U.S. patent application number 12/808741 was filed with the patent office on 2010-12-30 for immunogenic compositions and methods of use thereof.
Invention is credited to Jadranka Bozia, Richard W. Compans, Ioanna Skountzou, Baozhong Wang.
Application Number | 20100330190 12/808741 |
Document ID | / |
Family ID | 40796128 |
Filed Date | 2010-12-30 |
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United States Patent
Application |
20100330190 |
Kind Code |
A1 |
Compans; Richard W. ; et
al. |
December 30, 2010 |
IMMUNOGENIC COMPOSITIONS AND METHODS OF USE THEREOF
Abstract
Embodiments of the disclosure encompass compositions and methods
for generating immune responses in an animal or human host.
Embodiments of the compositions encompass proteins derived from the
surface proteins of bacteria and protozoa, and in particular the
flagellum component flagellin, and which have adjunctival
properties when administered in conjunction with an immunogen.
Embodiments of the compositions of the disclosure are modified to
incorporate a heterologous transmembrane-cytoplasmic domain
allowing the peptides to be incorporated into virus-like particles.
Embodiments of the methods of generating an immunological response
in an animal or human comprise exposing the immune system of an
animal or human host to an immunogen and a virus-like particle
comprising an adjuvant polypeptide including a host cell Toll-like
receptor ligand polypeptide having a transmembrane-cytoplasmic tail
polypeptide, and a heterologous signal peptide.
Inventors: |
Compans; Richard W.;
(Atlanta, GA) ; Wang; Baozhong; (Duluth, CN)
; Bozia; Jadranka; (Tucker, GA) ; Skountzou;
Ioanna; (Atlanta, GA) |
Correspondence
Address: |
THOMAS, KAYDEN, HORSTEMEYER & RISLEY, LLP
600 GALLERIA PARKWAY, S.E., STE 1500
ATLANTA
GA
30339-5994
US
|
Family ID: |
40796128 |
Appl. No.: |
12/808741 |
Filed: |
December 17, 2008 |
PCT Filed: |
December 17, 2008 |
PCT NO: |
PCT/US08/87194 |
371 Date: |
September 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61014130 |
Dec 17, 2007 |
|
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61078815 |
Jul 8, 2008 |
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Current U.S.
Class: |
424/499 ;
424/185.1; 435/320.1; 530/350; 536/23.4 |
Current CPC
Class: |
C07K 2319/03 20130101;
A61K 2039/5258 20130101; A61K 2039/5252 20130101; A61K 39/00
20130101; A61K 39/145 20130101; A61K 2039/543 20130101; C07K
2319/40 20130101; C12N 2760/16134 20130101; C12N 7/00 20130101;
C12N 2710/14043 20130101; A61K 2039/55516 20130101; A61K 39/39
20130101; C12N 2760/16123 20130101; C07K 2319/02 20130101; A61K
39/12 20130101; C07K 14/255 20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/499 ;
424/185.1; 435/320.1; 530/350; 536/23.4 |
International
Class: |
A61K 9/14 20060101
A61K009/14; A61K 39/00 20060101 A61K039/00; C12N 15/63 20060101
C12N015/63; C07K 14/00 20060101 C07K014/00; C07H 21/04 20060101
C07H021/04; C07K 14/195 20060101 C07K014/195; C07K 14/44 20060101
C07K014/44; C07K 14/255 20060101 C07K014/255; C07K 14/11 20060101
C07K014/11; C07K 14/435 20060101 C07K014/435; A61K 39/002 20060101
A61K039/002; A61K 39/02 20060101 A61K039/02; A61K 39/112 20060101
A61K039/112; A61K 39/145 20060101 A61K039/145 |
Goverment Interests
RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under NIH
Grant No. RO1 Al068003 awarded by the U.S. National Institutes of
Health of the United States government. The government has certain
rights in the invention.
Claims
1. An adjuvant polypeptide comprising: at least one domain capable
of selectively interacting with a Toll-like receptor of an animal
or human cell, and wherein the domain is capable of increasing an
immune response in a recipient host; and at least one heterologous
region selected from the group consisting of a signal peptide
region, a transmembrane-cytoplasmic tail region, and an immunogenic
peptide.
2. The adjuvant polypeptide of claim 1, comprising a first region,
wherein the first region comprises a signal peptide; a second
region, wherein the second region comprises at least one domain
capable of selectively interacting with a Toll-like receptor of an
animal or human cell, and wherein the domain is capable of
increasing an immune response in a recipient host; and a third
region, wherein the third region comprises a
transmembrane-cytoplasmic tail peptide.
3. The adjuvant polypeptide of claim 1, wherein the at least one
domain capable of selectively interacting with a Toll-like receptor
of an animal or human cell is derived from a surface protein of a
bacterial species, or of a protozoal species.
4. The adjuvant polypeptide of claim 3, wherein the surface protein
is a protein of a bacterial or a protozoal flagellum, or a fragment
thereof.
5. The adjuvant polypeptide of claim 4, wherein the surface protein
is a flagellin of the bacterial species Salmonella enteritidis.
6. The adjuvant polypeptide of claim 1 having the amino acid
sequence SEQ ID NO.: 10, or a conservative variant thereof.
7. The adjuvant polypeptide of claim 1, further comprising a
peptide linker.
8. The adjuvant polypeptide of claim 6, wherein the peptide linker
has an amino acid sequence according to SEQ ID NO.: 18.
9. The adjuvant polypeptide of claim 1, wherein the signal peptide
is a signal peptide of a bee melittin polypeptide.
10. The adjuvant polypeptide of claim 1, wherein the
transmembrane-cytoplasmic tail is derived from a hemagglutinin A
polypeptide of an influenza virus.
11. The adjuvant polypeptide of claim 1, further comprising an
immunogenic peptide.
12. The adjuvant polypeptide of claim 1, wherein the immunogenic
peptide has an amino acid sequence according to SEQ ID NO.: 19.
13. A nucleic acid molecule, comprising: a region encoding a
bacterial flagellin polypeptide, or a fragment thereof, wherein the
flagellin polypeptide or fragment thereof comprises at least one
domain capable of specifically interacting with a Toll-like
receptor of an animal or human cell; and at least one region
selected from the group consisting of: a region encoding a
heterologous signal peptide and a region encoding a
transmembrane-cytoplasmic tail capable of being incorporated into a
virus-like particle or virosome.
14. The nucleic acid molecule of claim 13, comprising: a region
encoding a bacterial flagellin polypeptide, or a fragment thereof,
wherein the flagellin polypeptide or fragment thereof comprises at
least one domain capable of specifically interacting with a
Toll-like receptor of an animal or human cell; a region encoding a
heterologous signal peptide; and a region encoding a
transmembrane-cytoplasmic tail capable of being incorporated into a
virus-like particle or virosome.
15. The nucleic acid molecule of claim 13, wherein the heterologous
signal peptide is a bee melittin signal peptide, and the
transmembrane-cytoplasmic tail is from an influenza virus
hemagglutinin.
16. The nucleic acid molecule of claim 13, comprising: a first
nucleotide sequence encoding an amino acid sequence from amino acid
positions of about 1 to about 305 of sequence SEQ ID NO.: 10, or a
conservative variant thereof; a second nucleotide sequence encoding
an amino acid sequence from amino acid positions about 430 to about
565 of sequence SEQ ID NO.: 10, or a conservative variant thereof;
and a third nucleotide sequence disposed between the first
nucleotide sequence and the second nucleotide sequence, wherein the
third nucleotide sequence encodes a region selected from the group
consisting of: a region of a bacterial or protozoal surface protein
polypeptide, a peptide linker, an immunogenic peptide, and an
antigenic peptide.
17. The nucleic acid molecule of claim 16, wherein the first
nucleotide sequence is according to about position 1 to about
position 615 of nucleotide sequence SEQ ID NO.: 9, or a
conservative variant thereof; and the second nucleotide sequence is
according to about position 1293 to about position 1695 of
nucleotide sequence SEQ ID NO.: 9, or a conservative variant
thereof.
18. The nucleic acid molecule of claim 16, comprising the nucleic
acid sequence according to SEQ ID NO.: 9, or a conservative variant
thereof.
19. The nucleic acid molecule of claim 16 having the nucleic acid
sequence according to SEQ ID NO.: 9.
20. The nucleic acid molecule of claim 13, wherein the nucleic acid
molecule is operably inserted into a nucleic acid expression
vector.
21. The nucleic acid molecule of claim 20, wherein the nucleic acid
expression vector is selected from the group consisting of: a
plasmid, a baculovirus vector, a cosmid, a viral vector, a
chromosome, a mini-chromosome, a modified vaccinia Ankara (MVA)
vector, a recombinant poxvirus vector, a recombinant adenovirus
vector, an alphavirus vector, and a paramyxovirus vector.
22. An immunogenic composition comprising: an adjuvant polypeptide
comprising at least one region capable of selectively interacting
with a Toll-like receptor protein of a host; and an immunogen
capable of producing an immune response in a recipient host.
23. The immunogenic composition of claim 22, further comprising a
virus-like carrier, wherein the virus-like carrier is selected from
the group consisting of a virus-like particle and a virosome, and
wherein the adjuvant polypeptide and the immunogen are incorporated
in the virus-like particle or the virosome.
24. The immunogenic composition of claim 22, wherein the adjuvant
polypeptide is a surface polypeptide of a bacterial species or of a
protozoal species, or a modified variant of the surface
polypeptide.
25. The immunogenic composition of claim 24, wherein the surface
polypeptide is a bacterial flagellin.
26. The immunogenic composition of claim 25, wherein the flagellin
is of the bacterial species Salmonella enteritidis.
27. The immunogenic composition of claim 24, wherein the modified
variant of the adjuvant polypeptide comprises at least one
heterologous peptide region, wherein the at least heterologous
region is selected from the group consisting of a signal peptide
region and a transmembrane-cytoplasmic tail region.
28. The immunogenic composition of claim 22, wherein the modified
variant of the adjuvant polypeptide comprises a first heterologous
peptide region, wherein the first heterologous peptide region is a
signal peptide, a second heterologous peptide region, wherein the
second heterologous peptide region is a transmembrane-cytoplasmic
tail peptide, and at least one region capable of selectively
interacting with a Toll-like receptor protein of a host.
29. The immunogenic composition of claim 25, wherein the bacterial
flagellin polypeptide is a modified bacterial flagellin polypeptide
modified by deletion of a region of a full-length bacterial
flagellin polypeptide.
30. The immunogenic composition of claim 22, wherein the adjuvant
polypeptide further comprises a heterologous peptide region,
wherein said region is disposed between two domains of the adjuvant
polypeptide, wherein each of said domains is capable of selectively
targeting a toll-like receptor protein of a host.
31. The immunogenic composition of claim 22, wherein the adjuvant
polypeptide further comprises a heterologous peptide region,
wherein said region is antigenic.
32. The immunogenic composition of claim 31, wherein the
heterologous peptide region is antigenic and has the amino acid
sequence according to SEQ ID NO.: 19.
33. The immunogenic composition of claim 23, wherein the virus-like
carrier is a virosome, comprising: at least one viral surface
envelope glycoprotein expressed on the surface of the virosome; and
at least one adjuvant molecule expressed on the surface of the
virosome, wherein the at least one adjuvant molecule comprises a
membrane-anchored form of a bacterial or protozoal surface
component that is a mammalian toll-like receptor (TLR) ligand
molecule.
34. The immunogenic composition of claim 33, wherein the virus-like
carrier is a virus-like particle and further comprises a viral core
protein capable of self-assembling into a virus-like particle
core.
35. The immunogenic composition of claim 34, wherein the viral core
protein and the at least one viral surface envelope glycoprotein
are from different viruses.
36. The immunogenic composition of claim 34, wherein the viral core
protein is selected from the group consisting of: a retrovirus Gag
protein, a retrovirus matrix protein, a rhabdovirus M protein, a
filovirus viral core protein, a coronavirus M protein, a
coronavirus E protein, a coronavirus NP protein, a bunyavirus N
protein, an influenza M1 protein, a paramyxovirus M protein, an
arenavirus Z protein, a cytomegalovirus (CMV) core protein, a
herpes simplex virus (HSV) core protein, Vesicular Stomatitis Virus
(VSV) M protein, an Ebola Virus VP40 protein, a Lassa Fever Virus Z
protein, and a combination thereof.
37. The immunogenic composition of claim 36, wherein the retrovirus
gag protein is selected from the group consisting of; a Human
Immunodeficiency Virus (HIV) Gag protein, a Simian Immunodeficiency
Virus (SIV) Gag protein, a human foamy virus Gag protein, and a
Murine Leukemia Virus (MuLV) Gag protein.
38. The immunogenic composition of claim 33, wherein the at least
one viral surface envelope surface glycoprotein is selected from
the group consisting of: a retrovirus/lentivirus glycoprotein, a
bunyavirus glycoprotein, a coronavirus glycoprotein, an arenavirus
glycoprotein, a filovirus glycoprotein, an influenza virus
glycoprotein, a paramyxovirus glycoprotein, a rhabdovirus
glycoprotein, an alphavirus glycoprotein, a flavivirus
glycoprotein, a cytomegalovirus glycoprotein, a herpes virus
glycoprotein, and a combination thereof.
39. The immunogenic composition of claim 38, wherein the retrovirus
glycoprotein is selected from the group consisting of: a human
immunodeficiency virus (HIV) glycoprotein, a simian
immunodeficiency virus (SIV) glycoprotein, a simian-human
immunodeficiency virus (SHIV) glycoprotein, a feline
immunodeficiency virus (FIV) glycoprotein, a feline leukemia virus
glycoprotein, a bovine immunodeficiency virus glycoprotein, a
bovine leukemia virus glycoprotein, an equine infectious anemia
virus glycoprotein, a human T-cell leukemia virus glycoprotein, a
mouse mammary tumor virus envelope glycoprotein (MMTV), a human
foamy virus glycoprotein, and a combination thereof.
40. The immunogenic composition of claim 38, wherein the viral
surface envelope surface glycoprotein is further selected from the
group consisting of: an influenza virus glycoprotein, a Respiratory
syncytial virus (RSV) glycoprotien, a Lassa Fever virus
glycoprotein, an Ebola Virus glycoprotein, a Marburg virus
glycoprotein, a VSV glycoprotein, a rabies virus glycoprotein, a
hepatitis virus glycoprotein, a herpes virus glycoprotein, a CMV
glycoprotein, and a combination thereof.
41. The immunogenic composition of claim 23, wherein the virus-like
carrier comprises an influenza hemagglutinin, a matrix protein M1,
and a modified bacterial flagellin adjuvant polypeptide, wherein
the modified bacterial flagellin adjuvant comprises a heterologous
transmembrane-cytoplasmic tail and is incorporated into the
virus-like carrier, and wherein the virus-like carrier is a
virus-like particle or a virosome.
42. The immunogenic composition of claim 22, further comprising a
pharmacologically acceptable carrier.
43. A method of generating an immunological response in an animal
or human host, comprising: exposing an animal or human host to an
immunogen and a virus-like carrier, wherein the virus-like carrier
is a virus-like particle or a virosome, and wherein the virus-like
carrier comprises an adjuvant polypeptide comprising a host cell
Toll-like receptor ligand polypeptide derived from a bacterial or
protozoal flagellum polypeptide, and at least one heterologous
peptide selected from the group consisting of: a
transmembrane-cytoplasmic tail polypeptide and a heterologous
signal peptide; thereby generating in the recipient host an immune
response directed against the immunogen.
44. The method of claim 43, wherein the immunogen is incorporated
into the virus-like carrier.
45. The method of claim 43, further comprising: delivering to the
recipient host or host cell at least one expression vector, wherein
the at least one expression vector or a multiplicity of expression
vectors comprise at least one polynucleotide encoding at least one
polypeptide selected from the group consisting of: a viral core
protein, a viral surface envelope glycoprotein, and an adjuvant
molecule, wherein each of the polynucleotide or polynucleotides is
operably linked to an expression control region; expressing in the
recipient host or host cell at least one viral surface envelope
glycoprotein, and at least one adjuvant molecule, thereby
assembling a virosome virus-like carrier.
46. The method of claim 45, wherein an expression vector further
comprises a polynucleotide encoding a viral core protein, wherein
the viral core protein is incorporated into a virus-like
particle.
47. The method of claim 45, wherein an expression vector is
selected from the group consisting of: a plasmid, a cosmid, a viral
vector, an artificial chromosome, a mini-chromosome, a baculovirus
vector, a modified vaccinia Ankara (MVA) vector, a recombinant
poxvirus vector, a recombinant VSV vector, a recombinant adenovirus
expression systems, an alphavirus vector, a paramyxovirus vector,
and a combination thereof.
48. A method of immunizing a host comprising: co-expressing in one
or more host cells at least one viral surface envelope surface
glycoprotein, and at least one adjuvant molecule; whereby the at
least one viral surface envelope glycoprotein and the adjuvant
molecule assemble to form a virus-like carrier, and wherein the at
least one adjuvant molecule is a mammalian toll-like receptor
ligand molecule.
49. The method of claim 48, further comprising co-expressing a
viral core protein, wherein the viral core protein is assembled
into the virus-like carrier, thereby forming a virus-like
particle.
50. The method of claim 48, wherein the at least one adjuvant
molecule is a bacterial flagellin molecule.
51. The method of claim 48, wherein the virus-like particle is a
chimeric virus-like particle comprising an influenza hemagglutinin,
a matrix protein M1, and a modified bacterial flagellin adjuvant
polypeptide, wherein the modified bacterial flagellin comprises a
heterologous transmembrane-cytoplasmic tail and is incorporated
into the chimeric virus-like particle, the chimeric virus-like
particle inducing an immune response in the animal or human host
and thereby inhibiting the development of an influenza infection in
the host.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Applications No. 61/014,130 filed Dec. 17, 2007 (Ti:
IMMUNOGENIC COMPOSITIONS AND METHODS OF USE THEREOF), and
61/078,815 filed Jul. 8, 2008 (Ti: IMMUNOGENIC COMPOSITIONS AND
METHODS OF USE THEREOF), the complete disclosures of which are
incorporated herein by reference in their entireties.
BACKGROUND
[0003] The innate immune responses of a host are the first line of
defense against infectious disease. The principal challenge for the
host is to detect the pathogen and mount a rapid defensive
response. A group of proteins that comprise the Toll-like Receptor
(TLR) family performs this role in vertebrate organisms. The TLRs
are key proteins that allow mammals, whether immunologically naive
or experienced, to detect microbes. For instance, TLR5 recognizes
bacterial flagellin, the major structural protein of flagella.
Flagellin is a 55-kD protein monomer obtained from bacterial
flagella, polymeric rod-like appendages that extend from the outer
membrane of Gram-negative bacteria. Gram negative flagellin is a
potent inducer of innate immune effectors, such as cytokines and
nitric oxide. Vaccines that induce mucosal immunity are likely to
provide the most effective protection against pathogens, such as
influenza and other viruses.
[0004] Since the outbreak of a highly pathogenic avian influenza
virus (HPAI) H5N1 variant in 1997 in Hong Kong, there have been
increased concerns about the threat of a new pandemic that may
cause widespread fatal infection in humans. Although the
transmission of avian influenza viruses from birds to humans is a
rare event, both the continuing increase of infected human cases
and the high mortality rates suggest the persisting threat of an
H5N1 pandemic. There is evidence that the 1918 pandemic virus,
which caused an estimated 40 million deaths, was an avian virus
directly adapted to humans. Although two classes of antiviral drugs
targeting the viral matrix protein M2 and neuraminidase,
respectively, are available against influenza A viruses, financial
and supply limitations as well as frequent drug resistance may
limit the ability to utilize these drugs for preventing a new
pandemic. It is well recognized that an effective vaccine is the
primary strategy for protection against an emerging pandemic.
[0005] Currently, an inactivated influenza vaccine is the dominant
form used, although a live attenuated (cold-adapted) influenza
virus vaccine has also been introduced. However, the emergence of a
new pandemic strain could easily overwhelm the present capacity of
vaccine production, which is based on embryonic hens' eggs. There
are additional concerns that biosafety containment facilities may
be needed for virus-based vaccine production, and a period of 6 to
9 months would be required. A safe, convenient, and more reliable
alternative is needed as a countermeasure to the emerging
challenge.
[0006] As a new form of vaccine candidate, virus-like particles
(VLPs) have been reported to be potent vaccines for a variety of
pathogenic viruses (Koutsky et al., (2002) N. Engl. J. Med.
347:1645-1651; Leclerc et al., (2007) J. Virol. 81:1319-1326; Revaz
et al., (2007) Antivir. Res. 76:75-85; Skountzou et al., (2007) J.
Virol. 81:1083-1094; Zhang et al., (2007) Scand. J. Immunol.
