U.S. patent application number 16/044985 was filed with the patent office on 2019-01-31 for soluble forms of hendra and nipah virus g glycoprotein.
This patent application is currently assigned to THE HENRY M. JACKSON FOUNDATION FOR THE ADVANCEMENT OF MILITARY MEDICINE, INC.. The applicant listed for this patent is THE HENRY M. JACKSON FOUNDATION FOR THE ADVANCEMENT OF MILITARY MEDICINE, INC.. Invention is credited to Katharine N. Bossart, Christopher C. Broder.
Application Number | 20190031719 16/044985 |
Document ID | / |
Family ID | 36793510 |
Filed Date | 2019-01-31 |
![](/patent/app/20190031719/US20190031719A1-20190131-D00001.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00002.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00003.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00004.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00005.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00006.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00007.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00008.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00009.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00010.png)
![](/patent/app/20190031719/US20190031719A1-20190131-D00011.png)
View All Diagrams
United States Patent
Application |
20190031719 |
Kind Code |
A1 |
Broder; Christopher C. ; et
al. |
January 31, 2019 |
Soluble Forms of Hendra and Nipah Virus G Glycoprotein
Abstract
This invention relates to soluble forms of G glycoprotein from
Hendra and Nipah virus. In particular, this invention relates to
compositions comprising soluble forms of G glycoprotein from Hendra
and Nipah virus and also to diagnostic and therapeutic methods
using the soluble forms of G glycoprotein from Hendra and Nipah
virus. Further, the invention relates to therapeutic antibodies
including neutralizing antibodies, and vaccines for the prevention
and treatment of infection by Hendra and Nipah viruses.
Inventors: |
Broder; Christopher C.;
(Silver Spring, MD) ; Bossart; Katharine N.; (San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE HENRY M. JACKSON FOUNDATION FOR THE ADVANCEMENT OF MILITARY
MEDICINE, INC. |
BETHESDA |
MD |
US |
|
|
Assignee: |
THE HENRY M. JACKSON FOUNDATION FOR
THE ADVANCEMENT OF MILITARY MEDICINE, INC.
BETHESDA
MD
|
Family ID: |
36793510 |
Appl. No.: |
16/044985 |
Filed: |
July 25, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15395418 |
Dec 30, 2016 |
10053495 |
|
|
16044985 |
|
|
|
|
14701006 |
Apr 30, 2015 |
9533038 |
|
|
15395418 |
|
|
|
|
13530922 |
Jun 22, 2012 |
9045532 |
|
|
14701006 |
|
|
|
|
11629682 |
May 29, 2008 |
8865171 |
|
|
PCT/US2005/024022 |
Jul 7, 2005 |
|
|
|
13530922 |
|
|
|
|
60586843 |
Jul 9, 2004 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/18222
20130101; C07K 2319/40 20130101; A61K 2039/6075 20130101; C12N
2760/18234 20130101; A61P 31/00 20180101; G01N 33/6854 20130101;
G01N 33/56983 20130101; C12N 7/00 20130101; A61K 39/155 20130101;
C12N 2760/18271 20130101; A61K 2039/575 20130101; C07K 14/115
20130101; A61K 39/00 20130101; A61K 39/12 20130101; A61K 2039/505
20130101; C07K 14/005 20130101; C07K 14/07 20130101; C12N
2710/24141 20130101; C07K 16/1027 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; G01N 33/68 20060101 G01N033/68; G01N 33/569 20060101
G01N033/569; A61K 39/12 20060101 A61K039/12; A61K 39/155 20060101
A61K039/155; C07K 14/07 20060101 C07K014/07; C07K 14/115 20060101
C07K014/115; C07K 16/10 20060101 C07K016/10; C12N 7/00 20060101
C12N007/00 |
Goverment Interests
RIGHTS IN THE INVENTION
[0001] This invention was made with government support under
AI056423 awarded by National Institutes of Health (NIH). The
government has certain rights in the invention.
Claims
1. A peptide comprising a soluble form of the G protein, or a
fragment, analog or homolog thereof, which is derived from Hendra
virus.
2. The peptide of claim 1, which comprises only the ectodomain of G
protein.
3. The peptide of claim 1, comprising only amino acids 71-604 of a
native Hendra virus G protein.
4. The peptide of claim 1, which retains one or more
characteristics of a native Hendra virus G protein.
5. The peptide of claim 4, wherein the one or more characteristics
are selected from the group consisting of ability to interact with
a viral host receptor cell, ability to be produced in one or more
oligomeric forms, ability to elicit an antibody reaction, ability
to elicit a neutralizing antibody reaction, ability to block or
prevent infection of a host cell by Hendra virus, and combinations
thereof
6. A fusion protein containing the peptide of claim 1 linked to
another peptide.
7. The fusion protein of claim 6, wherein the another peptide
enhances the stability, immunogenicity or assist in the
purification of said peptide
8. The fusion protein of claim 6, wherein the another peptide is
derived from vaccinia virus.
9. A nucleic acid that contains a sequence which encodes the
peptide of claim 1.
10. An expression vector that contains the nucleic acid sequence of
claim 9.
11. An antibody specifically reactive to the polypeptide of claim
1.
12. The antibody of claim 11, which is a monoclonal antibody, a
polyclonal antibody, a humanized antibody, a recombinantly produced
antibody, or a fragment of any of the preceding.
13. A vaccine for preventing or treating infection of a patient by
Hendra virus comprising the antibody of claim 11.
14. The vaccine of claim 13, which is cross-protective against
infection by Nipah virus.
15. A pharmaceutical composition comprising the peptide of claim 1
and a pharmaceutically acceptable carrier.
16. A diagnostic kit for detecting an infection of a subject by
Hendra virus comprising the peptide of claim 1.
17. A diagnostic kit for detecting infection of a subject by Hendra
virus comprising the antibody of claim 11.
18. A method of detecting infection by a Hendra virus comprising
detecting Hendra virus antigens or antibodies to Hendra virus in a
sample obtained from a subject suspected of being infected.
19. A method of treating or preventing infection by a Hendra virus
in a subject comprising administering the peptide of claim 1 to
said subject.
20. A method of treating or preventing infection by a Hendra virus
in a subject comprising administering the antibody of claim 11 to
said subject.
21. A peptide comprising a soluble form of the G protein, or a
fragment, analog or homolog thereof, which is derived from Nipah
virus.
22. The peptide of claim 21, which comprises only the ectodomain of
G protein.
23. The peptide of claim 21, comprising only amino acids 71-602 of
a native Nipah virus G protein.
24. The peptide of claim 21, which retains one or more
characteristics of a native Nipah virus G protein.
25. The peptide of claim 24, wherein the one or more
characteristics are selected from the group consisting of ability
to interact with a viral host receptor cell, ability to be produced
in one or more oligomeric forms, ability to elicit an antibody
reaction, ability to elicit a neutralizing antibody reaction,
ability to block or prevent infection of a host cell by Nipah
virus, and combinations thereof.
26. A fusion protein containing the peptide of claim 21 linked to
another peptide.
27. The fusion protein of claim 26, wherein the another peptide
enhances the stability, immunogenicity or assist in the
purification of said peptide
28. The fusion protein of claim 26, wherein the another peptide is
derived from vaccinia virus.
29. A nucleic acid that contains a sequence which encodes the
peptide of claim 21.
30. An expression vector that contains the nucleic acid sequence of
claim 29.
31. An antibody specifically reactive to the polypeptide of claim
21.
32. The antibody of claim 31, which is a monoclonal antibody, a
polyclonal antibody, a humanized antibody, a recombinantly produced
antibody, or a fragment of any of the preceding.
33. A vaccine for preventing or treating infection of a patient by
Nipah virus comprising the antibody of claim 31.
34. The vaccine of claim 33, which is cross-protective against
infection by Hendra virus.
35. A pharmaceutical composition comprising the peptide of claim 1
and a pharmaceutically acceptable carrier.
36. A diagnostic kit for detecting an infection of a subject by
Nipah virus comprising the peptide of claim 21.
37. A diagnostic kit for detecting infection of a subject by Nipah
virus comprising the antibody of claim 31.
38. A method of detecting infection by a Nipah virus comprising
detecting Nipah virus antigens or antibodies to Nipah virus in a
sample obtained from a subject suspected of being infected.
39. A method of treating or preventing infection by a Nipah virus
in a subject comprising administering the peptide of claim 31 to
said subject.
40. A method of treating or preventing infection by a Nipah virus
in a subject comprising administering the antibody of claim 31 to
said subject.
Description
SEQUENCE LISTING SUBMISSION VIA EFS-WEB
[0002] A computer readable text file, entitled
"013306-5008-06-SequenceListing.txt" created on or about July 19,
2018 with a file size of about 17 KB contains the sequence listing
for this application and is hereby incorporated by reference in its
entirety.
1. Field of the Invention
[0003] This present invention relates to soluble forms of a G
glycoprotein from Hendra and Nipah virus, to compositions
comprising the soluble forms of G glycoprotein from Hendra and
Nipah virus, to antibodies reactive against soluble forms of G
glycoprotein from Hendra and Nipah virus, and to methods relating
thereto.
2. Description of the Background
[0004] Nipah virus and Hendra virus are emerging viruses that are
responsible for previously unrecognized fatal diseases in animals
and humans. These viruses are closely related members of a new
genus, Henipavirus, in the Paramyxoviridae family, a diverse group
of large, enveloped, negative-sense RNA viruses, that includes a
variety of important human and animal pathogens. The recent
emergence of these two viruses appears to have been the result of
exposure of new hosts precipitated by certain environmental and
behavioral changes. Hendra virus was identified first, from cases
of severe respiratory disease that fatally affected both horses and
man. Subsequent to that appearance, an outbreak of severe febrile
encephalitis associated with human deaths occurred in Malaysia.
Later studies identified a Hendra-like virus, now known as Nipah
virus, as the etiologic agent of that episode. These viruses are
unusual among the paramyxoviruses in their abilities to infect and
cause disease with high fatality rates in a number of host species,
including humans, and are zoonotic Biological Safety Level-4
agents. Presently, the cat appears to be the ideal small-animal
model capable of reproducing the pathology seen in infected
humans.
[0005] Nipah and Hendra virus are NIAID select, category C viruses
and possess several features which make them highly adaptable for
use as biowarfare agents. For example, both readily grow in cell
culture or embryonated chicken eggs, produce high un-concentrated
titers near 1x10.sup.8 TCID50/ml, (14), are highly infectious and
transmitted via the respiratory tract (22, 27), and can be
amplified and spread in livestock serving as a source for
transmission to humans. Recent evidence also indicates that
nosocomial transmissibility of NiV from patients with encephalitis
to healthcare workers is possible (45, 60).
[0006] Fusion of the membrane of enveloped viruses with the plasma
membrane of a receptive host cell is a prerequisite for viral entry
and infection and an essential step in the life cycle of all
enveloped viruses. Research towards dissecting and understanding
the mechanisms of this process is an important area of work. Not
only does it afford insights into the complex interactions between
viral pathogens and their host cells, but it can also shed light on
the complex and essential biochemical process of protein-mediated
membrane fusion, and also lead to the development of novel
intervention and vaccine strategies. This has been demonstrated in
the HIV research field, where the discovery of the long-sought
coreceptors involved in entry and infection has opened a broad new
era in the development of therapeutics to block the infection
process at the level of entry (reviewed in (3, 18)).
[0007] Paramyxoviruses are negative-sense RNA enveloped viruses and
encompass a variety of important human and animal pathogens,
including measles virus (MeV), mumps virus, Sendai virus (SeV),
Newcastle disease virus (NDV), rinderpest virus, canine distemper
virus (CDV), human parainfluenza viruses (hPIV) 1-4, respiratory
syncytial virus (RSV), and simian virus 5 (SV5) (reviewed in (36)).
In contrast to retroviruses, paramyxoviruses contain two principal
membrane-anchored glycoproteins, which appear as spikes projecting
from the envelope membrane of the viral particle when viewed under
the electron microscope. One glycoprotein is associated with virion
attachment to the host cell, and, depending on the particular
virus, has been designated as either the
hemagglutinin-neuraminidase protein (RN), the hemagglutinin (H), or
the G protein which has neither hemagglutinating nor neuraminidase
activities (reviewed in (44)). The other glycoprotein is the fusion
protein (F) which is directly involved in facilitating the fusion
of the viral and host cell membranes (reviewed in (36)). Following
virus attachment to a permissive host cell, fusion at neutral pH
(or independently of the pH) between the virion and plasma
membranes ensues, resulting in delivery of the nucleocapsid into
the cytoplasm. In a related process, cells expressing these viral
glycoproteins on their surfaces can fuse with receptor-bearing
cells, resulting in the formation of multinucleated giant cells
(syncytia) under physiological or cell culture conditions.
[0008] The Envelope Glycoproteins. The HN envelope glycoprotein is
responsible for attachment of the virion to its receptor, sialic
acid, on the target cell as is the case for the hPIVs, NDV, SV5 and
others. In contrast, the morbilliviruses, like MeV and CDV, have an
attachment protein (H) possessing only hemagglutinating activity
and do not bind to sialic acid. MeV was the first morbillivirus
shown capable of utilizing a cell-surface protein as a receptor
(19, 47), and was the demonstration of the predicted interaction
between the MeV H glycoprotein and the MeV receptor CD46 using
co-ip experiments and soluble CD46 (48). In addition, MeV field
isolates as well as vaccine strains have been shown capable of
utilizing signaling lymphocyte activation molecule (SLAM; CD150)
(61). SLAM is also capable of serving as a receptor for several
other morbilliviruses, including CDV (62).
[0009] A third class of paramyxovirus attachment glycoproteins,
which are possessed by the Pneumovirinae such as RSV, are
designated G, and have neither hemmagglutinating nor neuraminidase
activities (reviewed in (44)). The attachment glycoproteins are
type II membrane proteins where the molecule's amino (N)-terminus
is oriented towards the cytoplasm and the protein's carboxy
(C)-terminus is extracellular. The other major envelope
glycoprotein is the fusion (F) glycoprotein, and the F of these
viruses are more similar, where in all cases it is directly
involved in mediating fusion between the virus and host cell at
neutral pH.
[0010] The F glycoprotein of the paramyxoviruses is a type I
integral membrane glycoprotein with the protein's N-terminus being
extracellular. It shares several conserved features with other
viral fusion proteins, including the envelope glycoprotein (Env) of
retroviruses like gp120/gp41 of HIV-1, and hemagglutinin (HA) of
influenza virus (reviewed in (26)). The biologically active F
protein consists of two disulfide linked subunits, F.sub.1 and
F.sub.2, that are generated by the proteolytic cleavage of a
precursor polypeptide known as F.sub.0 (reviewed in (34, 55)).
Likewise, HIV-1 Env and influenza HA are proteolytically activated
by a host cell protease, leading to the generation of a membrane
distal subunit analogous to F.sub.2 and a membrane-anchored subunit
analogous to F.sub.1. In all cases, the membrane-anchored subunit
contains a new N-terminus that is hydrophobic and highly conserved
across virus families and is referred to as the fusion peptide
(reviewed in (30)). All paramyxoviruses studied to date require
both an attachment and F protein for efficient fusion, with the
exception of SV5 which can mediate some fusion in the absence of HN
(50). Evidence of a physical association between these
glycoproteins has been observed with only limited success and only
with NDV (57), hPIV (73), and recently with MeV (51), but these
observations have often been with the aid of chemical cross-linking
agents. It is hypothesized that following receptor engagement, the
attachment protein must somehow signal and induce a conformational
change in F leading to virion/cell fusion (35, 53). That
conformational distinctions existed in the HN and F of a
paramyxovirus depending on whether they were expressed alone or in
combination has been noted for quite sometime (13).
[0011] The Paramyxovirus F envelope glycoproteins, like those of
retroviruses, are considered class I membrane fusion-type proteins.
An important feature of the fusion glycoproteins of these viruses
is the presence of 2 a-helical domains referred to as heptad
repeats that are involved in the formation of a trimer-of-hairpins
structure during or immediately following fusion (29, 56). These
domains are also referred to as either the amino (N)-terminal and
the carboxyl (C)-terminal heptad repeats (or HR1 and HR2), and
peptides corresponding to either of these domains can inhibit the
activity of the viral fusion glycoprotein when present during the
fusion process, first noted with sequences derived from the gp41
subunit of HIV-1 envelope glycoprotein (32, 67). Indeed, HIV-1
fusion-inhibiting peptides have met with clinical success and are
likely to be the first approved fusion inhibitor therapeutics.
