U.S. patent application number 11/908474 was filed with the patent office on 2008-11-13 for compositions of hsp60 peptides and viral antigens for vaccination and diagnosis.
This patent application is currently assigned to B.G. NEGEV TECHNOLOGIES AND APPLICATIONS LTD.. Invention is credited to Irun R. Cohen, Johannes Herkel, Angel Porgador, Bracha Rager-Zisman.
Application Number | 20080279878 11/908474 |
Document ID | / |
Family ID | 36992117 |
Filed Date | 2008-11-13 |
United States Patent
Application |
20080279878 |
Kind Code |
A1 |
Cohen; Irun R. ; et
al. |
November 13, 2008 |
Compositions of Hsp60 Peptides and Viral Antigens for Vaccination
and Diagnosis
Abstract
The present invention provides improved vaccines comprising an
isolated viral antigenic peptide and a synthetic peptide derived
from a T cell epitope of HSP60. The invention includes mixtures
where the peptide serves as an adjuvant as well as conjugates where
the peptide is covalently linked to the viral antigen. The known
synthetic peptide carrier, p458, provides significantly improved
immunogenicity for synthetic viral epitopes and analogs. Ec27 is a
novel peptide derived from HSP60 which increases the immunogenicity
substantially of the viral antigen both as a mixture or a covalent
conjugate. Some of the isolated viral epitopes are novel and are
claimed for diagnostic as well as therapeutic or prophylactic
uses.
Inventors: |
Cohen; Irun R.; (Rehovot,
IL) ; Rager-Zisman; Bracha; (Beer Sheva, IL) ;
Porgador; Angel; (Lehavim, IL) ; Herkel;
Johannes; (Hamburg, DE) |
Correspondence
Address: |
FENNEMORE CRAIG
3003 NORTH CENTRAL AVENUE, SUITE 2600
PHOENIX
AZ
85012
US
|
Assignee: |
B.G. NEGEV TECHNOLOGIES AND
APPLICATIONS LTD.
Beer Sheva
IL
YEDA RESEARCH AND DEVELOPMENT CO. LTD. at The Weizmann Institute
of Science
Rehovot
IL
|
Family ID: |
36992117 |
Appl. No.: |
11/908474 |
Filed: |
February 21, 2006 |
PCT Filed: |
February 21, 2006 |
PCT NO: |
PCT/IL06/00222 |
371 Date: |
May 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60661017 |
Mar 14, 2005 |
|
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|
Current U.S.
Class: |
424/186.1 ;
424/185.1; 435/235.1; 435/320.1; 435/325; 435/5; 530/326;
536/23.72 |
Current CPC
Class: |
A61P 37/04 20180101;
Y02A 50/394 20180101; C07K 14/005 20130101; Y02A 50/30 20180101;
Y02A 50/388 20180101; Y02A 50/53 20180101; Y02A 50/60 20180101;
G01N 33/56983 20130101; Y02A 50/39 20180101; A61K 2039/6043
20130101; A61P 31/12 20180101; A61K 2039/64 20130101; A61K 47/646
20170801; C12N 2770/24122 20130101; A61P 31/22 20180101; Y02A
50/386 20180101 |
Class at
Publication: |
424/186.1 ;
424/185.1; 435/325; 536/23.72; 530/326; 435/235.1; 435/5;
435/320.1 |
International
Class: |
A61K 39/245 20060101
A61K039/245; A61K 39/12 20060101 A61K039/12; C12N 5/10 20060101
C12N005/10; C12N 15/31 20060101 C12N015/31; C07K 7/00 20060101
C07K007/00; C12N 7/00 20060101 C12N007/00; C12Q 1/70 20060101
C12Q001/70; C12N 15/85 20060101 C12N015/85; A61P 31/22 20060101
A61P031/22; A61P 31/12 20060101 A61P031/12 |
Claims
1-54. (canceled)
55. A vaccine comprising an isolated viral antigenic peptide and a
peptide comprising a T cell epitope of HSP60, wherein the HSP60
peptide enhances the immunogenicity of the viral antigenic peptide
by at least two fold compared to the peptide without the HSP60
peptide.