65:320-3). VLPs elicit immune responses including both
B-cell-mediated antibody and specific T-cell-mediated cellular
responses to protect experimental animals against lethal influenza
virus challenge (Bright et al., (2007) Vaccine 25:3871-3878;
Galarza et al., (2005) Viral Immunol. 18:244-251; Matassov et al.,
(2007) Viral Immunol. 20:441-452; Pushko et al., (2005) Vaccine
23:5751-5759; Quan et al., (2007) J. Virol. 81:3514-3524). However,
though VLPs provide an attractive platform for designing vaccines
against a possible new influenza virus pandemic strain, they
resemble the current vaccines in inducing immune responses that are
predominantly subtype specific. An important advance would be the
development of new vaccines with enhanced breadth of immunity,
which could potentially be used to prevent infection by newly
emerging variants, including influenza viruses of other
subtypes.
SUMMARY
[0007] Briefly described, embodiments of this disclosure, among
others, encompass compositions and methods for generating immune
responses in an animal or human host. Embodiments of the
compositions of the disclosure encompass proteins derived from the
surface proteins of bacteria and protozoa, and in particular the
flagellum component flagellin, and which have adjunctival
properties when administered in conjunction with an immunogen.
Embodiments of the compositions of the disclosure, therefore, are
modified surface proteins that incorporate a heterologous
transmembrane-cytoplasmic domain allowing the peptides to be
incorporated into virus-like particles.
[0008] An embodiment of the disclosure, therefore, provide adjuvant
polypeptides comprising a first region comprising a heterologous
signal peptide; a second region comprising at least one domain
capable of selectively interacting with a Toll-like receptor of an
animal or human cell, and wherein the domain is capable of
increasing an immune response in a recipient host; and at least one
heterologous region selected from the group consisting of a signal
peptide region and a transmembrane-cytoplasmic tail region.
[0009] An embodiment of the disclosure may comprise a first region,
wherein the first region comprises a signal peptide; a second
region, where the second region may comprise at least one domain
capable of selectively interacting with a Toll-like receptor of an
animal or human cell, and where the domain may be capable of
increasing an immune response in a recipient host; and a third
region, where the third region comprises a
transmembrane-cytoplasmic tail peptide. In embodiments of this
aspect of the disclosure, the at least one domain that is capable
of selectively interacting with a Toll-like receptor of an animal
or human cell may be derived from a surface protein of a bacterial
species, or of a protozoal species.
[0010] In some embodiments of the adjuvant polypeptide, the surface
protein may be a protein of a bacterial or a protozoal flagellum,
or a fragment thereof. In an embodiment, the surface protein is a
flagellin of the bacterial species Salmonella enteritidis.
[0011] Another aspect of the disclosure provides nucleic acid
molecules comprising a region encoding a bacterial flagellin
polypeptide, or a fragment thereof, where the flagellin polypeptide
or fragment thereof comprises at least one domain capable of
specifically interacting with a Toll-like receptor of an animal or
human cell; and at least one region selected from the group
consisting of: a region encoding a heterologous signal peptide, and
a region encoding a transmembrane-cytoplasmic tail capable of being
incorporated into a virus-like particle or virosome.
[0012] In embodiments of this aspect of the disclosure, the
heterologous signal peptide may be a bee melittin signal peptide,
and the trans-membrane-cytoplasmic tail may be from an influenza
virus hemagglutinin.
[0013] In some embodiments of the disclosure, the nucleic acid
molecule may be operably inserted into a nucleic acid expression
vector.
[0014] Another aspect of the disclosure provides immunogenic
compositions comprising: an adjuvant polypeptide comprising at
least one region capable of selectively interacting with a
Toll-like receptor protein of a host; and an immunogen capable of
inducing an immune response in a recipient host.
[0015] In an embodiment of this aspect of the disclosure, the
immunogenic compositions may further comprise a virus-like carrier
selected from a virus-like particle and a virosome, and wherein the
adjuvant polypeptide and the immunogen may be incorporated in the
virus-like particle or the virosome. In these embodiments of the
disclosure, the adjuvant polypeptide may be incorporated into the
virus-like particle or virosome.
[0016] In still other embodiments of the disclosure, the virus-like
carrier may comprise: a viral core protein capable of
self-assembling into a virus-like particle (VLP); at least one
viral surface envelope glycoprotein expressed on the surface of the
VLP; and at least one adjuvant molecule expressed on the surface of
the VLP, where the adjuvant molecule may comprise a
membrane-anchored form of a bacterial or protozoal surface
component that is a mammalian Toll-like receptor (TLR) ligand
molecule. In these embodiments, the viral core protein and at least
one viral surface envelope glycoprotein may be from different
viruses.
[0017] Still another aspect of the disclosure provides methods of
generating an immunological response in an animal or human
comprising: exposing the immune system of an animal or human host
to an immunogen and a virus-like carrier, wherein the virus-like
carrier is a virus-like particle or a virosome, and wherein the
virus-like carrier comprises an adjuvant polypeptide comprising a
host cell Toll-like receptor ligand polypeptide derived from a
bacterial or protozoal flagellum polypeptide, and at least one
heterologous peptide selected from the group consisting of a
transmembrane-cytoplasmic tail polypeptide and a heterologous
signal peptide; thereby generating in the recipient host an immune
response directed against the immunogen.
[0018] In embodiments of this aspect of the disclosure, delivering
to the recipient host or host cell at least one expression vector,
wherein the at least one expression vector or a multiplicity of
expression vectors comprise at least one polynucleotide encoding at
least one polypeptide selected from the group consisting of: a
viral core protein, a viral surface envelope glycoprotein, and an
adjuvant molecule, wherein each of the polynucleotide or
polynucleotides is operably linked to an expression control region;
expressing in the recipient host or host cell at least one viral
surface envelope glycoprotein, and at least one adjuvant molecule,
thereby assembling a virosome virus-like carrier.
[0019] In other embodiments, an expression vector may comprise a
polynucleotide encoding a viral core protein, wherein the viral
core protein is incorporated into a virus-like particle.
[0020] Yet another aspect of the disclosure are methods of
immunizing a host comprising: co-expressing a viral core protein,
at least one viral surface envelope surface glycoprotein, and at
least one adjuvant molecule in one or more host cells; whereby the
viral core protein, at least one viral surface envelope
glycoprotein, assemble to form a virus-like particle, and wherein
the at least one adjuvant molecule is mammalian toll-like receptor
ligand molecule.
DESCRIPTION OF THE DRAWINGS
[0021] Many aspects of the disclosure can be better understood with
reference to the following drawings. The components in the drawings
are not necessarily to scale, emphasis instead being placed upon
clearly illustrating the principles of the present disclosure.
Moreover, in the drawings, like reference numerals designate
corresponding parts throughout the several views.
[0022] FIG. 1A shows a digital image of a Coomassie Blue stained
electrophoretic gel analysis of Salmonella flagellin expressed and
isolated from E. coli. Lane 1: molecular weight markers; Lane 2:
soluble flagellin expressed by an E. coli culture and purified
therefrom with affinity chorography. FIG. 1B is a digital image of
a Western blot analysis showing soluble flagellin.
[0023] FIG. 2 shows a Coomassie Blue stained electrophoretic gel
analysis (10% SDS polyacrylamide gel) of flagellin proteins. The
samples shown were from individually prepared batches of flagellin
proteins. The band on the extreme left is monomeric flagellin
having a size of 50-60 kDa.
[0024] FIG. 3 is a bar graph illustrating the immune responses
generated by recombinant monomeric soluble flagellin from
Salmonella enteritidis gene expressed in an E. coli cell system,
and purified polymeric flagellin isolated from Salmonella
enteritidis bacteria. The flagellins were tested for immunogenicity
in female Balb/c 4-6 old mice. Plates were coated with anti-S.
enteritidis flagellin-specific monoclonal antibody.
[0025] FIG. 4A is a bar graph illustrating the results of
immunization of mice intranasally with 10 .mu.g of whole
inactivated influenza virus (A/PR/8/34, PR8) combined with 1 .mu.g
of polymeric (FliC poly), or monomeric (FliC mono) flagellin.
[0026] FIG. 4B is a bar graph of results from intramuscular
immunization illustrating that inactivated influenza virus mixed
with monomeric flagellin induced a 3.5-fold higher antibody titer
when compared to inactivated influenza virus vaccine alone.
Inactivated influenza virus mixed with the polymeric form of
flagellin induced a 6-fold higher IgG level than did the PR8 group,
and at least 1.5-fold higher titer than did the vaccine composition
of PR8 with monomeric flagellin after a single immunization.
[0027] FIG. 5 schematically illustrates an embodiment of a chimeric
flagellin nucleic acid molecule, and the domains therein.
[0028] FIG. 6 illustrates the nucleotide sequences SEQ ID NOS.: 1-8
of primers used in the construction of a nucleic acid molecule
encoding a membrane-anchored flagellin-having, at the N terminus of
the flagellin-encoding nucleic acid region, the coding sequence for
the signal peptide (SP) of the honeybee protein melittin and, at
the C-terminus of the flagellin-encoding nucleic acid, the
transmembrane-cytoplasmic tail (TM-CT) from influenza hemagglutinin
(HA).
[0029] FIG. 7 illustrates the nucleotide sequence SEQ ID NO.: 9 of
the nucleic acid molecule encoding a membrane-anchored
flagellin-having, at the N terminus of the flagellin-encoding
nucleic acid region, the coding sequence for the signal peptide
(SP) of the honeybee protein melittin (capitalized and underlined)
and, at the C-terminus of the flagellin-encoding nucleic acid, the
transmembrane-cytoplasmic tail (TM-CT) from influenza hemagglutinin
(HA).
[0030] FIG. 8 illustrates the amino acid sequence SEQ ID NO.: 10
derived from the nucleotide sequence SEQ ID NO.: 9. Potential
N-linked glycosylation sites are indicated by arrows.
[0031] FIG. 9 is a digital image of a gel electrophoretic analysis
of cellular and cell surface expression of membrane-anchored
flagellin. Surface expression of the membrane-anchored flagellin
was detected by cell surface biotinylation. Lane: 1, cell lysate
from 5' and 3' ends membrane-anchored flagellin; Lane 2, mock rBV
(rBV expressing human immunodeficiency virus Gag)-infected
cells.
[0032] FIG. 10 is a digital image illustrating the optimization of
VLP production: Sf9 cells were infected with rBVs expressing HA,
M1, and flagellin at different MOI as designated below the image.
HA and M1 bands were probed with mouse anti-influenza serum. The
band below M1 was variable in different VLP preparations and may
represent a degradation product. Membrane-anchored flagellin (FliC)
was probed with rabbit anti-flagellin-specific polyclonal
antibody.
[0033] FIG. 11 is a digital electron microscopy image of influenza
VLPs. Influenza VLPs containing flagellin, HA, and M were
negatively stained
[0034] FIG. 12 is a digital image of a Western blot where 10 .mu.g
aliquots of flagellin-containing VLPs were left untreated (lane 2)
or were digested by PNGase F (lane 3) or endo-H (lane 4). Lane 1 is
cell lysate from cells infected by membrane-anchored flagellin
expressing rBV.
[0035] FIG. 13 is a graph of TLR-5 agonist activity of
membrane-anchored flagellin. TLR-5-positive and -negative RAW264.7
cells were activated with soluble flagellin (sFliC) and
flagellin-containing VLPs (FliC/HA/M1 VLPs), respectively. Standard
HA/M1 VLPs were used as controls. TLR-5-specific bioactivity was
expressed by the production by TNF-.alpha. of TRL-5-positive cells,
from which was subtracted that of TLR-5-negative cells stimulated
by flagellin, flagellin-containing VLPs, or standard HA/M1 VLPs at
the same concentration. Data represent means.+-.standard errors
from triplicate repeats.
[0036] FIGS. 14A-14D are bar graphs illustrating serum IgG and IgG
isotype endpoint titers. Serum antibodies specific for influenza
A/PR8 virus were determined. The highest serum dilution (n-fold)
which gave an OD.sub.450 twice that of naive mice was designated as
the serum antibody endpoint titer. Data are the mean.+-.standard
deviation (SD) of six mice per group, and were analyzed by an
unpaired t test. A two-tailed P value of 0.05 is designated as a
significant difference. FIG. 14A: Serum IgG (*, P<0.05); FIG.
14B: IgG1; FIG. 14C: IgG2a (*, P<0.05); FIG. 14D: IgG2b (*,
P<0.05) (D). sFliC: soluble flagellin.
[0037] FIGS. 15A-15C are graphs illustrating neutralization and HI
titers against influenza A/PR8 virus, and the effect of
pre-existing anti-flagellin immunity.
[0038] FIG. 15A is a graph illustrating neutralization activities
determined using the capacity of sera to neutralize plaque
formation by influenza PR8 virus in MDCK cell cultures. Serial
dilutions of sera were incubated with influenza PR8 virus (about
100PFU) at 37.degree. C. for 1 h. A standard plaque reduction assay
was performed using MDCK cells.
[0039] FIG. 15B is a bar graph illustrating HI titers of sera
determined using the capacity of sera to inhibit virus
hemagglutination of chicken red blood cells (*, P<0.05).
[0040] FIG. 15C is a bar graph illustrating the preexisting
anti-flagellin IgG titer as determined with ELISA. Six mice were
pre-immunized twice intramuscularly at a 4-week interval with 10
.mu.g of soluble recombinant flagellin and subsequently immunized
twice with 10 .mu.g cVLPs at a 4-week interval. A six-mouse group
without pre-immunization was used as the control. Serum
anti-flagellin and anti-inactivated PR8 virus IgG titers were
determined by ELISA. For flagellin-specific IgG titers, microplates
were coated with 100 .mu.l of recombinant flagellin per well at 5
.mu.g/ml. Representative data are the mean.+-.SD from six mice in
each group. sFliC, soluble flagellin.
[0041] FIGS. 16A and 16B are bar graphs illustrating the serum IgG
endpoint and HI titers, respectively, against the heterosubtypic
virus A/Philippines (H3N2), respectively. Data depict the
mean.+-.SD from six mice per group (*, P<0.05). sFliC, soluble
flagellin.
[0042] FIG. 17 illustrates peptide sequences SEQ ID NOs.:
11-18.
[0043] FIGS. 18A-18D are bar graphs illustrating cytokine secretion
from immunized mouse splenocytes. Splenocytes were isolated from
immunized six-mouse groups 3 weeks after the boosting immunization.
Cells (1.times.10.sup.6) were seeded into 96-well cell culture
plates with 200 .mu.l RPMI 1640 medium. The MHC-I- or
MHC-II-specific HA peptides of A/PR8 virus were added into cell
culture medium, and secreted cytokines were determined. Data depict
the mean.+-.SD of six mice per group with similar results in
triplicate assays (*, +, or , P<0.05). FIG. 18A: IL-2; FIG. 18B:
IFN-.gamma.; FIG. 18C: TNF-.alpha.; FIG. 18D: IL-4. FliC:
flagellin.
[0044] FIGS. 19A-19D are graphs illustrating protection from
challenge with A/PR8 or A/Philippines virus. Mouse groups
containing six mice were challenged with 40.times.LD.sub.50 of
PR8(H.sub.1N.sub.1) or A/Philippines(H.sub.3N.sub.2) virus. Mice
were monitored daily for 14 days for body weight changes (FIG.
19A), or percentages of survival (FIG. 19B) after PR8 virus
challenge, or for body weight changes (FIG. 19C) and percentages of
survival (FIG. 19D) after A/Philippines virus challenge. sFliC:
soluble flagellin.
[0045] FIG. 20 is a bar graph illustrating lung viral load on day 4
post challenge. Six mice in each group were challenged with
40.times.LD.sub.50 of PR8 (H1N1) or A/Philippines(H3N2) virus.
Mouse lung samples were collected on day 4 post challenge. Six
lungs in each group were pooled, ground, and cleared in 6 ml of
DMEM. Lung virus loads were determined using a standard plaque
assay with MDCK cells. Bars represent mean virus titers standard
errors from three independent assays (*, P<0.05). FliC,
flagellin.
[0046] FIG. 21 illustrates modified flagellin variants with an
influenza HA membrane anchor peptide.
[0047] FIG. 22 illustrates modified flagellin variants with a mouse
mammary tumor virus (MMTV) Env membrane anchor.
[0048] FIG. 23 illustrates modified flagellin variants with an
influenza HA membrane anchor peptide and a hexahistidine tag is
fused to C-terminus.
[0049] FIG. 24 is an illustration of a representative virus like
particle (VLP) according to the present disclosure.
[0050] FIG. 25 is a schematic diagram of membrane-anchored
flagellin with a tandem of four repeats of M2e (SEQ ID NO.: 19)
inserted (tFliC-4.times.M2e).
[0051] FIG. 26 is a digital image of a stained gel analysis of
cellular and cell surface expression of membrane-anchored flagellin
with M2e inserted therein.
DETAILED DESCRIPTION
[0052] Before the present disclosure is described in greater
detail, it is to be understood that this disclosure is not limited
to particular embodiments described, and as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0053] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the disclosure.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the disclosure, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the disclosure.
[0054] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and materials similar or equivalent to those
described herein can also be used in the practice or testing of the
present disclosure, the preferred methods and materials are now
described.
[0055] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present disclosure
is not entitled to antedate such publication by virtue of prior
disclosure. Further, the dates of publication provided could be
different from the actual publication dates that may need to be
independently confirmed.
[0056] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present disclosure. Any recited
method can be carried out in the order of events recited or in any
other order that is logically possible.
[0057] Embodiments of the present disclosure will employ, unless
otherwise indicated, techniques of medicine, organic chemistry,
biochemistry, molecular biology, pharmacology, and the like, which
are within the skill of the art. Such techniques are explained
fully in the literature.
[0058] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an," and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a cell" includes a plurality or
multiplicity of cells. In this specification and in the claims that
follow, reference will be made to a number of terms that shall be
defined to have the following meanings unless a contrary intention
is apparent.
[0059] As used herein, the following terms have the meanings
ascribed to them unless specified otherwise. In this disclosure,
"comprises," "comprising," "containing," "having," and the like can
have the meaning ascribed to them in U.S. Patent law and can mean "
includes," "including," and the like; "consisting essentially of"
or "consists essentially" or the like, when applied to methods and
compositions encompassed by the present disclosure refers to
compositions like those disclosed herein, but which may contain
additional structural groups, composition components or method
steps (or analogs or derivatives thereof as discussed above). Such
additional structural groups, composition components or method
steps, etc., however, do not materially affect the basic and novel
characteristic(s) of the compositions or methods, compared to those
of the corresponding compositions or methods disclosed herein.
"Consisting essentially of" or "consists essentially" or the like,
when applied to methods and compositions encompassed by the present
disclosure have the meaning ascribed in U.S. Patent law and the
term is open-ended, allowing for the presence of more than that
which is recited so long as basic or novel characteristics of that
which is recited is not changed by the presence of more than that
which is recited, but excludes prior art embodiments.
[0060] It should be noted that ratios, concentrations, amounts, and
other numerical data may be expressed herein in a range format. It
is to be understood that such a range format is used for
convenience and brevity, and thus, should be interpreted in a
flexible manner to include not only the numerical values explicitly
recited as the limits of the range, but also to include all the
individual numerical values or sub-ranges encompassed within that
range as if each numerical value and sub-range is explicitly
recited. To illustrate, a concentration range of "about 0.1% to
about 5%" should be interpreted to include not only the explicitly
recited concentration of about 0.1 wt % to about 5 wt %, but also
include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and
the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the
indicated range. The term "about" can include .+-.1%, .+-.2%,
.+-.3%, .+-.4%, .+-.5%, .+-.6%, .+-.7%, .+-.8%, .+-.9%, or .+-.10%,
or more of the numerical value(s) being modified.
[0061] Prior to describing the various embodiments, the following
definitions are provided and should be used unless otherwise
indicated.
Definitions:
[0062] In describing and claiming the disclosed subject matter, the
following terminology will be used in accordance with the
definitions set forth below.
[0063] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art of molecular biology. Although methods
and materials similar or equivalent to those described herein can
be used in the practice or testing of the present disclosure,
suitable methods and materials are described herein.
[0064] Further definitions are provided in context below. Unless
otherwise defined, all technical and scientific terms used herein
have the same meaning as commonly understood by one of ordinary
skill in the art of molecular biology. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present disclosure, suitable
methods and materials are described herein.
[0065] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those of skill in the art to which
this disclosure pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
necessarily be construed to represent a substantial difference over
what is generally understood in the art. The techniques and
procedures described or referenced herein are generally well
understood and commonly employed using conventional methodology by
those skilled in the art, such as, for example, the widely utilized
molecular cloning methodologies described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and
Current Protocols in Molecular Biology (Ausbel et al., eds., John
Wiley & Sons, Inc. 2001). As appropriate, procedures involving
the use of commercially available kits and reagents are generally
carried out in accordance with manufacturer defined protocols
and/or parameters unless otherwise noted.
