U.S. patent application number 12/378558 was filed with the patent office on 2009-10-22 for influenza virus inhibiting peptides.
This patent application is currently assigned to The Administrators of the Tulane Educational Fund. Invention is credited to Robert F. Garry, Russell Wilson.
Application Number | 20090264362 12/378558 |
Document ID | / |
Family ID | 34572923 |
Filed Date | 2009-10-22 |
United States Patent
Application |
20090264362 |
Kind Code |
A1 |
Garry; Robert F. ; et
al. |
October 22, 2009 |
Influenza virus inhibiting peptides
Abstract
The present invention provides a pharmaceutical composition for
the treatment or prevention of an influenza infection. The
composition comprises an isolated polypeptide including a sequence
of at least 8 contiguous amino acid residues of the fusion
initiation region (FIR) of an influenza hemagglutinin 2 protein or
a peptide analog of the sequence. The FIR is a segment of the full
length hemagglutinin 2 protein which is bounded by an
amino-terminal region within the amino-terminal alpha-helix thereof
and a carboxy terminus within the carboxy-terminal alpha-helix
thereof, with a cysteine loop therebetween. The amino-terminal
region of the FIR comprises a portion of the final 10 to 20 amino
acid residues of the amino-terminal alpha-helix of the
hemagglutinin 2 protein, and includes 3 or 4 hydrophobic amino acid
residues, a positively-charged amino acid residue, a
negatively-charged amino acid residue, and an aromatic amino acid
residue. The carboxy terminus of the FIR is the carboxy terminus of
the first peptide sequence of the hemagglutinin 2 protein beyond
the amino terminal helix, which exhibits a positive Wimley-White
interfacial hydrophobicity.
Inventors: |
Garry; Robert F.; (New
Orleans, LA) ; Wilson; Russell; (Mandeville,
LA) |
Correspondence
Address: |
Olson & Cepuritis, LTD.
20 NORTH WACKER DRIVE, 36TH FLOOR
CHICAGO
IL
60606
US
|
Assignee: |
The Administrators of the Tulane
Educational Fund
|
Family ID: |
34572923 |
Appl. No.: |
12/378558 |
Filed: |
February 17, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10578013 |
May 3, 2006 |
7491793 |
|
|
PCT/US2004/036578 |
Nov 3, 2004 |
|
|
|
12378558 |
|
|
|
|
60517181 |
Nov 4, 2003 |
|
|
|
Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
C12N 7/00 20130101; C07K
7/06 20130101; C12N 2770/20022 20130101; C12N 2760/14122 20130101;
A61P 31/12 20180101; C07K 7/00 20130101; A61K 38/00 20130101; C07K
7/08 20130101; G01N 33/56988 20130101; A61K 38/04 20130101; C12N
2760/10022 20130101; C07K 14/005 20130101; A61P 31/16 20180101;
C12Q 1/18 20130101; A61K 38/162 20130101; C12N 2760/18433 20130101;
C12N 2760/18422 20130101 |
Class at
Publication: |
514/12 ; 514/16;
514/15; 514/14; 514/13 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 38/08 20060101 A61K038/08; A61K 38/10 20060101
A61K038/10; A61P 31/16 20060101 A61P031/16 |
Claims
1. A pharmaceutical composition for the treatment or prevention of
an influenza infection comprising: an isolated polypeptide
including at least 8 contiguous amino acid residues of the fusion
initiation region (FIR) of an influenza hemagglutinin 2 protein;
wherein the hemagglutinin 2 protein includes an amino-terminal
alpha-helix and a carboxy-terminal alpha-helix; and the FIR is a
segment of the full length hemagglutinin 2 protein which is bounded
by an amino-terminal region within the amino-terminal alpha-helix
and a carboxy terminus within the carboxy-terminal alpha-helix,
with a cysteine loop therebetween; and wherein: the amino-terminal
region of the FIR comprises a portion of the final 10 to 20 amino
acid residues of the amino-terminal alpha-helix of the
hemagglutinin 2 protein, and includes 3 or 4 hydrophobic amino acid
residues, a positively-charged amino acid residue, a
negatively-charged amino acid residue, and an aromatic amino acid
residue; and the carboxy terminus of the FIR is the carboxy
terminus of the first peptide sequence of the hemagglutinin 2
protein beyond the amino terminal helix, which exhibits a positive
Wimley-White interfacial hydrophobicity.
2. The composition of claim 1 wherein the polypeptide comprises 8
to 40 contiguous amino acid residues of the FIR of the influenza
hemagglutinin 2 protein.
3. The composition of claim 1 wherein the hemagglutinin 2 protein
is an influenza A hemagglutinin 2 protein.
4. The composition of claim 1 wherein the polypeptide includes a
hydrophobic group at the amino-terminus thereof.
5. The composition of claim 1 wherein the polypeptide includes a
macromolecular carrier at the amino-terminus thereof.
6. The composition of claim 5 wherein the macromolecular carrier is
selected from the group consisting of a lipid conjugate, a
polyethylene glycol moiety, and a carbohydrate moiety.
7. The composition of claim 1 wherein the polypeptide includes a
hydrophobic group at the carboxy-terminus thereof.
8. The composition of claim 1 wherein the polypeptide includes a
macromolecular carrier at the carboxy-terminus thereof.
9. The composition of claim 8 wherein the macromolecular carrier is
selected from the group consisting of a lipid conjugate, a
polyethylene glycol moiety, and a carbohydrate moiety.
10. The composition of claim 1 wherein the polypeptide is capable
of binding to the FIR of the influenza virus with a dissociation
constant, K.sub.d, of at least about 9.times.10.sup.-6.
11. A pharmaceutical composition for the treatment or prevention of
an influenza infection comprising: an isolated polypeptide
including a sequence of at least 8 contiguous amino acid residues
of the fusion initiation region (FIR) of an influenza A
hemagglutinin 2 protein or a peptide analog of the sequence;
wherein the hemagglutinin 2 protein includes an amino-terminal
alpha-helix and a carboxy-terminal alpha-helix; and the FIR is a
segment of the full length hemagglutinin 2 protein which is bounded
by an amino-terminal region within the amino-terminal alpha-helix
and a carboxy terminus within the carboxy-terminal alpha-helix,
with a cysteine loop therebetween; and wherein: the amino-terminal
region of the FIR comprises a portion of the final 10 to 20 amino
acid residues of the amino-terminal alpha-helix of the
hemagglutinin 2 protein, and includes 3 or 4 hydrophobic amino acid
residues, a positively-charged amino acid residue, a
negatively-charged amino acid residue, and an aromatic amino acid
residue; and the carboxy terminus of the FIR is the carboxy
terminus of the first peptide sequence of the hemagglutinin 2
protein beyond the amino terminal helix, which exhibits a positive
Wimley-White interfacial hydrophobicity.
12. The composition of claim 11 wherein the polypeptide comprises a
sequence of 8 to 40 contiguous amino acid residues of the FIR of
the influenza hemagglutinin 2 protein or a peptide analog of the
sequence.
13. The composition of claim 11 wherein the hemagglutinin 2 protein
is an influenza A hemagglutinin 2 protein.
14. The composition of claim 11 wherein the polypeptide includes a
hydrophobic group at the amino-terminus thereof.
15. The composition of claim 11 wherein the polypeptide includes a
macromolecular carrier at the amino-terminus thereof.
16. The composition of claim 15 wherein the macromolecular carrier
is selected from the group consisting of a lipid conjugate, a
polyethylene glycol moiety, and a carbohydrate moiety.
17. The composition of claim 11 wherein the polypeptide includes a
hydrophobic group at the carboxy-terminus thereof.
18. The composition of claim 11 wherein the polypeptide includes a
macromolecular carrier at the carboxy-terminus thereof.
19. The composition of claim 18 wherein the macromolecular carrier
is selected from the group consisting of a lipid conjugate, a
polyethylene glycol moiety, and a carbohydrate moiety.
20. The composition of claim 11 wherein the polypeptide includes a
peptide analog, wherein the analog includes one or more
conservative amino acid substitution in the sequence of the at
least 8 contiguous amino acid residues of the FIR.
21. The composition of claim 11 wherein the polypeptide includes a
peptide analog, wherein the analog includes one or more D-amino
acid substitutions in the amino acid residue sequence of the at
least 8 contiguous amino acid residues of the FIR.
22. The composition of claim 11 wherein the polypeptide is capable
of binding to the FIR of the influenza virus with a dissociation
constant, K.sub.d, of at least about 9.times.10.sup.-6.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/578,013, filed on May 3, 2006, now U.S. Pat. No. 7,491,793,
which is the National Stage of PCT/US2004/36578, filed on Nov. 3,
2004, which claims the benefit of U.S. Provisional Application Ser.
No. 60/517,181, filed Nov. 4, 2003, each of which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of preventing or
inhibiting viral infection of a cell and/or fusion between the
envelope of a virus and the membranes of a cell targeted by the
virus (thereby preventing delivery of the viral genome into the
cell cytoplasm, a step required for viral infection). The present
invention provides methods for identifying a fusion initiation
region, or FIR, of the viruses. The present invention provides for
a method of identifying the FIR in these viruses. The present
invention further provides for methods of preventing infection by a
Type I virus by interfering with its FIR.
INTRODUCTION
[0003] All viruses must bind to and invade their target cells to
replicate. For enveloped animal viruses, including RNA viruses
having Class I membrane fusion proteins (Type I viruses), the
process involves (a) binding of the virion to the target cell, (b)
fusion of the envelope of the virus with the plasma membrane or an
internal cellular membrane, (c) destabilisation of the viral
envelope and cellular membrane at the fused area to create a fusion
pore, (d) transfer of the viral RNA through the pore, and (e)
modification of cellular function by the viral RNA.
[0004] Fusion of the viral membrane and the cell envelope, steps
(b) and (c) above, is mediated by the interaction of a viral
transmembrane glycoprotein (fusion protein) with surface proteins
and membranes of the target cell. These interactions cause
conformational changes in the fusion protein that result in the
insertion of a viral fusion peptide into the target cell membrane.
This insertion is followed by further conformational changes within
the fusion protein that bring the viral envelope and cell membranes
into close proximity and results in the fusion of the two membrane
bilayers.
[0005] A virus is unable to spread and propagate within its host if
this fusion process is disrupted. Intentional disruption of this
fusion process can be achieved by directing peptides and peptide
mimics homologous to fusion protein sequences, antibodies that
recognize the fusion protein, and other factors that act against
the fusion protein.
BACKGROUND OF THE INVENTION
[0006] Structural Similarities among RNA Virus Class I Fusion
Proteins. Hemagglutinin 2 (HA2) of influenza virus, an
orthomyxovirus, is the prototypic RNA virus Class I fusion protein
and contains an amino terminal hydrophobic domain, referred to as
the fusion peptide, that is exposed during cleavage of the
hemagglutinin precursor protein. The membrane fusion proteins of
RNA viruses from several diverse families, including arenaviruses,
coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and
retroviruses, share several common structural features with HA2 and
have been referred to as Class I viral fusion proteins.
[0007] It has been observed that the fusion protein of HIV-1, the
transmembrane glycoprotein and other retroviral transmembrane
proteins, like those of orthomyxoviruses and paramyxoviruses,
possess a hydrophobic fusion peptide domain exposed during cleavage
of a precursor (gp160) (Gallaher, 1987; Gonzalez-Scarano et al.,
1987). Based on these similarities and computer algorithms that
predict protein configurations, it has been suggested (Gallaher et
al., 1989) that the external portion (ectodomain, amino terminus)
of HIV-1 transmembrane protein and the transmembrane proteins of
other retroviruses, all could fit the scaffold of HA2 structure as
determined by x-ray crystallography (Wilson, Skehel, and Wiley,
1981).
