Patent Application
20080027006
The invention may be further understood by the following non-limiting examples.
While not wishing to be bound by any particular theory, a possible explanation of events or mechanisms is as follows.
The spike (S) glycoprotein of coronaviruses mediates viral entry into host cells. It is a type 1 viral fusion protein which characteristically contains two heptad repeat regions, denoted HR-N and HR-C, that form coiled-coil structures within the ectodomain of the protein. The two heptad repeat regions can undergo a conformational change from their native state to a 6-helix bundle (trimer of dimers) which is involved in mediating fusion of viral and host cell membranes.
It is thought that the binding of S protein of murine coronavirus MHV to a specific soluble or cell surface glycoprotein receptor induces global changes in the conformation of S protein that converts S protein from a native pre-fusogenic state to a fusion active state. In the case of the MHV-JHM strain, interaction with the host cell receptor CEACAM1 causes the S1 domain to readily dissociate from the S1/S2 complex, resulting in display of a new hydrophobic surface area that allows the virions to bind to liposomes and exposes previously hidden trypsin cleavage sites in S2 (21-23). This receptor-induced conformational change is believed to facilitate the release of a hydrophobic fusion peptide from the interior of S2 and position it to interact with the host cell membrane.
The fusion-active state of type 1 viral fusion proteins resolves itself to a fusogenic or post-fusion state (24). This change is thought to arise from an association between the two coiled-coil regions within the S2 subunit; in which the N-terminal HR region from three S proteins form a parallel triple stranded coiled-coil and the interhelical polypeptide chain loops around to reverse the polypeptide chain direction, positioning the HR-C region antiparallel to HR-N.
The positions of a heptad repeat unit are denoted as (abcdefg)n, where n is a repeat number. The residues at the a and d positions (of the heptad repeat positions denoted (abcdefg)n) of HR-C then pack into grooves formed by the residues at the a, d, e and g positions of the HR-N core coiled-coil to complete the 6-stranded alpha-helical bundle structure also termed “trimer of hairpins” or “trimer of dimers”. This structure ultimately draws the viral and cellular membranes close together, destabilizing the lipid bilayers which surround the virus and target cell, causing fusion and release of the viral nucleocapsid into the host cell.
Whether or not the above possible explanations or any depiction of a general membrane fusion mechanism (25-36) is accurate for the SARS virus and other coronaviruses, compositions and methods of the present invention can operate effectively, for example to modify or prevent initial infectivity and the spread of infection.
We describe the biophysical analysis of two heptad repeat regions within the severe acute respiratory syndrome (SARS) coronavirus S protein. Our results show that in isolation, the HR-N region forms a stable alpha-helical coiled-coil which associates in a tetrameric state. The HR-C region in isolation formed a weakly stable trimeric coiled-coil. Thus we demonstrate that the HR-N and HR-C regions of the SARS-CoV S glycoprotein can independently form alpha-helical coiled-coil structures.
When mixed together, the two peptide regions (HR-N and HR-C) associated to form a stable alpha-helical 6-stranded structure (trimer of heterodimers). Systematic peptide mapping showed that a site of interaction between the HR-N and HR-C regions is between residues 916 to 950 of the HR-N domain and residues 1151 to 1185 of the HR-C domain. Additionally, interchain disulfide-bridge experiments showed that the relative orientation of the HR-N and HR-C helices in the complex was antiparallel.
Peptide synthesis. The HR-N and HR-C peptides of SARS CoV S glycoprotein were prepared by solid-phase synthesis methodology using 4-benzylhydrylamine hydrochloride (MBHA) resin with conventional N-t-butyloxycarbonyl (t-Boc) chemistry as described by Tripet et. al., (47). Peptides were N-terminally acetylated, cleaved from the resin and purified by reversed-phase high performance liquid chromatography (RP-HPLC) to homogeneity and characterized by amino acid analysis and electrospray mass spectroscopy.
Plasmid construction. Production of the peptide corresponding to SARS S protein amino acids 882-973 (HR-N1) was done using directional sub-cloning and bacterial expression techniques. PCR fragments were prepared from the plasmid SARS S #18 (containing the entire S protein from SARS, Urbani strain (Genbank Accession No. AY278741)). Primers were designed to incorporate an Ndel restriction site upstream (GTACGTACGCATATGATGCAAATGGCATATAG GTTC) (SEQ ID NO:103) and an EcoRI site downstream (GCGMTTCCCTTTGTCGTCGTCGTC GCAGCCGCCTACCTCCGCCTCGACTTTATCMG) (SEQ ID NO:104) of the amplified fragment. The Ndel site contains an ATG start codon just upstream of the SARS sequence. The reverse primer was engineered to incorporate the amino acid sequence Gly-Gly-Cys-Asp-Asp-Asp-Asp-Lys to insert an enterokinase site (DDDDK) just downstream of the synthesized sequence, and just upstream of the EcoRI site. The nucleotide fragment corresponding to residues 882-973 was amplified by PCR and subcloned into the EcoRI/Ndel site of the pT7SH6 plasmid, in frame with the 6-His tag directly downstream of the EcoRI site to create the plasmid pT7SH6 SARS 882-973. pT7SH6 contains an oligonucleotide-encoded 6-His tag followed by two stop codons cloned into the EcoRI-BamHI sites of pT7.7 so that cloning into the EcoRI site creates a C-terminal protein fusion encoding NSHHHHHHXX.
Plasmid pT7.7 (based on the plasmids of Tabor and Richardson (48)) enables genes to be expressed by T7 RNA polymerase using the highly efficient translation signals of T7 gene 10 by cloning into and Ndel (CATATG) site where the ATG is the initiation codon. Plasmid SARS S #18 was created by cloning the full length SARS S protein into pcDNA3.1V5-His TA cloning vector (Invitrogen, Carlsbad, Calif.). The SARS S sequence was amplified using reverse-transcription from SARS genomic RNA (W. Bellini, CDC, Atlanta, Ga.), followed by PCR using primers F21488 (CACCATGTTTATTTTCTTATTATTT) (SEQ ID NO:105) and R25259 STOP (TTATGTGTAATGTMT TTGACACC) (SEQ ID NO:106). The reverse primer contains a stop codon to halt transcription before the V5-6-His tag in the cloning vector.