[0066] As used herein, the term "adjuvant molecule" includes
bacterial surface proteins capable of eliciting an immune response
in a host. In particular the term includes bacterial surface
proteins capable of targeting a host Toll-like receptor (TLR)
protein, such as, but not limited to, a bacterial flagellin protein
that targets a host TLR5 protein. In particular embodiments, the
adjuvant molecule is a "membrane-anchored form" of the adjuvant
molecule which indicates that the adjuvant molecule has been
engineered to include a signal peptide (SP) and a membrane anchor
sequence to direct the transport and membrane orientation of the
protein. Thus, in embodiments, a membrane-anchored form of an
adjuvant molecule is a recombinant protein including a portion of
the bacterial protein (such as bacterial flagellin) fused to a SP
and membrane anchor sequence (e.g., the membrane-anchored form of
Salmonella flagellin described in Example 5 below).
[0067] The term "virus-like carrier" as used herein refers to
either a virus-like particle", a virosome, or both.
[0068] The term "virus-like particle" (VLPs) as used herein refers
to a membrane-surrounded viral core structure having viral envelope
proteins expressed on the VLP surface. In addition, adjuvant
molecules can be expressed on the VLP. Further, viral core proteins
are located within the membrane of the VLP. Additional components
of VLPs, as known in the art, can be included within or disposed on
the VLP. VLPs do not contain intact viral nucleic acids, and they
are non-infectious. Desirably, there is sufficient viral surface
envelope glycoprotein and/or adjuvant molecules expressed, at least
in part, on the surface of the VLP so that when a VLP preparation
is formulated into an immunogenic composition and administered to
an animal or human, an immune response (cell-mediated or humoral)
is raised.
[0069] The term "virosome" as used herein refers to a virus-like
carrier that is similar to a virus-like particle, except that a
virosome does not contain a viral core protein.
[0070] A "chimeric virus-like particle" as used herein, can be
defined as a VLP having at least one viral surface envelope
glycoprotein incorporated into the VLP, wherein the viral core
protein and at least one viral surface envelope glycoprotein are
from different viruses. A chimeric VLP, as used herein, may include
additional viral surface envelope glycoproteins that are from the
same or different virus as the viral core protein, so long as at
least one is different.
[0071] A "phenotypically mixed" VLP, as used herein, can be defined
as a VLP having at least two different surface molecules (e.g.,
surface envelope glycoproteins and/or adjuvant molecules)
incorporated into the VLP. A phenotypically mixed VLP, as used
herein, may include additional surface molecules that are from the
same or different source as the viral core protein, so long as at
least one is different.
[0072] A "truncated" viral surface envelope glycoprotein is one
having less than a full length protein (e.g., a portion of the
cytoplasmic domain has been removed), which retains surface
antigenic determinants against which an immune response is
generated, preferably a protective immune response, and it retains
sufficient envelope sequence for proper membrane insertion. The
skilled artisan can produce truncated virus envelope proteins using
recombinant DNA technology and virus coding sequences, which are
readily available to the public.
[0073] As used herein "chimeric" viral surface glycoproteins are
ones that contain at least a portion of the extracellular domain of
a viral surface glycoprotein of one virus and at least a portion of
the transmembrane and/or cytoplasmic domains and/or signal peptide
sequence of a different transmembrane glycoprotein from a different
virus or other organism. Such chimeric proteins retain surface
antigenic determinants against which an immune response is
generated, preferably a protective immune response, and retain
sufficient envelope sequence for proper precursor processing and
membrane insertion. The operably linked transmembrane and/or
cytoplasmic domains will serve to preferentially interact with the
desired viral core protein components in VLP assembly, and thus
increase the levels of viral surface glycoprotein in VLPs. The
skilled artisan can produce chimeric viral surface glycoproteins
using recombinant DNA technology and protein coding sequences,
techniques known to those of skill in the art and available to the
public. Such chimeric viral surface glycoproteins may be useful for
increasing the level of incorporation of viral glycoproteins in
VLPs for viruses that may naturally have low levels of
incorporation.
[0074] The term "polypeptides" includes proteins and fragments
thereof. Polypeptides are disclosed herein as amino acid residue
sequences. Those sequences are written left to right in the
direction from the amino to the carboxy terminus. In accordance
with standard nomenclature, amino acid residue sequences are
denominated by either a three letter or a single letter code as
indicated as follows: Alanine (Ala, A), Arginine (Arg, R),
Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C),
Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G),
Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine
(Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline
(Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W),
Tyrosine (Tyr, Y), and Valine (Val, V).
[0075] "Variant" refers to a polypeptide or polynucleotide that
differs from a reference polypeptide or polynucleotide, but retains
essential properties. A typical variant of a polypeptide differs in
amino acid sequence from another, reference polypeptide. Generally,
differences are limited so that the sequences of the reference
polypeptide and the variant are closely similar overall
(homologous) and, in many regions, identical. A variant and
reference polypeptide may differ in amino acid sequence by one or
more modifications (e.g., substitutions, additions, and/or
deletions). A substituted or inserted amino acid residue may or may
not be one encoded by the genetic code. A variant of a polypeptide
may be naturally occurring such as an allelic variant, or it may be
a variant that is not known to occur naturally.
[0076] Modifications and changes can be made in the structure of
the polypeptides of this disclosure and still result in a molecule
having similar characteristics as the polypeptide (e.g., a
conservative amino acid substitution). For example, certain amino
acids can be substituted for other amino acids in a sequence
without appreciable loss of activity. Because it is the interactive
capacity and nature of a polypeptide that defines that
polypeptide's biological functional activity, certain amino acid
sequence substitutions can be made in a polypeptide sequence and
nevertheless obtain a polypeptide with like properties.
[0077] In making such changes, the hydropathic index of amino acids
can be considered. The importance of the hydropathic amino acid
index in conferring interactive biologic function on a polypeptide
is generally understood in the art. It is known that certain amino
acids can be substituted for other amino acids having a similar
hydropathic index or score and still result in a polypeptide with
similar biological activity. Each amino acid has been assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics. Those indices are: isoleucine (+4.5); valine
(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine
(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4);
threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
(-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5);
glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine
(-3.9); and arginine (-4.5).
[0078] It is believed that the relative hydropathic character of
the amino acid determines the secondary structure of the resultant
polypeptide, which in turn defines the interaction of the
polypeptide with other molecules, such as enzymes, substrates,
receptors, antibodies, antigens, and the like. It is known in the
art that an amino acid can be substituted by another amino acid
having a similar hydropathic index and still obtain a functionally
equivalent polypeptide. In such changes, the substitution of amino
acids whose hydropathic indices are within .+-.2 is preferred,
those within .+-.1 are particularly preferred, and those within
.+-.0.5 are even more particularly preferred.
[0079] Substitution of like amino acids can also be made on the
basis of hydrophilicity, particularly where the biologically
functional equivalent polypeptide or peptide thereby created is
intended for use in immunological embodiments. The following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); proline (-0.5.+-.1); threonine (-0.4); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4). It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent,
and in particular, an immunologically equivalent polypeptide. In
such changes, the substitution of amino acids whose hydrophilicity
values are within .+-.2 is preferred, those within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0080] As outlined above, amino acid substitutions are generally
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions that take one
or more of the foregoing characteristics into consideration are
well known to those of skill in the art and include, but are not
limited to (original residue: exemplary substitution): (Ala: Gly,
Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln:
Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val),
(Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr:
Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu). Embodiments
of this disclosure thus contemplate functional or biological
equivalents of a polypeptide as set forth above. In particular,
embodiments of the polypeptides can include variants having about
50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the
polypeptide of interest. The term "substantially homologous" is
used herein to denote polypeptides of the present disclosure having
about 50%, about 60%, about 70%, about 80%, about 90%, and
preferably about 95% sequence identity to the reference sequence.
Percent sequence identity is determined by conventional methods as
discussed above. In general, homologous polypeptides of the present
disclosure are characterized as having one or more amino acid
substitutions, deletions, and/or additions.
[0081] "Identity," as known in the art, is a relationship between
two or more polypeptide sequences, as determined by comparing the
sequences. In the art, "identity" also refers to the degree of
sequence relatedness between polypeptides as determined by the
match between strings of such sequences. "Identity" and
"similarity" can be readily calculated by known methods, including,
but not limited to, those described in Computational Molecular
Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988;
Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed.,
Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New
Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje,
G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov,
M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and
Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073,
(1988).
[0082] Preferred methods to determine identity are designed to give
the largest match between the sequences tested. Methods to
determine identity and similarity are codified in publicly
available computer programs. The percent identity between two
sequences can be determined by using analysis software (i.e.,
Sequence Analysis Software Package of the Genetics Computer Group,
Madison, Wis.) that incorporates the Needelman & Wunsch, (J.
Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST and XBLAST).
The default parameters are used to determine the identity for the
polypeptides of the present disclosure.
[0083] By way of example, a polypeptide sequence may be identical
to the reference sequence, that is be 100% identical, or it may
include up to a certain integer number of amino acid alterations as
compared to the reference sequence such that the % identity is less
than 100%. Such alterations are selected from: at least one amino
acid deletion, substitution (including conservative and
non-conservative substitution), or insertion, and wherein said
alterations may occur at the amino- or carboxy-terminus positions
of the reference polypeptide sequence or anywhere between those
terminal positions, interspersed either individually among the
amino acids in the reference sequence, or in one or more contiguous
groups within the reference sequence. The number of amino acid
alterations for a given % identity is determined by multiplying the
total number of amino acids in the reference polypeptide by the
numerical percent of the respective percent identity (divided by
100) and then subtracting that product from said total number of
amino acids in the reference polypeptide.
[0084] Conservative amino acid variants can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methyl-glycine, allo-threonine,
methylthreonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine,
nitro-glutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenyl-alanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. (Robertson, et al., J. Am. Chem. Soc., 113: 2722,
1991; Ellman, et al., Methods Enzymol., 202: 301, 1991; Chung, et
al., Science, 259: 806-9, 1993; and Chung, et al., Proc. Natl.
Acad. Sci. USA, 90: 10145-9, 1993). In a second method, translation
is carried out in Xenopus oocytes by microinjection of mutated mRNA
and chemically aminoacylated suppressor tRNAs (Turcatti, et al., J.
Biol. Chem., 271: 19991-8, 1996). Within a third method, E. coli
cells are cultured in the absence of a natural amino acid that is
to be replaced (e.g., phenylalanine) and in the presence of the
desired non-naturally occurring amino acid(s) (e.g.,
2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or
4-fluorophenylalanine). The non-naturally occurring amino acid is
incorporated into the protein in place of its natural counterpart.
(Koide, et al., Biochem., 33: 7470-6, 1994). Naturally occurring
amino acid residues can be converted to non-naturally occurring
species by in vitro chemical modification. Chemical modification
can be combined with site-directed mutagenesis to further expand
the range of substitutions (Wynn, et al., Protein Sci., 2: 395-403,
1993).
[0085] Furthermore, unless the context demands otherwise, the term
peptide, polypeptide and protein are used interchangeably to refer
to amino acids in which the amino acid residues are linked by
covalent peptide bonds or alternatively (where post-translational
processing has removed an internal segment) by covalent disulphide
bonds, etc. The amino acid chains can be of any length and comprise
at least two amino acids, they can include domains of proteins or
full-length proteins. Unless otherwise stated the terms peptide,
polypeptide, and protein also encompass various modified forms
thereof, including but not limited to glycosylated forms,
phosphorylated forms, etc.
[0086] As used herein, the term "polynucleotide" generally refers
to any polyribonucleotide or polydeoxribonucleotide, which may be
unmodified RNA or DNA or modified RNA or DNA. Thus, for instance,
polynucleotides as used herein refers to, among others, single- and
double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA
that is a mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. The terms "nucleic acid," "nucleic acid
sequence," or "oligonucleotide" also encompass a polynucleotide as
defined above.
[0087] In addition, "polynucleotide" as used herein refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from
different molecules. The regions may include all of one or more of
the molecules, but more typically involve only a region of some of
the molecules. One of the molecules of a triple-helical region
often is an oligonucleotide.
[0088] As used herein, the term polynucleotide includes DNAs or
RNAs as described above that contain one or more modified bases.
Thus, DNAs or RNAs with backbones modified for stability or for
other reasons are "polynucleotides" as that term is intended
herein. Moreover, DNAs or RNAs comprising unusual bases, such as
inosine, or modified bases, such as tritylated bases, to name just
two examples, are polynucleotides as the term is used herein.
[0089] It will be appreciated that a great variety of modifications
have been made to DNA and RNA that serve many useful purposes known
to those of skill in the art. The term polynucleotide as it is
employed herein embraces such chemically, enzymatically, or
metabolically modified forms of polynucleotides, as well as the
chemical forms of DNA and RNA characteristic of viruses and cells,
including simple and complex cells, inter alia.
[0090] By way of example, a polynucleotide sequence of the present
disclosure may be identical to the reference sequence, that is be
100% identical, or it may include up to a certain integer number of
nucleotide alterations as compared to the reference sequence. Such
alterations are selected from the group including at least one
nucleotide deletion, substitution, including transition and
transversion, or insertion, and wherein said alterations may occur
at the 5' or 3' terminus positions of the reference nucleotide
sequence or anywhere between those terminus positions, interspersed
either individually among the nucleotides in the reference sequence
or in one or more contiguous groups within the reference sequence.
The number of nucleotide alterations is determined by multiplying
the total number of nucleotides in the reference nucleotide by the
numerical percent of the respective percent identity (divided by
100) and subtracting that product from said total number of
nucleotides in the reference nucleotide. Alterations of a
polynucleotide sequence encoding the polypeptide may alter the
polypeptide encoded by the polynucleotide following such
alterations.
[0091] As used herein, DNA may be obtained by any method. For
example, the DNA includes complementary DNA (cDNA) prepared from
mRNA, DNA prepared from genomic DNA, DNA prepared by chemical
synthesis, DNA obtained by PCR amplification with RNA or DNA as a
template, and DNA constructed by appropriately combining these
methods.
[0092] As used herein, an "isolated nucleic acid" is a nucleic
acid, the structure of which is not identical to that of any
naturally occurring nucleic acid or to that of any fragment of a
naturally occurring genomic nucleic acid spanning more than three
genes. The term therefore covers, for example, (a) a DNA which has
the sequence of part of a naturally occurring genomic DNA molecule
but is not flanked by both of the coding sequences that flank that
part of the molecule in the genome of the organism in which it
naturally occurs; (b) a nucleic acid incorporated into a vector or
into the genomic DNA of a prokaryote or eukaryote in a manner such
that the resulting molecule is not identical to any naturally
occurring vector or genomic DNA; (c) a separate molecule such as a
cDNA, a genomic fragment, a fragment produced by polymerase chain
reaction (PCR), or a restriction fragment; and (d) a recombinant
nucleotide sequence that is part of a hybrid gene, i.e., a gene
encoding a fusion protein. Specifically excluded from this
definition are nucleic acids present in random, uncharacterized
mixtures of different DNA molecules, transfected cells, or cell
clones, e.g., as these occur in a DNA library such as a cDNA or
genomic DNA library.
[0093] The term "substantially pure" as used herein in reference to
a given polypeptide means that the polypeptide is substantially
free from other biological macromolecules. For example, the
substantially pure polypeptide is at least 75%, 80, 85, 95, or 99%
pure by dry weight. Purity can be measured by any appropriate
standard method known in the art, for example, by column
chromatography, polyacrylamide gel electrophoresis, or HPLC
analysis.
[0094] The DNA encoding the protein disclosed herein can be
prepared by the usual methods: cloning cDNA from mRNA encoding the
protein, isolating genomic DNA and splicing it, chemical synthesis,
and so on.
[0095] cDNA can be cloned from mRNA encoding the protein by, for
example, the method described below:
[0096] First, the mRNA encoding the protein is prepared from the
above-mentioned tissues or cells expressing and producing the
protein. mRNA can be prepared by isolating total RNA by a known
method such as guanidine-thiocyanate method (Chirgwin et al.,
Biochemistry, 18:5294, 1979), hot phenol method, or AGPC method,
and subjecting it to affinity chromatography using oligo-dT
cellulose or poly-U Sepharose.
[0097] Then, with the mRNA obtained as a template, cDNA is
synthesized, for example, by a well-known method using reverse
transcriptase, such as the method of Okayama et al (Mol. Cell.
Biol. 2:161 (1982); Mol. Cell. Biol. 3:280 (1983)) or the method of
Hoffman et al. (Gene 25:263 (1983)), and converted into
double-stranded cDNA. A cDNA library is prepared by transforming E.
coli with plasmid vectors, phage vectors, or cosmid vectors having
this cDNA or by transfecting E. coli after in vitro packaging.
[0098] The plasmid vectors used herein are not limited as long as
they are replicated and maintained in hosts. Any phage vector that
can be replicated in hosts can also be used. Examples of usually
used cloning vectors are pUC19, .lamda.gt10, Igt11, and so on. When
the vector is applied to immunological screening as mentioned
below, a vector having a promoter that can express a gene encoding
the desired protein in a host is preferably used.
[0099] cDNA can be inserted into a plasmid by, for example, the
method of Maniatis et al. (Molecular Cloning, A Laboratory Manual,
second edition, Cold Spring Harbor Laboratory, p. 1.53, 1989). cDNA
can be inserted into a phage vector by, for example, the method of
Hyunh et al. (DNA cloning, a practical approach, 1, p. 49 (1985)).
These methods can be simply performed by using a commercially
available cloning kit (for example, a product from Takara Shuzo).
The recombinant plasmid or phage vector thus obtained is introduced
into an appropriate host cell such as a prokaryote (for example, E.
coli: HB101, DH5a, MC1061/P3, etc).
[0100] Examples of a method for introducing a plasmid into a host
are, calcium chloride method, calcium chloride/rubidium chloride
method and electroporation method, described in Molecular Cloning,
A Laboratory Manual (second edition, Cold Spring Harbor Laboratory,
p. 1.74 (1989)). Phage vectors can be introduced into host cells
by, for example, a method in which the phage DNAs are introduced
into grown hosts after in vitro packaging. In vitro packaging can
be easily performed with a commercially available in vitro
packaging kit (for example, a product from Stratagene or
Amersham).
[0101] An "expression vector" is useful for expressing the DNA
encoding the protein used herein and for producing the protein. The
expression vector is not limited as long as it expresses the gene
encoding the protein in various prokaryotic and/or eukaryotic host
cells and produces this protein. Examples thereof are pMAL C2,
pEF-BOS (Nucleic Acids Res. 18:5322 (1990) and so on), pME18S
(Experimental Medicine: SUPPLEMENT, "Handbook of Genetic
Engineering" (1992)), etc.
[0102] When bacteria, particularly E. coli are used as host cells,
an expression vector generally comprises, at least, a
promoter/operator region, an initiation codon, the DNA encoding the
protein termination codon, terminator region, and replicon.
[0103] When yeast, animal cells, or insect cells are used as hosts,
an expression vector is preferably comprising, at least, a
promoter, an initiation codon, the DNA encoding the protein and a
termination codon. It may also comprise the DNA encoding a signal
peptide, enhancer sequence, 5'- and 3'-untranslated region of the
gene encoding the protein, splicing junctions, polyadenylation
site, selectable marker region, and replicon. The expression vector
may also contain, if required, a gene for gene amplification
(marker) that is usually used.
[0104] A promoter/operator region to express the protein in
bacteria comprises a promoter, an operator, and a Shine-Dalgarno
(SD) sequence (for example, AAGG). For example, when the host is
Escherichia, it preferably comprises Trp promoter, lac promoter,
recA promoter, lambda.PL promoter, b 1pp promoter, tac promoter, or
the like. Examples of a promoter to express the protein in yeast
are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, and so
on. When the host is Bacillus, examples thereof are SL01 promoter,
SP02 promoter, penP promoter, and so on. When the host is a
eukaryotic cell such as a mammalian cell, examples thereof are
SV40-derived promoter, retrovirus promoter, heat shock promoter,
and so on, and preferably SV-40 and retrovirus-derived one. As a
matter of course, the promoter is not limited to the above
examples. In addition, using an enhancer is effective for
expression.
[0105] A preferable initiation codon is, for example, a methionine
codon (ATG).
[0106] A commonly used termination codon (for example, TAG, TAA,
TGA) is exemplified as a termination codon. Usually, used natural
or synthetic terminators are used as a terminator region.
[0107] A "replicon" means a DNA capable of replicating the whole
DNA sequence in host cells, and includes a natural plasmid, an
artificially modified plasmid (DNA fragment prepared from a natural
plasmid), a synthetic plasmid, and so on. Examples of preferable
plasmids are pBR322 or its artificial derivatives (DNA fragment
obtained by treating pBR322 with appropriate restriction enzymes)
for E. coli, yeast 2.mu. plasmid or yeast chromosomal DNA for
yeast, and pRSVneo ATCC 37198, pSV2dhfr ATCC 37145, pdBPV-MMTneo
ATCC 37224, pSV2neo ATCC 37149, and such for mammalian cells.
[0108] An enhancer sequence, polyadenylation site, and splicing
junction that are usually used in the art, such as those derived
from SV40 can also be used.
[0109] A selectable marker usually employed can be used according
to the usual method. Examples thereof are resistance genes for
antibiotics, such as tetracycline, ampicillin, or kanamycin.