[0008] Based on these observations, it was predicted that
retroviral transmembrane proteins contain several structural
features in addition to the fusion peptide in common with the known
structure of HA2, including an extended amino terminal helix
(N-helix, usually a "heptad repeat" or "leucine zipper"), a
carboxyl terminal helix (C-helix), and an aromatic motif proximal
to the transmembrane domain. The presence of at least four out of
these five domains defines a viral envelope protein as a Class I
fusion protein. This retroviral transmembrane protein model was
subsequently confirmed by structural determinations and mutational
analyses (Chan et al., 1997; Kowalski et al., 1991; Weissenhorn et
al., 1997). Common structural motifs are present not only in
orthomyxovirus and retrovirus fusion proteins, but also in those of
paramyxoviruses, filoviruses (such as Ebola virus, EboV) (Gallaher,
1996) and arenaviruses (Gallaher, DiSimone, and Buchmeier, 2001).
The Gallaher structural model of the EboV fusion protein (GP2) has
also been confirmed by x-ray crystallographic methods (Malashkevich
et al., 1999; Weissenhom et al., 1998).
[0009] FIG. 1 shows the five, previously-described, domains of the
fusion proteins of the six families of Type I viruses. The fusion
proteins originate in a hydrophobic fusion peptide, terminate in an
anchor peptide, and incorporate an extended amino terminal
alpha-helix (N-helix, usually a "heptad repeat" or "leucine
zipper"), a carboxyl terminal alpha-helix (C-helix) (Carr and Kim,
1993; Suarez et al., 2000; Wilson, Skehel, and Wiley, 1981), and
sometimes an aromatic motif proximal to the virion envelope. Also
shown is the sixth domain, the fusion initiation region (FIR),
discovered by the present inventors.
[0010] Fusion Inhibition in Type I Viruses. Previous attempts by
the present inventors (Garry) and others to design peptides and
peptide mimics, antibodies, and other factors that inhibit fusion
in Type I viruses have focused on the fusion peptide, the N-helix,
and the C-helix of the fusion proteins. In the case of fusion
peptides, analogs of the orthomyxoviruses and paramyxoviruses
(Richardson, Scheid, and Choppin, 1980) and HIV-1 fusion peptide
domains (Gallaher et al., 1992; Owens et al., 1990; Silburn et al.,
1998) have been found to block viral infection, presumably by
forming inactive heteroaggregates. Peptides corresponding to
portions of the N-helix and C-helix have also been found to be
effective in inhibiting viral infection both in vitro and in vivo.
For example, a 17-amino-acid peptide corresponding to the
carboxy-terminal portion of the N-helix of the HIV-1 fusion
protein, defined as the CS3 region, blocked HIV infection (Qureshi
et al., 1990). In addition, other N-helix and C-helix inhibitory
peptides were developed based on the fusion protein structural
model (Wild, Greenwell, and Matthews, 1993; Wild et al., 1992),
including the C-helix anti-HIV-1 peptidic drug DP178 (T-20 or
FUZEON.RTM.). DP178 overlaps the C-helix and the aromatic
anchor-proximal domain and inhibits HIV-1 virion: cell fusion at
very low concentrations (50% inhibition at 1.7 nM) achievable in
vivo following injection. In a clinical trial, 100 mg/day of DP178
caused an approximately 100-fold reduction in plasma HIV-1 load of
infected individuals (Kilby et al., 1998). This result has greatly
motivated the search for other HIV-1 inhibitory peptides based on
transmembrane protein structure (Pozniak, 2001; Sodroski, 1999).
Peptidic inhibitors of paramyxoviruses have also been shown to
inhibit viral replication (Lambert et al., 1996; Young et al.,
1999). Studies by Watanabe and coworkers suggest that a similar
approach of targeting the N-helix and the C-helix of EboV GP2 may
also lead to useful inhibitors (Watanabe et al., 2000).
Neutralizing antibodies directed against portions of the fusion
protein domains have also been shown to inhibit virion: cell
fusion.
[0011] Observations in HIV-1. A great deal of study has been
devoted to fusion inhibition in human immunodeficiency virus HIV-1,
one of the Type I RNA viruses. Bolognesi et al. (U.S. Pat. No.
5,464,933) and the present inventors (Garry, U.S. Pat. No.
5,567,805) teach that HIV-mediated cell killing can be inhibited by
introducing peptides that bind to portions of the transmembrane
fusion protein of the HIV-1 virion. The Bolognesi DP178 binding
region, labeled FUZEON.RTM. in FIG. 7, lies primarily on the
C-helix and is outside what is described in the present application
the fusion initiation region (FIR). Bolognesi demonstrates
inhibition but teaches no method of inhibition. The present
inventors (Garry) previously demonstrated inhibition at the CS3
region of HIV-1 .TM., labeled CS3 in FIG. 7, but identified no
method of inhibition, suggesting only that CS3: CS3-receptor
interaction is inhibited. The unexpected discovery of the FIR by
the present inventors (as currently described herein) and the fact
that the CS3 sequences lie within the FIR indicates that the CS3:
CS3-receptor binding described in U.S. Pat. No. 5,567,805 is in
fact binding that occurs between the CS3 portion of the FIR and
portions of the cell membrane for which the CS3 portion of the FIR
has an affinity. In addition, although Melikyan, Watanabe, Bewley,
and others have described fusion inhibition with introduced
peptides, they have not explained the mechanisms through which the
inhibition occurs. Correspondingly, the location of the FUZEON.RTM.
peptide is distant from the FIR, which strongly suggests that other
elements of the fusion process operate in the FUZEON.RTM.
region.
[0012] In view of the foregoing, it is clear that there exists a
need in the art for a more effective means for identifying those
regions of viruses that are involved in the infection process and
for compositions effective for preventing or inhibiting viral
infection. The invention described and disclosed herein provides an
effective solution to these needs.
SUMMARY OF THE INVENTION
[0013] Various embodiments of the instant invention provide for
methods of identifying "factors" (compounds) capable of inhibiting
membrane fusion between viruses and their host cells and, thereby,
preventing or inhibiting infection of the host cell by the virus.
Aspects of this embodiment of the invention provide for methods of
identifying these inhibitory "factors" where the method comprises
the steps of (a) identifying a virus having an envelope fusion
protein having two, or more, extended alpha helices, a fusion
peptide, and a fusion initiation region (FIR); (b) preparing a
"target" wherein the target comprises the amino acid sequence of
the FIR, (c) exposing the "target" to one or more test compounds,
and (d) identifying those test compounds that physically interact
with the "target". For example, physical interaction can be
detected using a "target" bound to a solid substrate and a
fluorescently or radioactively labeled test compound in a standard
binding assay. Target and test compounds having dissociation
coefficients (K.sub.d) in the micromolar range or lower (i.e.
.ltoreq.about 9.times.10.sup.-6) are considered to be positively
interacting.
[0014] Other aspects of the instant invention provide for
compositions comprising an isolated peptide having the amino acid
sequence of a viral fusion initiation region (FIR) or a functional
segment of the FIR or having an amino acid sequence which is
analogous to the sequence of a FIR or a functional segment of a
FIR. As used herein, an analogous amino acid or peptide sequence is
a sequence containing a majority of identical or chemically similar
amino acids in the same order as a primary sequence. Such chemical
similarities are well known to those skilled in the art.
[0015] Other aspects of this embodiment of the invention provide
for isolated, typically substantially purified, peptides or peptide
analogs that are capable of preventing or inhibiting viral
infection of a host cell and/or inhibiting membrane fusion of a
virus with a host cell, where the virus comprises a membrane fusion
protein having two (extended) alpha helices, a fusion peptide and a
FIR.
[0016] Additional embodiments of the instant invention provide for
methods of treating or preventing viral infection by administering
to a patient one or more of the compounds identified by the methods
described herein as capable of inhibiting viral infection. In
various aspects of this embodiment of the invention the compounds
administered are peptides or peptide analogs comprising all or a
functional segment of a viral FIR sequence. In any aspect of this
embodiment of the invention the administered compound is antigenic
and is administered in an amount sufficient to eliciting an immune
response.
[0017] Other embodiments of the instant invention provide for a
"molecular factor", such as a plasmid, recombinant virus, or other
substance which enables or stimulates a cell or organism to produce
a peptide or peptide analog that is capable of preventing or
inhibiting a viral infection of that cell or organism. In any
aspect of this embodiment the "molecular factor" is capable of
preventing or inhibiting a viral infection when administered to a
patient.
[0018] Another embodiment of the instant invention provides for
antibodies capable of inhibiting the virus:cell membrane fusion of
a virus having a fusion protein comprising two, extended
alpha-helices, a fusion peptide and a FIR. In any aspect of this
embodiment of the invention the antibodies are capable of binding
specifically to amino acid sequences comprising the FR sequence, or
fragments thereof of sufficient size to allow antibody recognition.
Various aspects of this embodiment of the invention provide for
methods of producing the antibodies. In certain aspects of this
embodiment, the method for producing antibodies comprises: (a)
providing as the antigen a peptide comprising a viral initiation
region (FIR) or an antigenic fragment of the FIR; (b) introducing
the antigen in to an animal so as to elicit an immune response; (c)
collecting antibodies from the animal; and optionally, (d)
purifying the collected antibodies to identify that fraction of the
collected antibodies having a high specificity for the antigen.
[0019] Other embodiments of the current invention provide methods
of treating patients, which methods comprise administering to the
patient antibodies that specifically recognize and bind to peptides
comprising a FIR region from a virus or comprising a functional
fragment of such a FIR region where the functional fragment is of
sufficient size to allow its specific recognition by an antibody
(that is, it is an antigenic fragment).
[0020] Other embodiments of the instant invention provide for
methods of producing antibodies specific for FIR or functional
fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1 shows the domains of the fusion proteins of one
member of each of these six viral families (namely, arenaviruses,
coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and
retroviruses). The circles in FIG. 1 show the approximate location
of the FIR in each virus illustrated.
[0022] FIGS. 2 through 7 show the amino acid sequences of these
fusion proteins (corresponding to SEQ ID NOs 16-21, respectively)
and a schematic representation of their ectopic structure.
Specifically shown are the five previously-described domains are
the fusion peptide, i.e., the N-helix, the C-helix, the aromatic
motif (if present), and the anchor peptide. The newly-discovered
sixth domain, the fusion initiation region, or FIR is also
identified. Each FIR is indicated by a polygon in FIGS. 2 through
7. The circled area behind the fusion proteins in each of FIGS. 2-7
represents the primary virus:cell binding protein (VCBP) of the
virus. The VCBP usually interacts with the portion of the fusion
protein which is most distal from the viral membrane and is thus
shown to be so positioned in the Figures. Unlike the
highly-conserved fusion protein, the VCBP of each virus family is
more divergent. It is usually the VCBP that dictates the host range
of the virus and determines which of the host's cell types are
targeted for infection. The VCBP acts in this capacity by
recognizing and binding with specific cell surface proteins. The
binding of the VCBP to the targeted cell proteins occurs prior to
and is typically a prerequisite for virus:cell fusion.
[0023] FIG. 8: Inhibition of coronavirus infectivity by fusion
initiation region peptides. Between 50 and 100 plaque forming units
(PFU) of mouse hepatitis virus strain A59 or SARS coronavirus
strain Urbani were pre-incubated with or without the indicated
peptides (approximately 100 .mu.M) in serum-free DMEM for 1 hour.