Bacterial Protein Expression and Purification. BL21 Al E. coli cells (Invitrogen, Carlsbad, Calif.) were transformed with pT7SH6 SARS 882-973. These cells were grown overnight in Luria Broth (LB) containing ampicillin (Sigma, St Louis, Mo.). The overnight culture was used to inoculate a flask of LB, containing ampicillin, and the cells were grown to an OD600 (optical density) of 0.7. The cells were induced by adding 10% L-Arabinose (Sigma) to a final concentration of 0.2%. Three hours later the cells were pelleted, resuspended in 1/10th volume of 20 mM tris/HCl pH 8.0 and protease inhibitors (Complete EDTA-free, Roche, Penzberg, Germany). The sample was frozen at −80° C. and thawed at 50° C. three times. A quantity of 1 mg DNAse (Promega, Madison, Wis.) was added to the sample and incubated at room temperature for 20 minutes. The sample was centrifuged at 14,000×g for 10 minutes. The soluble, supernatant medium, was purified with HiTrap nickel affinity columns (Pharmacia, Piscataway, N.J.) (49). The column was washed with 20 mM Tris/HCl pH 8.0, and bound proteins were eluted with a 0-500 mM gradient of Imidizole (Sigma) in 20 mM Tris/HCl pH 8.0. Fractions containing the SARS 882-973 (HR-N1) peptide were identified using immunoblot analysis with an anti-6-His polyclonal antibody (AbCam, Cambridge, Mass.). These fractions were pooled and placed over a reversed-phase chromatography column for purification.
Circular dichroism spectroscopy. Circular dichroism (CD) spectra were recoded on a Jasco J-810 spectropolarimeter (Jasco Inc., Easton, Md.). The CD wave scans were measured from 190 to 255 nm in benign buffer (0.1M KCl, 0.05M PO4, pH 7). For samples containing trifluoroethanol (TFE), the above buffer was diluted 1:1 (v/v) with TFE. Temperature denaturation midpoints (T1/2) for the peptides were determined by following the change in molar ellipticity at 222 nm from 4° C. to 95° C. in a 1 mm path length cell and a temperature increase rate of 1° C./min. Ellipticity readings were normalized to the fraction of peptide folded (ff) or the fraction of peptide unfolded (fu) using the standard equations:
ff=(]]−[
]u)/([
]n−[
]u); and fu=(1−ff); where [
]n and [
]u represent the elipticity values for the fully folded and fully unfolded species, respectively, and [
] is the observed ellipticity at 222 nm at any temperature.
Sedimentation analysis. Sedimentation equilibrium experiments were performed on the analytical ultracentrifuge (XLA from Beckman-Coulter) as described by Tripet et. al., (47). In general HR-N and HR-C peptides were dialyzed exhaustively against 50 mM K2PO4, 100 mM KCl, pH 7 buffer at 4° C. For full length HR-N1 peptide, HR-N1 was dialyzed against the low salt containing buffer 50 mM K2PO4, pH 6 for enhanced solubility. Three 100 μl aliquots of the sample were loaded into a 12 mm six-sector, charcoal-filled Epon cell and centrifugation proceeded for 48 hours with rotor speeds of 20,000, 26,000 and 30,000 rpm. The HR-N and HR-C peptides were each loaded at initial total peptide concentrations of 50, 100 and 250 μM whereas the full length HR-N1 peptide was loaded at 10, 20 and 50 μM. The partial specific volume of each peptide was calculated using the program SednTerp v. 1.08 (50). The density of the solvent was calculated to be 1.009 g/ml. The data were evaluated using a non-linear least squares curve-fitting algorithm contained in the WinNonLin analysis program v. 1.06 (51).
Gel electrophoresis. HR-N and HR-C region peptides singly or as an equimolar mixture (200 μM of each peptide) were dissolved in 100 mM KCl, 50 mM PO4, pH 7 buffer and incubated at room temperature. Samples were then diluted with 1 volume of 2×Laemmli sample buffer and analyzed by SDS-PAGE in a 15% Tris/Glycine gel.
Size-exclusion and reversed-phase chromatography. HR-N and HR-C region peptides singly or as an equimolar mixture (200 μM of each peptide) were dissolved in 100 mM KCl, 50 mM PO4, pH 7 and equilibrated at room temperature for 30 min. A 10 μl aliquot of the mixture was loaded onto a high-performance size-exclusion column, Superdex 75™ (1 cm×30 cm, Pharmacia, Uppsala, Sweden) equilibrated in a buffer with 50 mM PO4, 100 mM KCl, pH 7 at a flow rate of 0.75 ml/min and ambient temperature. For peptides which formed stable heterostranded complexes, the complex peak was collected and analyzed by reversed-phase chromatography on an analytical C8 column (Zorbax 300SB-C8, 15 cm×4.6 mm l.D., 6.5 μM particle size, 300 Å pore size; Agilent Technologies). The peptides were eluted from the column by employing a linear AB gradient of 2% B/min, where eluent A is 0.05% aqueous TFA and eluent B is 0.05% TFA in acetonitrile at a flow rate of 1.0 ml/min at room temperature. To calculate the peptide ratio in the complex, the peak areas of each component were compared with peak areas of known standard solutions of each peptide.
Formation of parallel and anti-parallel disulfide-bridged hetero-two-stranded molecules. Preferential disulfide-bridge formation between the HR-N9 and HR-C1 regions was performed similar to that described by Semchuck et. al., (52). In brief, 10 mg of 2,2′-dithiopyridine (DTDP) was dissolved in 100 μl of dimethylformamide (DMF) with sonication. A 10 μl (3.4 μmoles) aliquot of this solution was added to a solution of the HR-N9 peptide (2 mg, 0.8 μmoles) dissolved in a 3:1 (v/v) acetic acid/H2O buffer and stirred for 6 h. The solution was then extracted 3× with 500 μl of ether and the aqueous layer was applied to a Sephadex G-25 desalting column conditioned with 50 mM NH4Acetate, pH 5.5 running buffer. Fractions (1 ml) eluted from the column were collected and those with 220 nm absorbance were pooled. To one half of the pooled solution was added HR-C1 peptide with either a N-terminal CGG or a C-terminal GGC linker (1 mg dissolved in 1 ml of 50 mM NH4Ac, pH 5.5. buffer) in 100 μl aliquots over 30 min. The reaction were then stirred for 1 h and the final complex purified by RP-HPLC and freeze dried.