[0110] The expression vector used herein can be prepared by
continuously and circularly linking at least the above-mentioned
promoter, initiation codon, DNA encoding the protein, termination
codon, and terminator region, to an appropriate replicon. If
desired, appropriate DNA fragments (for example, linkers,
restriction sites, and so on), can be used by the usual method such
as digestion with a restriction enzyme or ligation using T4 DNA
ligase.
[0111] As used herein, "transformants" can be prepared by
introducing the expression vector mentioned above into host
cells.
[0112] As used herein, "host" cells are not limited as long as they
are compatible with an expression vector mentioned above and can be
transformed. Examples thereof are various cells such as wild-type
cells or artificially established recombinant cells usually used in
technical field (for example, bacteria (Escherichia and Bacillus),
yeast (Saccharomyces, Pichia, and such), animal cells, insect
cells, or plant cells).
[0113] By "administration" is meant introducing a composition
(e.g., a vaccine, adjuvant, or immunogenic composition) of the
present disclosure into a subject. The preferred route of
administration of the vaccine composition is intravenous. However,
any route of administration, such as oral, topical, subcutaneous,
peritoneal, intra-arterial, inhalation, vaginal, rectal, nasal,
introduction into the cerebrospinal fluid, or instillation into
body compartments can be used.
[0114] "Immunogenic compositions" are those which result in
specific antibody production or in cellular immunity when injected
into a host. Such immunogenic compositions or vaccines are useful,
for example, in immunizing hosts against infection and/or damage
caused by viruses, including, but not limited to, HIV, human T-cell
leukemia virus (HTLV) type I, SIV, FIV, SARS, RVFV, Filovirus,
Flavivirus, arenavirus, bunyavirus, paramyxovirus, influenza virus,
cytomegalovirus, herpesvirus, and alphavirus.
[0115] By "immunogenic amount" is meant an amount capable of
eliciting the production of antibodies directed against the virus,
in the host to which the vaccine has been administered. It is
preferred for HIV, influenza virus, RSV, and cytomegalovirus, among
others, that the route of administration and the immunogenic
composition is designed to optimize the immune response on mucosal
surfaces, for example, using nasal administration (via an aerosol)
of the immunogenic composition.
[0116] The term "pharmaceutically" or "pharmaceutically acceptable"
as used herein refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, or a human, as appropriate.
[0117] The term "pharmaceutically acceptable carrier" as used
herein refers to any carrier, excipient, diluents, adjuvants, or
vehicles, such as preserving or antioxidant agents, fillers,
disintegrating agents, wetting agents, emulsifying agents,
suspending agents, solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well-known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions as suitable therapeutic
combinations.
[0118] The term "host" or "organism" as used herein includes
humans, mammals (e.g., cats, dogs, horses, etc.), living cells, and
other living organisms. A living organism can be as simple as, for
example, a single eukaryotic cell or as complex as a mammal.
Typical hosts to which embodiments of the present disclosure may be
administered will be mammals, particularly primates, especially
humans. For veterinary applications, a wide variety of subjects
will be suitable, e.g., livestock such as cattle, sheep, goats,
cows, swine, and the like; poultry such as chickens, ducks, geese,
turkeys, and the like; and domesticated animals particularly pets
such as dogs and cats. For some applications, hosts may also
include plants. For diagnostic or research applications, a wide
variety of mammals will be suitable subjects, including rodents
(e.g., mice, rats, hamsters), rabbits, primates, and swine such as
inbred pigs and the like. Additionally, for in vitro applications,
such as in vitro diagnostic and research applications, body fluids
and cell samples of the above subjects will be suitable for use,
such as mammalian (particularly primate such as human) blood,
urine, or tissue samples, or blood, urine, or tissue samples of the
animals mentioned for veterinary applications. Hosts that are
"predisposed to" condition(s) can be defined as hosts that do not
exhibit overt symptoms of one or more of these conditions but that
are genetically, physiologically, or otherwise at risk of
developing one or more of these conditions.
[0119] The term "treat", "treating", and "treatment" are an
approach for obtaining beneficial or desired clinical results. For
purposes of embodiments of this disclosure, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilization (e.g.,
not worsening) of disease, preventing spread of disease, delaying
or slowing of disease progression, amelioration or palliation of
the disease state, and remission (partial or total) whether
detectable or undetectable. In addition, "treat", "treating", and
"treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment.
[0120] The term "condition" and "conditions" denote a state of
health that can be related to infection by a virus. The infections
that are discussed herein are to be included as conditions that can
be treated by embodiments of the present disclosure.
Discussion:
[0121] Embodiments of the present disclosure relates to novel
immunogenic compositions for stimulating systemic, and/or mucosal
immunity. Embodiments of the present disclosure provides novel
biological tools, chimeric virus-like particles (VLPs) and methods
of use, for inducing immunogenic responses in an animal or human
host. Briefly described, embodiments of the present disclosure
include immunogenic compositions comprising an adjuvant molecule
and an immunogen, where the adjuvant molecule may be a surface
protein, such as, but not limited to, a bacterial or protozoal
flagellin molecule. In certain embodiments of the present
disclosure, a bacterial surface protein may be a membrane-anchored
form of the protein, such as, but not limited to, a
membrane-anchored flagellin. In some embodiments of the present
disclosure, the surface protein may be a modified form of the
protein, where such modifications may include removal or
modification of certain immunogenic or antigenic amino acid
sequences, and/or addition of antigenic or immunogenic sequences
derived from a heterologous protein species, shown, for example in
FIGS. 25 and 26 and in Example 16, below. In these embodiments, the
surface polypeptide may be modified by the insertion or deletion of
amino acids of the native surface polypeptide or regions of the
surface polypeptide may be replaced by heterologous amino acids or
amino acid sequences.
[0122] Embodiments of the present disclosure also include methods
of using the immunogenic compositions of the present disclosure to
generate an immune response in an animal or human host, as well as
methods of making the immunogenic compositions of the present
disclosure. One embodiment, for example, provides a method of
presenting immunogenic moieties derived from an influenza virus,
thereby inducing a protective immune response in the recipient
animal or human host.
[0123] Surface protein adjuvant molecules. As described by McDonald
et al., (2007) J. Infect. Dis. 195:1607-1617, and by Eaves-Pyles et
al., (2001) J. Immunol. 167:7009-7016), a modified flagellin
protein with a removed hypervariable region did not lose the
ability to activate TLR5 activation in vitro. In some embodiments,
the removed fragment may be replaced with a linker peptide, while
in other embodiments it may be replaced with immunogenic fragments
isolated from other polypeptides, such as bacterial or viral
peptides. In some embodiments of the disclosure, the flagellin
polypeptide may also be modified by the addition of a
membrane-anchoring peptide sequence. It is contemplated that a
useful membrane-anchoring peptide may be a viral peptide (for
example, an influenza hemagglutinin (HA) membrane-anchoring
sequence, an MMTV Env membrane anchor sequence, and the like).
[0124] In other embodiments of the disclosure, modified flagellin
polypeptides may also comprise a signal peptide sequence. In some
of these embodiments the signal peptide sequence may be isolated
from a heterologous polypeptide. The signal sequence may be
attached at the N- or C-terminus of the modified flagellin and may
be included to help direct the incorporation of the flagellin
polypeptide into a VLP. Exemplary embodiments of such variants are
described in greater detail in the examples 11-13, and in and
figures below, particularly FIGS. 21-23.
[0125] Immunogenic compositions comprissions surface protein
adjuvant molecules. Embodiments of the present disclosure further
encompass flagellin and/or other cell surface proteins that may be
used as an adjuvant for vaccines. Flagellin has been demonstrated
to have a strong adjuvant activity for influenza vaccines, and for
vaccines including virus like particles (VLP). The immunogenic
compositions of the present disclosure may be used to enhance an
immune response, including antibody production, cytotoxic T cell
activity, and cytokine activity. The presently disclosed
immunogenic compositions may act as a prophylactic vaccine to
prevent the onset or establishment of a viral infection such as,
but not limited to, a viral infection caused by the human
immunodeficiency virus (HIV), the coronavirus, the influenza virus,
the paramyxovirus, the herpesvirus, the Ebola virus, the Rift
Valley Fever virus, the Hantavirus, the Lassa fever virus, the
Flavivirus, and the like. It is further contemplated, however, that
the modified flagellin adjuvants of the present disclosure may also
be used in vaccines directed against bacterial target infections
for the prevention or treatment of such bacterial diseases as
diphtheria, cholera, bacterial tuberculosis, and other bacterial
infections known to those in the art.
[0126] The embodiments of the present disclosure further relate to
novel vaccine compositions of chimeric virus-like particles for
administering to humans and animals. In particular, the present
disclosure demonstrates that soluble membrane-anchored flagellin
and/or other surface proteins can be cloned and expressed in such
as an insect cell protein expression system, and mixed with
antigens (VLP or others) to be used for immunization of hosts
against various pathogens.
[0127] In embodiments of the disclosure, membrane-anchored
flagellin or other surface protein may be incorporated into a VLP,
and in particular incorporated in a manner that exposes the
membrane-anchored flagellin externally to the VLP, allowing use as
a vaccine to stimulate an immunogenic response in a recipient host
receiving. Such VLPs, containing membrane-anchored flagellin or
other surface protein as a vaccine adjuvant can be used for
systemic, mucosal or other immunostimulatory routes.
[0128] As described in the below, the methods of the present
disclosure include making immunogenic compositions of the present
disclosure by cloning membrane-anchored flagellin of a bacterial
source and expressing the cloned membrane-anchored flagellin in
insect cell protein expression system. The resulting
membrane-anchored flagellin can then be incorporated into a VLP or
virosome together with a selected immunogen. In some embodiments,
to incorporate flagellin into VLPs, a signal peptide (SP) and a
membrane anchor are fused to direct the transport and membrane
orientation. In an exemplary embodiment, the resulting recombinant
gene was used for the generation of recombinant baculovirus
(rBV).
[0129] Modified surface polypeptide variants. Embodiments of the
compositions and methods of the present disclosure may include, but
are not limited to, a flagellin isolated from a bacterial source,
such as Salmonella enteritidis. The examples below demonstrate that
soluble monomeric flagellin and purified polymeric flagellin
isolated from S. enteritidis may contribute to the generation of an
immune response. In addition to flagellin from Salmonella spp.,
however, it is considered to be within the scope of the disclosure
for other motile bacterial or protozoal flagellar proteins to be
used in the constructs and compositions of the disclosure. In
particular, bacterial or protozoal surface polypeptides that may
cooperate with the TLR5 or other TLRs include, but are not limited
to polypeptides of: Proteus spp., Pseudomonas spp., Serratia spp.,
Morganella morganii (grown under 30.degree. C.), Providencia
stuartii, P. rettgeri, P. Alcalifaciens, Arcobacter spp., Aeromonas
spp., Acidivorax spp., Helicobacter spp., Flexispira rappine,
Wolinella spp., Rhizobium spp., Vibrio spp., Legionella spp.,
Edwardsiella spp., Shigella spp., Escherichia spp., Listeria spp.,
Bordetella spp., Burkholderia spp., Helicobacter spp., Butyrivibrio
spp., Roseburia spp., Thermotoga spp., Clostridium spp., Bacillus
spp., Oceanobacillus iheyensis, Thermotoga maritime, Leptospira
spp., Yersinia spp., Bordetella spp, Legionella spp., Trichomonas
spp., Bartonella bacilliformis, Caulobater crescentus, Campylobacer
spp., and Treponema spp flagellins. Modified forms of the
flagellins can be used, where portions of the flagellin polypeptide
has been deleted and replaced by a linker sequence and/or an
immunogenic or antigenic sequence.
[0130] In embodiments of the disclosure, a modified flagellin
polypeptide may be a Salmonella flagellin that has been modified to
delete a fragment located between the N- and C-terminii that are
known to interact with a TLR. For example, deletion of the region
of the Salmonella flagellin of amino acids about 176 to about 402
inclusive leaves regions about 1 to about 175 and about 403 to
about 495 that may interact with TLRs. It may be advantageous to
remove this antigenic fragment to reduce unfavorable host
responses, such as excessive inflammation, previously acquired
immune responses, and the like that may result in inactivation of
the administered adjuvant modified flagellin once administered to
an animal or human host. In embodiments, such a deleted fragment
may be replaced with a linker peptide, while in other embodiments
the fragment may be replaced with an antigenic or immunogenic
peptide derived from a heterologous source. In some embodiments,
the modified flagellin polypeptide may also include a heterologous
signal peptide sequence and/or a heterologous transmembrane and
cytoplasmic domain from other sources, such as viral sources,
including influenza hemagglutinin, MMTV Env peptides, and the like.
Such embodiments are described in additional detail in FIGS.
21-23.
[0131] The adjuvant molecules of the present disclosure may be
co-administered with an antigenic composition or may be physically
incorporated with the antigen or immunogen. For example, the
adjuvant molecule may be incorporated into, or otherwise physically
linked to, an antigenic compound, such as a VLP. Embodiments of the
adjuvant molecule, therefore, may be a membrane-anchored surface
protein that is incorporated into a VLP. In one embodiment of the
present disclosure, the adjuvant molecule may be a
membrane-anchored flagellin that is incorporated onto the surface
of a VLP, to produce a chimeric VLP (cVLP). In other embodiments of
the disclosure, the immunogenic compositions may comprise a VLP
co-administered with a flagellin as the adjuvant molecule, where
the flagellin is not incorporated into the VLP. Additional details
about VLPs and chimeric VLPs are described in U.S. patent
application Ser. Nos. 10/514,462 and 11/397,830, which are herein
incorporated by reference in their entireties. The adjuvant
molecule of the present disclosure can also be used with other
antigen-presenting systems, such as virosomes as described, for
example, in PCT Patent Application No. PCT/US2007/073342, which is
hereby incorporated herein by reference in its entirety.
[0132] Immunogenic compositions including VLPs. Embodiments of the
immunogenic compositions of the present disclosure include chimeric
virus-like particles (cVLPs) with adjuvanted characteristics as
described above, and derived from viruses selected from, but not
limited to, human immunodeficiency virus (HIV), feline
immunodeficiency virus (FIV), bovine immunodeficiency virus, bovine
leukemia virus, equine infectious anemia virus, human T-leukemia
virus, Bunya virus, Lassa fever virus, Rift Valley virus, ebola
virus, coronavirus, arenavirus, filovirus, influenza virus,
paramyxovirus, rhabdovirus, alphavirus, flavivirus, herpesvirus;
hanta virus; hepadna virus, cytomegalovirus, and the like.
[0133] Embodiments of the present disclosure, therefore, further
provide methods of using the virus-like particles, and methods of
making virus-like particles that can be used in immunogenic
compositions to treat conditions in a host, and the immunogenic
compositions that include virus-like particles.
[0134] Referring now to FIG. 24, a virus-like particle (VLP) 10
according to the present disclosure comprises at least a viral core
protein 12 (hereinafter "viral protein") and at least one viral
surface envelope glycoprotein 14. The viral surface envelope
glycoprotein 14 may be selected from, but is not limited to type 1
(14a) or type 2 (14b) viral surface envelope glycoproteins. In some
embodiments of the disclosure, the VLP 10 can include at least one
adjuvant molecule 16 according to the present disclosure
incorporated onto the lipid membrane 18 of the VLP 10. In other
embodiments, the adjuvant molecule may be co-administered with the
VLP, but is not incorporated into the VLP itself. The adjuvant
molecule 16 may be a surface protein (such as a bacterial
flagellin) that may selectively target a host cell TLR. It is
contemplated that the VLPs of the present disclosure may comprise
more than one type of adjuvant molecule (e.g. 16a, 16b, and so on).
In some embodiments, the adjuvant molecule 16 may be a
membrane-anchored form of a surface protein, such as but not
limited to, a membrane-anchored form of a bacterial flagellin such
as described in the examples below.
[0135] Furthermore, the VLP may comprise a lipid membrane 18, viral
glycoprotein transmembrane unit 20, and a matrix protein 22. In
particular, chimeric VLPs (cVLPs) are VLPs having at least one
viral surface envelope glycoprotein 14 incorporated into the VLP
10, wherein the viral core protein 12, and at least one viral
surface envelope glycoprotein 14, may be from different viral
sources. Thus, cVLPs also include VLPs wherein there are more than
one type of viral surface envelope glycoprotein 14 (14a, 14b, and
the like), and wherein one or both of 14a and 14b are from a
different virus than the viral core protein 12.
[0136] Such cVLPs may or may not have the adjuvant molecule 16
incorporated into the VLP 10. In embodiments of the disclosure, the
cVLPs may have at least one adjuvant molecule 16 incorporated into
the VLP, where the adjuvant molecule(s) 16 may comprise a bacterial
surface protein, such as a bacterial flagellin, or a fragment
thereof, that targets a host TLR. Embodiments of the disclosure
also include phenotypically mixed VLPs where there is more than one
type of adjuvant molecule 16, such as 16a and 16b, where one or
both of 16a and 16b are from a different bacterial source from each
other.
[0137] Viral core proteins 12 include proteins that are capable of
self-assembling into a VLP core, as described by Freed, E. O.,
(2002) J. Virol., 76: 4679-4687. The viral core proteins 12 can
include, but are not limited to, such as a viral Gag protein,
including a retrovirus gag protein (such as the HIV Gag viral
protein `HIV-1 NL43 Gag` (GenBank serial no. AAA44987), the simian
immunodeficiency virus (SIV) Gag viral protein `SIVmac239 Gag`
(GenBank serial no. CAA68379), the murine leukemia virus (MuLV) Gag
viral protein `MuLV Gag` (GenBank serial no. S70394), or the human
foamy virus Gag viral protein), a retrovirus matrix protein, a
rhabdovirus matrix protein M protein such as the vesicular stomatis
virus (VSV) M protein `VSV Matrix protein` (GenBank serial no.
NP041714), a filovirus viral core protein such as the Ebola VP40
viral protein `Ebola virus VP40` (GenBank serial no. AAN37506), the
Rift Valley Fever virus N protein `RVFV N Protein` (GenBank serial
no. NP049344), coronavirus M, E and NP proteins such as NP protein
(GenBank serial no. NP040838), M protein (GenBank serial no.
NP040835), E protein of Avian Infections Bronchitis Virus (GenBank
serial no. CAC39303), and E protein of the SARS virus (GenBank
serial no. NP828854), a bunyavirus N protein (GenBank serial no.
AAA47114), an influenza M1 protein, a paramyxovirus M protein,
arenavirus Z protein (e.g., a Lassa Fever Virus Z protein), a
cytomegalovirus (CMV) core protein, a herpes simplex virus (HSV)
core protein, and combinations thereof. Appropriate surface
glycoproteins and/or viral RNA may be included to form the VLP
10.
[0138] In general, the viral core protein 12 sequence and the
corresponding polynucleotide sequence can be found in GenBank and
the access numbers can be obtained online at the National Center
for Biotechnology Information (NCBI). In addition, the sequences
identified for the viral proteins 12 above are only illustrative
examples of representative viral proteins 12. Furthermore, variants
that are substantially homologous to the above referenced viral
proteins 12 and viral proteins 12 having conservative substitutions
of the above referenced viral proteins 12 can also be incorporated
into VLPs 10 of the present disclosure to enhance the immunogenic
characteristics of VLPs.
[0139] The viral surface envelope glycoprotein 14, or at least a
portion of the viral surface envelope glycoprotein 14, may be
disposed on the surface of the VLP so that it can interact with
target molecules or cells, such as the interaction between the HIV
surface envelope glycoprotein and the B cell receptor to activate
HIV envelope glycoprotein specific antibody-producing B cells, to
produce immunogenic responses including antibody production.
[0140] The viral surface envelope glycoproteins 14 of the VLPs
according to the present disclosure can include, but are not
limited to, a retroviral glycoprotein such as the human
immunodeficiency virus (HIV) envelope glycoprotein `HIVSF162
envelope glycoprotein` (GenBank serial no. M65024), the simian
immunodeficiency virus (SIV) envelope glycoprotein `SIVmac239
envelope glycoprotein` (GenBank serial no. M33262), the
simian-human immunodeficiency virus (SHIV) envelope glycoprotein
`SHIV-89.6p envelope glycoprotein` (GenBank serial no. U89134), the
feline immunodeficiency virus (FIV) envelope glycoprotein `feline
immunodeficiency virus envelope glycoprotein` (GenBank serial no.
L00607), the feline leukemia virus envelope glycoprotein `feline
leukemia virus envelope glycoprotein` (GenBank serial no. M12500),
the bovine immunodeficiency virus envelope glycoprotein `bovine
immunodeficiency virus envelope glycoprotein` (GenBank serial no.
NC001413), the bovine leukemia virus envelope glycoprotein (GenBank
serial no. AF399703), the equine infectious anemia virus envelope
glycoprotein (GenBank serial no. NC001450), the human T-cell
leukemia virus envelope glycoprotein (GenBank serial no.