Cells were then exposed to peptide-treated inoculum or a vehicle
control (no peptide). After 1 hour adsorption, the inoculum was
removed, cells were washed twice with 1.times. phosphate buffered
saline, and the cells were overlaid with DMEM containing 10% FBS
and 0.5% agarose. Forty eight hours after infection, infected
monolayers were fixed and stained with crystal violet to determine
plaque numbers.
[0024] FIG. 9: Inhibition of Lassa virus infectivity by fusion
initiation region peptides. Between 50 and 100 PFU Lassa virus was
pre-incubated with or without the indicated peptides (approximately
100 .mu.M) in serum-free BME for 1 hour. Cells were then exposed to
the peptide-treated inoculum or vehicle control (no peptide). After
1 hour adsorption, the inoculum was removed, cells were washed
twice with 1.times. phosphate buffered saline, and the cells were
overlaid with BME containing 5% FBS, 10 mM HEPES and 0.5% agarose.
Four days after infection a second overlay containing 5% neutral
red was applied, and plaques were counted 24 hours later.
ILLUSTRATIVE EMBODIMENTS OF THE INVENTION
[0025] The Sixth Domain of RNA Viruses Having Class I Membrane
Fusion Proteins. The arenaviruses, coronaviruses, filoviruses,
orthomyxoviruses, paramyxoviruses, and retroviruses are the six
families of RNA viruses currently identified that have Class I
membrane fusion envelope proteins. The fusion proteins of these
Type I viruses have previously been shown by the present inventors
(Garry) and others to incorporate five conserved motifs, or domains
(Carr and Kim, 1993; Gallaher et al., 1989; Suarez et al., 2000;
Wilson, Skehel, and Wiley, 1981). These domains comprise a fusion
peptide, an N-helix, a C-helix, and an aromatic motif, all of which
are ectodomains, and an anchor peptide, which is an endodomain.
[0026] Using computational analyses, secondary structure models,
interfacial hydrophobicity calculations and other techniques, the
present inventors have made the surprising discovery of a highly
conserved sixth domain that is present in the fusion proteins of a
wide variety of viruses (this sixth domain is described herein).
The viruses possessing this domain include, but are not necessarily
limited to the six classes of RNA viruses listed above. To
emphasize the critical function of this newly identified domain,
which is an ectodomain, the domain is referred to herein as the
fusion initiation region (FIR) of the viruses.
[0027] Various embodiments of the instant invention provide methods
of identifying the FIR in arenavirus, coronavirus, filovirus,
orthomyxovirus, paramyxovirus, and retrovirus families of viruses.
Also provided are methods of determining whether the FIR is present
in other known virus families or in any newly discovered virus
families.
[0028] As used herein the term "extended" alpha helix refers to an
alpha helix having more than four "alpha helix turns"
(specifically, more than 14 amino acids).
[0029] Other embodiments provide for "factors" that the inventors
have unexpectedly found are effective for preventing or inhibiting
viral infection and/or virus:cell fusion.
[0030] As used herein the term "factors" includes, but is not
limited to isolated peptides or functional peptide segments (or
peptide analogs thereof) of the newly described fusion initiation
region (FIR) domains, peptide mimics ("peptide mimic" refers to any
compound or substance that could serve as a substitute for a
peptide interacting with the FIR, that is any compound that mimics
the properties of a functional segment of the FIR), antibodies
specific for functional FIR domains (e.g. idiotypic or
anti-idiotypic antibodies) and other molecular compounds that
interfere with virus:cell binding and/or fusion.
[0031] As used herein the term "functional segment" or "functional
fragment" of a fusion initiation region (FIR) refers to a fragment
capable of inhibiting virus:cell fusion, inhibiting viral
infectivity, capable of eliciting an antibody capable of
recognizing and specifically binding to the FIR and/or interfering
with FIR-mediated cell infection.
[0032] As used herein, a "peptide analog" or "modified peptide" is
preferably defined as a FIR peptide modified to contain an amino
group, an acetyl group, a hydrophobic group (for example
carbobenzoxyl, dansyl, or t-butyloxycarbonyl) or a macromolecular
carrier group (for example lipid conjugate, polyethylene glycol, a
carbohydrate or a protein) at the amino terminus. An additional
class of FIR peptide analogs contains a carboxyl group, an amido
group, a hydrophobic group or a macromolecular carrier group at the
carboxyl terminus. Other peptide analogs are defined as FIR
peptides wherein at least one bond linking adjacent amino acids
residues is a non-peptide bond (for example an imido, ester,
hydrazine, semicarbazoide or azo bond), a peptide wherein at least
one amino acid residue is in a D-isomer configurations or a peptide
in which the order of the amino acids is inverted. Additional
peptide analogs are FIR peptides compromising at least one amino
acid substitution wherein a first amino acid residue is substituted
for a second, different amino acid residue (the amino acid
substitution can be a conserved substitution or a non-conserved
substitution). As used herein, such peptide analogs may comprise
analogous amino acid sequences in which the analogous sequences
contain a majority of identical or chemically similar amino acids
in the same order as the primary sequences.
[0033] As used herein, the term "fusion initiation region" (FIR)
generally refers to a region of a viral fusion protein involved in
the initial step or steps of viral infection and/or fusion with a
host cell.
[0034] As used herein the term "peptide mimic" includes, but is not
limited to organic compounds or other chemicals that mimic the
structure or function of the FIR peptide. Examples of peptide
mimics include, but are not limited to organic compounds comprising
the functional side-groups of an amino acid or peptide, but lacking
the carbon/nitrogen backbone or peptide bonds. Peptide mimic also
refers to compounds that mimic the action of these functional
side-groups with other moieties.
[0035] Other molecules, such as idiotype or anti-idiotype
antibodies or proteins selected via phage display methods, that
bind to the peptides, peptide analogs or peptide mimics described
in the present application may also function as inhibitors of viral
infection and/or virus:cell fusion. Also contemplated by the
instant invention are plasmids, or recombinant viruses, or other
molecules or compounds that enable or stimulate the patient to
produce an analog of the inhibitory compounds. For example, a
recombinant protein, produced in an engineered bacterial, fungal,
or mammalian cell, can be used to produce an immunogenic analog of
the FIR of a viral fusion protein. Similarly, an anti-idiotypic
response could be induced in the individual by using an engineered
protein comprising a sequence corresponding to the binding site of
a FIR-specific antibody.
[0036] As used herein the term "fusion peptide" preferably refers
to a hydrophobic sequence at or near the amino terminus of a class
I viral fusion protein (see, Gallaher et al., 1987; 1992).
[0037] As used herein the term "substantially purified" peptide or
peptide analog preferably refers to a peptide or peptide analog
that is greater than about 80% pure. More preferably,
"substantially purified" refers to a peptide or peptide analog that
is greater than about 90% or greater than about 95% pure. Most
preferably it refers to a peptide or peptide analog that is greater
than 96%, 97%, 98%, or 99% pure. Functionally, "substantially
purified" means that it is free from contaminants to a degree that
makes it suitable for the purposes provided herein. Methods for
assessing purity are well known to those of skill in the art.
Suitable methods include, but are not limited to gas chromatography
(GC) linked mass spectrophotometry, high performance liquid
chromatography (HPLC) analysis, and functional assays in cell
culture systems that, inter alia, assess cytotoxicity.
[0038] As used herein the term "stable analog" refers to a peptide
that has a pharmacologically active half-life in biological
systems. Biological half-lives of greater than 60 minutes are
contemplated.
[0039] As used herein the term "peptide derivative" refers to a
peptide that has substituted amino acids different from those in
the FIR sequence of a viral fusion protein. Wherein the
substitutions do not render the peptide useless for the instant
invention.
[0040] According to various aspects of the present embodiment of
the invention the peptides, peptide analogs, peptide mimics, and
other factors may be produced by any means known in the art,
including, but not limited to, chemical synthesis, recombinant DNA
methods and combinations thereof.
[0041] The present invention provides methods for identifying the
FIR of Type I, and other, viruses and for treating or preventing
infection by these viruses. One possible mechanism by which the
current invention may to prevent and/or inhibit infection is by
interfering with the FR mediated virus:cell fusion. The six
families of RNA viruses now known to have Class I membrane fusion
proteins (Type I viruses) and representative members of each family
are listed in Table 1 and Table 2.
TABLE-US-00001 TABLE 1 Representative RNA Viruses Having Class I
Membrane Fusion Proteins (Type I Viruses) Familv Representative
Virus Shown in Figures Arenaviruses Lassa Virus Yes Lymphocytic
Choriomeningitis Virus (LCMV) No Junin Virus No Machupo Virus No
Guanarito Virus No Sabia Virus No Coronaviruses Severe Acute
Respiratory Syndrome (SARS) Virus Yes Murine Hepatitis Virus (MHV)
No Bovine Coronavirus No Canine Coronavirus No Feline Infectious
Peritonitis Virus No Filoviruses Ebola Virus Yes Marburg Virus No
Orthomyxoviruses Influenza A Virus Yes Influenza B Virus No
Influenza C Virus No Paramyxoviruses Measles Virus Yes Mumps Virus
No Canine Distemper Virus No Newcastle Disease Virus No
Retroviruses Human Immunodeficiency Virus 1 (HIV-1) Yes Human
Immunodeficiency Virus 2 (HIV-2) No Human T-cell Lymphotrophic
Virus 1 (HTLV-1) No Human T-cell Lymphotrophic Virus 2 (HTLV-2) No
Human Intracisternal A-type Particle 1 (HIAP-1) No Human
Intracisternal A-type Particle 2 (HIAP-2) No
TABLE-US-00002 TABLE 2 Illustrated RNA Viruses Having Class I
Membrane Fusion Proteins (Type I Viruses) shown in the Figures.
Figure Family Virus Shown Protein Shown FIG. 2 Arenaviruses Lassa
Virus GP2 FIG. 3 Coronaviruses SARS Virus S FIG. 4 Filoviruses
Ebola Virus GP2 FIG. 5 Orthomyxoviruses Influenza A Virus HA2 FIG.
6 Paramyxoviruses Measles Virus F1 FIG. 7 Retroviruses HIV-1 TM
[0042] The sequences of illustrated Class I membrane fusion
proteins (Type I Viruses) shown in the figures are as follows:
LASSA GP2 (Genbank Accession Number: A43492, amino acids 257-490),
SEQ ID NO: 16; SARS S (Genbank Accession Number: AAQ9406, amino
acids 864-1256), SEQ ID NO: 17; EBOLA GP2 (Genbank Accession
Number: AAM76034, amino acids 502-676), SEQ ID NO: 18; INFLUENZA
HA2 (Genbank Accession Number: P03437, amino acids 346-566), SEQ ID
NO: 19; MEASLES F1 (Genbank Accession Number: VGNZMV, amino acids
116-553), SEQ ID NO: 20; HIV TM (Genbank Accession Number:
AAB50262, amino acids 512-710), SEQ ID NO: 21.
[0043] Method of Identifying the FIR. Certain embodiments of the
invention comprise a method of identifying within the fusion
proteins of viruses a conserved motif. The conserved motif of the
FIR regions from different viruses will have similar structure and
function. Additionally, the FIR regions of related viruses may, but
will not necessarily, have highly similar primary amino acid
sequences. The current invention provides means for identifying
these regions, either with or without relying on their
identity/similarity to known sequences.