Peptide sequences from either the N or C heptads of the F of SV5,
MeV, RSV, hPIV, NDV, and SeV have also been shown to be potent
inhibitors of fusion (33, 37, 52, 68, 74, 75). It is generally
accepted that significant conformational change would occur during
activation of paramyxovirus F fusogenic activity. Differential
antibody binding reactivities of precursor and proteolytically
processed forms of SV5 F (20) and in conjunction with the structure
of the post fusion 6-helix bundle of SV5 F (2), strongly support
the conformational change model, not only from the pre-fusion to
post-fusion structural change, but also from the Fo precursor to
the F.sub.2-F.sub.1 mature protein. That the post-fusion structure
of a paramyxovirus F core is likely conserved across other
paramyxoviruses has been further supported by the F core structures
of RSV (79) and MeV (80). However, recent structural studies on the
F glycoprotein of NDV have yielded some different and interesting
findings. The oligomeric trimer structure of NDV F (in perhaps the
pre-fusion or meta-stable state) has offered some alternative
information which distinguishes it from the classic influenza HA
structure, this is principally reflected in the completely opposite
orientation of the central coiled coils formed by the HR1 (also
termed HRA) segments of the trimer (9, 10). To date this is the
only structural information on the pre-fusion (or meta-stable) form
of a paramyxovirus F (in fact the only other meta-stable, class I,
structure other than influenza HA), and perhaps represents a
possible third-class of viral fusion proteins.
[0012] A precise understanding of how the fusion and attachment
glycoproteins function in concert in mediating fusion has yet to be
elucidated, but there are two central models proposed for the role
of the attachment glycoprotein in the paramyxovirus-mediated
membrane fusion process, which were recently detailed by Morrison
and colleagues (41), in the context of the HN glycoprotein of NDV.
In the first model, the fusion and attachment glycoproteins are not
physically associated in the membrane, but following receptor
engagement there is an alteration in the attachment glycoprotein
which facilitates its association with F and in so doing imparts or
facilitates F conformational change leading to membrane fusion. In
the second model, the F and attachment glycoprotein are
pre-associated and receptor engagement induces conformational
alteration in the attachment glycoprotein, and this process alters
or releases an interaction with F that allows F to proceed towards
its fusion active state--formation of the 6-helix bundle just prior
or concomitant with membrane merger. Findings on NDV demonstrate
the variable accessibility of the HR1 domain during the process,
where HR1 of F are accessible to specific fusion-inhibiting
antibody when F is presented in the context of HN, however
expression of F alone results in a non-fusogenic version of F with
distinctly altered conformation having an HR1 domain which is no
longer accessible to antibody (41). The second model is that the
attachment glycoprotein is holding F in its non-fusogenic
conformation and upon receptor engagement and conformational change
in the attachment glycoprotein F is released to undergo
conformational changes leading to 6-helix bundle formation and
facilitation of membrane fusion. This is supported by observations
that paramyxovirus F expressed alone neither mediates fusion (with
the exception of SV5 under certain conditions) and has variably
antibody accessibility of certain domains such as the NDV F HR1
domain (41). This is perhaps because F alone has transitioned to a
fusion triggered or intermediate conformation at an inappropriate
moment, which would be consistent with observations of fusion
defective or triggered HIV-1 gp41 also referred to as dead-spikes
(17). Preliminary findings with HeV and NiV support this second
model. Finally, that the attachment glycoprotein of a paramyxovirus
undergoes specific conformation alteration when bound to receptor
has been recently revealed at the molecular level from studies on
the HN glycoprotein of NDV (58, 59). These studies have revealed
clear differences in the structure of HN when the receptor-bound
glycoprotein is compared to the non-receptor-bound HN structure. In
addition, all known viral envelope glycoproteins are homo- or
heterooligomers in their mature and functional forms (reviewed in
(16)). Multimeric proteins, like these, generally interact over
large areas, making structural differences between monomeric
subunits and the mature oligomer likely (31). This feature can also
translate into differences in antigenic structure and has been
shown for a number of proteins, most notably the trimeric influenza
HA glycoprotein (69) and HIV-1 gp120/gp41 (7). Indeed, a
trimer-specific, potent neutralizing determinant has been mapped to
the interface between adjoining subunits of HA, and
oligomer-specific anti-HIV-1 Env antibodies have been identified
and mapped to conformation-dependent epitopes in gp41 (7). Thus
far, all paramyxoviruses, retroviruses, and influenza virus fusion
glycoproteins appear to be homotrimers (8, 9, 21, 54, 71), and
several HN attachment proteins have been shown to be tetrameric,
comprised of a dimer of homodimers. For example, the NDV HN can
form dimers and tetramers on the viral surface (40, 43), and
recently the crystal structure of the globular head region of the
HN dimer from NDV has been solved (15). Finally, and of importance
in understanding certain aspects of the immune response to these
viruses and the development of vaccines, it is these major envelope
glycoproteins of these viruses to which virtually all
virus-neutralizing antibodies are directed against.
[0013] Emerging Pathogenic Paramyxoviruses. In 1994, a new
paramyxovirus, was isolated from fatal cases of respiratory disease
in horses and humans, and was shown to be distantly related to MeV
and other members of the morbillivirus genus; it was provisionally
termed equine morbillivirus (EMV) but has since been re-named
Hendra virus (HeV) (46). The first outbreak of severe respiratory
disease in the Brisbane suburb of Hendra Australia resulted in the
death of 13 horses and their trainer, and the non-fatal infection
of a stable hand and a further 7 horses. At approximately the same
time, in an unrelated incident almost 100 km north of Hendra, a
35-year-old man experienced a brief aseptic meningitic illness
after caring for and assisting at the necropsies of two horses
subsequently shown to have died as a result of HeV infection.
Thirteen months later this individual suffered a recurrence of
severe encephalitis characterized by uncontrolled focal and
generalized epileptic-activity. A variety of studies that were
performed in the evaluation of this fatality, including serology,
PCR, electron microscopy (EM) and immunohistochemistry, strongly
suggested that HeV was indeed the cause of this patient's
encephalitis, and the virus was acquired from the HeV-infected
horses 13 months earlier (49). In all, fifteen horses and two
people died in the two episodes. At the time the source of the
emerging virus was undetermined, but more recently it has been
found that approximately 50% of certain Australian fruit bat
species, commonly known as flying foxes, have antibodies to HeV and
Hendra-like viruses have been isolated from bat uterine fluids. It
appears that these animals are the natural host for the virus (22,
24, 25, 76). Recently, the nucleic acid sequence of HeV genes has
been analyzed and compared with those of other paramyxoviruses (64,
77, 78). These studies have confirmed that HeV is a member of the
Paramyxoviridae, subfamily Paramyxovirinae.
[0014] Subsequent to these events, an outbreak of severe
encephalitis in people with close contact exposure to pigs in
Malaysia and Singapore occurred in 1998 (1). The outbreak was first
noted in September 1998 and by mid-June 1999, more than 265 cases
of encephalitis, including 105 deaths, had been reported in
Malaysia, and 11 cases of disease with one death reported in
Singapore. This outbreak had a tremendous negative economic impact,
which continues to date. Although successful, measures taken in the
early days of the outbreak resulted in the slaughter of
approximately 1.3 million pigs and the virtual closure of the pig
farming industry in peninsular Malaysia. EM, serologic, and genetic
studies have since indicated that this virus is also a
paramyxovirus, and was closely related to HeV. This virus was named
Nipah virus (NiV) after the small town in Malaysia from which the
first isolate was obtained from the cerebrospinal fluid of a fatal
human case (11, 12, 23, 38, 39).
[0015] Most human patients present with acute encephalitis, which
in the Malaysia outbreak of 1998-1999 ultimately resulted in a
mortality rate of approximately 40%, but infection can also present
as a nonencephalitic or asymptomatic episode with seroconversion.
Interestingly, infection with NiV can also take a more chronic
course with more serious neurological disease occurring late
(greater than 10 weeks) following a nonencephalitic or asymptomatic
infection. On the other hand, the recurrence of neurological
manifestations (relapsed encephalitis) has also been noted in
patients who had previously recovered from acute encephalitis.
Cases of relapsed-encephalitis presented from several months to
nearly two years after the initial infection (72) Taken together,
there was nearly a 10% incidence rate of late encephalitic
manifestations with a mortality rate of 18%. Thus, with both NiV
and HeV a prolonged period of infection is possible before serious
neurological disease occurs. The underlying mechanisms which allow
these viruses, especially NiV, to escape immunological clearance
for such an extended period are completely unknown.
[0016] In the case of NiV, the late presentation of neurological
disease and IgG subclass response showed similarities to subacute
sclerosing panencephalitis (SSPE), a rare late manifestation of MeV
infection (72). It was molecular characterization of HeV and NiV
which distinguished them as distinctly new paramyxoviruses. The
families Paramyxoviridae, Filoviridae, Rhabdoviridae, and
Bornaviridae are all negative-sense RNA enveloped viruses sharing
similar genome organization, replication strategies, and polymerase
domain structure (63). These families are grouped in the order
Mononegavirales, the first taxon above family level virus taxonomy.
The genome size in the Mononegavirales is wide ranging,
.about.8.9-19.1 kb. The genomes of paramyxoviruses, as a group, are
generally considered tightly spread, having sizes in the range of
15.1-15.9 kb, except HeV and NiV whose genomes of 18.2 kb, far
closer in size to the Filoviridae. Much of this added length is
untranslated regions at the 3' end in the six transcription units,
again quite similar to Marburg and Ebloa Filoviruses (63). Also,
the P protein is larger by 100 residues (longest known), and a
small basic protein (SB) in HeV of unknown function. Taken
together, the molecular features of both HeV and NiV make them
unusual paramyxoviruses, as does their ability to cause potentially
fatal disease in a number of species, including humans.
[0017] Pathogenesis. The development or characterization of animal
models to study these newly identified viral zoonoses is important
for understanding their pathogenic features and in the development
of therapeutics. Of the two fatal cases of HeV infection in humans,
the first was the result of severe respiratory disease following
several days of ventilated life-support. The patient's lungs had
gross lesions of congestion, hemorrhage and oedema associated with
histological chronic alveolitis with syncytia. The second fatal
case was one of leptomeningitis with lymphocytes and plasma cells
and foci of necrosis in various parts of the brain parenchyma,
after initial infection more than 1 year previously (reviewed in
(27)). Multinucleate endothelial cells were also seen in the
viscera as well as in the brain. In contrast, there were many more
human cases of infection with NiV. More than 30 individuals
resulting from the large NiV outbreak in Malaysia and Singapore
were autopsied, and the immuno- and histological features included
systemic endothelial infection accompanied by vasculitis,
thrombosis, ischaemia and necrosis (reviewed in (27)). These
changes were especially noted in the central nervous system (CNS).
Immunohistochemical analysis have also shown widespread presence of
NiV antigens in neurons and other parenchymal cells in necrotic
foci seen in the CNS as well as in endothelial cells and media of
affected vessels (27). In infected humans, evidence of vasculitis
and endothelial infection was also seen in most organs examined.
Disseminated endothelial cell infection, vasculitis, and CNS
parenchymal cell infection play an essential role in the fatal
outcome of NiV infection in humans (reviewed in (27)). The
principal zoonotic episodes in nature involved the horse in the HeV
cases and the pig in the case of NiV. Both these viruses have a
notable broad host cell tropism in in vitro studies (4, 5). These
observations correlated to what has been observed in natural and
experimental infection.
[0018] Experimental infections of the horse and pig have been
carried out with HeV and NiV respectively and one naturally
NiV-infected horse has been examined. The pathology caused by
either virus in horses appears to be more severe than that caused
by NiV in pigs. In addition to pigs, HeV infection of cats has also
been performed and in this case disease resembles that seen in
horses, characterized by generalized vascular disease with the most
severe effects seen in the lung (28). Guinea pigs have also been
experimentally infected with HeV (28) and the pathology seen
differed significantly in several respects in comparison to the
human cases as well as natural and experimentally infected horses.
In guinea pigs HeV caused generalized vascular disease but, unlike
horses and cats, there was little or no pulmonary oedema.
Histologically, vascular disease was prominent in arteries and
veins, and in many organs such as the lung, kidney, spleen, lymph
nodes, gastrointestinal tract and skeletal and intercostal muscles.
NiV infection of the guinea pig has not yet been well
described.
[0019] In regards to other small laboratory animal models, NiV and
HeV do not cause disease in mice even after subcutaneous
administration, however, and not surprisingly, they will kill mice
if administered intracranially. Further, there is also no
serological evidence for NiV in rodents in Malaysia, and several
hundred sera were tested during the outbreak. Evidence of natural
NiV infections were also noted in dogs and cats.
[0020] In experimental NiV infection of the cat, gross lesions in
animals with severe clinical signs strongly resembled those of cats
infected with HeV. These consisted of hydrothorax, oedema in the
lungs and pulmonary lymph nodes, froth in the bronchi, and dense
purple-red consolidation in the lung. There were also similar
features in the histological appearance, diffuse perivascular,
peribronchial and alveolar hemorrhage and oedema, vasculitis
affecting arteries and arterioles, alveolitis, syncytium formation
within endothelial cells and alveolar epithelial cells (reviewed in
(27)). Taken together, the evidence to date indicates that the cat
represents an animal model whereby the pathology seen most closely
resembles the lethal human disease course. In addition, infection
of cats with either NiV or HeV is uniformly fatal. NiV and HeV
appear to cause similar diseases but with some notable variations,
and although the basic pathologic processes have been well
described, less is known about the factors which clearly influence
disease course depending on the species infected. This is a special
concern in human infections, where there is a remarkable ability of
these viruses to persist in the host (up to 2 yrs) before causing a
recurrence of severe and often fatal disease. Cats succumb within
6-8 days to subcutaneous infection with 5,000, and subcutaneous or
oral administration of 50,000, TCID5o of a low passage, purified
HeV (65, 66, 70). Experimental infection of cats with NiV has
confirmed the susceptibility of this species to oronasal infection
with 50,000 TCID50 NiV (42). In summary, the clinical and
pathological syndrome induced by NiV in cats was comparable with
that associated with HeV infection in this species, except that in
fatal infection with NiV there was extensive inflammation of the
respiratory epithelium, associated with the presence of viral
antigen.
[0021] In summary, recurrent outbreaks of NiV resulting in
significant numbers of human fatalities have recently been
confirmed (Fatal fruit bat virus sparks epidemics in southern Asia.
Nature 429, 7, 06 May 2004). HeV is also know to cause fatalities
in human and animals and is genetically and immunologically closely
related to NiV. There are presently no vaccines or therapeutics for
prevention of infection or disease caused by Nipah virus or Hendra
virus. Both Nipah virus and Hendra virus are United States,
National Institute of Allergy and Infectious Disease, category C
priority agents of biodefense concern. Further, as these viruses
are zoonotic Biological Safety Level-4 agents (BSL-4), production
of vaccines and/or diagnostics, with safety is very costly and
difficult. Thus, there is a need for a Nipah virus or Hendra virus
vaccines and diagnostics that allow for high throughput production
of vaccines and/or diagnostics. All references cited herein,
including patent applications and publications, are incorporated by
reference in their entirety.
SUMMARY OF THE INVENTION
[0022] The present invention overcomes the problems and
disadvantages associated with current strategies and designs and
provides new tools and methods for the design, production and use
of soluble forms of the G envelope glycoprotein of Hendra virus and
Nipah virus.
[0023] One embodiment of the invention is directed to
polynucleotides and polypeptides or fragments thereof encoding a
soluble G protein derived from Hendra virus.
[0024] Another embodiment of the invention is directed to
polynucleotides or polypetides or fragments thereof encoding a
soluble G protein derived from Nipah virus.
[0025] Another embodiment is directed to methods of producing
soluble G protein derived from Hendra virus and/or Nipah virus.