AF0033817), the human foamy virus glycoprotein, the mouse mammary
tumor virus envelope glycoprotein (MMTV), a bunya virus
glycoprotein such as the Rift Valley Fever virus (RVFV)
glycoprotein `RVFV envelope glycoprotein` (GenBank serial no.
M11157), an arenavirus glycoprotein such as the Lassa fever virus
glycoprotein (GenBank serial no. AF333969), a filovirus
glycoprotein such as the Ebola virus glycoprotein (GenBank serial
no. NC002549), the coronavirus glycoprotein (GenBank serial no.
AAP13567), an influenza virus glycoprotein (GenBank serial number
V01085), a paramyxovirus glycoprotein such as the Nipah virus F and
G proteins (GenBank serial no. NC002728), a rhabdovirus
glycoprotein such as Vesicular Stomatitis Virus (VSV) glycoprotein
(GenBank serial no. NP049548), an alphavirus glycoprotein such as
Venezuelan equine encephalomyelitis (VEE) (GenBank serial no.
AAA48370), the flavivirus glycoprotein such as West Nile virus
(GenBank serial no. NC001563), a Hepatitis C Virus glycoprotein, a
Herpes Virus glycoprotein, a cytomegalovirus (CMV) glycoprotein, a
Respiratory Syncytial virus (RSV) glycoprotein, a rabies virus
glycoprotein, a Marburg virus glycoprotein, and combinations
thereof.
[0141] In general, the viral surface envelope glycoprotein 14
sequence and the corresponding polynucleotide sequence can be found
in GenBank and the access numbers can be obtained online at NCBI.
In addition, the sequences identified for the viral surface
envelope glycoproteins 14 above are only illustrative examples of
representative viral surface envelope glycoproteins 14. Further,
variants that are substantially homologous to the above referenced
viral surface envelope glycoproteins 14 and viral surface envelope
glycoproteins 14 having conservative substitutions of the above
referenced viral surface envelope glycoproteins 14 can also be
incorporated into VLPs 10 of the present disclosure to enhance the
immunogenic characteristics of VLPs.
[0142] In embodiments of the disclosure where the adjuvant molecule
16 is incorporated into the VLP, the adjuvant molecule 16, or at
least a portion of the adjuvant molecule 16, is disposed on the
surface of the VLP 10. The adjuvant molecule 16 may interact with
other molecules or cells, such as mucosal surfaces having sialic
acid residues thereon, and antigen-presenting cells such as
dendritic cells, follicular dendritic cells, and the like.
[0143] The adjuvant molecule 16 may be, but is not limited to,
monomeric or polymeric forms of a surface protein that targets a
host TLR, such as, but not limited to a bacterial flagellin. For
incorporation into the VLP, the adjuvant molecule should generally
be a membrane-anchored form of the surface protein. Examples of
membrane-anchored forms of mammalian TLR ligand molecules include,
but are not limited to, ligands listed in Akira & Takeda (2004)
Nature Revs/Immunol., 4: 499-511, which is incorporated by
reference herein in its entirety. In particular, exemplary TLR
ligand molecules include glycoproteins from Prevotella intermedia,
respiratory syncytial virus protein F, fibronectin A domain,
fibrinogen, flagellin, a measles virus HA protein, and Pam2Cys
lipoprotein/lipopeptide (MALP-2). In addition to the surface
protein adjuvant molecule, the VLPs of the present disclosure may
also include one or more additional adjuvant molecules which may
include, but are not limited to, an influenza hemagglutinin (HA)
molecule (GenBank access no. J02090), a parainfluenza
hemagglutinin-neuraminidase (HN) molecule (GenBank access no.
Z26523 for human parainfluenza virus type 3 HN sequence
information), a Venezuelan equine encephalitis (VEE) adjuvant
molecule (GenBank access no. NC001449), a fms-like tyrosine kinase
ligand (Flt3) adjuvant molecule (GenBank access no. NM013520), a
C3d adjuvant molecule (GenBank access no. NM009778 for mouse C3
sequence; access no. NM000064 for human C3 sequence), a mannose
receptor adjuvant molecule, a CD40 ligand adjuvant molecule
(GenBank access no. M83312 for mouse CD40), and combinations
thereof.
[0144] In general, the adjuvant molecule 16 sequence and the
corresponding polynucleotide sequence can be found in GenBank and
the access numbers can be obtained online at the NCBI. In addition,
the sequences identified for the adjuvant molecules 16 above are
only illustrative examples of representative adjuvant molecules 16.
Further, variants that are substantially homologous to the above
referenced adjuvant molecules 16 and adjuvant molecules 16 having
conservative substitutions of the above referenced adjuvant
molecules 16 can also be incorporated into VLPs 10 of the present
disclosure to enhance the immunogenic characteristics of VLPs.
[0145] Methods of making and using the immunogenic compositions.
Embodiments of the present disclosure further include methods of
inducing an immune response in a host by administering to the host
an effective amount of an immunogenic composition according to the
present disclosure. It is contemplated that the methods of the
present disclosure are useful in preventing a disease or disorder
by administering to an animal or human host in need thereof an
effective amount of an immunogenic composition according to the
present disclosure.
[0146] Embodiments of the disclosure further include methods of
immunizing an animal or human host by administering to such a host
an immunogenic composition of the present disclosure. In
embodiments where the immunogenic composition includes a VLP that
comprises a membrane-anchored form of an adjuvant molecule of the
present disclosure incorporated into the VLP, the method may
comprise expressing a viral core protein, at least one viral
surface envelope surface glycoprotein, and at least one adjuvant
molecule of the present disclosure in one or more host cells. It is
contemplated, for example, that nucleic acid molecules encoding
such proteins may be included in at least one expression vector
nucleic acid which may be delivered to a recipient host cell. The
viral core protein, at least one viral surface envelope
glycoprotein, and at least one adjuvant molecule thus expressed by
the host cell(s), may be assembled to form a VLP. The VLP can then
elicit an immune response from the host animal or human, thereby
providing future protection from infection by a pathogen
corresponding to the proteins expressed by the VLP.
[0147] Methods of making the immunogenic compositions of the
present disclosure. Embodiments of the present disclosure also
include methods of making the immunogenic compositions of the
present disclosure. Some exemplary methods of making the adjuvant
molecules of the present disclosure are described below, including
methods of making membrane-anchored forms of the adjuvant
molecules. Methods useful for the making VLPs of the present
disclosure for administration with the adjuvant molecules of the
present disclosure, or which incorporate adjuvant molecules into
the VLP, may be found in U.S. patent application Ser. Nos.
10/514,462 and 11/397,830, which are herein incorporated by
reference in their entireties, and which are also described
below.
[0148] VLPs for use in the immunogenic compositions of the present
disclosure can be produced by in vitro cell culture expression
systems such as, but not limited to, recombinant baculovirus
expression system (BEVS) (see, for example, Yamshchikov et al.,
(1995) Virology: 214, 50-58). Assembly of HIV or SIV virus-like
particles containing envelope proteins may be performed using
expression systems, such as, but not limited to, a baculovirus
expression system (Yamshchikov et al., (1995) Virology: 214,
50-58), recombinant poxvirus expression system (MVA) (Wyatt et al.,
(1996), Vaccine: 15, 1451-1458), recombinant VSV, recombinant
adenovirus, and recombinant DNA expression vectors. Preferably, the
VLPs are produced using recombinant BEVS and recombinant poxvirus
expression systems.
[0149] In general, VLPs can be produced by simultaneously
introducing into a cell a viral core protein expression vector, a
viral surface envelope glycoprotein expression vector, and/or an
adjuvant molecule expression vector. The expressed viral core
protein self-assembles into a VLP that incorporates the viral
surface envelope glycoprotein and/or the adjuvant molecule. The
viral surface envelope glycoprotein and/or the adjuvant molecule
are expressed and disposed on the VLP surface. Thereafter, the cell
produces the VLP (for example, Vero cells, chimeric and/or
phenotypically mixed VLPs). The cells may be selected from, but are
not limited to, insect cells (e.g., Spodoptera frugiperda Sf 9 and
Sf21cells), and mammalian cells such as, but not limited to, EL4
cells and HeLa cells. The expression elements for expressing the
viral core protein, viral surface envelope glycoprotein, and
adjuvant molecule can also be included together in a single
expression vector, or can be included in two or more expression
vectors.
[0150] In general, the viral protein expression vector can be
produced by operably linking a coding sequence for a viral protein
of a virus to an appropriate promoter (e.g., an early promoter,
late promoter, or hybrid late/very late promoter). The viral
protein expression vector can also be modified to form a viral
protein expression construct. In addition, the viral surface
envelope glycoprotein expression vector can be produced by operably
linking a coding sequence for a viral surface envelope glycoprotein
of a virus to an appropriate promoter (e.g., early promoter, late
promoter, or hybrid late/very late promoter). The viral surface
envelope glycoprotein expression vector can be modified to form a
viral surface envelope glycoprotein expression construct.
Similarly, the adjuvant molecule expression vector can be produced
by operably linking a coding sequence for an adjuvant molecule to
an appropriate promoter (e.g., early promoter, late promoter, or
hybrid late/very late promoter). The adjuvant molecule expression
vector can be modified to form an adjuvant molecule expression
construct.
[0151] In other embodiments of the disclosure, polynucleotide
sequences encoding for a viral core protein, at least one viral
surface envelope glycoprotein, and at least one adjuvant molecule
can be included in a single expression vector, or in two or more
expression vectors. The one or more expression vectors can be
introduced into a host cell, the proteins can be expressed in the
cell, whereby the cell forms the VLP. In embodiments, each of the
polynucleotide sequences encoding for the viral core protein, the
viral surface envelope glycoprotein, and the adjuvant molecule is
operably linked to an appropriate promoter (e.g., a baculovirus
promoter, a recombinant Modified Vaccinia Ankara (MVA) promoter, a
CMV promoter, an EF promoter, an adenovirus promoter, a recombinant
VSV promoter, a recombinant adenovirus promoter, a recombinant
alphavirus promoter, and a recombinant DNA expression vector).
Appropriate promoters include, but are not limited to, a
constitutive or inducible promoter; an early, late, or hybrid
late/very late promoter.
[0152] Compositions and immunogenic preparations of the present
disclosure, including vaccine compositions comprising the VLPs of
the present disclosure which are capable of inducing protective
immunity in a suitably treated host, and a suitable carrier
therefore are provided. The vaccine preparations of the present
disclosure can include an immunogenic amount of one or more VLPs,
fragment(s), or subunit(s) thereof. Such vaccines can include one
or more viral surface envelope glycoproteins and portions thereof,
and adjuvant molecule and portions thereof on the surfaces of the
VLPs, or in combination with another protein or other immunogen,
such as one or more additional virus components naturally
associated with viral particles or an epitopic peptide derived
therefrom.
[0153] The immunogenic compositions and/or vaccines of the present
disclosure may be formulated by any of the methods known in the
art. They can be typically prepared as injectables or as
formulations for intranasal administration, either as liquid
solutions or suspensions. Solid forms suitable for solution in, or
suspension in, liquid prior to injection or other administration
may also be prepared. The preparation may also, for example, be
emulsified, or the protein(s)/peptide(s) encapsulated in liposomes.
The immunogenic compositions can be used, but it is not limited to,
skin immunization, microneedle delivery, mucosal delivery, and/or
intramuscularly.
[0154] The active immunogenic ingredients are often mixed with
excipients or carriers, which are pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients include
but are not limited to water, saline, dextrose, glycerol, ethanol,
or the like and combinations thereof. The concentration of the
immunogenic polypeptide in injectable, aerosol, or nasal
formulations is usually in the range of about 0.2 to 5 mg/ml.
Similar dosages can be administered to other mucosal surfaces.
[0155] In addition, if desired, the vaccines may contain amounts of
auxiliary substances such as wetting or emulsifying agents, pH
buffering agents, and/or other agents, which enhance the
effectiveness of the vaccine. Examples of agents which may be
effective include, but are not limited to: aluminum hydroxide;
aluminum phosphate, N-acetyl-muramyl-L-threonyl-D-isoglutamine
(thr-MDP); N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,
referred to as nor-MDP);
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dip-
almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylanine (CGP 19835A,
referred to as MTP-PE); and RIBI, which contains three components
extracted from bacteria: monophosphoryl lipid A, trehalose
dimycolate, and cell wall skeleton (MPL+TDM+CWS) in a 2%
squalene/Tween 80 emulsion. The effectiveness of the auxiliary
substances may be determined by measuring the amount of antibodies
(especially IgG, IgM or IgA) directed against the immunogen
resulting from administration of the immunogen in vaccines which
comprise the adjuvant in question. Additional formulations and
modes of administration may also be used.
[0156] The immunogenic compositions and/or vaccines of the present
disclosure can be administered in a manner compatible with the
dosage formulation, and in such amount and manner as will be
prophylactically and/or therapeutically effective, according to
what is known to the art. The quantity to be administered, which is
generally in the range of about 1 to about 1,000 micrograms of
viral surface envelope glycoprotein per dose and/or adjuvant
molecule per dose, more generally in the range of about 5 to about
500 micrograms of glycoprotein per dose and/or adjuvant molecule
per dose, depends on the subject to be treated, the capacity of the
hosts immune system to synthesize antibodies, and the degree of
protection desired. Precise amounts of the active ingredient
required to be administered may depend on the judgment of the
physician or veterinarian and may be peculiar to each individual,
but such a determination is within the skill of such a
practitioner.
[0157] The vaccine or immunogenic composition may be given in a
single dose; two dose schedule, for example two to eight weeks
apart; or a multiple dose schedule. A multiple dose schedule is one
in which a primary course of vaccination may include 1 to 10 or
more separate doses, followed by other doses administered at
subsequent time intervals as required to maintain and/or reinforce
the immune response (e.g., at 1 to 4 months for a second dose, and
if needed, a subsequent dose(s) after several months). Humans (or
other animals) immunized with the compositions of the present
disclosure are protected from infection by the cognate virus.
[0158] It should also be noted that the vaccine or immunogenic
composition can be used to boost the immunization of a host having
been previously treated with a vaccine such as, but not limited to,
DNA vaccine and a recombinant virus vaccine.
[0159] Construction of the membrane-anchored flagellin coding
sequence. To incorporate flagellin into VLPs as a molecular
adjuvant according to an embodiment of the present disclosure, the
gene was modified to enable membrane translocation, transport, and
cell surface expression. As schematized in FIG. 5, a
membrane-anchored flagellin-encoding nucleic acid construct was
prepared by merging, at the N terminus of the flagellin-encoding
nucleic acid, the coding sequence for the signal peptide (SP) of
the honeybee protein melittin. The transmembrane-cytoplasmic tail
(TM-CT) from influenza hemagglutinin (HA) was added in-frame at the
C-terminus of the flagellin-encoding nucleic acid. As a
heterologous SP, melittin SP is known to improve glycoprotein cell
surface expression in an insect cell system. The TM-CT sequence
from HA provided a membrane anchor sequence to allow the modified
flagellin to assemble into influenza virus matrix
protein(M1)-derived VLPs (Ali et al., (2000) J. Virol.
74:8709-8719).
[0160] Recombinant baculovirus (rBV) was generated using the
resulting membrane-anchored flagellin-encoding sequence. As shown
in FIG. 9 (left panel), flagellin fused with the HA TM-CT region
could be expressed in rBV-infected Sf9 cells. The modified protein
yielded two major bands in cell lysates by autoradiography (FIG. 9,
left panel, lane 1). The lowest band is around 55 kDa and
corresponds to the molecular mass estimated according to its amino
acid composition in a non-glycosylated form. The top band is about
65 kDa, indicating that glycosylation of this modified protein
occurred in the insect cell protein expression system. Other bands
between the two main bands are possibly due to differences in
glycosylation of the membrane-anchored flagellin. As shown in FIG.
9, right panel, surface expressed flagellin corresponds to the top
band with a molecular mass of 65 kDa, suggesting that only
glycosylated flagellin is transported to cell surfaces.
[0161] Production of cVLPs containing flagellin. In an embodiment
VLPs were produced in an rBV-derived protein expression system in
Sf9 insect cells and purified by gradient centrifugation. To
optimize the production of cVLPs, rBVs expressing HA, M1, and
flagellin were compared at various multiplicities of infection
(MOI), as shown in FIG. 10, bottom. The results, in FIG. 10, top,
demonstrate that standard influenza VLPs with a high HA content
resulted from co-infection of HA- and M1-expressing rBVs at MOIs of
4 and 2, respectively, and the VLPs (total protein concentration, 1
mg/ml) have an HA titer as high as 2048 U when titrated with
chicken blood cells.
[0162] The cVLPs containing flagellin were produced by co-infection
of rBVs expressing HA, M1, and flagellin at MOIs of 6, 2, and 6,
respectively. These cVLPs had HA and M1 contents comparable to
those of standard HA and M1 VLPs, as shown by the Western blot
results in FIG. 10 (top). The HA titer of the cVLPs (protein
concentration, 1 mg/ml) was 2048 U, the same as that for standard
VLPs. When purified recombinant flagellin was used as a standard, a
Western blot comparison showed that the cVLPs have a flagellin
content of about 8 .mu.g/100 .mu.g VLPs. To further confirm the
morphology and integrity of these cVLPs, the cVLP samples were
examined by electron microscopy after negative staining. As shown
in FIG. 11, enveloped VLPs with projections on the surface were
observed, with diameters of about 80 to about 100 nm. In addition,
the cVLPs had morphological characteristics similar to those of
standard HA/M1 VLPs, as described previously (Quan et al., (2007).
J. Virol. 81:3514-3524). Membrane-anchored flagellin variants of
the present disclosure, therefore, together with HA, is
incorporated into M1-derived VLPs with a morphology and size
similar to those of standard HA/M1VLPs and influenza virions.
Characterization of the Membrane-Anchored Flagellin incVLPs. [0163]
(i) In an embodiment there are six potential N-linked glycosylation
sites in the flagellin sequence itself, with an NXT/S motif (Asn
19, 101, 200, 346, 446, and 465, respectively, and identified by
arrows in FIG. 8). Four of them are located in the
TLR-5-recognizing regions (Asn 19, 101, 446, and 465). To further
characterize the possible glycosylation of the membrane-anchored
flagellin in cVLPs, VLPs containing flagellin were treated with
PNGase F or endo-H. PNGase F is an amidase that can remove N-linked
oligosaccharides from glycoproteins, whereas endo-H cleaves the
chitobiose core of high-mannose and hybrid oligosaccharides from
N-linked glycoproteins.
[0164] As shown in FIG. 12, most of the modified flagellin in cell
lysates is seen as two main portions on the blot with molecular
masses of 55 and 65 kDa (lane 1). The flagellin incorporated into
cVLPs corresponds to the upper band (65kDa) (lane 2). After
treatment of VLPs with PNGase F, flagellin bands of faster mobility
at 55 kDa were observed (FIG. 12, lane 3), demonstrating that the
membrane-anchored flagellin is glycosylated by N-linked
oligosaccharides. When the glycosylated flagellin in VLPs was
treated by endo-H, an intermediate band (around 60 kDa) was
observed (FIG. 12, lane 4), revealing the partial sensitivity of
the flagellin in VLPs to endo-H. These results indicate that at
least some of the oligosaccharides are of the high-mannose type. In
conclusion, these results indicate that flagellin in VLPs is
glycosylated and that the oligosaccharides are linked to the
flagellin peptide backbone by N-type glycosidic linkages. [0165]
(ii) In an embodiment the adjuvant properties of flagellin are
based on its TLR-5-activating activity. To evaluate the ability of
the membrane-anchored flagellin in VLPs to function as a TLR-5
ligand, flagellin-containing VLPs were analyzed by a mouse
macrophage cell line RAW264.7-based assay, and results were
compared to those from purified soluble flagellin. As shown in FIG.
13, flagellin-containing cVLPs stimulated TLR-5-positive RAW264.7
cells to produce TNF-.alpha. over a broad concentration spectrum,
similar to that seen with soluble flagellin. The 50% effective
concentration (concentration which produces 50% of maximal
activity) of flagellin-containing VLPs was about 8 ng/ml, whereas
the 50% effective concentration of soluble flagellin was about 1
ng/ml. Because the flagellin content in cVLPs is about 8% by
weight, the results indicate that the TLR-5 agonist activity of
membrane-anchored flagellin in cVLPs is comparable to that of
soluble flagellin.
[0166] cVLPs containing flagellin induce enhanced humoral immune
responses. It is well recognized that flagellin in full length,
truncated, or fusion protein forms enhances antigen specific
antibody responses (Cuadros et al., (2004) Infect. Immun.
72:2810-28168; Honko et al., (2006). Infect. Immun. 74:1113-1120;
McDonald et al., (2007) J. Infect. Dis. 195:1607-1617).