Sequence Analysis.
To identify the heptad repeat (HR) regions within the SARS-CoV S protein, we utilized the coiled-coil prediction algorithm STABLECOIL (53).
Circular Dichroism analysis of the HR-N and HR-C Regions.
To examine the structural characteristics of the HR-N and HR-C regions of the SARS CoV S protein, peptides (see
−8,700f
−7,600f
−6,200f
aName of each peptide studied. The sequence position of each peptide is shown in FIG. 1.
bThe mean residue molar ellipticities at 222 nm were measured at 22° C. in benign buffer (0.1 M KCl, 0.05 M K2PO4, pH 7). For samples containing TFE, the above buffer was diluted 1:1 (v/v) with TFE. Values are in units of (degrees · cm2 · dmol−1).
cThe number of α-helical residues was calculated based on the predicted molar ellipticity. The predicted molar ellipticity for a completely α-helical peptide was calculated using [theta]theoretical = 40,000 × (1-4.6/N) where N is the number of residues in the polypeptide chain. The number of α-helical residues in benign or TFE conditions was calculated as the observed [θ] divided by the theoretical [θ].
dthe percent α-helix in benign medium at the concentration used was calculated as the number of helical residues in benign divided by the maximum inducible α-helical residues in TFE. Note that in oligomerizing α-helices the helical content is concentration dependent. Higher concentrations shift the monomer to oligomer equilibrium increasing the α-helical content based on a two state unfolding mechanism (random coil to coiled-coil)
eT1/2 is the transition midpoint temperature at which there is a 50% decrease in molar ellipticity [θ]222 compared to the fully folded peptide as determined by CD at 5° C. All peptides in which T1/2 was determined are boxed, for example, HR-N1, HR-N2, HR-N3, HR-N12, HR-C1, and HR-C4.
fThese peptides all showed a characteristic α-helical spectrum with minima at 208 and 222 nm even though the [θ]222 was low.
+Peptides were studied in 0.05 M K2PO4 buffer, pH 7 due to low solubility.
Mapping of the HR-N region further with smaller peptides showed that HR-N5 and HR-N4 (region 1 and 3) were also largely insoluble, and the limited amount that did dissolve (15 μM) exhibited random coil spectra (
We analyzed the CD spectra of several overlapping 35-residue peptides shifted 1 heptad (7 residues) toward the C-terminus of the HR-N region beginning at HR-N5 and ending with HR-N17 (
The far UV CD spectrum of HR-C1 exhibited double minima at 208 and 222 nm (
To see if we could define further the α-helical region within HR-C (39-residues), we analyzed three truncated peptides of HR-C1. CD analysis of HR-C2 and HR-C3 (21 and 28 residue length, respectively) displayed random coil spectra in benign buffer, whereas HR-C4 (35 residue length) showed α-helical structure with a [θ]222 value of −17,500 degrees (Table 1). Additionally, the thermal melting profile of HR-C4 indicated the same temperature transition midpoint (33° C.) as full length HR-C1 (Table 1). Thus a possible minimal length required for folding of the HR-C region coiled-coil appears to be 35 residues corresponding to the sequence 1151 to 1185.
Oligomeric State of the HR Regions
Sedimentation equilibrium experiments were carried out to determine the oligomeric states of the HR-N and HR-C peptides. The HR-N1 peptide was studied at three different concentrations and three different rotor speeds. The data obtained fit well to a single-species model with a weight average molecular weight of 48,750 Da (
In contrast, sedimentation equilibrium experiments of the HR-C1 peptide at three different concentrations and three speeds showed that the data was best fit globally to an associating monomer to trimer equilibrium model.
Interaction between the HR-N and HR-C.
The HR-N and HR-C of the murine coronavirus MHV S glycoprotein self associate to form a 6-helix bundle structure typical of other type 1 viral fusion proteins (19). To identify the significant sequences within the SARS-CoV S glycoprotein, mixtures of the HR-N and HR-C peptides were screened by CD spectroscopy and size-exclusion chromatography (SEC). Initially, mixtures between the three HR-N peptides: HR-N5, HR-N3 and HR-N4 (which correspond to regions 1, 2 and 3 of HR-N,
74+
57+
aName of the peptides in the mixture. For sequence regions see FIG. 1. Names followed by “ox” indicate peptide mixtures which have been covalently linked by a disulfide bridge.
bThe mean residue molar ellipticities at 222 nm were measured at 22° C. in benign buffer (0.1 M KCl, 0.05 M K2PO4, pH 7). Peptide complexes scanned in a 0.05 M K2PO4 buffer are indicated with an asterisk (*). Peptide complex concentration was 100 μM and 15 μM (indicated with a cross, +). The predicted molar ellipticity for two non-interacting peptides was calculated by summing the two individual spectra. Values are in units of(degrees · cm2 · dmol−1).
cThe difference in molar ellipcity from the observed and predicted values (column 2 - column 3; observed minus predicted).
dT1/2 is the transition midpoint temperature at which there is a 50% decrease in molar ellipticity [θ]222 compared to the fully folded peptide as determined by CD at 5°C. When no change in molar ellipticity (column 3) was observed, the t1/2 was not determined. Peptide complex concentration was 100 μM and 15 μM (indicated with a cross, +).
eSummary of the analysis of the peptide complexes by size-exclusion chromatography (SEC). (−) denotes no complex formation; (+) denotes weak complex formation between the two peptides (about 30% of total peak arrea or less); (++) denotes strong complex formation between the two peptides (>95% of total peak area).
To confirm that the structure of the HR-N region is not a factor in binding to HR-C1, as HR-N10 was a random coil on its own (
We also analyzed the truncated peptides of HR-C1 in an attempt to localize the binding site for HR-N10. HR-C2 and HR-C3 peptides of 21 and 28 residues showed no ability to interact the HR-N10 region as assessed by CD spectroscopy and SEC (Table 2). In contrast, the HR-C4 peptide of 35-residues which folded independently into an α-helical structure showed strong complex formation with the HR-N1 peptide observed by CD and SEC (Table 2). For example, the temperature denaturation profile of HR-N10/HR-C4 displayed a similar temperature midpoint and molar ellipticity observed for HR-N10/HR-C1 (compare 57° C. with 56° C. and −17,700 with −17,900, respectively). Thus the binding interaction between the HR-N and HR-C regions appears to be localized to residues 916 to 950 in the center of HR-N and 1151 to 1185 within the HR-C region (based on the N-terminal truncations).