[0044] Other embodiments of the present invention provide for
methods useful for preventing or inhibiting viral infection and/or
virus:cell fusion using peptides, peptide mimics, antibodies or
other factors that are targeted to the specific virus' FIR and
interfere with the function of that FIR.
[0045] The FIR is typically between 50 and 100 amino acids in
length, although it may be longer in some viruses. Various aspects
of the current embodiments provide methods for identifying the FIR
of a viral fusion protein wherein the methods comprises the
following steps: (1) The sequence of the fusion protein is first
fitted to the HfV transmembrane fusion protein scaffold, which
comprises the N-helix, the C-helix, and other previously-described
domains, in order to identify the N-helix and the C-helix in the
subject fusion protein. This fitting process is facilitated by
searching the primary amino acid sequence of the protein for two or
more cysteines that have a propensity to form at least one
covalently bonded loop, which will be present in most but not all
of these sequences. The N-helix can then be identified in the
region preceding this cysteine loop by examining the region for
charged amino acids and other amino acids that have the propensity
to form an alpha helix (e.g., glutamine (Q), alanine (A),
tryptophane (W), lysine (K) and leucine (L)). (2) The amino
terminus of the FIR is then identified on the N-helix. This
terminus will usually lie within the final 10 to 20 amino acids of
the N-helix and will have a core typically comprising three or four
hydrophobic amino acids (such as leucine (L) or alanine (A)), a
positively-charged amino acid (such as lysine (K) or arginine (R)),
a negatively-charged amino acid (such as glutamate (E)), and an
aromatic amino acid (such as tyrosine (Y)). (3) The carboxy
terminus of the FIR is then identified. In the case of all of the
families except the coronaviruses and paramyxoviruses, this
terminus is the carboxy-terminus of the first peptide sequence with
positive interfacial-hydrophobicity that is found beyond the
N-helix. This terminus is usually located beyond the cysteine loop,
if the loop is present, and sometimes overlaps the C-helix or is
positioned on the C-helix. The positive interfacial-hydrophobicity
sequences have a high percentage of aromatic amino acids (such as
tryptophane (W), phenylalanine (F), and tyrosine (Y)) and small
hydrophobic amino acids (such as glycine (G)). The degree of
interfacial hydrophobicity of these sequences can be determined by
using the Wimley-White interfacial hydrophobicity scale, preferably
with a computer program such as the MPEX program that incorporates
this scale. "Interfacial hydrophobicity" is a measure of a
peptide's ability to transfer from an aqueous solution to the
membrane bilayer interface and is based on the experimentally
determined Wimley-White whole-residue hydrophobicity scale
(Jaysinghe, Hristova, and White, 2000). Computer programs using
this scale can identify a peptide sequence of a peptide chain
having positive interfacial hydrophobicity scores and are therefore
the most likely to associate with the surface of membranes. See
Example 1, as an example of the application of this method to the
identification of the FIR in the Ebola virus.
[0046] In the case of the coronaviruses, which have longer alpha
helices and a generally larger scale, and the paramyxoviruses, in
which the FIR is discontinuous because of a non-FIR sequence
insert, the carboxy terminus of the FIR is the carboxy-terminus of
the second peptide sequence with positive
interfacial-hydrophobicity that is found beyond the N-helix. The
sequence between the N-helix and C-helix in the F1 protein of
paramyxoviruses is longer than the interhelical sequences of other
viruses with Class I viral fusion proteins. The F2 protein of
paramyxoviruses, which serves a receptor-binding function, is
correspondingly shorter. Upon inspection of computer models, it is
obvious to those skilled in the art that the F1 protein contains a
sequence insert between the N-helix and C-helix. Consequently, the
FIR of paramyxoviruses contains two cysteine loops and two
high-interfacial-hydrophobicity sequences and is discontinuous
because additional amino acids which are characteristic only of the
paramyxoviruses and appear between the N-helix and the first
high-interfacial-hydrophobicity sequence are excluded from the
FIR.
[0047] FIR SEQUENCES. The sequence of the fusion protein and FIR
for each of the six representative viruses shown in FIG. 2 through
FIG. 7 is given in the respective Figure and in the Sequence
Listing provided below (SEQ ID NO: 16 to SEQ ID NO: 21 provide the
respective fusion proteins; and SEQ ID NO: 1 to SEQ NO: 7 provide
the respective FIR). Although there is some minor sequence
variation among the sister viruses within each of these six
families, the FIR in any Type I virus can readily be identified
using the representative sequence given in the appropriate
figure.
[0048] Methods of Inhibiting Fusion in these Viruses. Other
embodiments of the present invention provide methods of inhibiting
virus:cell fusion by interfering with the function of the FIR.
Various aspects of these embodiments include targeting the FIR with
peptides, peptide mimics and other factors which may or may not be
analogs of the FIR, in order to interfere with virus:cell fusion.
In the various aspects of this embodiment of the present invention
the peptides, peptide mimics, and peptide analogs are between about
6 and 150 amino acid residues long. More preferably, they are from
about 8 to 50 residues long, even more preferably they are from
about 8 to 40 amino acids in length or of such length as is
necessary to provide effective inhibition of viral infection. As
used herein the term "of such length as necessary to provide
effective inhibition of the virus", preferably refers to a length
sufficient to provide a 5-fold or greater reduction in viral
infectivity, when used according to the instant invention. Methods
for quantifying reduction in viral infectivity are well known to
those of skill in the art. For example, reductions in viral
activity may be determined by plaque reduction, binding inhibition,
titer reduction assays, or by animal challenge studies.
[0049] FIR peptides, peptides of analogous sequences, or fragments
or derivatives thereof, contemplated as being part of the instant
invention include, but are not limited to, those comprising, as
primary amino acid sequences, all or an efficacious part of one or
more of the following: LASSA,
X-LIMKNHLRDIMGIPYCNYSRYWYLNHTSTGKTLPRCWLI-Z (SEQ ID NO: 1); SARS,
X-LIRAAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQAAPH
GVVFLHVTYVPSQERNFTTAPAICHEGKAYFPREGVFVFNGTSWFITQRNFFS-Z (SEQ ID NO:
2); EBOLA, X-LRTFSILNRKAIDFLLQRWGGTCHILGPDCCI-Z (SEQ ID NO: 3);
INFLUENZA, X-IQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLF-Z (SEQ ID
NO: 4), MEASLES, X-LGLKLLRYYTEILSLFG-Z (SEQ ID NO: 5) - - -
X-WYTTVPKYV
ATQGYLISNFDESSCTFMPEGTVCSQNALYPMSPLLQECLRGSTKSCARTLVSGSFGNRFILS
QGNLIANCASILCKCYTTGTII-Z (SEQ ID NO: 6), wherein the " - - - "
indicates that the Measles FIR is discontinuous; and HIV,
X-LQARILAVERYLKDQQLLGIWGCSGKLICTTAVP WNASWSNKSLE QIWNHTTWMEWD-Z
(SEQ ID NO: 7). In each of the foregoing sequences the "X" and the
"Z" respectively designate either the amino- or carboxy-terminus,
respectively, of the peptide or an additional moiety, as described
below.
[0050] Other peptides provided by the instant invention include
those having the sequence of a FIR region. In a preferred aspect of
this embodiment the FIR region is from a virus belonging to one of
the viral families selected from the group consisting of
arenaviruses, coronaviruses, filoviruses, orthomyxoviruses,
paramyxoviruses, and retroviruses. In a more preferred aspect of
this embodiment, the FIR is from a virus selected from the group
consisting of Lassa Virus, Lymphocytic Choriomeningitis Virus
(LCMV), Junin Virus, Machupo Virus, Guanarito Virus, Sabia Virus,
Severe Acute Respiratory Syndrome (SARS) Virus, Murine Hepatitis
Virus (MHV), Bovine Coronavirus, Canine Coronavirus, Feline
Infectious Peritonitis Virus, Ebola Virus, Marburg Virus, Influenza
A Virus, Influenza B Virus, Influenza C Virus, Measles Virus, Mumps
Virus, Canine Distemper Virus, Newcastle Disease Virus, Human
Ihmunodeficiency Virus 1 (HIV-1), Human Immunodeficiency Virus 2
(HIV-2), Human T-cell Lymphotrophic Virus 1 (HTLV-1), Human T-cell
Lymphotrophic Virus 2 (HTLV-2), Human Intracistemal A-type Particle
1 (HIAP-1), and Human Intracistemal A-type Particle 2 (HIAP-2).
[0051] Other aspects of this embodiment of the invention provide
for sequences comprising a functional fragment of a FIR sequence or
sequences analogous thereto, particularly from a virus belonging to
one of the viral families selected from the group consisting of
arenaviruses, coronaviruses, filoviruses, orthomyxoviruses,
paramyxoviruses, and retroviruses (with the exception of the HIV-1
.TM. CS3 peptide previously described by the present inventors
(Garry) and depicted in FIG. 7). In another preferred aspect of
this embodiment, the peptide comprises a functional fragment
(except the HIV-1 .TM. CS3 fragment) or a sequence analogous to a
functional fragment from a virus selected from the group consisting
of Lassa Virus, Lymphocytic Choriomeningitis Virus (LCMV), Junin
Virus, Machupo Virus, Guanarito Virus, Sabia Virus, Severe Acute
Respiratory Syndrome (SARS) Virus, Murine Hepatitis Virus (MHV),
Bovine Coronavirus, Canine Coronavirus, Feline Infectious
Peritonitis Virus, Ebola Virus, Marburg Virus, Influenza A Virus,
Influenza B Virus, Influenza C Virus, Measles Virus, Mumps Virus,
Canine Distemper Virus, Newcastle Disease Virus, Human
Immunodeficiency Virus 1 (HIV-1), Human Immunodeficiency Virus 2
(HIV-2), Human T-cell Lymphotrophic Virus 1 (HTLV-1), Human T-cell
Lymphotrophic Virus 2 (HTLV-2), Human Intracistemal A-type Particle
1 (HIAP-1), and Human Intracistemal A-type Particle 2 (HIAP-2).
[0052] As noted above the instant invention also contemplates
derivatives of the FIR peptides described above and analogous
sequences thereto. These derivative peptides may comprise altered
sequences in which functionally equivalent amino acid residues are
substituted for residues within the sequence resulting in a silent
change. For example, one or more amino acid residues within the
sequence can be substituted for by another amino acid of a similar
polarity that acts as a functional equivalent, resulting in a
silent alteration (e.g. substitution of leucine for isoleucine).
Substitutes for an amino acid within the sequence may be selected
from other members of the class to which the amino acid belongs.
For example, the nonpolar (hydrophobic) amino acids include
alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan and methionine. The polar neutral amino acids include
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine. The positively charged (basic) amino acids include
arginine, lysine and histidine. The negatively charged (acidic)
amino acids include aspartic acid and glutamic acid. By way of
further example, and not by way of limitation, such peptides may
also comprise D-amino acids, and/or the may comprise an inefficient
carrier protein, or no carrier protein at all.
[0053] FIR peptides may comprise peptides in which "X" comprises an
amino group, an acetyl group, a hydrophobic group or a
macromolecular carrier group; and/or "Z" comprises a carboxyl
group, an amido group a hydrophobic group or a macromolecular
carrier group. Various aspects of the instant invention are drawn
to peptides wherein the "X" moiety may also be selected from the
group comprising: a hydrophobic moiety, a carbobenzoxyl moiety,
dansyl moiety, or a t-butyloxycarbonyl moiety. In any of the
peptides of the instant invention the "Z" moiety may be selected
from the group comprising: a hydrophobic moiety, a
t-butyloxycarbonyl moiety.