[0167] To evaluate the ability of membrane-bound flagellin in
influenza cVLPs to function as an adjuvant, the humoral immune
response against influenza viral antigen was determined for mice
immunized with standard HA/M1 VLPs or flagellin/HA/M1 cVLPs. As
shown in FIG. 14A, high levels of serum antigen-specific IgG were
promoted by priming or priming plus boosting for mice immunized
with flagellin-containing cVLPs. A 2500-fold-higher IgG titer was
achieved by the flagellin-containing VLP group after only the
priming immunization compared with that of the standard HA/M1 VLP
group, and this IgG level was comparable to that of the standard
VLP group after two immunizations, demonstrating a significant
enhancement of responses promoted by the incorporated flagellin.
After two immunizations, the IgG level of the cVLP group remained
two times higher than that of the standard VLP group (P 0.05). In
contrast, when mice were immunized with mixtures of HA/M1 VLPs plus
soluble recombinant flagellin, no significant difference in
antibody response was detected compared to what was seen for HA/M1
VLPs alone. These results indicate that the incorporation of the
membrane-anchored flagellin into VLPs is significant for its
adjuvant effect.
[0168] Influenza VLP vaccines induce mixed Th1/Th2-type immune
responses. To further evaluate the serum antibody response induced
by flagellin, production levels of IgG subtypes IgG1, IgG2a, and
IgG2b were determined. As shown in FIGS. 14B-14D, both standard
VLPs and cVLPs promoted the production of all three IgG subtypes
compared to what was seen for the control (M1-only) VLP group,
demonstrating that both Th1 and Th2 immune responses were induced
by VLP vaccines. However, the flagellin-containing VLPs elicited a
level of IgG2a (IgG1/IgG2a ratio,0.5) significantly higher than
that seen for standard VLPs (IgG1/IgG2a ratio, 1.5; P 0.05), but
this was not the case for IgG1, demonstrating that Th1-biased
type-mixed responses and IgG2a-dominant class switching were
effectively promoted by the incorporation of flagellin compared to
standard VLPs.
[0169] Flagellin stimulates enhanced virus neutralization and HI
activity. Virus neutralization activity is an important serological
assay to reflect that functional antibodies provide protective
immunity. To determine the effects of flagellin on conferring
protective humoral responses, sera from mouse groups immunized with
HA/M1 VLPs or flagellin-containing HA/M1 VLPs were evaluated for
neutralization activities against PR8 virus. As shown in FIG. 15A,
sera from standard VLP-immunized mice 3 weeks after the boost
immunization showed a neutralization titer (50% plaque reduction)
of 1280. In contrast, the flagellin-containing cVLP group showed a
virus neutralization titer of 4000, more than threefold higher,
revealing the effectiveness of flagellin incorporated into VLPs as
an adjuvant. The enhanced responses were also demonstrated by the
HI titers, which are based on blocking the ability of influenza HA
to agglutinate erythrocytes by specific antibodies. As shown in
FIG. 15B, the flagellin-containing cVLP group achieved an HI titer
of 1080, three-fold higher than that of the standard VLP group (P
0.05), which had a mean HI titer of 360. The neutralization
activity and HI titers were highly consistent, demonstrating that
functional antibodies elicited by influenza VLPs are directed
against the HA. Immune sera from the group immunized with a mixture
of soluble flagellin plus HA/M1 VLPs also achieved levels of
neutralization and HI titers similar to those of the standard HA/M1
VLP group.
[0170] Modified flagellin antigenicity. A concern for using a
protein component as an adjuvant is the antigenicity of the protein
itself, and pre-existing immunity against flagellin might block its
further function as an adjuvant. To evaluate the effects of
preexisting anti-flagellin antibody, mice were pre-immunized
intramuscularly twice with 10 .mu.g of recombinant flagellin.
Subsequently, the same group was immunized twice with 10 .mu.g of
cVLPs at 4-week intervals. As shown in FIG. 15C, this resulted in a
significant mean anti-flagellin IgG titer of 2.7.times.10.sup.5,
stable for 8 weeks. Interestingly, the PR8-specific IgG titers of
flagellin-pre-immunized mice rose to levels similar to those of the
cVLP control group without flagellin pre-immunization, as shown in
FIG. 15C. Flagellin is an effective adjuvant to promote
antigen-specific humoral responses when incorporated into the VLP
embodiments of the present disclosure. Pre-existing flagellin
immunity did not decrease its adjuvant function.
[0171] Because cVLPs induced significantly higher humoral
responses, it was determined whether these immune sera could confer
a cross-reaction with a heterosubtypic virus, by determining the
serum IgG titer and HI titer against a heterosubtypic A/Philippines
virus (H.sub.3N.sub.2). Though the humoral responses against
A/Philippines were at low levels compared to those against A/PR8,
flagellin-containing cVLPs induced significantly high IgG (2800)
and HI (60) titers against A/Philippines compared to those induced
by standard VLPs (all P values were 0.05) (FIGS. 16A and 16B). The
membrane-anchored flagellin of the present disclosure, therefore,
broaden the spectrum of immunity to confer heterosubtypic immune
responses.
[0172] Flagellin promotes antigen-specific T-cell responses.
Several microbial products, such as lipopolysaccharide and CpG DNA,
induce dendritic cell (DC) maturation by binding to TLR family
molecules, including TLR-5 (Hemmi et al., (2000) Nature
408:740-745; Kaisho et al., (2001). J. Immunol. 166:5688-5694;
McSorley et al., (2002) J. Immunol. 169:3914-3919; Takeuchi et al.,
(1999) Immunity 11:443-451). DCs are found in physical contact with
naive T cells in vivo and, therefore, recognize microbial products
and activate antigen-specific T-cell clonal expansion. To test the
effects of membrane-anchored flagellin on enhancing the production
of cytokines, cytokine secretions from splenocytes of mice
immunized with the flagellin-containing cVLPs of the present
disclosure were compared to those of the standard VLP-immunized
mice. The results in FIGS. 18A-18D show that spleen T cells from
mice immunized with flagellin-containing VLP secreted high levels
of IL-2, IFN-.gamma., TNF-.alpha., and IL-4 when stimulated by
MHC-I or -II HA peptides, or inactivated PR8 virus, compared to
those for the standard VLP group (all P values were 0.05).
MHC-II-recognized HA peptides induced relatively higher levels of
cytokine secretion, demonstrating a CD4-dominant memory T-cell
population; both Th1 (IL-2 and IFN-.gamma.)- and Th2 (TNF-.alpha.
and IL-4)-type cytokine production was observed, but not detectable
TNF-.alpha. secretion from splenocytes from the standard VLP group
upon stimulation with either MHC-I or -IIHA peptides. However,
splenocytes from flagellin-containing cVLP-immunized mice produced
high levels of TNF-.alpha..
[0173] Flagellin-containing VLPs promote protective immunity
against lethal virus challenge. To determine whether the enhanced
antibody and T-cell responses as described above conferred
protection against lethal virus challenge, immunized mice were
challenged intranasally with mouse-adapted PR8 viruses at
40.times.LD.sub.50. As shown in FIGS. 18A and 18B, mice immunized
with standard VLPs, flagellin-incorporating VLPs, or a mixture of
standard VLPs plus soluble flagellin, retained their healthy status
as measured by body weight. These groups showed 100% protective
immunity to lethal PR8 virus challenge. By contrast, no protection
was observed using M1 VLPs, though mice immunized with M1 VLPs
showed a minor improvement clinical score, as represented by body
weight loss and a 1- to 2-day delay in reaching the endpoint upon
lethal virus challenge compared to what was seen for the negative
control group
[0174] Because the flagellin-containing cVLP group showed much
higher antibody levels and enhanced T cellular responses, we tested
whether their enhanced immunity also can provide cross-protection
to a heterologous virus challenge. Thus, immunized mice were
challenged with an A/Philippines/82 H.sub.3N.sub.2 virus at
40.times.LD.sub.50 per mouse. Data shown in FIG. 19C demonstrate
that all VLP groups lost weight, as did the naive group, but the
flagellin-containing cVLP-immunized group lost less weight. Though
there was a 1-day delay compared to the control group, all mice in
the standard VLP group or in the group immunized with the standard
VLPs plus soluble flagellin, as well as those of the M1 VLP group,
reached the endpoint by day 8 or 9. In the flagellin-containing
cVLP-immunized group, however, mice began to regain body weight
from day 9 (FIG. 19C), and 67% of mice survived the challenge with
the heterologous A/Philippines virus, as shown in FIG. 19D.
[0175] Clearance of virus load from an infected host. Rapid
clearance of virus from the body and low virus loads after
infection are important for decreased morbidity and mortality after
infection. Immunized mice, therefore, were intranasally infected
with A/PR8 or A/Philippines virus at the same dose as used in the
challenge experiment (above). Mice were sacrificed on day 4, and
the virus loads in lungs were determined by plaque assay on MDCK
cells. As shown in FIG. 20, virus titers were not detected for
either the standard or the flagellin-containing VLP groups in the
PR8-infected mice. In contrast, naive mice and mice of the M1 VLP
group were carrying high virus loads of 1.5.times.10.sup.9 and
8.5.times.10.sup.8 PFU/ml of lung extract, respectively. For
A/Philippines virus-infected mice, the standard VLP group showed a
lung virus titer of 4.2.times.10.sup.6 PFU/ml of lung extract,
whereas the flagellin-containing VLP group showed
1.6.times.10.sup.4 PFU/ml of lung extract, a low titer when
compared to that of standard cVLP group (P 0.05). The naive and M1
VLP groups showed titers of 1.5.times.10.sup.9 and
8.7.times.10.sup.8 PFU/ml of lung extract, respectively. These
results demonstrated that by incorporating flagellin into VLPs as
an adjuvant as disclosed in the present disclosure, influenza VLPs
induce enhanced immunity, providing complete protection against a
homologous virus challenge, and significant cross-protection
against a heterosubtypic virus challenge.
[0176] The present disclosure, therefore, encompasses adjuvants for
VLP and virosomal vaccines where the membrane-anchored form of
Salmonella flagellin is incorporated as a modified flagellin into
VLPs. By also fusing the SP-encoding DNA from honeybee protein
melittin and the influenza HA TM-CT region to the Salmonella
flagellin, the modified flagellin was efficiently expressed in
insect cells with a baculovirus-derived protein expression system,
and the expressed modified flagellin protein was presented on the
cell surface. This membrane-anchored flagellin, together with HA,
was also incorporated into M1-derived influenza VLPs.
[0177] The adjuvant function of flagellin is derived from its
capacity for binding TLR-5 and several reports have shown that
soluble chimeric flagellins in-frame fused to an antigen at the C
terminus of the flagellin region retain both TLR-5-binding capacity
and can promote the immunogenicity of the antigen itself (Cuadros
et al., (2004) Infect. Immun. 72:2810-2816; Huleatt et al., (2008)
Vaccine 26:201-214; McDonald et al., (2007) J. Infect. Dis.
195:1607-1617). Embodiments of the present disclosure introduce the
HA membrane anchor at the C terminus of flagellin. The TLR-5
agonist activity of membrane-anchored flagellin in VLPs was
verified by a TLR-5 bioactivity assay, and the ability of the
membrane-anchored flagellin to act as an adjuvant for VLP vaccines
was shown in the immunization results.
[0178] Previously, the immune responses induced by influenza VLPs
containing HA and M1 were shown (Quan et al., (2007) J Virol
81:3514-3524) and it was found that the influenza VLPs induced high
levels of antibody responses. However, using the methods
encompassed by the present disclosure, flagellin may be
incorporated into VLPs. The cVLP embodiments of the disclosure
induce antibody responses more rapidly, and to a greater extent,
than those induced by standard VLPs.
[0179] Flagellin-incorporated cVLPs of the present disclosure also
activated a Th1-preferred Th1/Th2 profile, as demonstrated by a
lower IgG1/IgG2a ratio. Standard VLPs and flagellin-containing
cVLPs induced comparable levels of IgG1. However, embodiments of
the flagellin-containing VLPs of the disclosure induced a
fourfold-higher level of IgG2a production. Therefore,
membrane-anchored flagellin variant embodiments of the present
disclosure differ significantly from recombinant soluble flagellin,
which induces a Th2 phenotype (Didierlaurent et al., (2004) J.
Immunol. 172:6922-6930). The response induced by the
membrane-anchored flagellin variants as used in the embodiments of
the present disclosure are similar to flagellin in its native
surface-bound context on live Salmonella bacteria, as described by
Cunningham et al., (2004) Eur. J. Immunol. 34:2986-2995.
[0180] Splenocytes from mice immunized with embodiments of the
flagellin-containing VLPs of the present disclosure produced high
levels of IL-2, IFN-.gamma., TNF-.alpha., and IL-4 when stimulated
by HA-specific MHC-I or-II-restricted peptides, indicating the
induction of antigen-specific T cells after immunization. Higher
levels of cytokine production stimulated by an MHC-II HA-specific
peptide demonstrated that flagellin primed a CD4-dominant T-cell
response.
[0181] The flagellin-containing VLPs of the present disclosure
primed a high level of TNF-.alpha. secretion, whereas TNF-.alpha.
secretion of splenocytes as induced by standard VLPs remained at
the background level. The high-level production of TNF-.alpha.
induced by flagellin-containing VLPs and the enhanced specific
immunity show that TNF-.alpha. plays an important role in
activating adaptive immunity in flagellin-containing VLP
immunization.
[0182] Mice pre-immunized with soluble recombinant flagellin
produce high levels of flagellin-specific IgG responses. However,
such pre-existing immunity against flagellin did not block the
adjuvant function of membrane-anchored flagellin in VLPs,
consistent with flagellin being an effective adjuvant even in the
presence of pre-existing anti-flagellin immunity. While not wishing
to be bound by any one theory, pre-existing anti-flagellin antibody
might enhance the targeting of cVLPs to antigen-presenting cells
(APCs) by the Fc portion of the cVLP-bound anti-flagellin IgG,
since APCs express Fc.gamma. receptors. However, significant
differences in responses with or without pre-existing
anti-flagellin immunity were not observed.
[0183] In its native state, flagellin is glycosylated at six sites
by O-linked .beta.-acetylglucosamine (Schirm et al., (2004). J.
Bacteriol. 186:2523-2531). Glycosylation has roles for both
flagellar assembly and biological function (Logan, S. M. (2006)
Microbiology 152:1249-1262). In the N- and C-termini of Salmonella
flagellin, which are both recognized by TLR-5, four N-linked
glycosylation sites are located, at Asn19 and Asn101 in the
N-terminal region, and at Asn446 and Asn465 in the C-terminal
region. For the insect cell-derived membrane-anchored flagellins of
the disclosure, though the whole-cell lysate showed two bands at 55
and 65 kDa, the VLPs showed only the higher (65 kDa) molecular-mass
form. It is known that insect cells mostly produce simple N-glycans
with terminal mannose residues (Harrison & Jarvis (2006). Adv.
Virus Res. 68:159-191). The glycosylated forms of flagellin
variants of the present disclosure do retain potent adjuvant
activity.
[0184] Mixtures of soluble flagellin plus antigen do not promote
high antigen-specific immune responses, and the physical
association of an antigen with flagellin (fusion protein of antigen
with flagellin) may be necessary for the promotion of the specific
immune responses (Huleatt et al., (2008) Vaccine 26:201-214;
McDonald et al., (2007) J. Infect. Dis. 195:1607-1617). However,
embodiments of membrane-anchored flagellin incorporated into VLPs,
as disclosed in the present disclosure, boosted a strong specific
immune response; a mixture of soluble flagellin and standard VLPs,
however, failed to show a similar adjuvant effect. While not
wishing to be bound by any one theory, because TLR-5 is expressed
on the surfaces of APCs, an association with flagellin may result
in the TLR-5-mediated uptake of antigens and consequent processing
and presentation.
[0185] The goal of vaccines is to elicit protection against
pathogens and an effective adjuvant should extend this protective
effect by promoting the immunogenicity of specific antigens in
vaccines, thereby increasing the magnitude and the duration of
immunity. The flagellin-containing VLPs of embodiments of the
present disclosure induced apparently complete protection against
an homologous virus challenge, and partially cross-protected
against heterologous virus challenge, a broader-spectrum
immunity.
[0186] Except as noted hereafter, standard techniques for peptide
synthesis, cloning, DNA isolation, amplification and purification,
for enzymatic reactions involving DNA ligase, DNA polymerase,
restriction endonucleases, and the like, and various separation
techniques are those known and commonly employed by those skilled
in the art. A number of standard techniques are described in
Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold
Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982)
Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.;
Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth.
Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101;
Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972)
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y., Old Primrose (1981) Principles of Gene
Manipulation, University of California Press, Berkeley; Schleif and
Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.)
(1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and
Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press,
Oxford, UK; Setlow and Hollaender (1979) Genetic Engineering:
Principles and Methods, Vols. 1-4, Plenum Press, N.Y.
[0187] One aspect of the disclosure, therefore, provides adjuvant
polypeptides comprising at least one domain capable of selectively
interacting with a Toll-like receptor of an animal or human cell,
and where the domain may be capable of increasing an immune
response in a recipient host; and at least one heterologous region
selected from the group consisting of a signal peptide region and a
transmembrane-cytoplasmic tail region.
[0188] In one embodiment of the disclosure, the adjuvant
polypeptide may comprise a first region, where the first region
comprises a signal peptide; a second region, where the second
region may comprise at least one domain capable of selectively
interacting with a Toll-like receptor of an animal or human cell,
and where the domain is capable of increasing an immune response in
a recipient host; and a third region, where the third region may
comprise a transmembrane-cytoplasmic tail peptide.
[0189] In embodiments of this aspect of the disclosure, in the
adjuvant polypeptides thereof, at least one domain is capable of
selectively interacting with a Toll-like receptor of an animal or
human cell, and may be derived from a surface protein of a
bacterial species, or of a protozoal species.
[0190] In some embodiments of the adjuvant polypeptide, the surface
protein may be a protein of a bacterial or a protozoal flagellum,
or a fragment thereof.
[0191] In one embodiment, the surface protein is a flagellin of the
bacterial species Salmonella enteritidis.
[0192] In one embodiment of the adjuvant polypeptides of the
disclosure, the adjuvant polypeptide may have the amino acid
sequence SEQ ID NO.: 10, or a conservative variant thereof.
[0193] In another embodiment, the second region thereof may further
comprise a peptide linker.
[0194] In yet another embodiment, the peptide linker has the amino
acid sequence according to SEQ ID NO.: 18.
[0195] In one embodiment of the disclosure, the signal peptide may
be a signal peptide of a bee melittin polypeptide.
[0196] In another embodiment of the disclosure, the
transmembrane-cytoplasmic region may be derived from a
hemagglutinin A polypeptide of the influenza virus.
[0197] Another aspect of the disclosure is nucleic acid molecules
comprising; a region encoding a bacterial flagellin polypeptide, or
a fragment thereof, wherein the flagellin polypeptide or fragment
thereof may comprise at least one domain capable of specifically
interacting with a Toll-like receptor of an animal or human cell;
and at least one region selected from the group consisting of: a
region encoding a heterologous signal peptide, and a region
encoding a transmembrane-cytoplasmic tail capable of being
incorporated into a virus-like particle or virosome.
[0198] One embodiment of this aspect of the disclosure may
comprise: a region encoding a bacterial flagellin polypeptide, or a
fragment thereof, wherein the flagellin polypeptide or fragment
thereof comprises at least one domain capable of specifically
interacting with a Toll-like receptor of an animal or human cell; a
region encoding a heterologous signal peptide; and a region
encoding a transmembrane-cytoplasmic tail capable of being
incorporated into a virus-like particle or virosome.
[0199] In embodiments of this aspect of the disclosure, the
heterologous signal peptide may be a bee melittin signal peptide,
the trans-membrane-cytoplasmic tail may be from an influenza virus
hemagglutinin.
[0200] In other embodiments, the nucleic acid molecules may
comprise: a first nucleotide sequence encoding the amino acid
sequence from amino acid positions about 1 to about 305 of sequence
SEQ ID NO.: 10, or a conservative variant thereof; a second
nucleotide sequence encoding the amino acid sequence from amino
acid positions about 430 to about 565 of sequence SEQ ID NO.: 10,
or a conservative variant thereof; and a third nucleotide sequence
disposed between the first and the second nucleotide sequences,
where the third nucleotide sequence encodes a region selected from
the group consisting of; a region of a bacterial or protozoal
surface protein polypeptide, a peptide linker, and an immunogenic
peptide.
[0201] In one embodiment of this aspect of the disclosure, the
first nucleotide sequence may be according to about position 1 to
about position 615 of nucleotide sequence SEQ ID NO.: 9, or a
conservative variant thereof; and the second nucleotide sequence
may be according to about position 1293 to about position 1695 of
nucleotide sequence SEQ ID NO.: 9, or a conservative variant
thereof.
[0202] In one embodiment of the disclosure, the nucleic acid
molecule may comprise the nucleic acid sequence SEQ ID NO.: 9, or a
conservative variant thereof.
[0203] In one embodiment of the disclosure, the nucleic acid
molecule may have the nucleic acid sequence SEQ ID NO.: 9.
[0204] In some embodiments of the disclosure, the nucleic acid
molecule may be operably inserted into a nucleic acid expression
vector.