[0026] Another embodiment is directed to expression vectors
comprising the polynucleotides encoding a soluble G protein derived
from Hendra and/or Nipah virus.
[0027] Another embodiment is directed to a fusion protein
comprising a polypeptide of the invention and one or more other
polypetides that enhance the stability of a polypeptide of the
invention, enhance the immunogenicity of a polypeptide of the
present invention and/or assist in the purification of a
polypeptide of the present invention.
[0028] Another embodiment is directed to antibodies and fragments
thereof, such as neutralizing antibodies, specific for a soluble G
protein derived from Hendra and/or Nipah virus and diagnostic
and/or therapeutic application of such antibodies.
[0029] Another embodiment is directed to subunit vaccine comprising
the polynucleotides or polypeptides of the invention.
[0030] Another embodiment of the invention is directed to methods
of preventing infection with Hendra and/or Nipah virus in a subject
or mitigating an infection of Hendra and/or Nipah virus in a
subject.
[0031] Another embodiment of this invention is directed to
diagnostic kits comprising the polynucleotides, polypeptides and/or
antibodies of the invention.
[0032] Other embodiments and advantages of the invention are set
forth in part in the description, which follows, and in part, may
be obvious from this description, or may be learned from the
practice of the invention.
DESCRIPTION OF THE FIGURES AND TABLE
[0033] FIG. 1 shows expression of soluble HeV G envelope
glycoprotein. Vaccinia virus encoding either a myc-tag or S-tag
soluble HeV G was produced by metabolic labeling in HeLa cells.
Control is wild-type HeV G. Specific precipitation of each sG
construct is shown by precipitation from either lysates or
supernatants using myc MAb or S-beads.
[0034] FIGS. 2A-2F shows inhibition of HeV and NiV-mediated fusion
by sG S-tag. HeLa cells were infected with vaccinia recombinants
encoding HeV F and HeV G or NiV F and NiV G glycoproteins, along
with a vaccinia recombinant encoding T7 RNA polymerase (effector
cells). Each designated target cell type was infected with the E.
coli LacZ-encoding reporter vaccinia virus vCB21R. Each target cell
type (1.times.10.sup.5) was plated in duplicate wells of a 96-well
plate. sG S-tag or control supernatants were added and incubated
for 30 minutes at 37.degree. C. The HeV or NiV
glycoprotein-expressing cells (1.times.10.sup.5) were then mixed
with each target cell type. After 2.5 hr at 37.degree. C., Nonidet
P-40 was added and (3-Gal activity was quantified. FIGS. 2A and 2B:
Inhibition of HeV-mediated fusion by sG S-tag-infected supernatant
or WR-infected supernatant in U373 cells (FIG. 2A) or PCI 13 cells
(FIG. 2B). FIGS. 2C and 2D: Inhibition of HeV- and NiV-mediated
fusion by sG S-tag in U373 cells (FIG. 2C) or PCI 13 cells (FIG.
2D). FIGS. 2E and 2F: Inhibition of HeV- and NiV-mediated fusion by
purified sG myc-tag in U373 cells (FIG. 2E) or PCI 31 cells (FIG.
2F).
[0035] FIGS. 3A and 3B shows indirect immunofluorescence of
permissive and non-permissive cell lines stained with sHeV G
envelope glycoprotein. Cells were plated into 8 well Lab-Tek II
chamber slides in the appropriate medium and incubated for 3 days.
The cells were fixed with acetone for 2 minutes. HeLa cells
represent a fusion non-permissive cell line whereas U373, PCI 13,
and Vero represent fusion permissive cell lines. The cells were
stained with sG S-tag followed by an anti-HeV G specific rabbit
antiserum and a donkey anti-rabbit Alexa Fluor 488 conjugate.
Samples were examined with an Olympus microscope with a reflected
light fluorescence attachment and an Olympus U-M41001 filter. All
images were obtained with a SPOT RT CCD digital camera at an
original magnification of 40x. FIG. 3A: sG S-tag and donkey
anti-rabbit Alexa Fluor 488 conjugate. FIG. 3B: sG S-tag, anti-HeV
G antiserum, and donkey anti-rabbit Alexa Fluor 488 conjugate.
[0036] FIG. 4 shows expression of soluble NiV G envelope
glycoprotein. Vaccinia virus encoding S-tag soluble HeV G, or
plasmid expression vectors encoding S-tag soluble HeV G or NiV G
were produced by metabolic labeling in HeLa cells. Specific
precipitation of each sG construct is shown by precipitation from
either lysates or supernatants using S-beads.
[0037] FIG. 5 shows analysis of the oligomeric structures of
soluble HeV G envelope glycoprotein. HeLa cells were infected with
sHeV G (S-tag) encoding vaccinia virus and incubated 16 h at
37.degree. C. (4 wells of a 6 well plate). Beginning at 6 h
post-infection, the cells were metabolically labeled overnight with
[.sup.35S]-met/cys. Supernatants were removed, clarified by
centrifugation, concentrated, buffer exchanged into PBS. One half
(400 .mu.l) of the sHeV G was then cross-linked with DTS SP
[3,3'-Dithiobis(sulfosuccinimidylpropionate)] (4 mM/RT.degree./15
min) quenched with 100 mM Tris pH 7.5. The cross-linked and
un-cross linked preparations were then divided into two equal
portions and layered onto continuous (5-20%) sucrose gradients (4
gradients) and fractioned. All fractions were then precipitated
with S-protein agarose, and the samples of metabolically labeled sG
were resolved by 10% SDS-PAGE under reducing with no cross-linking
(FIG. 5B), non-reducing with no crosslinking (FIG. 5A), reducing
with cross-linking (FIG. 5D), and non-reducing with cross-linking
(FIG. 5C) conditions and detected by autoradiography.
[0038] FIG. 6 shows a schematic of soluble Hey G glycoprotein
constructs. IgK-chain linker (SEQ ID NO. 10); 15 aa linker (SEQ ID
NO. 11); S-peptide tag (SEQ ID NO. 12); c-myc-epitope tag (SEQ ID
NO 13); 15 aa linker (SEQ ID NO. 14).
[0039] FIG. 7 shows a molecular weight profile of HeV sG. A panel
of high molecular weight standards was separated on a Superdex 200
size exclusion column and a calibration curve was generated.
Samples of purified .sub.SG.sub.S-tag and .sub.SG.sub.myc-tag were
separated on the calibrated Superdex 200 column and fractionated.
The Kay values of major sG peaks were calculated and their apparent
molecular weights were determined using the calibration curve from
the molecular weight standards. The figure shows the three
principal peaks observed with .sub.SG.sub.S-tag. The molecular
estimates shown associated with each of the three peaks (peak 1, 2
and 3) are the averages of seven independent separations of three
different .sub.SG.sub.S-tag preparations.
[0040] FIGS. 8A-8D shows oligomeric forms of .sub.SG.sub.S-tag.
HeLa cells were infected with .sub.SG.sub.S-tag encoding vaccinia
virus and incubated 16 h at 37.degree. C. Beginning at 6 h
post-infection, the cells were metabolically labeled overnight with
[.sup.35S]-methionine/cysteine. Supernatants were removed,
clarified by centrifugation, concentrated, buffer exchanged into
PBS. One half (200 .mu.l) of the .sub.SG.sub.S-tag was then
cross-linked with DTS SP
[3,3'-Dithiobis(sulfosuccinimidylpropionate)] (4 mM/RT.degree./30
min) quenched with 100 mM Tris pH 7.5. The cross-linked and
un-cross linked preparations were layered onto continuous (5-20%)
sucrose gradients (2 gradients) and fractioned. All fractions were
split into 2 tubes (for reducing and non-reducing conditions),
fractions were then precipitated with S-protein agarose, and the
samples of metabolically labeled sG were resolved by 7.5% SDS-PAGE
under reducing and non-reducing conditions and detected by
autoradiography. Bottom and top of gradients are indicated. FIG.
8A: non-cross-linked and unreduced, FIG. 8B: non-cross-linked and
reduced, FIG. 8C: cross-linked and unreduced, FIG. 8D: cross-linked
and reduced.
[0041] FIGS. 9A and 9B shows the oligomeric form of full length HeV
G. HeLa cells were infected with HeV G encoding vaccinia virus and
incubated 16 h at 37.degree. C. Beginning at 6 h post-infection,
the cells were metabolically labeled overnight with {.sup.35
S}-methionine/cysteine. Supernatants were removed and cells were
chased for 2 h in complete medium, washed twice in PBS and
recovered. The non-cross-linked HeV G-expressing cells were lysed
in Triton-X containing buffer, clarified by centrifugation, and
layered onto a continuous sucrose gradient (5-20%) and fractioned.
All fractions were split into 2 tubes (for reducing and
non-reducing conditions), fractions were then precipitated with
anti-HeV antiserum followed by Protein G-Sepharose, and the samples
of metabolically labeled HeV G were resolved by 7.5% SDS-PAGE under
reducing and non-reducing conditions and detected by
autoradiography. Bottom and top of gradients are indicated. FIG.
9A: non-cross-linked and unreduced, FIG. 9B: non-cross-linked and
reduced.
[0042] FIG. 10 shows immunofluorescence-based syncytia assay of HeV
and NiV. Vero cells were plated into 96 well plates and grown to
90% confluence. Cells were pretreated with .sub.SG.sub.S-tag for 30
min at 37.degree. C. prior to infection with 1.5.times.10.sup.3
TCID.sub.50/ml and 7.5.times.10.sup.2 TCID.sub.50/ml of live HeV or
NiV (combined with .sub.SG.sub.S-tag). Cells were incubated for 24
hours, fixed in methanol and immunofluorescently labeled for P
protein prior to digital microscopy. Images were obtained using an
Olympus IX71 inverted microscope coupled to an Olympus DP70 high
resolution color camera and all images were obtained at an original
magnification of 85.times.. Representative images of FITC
immunofluorescence of anti-P labeled HeV and NR. FIG. 10A:
untreated control for HeV, FIG. 10B: untreated control infection
for NiV, FIG. 10C: infection for HeV in the presence of 100
.mu.g/ml .sub.SG.sub.S-tag, FIG. 10D: infection for NiV in the
presence of 100 .mu.g/ml .sub.SG.sub.S-tag.
[0043] FIG. 11 shows inhibition of HeV and NiV infection by
.sub.SG.sub.S-tag. Vero cells were plated into 96 well plates and
grown to 90% confluence. Cells were pretreated with
.sub.SG.sub.S-tag for 30 min at 37.degree. C. prior to infection
with 1.5.times.10.sup.3 TCID.sub.50/ml and 7.5.times.10.sup.2
TCID.sub.50/ml of live HeV or NiV (combined with
.sub.SG.sub.S-tag). Cells were incubated for 24 hours, fixed in
methanol and immunofluorescently labeled for P protein prior to
digital microscopy and image analysis to determine the relative
area of each syncytium. Figure shows the relative syncytial area
(pixel.sup.2) versus .sub.SG.sub.S-tag concentration for HeV
(circles) and NiV (triangles).
[0044] FIG. 12 shows the linear structure of the sG protein.
[0045] FIG. 13 shows a three dimensional structure of the sG
protein.
[0046] FIG. 14 shows the amino acid sequence of the sG protein (SEQ
ID NO 15).
[0047] Table 1 shows neutralization of HeV and NiV infection.
Anti-HeV G antisera were generated in rabbits by 3 inoculations
with purified .sub.SG.sub.S-tag. Sera collected 2 weeks after the
third injection were analyzed in a virus-neutralization assay
against HeV and NiV. Serum neutralization titers were determined by
presence of CPE (indicated by +) and recorded as the serum dilution
where at least one of the duplicate wells showed no CPE.
DESCRIPTION OF THE INVENTION
[0048] General Techniques
[0049] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are within the skill of the art.
Such techniques are explained fully in the literature, such as, for
example, Molecular Cloning: A Laboratory Manual, second edition
(Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide
Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology,
Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis,
ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,
ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather
and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:
Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,
eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic
Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and
C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells
(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology (F. M. Ausubel, et al., eds., 1987); PCR: The
Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short
Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.
Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL
Press, 1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
[0050] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise. For example,
"a" G glycoprotein includes one or more G glycoproteins.
[0051] Generally this invention provides soluble forms of HeV and
NiV G glycoprotein envelope protein, the polynucleotides encoding
the proteins and to methods for using these proteins in diagnosis,
detection and treatment. Specifically this invention provides
soluble forms of HeV and NiV G glycoprotein envelope proteins which
retain characteristics of the native viral G glycoprotein allowing
for rapid high throughput production of vaccines, diagnostics and
screening.
[0052] Generally, the soluble forms of the HeV and NiV G
glycoproteins comprise all or part of the ectodomain (e.g.
extracellular) of the G glycoprotein of a HeV or NiV and are
generally produced by deleting all or part of the transmembrane
domain of the G glycoprotein and all or part of the cytoplasmic
tail of the G glycoprotein. By way of example, a soluble G
glycoprotein may comprise the complete ectodomain of an HeV or NiV
G glycoprotein. Also by way of example, and not limitation a
soluble G glycoprotein may comprise all or part of the ectodomain
and part of the transmembrane domain of an HeV or NiV G
glycoprotein.
[0053] The soluble HeV or NiV G glycoproteins of the invention,
generally retain one or more characteristics of the corresponding
native viral glycoprotein, such as, ability to interact or bind the
viral host cell receptor, can be produced in oligomeric form or
forms, or the ability to elicit antibodies (including, but not
limited to, viral neutralizing antibodies) capable of recognizing
native G glycoprotein. Examples of additional characteristics
include, but are not limited to, the ability to block or prevent
infection of a host cell. Conventional methodology may be utilized
to evaluate soluble HeV or NiV G glycoproteins for one of more of
the characteristics. Examples of methodology that may be used
include, but are not limited to, the assays described herein in the
Examples.
[0054] Polynucleotides
[0055] The term polynucleotide is used broadly and refers to
polymeric nucleotides of any length (e.g., oligonucleotides, genes,
small inhibiting RNA, fragments of polynucleotides encoding a
protein etc). By way of example, the polynucleotides of the
invention may comprise all or part of the ectodomain or all or part
of the ectodomain and part of the transmembrane domain. The
polynucleotide of the invention may be, for example, linear,
circular, supercoiled, single stranded, double stranded, branched,
partially double stranded or single stranded. The nucleotides
comprising the polynucleotide may be naturally occurring
nucleotides or modified nucleotides. Generally the polynucleotides
of the invention encode for all or part of the ectodomain (e.g.
extracellular) of the G glycoprotein of a HeV or NiV.
[0056] Non-limiting examples of sequences that may be used to
construct a soluble HeV G glycoprotein can be found in Wang, L. F.
et al., J. Virol. 74 (21), 9972-9979 (2000) and Yu, M. et al.,
Virology 251 (2), 227-233 (1998) (herein incorporated by reference
in their entirety). Non-limiting examples of sequences that may be
used to construct a soluble NiV G glycoprotein can be found in
Harcourt, B H et al., Virology 271: 334-349, 2000 and Chua, K. B.
et al, Science 288 (5470), 1432-1 (herein incorporated by reference
in their entirety). Generally, G glycoprotein sequences from any
Hendra virus and Nipah virus isolate or strain may be utilized to
derive the polynucleotides and polypeptides of the invention.
[0057] By way of example, and not limitation, a polynucleotide
encoding a soluble HeV G Glycoprotein may comprise a polynucleotide
sequence encoding about amino acids 71-604 of the amino acid
sequence for an HeV G Glycoprotein in Wang, L. F. et al., J. Virol.
74 (21), 9972-9979 (2000) (SEQ ID NO: 16) (see also, e.g., Yu, M.
et al., Virology 251 (2), 227-233 (1998)). Also by way of example,
and not limitation, a polynucleotide encoding a soluble HeV G
glycoprotein may comprise nucleotides 9048 to 10727 of the
polynucleotide sequence for an HeV G glycoprotein in Wang, L. F. et
al., J. Virol. 74 (21), 9972-9979 (2000) (see also, e.g., Yu, M. et
al., Virology 251 (2), 227-233 (1998)).