[0054] In other aspects of this embodiment of the invention the "X"
moiety may comprise a macromolecular carrier group. Such
macromolecular carrier group may be selected from the group
comprising, but not limited to: a lipid conjugate, a polyethylene
glycol moiety, or a carbohydrate moiety. Similarly the "Z" may also
comprise a macromolecular carrier group; wherein said
macromolecular carrier is selected from the group comprising, but
not limited to: a lipid conjugate, polyethylene glycol moiety, or a
carbohydrate moiety.
[0055] Various embodiments of this aspect of the invention also
contemplate peptides wherein one or more of the molecular bonds
linking adjacent amino acid residues is a non-peptide bond. Such
non-peptide bonds include, but are not limited to: imido, ester,
hydrazine, semicarbazoide and azo bonds.
[0056] Yet other aspects of the instant invention provide for
peptides wherein the peptide comprises one or more amino acid
residues that is/are in a D-isomer amino acid.
[0057] Other aspects of the instant invention provide for peptides
comprising one or more amino acid substitution wherein a first
amino acid residue is substituted for a second, different amino
acid residue, in the sequences provided above (or a functional
segment thereof). In various aspects of this embodiment, the amino
acid substitution is a conservative substitution. In other aspects
of this embodiment the amino acid substitution is a
non-conservative substitution. Yet other aspects of this embodiment
of the invention provide for peptides as described above except
that one or more amino acid residues have been deleted.
[0058] In various preferred aspects of the instant embodiments the
FIR peptides comprise at least three contiguous residues of a FIR.
More preferably the FIR peptide comprises at least 8 contiguous
residues of a FIR. As used herein the term "FIR inhibitory
peptide(s)" preferably refers to a peptide or peptides having the
sequence of a FIR (or functional segment thereof) and to such FIR
peptides or functional segments in which one or more amino acids
is/are substituted for by functionally equivalent or chemically
similar amino acids (see infra). It also refers to derivatives of
these peptides, including but not limited to, benzylated
derivatives, glycosylated derivatives, and peptides that include
enantiomers of naturally occurring amino acids. In a preferred
aspect of this embodiment the peptide is selected from those having
the sequence of any of SEQ ID NOs 1-7, 8-15, 22-25, and 30. In
particularly preferred aspects of this embodiment the peptide has a
sequence selected from the group consisting of SEQ ID NOs 22-25 and
30.
[0059] In yet other aspects of this embodiment of the invention,
the FIR peptides may be linked to a carrier molecule such as a
protein, including but not limited to, human serum albumin
(HSA).
[0060] Furthermore, the instant invention contemplates molecules
comprising any combination of the X and Z moieties and/or other
peptide modifications described above.
[0061] Peptides according to the instant invention may be produced
from naturally occurring or recombinant viral proteins. They may
also be produced using standard recombinant DNA techniques (e.g.
the expression of peptide by a microorganism that contains
recombinant nucleic acid molecule encoding the desired peptide,
expressed under the control of a suitable transcriptional promoter,
and the harvesting of desired peptide from said microorganism). In
a preferred aspect of the invention, any of the peptides of the
invention may be prepared using any chemical synthesis methodology
known in the art including, but not limited to, Merrifield solid
phase synthesis (Clark-Lewis et al., 1986, Science 231:
134-139).
[0062] Embodiments of the instant invention also provide for other
compounds useful for treating or preventing infection of a cell by
a virus. These include antibodies (or active segments thereof,
meaning portions of antibodies capable of specifically recognizing
a FIR region or a functional segment thereof) and other molecules.
Certain aspects of this embodiment of the invention provide for
antibodies that specifically recognize a FIR, or antigenic fragment
thereof and/or are capable of interfering with virus:cell
interaction sufficiently to prevent or reduce infection of the cell
by the virus. Antibodies according to these embodiments of the
invention may be monoclonal or polyclonal.
[0063] Various embodiments of the invention provide for methods of
producing antibodies capable of specifically recognizing a FIR
and/or preventing or reducing infection of the cell by the virus.
General methods for producing antibodies are well known to those of
skill in the art. Methods for producing antibodies according to the
instant invention comprise the steps of (1) providing an antigen
comprising a FIR or an antigenic fragment thereof (such antigen may
be an unmodified peptide, a peptide mimic, a peptide analog, or a
peptide derivative); (ii) exposing the immune system of an animal
to the antigen so as to induce an immune response; (iii) collecting
antibodies from the animal and identifying those antibodies that
either specifically recognize a FIR (or functional segment thereof)
and/or are capable of inhibiting or reducing virus:cell infection
in a dose responsive manner in assays that measure viral
infectivity.
[0064] Other embodiment of the instant invention provide for
methods of identifying compounds capable of preventing or
inhibiting infection by a virus comprising a FIR or that are useful
as drug leads for the development of drugs for preventing or
inhibiting viral infection. Such methods comprise the steps of: (I)
identifying a virus having at least one membrane fusion protein
comprising a fusion initiation region that is requisite for
virus:cell fusion; (ii) preparing a target, where the target
comprises the amino acid sequence of a FIR, or a functional segment
of a FIR; (iii) screening a plurality of compounds to identify at
least one compound that binds to the target, thereby identifying a
target-binding compound; (iv) screening at least one target-binding
compound to identify a target-binding compound that is capable of
specifically preventing or reducing viral infection by the virus
from which the target was obtained or that us useful as a drug lead
for the development of a drug for specifically preventing or
reducing infection by such a virus. As used herein the phrase
"specifically preventing or reducing viral infection" means that
the compound specifically prevents infection by the target virus,
without any substantial effect on an unrelated virus. For example,
if a compound that specifically prevented infection by the SARS
virus would not prevent infection by the HIV-1 virus.
[0065] As used herein the compounds (e.g. drugs or drug leads)
identified by the methods described above may be of any type, by
way of non-exclusive list they may be any peptide (or derivative,
analog, or mimic thereof) this includes short peptides as are
typically employed in phage display libraries, any antibody or
active fragment thereof (i.e., any fragment, such as an Fab that is
capable of specifically recognizing the target) or any other
organic or inorganic molecule.
[0066] In any embodiment of the instant invention the FIR may be
from any virus having a membrane fusion protein comprising at least
extended two alpha-helices, a fusion peptide, and a fusion
initiation region. Preferably, the virus is selected from a virus
family, wherein the virus family is selected from the group
consisting of: arenaviruses, coronaviruses, filoviruses,
orthomyxoviruses, paramyxoviruses, and retroviruses. More
preferably, the virus is selected from the group consisting of:
Lassa virus, SARS (severe acute respiratory syndrome) virus, Ebola
virus, influenza virus, measles virus, and HIV-1 (human
immunodeficiency virus type 1).
[0067] According to various aspects of the instant invention, the
peptides and/or factors of the instant invention useful for
treating or preventing viral infection of a cell can target the
amino acids surrounding and within the FIR cysteine loop, the
distal portion of the FIR N-helix, any of the interfacial
hydrophobicity regions of the FIR, other areas of the FIR, or any
combination of thereof. These factors, antibodies, peptides or
peptide analogs (collectively compounds) may be used individually;
alternatively they may be used in combinations of two or more to
prevent or inhibit infection of the cell by the virus. The methods
of preventing or inhibiting viral infection of the cell by
interfering with the function of the FIR provided by the instant
invention also include the use of neutralizing antibodies, produced
exogenously or endogenously, against all or portions of the FIR.
The purpose of such use is to interfere with the function of the
FIR, thereby inhibiting viral infection of the cell and/or
virus:cell membrane fusion.
[0068] Other embodiments of the instant invention provide for
compositions, including pharmaceutical compositions, comprising any
and all of the compounds, peptides (including analogs, derivatives,
and mimics thereof), antibodies, or any other molecule of the
instant invention or identified by the methods of instant
invention. This includes, but is not limited to, compositions
containing any molecule that comprises, consists essentially of, or
consists of a FIR, or a functional segment of a FIR. It further
includes, but is not limited to compositions comprising any
compound that specifically recognizes, binds to, or interferes with
the function of a viral FIR. As used herein, the phrase
"interfering with the function of the FIR" means that a compound
interacts with the FIR or with the cellular protein that serves as
the receptor that recognizes the FIR so as to prevent or reduce
infection of the cell by the virus. Additionally, it is
contemplated that the compositions may comprise either one of the
molecules described or mixtures of two or more of the
molecules.
[0069] Further embodiments of the instant invention provide for
methods of treating or preventing infection of a cell by a virus
(where the virus comprises a FIR) using any of the compounds of the
instant invention and/or any compound identified by any of the
methods of the instant invention. Various aspects of this
embodiment of the invention provide for administering an effective
amount of any of the pharmaceutical compositions described herein
to a patient suspected of being exposed to a virus (or having
potential for being exposed to a virus) wherein the virus comprises
a FIR. In various aspects of the invention the pharmaceutical
composition comprises an antibody that specifically recognizes and
binds to a FIR (or functional segment of a FIR) or a fragment of
such antibody that specifically recognizes and binds to a FIR, or
functional segment of a FIR.
[0070] Still other aspects of this embodiment of the invention
provide for methods that comprise administering to a patient an
effective amount of a composition comprising at least one
recombinant DNA or RNA molecule; where the RNA or DNA encodes a FIR
(or functional segment thereof) or a molecule capable of
specifically binding to a FIR or a cellular receptor that
recognizes a FIR so as to prevent or reduce infection by the virus.
In a preferred aspect of this embodiment the recombinant RNA or DNA
molecule and or pharmaceutical composition further comprises the
elements necessary to allow the protein encoded by the RNA or DNA
molecule to be expressed in a human cell. By way of non-exclusive
example, in certain aspects of this embodiment of the invention the
recombinant RNA or DNA molecule is part of a recombinant plasmid or
a recombinant virus.
Example 1
[0071] Identification of the FIR in Ebola virus. The method to
identify the FIR of Class I viral fusion proteins can be
illustrated by two examples. The first example is identification of
the FIR in the minimal class I fusion protein glycoprotein 2 (GP2)
of Ebola virus, a filovirus. The boundaries of the N-helix and the
C-helix of Ebola virus GP2 have been determined by x-ray
crystallographic methods (Malashkevich et al., 1999). The terminal
amino acids of the N-helix contain the sequence ILNRKAIDF (SEQ ID
NO: 8) that fits the consensus of a core comprising three or four
hydrophobic amino acids, a positively-charged amino acid, a
negatively-charged amino acid, and an aromatic amino acid. Between
these two helices are two cysteines in the sequence CHILGPDC (SEQ
ID NO: 9). Defining the ends of the Ebola virus GP2 FHR is the
sequence FLLQRWGGTCHILGPDCCI (SEQ ID NO: 10), which has a
Wimley-White interfacial hydrophobicity score of 2.59 as determined
by the MPEX program (Jaysinghe et al, 2002). Thus, the FIR of Ebola
virus GP2 extends from amino acids 579 to 610.