HR-N and HR-C form a 3:3 Hexameric Complex.
To determine the ratio of the peptides within the HR-N10/HR-C1 complex, mixtures of HR-N10 and HR-C1 peptides at molar ratios of 1.5:1, 1:1 and 1:1.5, respectively, were pre-incubated for 30 min and then applied to a size exclusion column (
Next, we analyzed the molecular mass of the complex. Bosch et. al., (19) had shown for MHV-A59 that complexes of HR-1 and HR-2 are stable during SDS-PAGE as long as the samples are not heated before loading. Therefore, HR-C1, HR-N10 or HR-N2 peptides separately or as pre-incubated equimolar mixtures were subject to Tris SDS-15% PAGE (
Similar results were also seen for the HR-N2/HR-C1 peptides (
Sedimentation equilibrium analysis was carried out on the more stable HR-N2/HR-C1 complex. As shown in
Antiparallel Helix Orientation.
To investigate the orientation of the helices within the HR-N10/HR-C1 complex, we chose to form disulfide-bridged heterodimers between HR-N10 and HR-C1 in the parallel and antiparallel polypeptide chain orientation. The premise of this experiment was that the correct polypeptide chain orientation should form a tightly folded helical structure of high solubility and greater stability due to elimination of the concentration dependence between the two peptides, whereas the incorrect orientation would form head-to-tail complexes, which would aggregate and show little or no increase in stability (
Table 2 and
Here we used CD spectroscopy, SEC and sedimentation equilibrium analysis to characterize predicted HR-N and HR-C regions in the ectodomain of the spike S glycoprotein of the SARS-COV. In isolation, the HR-N region folds as an independent α-helical coiled-coil structure of high stability, but with only about 40 helical residues of a possible 92 residues. Analysis of smaller peptides of HR-N showed that the helical content is largely localized to residues 927 to 973. The central portion of HR-N and the N- and C-termini were unfolded.
It is interesting that the N-terminal region closest to the predicted fusion peptide sequence (residues 851 to 882,
Studies on the HR regions of the S protein of the mouse hepatitis virus strain A59 showed the α-helical content of the separate HR-N and HR-C peptides were calculated to be about 89%, and that the helical content of their equimolar mixture was calculated to be about 82% (19). Further, electron microscopy showed rod-like structures of about 14.5 nm, correlating well with the length predicted for an α-helix the size of HR-N (96 residues). Since the sequences between coiled-coil domains of the SARS-CoV Urbani strain analyzed here and MHV-A59 have a high degree of sequence identity, this difference was quite surprising. One possibility for the difference may be due to the buffer conditions in which the samples were analyzed. The MHV-A59 peptide was analyzed in H2O versus physiological conditions used in this study (100 mM KCl, 50 mM PO4, pH 7 buffer). Alternatively, the difference could be due to the low concentration at which we analyzed SARS HR-N1 (15 μM). The α-helical content of peptides that form coiled-coil structures are affected by concentration due to the monomer-oligomer equilibrium (two-state folding mechanism random coil to coiled-coil). We observed a strong α-helical coiled-coil in HR-N between residues 927 to 973. This region correlates with the protease resistant portion of the HR-N region of MHVS, residues 976 to 1040 (19), corresponding to residues 909 to 973 of the SARS-CoV S protein sequence.
Sedimentation equilibrium analysis showed that both the long and short folded HR-N region peptides associated as a tetramer; however, in SDS-PAGE gel electrophoresis the shorter HR-N region (HR-N2) displayed a smear between tetramer and trimer with the more dominant band displaying a trimeric molecular loss in the ability to interact with HR-C1. The HR-N10 region is a significant site of interaction with HR-C1.
Despite CD analysis of the HR-N10/HR-C1 complex showing a significant increase in thermal stability and α-helical induction, the complex did not adopt a fully helical structure (−17,700° molar ellipticity observed). Some loss can be attributed to end fraying and concentration dependence (66-68). However, when HR-N10 and HR-C1 were disulfide-bridged, removing the concentration dependence between these two peptides, the molecule only displayed −19,200 degrees molar ellipticity. One possible explanation may be that the interaction between HR-C and HR-N10 is not fully helical, but more akin to the binding interactions observed for HR-C to HR-N of the F protein of the paramyoxovirus SV5 (25). The structure of the fusion-active core region of the SV5 F protein shows that the HR-C (HR2) domain contains both an α-helix and an extended conformation that stretches out along the HR-N (HR2) region. Further characterization of the binding interactions for S of SARS-COV is resolved by high resolution structural studies.
Viral fusion proteins can adopt at least two different conformations (i.e., native and fusogenic states), and the change in conformation is thought to be required for fusion of the viral and cellular membranes during virus entry (24). The α-helical hetero-trimeric (6-helix) conformation of the complex of HR-N10 and HR-C1 peptides of SARS-COV S protein is a structure that can represent the core of the fusion competent state of the SARS-CoV S protein. Without wishing to be bound by any particular theory, a possible explanation is that the 6-helix form of the HR-N10 and HR-C1 peptides displays greater stability than the separate domains, and an energy release from conformational change to a more stable form may provide the energy to overcome the activation energy needed to bring the two lipid bilayers close together at the fusion site (60, 62, 63).
Also without wishing to be bound by any particular theory, a possible explanation of a fusion mechanism is as follows. In the native state, the two HR regions of the fusion protein ectodomain oligomerize to form homo-trimeric coiled-coil cores, which when stimulated by receptor interaction undergo a conformational change to an antiparallel 6-stranded α-helical structure (trimer of dimers) in the fusion active state to initiate the fusion of viral and cellular membranes. Our observation of the 6-helix bundle conformation in connection with SARS therefore leads to compositions such as peptides corresponding to the SARS-CoV S protein HR regions that can modify viral fusion. For example, a peptide composition can inhibit viral fusion for SARS and optionally other coronaviruses. A composition can comprise free HR-N or HR-C peptides, complexes of each peptide, or complexes of a peptide mixture. Such compositions can compete for or affect binding to the HR-N and HR-C coiled-coils in the native state, thereby blocking the conformational transition to the fusion-active form. For example, a homotrimer of HR-N10 can function to disrupt the ability of a SARS S protein to adopt or maintain a fusion competent conformation, thereby inhibiting infectivity or spread of a SARS coronavirus.
