primer 5' GATCGGATCCGGTACAATCACAG 3' (SEQ ID NO:64) and primer 5' GATCGGGCCCGACACACTGGTTC 3' (SEQ ID NO:65).
- the amplified fragment was digested with BamHI and Apal and ligated into pSecTag2B digested with the same restriction enzymes.
- FIG. IB A schematic diagram of the position of many of the soluble spike protein fragments within the full-length spike protein is provided in Fig. IB.
- nucleic acids encoding the S fragments and full-length S polypeptides had their native leader sequence (spike protein amino acids 1-16, MFIFLLFLTLTSGSDL (SEQ ID NO:60)) replaced with a mouse k chain leader sequence (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO: 16) to permit secretion, as described below.
- Example 3 Generation of the whole soluble spike protein (sS) lacking the cvtoplasmic tail and the transmembrane domain
- the following pair of primers were used to generate a nucleic acid segment encoding a fragment of the spike protein (sS) lacking the cytoplasmic tail having amino acids 17-1189 of SEQ ID NO: 1: SI Forward: 5'- AGTC
- Example 4 Expression ofan amino-terminal and carboxyl-terminal fragment of a spike protein Expression will be done by transfecting an expression constract containing the pSecTag2B or pCDNA3.1 (+) plasmid and a nucleic acid insert that encodes an amino-terminal (SI), a carboxyl-terminal (S2) fragment, or a fragment of the spike protein of SARS-CoV that lacks the cytoplasmic tail and the transmembrane domain, into 293 or Vero E6 cells.
- SI amino-terminal
- S2 carboxyl-terminal
- S2 carboxyl-terminal
- Amino acid sequence of a soluble amino-terminal fragment of the spike protein (amino acids 17-757)
- Example 5 Generation of additional soluble fragments of the spike protein
- the nucleic acid sequence encoding a polypeptide containing amino acids 17-757 of SEQ ID NO: 1 was obtained through use of polymerase chain reaction (PCR).
- PCR polymerase chain reaction
- the following primers were used during the PCR reactions to amplify the nucleic acid sequence: Forward primer: 5' AGCT GGA TCC GAC CGG TGC ACC ACT TTT G 3' (SEQ ID NO: 9); and Reverse primer: 5' AGCT GGG CCC CTG TTC AGC AGC AAT ACC 3' (SEQ ID NO: 10).
- the resulting PCR product was digested with BamHI and Apal and, encodes a polypeptide having an amino acid sequence corresponding to SEQ ID NO: 43.
- the digested PCR product was then ligated to pSecTag2B (Invitrogen, Carlsbad, California) that was digested with the same enzymes.
- the pSecTag2B construct containing the PCR product insert encodes a polypeptide having SEQ ID NO: 46 with the mouse k chain leader sequence (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO: 16) at the N-terminus for secretion, and a myc epitope
- EQKLISEEDL SEQ ID NO: 17
- HHHHHH histidine tag
- SEQ ID NO: 18 The nucleic acid sequence encoding a polypeptide containing amino acids 17-276 of SEQ ID NO: 1 was obtained through use of polymerase chain reaction (PCR). The following primers were used during the PCR reactions to amplify the nucleic acid sequence: Forward primer: 5' AGCT GGA TCC GAC CGG TGC ACC ACT TTT G 3' (SEQ ID NO: 9); and Reverse primer: 5' CTAG CTC GAG CAA CAG CAT CTG TG 3' (SEQ ID NO: 19).
- the resulting PCR product was digested with BamHI and Xhol and, encodes an amino acid having SEQ ID NO: 44.
- the digested PCR product was then ligated to pSecTag2B (Invitrogen, Carlsbad, California) that was digested with the same enzymes.
- the pSecTag2B constract containing the PCR product insert encodes a polypeptide having SEQ ID NO: 47 with the mouse k chain leader sequence (METDTLLLWNLLLWNPGSTGD) (SEQ ID NO: 16) at the N-tenninus for secretion, and a myc epitope (EQKLISEEDL) (SEQ ID NO: 17) plus a histidine tag (HHHHHH) (SEQ ID NO: 18) at the C-terminus for affinity purification.
- the nucleic acid sequence encoding a polypeptide containing amino acids 17-537 of SEQ ID NO: 1 was obtained by digesting the nucleic acid sequence that encodes SEQ ID NO: 43 (as described above) with BamHI and Hindi.
- the nucleic acid segment produced encodes a polypeptide having SEQ ID NO: 45.