[0058] By way of example, and not limitation, a polynucleotide
encoding a soluble NiV G glycoprotein may comprise a polynucleotide
sequence encoding about amino acids 71-602 of the amino acid
sequence for an NiV G Glycoprotein in Harcourt, B H et al.,
Virology 271: 334-349, 2000 (SEQ ID NO: 17) (see also Chua, K. B.
et al., Science 288 (5470), 1432-1). Also by way of example, and
not limitation, a polynucleotide encoding a soluble NiV G
glycoprotein may comprise nucleotides 9026 to 10696 of the
polynucleotide sequence for an HeV G glycoprotein in Harcourt, B H
et al., Virology 271: 334-349, 2000 (see also Chua, K. B. et al.,
Science 288 (5470), 1432-1).
[0059] Functional equivalents of these polynucleotides are also
intended to be encompassed by this invention. By way of example and
not limitation functionally equivalent polynucleotides encode a
soluble G glycoprotein of a HeV or NiV and possess one or more of
the following characteristics: ability to interact or bind the
viral host cell receptor, can be produced in oligomeric form or
forms, the ability to elicit antibodies (including, but not limited
to, viral neutralizing antibodies) capable of recognizing native G
glycoprotein and/or the ability to block or prevent infection of a
host cell.
[0060] Polynucleotide sequences that are functionally equivalent
may also be identified by methods known in the art. A variety of
sequence alignment software programs are available in the art to
facilitate determination of homology or equivalence. Non-limiting
examples of these programs are BLAST family programs including
BLASTN, BLASTP, BLASTX, TBLASTN, and TBLASTX (BLAST is available
from the worldwide web at ncbi.nlm.nih.gov/BLAST/), FastA, Compare,
DotPlot, BestFit, GAP, FrameAlign, ClustalW, and PileUp. These
programs are obtained commercially available in a comprehensive
package of sequence analysis software such as GCG Inc.'s Wisconsin
Package. Other similar analysis and alignment programs can be
purchased from various providers such as DNA Star's MegAlign, or
the alignment programs in GeneJockey. Alternatively, sequence
analysis and alignment programs can be accessed through the world
wide web at sites such as the CMS Molecular Biology Resource at
sdsc.edu/ResTools/cmshp.html. Any sequence database that contains
DNA or protein sequences corresponding to a gene or a segment
thereof can be used for sequence analysis. Commonly employed
databases include but are not limited to GenBank, EMBL, DDBJ, PDB,
SWISS-PROT, EST, STS, GSS, and HTGS.
[0061] Parameters for determining the extent of homology set forth
by one or more of the aforementioned alignment programs are well
established in the art. They include but are not limited top value,
percent sequence identity and the percent sequence similarity. P
value is the probability that the alignment is produced by chance.
For a single alignment, the p value can be calculated according to
Karlin et al. (1990) Proc. Natl. Acad. Sci. (USA) 87: 2246. For
multiple alignments, the p value can be calculated using a
heuristic approach such as the one programmed in BLAST. Percent
sequence identify is defined by the ratio of the number of
nucleotide or amino acid matches between the query sequence and the
known sequence when the two are optimally aligned. The percent
sequence similarity is calculated in the same way as percent
identity except one scores amino acids that are different but
similar as positive when calculating the percent similarity. Thus,
conservative changes that occur frequently without altering
function, such as a change from one basic amino acid to another or
a change from one hydrophobic amino acid to another are scored as
if they were identical.
[0062] Polypeptides
[0063] Another aspect of this invention is directed to soluble G
glycoprotein polypeptides of HeV or NiV. The term polypeptide is
used broadly herein to include peptide or protein or fragments
thereof. By way of example, and not limitation, a soluble HeV G
glycoprotein may comprise amino acids 71-604 of the amino acid
sequence for a HeV G glycoprotein in Wang, L. F. et al., J. Virol.
74 (21), 9972-9979 (2000) (see also, e.g., Yu, M. et al., Virology
251 (2), 227-233 (1998)). Also by way of example and not
limitation, a soluble NiV G glycoprotein may comprise amino acids
71-602 of the amino acid sequence for a NiV G glycoprotein in
Harcourt, B H et al., Virology 271: 334-349, 2000 (see also Chua,
K. B. et al., Science 288 (5470), 1432-1).
[0064] Functional equivalents of these polypeptides are also
intended to be encompassed by this invention. By way of example and
not limitation functionally equivalent polypeptides possess one or
more of the following characteristics: ability to interact or bind
the viral host cell receptor, can be produced in oligomeric form or
forms, the ability to elicit antibodies (including, but not limited
to, viral neutralizing antibodies) capable of recognizing native G
Glycoprotein and/or the ability to block or prevent infection of a
host cell.
[0065] Also intended to be encompassed are peptidomimetics, which
include chemically modified peptides, peptide-like molecules
containing non-naturally occurring amino acids, peptides and the
like, and retain the characteristics of the soluble G glycoprotein
polypeptides provided herein. ("Burger's Medicinal Chemistry and
Drug Discovery" 5th ed., vols. 1 to 3 (ed. M. E. Wolff; Wiley
Interscience 1995).
[0066] This invention further includes polypeptides or analogs
thereof having substantially the same function as the polypeptides
of this invention. Such polypeptides include, but are not limited
to, a substitution, addition or deletion mutant of the polypeptide.
This invention also encompasses proteins or peptides that are
substantially homologous to the polypeptides. A variety of sequence
alignment software programs described herein above are available in
the art to facilitate determination of homology or equivalence of
any protein to a protein of the invention.
[0067] The term "analog" includes any polypeptide having an amino
acid residue sequence substantially identical to a polypeptide of
the invention in which one or more residues have been
conservatively substituted with a functionally similar residue and
which displays the functional aspects of the polypeptides as
described herein. Examples of conservative substitutions include
the substitution of one non-polar (hydrophobic) residue such as
isoleucine, valine, leucine or methionine for another, the
substitution of one polar (hydrophilic) residue for another such as
between arginine and lysine, between glutamine and asparagine,
between glycine and serine, the substitution of one basic residue
such as lysine, arginine or histidine for another, or the
substitution of one acidic residue, such as aspartic acid or
glutamic acid or another.
[0068] The phrase "conservative substitution" also includes the use
of a chemically derivatized residue in place of a non-derivatized
residue. "Chemical derivative" refers to a subject polypeptide
having one or more residues chemically derivatized by reaction of a
functional side group. Examples of such derivatized molecules
include for example, those molecules in which free amino groups
have been derivatized to form amine hydrochlorides, p-toluene
sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or formyl groups. Free carboxyl groups may be
derivatized to form salts, methyl and ethyl esters or other types
of esters or hydrazides. Free hydroxyl groups may be derivatized to
form O-acyl or 0-alkyl derivatives. The imidazole nitrogen of
histidine may be derivatized to form N-im-benzylhistidine. Also
included as chemical derivatives are those proteins or peptides
which contain one or more naturally-occurring amino acid
derivatives of the twenty standard amino acids. For examples:
4-hydroxyproline may be substituted for proline; 5-hydroxylysine
may be substituted for lysine; 3-methylhistidine may be substituted
for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine. Polypeptides of the
present invention also include any polypeptide having one or more
additions and/or deletions or residues relative to the sequence of
a any one of the polypeptides whose sequences is described
herein.
[0069] Two polynucleotide or polypeptide sequences are said to be
"identical" if the sequence of nucleotides or amino acids in the
two sequences is the same when aligned for maximum correspondence
as described below. Comparisons between two sequences are typically
performed by comparing the sequences over a comparison window to
identify and compare local regions of sequence similarity. A
"comparison window" as used herein, refers to a segment of at least
about 20 contiguous positions, usually 30 to about 75, 40 to about
50, in which a sequence may be compared to a reference sequence of
the same number of contiguous positions after the two sequences are
optimally aligned.
[0070] Optimal alignment of sequences for comparison may be
conducted using the Megalign program in the Lasergene suite of
bioinformatics software (DNASTAR, Inc., Madison, Wis.), using
default parameters. This program embodies several alignment schemes
described in the following references: Dayhoff, M. O. (1978) A
model of evolutionary change in proteins-Matrices for detecting
distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein
Sequence and Structure, National Biomedical Research Foundation,
Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990,
Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in
Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;
Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E.
W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971,
Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol.
4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical
Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman
Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J.,
1983, Proc. Natl. Acad. Sci. USA 80:726-730.
[0071] Preferably, the "percentage of sequence identity" is
determined by comparing two optimally aligned sequences over a
window of comparison of at least 20 positions, wherein the portion
of the polypeptide sequence in the comparison window may comprise
additions or deletions (i.e. gaps) of 20 percent or less, usually 5
to 15 percent, or 10 to 12 percent, as compared to the reference
sequences (which does not comprise additions or deletions) for
optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical r amino acid residue occurs in both sequences to yield
the number of matched positions, dividing the number of matched
positions by the total number of positions in the reference
sequence (i.e. the window size) and multiplying the results by 100
to yield the percentage of sequence identity.
[0072] Expression Vectors
[0073] This invention also relates to expression vectors comprising
at least one polynucleotide encoding a soluble G glycoprotein
protein of the invention. Expression vectors are well known in the
art and include, but are not limited to viral vectors or plasmids.
Viral-based vectors for delivery of a desired polynucleotide and
expression in a desired cell are well known in the art. Exemplary
viral-based vehicles include, but are not limited to, recombinant
retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO
94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO
91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127), alphavirus-based
vectors (e.g., Sindbis virus vectors, Semliki forest virus), Ross
River virus, adeno-associated virus (AAV) vectors (see, e.g., PCT
Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO
94/28938; WO 95/11984 and WO 95/00655), vaccinia virus (e.g.,
Modified Vaccinia virus Ankara (MVA) or fowlpox), Baculovirus
recombinant system and herpes virus.
[0074] Nonviral vectors, such as plasmids, are also well known in
the art and include, but are not limited to, yeast and bacterial
based plasmids.
[0075] Methods of introducing the vectors into a host cell and
isolating and purifying the expressed protein are also well known
in the art (e.g., Molecular Cloning: A Laboratory Manual, second
edition (Sambrook, et al., 1989) Cold Spring Harbor Press).
Examples of host cells include, but are not limited to, mammalian
cells, such as HeLa and CHO cells.
[0076] By way of example the vector comprising the polynucleotide
encoding the soluble G protein may further comprise a tag
polynucleotide sequence to facilitate isolation and/or
purification. Examples of tags include but are not limited to,
myc-eptiope, S-tag, his-tag, HSV-epitope, V5-epitope, FLAG and CBP.
Such tags are commercially available or readily made by methods
known to the art.
[0077] The vector may further comprise a polynucleotide sequence
encoding a linker sequence. Generally the linking sequence is
positioned in the vector between the soluble G protein
polynucleotide sequence and the polynucleotide tag sequence.
Linking sequences can encode random amino acids or can contain
functional sites. Examples of linking sequences containing
functional sites include but are not limited to, sequences
containing the thrombin cleavage site or the enterokinase cleavage
site.
[0078] By way of example, and not limitation, a soluble G
glycoprotein may be generated as described herein using vaccinia
virus recombinants in a mammalian cell culture system. Examples of
primers that may be used to amplify the desired ectodomain sequence
from a Hendra virus or Nipah virus cDNA template, include, but are
not limited to, the primers in the Examples.
[0079] Antibodies
[0080] Examples of antibodies encompassed by the present invention,
include, but are not limited to, antibodies specific for HeV G
glycoprotein, antibodies specific for NiV G glycoprotein,
antibodies that cross react with HeV G glycoprotein and NiV G
Glycoprotein and neutralizing antibodies. By way of example a
characteristic of a neutralizing antibody includes, but is not
limited to, the ability to block or prevent infection of a host
cell. The antibodies of the invention may be characterized using
methods well known in the art.
[0081] The antibodies useful in the present invention can encompass
monoclonal antibodies, polyclonal antibodies, antibody fragments
(e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies,
bispecific antibodies, heteroconjugate antibodies, single chain
(ScFv), mutants thereof, fusion proteins comprising an antibody
portion, humanized antibodies, and any other modified configuration
of the immunoglobulin molecule that comprises an antigen
recognition site of the required specificity, including
glycosylation variants of antibodies, amino acid sequence variants
of antibodies, and covalently modified antibodies. Preferred
antibodies are derived from murine, rat, human, primate, or any
other origin (including chimeric or humanized antibodies).
[0082] Methods of preparing monoclonal and polyclonal antibodies
are well know in the art. Polyclonal antibodies can be raised in a
mammal, for example, by one or more injections of an immunizing
agent and, if desired an adjuvant. Examples of adjuvants include,
but are not limited to, keyhole limpet, hemocyanin, serum albumin,
bovine thryoglobulin, soybean trypsin inhibitor, Freund complete
adjuvant and MPL-TDM adjuvant. The immunization protocol can be
determined by one of skill in the art.
[0083] The antibodies may alternatively be monoclonal antibodies.
Monoclonal antibodies may be produced using hybridoma methods (see,
e.g., Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as
modified by Buck, D. W., et al., In Vitro, 18:377-381 (1982).
[0084] If desired, the antibody of interest may be sequenced and
the polynucleotide sequence may then be cloned into a vector for
expression or propagation. The sequence encoding the antibody of
interest may be maintained in vector in a host cell and the host
cell can then be expanded and frozen for future use. In an
alternative, the polynucleotide sequence may be used for genetic
manipulation to "humanize" the antibody or to improve the affinity,
or other characteristics of the antibody (e.g., genetically
manipulate the antibody sequence to obtain greater affinity to the
G glycoprotein and/or greater efficacy in inhibiting the fusion of
the Hendra or Nipah virus to the host cell receptor.).
[0085] The antibodies may also be humanized by methods known in the
art. (See, for example, U.S. Pat. Nos. 4,816,567; 5,807,715;
5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089;
and 6,180,370). In yet another alternative, fully human antibodies
may be obtained by using commercially available mice that have been
engineered to express specific human immunoglobulin proteins.
[0086] In another alternative, antibodies may be made recombinantly
and expressed using any method known in the art. By way of example,
antibodies may be made recombinantly by phage display technology.
See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743;
and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455
(1994). Alternatively, the phage display technology (McCafferty et
al., Nature 348:552-553 (1990)) can be used to produce human
antibodies and antibody fragments in vitro. Phage display can be
performed in a variety of formats; for review see, e.g., Johnson,
Kevin S. and Chiswell, David J., Current Opinion in Structural
Biology 3:564-571 (1993). By way of example, a soluble G
glycoprotein as described herein may be used as an antigen for the
purposes of isolating recombinant antibodies by these
techniques.
[0087] Antibodies may be made recombinantly by first isolating the
antibodies and antibody producing cells from host animals,
obtaining the gene sequence, and using the gene sequence to express
the antibody recombinantly in host cells (e.g., CHO cells). Another
method which may be employed is to express the antibody sequence in
plants (e.g., tobacco) or transgenic milk. Methods for expressing
antibodies recombinantly in plants or milk have been disclosed.
See, for example, Peeters, et al. Vaccine 19:2756 (2001); Lonberg,
N. and D. Huszar Int. Rev. Immunol 13:65 (1995); and Pollock, et
al., J Immunol Methods 231:147 (1999). Methods for making
derivatives of antibodies, e.g., humanized, single chain, etc. are
known in the art.
[0088] The antibodies of the invention can be bound to a carrier by
conventional methods, for use in, for example, isolating or
purifying Hendra or Nipah G glycoproteins or detecting Hendra or
Nipah G glycoproteins in a biological sample or specimen.
Alternatively, by way of example, the neutralizing antibodies of
the invention may be administered as passive immunotherapy to a
subject infected with or suspected of being infected with Hendra or
Nipah virus. A "subject," includes but is not limited to humans,
simians, farm animals, sport animals and pets. Veterinary uses are
also encompassed by the invention.
[0089] Diagnostics
[0090] The soluble G glycoproteins and/or antibodies of the
invention may be used in a variety of immunoassays for Hendra and
Nipah virus. The recombinant expressed soluble G glycoproteins of
the invention can be produced with high quality control and are
suitable as a antigen for the purposes of detecting antibody in
biological samples. By way of example, and not limitation, a
soluble HEV or NiV G glycoprotein or combinations thereof could be
used as antigens in an ELISA assay to detect antibody in a
biological sample from a subject.