The peptides are prepared by synthetic chemistry methods. Methods known in the art such as those utilizing t-Boc/Benzyl or Fmoc/t-Boc chemistry are used. See, e.g., Solid phase peptide synthesis, John M Stewart and Janis D Young, Second edition, 1984, Pierce Chemical Company; Fmoc solid phase peptide synthesis, A practical approach, W. C. Chan and P. D. White, Editors, 2000, Oxford University Press.
The peptides are prepared using recombinant expression technology. For example, a desired gene sequence is used as an insert in connection with an expression vector and transformed into an appropriate host for expression.
Standard techniques for cloning, isolation, amplification and purification of DNA, RNA, and protein, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; and Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York.
Also see current editions in the series of Current Protocols titles (all generally published by John Wiley and Sons, New York), e.g. Current Protocols in Molecular Biology (edited by Frederick M. Ausubel et al., 1991-2004, New York: Greene Pub. Associates and Wiley-lnterscience: J. Wiley); Current Protocols in Cell Biology, Current Protocols in Cytometry, Current Protocols in Immunology (edited by John E. Coligan, et al., New York: John Wiley and Sons, 1994-1998), Current Protocols in Pharmacology, Current Protocols in Protein Science; and Current Protocols in Toxicology.
In one example, a recombinant peptide is prepared comprising a single peptide such as HR-N10 or HR-C4. In another example, a recombinant peptide is prepared comprising a first peptide and a second peptide. A spacer sequence of from about three to about thirty amino acids is optionally inserted between the first and second peptide. In a particular embodiment, the first peptide is HR-N10 and the second peptide is HR-C4, or vice versa.
Other than natural peptides corresponding to fragments of the native S protein of SARS-CoV, variant compounds or analogs are also prepared. For variant peptides produced via recombinant technology, site-directed mutagenesis or random mutagenesis techniques are employed. Peptides are optionally produced by chemical synthetic methods.
A peptide of the invention is used as an inhibitor to block entry of a SARS coronavirus in a target cell. Such inhibition can be by prevention of fusion of a viral membrane and a target cell membrane. In a particular application, a peptide is used as a therapeutic agent that can reduce viral infectivity.
In an example, a first peptide is derived from or designed in relation to an S protein of SARS CoV or fragment thereof, for example an HR-N or HR-C domain peptide. For example, such first peptide is HR-N10 or HR-C4. A sequence of a first peptide can be modified so as to achieve a desirable functional attribute or to provide an alternative without a change in attribute. An attribute can include one or more of modified efficacy, specific activity, solubility, toxicity, or stability. The attribute of stability, for example, can relate to the ability to maintain a particular conformation, susceptibility to degradation such as by proteolysis, or to host effects such as clearance or parameters of distribution, metabolism, and excretion.
A peptide sequence or peptide product is modified in one or more of several ways. A peptide is modified so as to affect the alpha-helical propensity of the peptide product. A peptide sequence is modified by introduction of one or more cyclic bridges in a peptide sequence or peptide product. For example, a lactam bridge can be introduced into a peptide. A peptide is modified by modulation of the “a” and “d” position residues. A peptide is modified by chemical conjugation to polyethylene glycol (PEG) or pegylation of the sequence. A peptide is modified by removal and/or chemical alteration of sites sensitive to proteolysis. In a specific example, a proteolytically sensitive site is modified by acetylation.
Helical Propensity Changes.
Regarding helical propensity changes, residues in the amino acid sequence of HR-N and HR-C can be substituted to increase the helical propensity (ability to form a helix) of the sequence and thus affect one or more attributes of the peptide such as stability, efficacy, or other attribute. One or more substitutions are made to residues occurring in the b, c, and f heptad positions with a molecule such as Ala, Lys, Arg, Aib (aminoisobutyric acid) or Dxg (dipropyl or dibutyl glycine). See
Other examples of HR-C analogs with ability for change of helical propensity include SEQ ID NO:68 and SEQ ID NO:70 (See
Introduction of Lactam and other Cyclic Bridges.
The formation of covalent bonds between side-chains of residues (lactam bridges) occurring on the outer helical face can significantly stabilize the helical conformation. For example, lactam bridge stabilization of alpha-helices in connection with the present invention is investigated. Parameters such as ring size, orientation, and positional effects are evaluated. Modified peptides are generated with one or more lactam bridges incorporated, for example into HR-N and HR-C sequences according to
An example of an HR-C analog with a lactam bridge includes SEQ ID NO:71. An example of an HR-C analog with a salt bridge introduced includes SEQ ID NO:72. An example of an HR-C analog with multiple salt bridges includes SEQ ID NO:74. An example of an additional analog of an HR-C peptide is SEQ ID NO:73.
Modulation of the a and/or d residue positions. The residues occurring in the a and d heptad positions are significant for the folding and stability of the coiled-coil structure. By substituting the present a and d residues to those which are optimal for stability, for example, IIe (isoleucine) at position a and Leu (leucine) at position d, inhibitor analogs of HR-N and HR-C are produced which bind with higher affinity than the native sequences. See
Another example of an HR-C analog with modulation of the “a” and/or “d” residue positions includes SEQ ID NO:67 (see
An example of an HR-C analog with modulation of the “a” and/or “d” residue positions and an ability for change of helical propensity includes SEQ ID NO:69 (see
Generation of inhibitor peptides less than 35 amino acids. The chain length of a peptide compound is decreased from about 35 residues to a chain length of about 14 residues. Inhibitor compounds are also generated comprising from about 14 residues to about 35 residues. For example, a peptide is synthesized or expressed as a recombinant comprising any two heptad units, any three heptad units, or any four heptad units of the HR-N10 peptide, thereby generating 14 mers, 21 mers, and 28 mers. Therefore we generate inhibitor compounds, capable of demonstrating coiled-coil dimerization, with as few as about two heptads or a chain length of about 14 residues. See Table 3.