- This nucleic acid segment was ligated into a pSecTag2B vector that was digested with BamHI and EcoRV.
- the pSecTag2B constract containing the PCR product insert encodes a polypeptide having SEQ ID NO: 48 with the mouse k chain leader sequence (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO: 16) at the N-terminus for secretion, and a myc epitope (EQKLISEEDL) (SEQ ID NO: 17) plus a histidine tag (HHHHHH) (SEQ ID NO: 18) at the C- terminus for affinity purification.
- Fig. 3 The expression of these peptide fragments in mammalian cells is illustrated in Fig. 3. This figure shows that the peptide fragments can be secreted into medium in which cells that express the peptide fragments are grown. Fig. 3 also indicates that the peptide fragments are soluble in aqueous medium.
- Example 6 Structure of the Spike Protein To characterize the properties and function of the SARS-CoV S protein, nucleic acids encoding the full-length Tor2 isolate were cloned into expression vectors as described above. The Tor2 isolate is further described in Marra et al Tiie genome sequence of the SARS-associated coronavirus, Science 300:1399- 1404 (2003).
- the SI subunit is approximately encompassed within the S756 fragment.
- Example 7 Expression of peptide fragments in Escherichia coli
- a nucleic acid segment encoding a SEQ ID ⁇ O:51 peptide fragment containing amino acid residues 17-446 of SEQ ID NO: 1 was cloned into the pRSET vector (Invitrogen, San Diego, CA) to create the plasmid pRSET-S(17- 446).
- E. coli BL21DE3 cells were transformed with pRSET-S(l 7-446) and then induced with IPTG. The results of the induction are shown in Fig. 2.
- Example 8 Use of the T7 promoter to drive expression of a cloned peptide fragment of the invention
- Human 293 cells or Monkey Nero E6 cells were grown to a density of 1.2X10 6 cells/T25 flask (60 mm dish) in 5 ml of DMEM+10% FBS medium the day prior to transfection.
- the cells were then transfected, using the Polyfect (Qiagen) transfection kit according to the manufacturer's protocol, with pSecTag2B constructs (6 ug each) containing inserts coding for the various peptide fragments of the spike protein. These constructs were prepared as described above.
- a NTF7.3 vaccinia virus carrying a T7 polymerase was used to infect the transfected cells at a MOI (multiplicity of infection) of 20 (Fuerst et al, Proc. ⁇ atl Acad. Sci., 93:11371 (1986)).
- This procedure provided for the use of the T7 promoter in the pSecTag2B vector instead of the CMN promoter, which is much weaker (Nussbaum et al, J. Virol. 68:5411 (1994)).
- 1.5 ml of fresh medium was added to the cells and then the cells were transferred to a 31°C incubator.
- Example 9 Spike-Specific -Antibodies New Zealand rabbits were immunized with 0.1 mg of various peptides selected by a computer program for their immunogenicity. Serum from the immunized rabbits was tested in ELISA and Western blot for reactivity. Sera from rabbits immunized with two peptides exhibited the highest and specific activity against the spike glycoprotein and were selected for further study. Antibodies denoted D24 and P540 were elicited by the peptides DVQAPNYTQH TSSMRGC (SEQ ID NO:58) and PSSKRFQPFQQFGRDC (SEQ ID NO:59), respectively.
- the cleared medium was incubated with either Ni-NTA agarose beads (Qiagen, Valencia, CA) or an immunoprecipitating antibody plus glycoprotein G-Sepharose beads (Sigma, St. Louis, MO) for 2 h at 4 °C.
- the beads were then mixed with an equal volume of SDS gel sample buffer, boiled for 3 min, and subjected to gel analysis.
- SDS gel sample buffer For full-length S glycoprotein, cells were lysed first in PBS supplemented with 1% NP-40 and 0.5mM PMSF for 1 h at 4 °C, and centrifuged at 14,000 rpm in a table-top Eppendorf centrifuge for 20 min.
- the cleared lysate was either immunoprecipitated first or used directly in Western blotting.
- Example 11 Western blotting and slot blots
- Cells expressing the S glycoprotein were lysed first with a PBS-based NP40 lysis buffer as described above, and the debris was cleared by centrifugation.
- the medium was collected and cleared as described above.
- slot blots the cleared lysate or medium from supernatant was used directly to blot the nitrocellulose membrane following the protocol suggested by the manufacturer (Bio-Rad, Hercules, CA) and the membrane was subjected to antibody detection as in conventional Western blotting.