[0091] Vaccines
[0092] This invention also relates to vaccines for Hendra and Nipah
virus. In one aspect the vaccines are DNA based vaccines. One
skilled in the art is familiar with administration of expression
vectors to obtain expression of an exogenous protein in vivo. See,
e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471.
Viral-based vectors for delivery of a desired polynucleotide and
expression in a desired cell are well known in the art and
non-limiting examples are described herein. In another aspect, the
vaccines are protein-based and comprises one or more fragments of
the G protein of Hendra or Nipah virus. Preferred fragments are the
ectodomain, and functional portions thereof, and also, portions
that are specifically reactive to neutralizing antibodies. Portions
that are so reactive are depicted in FIG. 14. Vaccines may also be
antibody-based vaccines for more immediate treatment as well as
prophylaxis against infection.
[0093] Administration of expression vectors includes local or
systemic administration, including injection, oral administration,
particle gun or catheterized administration, and topical
administration. Targeted delivery of therapeutic compositions
containing an expression vector, or subgenomic polynucleotides can
also be used. Receptor-mediated DNA delivery techniques are
described in, for example, Findeis et al., Trends Biotechnol.
(1993) 11:202; Chiou et al., Gene Therapeutics: Methods And
Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu
et al., J . Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem.
(1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990)
87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.
[0094] Non-viral delivery vehicles and methods can also be
employed, including, but not limited to, polycationic condensed DNA
linked or unlinked to killed adenovirus alone (see, e.g., Curiel,
Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J.
Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles
cells (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO
95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic
charge neutralization or fusion with cell membranes. Naked DNA can
also be employed. Exemplary naked DNA introduction methods are
described in PCT Publication No. WO 90/11092 and U.S. Pat. No.
5,580,859. Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO
95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional
approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411,
and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.
[0095] For human administration, the codons comprising the
polynucleotide encoding a soluble G glycoprotein may be optimized
for human use.
[0096] In another aspect of the invention, a soluble HeV or NiV G
glycoprotein or combinations thereof are used as a subunit vaccine.
The soluble HeV or NiV G glycoprotein or combination thereof may be
administered by itself or in combination with an adjuvant. Examples
of adjuvants include, but are not limited, aluminum salts,
water-in-soil emulsions, oil-in-water emulsions, saponin, QuilA and
derivatives, iscoms, liposomes, cytokines including gamma
interferon or interleukin 12, DNA, microencapsulation in a solid or
semi-solid particle, Freunds complete and incomplete adjuvant or
active ingredients thereof including muramyl dipeptide and
analogues, DEAE dextran/mineral oil, Alhydrogel, Auspharm adjuvant,
and Algammulin.
[0097] The subunit vaccine comprising soluble HeV or NiV G
glycoprotein or combinations thereof can be administered orally,
intravenously, subcutaneously, intraarterially, intramuscularly,
intracardially, intraspinally, intrathoracically,
intraperitoneally, intraventricularly, sublingually, and/or
transdermally.
[0098] Dosage and schedule of administration can be determined by
methods known in the art. Efficacy of the soluble HeV or NiV G
glycoprotein or combinations thereof as a vaccine for Hendra, Nipah
or related Henipavirus viruses may also be evaluated by methods
known in the art.
[0099] Pharmaceutical Compositions
[0100] The polynucleotides, polypetides and antibodies of the
invention can further comprise pharmaceutically acceptable
carriers, excipients, or stabilizers known in the art (Remington:
The Science and practice of Pharmacy 20th Ed. (2000) Lippincott
Williams and Wilkins, Ed. K. E. Hoover.), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations, and may comprise buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrans; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
Pharmaceutically acceptable excipients are further described
herein.
[0101] The compositions used in the methods of the invention
generally comprise, by way of example and not limitation, and
effective amount of a polynucleotide or polypeptide (e.g., an
amount sufficient to induce an immune response) of the invention or
antibody of the invention (e.g., an amount of a neutralizing
antibody sufficient to mitigate infection, alleviate a symptom of
infection and/or prevent infection).
[0102] The pharmaceutical composition of the present invention can
further comprise additional agents that serve to enhance and/or
complement the desired effect. By way of example, to enhance the
immunogenicity of a soluble G polypeptide of the invention being
administered as a subunit vaccine, the pharmaceutical composition
may further comprise an adjuvant. Examples of adjuvants are
provided herein.
[0103] Also by way of example, an not limitation, if a soluble G
protein polypeptide of the invention is being administered to
augment the immune response in a subject infected with or suspected
of being infected with Hendra or Nipah and/or if antibodies of the
present invention are being administered as a form of passive
immunotherapy the composition can further comprise, for example,
other therapeutic agents (e.g., anti-viral agents)
[0104] Diagnostic Kits
[0105] The invention also provides diagnostic kits for use in the
instant methods. Kits of the invention include one or more
containers comprising by way of example, and not limitation,
polynucleotides encoding a soluble G HeV or NiV G glycoprotein or
combinations thereof, a soluble G HeV or NiV G glycoprotein or
combinations thereof and/or antibodies of the invention and
instructions for use in accordance with any of the methods of the
invention described herein.
[0106] Generally, these instructions comprise a description of
administration or instructions for performance of an assay. The
containers may be unit doses, bulk packages (e.g., multi-dose
packages) or sub-unit doses. Instructions supplied in the kits of
the invention are typically written instructions on a label or
package insert (e.g., a paper sheet included in the kit), but
machine-readable instructions (e.g., instructions carried on a
magnetic or optical storage disk) are also acceptable.
[0107] The kits of this invention are in suitable packaging.
Suitable packaging includes, but is not limited to, vials, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. Also contemplated are packages for use in combination
with a specific device, such as an inhaler, nasal administration
device (e.g., an atomizer) or an infusion device such as a
minipump. A kit may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). The container
may also have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). Kits may optionally
provide additional components such as buffers and interpretive
information. Normally, the kit comprises a container and a label or
package insert(s) on or associated with the container.
[0108] The following examples illustrate only certain and not all
embodiments of the invention, and thus, should not be viewed as
limiting the scope of the invention.
EXAMPLES
Example 1
Vector Constructs
[0109] Vectors were constructed to express
transmembrane/cytoplasmic tail-deleted HeV G or NiV G. The cloned
cDNAs of full-length HeV or NiV G protein were amplified by PCR to
generate fragments about 2600 bp encoding the transmembrane
domain/cytoplasmic tail-deleted HeVG or NiV G protein.
[0110] The following oligonucleotide primers were synthesized for
amplification of HeV G. sHGS:
5'-GTCGACCACCATGCAAAATTACACCAGAACGACTGATAAT-3' (SEQ ID NO 1).
sHGAS: 5'-GTTTAAACGTCGACCAATCAACTCTCTGAACATTG GGCAGGTATC-3'. (SEQ
ID NO 2).
[0111] The following oligonucleotide primers were synthesized for
amplification of NiV G. sNGS:
5'-CTCGAGCACCATGCAAAATTACACAAGATCAACAGACAA-3' (SEQ ID NO 3). sNGAS:
5'-CTCGAGTAGCAGCCGGATCAAGCTTATGTACATT GCTCTGGTATC-3'. (SEQ ID NO
4).
All PCR reactions were done using Accupol DNA polymerase (PGS
Scientifics Corp., Gaithersburg, Md.) with the following settings:
94.degree. C. for 5 min initially and then 94.degree. C. for 1
minute, 56.degree. C. for 2 minutes, 72.degree. C. for 4 minutes;
25 cycles. These primers generated a PCR product for the sHeV G ORF
flanked by Sal 1 sites and the sNiV G ORF flanked by Xho 1 sites.
PCR products were gel purified (Qiagen, Valencia, Calif). After gel
purification, sHeV G and sNiV G were subcloned into a TOPO vector
(Invitrogen Corp., Carlsbad, Calif).
[0112] PSectag2B (Invitrogen Corp.) was purchased and modified to
contain a S-peptide tag or a myc-epitope tag. Overlapping
oligonucleotides were synthesized that encoded the sequence for the
S-peptide and digested Kpn 1 and EcoR1 overhangs. SPEPS:
5'-CAAGGAGACCGCTGCTGCTAAGTTCGAACGCCAGCACATGGATT CT-3' (SEQ ID NO
5). SPEPAS: 5' AATTAGAATCCATGTGCTGGCGTTCGAACTTAGCAGCAGCGGTCT
CCTTGGTAC-3' (SEQ ID NO 6).
[0113] Overlapping oligonucleotides were synthesized that encoded
the sequence for the myc-epitope tag and digested Kpn 1 and EcoR1
overhangs. MTS: 5'-CGAACAAAAGCTCATCTCAGAAGAGGATCTG-3' (SEQ ID NO
7). MTAS 5'-AATTCAGATCCTCTTCTGAGATGAGCTTTTGTTCGGTAC-3' (SEQ ID NO
8).
[0114] 64 pmol SPEPS and 64 pmol SPEPAS were mixed and heated to
65.degree. C. for 5 minutes and cooled slowly to 50.degree. C. 64
pmol MTS and 64 pmol MTAS were mixed and heated to 65.degree. C.
for 5 minutes and cooled slowly to 50.degree. C. The two mixtures
were diluted and cloned into Kpn1-EcoR1 digested pSecTag2B to
generate S-peptide modified pSecTag2B or myc-epitope modified
pSecTag2B. All constructs were initially screened by restriction
digest and further verified by sequencing.
[0115] The TOPO sG construct was digested with Sal 1 gel purified
(Qiagen) and subcloned in frame into the Xho 1 site of the
S-peptide modified pSecTag2B or myc-epitope modified pSecTag2B. All
constructs were initially screened by restriction digest and
further verified by sequencing.
[0116] The Ig.kappa. leader-5-peptide-s HeVG (.sub.SG.sub.S-tag)
and the Ig.kappa. leader-myc tag-sHeVG (sG.sub.myc-tag) constructs
were then subcloned into the vaccinia shuttle vector pMCO2
[Carroll, 1995]. Oligonucleotide SEQS:
5'-TCGACCCACCATGGAGACAGACACACTCCTGCTA-3' (SEQ ID NO 9) was
synthesized and used in combination with oligonucleotide sHGAS to
amplify by PCR the .sub.SG.sub.S-tag and .sub.SG.sub.myc-tag. All
PCR reactions were done using Accupol DNA polymerase (PGS
Scientifics Corp.) with the following settings: 94.degree. C. for 5
min initially and then 94.degree. C. for 1 minute, 56.degree. C.
for 2 minutes, 72.degree. C. for 4 minutes; 25 cycles. These
primers generated PCR products flanked by Sal 1 sites. PCR products
were gel purified (Qiagen). After gel purification,
.sub.SG.sub.S-tag and .sub.SG.sub.myc-tag were subcloned into a
TOPO vector (Invitrogen Corp.). sG S-tag and sG myc-tag were
digested with Sal 1 and subcloned into the Sal 1 site of pMCO2. All
constructs were initially screened by restriction digest and
further verified by sequencing. The polypeptide structures of HeV
sG S-tag and HeV sG myc-tag are depicted in a representative
drawing in FIG. 6.
Example 2
Protein Production of Soluble G Protein
[0117] For protein production the genetic constructs were used to
generate recombinant poxvirus vectors (vaccinia virus, strain WR).
Recombinant poxvirus was then obtained using standard techniques
employing tk-selection and GUS staining (6). Briefly, CV-1 cells
were transfected with either pMCO2 sHeV G fusion or pMCO2 sNiV G
fusion using a calcium phosphate transfection kit (Promega, Corp.,
Madison, Wis.). These monolayers were then infected with Western
Reserve (WR) wild-type strain of vaccinia virus at a multiplicity
of infection (MOI) of 0.05 PFU/cell. After 2 days the cell pellets
were collected as crude recombinant virus stocks. TK.sup.- cells
were infected with the recombinant crude stocks in the presence of
25 .mu.g/ml 5-Bromo-2'-deoxyuridine (BrdU) (Calbiochem, La Jolla,
Calif.). After 2 hours the virus was replaced with an EMEM-10
overlay containing 1% low melting point (LMP) agarose (Life
Technologies, Gaithersburg, Md.) and 25 .mu.g/ml BrdU. After 2 days
of incubation an additional EMEM-10 overlay containing 1% LMP
agarose, 25 .mu.g/ml BrdU, and 0.2 mg/ml
5-Bromo-4-chloro-3-indolyl-.beta.-D-glucuronic acid (X-GLUC)
(Clontech, Palo Alto, Calif.) was added. Within 24-48 hours blue
plaques were evident, picked and subject to two more rounds of
double selection plaque purification. The recombinant vaccinia
viruses vKB16 (sHeV G fusion) and vKB22 (sNiV G fusion) were then
amplified and purified by standard methods. Briefly, recombinant
vaccinia viruses are purified by plaque purification, cell-culture
amplification, sucrose cushion pelleting in an ultracentrifuge and
titration by plaque assay. Expression of sHeV G was verified in
cell lysates and culture supernatants (FIG. 1).
[0118] As shown in FIG. 1, vaccinia virus encoding either a myc-tag
or S-tag soluble HeV G was produced by metabolic labeling in HeLa
cells. Control is wild-type HeV G. Specific precipitation of each
sG construct is shown by precipitation from either lysates or
supernatants using myc MAb or S-beads.
Example 3
Properties of Soluble G Protein
[0119] To demonstrate that the recombinant expressed, soluble,
purified G (sHeV G) retained desirable properties (e.g. native
structural features such as receptor binding competence), it has
been demonstrated that pre-incubation of target cells with
affinity-purified sHeV G results in a dose-dependent inhibition of
virus-mediated fusion in several different cell lines that are
susceptible to virus-mediated fusion and infection (FIGS.
2A-2F).
[0120] For purification of soluble G glycoproteins, HeLa cells were
infected with vKB15 or vKB16 (moi=3) for 2 hours. After infection
the virus was removed and serum-free OptiMem medium (Invitrogen,
Corp.) was added. After 36 hours, the supernatants were removed and
clarified by centrifugation. A S-protein column was poured with 15
ml of S-protein agarose (Novagen) in a XK26 column (Amersham
Pharmacia Biotech, Piscataway, N.J.). The S-protein column was
washed with 10 bed volumes of PBS. The supernatant from
vKB16-infected cells was passed over S-protein agarose column, the
column was washed with 10 bed volumes of PBS, and the
.sub.SG.sub.S-tag was eluted with 1 bed volume of 0.2M citrate pH=2
into 20 ml 1M Tris pH=8. Lentil lectin Sepharose B was purchased
(Amersham Pharmacia Biotech) and a 25 ml XK26 column was poured.
The supernatants from vKB15-infected cells were passed over the
lentil lectin column, the column was washed with 10 bed volumes
PBS, and the .sub.SG.sub.myc-tag was eluted with 1 bed volume of
0.2M glycine pH=2.5 into 2 ml 1M Tris pH=8. Both eluates were then
concentrated using 30 kDA Centricon centrifugal filter units
(Millipore, Billerica, Mass.) and filter sterilized. Protein
concentrations were calculated using SDS/PAGE, Commassie brilliant
blue R-250 staining and densitometry analysis with NIH image 1.62
software.
[0121] As shown in FIGS. 2A-2F, soluble HeV G envelope glycoprotein
(either the S-tag or myc-tag versions) blocks both HeV and
NiV-mediated cell-cell fusion. Dose response inhibition of HeV and
NiV cell-cell fusion was conducted by pre-incubating target cells
with the indicated amount of purified sG for 30 min at room
temperature. Effector cells expressing either HeV or NiV F and G
were added and fusion was allowed to proceed for 2.5 h at
37.degree. C. Reaction mixes were processed for .beta.-gal
production using the .beta.-gal reporter gene assay. Assays were
carried out in duplicate. Panels A and B show that crude sHeV G
(S-tag) containing supernatant can potently block HeV-mediated
fusion in two alternant cell types, while a control supernatant
from WR infected cells (non-recombinant vaccinia virus infected)
has no effect. Panels C and D: Inhibition of HeV and NiV-mediated
fusion by sG S-tag in U373 cells (Panel C) or PCI 13 cells (Panel
D). Panels E and F: Inhibition of HeV- and NiV-mediated fusion by
purified sG myc-tag in U373 cells (Panel E) or PCI 31 cells (Panel
F).