Pegylation. Molecules are modified by the addition of the polymer polyethylene glycol (PEG). PEG addition or conjugation is performed as known in the art. Peptides with PEG can have one or more properties changed. For example, a PEG-peptide can be less prone to degradation such as by proteolysis. The weight. This difference may indicate that the HR-N region naturally associates as a trimer, but in the absence of the HR-C region (or other parts of the native sequence) the hydrophobic residues occurring in the e and g positions are exposed. To sequester these residues from the aqueous environments, the protein can switch from a trimer to a tetramer state. In agreement with this proposal is the observation that more interfacial surface area, in particular the e and g positions are buried in a tetrameric coiled-coil structure relative to a trimeric or dimeric coiled-coil (64).
The HR-C region in isolation showed a very concentration-dependent alpha-helical coiled-coil structure which at very high concentration (>500 μM) indicated only about 24 helical residues. The truncation studies showed that the N-terminal four residues could be deleted without adversely affecting the structure. Additionally, both sedimentation equilibrium and gel electrophoresis showed that the entire HR-C region alone associates as a homotrimer. Consistent with the helicity data, stability analysis showed that HR-C1 was of low stability with 50% of its structure unfolded at 33° C. This and the above characteristic is surprising, considering that 9 out of the 10 heptad a and d core positions contain large optimal hydrophobic residues (e.g. I, I, V, I, I, L, L, L, L), and no apparent intermolecular electrostatic repulsions can be predicted. The low stability, however, does fit with a general mechanism of type-1 viral fusion proteins, where the C-terminal HR separates in order to form a complex with the N-terminal HR. In agreement with this is the observation that there are no possible inter-chain electrostatic interactions that can occur between HR-C peptide chains. Such electrostatic interactions can add considerable stability to coiled-coils and stabilize their ability to stay associated. See (65) and references therein.
Upon mixing the HR-C1 peptide with several truncated HR-N region peptides, HR-C1 associated preferentially with HR-N10 to form a stable α-helical 6-stranded complex, comprising of a 3:3 mole ratio of HR-N10:HR-C1. Additionally, the selective disulfide bridge experiments indicated that the relative orientation of HR-N10 and HR-C1 peptides in the complex were anti-parallel relative to one another. The peptide mapping study revealed that a 35-residue length is desirable and may be optimal for the binding interaction with HR-C1, as shifting either 7 residues toward the N or C-terminus of HR-N (HR-9 or HR-11) caused significant effective lifetime, for example the half-life, of a PEG-peptide can be enhanced. The immunogenicity of a PEG-peptide can be decreased. In a specific example, modified HR-N and HR-C inhibitor peptides are generated wherein PEG is acetylated onto lysine residues substituted into the b, c, or f heptad positions along the sequence. See
Acetylation/mutation of sensitive sites. Residues which are prone to proteolytic cleavage, for example lysine in connection with trypsin, are optionally modified by acetylation using acetic anhydride or its equivalent. Such residues can also be mutated.
Use of Natural Amino Acids and other Building Blocks.
Peptides can be prepared using natural amino acids as building blocks for either recombinant expression or synthetic production. It is well known in the biological arts that certain amino acid substitutions can be made in protein sequences without affecting the function of the protein. Generally, conservative amino acids are tolerated without affecting protein function. Similar amino acids can be those that are similar in size and/or charge properties, for example, aspartate and glutamate and isoleucine and valine are both pairs of similar amino acids. Here, however, substituted peptides are further evaluated and selected regarding the affect of a substitution on the ability to maintain an alpha-helical coil structure or so as to facilitate a capability of forming a trimeric conformation, including a homotrimer, or a six helix bundle conformation, including a heterologous bundle.
Peptides are also prepared using at least one chemical moiety such as a non-proteinogenic amino acid, unnatural amino acid, peptidomimetic unit, peptoid, beta amino acid, or derivatized amino acid molecule. The moiety is selected so as to maintain an alpha-helical coil structure or so as to facilitate a capability of forming a trimeric conformation, including a homotrimer, or a six helix bundle conformation, including a heterologous bundle. For example, the moiety and/or substitution is selected and integrated in an HR-N10 or HR-C4 peptide derivative so as to maintain an alpha-helical structure. Alternatively, the moiety and/or substitution is selected and a peptide is prepared so as to maintain a functional ability to inhibit infectivity or cell-cell fusion in connection with a SARS coronavirus or a SARS coronavirus S protein.
Any of the peptides described herein may, additionally, have a non-peptide macromolecular carrier group covalently attached to their amino and/or carboxy termini. Such macromolecular carrier groups may include, for example, lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates.
Biophysical Properties.
The peptides or modified peptides are evaluated or screened regarding one or more biophysical properties. Modified peptides are evaluated with respect to stability. In a particular example, compounds are selected that demonstrate improved stability relative to that of peptides with native sequences. Stability can be defined here by the temperature midpoint unfolding transitions or Gdn-HCl denaturation unfolding transitions. See, e.g., Pace, C. N. 1986, Determination and analysis of urea and guanidine hydrochloride denaturation curves, Methods Enzymol. 131:266-280; Weber, P. C. and F. R. Salemme, 2003 Feb, Applications of calorimetric methods to drug discovery and the study of protein interactions, Curr. Opin. Struct. Biol. 13: 115-121.
Bioassays. Bioassays are performed to assess attributes of compounds such as peptides, for example relating to the ability to modify fusion and infectivity.
Cell-cell fusion assay. The inhibitory ability of the peptides (and analogs) is determined using a cell-to-cell fusion assay. Cell-to-cell fusion assays are performed by transfecting a receptor negative cell line with recombinantly expressed anchored S protein. When overlaid with cells expressing receptor, at least partial cell fusion occurs. Cell fusion is detected with a calorimetric assay. A first cell line expresses T7 polymerase, and a second cell line expresses beta-galactosidase under control of the T7 promoter; fusion results in blue cells. Peptides are added in increasing concentrations to determine the inhibition of the membrane fusion step. For another cell fusion assay system, see that of Bosch (19) which is adapted as necessary.