- a monoclonal anti-c-Myc epitope antibody (Invitrogen, Carlsbad, CA) or anti-spike protein rabbit polyclonal antibodies obtained by immunization of rabbits with spike peptides were diluted in TBST buffer. Antibodies were incubated with the membrane for 2 h, washed and then the membrane was incubated with a secondary antibody conjugated with HRP for 1 h, washed four times (each time for 15 min), and then developed using the ECL reagent (Pierce, Rockford, IL).
- Example 12 Cell-binding assay and ELISA Medium containing soluble S fragments was collected and cleared by centrifugation. Vero E6 or other cells (5x10 6 ) were incubated with 0.5 ml of cleared medium containing soluble S fragments and 2 ⁇ g of anti-c-Myc epitope antibody conjugated with HRP at 4 °C for 2 h. Cells were then washed three times with ice-cold PBS and collected by centrifugation. The cell pellets were incubated with ABTS substrate from Roche (Indianapolis, IN) at RT for 10 min, the substrate was cleared by centrifugation, and the optical density at 405 nm was measured. The result of the slot blot analysis is presented in Fig.
- ACE2 purified ACE2 (R&D, Minneapolis, MN) was adsorbed onto Maxisorp ELISA plates in pH 9.6 buffer at a concentration of 100 ng per well.
- Wells were washed and 60 ⁇ l of ABTS substrate was added to each well. The optical density (OD 4 o 5 ) was measured 20 min later.
- Example 13 Fluorescent dye redistribution cell fusion assay HeLa or 293T cells, transfected with plasmids encoding the S glycoprotein, were loaded with Calcein AM (Molecular Probes), which is converted within the cells to calcein green. The cells were incubated in medium containing 1 ⁇ g/ml Calcein AM for 1 h at 37 °C and 5% CO2, and then washed and re-suspended in fresh medium. Plated target cells, Vero E6, were stained with CMAC (Molecular Probes) by incubation in 1 ⁇ g/ml CMAC in medium for
- Example 14 /..-Galactosidase reporter gene-based cell-cell fusion assay 293T cells (1.5xl0 6 ) were plated in T25 flasks. The next day, these cells were separately transfected with pCDNA3-S, ⁇ Sectag2B-S, ⁇ CDNA3-ACE2, and pCDNA3-ACE2-Ecto using the Polyfect transfection kit (Qiagen, Valencia, CA) following the manufacturer's suggested protocol.
- Example 15 Expression of Spike Polypeptides in Mammalian Cells
- all proteins except the full-length S glycoprotein were tagged with a c-Myc epitope and a histidine tag. These proteins were expressed in 293 and Vero E6 cells after transfection with the corresponding plasmids followed by infection with vaccinia virus-expressing T7 polymerase. The tagged proteins were detected by using an anti-c-Myc monoclonal antibody (Fig. 4).
- the T7 promoter was a highly efficient promoter for expression of the S glycoprotein.
- the T7 promoter gave rise to higher levels of expression than the CMV promoter, which under most circumstances is a strong promoter (Fig. 4A).
- the S fragments were soluble and their concentration in the culture supematants was inversely proportional to their size.
- Example 16 Anti-Spike Antibodies To be able to detect unlabeled proteins, validate the data obtained by the anti-c-Myc antibody, and localize possible antigenic sites rabbit polyclonal antibodies were developed. Two of these antibodies, D24 and P540, were raised against peptides starting at residues 24 and 540, respectively. The D24 and P540 antibody preparations specifically recognized certain soluble fragments (Fig. 4C). As expected, D24 recognized all fragments; P540 recognized S756, Se, and S but not the smaller fragments (Fig. 4C; some data not shown). The D24 antibody preparation was relatively weak. However, the P540 preparation was very sensitive even at dilution 1:10,000 and was used extensively in the experiments described herein.
- the P540 antibody preparation was used to detect whether the S glycoprotein was expressed intracellularly, exfracellularly or on the cell surface. As shown in Fig. 5, the full-length S glycoprotein was expressed at the cell surface, although at low levels, as measured by flow cytometry.
- Example 17 Spike Protein Mediates Cell Fusion The full-length S glycoprotein mediates fusion at neutral pH with cells expressing receptor molecules. Cell-cell fusion assays were performed to confirm that the full-length recombinant S glycoprotein was functional, and to ascertain whether the S protein requires other viral proteins and/or low pH for its fusion activity.