[0122] As additional evidence, indirect immunofluorescence was
performed that demonstrated sHeV G can specifically bind to cell
lines that are susceptible to virus-mediated fusion and infection
(FIGS. 3A and 3B). sHeV G is unable to bind to HeLa cells, a
non-permissive cell line for HeV and NiV-mediated fusion and virus
infection. These data suggest that sHeV G inhibits HeV-mediated
fusion by binding to the putative receptor on target cells thus
blocking subsequent attachment and fusion of HeV G and F expressing
effector cells. The interaction of sHeV G with the putative HeV
receptor may be a useful tool for receptor purification and
identification. Since the soluble G glycoprotein can be expressed
and purified and also exhibits biochemical features similar to that
what would be expected from the native G glycoprotein making it an
ideal subunit immunogen for the elicitation of virus-neutralizing
antibodies. A similar sG construct has been made using the same
S-tag approach (see methods above) using the coding sequence of the
G envelope glycoprotein of Nipah virus. This sNiV G (S-tag) has
been cloned and expressed and is shown in FIG. 4.
[0123] A final analysis of the sHeV G (S-tag) envelope glycoprotein
was made to evaluate the predicted oligomeric nature of the
protein. The retention of some oligomeric properties of a soluble
and secreted G glycoprotein may be important in retaining critical
immunological or biochemical features as discussed in the
introduction above. FIGS. 5A-5D show an analysis of secreted sHeV G
(S-tag) glycoprotein by sucrose gradient fractionation which
identifies monomeric, dimeric and tetrameric forms of the G
glycoprotein. Both cross-linked and non-cross-linked materials were
analyzed. The results indicate that monomeric, dimeric, and some
tetrameric sG comprises the sG preparation, and this is in
agreement with findings on soluble and full-length versions of
other paramyxovirus H and HN attachment glycoproteins (discussed
above). These three species could be separated by preparative size
exclusion chromatography techniques if desired.
Example 4
Characterization of Soluble and Secreted HeV G
[0124] It was next sought to determine if the secreted sG was
oligomeric in nature. The apparent molecular weight of purified sG
material was first examined using size exclusion chromatography
with a calibrated Superdex 200 analytical grade column 10/300. A
500 .mu.g aliquot of either .sub.SG.sub.S-tag or
.sub.SG.sub.myc-tag was passed over the Superdex 200 and fractions
were collected using the same methods employed for the high
molecular weight standards. Essentially identical results were
observed with both the .sub.SG.sub.S-tag and .sub.SG.sub.myc-tag
glycoproteins, and the results shown in FIG. 7 are those for
.sub.SG.sub.S-tag. The locations of the protein standards and the
three major species of sG are indicated in the figure. The inset
shows the profile of separated sG. The analysis of purified
.sub.SG.sub.S-tag in seven independent separation experiments
indicated two major peaks with apparent molecular weights of
.about.372 KDa+/-19 KDa (.about.60% of the material) and .about.261
KDa+/-47 KDa (.about.35% of the material), and one minor peak of
.about.741 KDa+/-40 KDa (.about.5% of the material). These results
indicated that at least some of the material would be oligomeric in
nature, consistent with the glycoprotein's expected structure.
However, from prior experience in the preparation and analysis of
soluble virus-derived membrane glycoproteins, such as gp120 from
HIV-1, such molecular weight calculations derived from
size-exclusion chromatography analysis may be over-estimated.
[0125] To further characterize the apparent oligomeric species of
sG, .sub.SG.sub.S-tag was analyzed using sucrose gradient
densitometry. For this analysis, the .sub.SG.sub.S-tag glycoprotein
was chosen because it can be affinity-precipitated with S-protein
agarose circumventing the need for specific MAb. FIGS. 8A-8D depict
the oligomeric profiles of metabolically labeled .sub.SG.sub.S-tag
isolated from the supernatant of expressing cells. Following brief
centrifugation to remove any cellular debris the supernatant was
concentrated and buffer replaced with PBS as described herein.
Prior to separation in the sucrose gradient, half of the
supernatant was cross-linked with DTSSP, a reducible cross-linker,
and uncross-linked and cross-linked material were loaded onto two
separate sucrose gradients. After fractionation of each gradient,
the fractions were split into 2 tubes, precipitated with S-protein
agarose, washed and resuspended in SDS sample buffer, one set with
and one set without .beta.-mercaptoethanol and all four sets of
fractions then analyzed by SDS-PAGE. FIGS. 8A and 8B are
uncross-linked .sub.SG.sub.S-tag separated on the sucrose gradient,
fractionated, and immunoprecipitated. In FIG. 8A, the fractions
were resolved on SDS-PAGE in the absence of .beta.-mercaptoethanol,
whereas in FIG. 8B, the fractions were resolved on SDS-PAGE in the
presence of .beta.-mercaptoethanol. FIGS. 8C and 8D are
cross-linked .sub.SG.sub.S-tag separated on the sucrose gradient,
fractionated, and immunoprecipitated. In FIG. 8C, the fractions
were resolved on SDS-PAGE in the absence of .beta.-mercaptoethanol
whereas in FIG. 8D, the fractions were resolved on SDS-PAGE in the
presence of .beta.-mercaptoethanol. The starting material for each
gradient was also run on each gel in the presence or absence
.beta.-mercaptoethanol and is illustrated as control. From the data
shown in FIGS. 8A and 8C, it was determined that for both the
uncross-linked and cross-linked .sub.SG.sub.S-tag, there are three
distinct species of sG present. Based on the apparent molecular
weights of sG in each of the fractions across each of the
gradients, these three species likely represent monomer, dimer and
tetramer. In addition, the immediate cross-linking of the sG with
DTSSP prior to gradient centrifugation did not significantly
increase the amount of either the tetrameric or dimeric species.
The analysis of the non-cross-linked, un-reduced and reduced sG
clearly indicates that the dimeric oligomer is disulfide linked,
which was anticipated based on data derived from other
paramyxovirus attachment glycoproteins (36, 44). The dimers of
other paramyxovirus attachment glycoproteins have been shown to
form a tetramer on the surface of infected cells, and it is
generally believed that the native oligomeric structure is a dimer
of dimers. To analyze this possibility here, full-length HeV G was
expressed and metabolically labeled in HeLa cells (a
receptor-negative cell line) and performed a similar experiment and
sucrose gradient analysis. Following either a cross-linking
procedure, or no treatment, of surface-expressed HeV G on intact
cells, the cells were lysed with non-ionic detergent, lysates
clarified by centrifugation, and the surface-expressed HeV G
preparations analyzed by sucrose gradient centrifugation. Fractions
were analyzed by immunoprecipitation with polyclonal anti-HeV
rabbit sera followed by Protein G-Sepharose and resolved on
SDS-PAGE under reducing and non-reducing conditions as before.
Shown in FIGS. 9A and 9B is sucrose gradient analysis of surface
expressed uncross-linked full length radiolabeled HeV G. Here it is
observed that in the non-reduced fractions, >95% of the
full-length HeV G exists as the apparent tetrameric species (FIG.
9A, lanes 2-5) and this oligomeric species is clearly dependent on
disulfide bonds as illustrated by the corresponding reduced
fractions which are monomeric (FIG. 9B, lanes 2-5). In addition,
identical sucrose gradient profiles were seen regardless of whether
cross-linking reagent was used or not indicating that the native
cell-surface expressed G forms a very stable tetrameric oligomer.
The protein's natural membrane anchor domain and or its cytoplasmic
tail may contribute to this stability. Nevertheless, the majority
(.about.60-70%) of the .sub.SG.sub.S-tag glycoprotein product
produced here is an oligomeric dimer which indicates that it may
retain important and useful native structural features.
Example 5
Inhibition of HeV and NiV Infection by Soluble HeV G
[0126] It was next evaluated if .sub.SG.sub.S-tag effects on live
virus infection of Vero cells in culture. Here, following
preincubation of Vero cells with various concentrations of
.sub.SG.sub.S-tag, the cells were infected with 1.5.times.10.sup.3
TCID.sub.50/ml and 7.5.times.10.sup.2 TCID.sub.50/ml of live HeV or
NiV, respectively, in the presence of .sub.SG.sub.S-tag for 30 min,
followed by removal of the virus inoculum and incubation with
.sub.SG.sub.S-tag. After 24 hrs in culture, the number of HeV and
NiV infection foci was quantified by specific immunostaining of
cell monolayers with an anti-phosphoprotein (P) as detailed in the
methods. Representative examples of infected Vero cells in the
presence or absence of .sub.SG.sub.S-tag are shown in FIGS.
10A-10D. Typically, infection of Vero cells with live HeV or NiV
produces characteristic syncytium morphologies for each virus.
Immunofluorescence for HeV P protein in HeV syncytia demonstrated
that HeV reproducibly infects and incorporates surrounding cells
into each syncytium with cell nuclei and viral protein equally
detectable throughout the majority of infected cells (FIG. 10A).
NiV infected cells initially show a similar appearance to HeV
syncytia, but ultimately incorporated nuclei within each giant cell
are sequestered together towards the periphery while the remaining
cellular debris is also arranged around the outside leaving the
central region largely empty. Thus, immunofluorescence for HeV P
protein in NiV syncytia often appear as hollow spheres coated in
viral antigen (FIG. 10B) and by comparison, the untreated control
HeV infections produce smaller syncytium relative to the untreated
NiV control (FIGS. 10A and 10B). FIGS. 10C and 10D are
representative examples of HeV and NiV-infected Vero cells in the
presence of 100 .mu.g/ml. .sub.SG.sub.S-tag. Although there were
still some infected cells present as detected by
immunofluorescence, syncytia formation was completely blocked in
both HeV and NiV-infected cells (FIGS. 10C and 10D, respectively).
Furthermore, quantitative analysis of the inhibition of HeV and NiV
infection by purified .sub.SG.sub.S-tag glycoprotein revealed a
dose-dependent response, further demonstrating its specificity, as
shown in FIG. 11. Together these data provide strong evidence that
HeV and NiV utilize a common receptor on the surface of the host
cell. Additionally, the specific inhibition of both viruses by
.sub.SG.sub.S-tag further demonstrated that the .sub.SG.sub.S-tag
construct maintains important native structural elements.
Interestingly, HeV infection was inhibited significantly better
than NiV such that the IC.sub.50 determined for .sub.SG.sub.S-tag
was four-fold greater for NiV (13.20 .mu.g/ml) than for HeV (3.3
.mu.g/ml) (FIG. 11). Given the current evidence suggesting both
viruses utilize a common receptor, the reasons for the differences
observed in .sub.SG.sub.S-tag inhibition of virus infection versus
cell-fusion remain unknown. A similar difference in the ability of
.sub.SG.sub.S-tag to inhibit HeV and NiV-mediated cell-fusion was
not observed, as demonstrated in FIGS. 3A and 3B. Although
HeV-mediated fusion was more potent than NiV-mediated fusion,
illustrated by the higher levels of substrate turnover, the
.sub.SG.sub.S-tag IC.sub.50 in both cell-fusion assays remained
constant. In previous reports, it has been demonstrated through
heterotypic function that the difference in cell-fusion rates
between HeV and NiV was dependent on the fusion protein. Here, it
is demonstrated that natural NiV infection appears to be more
vigorous than HeV infection. Perhaps other viral proteins present
during infection are influencing the kinetics of infection thus
altering the inhibition susceptibility, or they may be differences
in the affinity of HeV sG versus NiV G to the cell surface
expressed receptor.
Example 6
Soluble HeV G Elicits a Potent Virus-Neutralizing Polyclonal
Antibody Response
[0127] With few exceptions, it is the envelope glycoproteins of
viruses to which virtually all neutralizing antibodies are directed
and all successful human viral vaccines induce neutralizing
antibodies that can cross-react with immunologically relevant
strains of a virus. More specifically, virus-neutralizing
antibodies are the key vaccine-induced protective mechanism in the
case of the paramyxoviruses mumps and measles, and it has been
shown that vaccinia virus expressed full-length envelope
glycoproteins from NiV can elicit virus-neutralizing antibodies.
Data indicate that the .sub.SG.sub.S-tag glycoprotein retains
important structural features based on its abilities to
specifically bind receptor positive cells and block both HeV and
NiV-mediated fusion and infection. Thus, the immunization of
animals with sG should potentially generate potent
virus-neutralizing antibodies. To test this possibility, purified
.sub.SG.sub.S-tag was used to immunize rabbits and the resulting
anti-G antiserum evaluated in virus neutralization assays with both
HeV and NiV. Table 1 summarizes the neutralization of HeV and NiV
infection by the polyclonal rabbit anti-G sera. The sera from both
rabbits were capable of complete neutralization of HeV at a
dilution of 1:1280. NiV was also neutralized by the
.sub.SG.sub.S-tag antiserum, with complete neutralization at a
dilution of 1:640. A two fold difference in titer is consistent
with partial antibody cross-reactivity of the HeV and NiV G
glycoproteins. Pre-bleeds from both rabbits were also tested for
their ability to neutralize HeV and NiV. Although there was slight
neutralization at the highest concentration, this activity was
completely abrogated upon dilution of the sera. Previous studies
have demonstrated that HeV and NiV antisera do cross neutralize,
with each serum being slightly less effective against the
heterotypic virus (14). Moreover, it has been demonstrated a
similar trend in cross-neutralization using the cell-fusion assay
for HeV and NiV (4,81). Because .sub.SG.sub.S-tag was able to
elicit such a potent immune response with high levels of
neutralizing antibodies, it may provide an avenue for vaccine
development strategies.
[0128] Other embodiments and uses of the invention will be apparent
to those skilled in the art from consideration of the specification
and practice of the invention disclosed herein. All references
cited herein, including all publications, U.S. and foreign patents
and patent applications, are specifically and entirely incorporated
by reference. It is intended that the specification and examples be
considered exemplary only with the true scope and spirit of the
invention indicated by the following claims.
CITED REFERENCES
[0129] 1. Anonymous. 1999. From the Centers for Disease Control and
Prevention. Outbreak of Hendra-like virus-Malaysia and Singapore,
1998-1999. Jama 281:1787-8. [0130] 2. Baker, K. A., R. E. Dutch, R.
A. Lamb, and T. S. Jardetzky. 1999. Structural basis for
paramyxovirus-mediated membrane fusion. Mol Cell 3:309-19. [0131]
3. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine
receptors as HIV-1 coreceptors: roles in viral entry, tropism, and
disease. Annu Rev Immunol 17:657-700. [0132] 4. Bossart, K. N.,
L.-F. Wang, B. T. Eaton, and C. C. Broder. 2001. Functional
expression and membrane fusion tropism of the envelope
glycoproteins of Hendra virus. Virology 290:121-35. [0133] 5.
Bossart, K. N., L. F. Wang, M. N. Flora, K. B. Chua, S. K. Lam, B.
T. Eaton, and C. C. Broder. 2002. Membrane fusion tropism and
heterotypic functional activities of the nipah virus and hendra
virus envelope glycoproteins. J Virol 76:11186-98. [0134] 6.
Broder, C. C., and P. L. Earl. 1999. Recombinant vaccinia viruses.
Design, generation, and isolation. Mol Biotechnol 13:223-45.
Broder, C. C., P. L. Earl, D. Long, S. T. Abedon, B. Moss, and R.
W. Doms. 1994. Antigenic implications of human immunodeficiency
virus type 1 envelope quaternary structure: oligomer-specific and
-sensitive monoclonal antibodies. Proc Natl Acad Sci USA
91:11699-703. [0135] 8. Chan, D. C., D. Fass, J. M. Berger, and P.
S. Kim. 1997. Core structure of gp41 from the HIV envelope
glycoprotein. Cell 89:263-73. [0136] 9. Chen, L., P. M. Colman, L.
J. Cosgrove, M. C. Lawrence, L. 3. Lawrence, P. A. Tulloch, and J.
J. Gorman. 2001. Cloning, expression, and crystallization of the
fusion protein of Newcastle disease virus. Virology 290:290-9.
[0137] 10. Chen, L., 3.3. Gorman, 3. McKimm-Breschkin, L. J.
Lawrence, P. A. Tulloch, B. J. Smith, P. M. Colman, and M. C.