[0205] In embodiments of the disclosure, the nucleic acid
expression vector may be selected from the group consisting of: a
plasmid, a baculovirus vector, a cosmid, a viral vector, a
chromosome, a mini-chromosome, a modified vaccinia Ankara (MVA)
vector, a plasmid, a recombinant poxvirus vector, and a recombinant
adenovirus vector, an alphavirus vector, and a paramyxovirus
vector.
[0206] Another aspect of the disclosure provides immunogenic
compositions comprising: an adjuvant polypeptide comprising at
least one region capable of selectively interacting with a
Toll-like receptor protein of a host; and an immunogen capable of
producing an immune response in a recipient host.
[0207] In one embodiment of this aspect of the disclosure, the
immunogenic compositions may further comprise a virus-like carrier,
wherein the virus-like carrier is selected from the group
consisting of a virus-like particle and a virosome, and wherein the
adjuvant polypeptide and the immunogen may be incorporated in the
virus-like particle or the virosome. In these embodiments of the
disclosure, the adjuvant polypeptide may be incorporated into the
VLP or virosome.
[0208] In another embodiment, the adjuvant polypeptide may be a
surface polypeptide of a bacterial species or of a protozoal
species, or a modified variant of the surface polypeptide.
[0209] In yet another embodiment, the surface polypeptide is a
bacterial flagellin.
[0210] In one embodiment of the disclosure, in the immunogenic
composition, the flagellin may be of the bacterial species
Salmonella enteritidis.
[0211] In embodiments of this aspect of the disclosure, the
modified variant of the adjuvant polypeptide may comprise at least
one heterologous peptide region selected from the group consisting
of a signal peptide region and a transmembrane-cytoplasmic tail
region.
[0212] In other embodiments of the disclosure, the modified variant
of the adjuvant polypeptide may comprise a first heterologous
peptide region, where the first heterologous peptide region may be
a signal peptide, and a second heterologous peptide region, wherein
the second heterologous peptide region may be a
transmembrane-cytoplasmic tail peptide.
[0213] In the embodiments of this aspect of the disclosure, the
bacterial flagellin polypeptide may be a modified bacterial
flagellin polypeptide modified by deletion of a region of a
full-length bacterial flagellin polypeptide.
[0214] In other embodiments, the modified adjuvant polypeptide may
further comprise a heterologous peptide region, where said region
may be disposed between two domains of the adjuvant polypeptide,
where each domain may be capable of selectively targeting a
toll-like receptor protein of a host.
[0215] In yet other embodiments of the immunogenic compositions of
the disclosure, the adjuvant polypeptide may further comprise a
heterologous peptide region, wherein said region is antigenic.
[0216] In still other embodiments of the disclosure, the virus-like
carrier may be a virosome, comprising: at least one viral surface
envelope glycoprotein expressed on the surface of the virosome; and
at least one adjuvant molecule expressed on the surface of the
virosome, wherein the adjuvant molecule comprises a
membrane-anchored form of a bacterial or protozoal surface
component that is a mammalian toll-like receptor ligand molecule.
In another embodiment of the disclosure, the virus-like carrier may
be a virus-like particle and further comprises a viral core protein
capable of self-assembling into a virus-like particle core.
[0217] In these embodiments, the viral core protein and at least
one viral surface envelope glycoprotein may be from different
viruses.
[0218] In these embodiments of the disclosure, the viral core
protein may be selected from: a retrovirus Gag protein, a
retrovirus matrix protein, a rhabdovirus M protein, a filovirus
viral core protein, a coronavirus M protein, a coronavirus E
protein, a coronavirus NP protein, a bunyavirus N protein, an
influenza M1 protein, a paramyxovirus M protein, an arenavirus Z
protein, a cytomegalovirus (CMV) core protein, a herpes simplex
virus (HSV) core protein, or a combination thereof.
[0219] In these embodiments, also, the retrovirus gag protein may
be selected from; a Human Immunodeficiency Virus (HIV) Gag protein,
a Simian Immunodeficiency Virus (SIV) Gag protein, a human foamy
virus Gag protein, or a Murine Leukemia Virus (MuLV) Gag
protein.
[0220] In other embodiments, the viral core protein may be selected
from: a Vesicular Stomatitis Virus (VSV) M protein, an Ebola Virus
VP40 protein, a Lassa Fever Virus Z protein, or a combination
thereof.
[0221] In still other embodiments, the viral surface envelope
surface glycoprotein is selected from: a retrovirus/lentivirus
glycoprotein, a bunyavirus glycoprotein, a coronavirus
glycoprotein, an arenavirus glycoprotein, a filovirus glycoprotein,
an influenza virus glycoprotein, a paramyxovirus glycoprotein, a
rhabdovirus glycoprotein, an alphavirus glycoprotein, a flavivirus
glycoprotein, a cytomegalovirus glycoprotein, a herpes virus
glycoprotein, or a combination thereof.
[0222] In other embodiments of the disclosure, the retrovirus
glycoprotein may be selected from: a human immunodeficiency virus
(HIV) glycoprotein, a simian immunodeficiency virus (SIV)
glycoprotein, a simian-human immunodeficiency virus (SHIV)
glycoprotein, a feline immunodeficiency virus (FIV) glycoprotein, a
feline leukemia virus glycoprotein, a bovine immunodeficiency virus
glycoprotein, a bovine leukemia virus glycoprotein, an equine
infectious anemia virus glycoprotein, a human T-cell leukemia virus
glycoprotein, a mouse mammary tumor virus envelope glycoprotein
(MMTV), a human foamy virus glycoprotein, or a combination
thereof.
[0223] In other embodiments, the viral surface envelope surface
glycoprotein may be selected from: an influenza virus glycoprotein,
a Respiratory syncytial virus (RSV) glycoprotien, a Lassa Fever
virus glycoprotein, an Ebola Virus glycoprotein, a Marburg virus
glycoprotein, a VSV glycoprotein, a rabies virus glycoprotein, a
hepatitis virus glycoprotein, a herpes virus glycoprotein, a CMV
glycoprotein, or a combination thereof.
[0224] In other embodiments of the disclosure, the virus-like
particle may be a chimeric virus-like particle comprising an
influenza hemagglutinin, a matrix protein M1, and a modified
bacterial flagellin adjuvant polypeptide, wherein the modified
bacterial flagellin comprises a heterologous
transmembrane-cytoplasmic tail and is incorporated into the
chimeric virus-like carrier, and wherein the virus-like carrier is
a virus-like particle or a virosome.
[0225] In the embodiments of this aspect of the disclosure, the
immunogenic compositions may further comprise a pharmacologically
acceptable carrier.
[0226] Still another aspect of the disclosure are methods of
generating an immunological response in an animal or human
comprising: exposing the immune system of an animal or human host
to an immunogen and a virus-like carrier, wherein the virus-like
carrier may be a virus-like particle or a virosome, and where the
virus-like carrier may comprise an adjuvant polypeptide comprising
a host cell Toll-like receptor ligand polypeptide derived from a
bacterial or protozoal flagellum polypeptide, and at least one
heterologous peptide selected from the group consisting of a
transmembrane-cytoplasmic tail polypeptide and a heterologous
signal peptide; thereby generating in the recipient host an immune
response directed against the immunogen.
[0227] In embodiments of this aspect of the disclosure, exposing
the host to the virus-like particle may comprise: delivering to the
recipient host or host cell at least one expression vector, where
the at least one expression vector or a multiplicity of expression
vectors comprise at least one polynucleotide encoding at least one
polypeptide selected from the group consisting of: a viral core
protein, a viral surface envelope glycoprotein, and an adjuvant
molecule, where each of the polynucleotide or polynucleotides is
operably linked to an expression control region; expressing in the
recipient host or host cell at least one viral surface envelope
glycoprotein, and at least one adjuvant molecule, thereby
assembling a virosome virus-like carrier.
[0228] In one embodiment of this aspect of the disclosure, an
expression vector may further comprise a polynucleotide encoding a
viral core protein, wherein the viral core protein is incorporated
into a virus-like particle.
[0229] In various embodiments of this aspect of the disclosure, one
or more vectors may be selected from the group consisting of: a
plasmid, a cosmid, a viral vector, an artificial chromosome, a
mini-chromosome, a baculovirus vector, a modified vaccinia Ankara
(MVA) vector, a recombinant poxvirus vector, a recombinant VSV
vectors, a recombinant adenovirus expression systems, an alphavirus
vector, a paramyxovirus vector, or a combination thereof.
[0230] Yet another aspect of the disclosure provides methods of
immunizing a host comprising: co-expressing in one or more host
cells at least one viral surface envelope surface glycoprotein, and
at least one adjuvant molecule; whereby the at least one viral
surface envelope glycoprotein and the adjuvant molecule assemble to
form a virus-like carrier, and wherein the at least one adjuvant
molecule is a mammalian toll-like receptor ligand molecule.
[0231] Embodiments of this aspect of the disclosure may further
comprise co-expressing a viral core protein, wherein the
co-expressed viral core protein is assembled into the virus-like
carrier, thereby forming a virus-like particle.
[0232] In embodiments of this aspect of the disclosure, the at
least one adjuvant molecule may be a bacterial flagellin
molecule.
[0233] In other embodiments of the disclosure, the at least one
adjuvant molecule may assemble with the viral core protein and the
at least one viral surface envelope glycoprotein to form a VLP,
wherein the at least one adjuvant molecule may be a
membrane-anchored variant of a bacterial surface protein that is a
mammalian toll-like receptor (TLR) ligand molecule.
[0234] In other embodiments, the virus-like particle may be a
chimeric virus-like particle comprising an influenza hemagglutinin,
a matrix protein M1, and a modified bacterial flagellin adjuvant
polypeptide, wherein the modified bacterial flagellin comprises a
heterologous transmembrane-cytoplasmic tail and is incorporated
into the chimeric virus-like particle, the chimeric virus-like
particle inducing an immune response in the animal or human host
and thereby inhibiting the development of influenza in the
host.
[0235] The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present disclosure to its fullest extent. All
publications recited herein are hereby incorporated by reference in
their entirety.
[0236] It should be emphasized that the embodiments of the present
disclosure, particularly, any "preferred" embodiments, are merely
possible examples of the implementations, merely set forth for a
clear understanding of the principles of the disclosure. Many
variations and modifications may be made to the above-described
embodiment(s) of the disclosure without departing substantially
from the spirit and principles of the disclosure. All such
modifications and variations are intended to be included herein
within the scope of this disclosure, and the present disclosure and
protected by the following claims.
[0237] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to perform the methods and use the compositions
and compounds disclosed and claimed herein. Efforts have been made
to ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.), but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C., and pressure is at or near
atmospheric. Standard temperature and pressure are defined as
20.degree. C. and 1 atmosphere.
Examples
Example 1
[0238] Recombinant Monomeric Soluble Flagellin from Salmonella
Enteritidis expressed in E. Coli
[0239] A construct was generated to express wild type flagellin
from the flagella filament protein gene FliC of S. enteritidis, and
the protein was purified from E. coli. The flagellin gene was
cloned into expression vector pET-22b(+) (Novigen) with NdeI/Xho1
in-frame with a polyhistidine tag. E. coli strain BL(21) was used
for the production of flagellin by IPTG induction. Five hours
post-induction, cells were harvested and used for the isolation of
flagellin with Nickle-bead (Qiagen) affinity purification following
the manufacturer's instruction. Purified soluble flagellin was
characterized by gel electrophoresis and Coomassie Blue staining
(as shown in FIG. 1A) and western blot analysis (as shown in FIG.
1B). Removal of LPS was determined as described.
Example 2
[0240] Isolating Polymeric Flagellin from Salmonella
Enteritidis
[0241] Flagella filament protein (FliC) from S. enteritidis wild
type was prepared as described by Ibrahim et al., (1985) J. Clin.
Microbiol. 22: 1040-1044; Vorderviszt, F., (1989) J. Mol. Biol.
209: 127-133; and Ogushi et al., (2001) J. Biol. Chem. 32:
30521-30526, incorporated herein by reference in their entireties,
with some modifications. FIG. 2 is a gel analysis showing the
degree of purity of the isolated flagellin.
[0242] Immunogenic activity was tested in Balb/c mice.
Anti-flagellin-specific IgG antibodies were assessed by Elisa.
Plates were coated with either recombinant flagellin (prepared as
described in Example 1 above) or purified bacterial flagellin, and
antibodies were detected with anti-Salmonella enteritidis flagellin
monoclonal antibody at a 1:100 dilution.
[0243] One immunization administered intramuscularly induced very
high IgG titers, even at administered concentrations as low as 1
.mu.g. In contrast, intranasal administration demonstrated low,
although still detectable, immune responses, as shown in FIG.
3.
Example 3
[0244] Flagellin (Monomeric and Polymeric) is an Effective Adjuvant
in Vaccine Composition with a Viral Antigen.
[0245] Balb/c female mice were immunized intramuscularly or
intranasally with either 1 .mu.g of soluble recombinant (monomeric)
flagellin, or 1 .mu.g of soluble polymeric flagellin (isolated from
S. enteritidis wild type), and co-administered with 10 .mu.g of
inactivated H1N1 influenza virus (A/PR/8/34, hereinafter
abbreviated as PR8) in a vaccine composition. Inactivated influenza
virus alone, administered intramuscularly or intranasally, was used
as a control group. Sera were collected retro-orbitally 14 days
after immunization and tested for influenza-specific binding IgG
antibodies.
[0246] The results from the intramuscular immunization demonstrated
that inactivated influenza virus mixed with monomeric flagellin
induced 3.5-fold higher antibody titers when compared to
inactivated influenza virus vaccine, as shown in FIG. 4B. The data
also demonstrated that inactivated influenza virus mixed with the
polymeric form of flagellin induced a 6-fold higher IgG level than
did the PR8 group, and at least 1.5-fold higher titer than did the
vaccine composition of PR8 with monomeric flagellin after a single
immunization (FIG. 4B).
[0247] The same vaccine compositions administered intranasally with
10 .mu.g of whole inactivated influenza virus (A/PR/8/34, PR8) with
1 .mu.g of polymeric (FliC poly), or monomeric (FliC mono),
flagellin, gave about a 1.5 to about 2-fold higher PR8-specific IgG
titer than did immunization with influenza antigen alone after 2
immunizations, 4 weeks apart, as shown in FIG. 4A.
Example 4
Cell Lines and Viruses.
[0248] Spodoptera frugiperda Sf9 cells were maintained as
suspension cultures in flasks with serum-free SF900 II medium
(Gibco-BRL) at 27.degree. C. with stirring at a speed of 80 rpm.
Madin-Darby canine kidney (MDCK) cells were cultured in Dulbecco's
Modified Eagle's Medium (DMEM) plus 10% fetal bovine serum.
Influenza A/PR8 (H1N1) virus was grown in and purified from hen egg
embryonic fluid as described by Quan et al., (2007) J. Virol.
82:1350-1359, incorporated herein by reference in its entirety.
Mouse-adapted PR8 and A/Philippines/2/82/X-79 (H3N2)
(A/Philippines) viruses were prepared as described by Quan et al.,
(2007) J. Virol. 81:3514-3524, incorporated herein by reference in
its entirety.
Example 5
Construction of a Membrane-Anchored Flagellin Gene.
[0249] A full-length membrane-anchored flagellin encoding nucleic
acid molecule was generated (as schematically illustrated in FIG.
5) by fusing in-frame (i) a signal peptide(SP)-encoding from
honeybee melittin, and (ii) the transmembrane (TM) and cytoplasmic
tail (CT) regions from the influenza A virus PR8 hemagglutinin
(HA), to the 5' and 3' termini of the flagellin gene, respectively.
The melittin SP-encoding fragment was PCR amplified from the
plasmid M-TM.CT.sub.MMTV (described by Wang et al., (2007) J.
Virol. 81:10869-10878, incorporated herein by reference in its
entirety) by use of primers 5'-GGTTCTAGAATGAAATTCTTAGTC-3' (SEQ ID
NO.: 1) and 5'-GTGGGATCCT TTCATGTTGATCGG-3' (SEQ ID NO.: 2) (XbaI
and BamHI sites are underlined) and cloned into the XbaI/BamHI
sites of cloning vector pBluescript (-), resulting in plasmid
pBluescript-SP. The Salmonella enterica serovar Typhimurium
flagellin gene (fliC; GenBank accession no. D13689) was amplified
from plasmid pEM045 pEF6 FliC stop (Alan Aderem, Institute of
Systems Biology, Seattle, Wash.) by using primers
5'-GCAGGATCCATGGCACAAGTCAT-3' (SEQ ID NO.: 3) and
5'-CGCGAATTCACGCAGTAAAGAGAG-3' (SEQ ID NO.: 4) (underlined sites
are BamHI and EcoRI, respectively) and inserted into
pBluescript-SP, resulting in construct pBluescript-SP-FliC. The
nucleic acid sequence encoding the HA TM-CT region was amplified
from plasmid pc/pS1 containing the full-length HA gene by using
primers 5'-GCTAGAATTCCAGATTCTGG CGATC-3' (SEQ ID NO.: 5) and
5'-GCTAGGGCCCTTATCAGATGCATATTCT-3' (SEQ ID NO.: 6) (underlined
sites are EcoRI and ApaI, respectively) and inserted into
pBluescript-SP-FliC to produce pBluescript-SP-FliC-HA tail.
[0250] The full-length membrane anchored flagellin gene was
amplified from pBluescript-SP-FliC-HA tail by using primers
5'-GCTCGTCGACATGAAATTCTTAG-3' (SEQ ID NO.: 7) and 5'-GCTACTCGAGT
TATCAGATGCATATTC-3' (SEQ ID NO.: 8) (SalI and XhoI sites,
respectively, are underlined) and inserted into pFastBac 1 under
the control of the polyhedrin promoter.
[0251] Primer sequences used in the generation of the
membrane-anchored flagellin-encoding nucleic acid constructs are
shown in FIG. 6. The nucleotide sequence (SEQ ID NO.: 9) of the
membrane-anchored flagellin-encoding nucleic acid was verified, and
is shown in FIG. 7. The amino acid sequence (SEQ ID NO.: 10)
encoded by the membrane-anchored flagellin-encoding nucleic acid
(SEQ ID NO.: 9) is shown in FIG. 8.
Example 6
[0252] Generation of rBVs.
[0253] A recombinant baculovirus vector (rBV) expressing membrane
anchored flagellin was derived from the transfer plasmid pFastBac1
(described above in Example 1) by using a Bac-to-Bac expression
system (Invitrogen) according to the manufacturer's instructions.
rBVs expressing PR8 HA and M1 were described by Quan et al.,
(2007). J. Virol. 81:3514-3524, incorporated herein by reference in
its entirety.
Example 7
Determination of the Cell Surface Expression of Membrane-Anchored
Flagellin.
[0254] The presence of the membrane-anchored flagellin on cell
surfaces was determined by a cell surface expression assay. Sf9
cells were seeded in six-well plates at 10.sup.6 cells/well. The
infection with the flagellin-expressing rBV and isotopic labeling
were performed as described by Wang et al., (2007) J. Virol.
81:10869-10878, incorporated herein by reference in its entirety.
The biotinylation of cell surface proteins and immunoprecipitation
were carried out according to Yang & Compans, Virol. (1996)
221:87-97, incorporated herein by reference in its entirety). Final
samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Gels were dried and then used for
autoradiography.
Example 8
[0255] Production and Characterization of Chimeric Virus-Like
Particles (cVLPs).
[0256] Flagellin-containing influenza cVLPs were produced by
infection of Sf9 cells with rBVs expressing PR8 HA, M1, and
membrane-anchored flagellin. Standard VLPs containing HA/M1, and
control VLPs containing M1 only, were produced by infecting Sf9
cell with rBVs expressing PR8 HA and M1 as described by Quan et
al., (2007). J. Virol. 81:3514-3524, incorporated herein by
reference in its entirety. Cell culture supernatants were collected
on day 3 post-infection and cleared by a brief centrifugation
(4,000.times.g for 20 min at 4.degree. C.). VLPs were pelleted by
ultracentrifugation at 100,000.times.g for 1 h at 4.degree. C. The
pellets were resuspended in phosphate-buffered saline (PBS) at
4.degree. C. overnight. VLPs were further purified through a
20%-35%-60% discontinuous sucrose gradient at 100,000.times.g for 1
h at 4.degree. C. The VLP band between 35% and 60% was collected
and then diluted with PBS and pelleted at 100,000.times.g for 1 h
at 4.degree. C. VLPs were resuspended in PBS overnight at 4.degree.
C. The resulting VLPs were characterized by Western blot analysis
(as shown in FIG. 10), hemagglutination activity analysis, and
electric microscopic observation (as shown in FIG. 11). For Western
blot analysis, HA and M1 bands were probed by mouse anti-HA or M1
polyclonal antibodies. Membrane-anchored flagellin was detected by
rabbit antiflagellin polyclonal antibodies. The flagellin content
in cVLPs was estimated by comparison with a standard purified
soluble standard flagellin in Western blotting. The
hemagglutination activity of VLPs was determined by the capacity to
hemagglutinate chicken red blood cells (Quan et al., (2007). J.