- This new assay was based on fluorescent dye redistribution that is able to detect fusion of single cells. Even with this fluorescent-based assay statistically significant differences between cells transfected with plasmids encoding the full-length S glycoprotein and various negative controls were not detected. Some of the negative controls included transfection with plasmids encoding soluble S fragments at different pH (data not shown). Significant cell-cell fusion was only detected when the cells were transfected with plasmids encoding ACE2, suggesting that the higher levels of receptor expression achieved by expression of recombinant ACE2 could be important for cell-cell fusion.
- S polypeptide fragments that have the receptor binding domain could inhibit SARS-CoN fusion with animal cells, thereby inhibiting or preventing SARS-CoN infection.
- blocking, modulating or inhibiting the activity of the spike protein receptor binding domain, with an anti-RBD antibody, S polypeptide, S peptide or aptamer may be an effective preventive or treatment for SARS-CoN infection.
- Example 18 Identification of Spike Protein Receptor-Binding Domain This Example illustrates that the Spike protein receptor-binding domain is localized within residues 272 to 537 (SEQ ID ⁇ O:57), and likely within residues 303-537 (SEQ ID NO:61). Later experiments have shown that a fragment containing residues 319-517 (SEQ ID NO: 62) also has receptor binding activity.
- An assay based on the binding of various soluble fragments to receptor expressing Nero E6 cells was developed to localize the receptor-binding domain (RBD) of the S glycoprotein. This assay involved measurement of fluorescence associated with binding of antibodies directed against the S polypeptides to Nero E6 cells and was developed prior to the identification of the SARS-CoN receptor.
- Nero E6 cells that are susceptible to SARS-CoN infection were incubated with full-length S polypeptide and various soluble S fragments.
- Several cell lines that are not susceptide to SARS-CoN infection were similarly incubated with full-length S polypeptides and soluble fragments thereof.
- ACE2 as a functional receptor for the SARS-CoV.
- Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus, Nature 426: 450-54 (2003).
- the identification of ACE2 as receptor permitted further validation that the results provided above are correct.
- Fig. 8C when purified ACE2 is used in an ELISA to test for binding, the same binding pattern was observed as for the cell-binding assay. This was true for all of the S fragments tested (Fig. 8C).
- ACE2 is a receptor for the SARS-CoV SI glycoprotein and localize the RBD but also facilitate development of novel vaccine immunogens and therapeutics for prevention and treatment of SARS.
- Example 19 N-terminal and C-terminal Oligomerization of the S glycoprotein This Example illustrates that the extreme N-terminal fragment of the S glycoprotein, upstream from the RBD, may play a role in fusion, and the S ectodomain forms trimers that could mediate fusion through six-helix bundle intermediates.
- the rabbit anti-S serum used in Western and FACS analyses, P540 was developed by the inventors as described above. See also, Xiao et al. Biochem. Biophys. Res. Comm. 312: 1159-65 (2003).
- the anti- Myc epitope antibody was purchased from Invitrogen (Carlsbad, CA).
- the anti- ACE2 goat polyclonal antibody was purchased from R&D system (Minneapolis, MN) and used for detection by Western blotting.
- Site directed mutagenesis was used to create the consensus cleavage sites corresponding to that of the HIV-1 envelope glycoprotein (Env) and some coronaviruses within the full length SARS-CoV S glycoprotein gene in pCDNA3.
- the QuickChange Kit from Sfratagene (La JoUa, CA) was employed using the protocol provided by manufacturer.
- the corresponding gene fragments were amplified by PCR and cloned into the pSecTag2 expression vector (Invitrogen, Carlsbad).
- the plasmid pCDNA3-ACE2-ecto, wliich expresses the ACE2 soluble ectodomain tagged with C9 peptide was kindly provided by Michael Farzan (Harvard University, Boston MA). Protein expression and purification. Various N terminal fragments of the S glycoprotein were sub-cloned in pSecTag2 expression vector and transfected into 293T cells followed by infection with VTF7.3 as described in Xiao et al. Biochem. Biophys. Res. Comm. 312: 1159-65 (2003). The protein expressed and secreted into the medium was purified using the HiTrap Ni ⁇ -Chelating column (Pharmacia) under native conditions.
- the purified protein was dialyzed against PBS buffer and stored for further analysis.
- S glycoprotein dimerization and its interaction with ACE2 examined by co-immunoprecipitation.
- S fragment dimerization different S glycoprotein constructs, alone or in combination, were transfected to 293T cells as described in Xiao et al. Biochem. Biophys. Res. Comm. 312: 1159-65 (2003).
- Medium containing S fragments was subjected to immunoprecipitation with rabbit anti-S polyclonal antiserum P540.