Lawrence. 2001. The structure of the fusion glycoprotein of
Newcastle disease virus suggests a novel paradigm for the molecular
mechanism of membrane fusion. Structure (Camb) 9:255-66. [0138] 11.
Chua, K. B., W. J. Bellini, P. A. Rota, B. H. Harcourt, A. Tamin,
S. K. Lam, T. G.
[0139] Ksiazek, P. E. Rollin, S. R. Zaki, W. Shieh, C. S.
Goldsmith, D. J. Gubler, J. T. Roehrig, B. Eaton, A. R. Gould, J.
Olson, H. Field, P. Daniels, A. E. Ling, C. J. Peters, L. J.
Anderson, and B. W. Mahy. 2000. Nipah virus: a recently emergent
deadly paramyxovirus. Science 288:1432-5. [0140] 12. Chua, K. B.,
K. J. Goh, K. T. Wong, A. Kamarulzaman, P. S. Tan, T. G. Ksiazek,
S. R. Zaki, G. Paul, S. K. Lam, and C. T. Tan. 1999. Fatal
encephalitis due to Nipah virus among pig-farmers in Malaysia [see
comments]. Lancet 354:1257-9. [0141] 13. Citovsky, V., P. Yanai,
and A. Loyter. 1986. The use of circular dichroism to study
conformational changes induced in Sendai virus envelope
glycoproteins. A correlation with the viral fusogenic activity. J
Biol Chem 261:2235-9.
[0142] 14. Crameri, G., L. F. Wang, C. Morrissy, J. White, and B.
T. Eaton. 2002. A rapid immune plaque assay for the detection of
Hendra and Nipah viruses and anti-virus antibodies. J Virol Methods
99:41-51. [0143] 15. Crennell, S., T. Takimoto, A. Portner, and G.
Taylor. 2000. Crystal structure of the multifunctional
paramyxovirus hemagglutinin-neuraminidase. Nat Struct Biol
7:1068-74. [0144] 16. Doms, R. W., R. Lamb, J. K. Rose, and A.
Helenius. 1993. Folding and assembly of viral membrane proteins.
Virology 193:545-562. [0145] 17. Doms, R. W., and J. P. Moore.
2000. HIV-1 membrane fusion. Targets Of opportunity. J Cell Biol
151:F.sub.9-F.sub.14. [0146] 18. Doms, R. W., and D. Trono. 2000.
The plasma membrane as a combat zone in the HIV battlefield [In
Process Citation]. Genes Dev 14:2677-88. [0147] 19. Dorig, R. E.,
A. Marcil, A. Chopra, and C. D. Richardson. 1993. The human CD46
molecule is a receptor for measles virus (Edmonston strain). Cell
75:295-305. [0148] 20. Dutch, R. E., R. N. Hagglund, M. A. Nagel,
R. G. Paterson, and R. A. Lamb. 2001. Paramyxovirus fusion (F)
protein: a conformational change on cleavage activation. Virology
281:138-50. [0149] 21. Fass, D., S. C. Harrison, and P. S. Kim.
1996. Retrovirus envelope domain at 1.7 angstrom resolution. Nat
Struct Biol 3:465-9. [0150] 22. Field, H., P. Young, J. M. Yob, J.
Mills, L. Hall, and J. Mackenzie. 2001. The natural history of
Hendra and Nipah viruses. Microbes Infect 3:307-14. [0151] 23. Goh,
K. J., C. T. Tan, N. K. Chew, P. S. Tan, A. Kamarulzaman, S. A.
Sarji, K. T. Wong, B. J. Abdullah, K. B. Chua, and S. K. Lam. 2000.
Clinical features of Nipah virus encephalitis among pig farmers in
Malaysia [see comments]. N Engl J Med 342:1229-35. 24. Halpin, K.,
P. L. Young, H. Field, and J. S. Mackenzie. 1999. Newly discovered
viruses of flying foxes. Vet Microbiol 68:83-7. [0152] 25. Halpin,
K., P. L. Young, H. E. Field, and J. S. Mackenzie. 2000. Isolation
of Hendra virus from pteropid bats: a natural reservoir of Hendra
virus. J Gen Virol 81:1927-1932. [0153] 26. Hernandez, L. D., L. R.
Hoffman, T. G. Wolfsberg, and J. M. White. 1996. Virus-cell and
cell-cell fusion. Annu Rev Cell Dev Biol 12:627-61. [0154] 27.
Hooper, P., S. Zaki, P. Daniels, and D. Middleton. 2001.
Comparative pathology of the diseases caused by Hendra and Nipah
viruses. Microbes Infect 3:315-22. [0155] 28. Hooper, P. T., H. A.
Westbury, and G. M. Russell. 1997. The lesions of experimental
equine morbillivirus disease in cats and guinea pigs. Vet Pathol
34:323-9. [0156] 29. Hughson, F. M. 1997. Enveloped viruses: a
common mode of membrane fusion? Curr Biol 7:R565-9. [0157] 30.
Hunter, E. 1997. Viral entry and receptors, p. 71-119. In S. H.
Coffin, S. H. Hughes, and
[0158] H. E. Varmus (ed.), Retroviruses. Cold Spring Harbor
Laboratory Press, New York. [0159] 31. Hurtley, S. M., and A.
Helenius. 1989. Protein oligomerization in the endoplasmic
reticulum. Ann. Rev. Cell Biol. 5:277-307. [0160] 32. Jiang, S., K.
Lin, N. Strick, and A. R. Neurath. 1993. HIV-1 inhibition by a
peptide. Nature 365:113. [0161] 33. Joshi, S. B., R. E. Dutch, and
R. A. Lamb. 1998. A core trimer of the paramyxovirus fusion
protein: parallels to influenza virus hemagglutinin and HIV-1 gp41.
Virology 248:20-34. [0162] 34. Klenk, H. D., and W. Garten. 1994.
Host cell proteases controlling virus pathogenicity. Trends
Microbiol 2:39-43. [0163] 35. Lamb, R. A. 1993. Paramyxovirus
fusion: A hypothesis for changes. Virology 197:1-11. [0164] 36.
Lamb, R. A., and D. Kolakofsky. 2001. Paramyxoviridae: The viruses
and their replication., p. 1305-1340. In D. M. Knipe and P. M.
Howley (ed.), Fields Virology, 4 ed. Lippincott Williams &
Wilkins, Philadelphia. [0165] 37. Lambert, D. M., S. Barney, A. L.
Lambert, K. Guthrie, R. Medinas, D. E. Davis, T. Bucy, J. Erickson,
G. Merutka, and S. R. Petteway, Jr. 1996. Peptides from conserved
regions of paramyxovirus fusion (F) proteins are potent inhibitors
of viral fusion. Proc Natl Acad Sci USA 93:2186-91. [0166] 38. Lee,
K. E., T. Umapathi, C. B. Tan, H. T. Tjia, T. S. Chua, H. M. Oh, K.
M. Fock, A. Kurup, A. Das, A. K. Tan, and W. L. Lee. 1999. The
neurological manifestations of Nipah virus encephalitis, a novel
paramyxovirus. Ann Neurol 46:428-32. [0167] 39. Lim, C. C., Y. Y.
Sitoh, F. Hui, K. E. Lee, B. S. Ang, E. Lim, W. E. Lim, H. M. Oh,
P. A. Tambyah, J. S. Wong, C. B. Tan, and T. S. Chee. 2000. Nipah
viral encephalitis or Japanese encephalitis? MR findings in a new
zoonotic disease. AJNR Am J Neuroradiol 21:455-61. [0168] 40.
Markwell, M. A., and C. F. Fox. 1980. Protein-protein interactions
within paramyxoviruses identified by native disulfide bonding or
reversible chemical cross-linking. J Virol 33:152-66. [0169] 41.
McGinnes, L. W., K. Gravel, and T. G. Morrison. 2002. Newcastle
disease virus HN protein alters the conformation of the F protein
at cell surfaces. J Virol 76:12622-33. [0170] 42. Middleton, D. J.,
H. A. Westbury, C. J. Morrissy, B. M. van der Heide, G. M. Russell,
M. A. Braun, and A. D. Hyatt. 2002. Experimental nipah virus
infection in pigs and cats. J Comp Pathol 126:124-36. [0171] 43.
Morrison, T. G. 1988. Structure, function, and intracellular
processing of paramyxovirus membrane proteins. Virus Res 10:113-35.
[0172] 44. Morrison, T. G. 2001. The three faces of paramyxovirus
attachment proteins. Trends Microbiol 9:103-5. [0173] 45. Mounts,
A. W., H. Kaur, U. D. Parashar, T. G. Ksiazek, D. Cannon, J. T.
Arokiasamy, L. J. Anderson, and M. S. Lye. 2001. A cohort study of
health care workers to assess nosocomial transmissibility of Nipah
virus, Malaysia, 1999. J Infect Dis 183:810-3. 46. Murray, K., P.
Selleck, P. Hooper, A. Hyatt, A. Gould, L. Gleeson, H. Westbury, L.
Hiley, L. Selvey, B. Rodwell, and et al. 1995. A morbillivirus that
caused fatal disease in horses and humans. Science 268:94-7. [0174]
47. Naniche, D., G. Varior-Krishnan, F. Cervoni, T. F. Wild, B.
Rossi, C. Rabourdin-Combe, and D. Gerlier. 1993. Human membrane
cofactor protein (CD46) acts as a cellular receptor for measles
virus. J Virol 67:6025-32. [0175] 48. Nussbaum, O, C. C. Broder, B.
Moss, L. B. Stem, S. Rozenblatt, and E. A. Berger. 1995. Functional
and structural interactions between measles virus hemagglutinin and
CD46. J Virol 69:3341-9. [0176] 49. O'Sullivan, J. D., A. M.
Allworth, D. L. Paterson, T. M. Snow, R. Boots, L. J. Gleeson, A.
R. Gould, A. D. Hyatt, and J. Bradfield. 1997. Fatal encephalitis
due to novel paramyxovirus transmitted from horses. Lancet
349:93-5. [0177] 50. Paterson, R. G., M. L. Johnson, and R. A.
Lamb. 1997. Paramyxovirus fusion (F) protein and
hemagglutinin-neuraminidase (HN) protein interactions:
intracellular retention of F and HN does not affect transport of
the homotypic HN or F protein. Virology 237:1-9. [0178] 51.
Plemper, R. K., A. L. Hammond, and R. Cattaneo. 2001. Measles virus
envelope glycoproteins hetero-oligomerize in the endoplasmic
reticulum. J Biol Chem 276:44239-46. [0179] 52. Rapaport, D., M.
Ovadia, and Y. Shai. 1995. A synthetic peptide corresponding to a
conserved heptad repeat domain is a potent inhibitor of Sendai
virus-cell fusion: an emerging similarity with functional domains
of other viruses. Embo J 14:5524-31. [0180] 53. Russell, C. J., T.
S. Jardetzky, and R. A. Lamb. 2001. Membrane fusion machines of
paramyxoviruses: capture of intermediates of fusion. Embo J
20:4024-34. [0181] 54. Russell, R., R. G. Paterson, and R. A. Lamb.
1994. Studies with cross-linking reagents on the oligomeric form of
the paramyxovirus fusion protein. Virology 199:160-8. [0182] 55.
Scheid, A., and P. W. Choppin. 1974. Identification of biological
activities of paramyxovirus glycoproteins. Activation of cell
fusion, hemolysis, and infectivity of proteolytic cleavage of an
inactive precursor protein of Sendai virus. Virology 57:475-90.
[0183] 56. Singh, M., B. Berger, and P. S. Kim. 1999.
LearnCoil-VMF: computational evidence for coiled-coil-like motifs
in many viral membrane-fusion proteins. J Mol Biol 290:1031-41.
[0184] 57. Stone-Hulslander, J., and T. G. Morrison. 1997.
Detection of an interaction between the HN and F proteins in
Newcastle disease virus-infected cells. J Virol 71:6287-95. [0185]
58. Takimoto, T., G. L. Taylor, H. C. Connaris, S. 3. Crennell, and
A. Portner. 2002. Role of the hemagglutinin-neuraminidase protein
in the mechanism of paramyxovirus-cell membrane fusion. J Virol
76:13028-33. [0186] 59. Takimoto, T., G. L. Taylor, S. J. Crennell,
R. A. Scroggs, and A. Portner. 2000. Crystallization of Newcastle
disease virus hemagglutinin-neuraminidase glycoprotein. Virology
270:208-14. [0187] 60. Tan, C. T., and K. S. Tan. 2001. Nosocomial
transmissibility of Nipah virus. J Infect Dis 184:1367. [0188] 61.
Tatsuo, H., N. Ono, K. Tanaka, and Y. Yanagi. 2000. SLAM (CDw150)
is a cellular receptor for measles virus. Nature 406:893-7. [0189]
62. Tatsuo, H., N. Ono, and Y. Yanagi. 2001. Morbilliviruses use
signaling lymphocyte activation molecules (CD150) as cellular
receptors. I Virol 75:5842-50. [0190] 63. Wang, L., B. H. Harcourt,
M. Yu, A. Tamin, P. A. Rota, W. J. Bellini, and B. T. Eaton. 2001.
Molecular biology of Hendra and Nipah viruses. Microbes Infect
3:279-87. [0191] 64. Wang, L. F., W. P. Michalski, M. Yu, L. I.
Pritchard, G. Crameri, B. Shiell, and B. T. Eaton. 1998. A novel
P/V/C gene in a new member of the Paramyxoviridae family, which
causes lethal infection in humans, horses, and other animals. J
Virol 72:1482-90. [0192] 65. Westbury, H. A., P. T. Hooper, S. L.
Brouwer, and P. W. Selleck. 1996. Susceptibility of cats to equine
morbillivirus. Aust Vet J 74:132-4. [0193] 66. Westbury, H. A., P.
T. Hooper, P. W. Selleck, and P. K. Murray. 1995. Equine
morbillivirus pneumonia: susceptibility of laboratory animals to
the virus. Aust Vet J 72:278-9. [0194] 67. Wild, C. T., D. C.
Shugars, T. K. Greenwell, C. B. McDanal, and T. J. Matthews. 1994.
Peptides corresponding to a predictive alpha-helical domain of
human immunodeficiency virus type 1 gp41 are potent inhibitors of
virus infection. Proc Natl Acad Sci USA 91:9770-4. [0195] 68. Wild,
T. F., and R. Buckland. 1997. Inhibition of measles virus infection
and fusion with peptides corresponding to the leucine zipper region
of the fusion protein. J Gen Virol 78:107-11. [0196] 69. Wiley, D.
C., and J. J. Skehel. 1987. The structure and function of the
hemagglutinin membrane glycoprotein of influenza virus. Ann. Rev.
Biochem. 56:365-394. [0197] 70. Williamson, M. M., P. T. Hooper, P.
W. Selleck, L. J. Gleeson, P. W. Daniels, H. A. Westbury, and P. K.
Murray. 1998. Transmission studies of Hendra virus (equine
morbillivirus) in fruit bats, horses and cats. Aust Vet J 76:813-8.
[0198] 71. Wilson, I. A., J. J. Skehel, and D. C., Wiley. 1981.
Structure of the haemagglutinin membrane glycoprotein of influenza
virus at 3 A resolution. Nature 289:366-73.
[0199] 72. Wong, S. C., M. H. Ooi, M. N. Wong, P. H. Tio, T.
Solomon, and M. J. Cardosa. 2001. Late presentation of Nipah virus
encephalitis and kinetics of the humoral immune response. J Neurol
Neurosurg Psychiatry 71:552-4. [0200] 73. Yao, Q., X. Hu, and R. W.
Compans. 1997. Association of the parainfluenza virus fusion and
hemagglutinin-neuraminidase glycoproteins on cell surfaces. J Virol
71:650-6. [0201] 74. Young, J. K., R. P. Hicks, G. E. Wright, and
T. G. Morrison. 1997. Analysis of a peptide inhibitor of
paramyxovirus (NDV) fusion using biological assays, NMR, and
molecular modeling. Virology 238:291-304. [0202] 75. Young, J. K.,
D. Li, M. C. Abramowitz, and T. G. Morrison. 1999. Interaction of
peptides with sequences from the Newcastle disease virus fusion
protein heptad repeat regions. J Virol 73:5945-56. [0203] 76.