Cell infectivity assay. Cell infectivity assays are performed. Vero cells are inoculated with SARS-COV at a multiplicity of infection of 0.2 PFU/cell in the absence of inhibitor. After 2 hours when the virus is into the cells, a putative inhibitor compound is added. Cells are fixed at 12 hours and 24 hours, and immunofluorescence is performed to detect cells that have synthesized viral antigens. The results are evaluated to identify inhibitor compounds by determining if the S protein on the cell membrane is inhibited, for example in the ability to achieve cell to cell spread of infection or cell fusion. The results are also evaluated regarding whether newly made virus is prevented from infecting other cells which depends on fusion of the viral envelope with the plasma membrane of an uninfected cell. In addition, Vero cells are inoculated with SARS-CoV at a multiplicity of infection of 10 PFU/cell in the absence of inhibitor. Under these conditions (with no inhibition) infected cells are antigen positive by 6 hours post-infection. The percent of infected cells in the presence of an inhibitor relative to control (absence of an inhibitor) is determined to assess inhibition of initial virus infection.
For peptide compounds where inhibition is detected, a dose-response inhibitory activity is determined. Peptides having substantial inhibitory activity are used to generate additional analogs with further modification. These analogs are optimized for one or more properties such as polypeptide chain length, hydrophobicity, helical propensity and electrostatic interactions.
Peptides and analogs are optionally generated by providing one or more constraints such as a covalent constraint. A covalent constraint can enhance the stability of a conformation such as the helical conformation. Constrained peptides can exhibit a desirable conformational entropy profile, for example an entropy that is lower than that for an unconstrained counterparts or peptide. A constrained peptide can adopt a single specific bioactive conformation and show a smaller decrease in entropy on binding. A constrained peptide can demonstrate an enhancement biological activity such as receptor binding avidity and affinity. Such enhancement can occur by one or more of (1) stabilizing the biologically active conformer; (2) decreasing degradation; and (3) improving biological selectivity through reduction or elimination of bioactive conformers in connection with undesired biological responses.
Peptides herein are used to stimulate an immune response in a host organism. For example, a peptide is used to act as a vaccine in a human to aid in preventing or blocking fusion, entry, infection, or disease in connection with SARS coronavirus. An immune response comprising an antibody response and a cellular response is thereby generated upon introduction of an effective amount of an immunogenic form of a peptide. For example, antibodies raised against SARS-CoV HR-N and HR-C peptide antigens are useful in at least partially blocking SARS-COV entry by binding to the corresponding conserved sequences in the SARS-COV S protein, thereby neutralizing free virus and preventing fusion of receptor bound virus.
In an embodiment, a peptide is conjugated to a carrier protein. A carrier protein is selected from those known in the art. Diphtheria toxin is optionally the carrier protein. The conjugated peptide is used directly to immunize a subject. An adjuvant is optionally used with the conjugated peptide as known in the art for potentiation of an immune response. For example, an adjuvant is selected so as to enhance an IgG response. In another approach, the peptides are linked to a carrier protein, for example through an amino or carboxyl group of the peptide sequence or via a cysteine residue added to a peptide sequence terminus. See
Immunogens comprising a trimer structure. Each of the peptide regions, for example HR-N or HR-C, is cross-linked together in a trimeric oligomeric state. This trimeric structure is used as a native-like coiled-coil antigen. For example, an HR-10 peptide is used to generate a homotrimer. The cross-linked molecules are optionally conjugated to a carrier protein. Such a conjugated composition is further optionally formulated or administered with an adjuvant. It is believed that the stabilized trimer antigen can induce an immune response such as an antibody response that is directed towards the relevant fusion competent conformation of a SARS coronavirus S protein. See
Immunogens comprising a six-helix bundle structure. A covalently linked six-stranded molecule of three HR-N and three HR-C peptides is produced. This six-stranded structure is optionally conjugated to a carrier protein for immunization. See
Immunogens comprising a helix template. A method is developed for the design of stabilized α-helical immunogens as a means of generating antibodies specific for helical protein segments. The method employs a two-stranded α-helical parallel coiled-coil as a template. The template and its preparation are shown in
Tandem repeat antigens. For peptides developed as immunogens, each of the sequences can be repeated. For example, a tandem peptide repeat can have the form of (peptide)n, wherein the peptide is present a total of n times. The repeat total is selected as is known in the art. In a particular example, n is from about two to about eight. In another example, an upper bound for n is determined empirically and is estimated as known in the art. It is believed that a tandem repeat approach can further increase the immunogenicity or antigenicity of a peptide.
Any peptide or other structure comprising a peptide (for example, a trimeric structure, six-stranded structure, or other structure) is optionally conjugated to a carrier protein. Such a conjugated composition is further optionally formulated with or administered in connection with an adjuvant.
The peptides may be formulated with a suitable adjuvant in order to enhance the immunological response. Such adjuvants may include, but are not limited to mineral gels such as aluminum hydroxide; surface active substances such as lysolecithin, pluronic polyols, polyanions; other peptides; oil emulsions; and potentially useful human adjuvants such as BCG and Corynebacterium parvum. Many methods may be used to introduce the vaccine formulations described here. These methods include but are not limited to oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, mucosal, respiratory, and intranasal routes.
A polyclonal or monoclonal antibody molecule or population generated in connection with a peptide of the present invention is evaluated regarding one or more properties such as specificity, affinity, avidity, kinetic parameter, and biological activity. Techniques are used as known in the art, for example Western blotting, ELISA, and analysis using surface plasmon resonance technology (such as with a BiaCore device). For example, the biological activity of an antibody is tested for the ability to neutralize or inhibit infectivity or fusion by a SARS coronavirus or a SARS coronavirus protein in the context of a cell-cell fusion assay or viral infectivity assay. Antibodies are optionally raised initially in mice and rabbits for analysis.
A nucleotide molecule encoding a peptide of the invention can also be used for a genetic immunization approach as is known in the art.
The SARS-CoV HR-N and HR-C peptides are used to generate humanized monoclonal and polyclonal antibodies for passive immunotherapy treatment of SARS. An effective amount of an antibody preparation is provided and administered to a human subject suspected of a SARS coronavirus infection. The antibody can operate to reduce the ability of a SARS coronavirus S protein to make a conformation that is competent for fusion or entry in a target cell. Therefore an effective concentration of polyclonal or monoclonal antibodies raised against the peptides of the invention is administered to a host so that uninfected cells are less prone to becoming infected by SARS. The exact concentration of such antibodies will vary according to each specific antibody preparation, but may be determined using standard techniques well known to those of ordinary skill in the art. Administration of the antibodies may be accomplished using a variety of techniques, including, but not limited to those described herein.