- DTT was added to create reducing condition to eliminate inter-molecule interactions through disulfide bonds.
- ACE2 was immunoprecipitated by incubating with 1D4 anti-C9 monoclonal antibody and protein G-Sepharose beads at 4°C for one hour. The beads were washed four times with PBS, suspended in SDS-PAGE sample buffer, boiled for 3 min and subjected to gel separation. The presence of either ACE2 or S in the sample was examined by Western as described in Xiao et al. Biochem. Biophys. Res. Comm. 312: 1159-65 (2003). Flow cytometry.
- BS 3 (Pierce, Rockford, IL) was added to the S537 solution to a final concenfration of 1 mg/ml and incubated on ice for 1 min. The samples were then mixed with an equal volume of 4X SDS-PAGE loading buffer and analyzed by Western blot.
- Cell fusion ⁇ -gal reporter gene assay Cells transfected with pSecTag2B-S or pCDNA3-ACE2 and infected with VTF7.3 and VCB21R respectively were collected by trypsin digestion and washed once with PBS . Cells were then suspended in regular DMEM medium at pH 7.4 and mixed. Cells were lysed after four hours of incubation and ⁇ -gal activity was measured using CPRG as the substrate (Roche) as described in Xiao et al. Biochem.
- ELISA Two ELISA assays were used. In the sandwich ELISA the plate was coated with an anti-His tag antibody, then the S fragment were added and detected with an anti-c-Myc epitope antibody. This assay was used for detection of the S fragments. In the second ELISA assay the C9 tagged receptor ACE2 was coated on the plates through an anti-C9 antibody (ID4) and the S fragments were added and after washing detected with an anti-c-Myc epitope antibody. In all experiments the incubations with the c-Myc epitope antibody were for 2 hours at room temperature. The optical density (OD) was measured and normalized to the highest value.
- S fragments were tested for oligomerization. These S fragments included the extreme N-terminal fragment (residues 17 through 276 denoted as S276, SEQ ID NO:50) that does not bind the receptor ACE2, several S fragments (S756, S537, S272-537) that bind ACE2, as well as a fragment including residues 319 through 517 (denoted as S319-517, SEQ ID NO:62) that retains receptor binding activity.
- this rabbit polyclonal antibody preparation was developed against a peptide containing residues 540-555 (SEQ ID NO:59) of the S glycoprotein.
- the P450 antibody binds the S756 polypeptide but not the other fragments (Fig. 9B, left). All N-terminal fragments except the smallest fragment (S319-517) containing the receptor binding domain were coimmunoprecipated with S756 by P540 (Fig. 9B, right).
- DTT was included in one of the coimmunoprecipitation experiments.
- the soluble S ectodomain is a trimer.
- Viral envelope glycoproteins of class I fusion proteins such as hemagglutinin (HA) of influenza are trimeric through the transmembrane domain.
- HA hemagglutinin
- the S2 subunit may facilitate trimerization of the whole S glycoprotein.
- a dimeric SI with a trimeric S2 could lead to higher order oligomers whose formation depends on the availability of the dimerization binding site in the native S glycoprotein.
- the size of the soluble S ectodomains (Se) was approximated by gel filtration, where the transmembrane domain and the cytoplasmic tail were deleted. As shown in Fig.
- CoV S glycoprotein (SI) forms dimers, 2) the dimerization domain does not overlap and is upstream of the receptor binding domain, 3) deletion of the dimerization domain abolishes fusion, 4) dimeric SI binds receptor molecules much more efficiently than monovalent fragments containing the receptor binding domain, and 5) the soluble S ectodomain forms trimers under gel filtration conditions.
- some SU subunits of class I fusion proteins that bind receptor molecules
- both the HIV-1 Env and the MHV S glycoproteins are cleaved and the SU can dissociate from the transmembrane subunit, however, such dissociation may not be important for fusion.
- the SARS-CoV S is not cleaved when expressed in membrane associated or soluble form and cleavage may not be required for fusion.
- the SARS-CoV S glycoprotein is a class I fusion protein, the lack of cleavage is an exception from the rale that the Envs of class I fusion proteins are cleaved presumably to confer a metastable high- energy state that could drive the fusion reaction.
- the three-dimensional structure of the trimer may not allow any interactions of the monomer dimerization sites with other monomers in the same or different trimer.