Example 2
[0072] Identification of the FIR in measles virus. The second
example is a complex class I fusion protein, the F1 protein of
measles virus, a paramyxovirus. The N- and C-helices of measles
virus F1 can be identified by examining the primary sequence for
amino acids with the propensity to form helices. Alignment of the
primary sequence of measles virus F1 with the primary amino acid
sequence of the F1 protein of another paramyxovirus, Newcastle
disease virus F1, can also aid in the identification of the helix
boundaries. The structure of the Newcastle disease virus F1 protein
has been determined by x-ray crystallographic methods (Chen et al.,
2001). The boundaries of the N- and C-helices can thus be predicted
to be amino acids 131-217 and 455-491 respectively. In contrast to
Ebola virus GP2 and most other viral class I fusion proteins, the
primary sequence between the N- and C-helices in the measles virus
is longer than 100 amino acids. The FIR region of measles virus F1
contains an insertion which, upon inspection of computer models, is
obvious to those skilled in the art, and thus the FIR structure is
formed by a secondary arrangement that brings together two parts of
the primary sequence. The inserted sequence forms a loop external
to the FIR. The terminal amino acids of the N-helix contain the
sequence LKLLRYYTE (SEQ ID NO: 11) which fits the consensus of a
core comprising three or four hydrophobic amino acids, a
positively-charged amino acid, a negatively-charged amino acid, and
an aromatic amino acid. There are eight cysteine residues in
measles virus F1 between the N- and C-helices. On the basis of the
alignment with Newcastle disease virus F1 it can be determined that
the first two cysteines and the second two cysteines form
disulfide-linked loops. The first pair of cysteines in the
sequence, CTFMPEGTVC (SEQ ID NO: 12), is part of the FIR because it
is bounded by a sequence WYTTVPKYVATQGYLISNF (SEQ ID NO: 13) with a
Wimley-White interfacial hydrophobicity score of 3.36, as
determined by the MPEX program. The second pair of cysteines in the
sequence, CLRGSTKSC (SEQ ID NO: 14), is also part of the FIR
because it is adjacent to a sequence
TLVSGSFGNRFWLSQGNLIANCASILCKCYTTGTII (SEQ ID NO: 15) with a
Wimley-White interfacial hydrophobicity score of 2.54, as
determined by the MPEX program. Thus, the FIR of measles virus F1
extends from amino acids 205 to 407, with amino acids 221 to 314
representing an insertion that does not participate in FIR
function.
Example 3
[0073] Identification Of Coronavirus Fusion Inhibitory Peptides.
Background. Severe acute respiratory syndrome (SARS) is a newly
recognized illness that spread from southern China in late
2002/early 2003 to several countries in Asia, Europe and North
America (Guan et al., 2004). SARS usually begins with a fever
greater than 38.degree. C. Initial symptoms can also include
headache, malaise and mild respiratory symptoms. Within two days to
a week, SARS patients may develop a dry cough and have trouble
breathing. Patients in more advanced stages of SARS develop either
pneumonia or respiratory distress syndrome. In the initial outbreak
there were 8098 cases worldwide, with an overall mortality of 9.6%.
A previously unrecognized coronavirus (CoV) has been demonstrated
to be the cause of the new disease (Poutanen et al., 2003; Peiris
et al., 2003; Drosten et al., 2003; Rota et al., 2003; Mara et al.,
2003). Public health interventions, such as surveillance, travel
restrictions and quarantines, contained the original spread of SARS
CoV in 2003 and again appear to have stopped the spread of SARS
after the appearance of a few new cases in 2004. It is unknown,
however, whether these draconian containment measures can be
sustained with each appearance of the SARS CoV in humans.
Furthermore, the potential of this new and sometimes lethal CoV as
a bio-terrorism threat is obvious.
[0074] Coronaviruses are large positive-stranded RNA viruses
typically with a broad host range. Like other enveloped viruses,
CoV enter target cells by fusion between the viral and cellular
membranes, a process mediated by the viral spike (S) protein. CoV S
proteins, characterized to date, appear to consist of two
non-covalently associated subunits, S1 and S2. Using computational
analysis, Garry and Gallaher (2003) first proposed that the portion
of the SARS--CoV S protein corresponding to the S2 subunit fit the
prototypical model of a class I viral fusion protein based on the
presence of two predicted alpha helical regions at the N- and
C-terminal regions of S2 (N-helix, C-helix) and an aromatic amino
acid-rich region just prior to the transmembrane anchor domain.
[0075] Materials And Methods. L2 cells or Vero E6 cells were
maintained as monolayers in complete Dulbecco's modified Eagle's
medium (DMEM) containing 0.15% HCO.sub.3-supplemented with 10%
fetal bovine serum (FBS), penicillin G (100 U/ml), streptomycin
(100 mg/ml), and 2 mM of L-glutamine at 37.degree. C. in a 5%
CO.sub.2 incubator. Mouse hepatitis virus (MHV) strain A59 or SARS
CoV strain Urbani or HK was propagated on L2 cells. For plaque
assays, L2 cells or Vero E6 cells were seeded at a density of
1.times.10.sup.6 cells in each well of a 6-well plate. Fifty to
100-plaque forming units (PFU) of MHV or SARS CoV were
pre-incubated with or without approximately 100, mg/ml of peptide
in serum-free DMEM for 1 hour. Cells were then infected with
peptide-treated inoculum or vehicle control inoculum. After 1 hour
adsorption, the inoculum was removed, cells were washed twice with
1.times. phosphate buffered saline, and the cells were overlaid
with 10% FBS/DMEM containing 0.5% SEAPLAQUE.RTM. agarose (Cambrex
Bio Science Rockland, Inc., Rockland, Me.). Monolayers were fixed
with 3.7% formalin and stained with IX crystal violet 2 days
post-infection, and plaque numbers were determined by light
microscopy.
[0076] Results And Discussion. Synthetic peptides corresponding to
the FIR domains of the MHV or SARS CoV S protein were tested for
their ability to inhibit infection by these coronaviruses. The
ability to inhibit formation of plaques in cell monolayers is the
most stringent in vitro test of a potential infection inhibitor
drug. Two peptides (GNHILSLVQNAPYGLYFIHFSW, SEQ ID NO: 22 and
GYFVQDDGEWKFRGSSYYY, SEQ ID NO: 23) from the MHV FIR can inhibit
plaque formation by MHV, though the first MHV FIR peptide is more
efficient (see FIG. 8A). Two peptides from the FIR of SARS, CoV
(GYHLMSFPQAAPHGVVFLHVTY, SEQ ID NO: 24 and GVFVFNGTSWFITQRNFFS, SEQ
ID NO: 25) inhibited plaque formation by this coronavirus (see FIG.
8B). There was also a significant reduction (-50%) in the average
diameter of the residual plaques. These results suggest that this
peptide inhibits both entry and spread of MHV. Similar results with
these inhibitory peptides were obtained in independent experiments,
with 50% plaque inhibition observed at concentrations of <5
.mu.M. These results are unlikely to be explained by non-specific
cytotoxic effects of the peptides. Except for the plaques, cells in
the monolayers were intact and viable. The low number of plaques
grew were similar in size to control plaques. Peptides from other
regions also inhibited infection by these viruses, but to a lesser
extent than the most active FIR peptides (FIG. 8). For example,
peptides from the fusion peptide region and the carboxyl terminal
helix (C-helix) of the MHV S and SARS CoV S provided some
inhibition (MHV S fusion peptide=MFPPWSAAAGVPFSLSVQY, SEQ ID NO:
26; MHV S C-helix=QDAIKKLNESYINLKEVGTYEMYVKW, SEQ ID NO: 27; SARS
CoV S fusion peptide=MYKTPTLKYFGGFNFSQIL, SEQ ID NO: 28; SARS CoV S
C-helix=AACEVAKNLNESLIDLQELGKYEQYIKW, SEQ ID NO: 29. Inhibitory
activities in the, mM range were recently reported with coronavirus
C-helix peptides by Bosch et al., (2003) and others (Bosch et al.,
2004; Lui et al., 2004; Yuan et al., 2004; Zhu et al., 2004).
However, no FIR coronavirus inhibitory peptides have been reported.
Nevertheless, in view of the current invention, the cited
references collectively, provide support for the tremendous
advantages of the currently disclosed and claimed inventions. That
is, these references are consistent with the inventors' assertion
that the methods of the present invention can be advantageously
used to identify synthetic peptides that inhibit fusion/infectivity
by members of the Coronaviridae family.
Example 4
[0077] Identification Of Arenavirus Fusion Inhibitory Peptides.
Background. Lassa fever is an often-fatal hemorrhagic illness named
for the town in the Yedseram River valley of Nigeria in which the
first described cases occurred in 1969 (Buckley and Casals, 1970).
Parts of Guinea, Sierra Leone, Nigeria, and Liberia are endemic for
the etiologic agent, Lassa virus (LasV). The public health impact
of LasV in endemic areas is immense. The Centers for Disease
Control, and Prevention (CDC) have estimated that there are
100,000-300,000 cases of Lassa per year in West Africa and 5,000
deaths. In some parts of Sierra Leone, 10-15% of all patients
admitted to hospitals have Lassa fever. Case fatality rates for
Lassa fever are typically 15% to 20%, although in epidemics overall
mortality can be as high as 45%. The mortality rate for women in
the last month of pregnancy is always high, about 90%, and LasV
infection causes high rates of fetal death at all stages of
gestation. Mortality rates for Lassa appear to be higher in
non-Africans, which is of concern because Lassa is the most
commonly exported hemorrhagic fever. Because of the high case
fatality rate and the ability to spread easily by human-human
contact, LasV is classified as a Biosafety Level 4 and NIAID
Biodefense category A agent.
[0078] LasV is a member of the Arenaviridae family. The genome of
arenaviruses consists of two segments of single-stranded, ambisense
RNA. When viewed by transmission electron microscopy, the enveloped
spherical virions (diameter: 110-130 nm) show grainy particles that
are ribosomes acquired from the host cells (Murphy and Whitfield,
1975). Hence, the use for the family name of the Latin "arena",
which means "sandy". In addition to LasV, other arenaviruses that
cause illness in humans include Junin virus (Argentine hemorrhagic
fever), Machupo virus (Bolivian hemorrhagic fever), Guanarito virus
(Venezuelan hemorrhagic fever) and Sabia virus (Brazilian
hemorrhagic fever). Arenaviruses are zoonotic; each virus is
associated with a specific species of rodent (Bowen, Peters, and
Nichol, 1997). The reservoir of LasV is the "multimammate rat" of
the genus Mastomys (Monath et al., 1974). The wide distribution of
Mastomys in Africa makes eradication of this rodent reservoir
impractical and ecologically undesirable.
[0079] Signs and symptoms of Lassa fever, which occur 1-3 weeks
after virus exposure, are highly variable, but can include fever,
retrosternal, back or abdominal pain, sore throat, cough, vomiting,
diarrhea, conjunctival injection, and facial swelling. LasV infects
endothelial cells, resulting in increased capillary permeability,
diminished effective circulating volume, shock, and multi-organ
system failure. Frank bleeding, usual mucosal (gums, etc.), occurs
in less than a third of cases, but confers a poor prognosis.
Neurological problems have also been described, including hearing
loss, tremors, and encephalitis. Patients who survive begin to
defervesce 2-3 weeks after onset of the disease. The most common
complication of Lassa fever is deafness. Temporary or permanent
unilateral or bilateral deafness occurs in approximately 30% of
Lassa fever patients during convalescence, and is not associated
with the severity of the acute disease. The antiviral drug
ribavirin is effective in the treatment of Lassa fever, but only if
administered early (up to six days) in the course of illness
(Johnson et al., 1987; McCormick et al., 1986). It is unknown
whether ribavirin is effective against other arenaviruses, such as
Junin, Machupo, Guanarito or Sabia viruses. No LasV vaccine is
currently available.
[0080] Materials And Methods. Vero cells were maintained as
monolayers in Basal Medium Eagle (BME) containing 10 mM HEPES and
5% FBS. Lassa virus (Josiah strain) was propagated on Vero cells.