Antibodies raised against the SARS HR-N and HR-C peptides are used in diagnosis to test for the presence of free virus in tissue or fluid samples.
Antibodies are used in a drug screening procedure to identify compounds that inhibit the formation of the 6-helix bundle fusion active core or the function of fusion or entry. The conformation-specific antibodies are used to detect the presence of the HR-N/HR-C 6-helix bundle structure. The binding of a test compound such as a drug to this structure, for example so as to disrupt this structure, is associated with the loss of an antibody reagent. Such loss can be by competitive inhibition due to displacement of the antibody by the test compound. Therefore the identification of a test compound capable of relevant binding is conducted using immunological procedures as known in the art.
An HR peptide such as HR-N10 is extended in either direction of the N/amino terminus or C/carboxy terminus. A peptide derived from HR-N10 is prepared by synthesizing a peptide beginning with any of HR-N amino acids 909 to 915 and ending with any of HR-N amino acids 951 to 957. For example, a derivative of HR-N10 is prepared by addition of about one heptad unit at one terminus or both termini. Other derivatives are prepared using less than a full heptad unit, for example from one to six amino acids.
An HR peptide such as HR-C4 is deleted from its N terminus so that from one to six amino acids are deleted. A peptide derived from HR-C4 is prepared by synthesizing a peptide beginning with any of HR-C amino acids 1152 to 1157. Such a peptide can also be perceived as being derived from HR-C3 with an earlier beginning point of the amino acid sequence.
The peptide derivative is tested for stability. The peptide is tested for ability to form a trimeric conformation or a six helix bundle conformation in a complex with an HR-N or HR-C peptide or native protein. The peptide is tested for ability to inhibit cell-cell fusion or infectivity.
A further object of the invention is to provide therapeutic compositions, suitable for human or veterinary pharmaceutical use, comprising a peptide, peptides, or complexes of a peptide or peptides of the present invention and a suitable pharmacological carrier. Such therapeutic compositions can be formulated as understood in the art for administration. For example, administration may be applied via oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, topical, mucosal, respiratory, and intranasal routes. For a respiratory route, an aerosol can be delivered from a nebulization formula, or an atomization spray formulation can be prepared. A therapeutic composition can control and/or prevent infection by a coronavirus, particularly a SARS coronavirus.
Effective dosages of the peptides of the invention to be administered may be determined through procedures well known to those in the art which address such parameters as biological half-life, bioavailability, and toxicity. For example, peptide HR-N10 may prove efficacious in vivo at doses required to achieve circulating levels of about 10 ng per ml of peptide.
Pharmaceutical compositions are formulated to contain a therapeutically effective amount of a composition of the invention and a pharmaceutically acceptable carrier appropriate for the route of administration. A composition of the invention can include, for example, a peptide of the invention or salt thereof.
A therapeutic form can comprise a carrier hydrogel composition of a polymer material and an effective dose of a peptide. The polymer materials used in the carrier hydrogel composition can preferably have reverse gelation properties and exist as a liquid, aqueous solution at temperatures below physiological temperatures (e.g., below the body temperature of a patient) but form hydrogels under physiological conditions (e.g., at temperatures at or near the body temperature of a patient). The carrier hydrogel compositions may thus be administered to a patient by injection while they are in a liquid state. Upon administration the carrier hydrogel compositions then form hydrogels with a peptide embedded therein. The peptide or peptides are thereby released with improved pharmacokinetic properties and bioavailability.
Compositions and methods of the invention are applicable to SARS coronaviruses generally. For example, the nucleotide sequences of NCBI Accession Numbers in Table 4 and protein sequences of NCBI Accession Numbers in Table 5 are used in embodiments of the invention.
Kliger Y and Levanon E Y, Cloaked similarity between HIV-1 and SARS CoV suggests an anti-SARS strategy, BMC Microbiology 2003 Sep 21; 3(1):20. U.S. Pat. No.: 5,464,933 by Bolognesi, et al.; U.S. Pat. Nos. 6,054,2651; 6,541,020; 6,518,013 Tripet B et al., 2004; Structural characterization of the SARS-Coronavirus spike S fusion protein core; J. Biol. Chem. 279 (20): 20836-20849.
All references throughout this application, for example publications, patents, and patent documents, are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
Any appendix or appendices hereto are incorporated by reference as part of the specification and/or drawings.
Where the terms “comprise”, “comprises”, “comprised”, or “comprising” are used herein, they are to be interpreted as specifying the presence of the stated features, integers, steps, or components referred to, but not to preclude the presence or addition of one or more other feature, integer, step, component, or group thereof. Separate embodiments of the invention are also intended to be encompassed wherein the terms “comprising” or “comprise(s)” or “comprised” are optionally replaced with the terms, analogous in grammar, e.g.; “consisting/consist(s)” or “consisting essentially of/consist(s) essentially of” to thereby describe further embodiments that are not necessarily coextensive.
The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. It will be apparent to one of ordinary skill in the art that compositions, methods, devices, device elements, materials, procedures and techniques other than those specifically described herein can be applied to the practice of the invention as broadly disclosed herein without resort to undue experimentation. All art-known functional equivalents of compositions, methods, devices, device elements, materials, procedures and techniques described herein are intended to be encompassed by this invention. Whenever a range is disclosed, all subranges and individual values are intended to be encompassed. This invention is not to be limited by the embodiments disclosed, including any shown in the drawings or exemplified in the specification, which are given by way of example or illustration and not of limitation. The scope of the invention shall be limited only by the claims.
This application claims the benefit of U.S. Provisional Application No. 60/544,410, filed Feb. 12, 2004, which is incorporated herein by reference in entirety.
This invention was made with government support under Grant/Contract Nos. NIH R01AI48717, NIH R01AI31948, and NIH R01AI25231 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US05/04408 | 2/14/2005 | WO | 00 | 8/3/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60544410 | Feb 2004 | US |