Young, P. L., K. Halpin, P. W. Selleck, H. Field, J. L. Gravel, M.
A. Kelly, and J. S. Mackenzie. 1996. Serologic evidence for the
presence in Pteropus bats of a paramyxovirus related to equine
morbillivirus. Emerg Infect Dis 2:239-40. [0204] 77. Yu, M., E.
Hansson, J. P. Langedijk, B. T. Eaton, and L. F. Wang. 1998. The
attachment protein of Hendra virus has high structural similarity
but limited primary sequence homology compared with viruses in the
genus Paramyxovirus. Virology 251:227-33. [0205] 78. Yu, M., E.
Hansson, B. Shiell, W. Michalski, B. T. Eaton, and L. F. Wang.
1998. Sequence analysis of the Hendra virus nucleoprotein gene:
comparison with other members of the subfamily Paramyxovirinae. J
Gen Virol 79:1775-80. [0206] 79. Zhao, X., M. Singh, V. N.
Malashkevich, and P. S. Kim. 2000. Structural characterization of
the human respiratory syncytial virus fusion protein core. Proc
Natl Acad Sci USA 97:14172-7. [0207] 80. Zhu, J., C. W. Zhang, Y.
Qi, P. Tien, and G. F. Gao. 2002. The fusion protein core of
measles virus forms stable coiled-coil trimer. Biochem Biophys Res
Commun 299:897-902. [0208] 81. Bossart, K. N., and C. C. Broder.
2004. Viral glycoprotein-mediated cell fusion assays using vaccinia
virus vectors. Methods Mol. Biol. 269:309-332.
TABLE-US-00001 [0208] TABLE 1 HeV NiV Dilution Rabbit 405 Rabbit
406 Rabbit 405 Rabbit 406 1:10 - - - - - - - - 1:20 - - - - - - - -
1:40 - - - - - - - - 1:80 - - - - - - - - 1:160 - - - - - - - -
1:320 - - - - - - - - 1:640 - - - - - - - - 1:1,280 - - - - + + - +
1:2,560 - - - + + + + + 1:5,120 - - + + + + + + 1:10,240 + + + + +
+ + + 1:20,480 + + + + + + + +
Sequence CWU 1
1
17140DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1gtcgaccacc atgcaaaatt acaccagaac
gactgataat 40245DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 2gtttaaacgt cgaccaatca
actctctgaa cattgggcag gtatc 45339DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 3ctcgagcacc
atgcaaaatt acacaagatc aacagacaa 39445DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4ctcgagtagc agccggatca agcttatgta cattgctctg gtatc
45546DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 5caaggagacc gctgctgcta agttcgaacg
ccagcacatg gattct 46654DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 6aattagaatc
catgtgctgg cgttcgaact tagcagcagc ggtctccttg gtac 54731DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7cgaacaaaag ctcatctcag aagaggatct g
31839DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 8aattcagatc ctcttctgag atgagctttt
gttcggtac 39934DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 9tcgacccacc atggagacag
acacactcct gcta 341021PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 10Met Glu Thr Asp Thr Leu Leu
Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Asp
20 1115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Ala Ala Gln Pro Ala Arg Arg Ala Arg Arg Thr Lys
Leu Gly Thr 1 5 10 151215PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Lys Glu Thr Ala Ala Ala Lys
Phe Glu Arg Gln His Met Asp Ser 1 5 10 151310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Glu
Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 101415PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Asn
Ser Ala Asp Ile Gln His Ser Gly Gly Arg Ser Thr Thr Met 1 5 10
1515421PRTHendra virus 15Gly Leu Pro Asn Gln Ile Cys Leu Gln Lys
Thr Thr Ser Thr Ile Leu 1 5 10 15 Lys Pro Arg Leu Ile Ser Tyr Thr
Leu Pro Ile Asn Arg Glu Gly Val 20 25 30 Cys Ile Thr Asp Pro Leu
Leu Ala Val Asp Asn Gly Phe Phe Ala Tyr 35 40 45 Ser His Leu Glu
Lys Ile Gly Ser Cys Thr Arg Gly Ile Ala Lys Gln 50 55 60 Arg Ile
Ile Gly Val Gly Glu Val Leu Asp Arg Gly Asp Lys Val Pro 65 70 75
80Ser Met Phe Met Thr Asn Val Trp Thr Pro Pro Asn Pro Ser Thr Ile
85 90 95 His His Cys Ser Ser Thr Tyr His Glu Asp Phe Tyr Tyr Thr
Leu Cys 100 105 110 Ala Val Ser His Val Gly Asp Pro Ile Leu Asn Ser
Thr Ser Trp Thr 115 120 125 Glu Ser Leu Ser Leu Ile Arg Leu Ala Val
Arg Pro Lys Ser Asp Ser 130 135 140 Gly Asp Tyr Asn Gln Lys Tyr Ile
Ala Ile Thr Lys Val Glu Arg Gly 145 150 155 160Lys Tyr Asp Lys Val
Met Pro Tyr Gly Pro Ser Gly Ile Lys Gln Gly 165 170 175 Asp Thr Leu
Tyr Phe Pro Ala Val Gly Phe Leu Pro Arg Thr Glu Phe 180 185 190 Gln
Tyr Asn Asp Ser Asn Cys Pro Ile Ile His Cys Lys Tyr Ser Lys 195 200
205 Ala Glu Asn Cys Arg Leu Ser Met Gly Val Asn Ser Lys Ser His Tyr
210 215 220 Ile Leu Arg Ser Gly Leu Leu Lys Tyr Asn Leu Ser Leu Gly
Gly Asp 225 230 235 240Ile Ile Leu Gln Phe Ile Glu Ile Ala Asp Asn
Arg Leu Thr Ile Gly 245 250 255 Ser Pro Ser Lys Ile Tyr Asn Ser Leu
Gly Gln Pro Val Phe Tyr Gln 260 265 270 Ala Ser Tyr Ser Trp Asp Thr
Met Ile Lys Leu Gly Asp Val Asp Thr 275 280 285 Val Asp Pro Leu Arg
Val Gln Trp Arg Asn Asn Ser Val Ile Ser Arg 290 295 300 Pro Gly Gln
Ser Gln Cys Pro Arg Phe Asn Val Cys Pro Glu Val Cys 305 310 315
320Trp Glu Gly Thr Tyr Asn Asp Ala Phe Leu Ile Asp Arg Leu Asn Trp
325 330 335 Val Ser Ala Gly Val Tyr Leu Asn Ser Asn Gln Thr Ala Glu
Asn Pro 340 345 350 Val Phe Ala Val Phe Lys Asp Asn Glu Ile Leu Tyr
Gln Val Pro Leu 355 360 365 Ala Glu Asp Asp Thr Asn Ala Gln Lys Thr
Ile Thr Asp Cys Phe Leu 370 375 380 Leu Glu Asn Val Ile Trp Cys Ile
Ser Leu Val Glu Ile Tyr Asp Thr 385 390 395 400Gly Asp Ser Val Ile
Arg Pro Lys Leu Phe Ala Val Lys Ile Pro Ala 405 410 415 Gln Cys Ser
Glu Ser 420 16534PRTHendra virus 16Gln Asn Tyr Thr Arg Thr Thr Asp
Asn Gln Ala Leu Ile Lys Glu Ser 1 5 10 15 Leu Gln Ser Val Gln Gln
Gln Ile Lys Ala Leu Thr Asp Lys Ile Gly 20 25 30 Thr Glu Ile Gly
Pro Lys Val Ser Leu Ile Asp Thr Ser Ser Thr Ile 35 40 45 Thr Ile
Pro Ala Asn Ile Gly Leu Leu Gly Ser Lys Ile Ser Gln Ser 50 55 60
Thr Ser Ser Ile Asn Glu Asn Val Asn Asp Lys Cys Lys Phe Thr Leu 65
70 75 80 Pro Pro Leu Lys Ile His Glu Cys Asn Ile Ser Cys Pro Asn
Pro Leu 85 90 95 Pro Phe Arg Glu Tyr Arg Pro Ile Ser Gln Gly Val
Ser Asp Leu Val 100 105 110 Gly Leu Pro Asn Gln Ile Cys Leu Gln Lys
Thr Thr Ser Thr Ile Leu 115 120 125 Lys Pro Arg Leu Ile Ser Tyr Thr
Leu Pro Ile Asn Thr Arg Glu Gly 130 135 140 Val Cys Ile Thr Asp Pro
Leu Leu Ala Val Asp Asn Gly Phe Phe Ala 145 150 155 160 Tyr Ser His
Leu Glu Lys Ile Gly Ser Cys Thr Arg Gly Ile Ala Lys 165 170 175 Gln
Arg Ile Ile Gly Val Gly Glu Val Leu Asp Arg Gly Asp Lys Val 180 185
190 Pro Ser Met Phe Met Thr Asn Val Trp Thr Pro Pro Asn Pro Ser Thr
195 200 205 Ile His His Cys Ser Ser Thr Tyr His Glu Asp Phe Tyr Tyr
Thr Leu 210 215 220 Cys Ala Val Ser His Val Gly Asp Pro Ile Leu Asn
Ser Thr Ser Trp 225 230 235 240 Thr Glu Ser Leu Ser Leu Ile Arg Leu
Ala Val Arg Pro Lys Ser Asp 245 250 255 Ser Gly Asp Tyr Asn Gln Lys
Tyr Ile Ala Ile Thr Lys Val Glu Arg 260 265 270 Gly Lys Tyr Asp Lys
Val Met Pro Tyr Gly Pro Ser Gly Ile Lys Gln 275 280 285 Gly Asp Thr
Leu Tyr Phe Pro Ala Val Gly Phe Leu Pro Arg Thr Glu 290 295 300 Phe
Gln Tyr Asn Asp Ser Asn Cys Pro Ile Ile His Cys Lys Tyr Ser 305 310
315 320 Lys Ala Glu Asn Cys Arg Leu Ser Met Gly Val Asn Ser Lys Ser
His 325 330 335 Tyr Ile Leu Arg Ser Gly Leu Leu Lys Tyr Asn Leu Ser
Leu Gly Gly 340 345 350 Asp Ile Ile Leu Gln Phe Ile Glu Ile Ala Asp
Asn Arg Leu Thr Ile 355 360 365 Gly Ser Pro Ser Lys Ile Tyr Asn Ser
Leu Gly Gln Pro Val Phe Tyr 370 375 380 Gln Ala Ser Tyr Ser Trp Asp
Thr Met Ile Lys Leu Gly Asp Val Asp 385 390 395 400 Thr Val Asp Pro
Leu Arg Val Gln Trp Arg Asn Asn Ser Val Ile Ser 405 410 415 Arg Pro
Gly Gln Ser Gln Cys Pro Arg Phe Asn Val Cys Pro Glu Val 420 425 430
Cys Trp Glu Gly Thr Tyr Asn Asp Ala Phe Leu Ile Asp Arg Leu Asn 435
440 445 Trp Val Ser Ala Gly Val Tyr Leu Asn Ser Asn Gln Thr Ala Glu
Asn 450 455 460 Pro Val Phe Ala Val Phe Lys Asp Asn Glu Ile Leu Tyr
Gln Val Pro 465 470 475 480 Leu Ala Glu Asp Asp Thr Asn Ala Gln Lys
Thr Ile Thr Asp Cys Phe 485 490 495 Leu Leu Glu Asn Val Ile Trp Cys
Ile Ser Leu Val Glu Ile Tyr Asp 500 505 510 Thr Gly Asp Ser Val Ile
Arg Pro Lys Leu Phe Ala Val Lys Ile Pro 515 520 525 Ala Gln Cys Ser
Glu Ser 530 17532PRTNipah virus 17Gln Asn Tyr Thr Arg Ser Thr Asp
Asn Gln Ala Val Ile Lys Asp Ala 1 5 10 15 Leu Gln Gly Ile Gln Gln
Gln Ile Lys Gly Leu Ala Asp Lys Ile Gly 20 25 30 Thr Glu Ile Gly
Pro Lys Val Ser Leu Ile Asp Thr Ser Ser Thr Ile 35 40 45 Thr Ile
Pro Ala Asn Ile Gly Leu Leu Gly Ser Lys Ile Ser Gln Ser 50 55 60
Thr Ala Ser Ile Asn Glu Asn Val Asn Glu Lys Cys Lys Phe Thr Leu 65
70 75 80 Pro Pro Leu Lys Ile His Glu Cys Asn Ile Ser Cys Pro Asn
Pro Leu 85 90 95 Pro Phe Arg Glu Tyr Arg Pro Gln Thr Glu Gly Val
Ser Asn Leu Val 100 105 110 Gly Leu Pro Asn Asn Ile Cys Leu Gln Lys
Thr Ser Asn Gln Ile Leu 115 120 125 Lys Pro Lys Leu Ile Ser Tyr Thr
Leu Pro Val Val Gly Gln Ser Gly 130 135 140 Thr Cys Ile Thr Asp Pro
Leu Leu Ala Met Asp Glu Gly Tyr Phe Ala 145 150 155 160 Tyr Ser His
Leu Glu Arg Ile Gly Ser Cys Ser Arg Gly Val Ser Lys 165 170 175 Gln
Arg Ile Ile Gly Val Gly Glu Val Leu Asp Arg Gly Asp Glu Val 180 185
190 Pro Ser Leu Phe Met Thr Asn Val Trp Thr Pro Pro Asn Pro Asn Thr
195 200 205 Val Tyr His Cys Ser Ala Val Tyr Asn Asn Glu Phe Tyr Tyr
Val Leu 210 215 220 Cys Ala Val Ser Thr Val Gly Asp Pro Ile Leu Asn
Ser Thr Tyr Trp 225 230 235 240 Ser Gly Ser Leu Met Met Thr Arg Leu
Ala Val Lys Pro Lys Ser Asn 245 250 255 Gly Gly Gly Tyr Asn Gln His
Gln Leu Ala Leu Arg Ser Ile Glu Lys 260 265 270 Gly Arg Tyr Asp Lys
Val Met Pro Tyr Gly Pro Ser Gly Ile Lys Gln 275 280 285 Gly Asp Thr
Leu Tyr Phe Pro Ala Val Gly Phe Leu Val Arg Thr Glu 290 295 300 Phe
Lys Tyr Asn Asp Ser Asn Cys Pro Ile Thr Lys Cys Gln Tyr Ser 305 310
315 320 Lys Pro Glu Asn Cys Arg Leu Ser Met Gly Ile Arg Pro Asn Ser
His 325 330 335 Tyr Ile Leu Arg Ser Gly Leu Leu Lys Tyr Asn Leu Ser
Asp Gly Glu 340 345 350 Asn Pro Lys Val Val Phe Ile Glu Ile Ser Asp
Gln Arg Leu Ser Ile 355 360 365 Gly Ser Pro Ser Lys Ile Tyr Asp Ser
Leu Gly Gln Pro Val Phe Tyr 370 375 380 Gln Ala Ser Phe Ser Trp Asp
Thr Met Ile Lys Phe Gly Asp Val Leu 385 390 395 400 Thr Val Asn Pro
Leu Val Val Asn Trp Arg Asn Asn Thr Val Ile Ser 405 410 415 Arg Pro
Gly Gln Ser Gln Cys Pro Arg Phe Asn Thr Cys Pro Glu Ile 420 425 430
Cys Trp Glu Gly Val Tyr Asn Asp Ala Phe Leu Ile Asp Arg Ile Asn 435
440 445 Trp Ile Ser Ala Gly Val Phe Leu Asp Ser Asn Gln Thr Ala Glu
Asn 450 455 460 Pro Val Phe Thr Val Phe Lys Asp Asn Glu Ile Leu Tyr
Arg Ala Gln 465 470 475 480 Leu Ala Ser Glu Asp Thr Asn Ala Gln Lys
Thr Ile Thr Asn Cys Phe 485 490 495 Leu Leu Lys Asn Lys Ile Trp Cys
Ile Ser Leu Val Glu Ile Tyr Asp 500 505 510 Thr Gly Asp Asn Val Ile
Arg Pro Lys Leu Phe Ala Val Lys Ile Pro 515 520 525 Glu Gln Cys Thr
530
* * * * *