For plaque assays, Vero cells were seeded at a density of
1.times.10.sup.6 cells in each well of a 6-well plate. Fifty to 100
p.f.u. of LasV were pre-incubated with or without peptide in
serum-free BME for 1 hour. Cells were then infected with
peptide-treated inoculum or vehicle control inoculum. After 1 hour
adsorption, the inoculum was removed, cells were washed twice with
1.times. phosphate buffered saline, and the cells were overlaid
with 2 ml of 0.5% agarose in BME containing 10 mM HEPES and 5% FBS,
and incubated for 4 days. A second overlay containing 5% neutral
red was applied, and plaques were counted 24 hours later.
[0081] Results And Discussion. Synthetic peptides corresponding to
the FIR domains of LasV glycoprotein 2 (GP2) were tested for their
ability to inhibit infection by this arenavirus. A peptide
(NYSKYWYLNHTTTGR, SEQ ID NO: 30) analogous to the sequence
NYSRYWYLNHTSTGK from SEQ ID NO: 1 (LASSA FIR) can inhibit plaque
formation by LasV (FIG. 9). A peptide analogous to another GP2
region, the fusion peptide, (GTFTWTLSDSEGKDTPGGY, SEQ ID NO: 31)
also inhibited infection by LasV, but to a lesser extent (FIG. 9).
No arenavirus inhibitory peptides have been reported. Collectively,
these results suggest that our approaches can identify synthetic
peptides that inhibit fusion/infectivity by members of the
Arenaviridae. These results, in combination with our results with
coronavirus FIR inhibitory peptides, establish proof of the
principle that FIR regions peptides can function as viral
inhibitors
[0082] Each of the following documents is herein incorporated by
reference. [0083] Bolognesi et al. U.S. Pat. No. 5,464,933. [0084]
Bosch et al. Proc Natl Acad Sci USA 101: 8455-8460. [0085] Bosch et
al. J Virol 77: 8801-8811. [0086] Bowen et al. (1997) Mol Phylogen
Evol 8 (3), 301-16. [0087] Buckley (1970) Am J Trop Med Hyg 19 (4),
680-91. [0088] Carr et al. (1993) Cell 73 (4), 823-32. [0089] Chan
et al. Cell 89 (2), 263-73. [0090] Chen et al. Structure 9 (3),
255-266. [0091] Clark-Lewis et al. Science 231: 134-9. [0092]
Drosten et al. New England J Med 348, 1967-76. [0093] Gallaher et
al. Adv. Membrane Fluidity 6, 113-142. [0094] Gallaher (1987) Cell
50 (3), 327-8. [0095] Gallaher (1996) Cell 85, 1-2. [0096] Gallaher
et al. AIDS Res Human Retroviruses 5 (4), 431-40. [0097] Gallaher
et al. BMC Microbiol 1 (1), 1. [0098] Gallaher et al., www(dot)
virology(dot) net/Articles/sars/s2model. html; May 1, 2003. [0099]
Gonzalez-Scarano et al. (1987) AIDS Res Hum Retroviruses 3 (3)
245-52. [0100] Guan et al. (2004) Lancet 363, 99-104. [0101] Guan
et al. (2003) Science 302, 276-278. [0102] Henderson et al. U.S.
Pat. No. 5,567,805. [0103] Jaysinghe et al. (2000)
www(dot)blanco(dot)biomol(dot)uci (dot)edu/index. [0104] Johnson et
al. J Infect Dis 155 (3), 456-64. [0105] Kilby et al. (1998) Nat
Med 4 (11), 1302-7. [0106] Kowalski et al. (1991) J Virol 65,
281-291. [0107] Ksiazek et al. (2003) N Engl J Med 348, 1953-66.
[0108] Lambert et al. (1996) Proc Natl Acad Sci USA 93 (5),
2186-91. [0109] Liu et al. (2004) Lancet 363: 938-947. [0110]
Malashkevich et al. (1999) Proc Natl Acad Sci USA 96 (6), 2662-7.
[0111] Marra et al. (2003) Science 300, 1399-1404. [0112] McCormick
et al. (1986) N EngI J Med 314 (1), 20-6. [0113] Monath et al.
(1974) Science 185 (147), 263-5. [0114] Murphy et al. (1975) Bull
World Health Organ 52 (4-6), 409-19. [0115] Owens et al. (1990)
AIDS Res Hum Retroviruses 6 (11), 1289-96. [0116] Peiris et al.
(2003) Lancet 361, 1319-25. Pozniak (2001) JHIV Ther 6 (4), 91-4.
[0117] Poutanen et al. (2003) New England J Med 348, 1995-2005.
[0118] Qureshi et al. (1990) AIDS 4, 553-558. [0119] Richardson et
al. (1980) Virology 105 (1), 205-22. [0120] Rota et al. (2003)
Science 300, 1394-1399. [0121] Silbum et al. (1998) AIDS Res Hum
Retroviruses 14 (5), 385-92. [0122] Sodroski et al. (1999) Cell 99
(3), 243-6. [0123] Suarez et al. (2000) J Virol 74 (17), 8038-47.
[0124] Watanabe et al. (2000) J Virol 74 (21), 10194-201. [0125]
Weissenhom et al. (1998) Mol Cell 2 (5), 605-16. [0126] Weissenhom
et al. (1997) Nature 387 (6631), 426-30. [0127] Wild et al. (1993)
Human Retroviruses 9 (11), 1051-3. [0128] Wild et al. (1992) Proc
Natl Acad Sci USA 89 (21), 10537-41. [0129] Wilson et al. (1981)
Nature 289 (5796), 366-73. [0130] Young et al. (1999) J Virol 73
(7), 5945-56. [0131] Yuan et al. (2004) Biochem Biophys Res Commun
319: 746-752. [0132] Zhu et al. (2004) Biochem Biophys Res Commun
319: 283-288.
Sequence CWU 1
1
31139PRTArtificial SequenceSynthetic peptide 1Leu Ile Met Lys Asn
His Leu Arg Asp Ile Met Gly Ile Pro Tyr Cys1 5 10 15Asn Tyr Ser Arg
Tyr Trp Tyr Leu Asn His Thr Ser Thr Gly Lys Thr 20 25 30Leu Pro Arg
Cys Trp Leu Ile 352100PRTArtificial SequenceSynthetic peptide 2Leu
Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr1 5 10
15Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys
20 25 30Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ala Ala Pro His
Gly 35 40 45Val Val Phe Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg
Asn Phe 50 55 60Thr Thr Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr
Phe Pro Arg65 70 75 80Glu Gly Val Phe Val Phe Asn Gly Thr Ser Trp
Phe Ile Thr Gln Arg 85 90 95Asn Phe Phe Ser 100332PRTArtificial
SequenceSynthetic peptide 3Leu Arg Thr Phe Ser Ile Leu Asn Arg Lys
Ala Ile Asp Phe Leu Leu1 5 10 15Gln Arg Trp Gly Gly Thr Cys His Ile
Leu Gly Pro Asp Cys Cys Ile 20 25 30443PRTArtificial
SequenceSynthetic peptide 4Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp
Thr Lys Ile Asp Leu Trp1 5 10 15Ser Tyr Asn Ala Glu Leu Leu Val Ala
Leu Glu Asn Gln His Thr Ile 20 25 30Asp Leu Thr Asp Ser Glu Met Asn
Lys Leu Phe 35 40517PRTArtificial SequenceSynthetic peptide 5Leu
Gly Leu Lys Leu Leu Arg Tyr Tyr Thr Glu Ile Leu Ser Leu Phe1 5 10
15Gly694PRTArtificial SequenceSynthetic peptide 6Trp Tyr Thr Thr
Val Pro Lys Tyr Val Ala Thr Gln Gly Tyr Leu Ile1 5 10 15Ser Asn Phe
Asp Glu Ser Ser Cys Thr Phe Met Pro Glu Gly Thr Val 20 25 30Cys Ser
Gln Asn Ala Leu Tyr Pro Met Ser Pro Leu Leu Gln Glu Cys 35 40 45Leu
Arg Gly Ser Thr Lys Ser Cys Ala Arg Thr Leu Val Ser Gly Ser 50 55
60Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly Asn Leu Ile Ala Asn Cys65
70 75 80Ala Ser Ile Leu Cys Lys Cys Tyr Thr Thr Gly Thr Ile Ile 85
90757PRTArtificial SequenceSynthetic peptide 7Leu Gln Ala Arg Ile
Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln1 5 10 15Leu Leu Gly Ile
Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala 20 25 30Val Pro Trp
Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp 35 40 45Asn His
Thr Thr Trp Met Glu Trp Asp 50 5589PRTArtificial SequenceSynthetic
peptide 8Ile Leu Asn Arg Lys Ala Ile Asp Phe1 598PRTArtificial
SequenceSynthetic peptide 9Cys His Ile Leu Gly Pro Asp Cys1
51019PRTArtificial SequenceSynthetic peptide 10Phe Leu Leu Gln Arg
Trp Gly Gly Thr Cys His Ile Leu Gly Pro Asp1 5 10 15Cys Cys
Ile119PRTArtificial SequenceSynthetic peptide 11Leu Lys Leu Leu Arg
Tyr Tyr Thr Glu1 51210PRTArtificial SequenceSynthetic peptide 12Cys
Thr Phe Met Pro Glu Gly Thr Val Cys1 5 101319PRTArtificial
SequenceSynthetic peptide 13Trp Tyr Thr Thr Val Pro Lys Tyr Val Ala
Thr Gln Gly Tyr Leu Ile1 5 10 15Ser Asn Phe149PRTArtificial
SequenceSynthetic peptide 14Cys Leu Arg Gly Ser Thr Lys Ser Cys1
51536PRTArtificial SequenceSynthetic peptide 15Thr Leu Val Ser Gly
Ser Phe Gly Asn Arg Phe Ile Leu Ser Gln Gly1 5 10 15Asn Leu Ile Ala
Asn Cys Ala Ser Ile Leu Cys Lys Cys Tyr Thr Thr 20 25 30Gly Thr Ile
Ile 3516234PRTLASSA VIRUS 16Leu Leu Gly Thr Phe Thr Trp Thr Leu Ser
Asp Ser Glu Gly Asn Glu1 5 10 15Thr Pro Gly Gly Tyr Cys Leu Thr Arg
Trp Met Leu Ile Glu Ala Glu 20 25 30Leu Lys Cys Phe Gly Asn Thr Ala
Val Ala Lys Cys Asn Glu Lys His 35 40 45Asp Glu Glu Phe Cys Asp Met
Leu Arg Leu Phe Asp Phe Asn Lys Gln 50 55 60Ala Ile Arg Arg Leu Lys
Thr Glu Ala Gln Met Ser Ile Gln Leu Ile65 70 75 80Asn Lys Ala Val
Asn Ala Leu Ile Asn Asp Gln Leu Ile Met Lys Asn 85 90 95His Leu Arg
Asp Ile Met Gly Ile Pro Tyr Cys Asn Tyr Ser Arg Tyr 100 105 110Trp
Tyr Leu Asn His Thr Ser Thr Gly Lys Thr Ser Leu Pro Arg Cys 115 120
125Trp Leu Ile Ser Asn Gly Ser Tyr Leu Asn Glu Thr Lys Phe Ser Asp
130 135 140Asp Ile Glu Gln Gln Ala Asp Asn Met Ile Thr Glu Met Leu
Gln Lys145 150 155 160Glu Tyr Ile Asp Arg Gln Gly Lys Thr Pro Leu
Gly Leu Val Asp Leu 165 170 175Phe Val Phe Ser Thr Ser Phe Tyr Leu