- the later possibility is supported by the preliminary data provided herein where higher order oligomers were not detected using the described gel filtration conditions. Under those conditions either intratrimer dimerization occurs but the third monomer conformation does not allow interactions with monomers from other trimers or such interactions are too weak to be detected, or the trimer three- dimensional structure is such that it does not allow dimerization interactions.
- Data provided herein demonstrate lack of fusion after deletion of portions of the dimerization domain and indicate that the dimerization region may play a role in fusion although its mechanism may not be through dimerization interactions.
- Example 20 Sera from Mice Immunized with DNA Encoding RBD Polypeptides frihibits S-Mediated Cell Fusion This Example illustrates that immunizing mammals with DNA encoding receptor binding domain polypeptides may prevent SARS infection.
- mice were divided into three groups: group A of mice # 1 through 5 were immunized with plasmid pSecTag-SRBD that encodes for the S319-518 fragment that includes the receptor binding domain (RBD) of the spike protein; group B of mice #1 to #5 were immunized with the plasmid pEAK-10-RBD-Fc that encodes for a fusion protein of RBD (S319-518) fragment fused to Fc and group C mice #1 to #3 which were immunized with a control plasmid. Five BALB/C mice per group were immunized at day 0, day 14 and day 28. Mice received less than 2 ug DNA per immunization with a gene gun.
- group A of mice # 1 through 5 were immunized with plasmid pSecTag-SRBD that encodes for the S319-518 fragment that includes the receptor binding domain (RBD) of the spike protein
- group B of mice #1 to #5 were immunized with the plasmi
- Sera were collected at day 56.
- the first number denotes an individual mouse, the letter denotes the respective immunization group, and the last number denotes the dilution used.
- Cells (293T) were incubated with anti-sera from the immunized mice and then mixed with cells expressing S protein. Fusion was measured as described in previous Examples (see also, Xiao et al. BBRC 2003).
- PC denotes positive control where no serum was added.
- serum dilution factors 10 100, and 1000 were used.
- mice #3-#5 in groups A and B, and #3 in the control group dilution factors of 20 and 100 were used.
- mice immunized with DNA encoding the spike protein receptor binding domain S319-518, groups A and B
- had very high titer antisera - dilutions up to 1 :7250 still reacted strongly to antigen in ELISA assays.
- anti-sera from mice immunized with DNA encoding the spike protein receptor binding domain inhibited fusion of cells that express the S protein in a dose dependent manner.
- anti-sera from mouse 1 A and 2 A which were immunized with DNA encoding the S receptor binding domain, substantially eliminated S-protein mediated cell fusion when used at a 1:10 dilution. Higher dilutions ( 1 : 100 and 1:1000) of this anti-sera were less effective. Similar results were observed on cell fusion inhibited by anti-sera from mouse 3 A (1 :20 dilution), from mouse 4A (1 :20 dilution), and from mouse 5A (1:20 dilution). These data indicate that immunizing mammals with DNA encoding S protein receptor binding domain polypeptides can raise a strong immune response against the spike protein and could prevent SARS infection. As described above, soluble fragments of the S glycoprotein that have the receptor binding domain inhibit S-mediated cell fusion (see Fig. 15).
- Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronaviras, Nature 426: 450-54 (2003).
- P.L. Earl, B. Moss Mutational analysis of the assembly domain of the HIV-1 envelope glycoprotein, AIDS Res.Hum.Retrovirases. 9 (1993) 589-94.
- R.J. Center P. Schuck, R.D. Leapman, L.O. Arthur, P.L. Earl, B. Moss, J.
- the coronavirus spike protein is a class I viras fusion protein: structural and functional characterization of the fusion core complex, J.Nirol 77 (2003) 8801- 8811. H. Bisht, A. Roberts, L. Nogel, A. Bukreyev, P.L. Collins, B.R. Murphy, K. Subbarao, B. Moss, Severe acute respiratory syndrome coronaviras spike protein expressed by attenuated vaccinia viras protectively immunizes mice, Proc. ⁇ atl Acad. Sci. U.S.A 101 (2004) 6641-6646.
- SARS-CoV severe acute respiratory syndrome-associated coronaviras
- a reference to "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.
- 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.
Abstract
Description
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CN113633764A (en) * | 2021-09-02 | 2021-11-12 | 中国食品药品检定研究院 | Novel corona DNA vaccine containing adjuvant |
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EP1648927A1 (en) | 2006-04-26 |
AU2004259750A1 (en) | 2005-02-03 |
AU2004259750A2 (en) | 2005-02-03 |
CA2533113A1 (en) | 2005-02-03 |
TW200510450A (en) | 2005-03-16 |
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