Virol. 81:3514-3524, incorporated herein by reference in its
entirety). For electron microscopy, VLP samples (5 .mu.l to 10
.mu.l; 0.1 mg/ml protein) were examined as described by Wang et
al., (2007) J. Virol. 81:10869-10878, incorporated herein by
reference in its entirety.
Example 9
[0257] Treatment with Glycosidases.
[0258] N-glycosidase F (PNGase F) and endoglycosidase H (endo-H)
(New England Biolabs) were used to determine the glycosylation of
membrane-anchored flagellin by following the manufacturer's
instructions. Flagellin-containing VLPs (10 .mu.g in a volume of 10
.mu.l) were mixed with 1 .mu.l of denaturing buffer and heated at
100.degree. C. for 10 min. After being cooled in an ice bath for 2
min, samples were mixed with reaction buffer and PNGase F or endo-H
and incubated at 37.degree. C. for 1 h. The reaction was terminated
by adding sodium dodecyl sulfate-polyacrylamide gel electrophoresis
loading buffer, and the mixture was heated at 100.degree. C. for 5
min. Samples were subjected to Western blotting analysis, as shown
in FIG. 12.
Example 10
TLR-5-Specific Bioactivity Assay.
[0259] A RAW264.7 cell-based assay (McDonald et al., (2007). J.
Infect. Dis. 195:1607-1617, incorporated herein by reference in its
entirety) with modifications was used to determine the bioactivity
of membrane-anchored flagellin in VLPs. The RAW264.7 cell line was
a mouse macrophage cell line that expressed TLR-2 and -4, but not
TLR-5. In brief, 80%-confluent RAW264.7 (TLR-5.sup.- negative)
cells in a 75-cm.sup.2 T flask were transfected with 10 .mu.g of
plasmid pUNO-hTLR5 expressing the human TLR-5 protein (InvivoGen)
by use of the transfection reagent LIPOFECTAMINE.TM. (Invitrogen)
by following the manufacturer's instructions. Six hours
post-transfection, cells were removed from the T flask with a cell
scraper and seeded into 96-well plates by using 5.times.10.sup.4
cells/well in 100 .mu.l of fresh medium. Non-transfected RAW264.7
cells (TLR-5 negative) were also seeded into 96-well plates for
comparison. The TLR-5-positive and TLR-5-negative cells were
incubated with 100 .mu.l of serially diluted purified soluble
flagellin, flagellin-containing VLPs, or standard HA/M1 VLPs in
DMEM, and supernatants were collected after 24 hr. Levels of tumor
necrosis factor alpha (TNF-.alpha.) production stimulated by
soluble flagellin, flagellin-containing VLPs, or standard HA/M1
VLPs in both TLR-5-positive and TLR-5-negative cell cultures were
determined by enzyme-linked immunosorbent assay (ELISA), the
results of which are shown in FIG. 13. TLR-5 bioactivity was
expressed as the level of TNF-.alpha. production of TLR-5-positive
cells, from which was subtracted that of TLR-5-negative cells
stimulated by flagellin, flagellin-containing VLPs, or standard
HA/M1 VLPs.
Example 11
Immunization and Challenge.
[0260] Inbred female BALB/c mice were obtained from Charles River
Laboratory. Mouse groups (six mice per group) were immunized twice
with 10 .mu.g/mouse of VLPs at 4-week intervals (weeks 1 and 4).
For virus challenge, mice were anesthetized with isoflurane and
infected with 40 times the 50% lethal dose (40.times.LD.sub.50)
(LD.sub.50.times.50 PFU/mouse) of mouse-adapted A/PR8 virus (2,000
PFU) or 40.times.LD.sub.50 (LD50.times.25 PFU/mouse) of
mouse-adapted A/Philippines virus (1,000 PFU) in 50 .mu.l of PBS
per mouse 4 weeks after the boosting immunization. For the
determination of lung virus titers, six mice from each group were
sacrificed on day 4 post-challenge. Blood samples were collected on
weeks 0, 3, and 7 by retro-orbital plexus puncture. After clotting
and a brief centrifugation, serum samples were collected and stored
at -80.degree. C. prior to use for assays.
Example 12
Antibody Titration.
[0261] The influenza virus-specific serum antibody endpoint titers,
including those for immunoglobulin G (IgG) and subtypes (IgG1,
IgG2, and IgG2b), were determined by ELISA as described by Quan et
al., (2007). J. Virol. 81:3514-3524, incorporated herein by
reference in its entirety. In brief, 96-well microtiter plates
(Nunc Life Technologies) were coated with 100 .mu.l/well of
inactivated PR8 virus (5 .mu.g/ml) in PBS overnight at 4.degree. C.
For serum IgG titers against the heterologous A/Philippines virus
(H3N2), plates were coated with 100 .mu.l/well of inactivated
A/Philippines virus (5 .mu.g/ml). The serum samples were serially
diluted in twofold steps. After being washed and blocked with 1.5%
bovine serum albumin, plates were used to bind antibody with the
diluted sera. The detection color was developed by binding
horseradish peroxidase-labeled goat anti-mouse IgG, IgG1, IgG2a, or
IgG2b (Southern Biotechnology) at 37.degree. C. for 1 h. After
extensive washing, the substrate TMB (Zymed, Invitrogen) was
applied. The optical density at 450 nm (OD.sub.450) was read using
an ELISA reader (model 680; Bio-Rad). The highest dilution which
gave an OD.sub.450 twice that of the naive group without dilution
was designated as the antibody endpoint titer. Results are shown in
FIGS. 14A-14D.
Example 13
Virus Neutralization and HI Assays.
[0262] A neutralization assay was performed using MDCK cells as
described by Quan et al., (2007). J. Virol. 81:3514-3524,
incorporated herein by reference in its entirety. Hemagglutination
inhibition (HI) assays were carried out as described by Compans, R.
W. (1974) J. Virol. 14:1307-1309, incorporated herein by reference
in its entirety, with modifications. Briefly, 8 hemagglutination
units of PR8 or A/Philippines virus were mixed with serially
diluted receptor-destroying enzyme-pretreated serum samples in a
total volume of 50 .mu.l and incubated at 37.degree. C. for 1 h. An
equal volume of chicken blood cells (0.5%) was mixed with the
virus-serum mixture and incubated at 25.degree. C. for 30 min. HI
titers were recorded. Results are shown in FIGS. 15A-16B.
Example 14
Cytokine Assays.
[0263] Interleukin-2 (IL-2), gamma interferon (IFN-.gamma.),
TNF-.alpha., and IL-4 levels were determined by ELISA (eBioscience,
San Diego, Calif.) according to the manufacturer's instructions.
Splenocytes (1.times.10.sup.6 cells) isolated from immunized mice
were seeded into 96-well issue culture plates and stimulated with a
mixture of two major histocompatibility complex class I (MHC-I) HA
peptides (IYSTVASSL (SEQ ID NO.: 11) and LYEKVKSQL (SEQ ID NO.:
12)) or a pool of five MHC-II HA peptides (SFERFEIFPKE (SEQ ID NO.:
13), HNTNGVTAACSH (SEQ ID NO.: 14), CPKYVRSAKLRM (SEQ ID NO.: 15),
KLKNSYV NKKGK (SEQ ID NO.: 16), and NAYVSWTSKYN RRF (SEQ ID NO.:
17)) (shown in FIG. 17) at a concentration of 10 The plates were
incubated at 37.degree. C. for 2 days, and cell culture
supernatants were collected for cytokine assays, the results of
which are shown in FIGS. 18A-18D.
Example 15
Modified Bacterial Flagellin Adjuvants
[0264] (i) Modified Flagellin with influenza HA membrane anchor, as
shown in FIG. 21. Referring to FIG. 21, a full-length native
Salmonella flagellin polypeptide contains 495 amino acids. Modified
flagellins and have a deletion of amino acids 176 to 402 inclusive,
but include, as heterologous polypeptide sequences fused in-frame,
a melittin signal peptide, and an influenza HA
transmembrane-cytoplasmic domain (TM-CT). In the variant
M-dFlagellin-HA tail (2, FIG. 21), amino acids from 176-402 are
deleted and replaced with a hinge peptide having the sequence
GAPYPYDVPDYASPW (SEQ ID NO.: 18). In (3), a heterologous peptide
sequence replaces the deleted fragment.
[0265] (ii) Referring to FIG. 22, another modified flagellin
includes a mouse mammary tumor virus (MMTV) Env membrane anchor.
Full-length native Salmonella flagellin polypeptide (1) contains
495 amino acids. Modified flagellins (2) and (3) have a deletion of
amino acids 176 to 402 inclusive, but include, as heterologous
polypeptide sequences fused in-frame, a melittin signal peptide,
and MMMTV Env transmembrane and cytoplasmic domains. In the
M-dFlagellin-MMTV tail (2), amino acids from 176-402 are deleted
and replaced with a flexible hinge GAPYPYDVPDYASPW (SEQ ID NO.:
18). In (3), a heterologous peptide replaces the deleted
fragment.
[0266] (iii) Referring to FIG. 23, a full-length native Salmonella
flagellin polypeptide (1) contains 495 amino acids. Modified
flagellins (2) and (3) have a deletion of amino acids 176 to 402
inclusive, but include, as heterologous polypeptide sequences fused
in-frame, a melittin signal peptide, and an influenza HA
transmembrane-cytoplasmic domain (TM-CT). In the variant
M-dFlagellin-HA tail (2, FIG. 21), amino acids from 176-402 are
deleted and replaced with a hinge peptide having the sequence
GAPYPYDVPDYASPW (SEQ ID NO.: 18). In (3), a heterologous peptide
sequence replaces the deleted fragment. Additionally, the variant
flagellins are His-tagged modified flagellins that further include
a hexahistidine tag fused to the C-terminus of the modified
flagellin.
Example 16
[0267] As shown in FIGS. 25 and 26, a membrane-anchored flagellin
was constructed having a tandem of four repeats of M2e having
sequence SEQ ID NO.: 19 and substituting for the region of the
flagellin between the amino acid positions of 176 and 402
(tFliC-4.times.M2e). Expression of the inserted region, as shown in
FIG. 26, was shown in insect cells.
[0268] Insect cells were infected with rBV expressing
membrane-anchored flagellin with tandem M2e sequences inserted
(lanes 2 and 3) or mock rBV (lanes 1 and 4). Lanes 1 and 2,
infected cells were lysed and applied to a Western blot 2 days
postinfection. Lanes 3 and 4, infected cells were labeled by
surface-protein biotinylation 2 days postinfection. Cell lysates
were precipitated by immobilized neutravidin. Precipitates were
dissolved in SDS-PAGE loading buffer and applied to Western blot.
A, protein bands were probed by anti-flagellin antibody; B, Protein
bands were probed with anti-M2e antibody.
Sequence CWU 1
1
19124DNAArtificial sequenceForward primer 1ggttctagaa tgaaattctt
agtc 24224DNAArtificial sequenceReverse primer 2gtgggatcct
ttcatgttga tcgg 24323DNAArtificial sequenceForward primer
3gcaggatcca tggcacaagt cat 23424DNAArtificial sequenceReverse
primer 4cgcgaattca cgcagtaaag agag 24525DNAArtificial
sequenceForward primer 5gctagaattc cagattctgg cgatc
25628DNAArtificial sequenceReverse primer 6gctagggccc ttatcagatg
catattct 28723DNAArtificial sequenceForward primer 7gctcgtcgac
atgaaattct tag 23827DNAArtificial sequenceReverse primer
8gctactcgag ttatcagatg catattc 2791695DNAArtificial
sequenceNucleotide sequence of modified flagellin adjuvunctal
polypeptide with melltinin signal peptide and influenza
hemagglutinin Atransmembrane-cytoplasmic tail 9atgaaattct
tagtcaacgt tgcccttgtt tttatggtcg tgtacatttc ttacatctat 60gcggacccga
tcaacatgac cggatccatg gcacaagtca ttaatacaaa cagcctgtcg
120ctgttgaccc agaataacct gaacaaatcc cagtccgctc tgggcaccgc
tatcgagcgt 180ctgtcttccg gtctgcgtat caacagcgcg aaagacgatg
cggcaggtca ggcgattgct 240aaccgtttta ccgcgaacat caaaggtctg
actcaggctt cccgtaacgc taacgacggt 300atctccattg cgcagaccac
tgaaggcgcg ctgaacgaaa tcaacaacaa cctgcagcgt 360gtgcgtgaac
tggcggttca gtctgctaac agcaccaact cccagtctga cctcgactcc
420atccaggctg aaatcaccca gcgcctgaac gaaatcgacc gtgtatccgg
ccagactcag 480ttcaacggcg tgaaagtcct ggcgcaggac aacaccctga
ccatccaggt tggtgccaac 540gacggtgaaa ctatcgatat cgatctgaag
cagatcaact ctcagaccct gggtctggat 600acgctgaatg tgcaacaaaa
atataaggtc agcgatacgg ctgcaactgt tacaggatat 660gccgatacaa
cgattgcttt agacaatagt acttttaaag cctcggctac tggtcttggt
720ggtactgacc agaaaattga tggcgattta aaatttgatg atacgactgg
aaaatattac 780gccaaagtta ccgttacggg gggaactggt aaagatggct
attatgaagt ttccgttgat 840aagacgaacg gtgaggagac tcttgctggc
ggtgcgactt ccccgcttac aggtggacta 900cctgcgacag caactgagga
tgtgaaaaat gtacaagttg caaatgctga tttgacagag 960gctaaagccg
cattgacagc agcaggtgtt accggcacag catctgttgt taagatgtct
1020tatactgata ataacggtaa aactattgat ggtggtttag cagttaaggt
aggcgatgat 1080tactattctg caactcaaaa taaagatggt tccataagta
ttaatactac gaaatacact 1140gcagatgacg gtacatccaa aactgcacta
aacaaactgg gtgacgcaga cggcaaaacc 1200gaagttgttt ctattggtgg
taaaacttac gctgcaagta aagccgaagg tcacaacttt 1260aaagcacagc
ctgatctggc ggaagcggct gctacaacca ccgaaaaccc gctgcagaaa
1320attgatgctg ctttggcaca ggttgacacg ttacgttctg acctgggtgc
ggtacagaac 1380cgtttcaact ccgctattac caacctgggc aacaccgtaa
acaacctgac ttctgcccgt 1440agccgtatcg aagattccga ctacgcgacc
gaagtttcca acatgtctcg cgcgcagatt 1500ctgcagcagg ccggtacctc
cgttctggcg caggcgaacc aggttccgca aaacgtcctc 1560tctttactgc
gtgaattcca gattctggcg atctactcaa ctgtcgccag ttcactggtg
1620cttttggtct ccctgggggc aatcagtttc tggatgtgtt ctaatggatc
tttgcagtgc 1680agaatatgca tctga 169510564PRTArtificial
sequenceAmino acid sequence of modified flagellin adjuvunctal
polypeptide with melltinin signal peptide and influenza
hemagglutinin Atransmembrane-cytoplasmic tail 10Met Lys Phe Leu Val
Asn Val Ala Leu Val Phe Met Val Val Tyr Ile1 5 10 15Ser Tyr Ile Tyr
Ala Asp Pro Ile Asn Met Thr Gly Ser Met Ala Gln 20 25 30Val Ile Asn
Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn Asn Leu Asn 35 40 45Lys Ser
Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg Leu Ser Ser Gly 50 55 60Leu
Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala Ile Ala65 70 75
80Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala Ser Arg Asn
85 90 95Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala Leu
Asn 100 105 110Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala
Val Gln Ser 115 120 125Ala Asn Ser Thr Asn Ser Gln Ser Asp Leu Asp
Ser Ile Gln Ala Glu 130 135 140Ile Thr Gln Arg Leu Asn Glu Ile Asp
Arg Val Ser Gly Gln Thr Gln145 150 155 160Phe Asn Gly Val Lys Val
Leu Ala Gln Asp Asn Thr Leu Thr Ile Gln 165 170 175Val Gly Ala Asn
Asp Gly Glu Thr Ile Asp Ile Asp Leu Lys Gln Ile 180 185 190Asn Ser
Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln Gln Lys Tyr 195 200
205Lys Val Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala Asp Thr Thr
210 215 220Ile Ala Leu Asp Asn Ser Thr Phe Lys Ala Ser Ala Thr Gly
Leu Gly225 230 235 240Gly Thr Asp Gln Lys Ile Asp Gly Asp Leu Lys
Phe Asp Asp Thr Thr 245 250 255Gly Lys Tyr Tyr Ala Lys Val Thr Val
Thr Gly Gly Thr Gly Lys Asp 260 265 270Gly Tyr Tyr Glu Val Ser Val
Asp Lys Thr Asn Gly Glu Glu Thr Leu 275 280 285Ala Gly Gly Ala Thr
Ser Pro Leu Thr Gly Gly Leu Pro Ala Thr Ala 290 295 300Thr Glu Asp
Val Lys Asn Val Gln Val Ala Asn Ala Asp Leu Thr Glu305 310 315
320Ala Lys Ala Ala Leu Thr Ala Ala Gly Val Thr Gly Thr Ala Ser Val
325 330 335Val Lys Met Ser Tyr Thr Asp Asn Asn Gly Lys Thr Ile Asp
Gly Gly 340 345 350Leu Ala Val Lys Val Gly Asp Asp Tyr Tyr Ser Ala
Thr Gln Asn Lys 355 360 365Asp Gly Ser Ile Ser Ile Asn Thr Thr Lys
Tyr Thr Ala Asp Asp Gly 370 375 380Thr Ser Lys Thr Ala Leu Asn Lys
Leu Gly Asp Ala Asp Gly Lys Thr385 390 395 400Glu Val Val Ser Ile
Gly Gly Lys Thr Tyr Ala Ala Ser Lys Ala Glu 405 410 415Gly His Asn
Phe Lys Ala Gln Pro Asp Leu Ala Glu Ala Ala Ala Thr 420 425 430Thr
Thr Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala Gln Val 435 440
445Asp Thr Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asn Ser
450 455 460Ala Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr Ser
Ala Arg465 470 475 480Ser Arg Ile Glu Asp Ser Asp Tyr Ala Thr Glu
Val Ser Asn Met Ser 485 490 495Arg Ala Gln Ile Leu Gln Gln Ala Gly
Thr Ser Val Leu Ala Gln Ala 500 505 510Asn Gln Val Pro Gln Asn Val
Leu Ser Leu Leu Arg Glu Phe Gln Ile 515 520 525Leu Ala Ile Tyr Ser
Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser 530 535 540Leu Gly Ala
Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys545 550 555
560Arg Ile Cys Ile119PRTArtificial sequenceMHC-1 HA peptide 1 11Ile
Tyr Ser Thr Val Ala Ser Ser Leu1 5129PRTArtificial sequenceMHC-1 HA
peptide 2 12Leu Tyr Glu Lys Val Lys Ser Gln Leu1 51311PRTArtificial
sequenceMHC-II HA peptide 1 13Ser Phe Glu Arg Phe Glu Ile Phe Pro
Lys Glu1 5 101412PRTArtificial sequenceMHC-II HA peptide 2 14His
Asn Thr Asn Gly Val Thr Ala Ala Cys Ser His1 5 101512PRTArtificial
sequenceMHC-II HA peptide 3 15Cys Pro Lys Tyr Val Arg Ser Ala Lys
Leu Arg Met1 5 101612PRTArtificial sequenceMHC-II HA peptide 4
16Lys Leu Lys Asn Ser Tyr Val Asn Lys Lys Gly Lys1 5
101715PRTArtificial sequenceMHC-II HA peptide 5 17Asn Ala Tyr Val
Ser Val Val Thr Ser Lys Tyr Asn Arg Arg Phe1 5 10
151816PRTArtificial sequenceFlexible linker sequence 18Gly Ala Pro
Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Pro Trp1 5 10
1519135PRTArtificial sequenceInsert antigenic sequence 19Val Asp
His Met Ala Ala Ala Ala Ser Leu Leu Thr Glu Val Glu Thr1 5 10 15Pro
Ile Arg Asn Glu Trp Gly Ser Arg Ser Asn Asp Ser Ser Asp Pro 20 25
30Ala Ala Gly Thr Ser Ala Ala Ala Ser Leu Leu Thr Glu Val Glu Thr
35 40 45Pro Ile Arg Asn Glu Trp Gly Ser Arg Ser Asn Asp Ser Ser Asp
Pro 50 55 60Ala Ala Ala Leu Gln Ala Ala Ala Ser Leu Leu Thr Glu Val
Glu Thr65 70 75 80Pro Ile Arg Asn Glu Trp Gly Ser Arg Ser Asn Asp
Ser Ser Asp Pro 85 90 95Ala Ala Ala Ala Cys Ala Ala Ala Ser Leu Leu
Thr Glu Val Glu Thr 100 105 110Pro Ile Arg Asn Glu Trp Gly Ser Arg
Ser Asn Asp Ser Ser Asp Pro 115 120 125Ala Ala Ala Ala Ala Lys Leu
130 135
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