Ile Ser Ile Phe Leu His Leu 180 185 190Val Lys Ile Pro Thr His Arg
His Ile Val Gly Lys Pro Cys Pro Lys 195 200 205Pro His Arg Leu Asn
His Met Gly Ile Cys Ser Cys Gly Leu Tyr Lys 210 215 220Gln Pro Gly
Val Pro Val Arg Trp Lys Arg225 23017388PRTSARS VIRUS 17Trp Thr Phe
Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln1 5 10 15Met Ala
Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr 20 25 30Glu
Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln 35 40
45Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln
50 55 60Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys
Gln65 70 75 80Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn
Asp Ile Leu 85 90 95Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile
Asp Arg Leu Ile 100 105 110Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr
Val Thr Gln Gln Leu Ile 115 120 125Arg Ala Ala Glu Ile Arg Ala Ser
Ala Asn Leu Ala Ala Thr Lys Met 130 135 140Ser Glu Cys Val Leu Gly
Gln Ser Lys Arg Val Asp Phe Cys Gly Lys145 150 155 160Gly Tyr His
Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val 165 170 175Phe
Leu His Val Thr Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr 180 185
190Ala Pro Ala Ile Cys His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly
195 200 205Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg
Asn Phe 210 215 220Phe Ser Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe
Val Ser Gly Asn225 230 235 240Cys Asp Val Val Ile Gly Ile Ile Asn
Asn Thr Val Tyr Asp Pro Leu 245 250 255Gln Pro Glu Leu Asp Ser Phe
Lys Glu Glu Leu Asp Lys Tyr Phe Lys 260 265 270Asn His Thr Ser Pro
Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 275 280 285Ala Ser Val
Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val 290 295 300Ala
Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys305 310
315 320Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp Leu Gly Phe
Ile 325 330 335Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu Leu
Cys Cys Met 340 345 350Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys
Ser Cys Gly Ser Cys 355 360 365Cys Lys Phe Asp Glu Asp Asp Ser Glu
Pro Val Leu Lys Gly Val Lys 370 375 380Leu His Tyr
Thr38518175PRTEBOLA VIRUS 18Glu Ala Ile Val Asn Ala Gln Pro Lys Cys
Asn Pro Asn Leu His Tyr1 5 10 15Trp Thr Thr Gln Asp Glu Gly Ala Ala
Ile Gly Leu Ala Trp Ile Pro 20 25 30Tyr Phe Gly Pro Ala Ala Glu Gly
Ile Tyr Thr Glu Gly Leu Met His 35 40 45Asn Gln Asp Gly Leu Ile Cys
Gly Leu Arg Gln Leu Ala Asn Glu Thr 50 55 60Thr Gln Ala Leu Gln Leu
Phe Leu Arg Ala Thr Thr Glu Leu Arg Thr65 70 75 80Phe Ser Ile Leu
Asn Arg Lys Ala Ile Asp Phe Leu Leu Gln Arg Trp 85 90 95Gly Gly Thr
Cys His Ile Leu Gly Pro Asp Cys Cys Ile Glu Pro His 100 105 110Asp
Trp Thr Lys Asn Ile Thr Asp Lys Ile Asp Gln Ile Ile His Asp 115 120
125Phe Val Asp Lys Thr Leu Pro Asp Gln Gly Asp Asn Asp Asn Trp Trp
130 135 140Thr Gly Trp Arg Gln Trp Ile Pro Ala Gly Ile Gly Val Thr
Gly Val145 150 155 160Ile Ile Ala Val Ile Ala Leu Phe Cys Ile Cys
Lys Phe Val Phe 165 170 17519191PRTINFLUENZA VIRUS 19Gly Leu Phe
Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly1 5 10 15Met Ile
Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr 20 25 30Gly
Gln Ala Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile 35 40
45Asn Gly Lys Leu Asn Arg Val Ile Glu Lys Thr Asn Glu Lys Phe His
50 55 60Gln Ile Glu Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp
Leu65 70 75 80Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu Trp Ser
Tyr Asn Ala 85 90 95Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile
Asp Leu Thr Asp 100 105 110Ser Glu Met Asn Lys Leu Phe Glu Lys Thr
Arg Arg Gln Leu Arg Glu 115 120 125Asn Ala Glu Glu Met Gly Asn Gly
Cys Phe Lys Ile Tyr His Lys Cys 130 135 140Asp Asn Ala Cys Ile Glu
Ser Ile Arg Asn Gly Thr Tyr Asp His Asp145 150 155 160Val Tyr Arg
Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val 165 170 175Glu
Leu Lys Ser Gly Tyr Lys Asp Trp Arg Cys Asn Ile Cys Ile 180 185
19020438PRTMEASLES VIRUS 20Phe Ala Gly Val Val Leu Ala Gly Ala Ala
Leu Gly Val Ala Thr Ala1 5 10 15Ala Gln Ile Thr Ala Gly Ile Ala Leu
His Gln Ser Met Leu Asn Ser 20 25 30Gln Ala Ile Asp Asn Leu Arg Ala
Ser Leu Glu Thr Thr Asn Gln Ala 35 40 45Ile Glu Ala Ile Arg Gln Ala
Gly Gln Glu Met Ile Leu Ala Val Gln 50 55 60Gly Val Gln Asp Tyr Ile
Asn Asn Glu Leu Ile Pro Ser Met Asn Gln65 70 75 80Leu Ser Cys Asp
Leu Ile Gly Gln Lys Leu Gly Leu Lys Leu Leu Arg 85 90 95Tyr Tyr Thr
Glu Ile Leu Ser Leu Phe Gly Pro Ser Leu Arg Asp Pro 100 105 110Ile
Ser Ala Glu Ile Ser Ile Gln Ala Leu Ser Tyr Ala Leu Gly Gly 115 120
125Asp Ile Asn Lys Val Leu Glu Lys Leu Gly Tyr Ser Gly Gly Asp Leu
130 135 140Leu Gly Ile Leu Glu Ser Arg Gly Ile Lys Ala Arg Ile Thr
His Val145 150 155 160Asp Thr Glu Ser Tyr Phe Ile Val Leu Ser Ile
Ala Tyr Pro Thr Leu 165 170 175Ser Glu Ile Lys Gly Val Ile Val His
Arg Leu Glu Gly Val Ser Tyr 180 185 190Asn Ile Gly Ser Gln Glu Trp
Tyr Thr Thr Val Pro Lys Tyr Val Ala 195 200 205Thr Gln Gly Tyr Leu
Ile Ser Asn Phe Asp Glu Ser Ser Cys Thr Phe 210 215 220Met Pro Glu
Gly Thr Val Cys Ser Gln Asn Ala Leu Tyr Pro Met Ser225 230 235
240Pro Leu Leu Gln Glu Cys Leu Arg Gly Ser Thr Lys Ser Cys Ala Arg
245 250 255Thr Leu Val Ser Gly Ser Phe Gly Asn Arg Phe Ile Leu Ser
Gln Gly 260 265 270Asn Leu Ile Ala Asn Cys Ala Ser Ile Leu Cys Lys
Cys Tyr Thr Thr 275 280 285Gly Thr Ile Ile Asn Gln Asp Pro Asp Lys
Ile Leu Thr Tyr Ile Ala 290 295 300Ala Asp His Cys Pro Val Val Glu
Val Asn Gly Val Thr Ile Gln Val305 310 315 320Gly Ser Arg Arg Tyr
Pro Asp Ala Val Tyr Leu His Arg Ile Asp Leu 325 330 335Gly Pro Pro
Ile Ser Leu Glu Arg Leu Asp Val Gly Thr Asn Leu Gly 340 345 350Asn
Ala Ile Ala Lys Leu Glu Asp Ala Lys Glu Leu Leu Glu Ser Ser 355 360
365Asp Gln Ile Leu Arg Ser Met Lys Gly Leu Ser Ser Thr Ser Ile Val
370 375 380Tyr Ile Leu Ile Ala Val Cys Leu Gly Gly Leu Ile Gly Ile
Pro Ala385 390 395 400Leu Ile Cys Cys Cys Arg Gly Arg Cys Asn Lys
Lys Gly Glu Gln Val 405 410 415Gly Met Ser Arg Pro Gly Leu Lys Pro
Asp Leu Thr Gly Thr Ser Lys 420 425 430Ser Tyr Val Arg Ser Leu
43521199PRTHIV 21Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu
Gly Ala Ala Gly1 5 10 15Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr
Val Gln Ala Arg Gln 20 25 30Leu Leu Ser Gly Ile Val Gln Gln Gln Asn
Asn Leu Leu Arg Ala Ile 35 40 45Glu Ala Gln Gln His Leu Leu Gln Leu
Thr Val Trp Gly Ile Lys Gln 50 55 60Leu Gln Ala Arg Ile Leu Ala Val
Glu Arg Tyr Leu Lys Asp Gln Gln65 70 75 80Leu Leu Gly Ile Trp Gly
Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala 85 90 95Val Pro Trp Asn Ala
Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp 100 105 110Asn His Thr
Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr 115 120 125Ser
Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys 130 135
140Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp
Asn145 150 155 160Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys
Leu Phe Ile Met 165 170 175Ile Val Gly Gly Leu Val Gly Leu Arg Ile
Val Phe Ala Val Leu Ser 180 185 190Ile Val Asn Arg Val Arg Gln
1952222PRTArtificial sequenceSynthetic peptide 22Gly Asn His Ile
Leu Ser Leu Val Gln Asn Ala Pro Tyr Gly Leu Tyr1 5 10 15Phe Ile His
Phe Ser Trp 202319PRTArtificial sequenceSynthetic peptide 23Gly Tyr
Phe Val Gln Asp Asp Gly Glu Trp Lys Phe Thr Gly Ser Ser1 5 10 15Tyr
Tyr Tyr2422PRTArtificial sequenceSynthetic peptide 24Gly Tyr His
Leu Met Ser Phe Pro Gln Ala Ala Pro His Gly Val Val1 5 10 15Phe Leu
His Val Thr Tyr 202519PRTArtificial sequenceSynthetic peptide 25Gly
Val Phe Val Phe Asn Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn1 5 10
15Phe Phe Ser2619PRTArtificial sequenceSynthetic peptide 26Met Phe
Pro Pro Trp Ser Ala Ala Ala Gly Val Pro Phe Ser Leu Ser1 5 10 15Val
Gln Tyr2726PRTArtificial sequenceSynthetic peptide 27Gln Asp Ala
Ile Lys Lys Leu Asn Glu Ser Tyr Ile Asn Leu Lys Glu1 5 10 15Val Gly
Thr Tyr Glu Met Tyr Val Lys Trp 20 252819PRTArtificial
sequenceSynthetic peptide 28Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe
Gly Gly Phe Asn Phe Ser1 5 10 15Gln Ile Leu2928PRTArtificial
sequenceSynthetic peptide 29Ala Ala Cys Glu Val Ala Lys Asn Leu Asn
Glu Ser Leu Ile Asp Leu1 5 10 15Gln Glu Leu Gly Lys Tyr Glu Gln Tyr
Ile Lys Trp 20 253015PRTArtificial sequenceSynthetic peptide 30Asn
Tyr Ser Lys Tyr Trp Tyr Leu Asn His Thr Thr Thr Gly Arg1 5 10
153119PRTArtificial sequenceSynthetic peptide 31Gly Thr
Phe Thr Trp Thr Leu Ser Asp Ser Glu Gly Lys Asp Thr Pro1 5 10 15Gly
Gly Tyr
* * * * *