56. The vaccine of claim 55, wherein the peptide comprising the T
cell epitope of HSP60 is selected from the group consisting of: (a)
NEDQKIGIEIIKRTLKI (p458h; SEQ ID NO: 1), (b) NEDQKIGIEIIKRALKI
(p458; SEQ ID NO:2), (c) EGDEATGANIVKVALEA (p458mt; SEQ ID NO:3),
(d) NEDQNVGIKVALRAMEA (p458e; SEQ ID NO:4), (e) an analog or
derivative of p458h (SEQ ID NO: 1) that has at least 70% of the
electric and hydrophilicity/hydrophobicity characteristic of human
HSP60 from position 458 to position 474, said peptide, analog or
derivative being capable of increasing substantially the
immunogenicity of the viral antigen when the conjugate is
administered in vivo, (f) KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76)
and analogs and derivatives thereof, (g) KKDRVTDALNATRAAVEEGI
(Ec27h; SEQ ID NO:86) and analogs and derivatives thereof.
57. A conjugate comprising a viral antigen covalently attached to a
synthetic peptide carrier comprising a T cell epitope of HSP60 in
which said synthetic peptide carrier is selected from the group of
peptides consisting of: (a) NEDQKIGIEIIKRTLKI (p458h; SEQ ID NO:
1), (b) NEDQKIGIEIIKRALKI (p458; SEQ ID NO:2), (c)
EGDEATGANIVKVALEA (p458mt; SEQ ID NO:3), (d) NEDQNVGIKVALRAMEA
(p458e; SEQ ID NO:4), (e) an analog or derivative of p458h (SEQ ID
NO: 1) that has at least 70% of the electric and
hydrophilicity/hydrophobicity characteristic of human HSP60 from
position 458 to position 474, said peptide, analog or derivative
being capable of increasing substantially the immunogenicity of the
viral antigen when the conjugate is administered in vivo, wherein
the viral antigen is derived from a virus belonging to a virus
family selected from the group consisting of flaviviridae and
herpesviridae.
58. The conjugate of claim 57, wherein the synthetic peptide is an
analog of p458h (SEQ ID NO: 1): .sup.458NEDQKIGIEIIKRTLKI.sup.474
in which the residue E.sup.459 is either E or D; the residue
D.sup.460 is either D or E; the residue K.sup.462 is either K or R
or ornithine (Orn); the residue I.sup.463 is either I or L, V, M,
F, norleucine (Nle) or norvaline (Nva); the residue I.sup.465
residue is either I or L, V, M, F, Nle or Nva; the residue
E.sup.466 is either E or D; the residue I.sup.467 is either I or L,
V, M, F, Nle or Nva; the residue I.sup.468 is either I or L, V, M,
F, Nle or Nva; the residue K.sup.469 is either K or R or Orn; the
residue R.sup.470 is either R, K or Orn; the residue L.sup.472 in
either L or I, V, M, F, Nle or Nva; the residue K.sup.473 is either
K or R or Orn; and the residue I.sup.474 is either I or L, V, M, F,
Nle or Nva.
59. The conjugate of claim 57, wherein the viral antigen is derived
from a betaherpesvirus.
60. The conjugate of claim 57, wherein the viral antigen is derived
from human cytomegalovirus.
61. The conjugate of claim 60, wherein the viral antigen is derived
from IE-1 protein.
62. The conjugate of claim 61, wherein the viral antigen comprises
a CTL epitope.
63. The conjugate of claim 57, wherein the viral antigen is derived
from a flavivirus.
64. The conjugate of claim 63, wherein the viral antigen is
selected from the group consisting of: West Nile virus (WNV),
Yellow fever virus, St. Louis encephalitis virus, Murray Valley
encephalitis virus, Kunjin virus, Japanese encephalitis virus,
Dengue virus type 1, Dengue virus type 2, Dengue virus type 3 and
Dengue virus type 4.
65. The conjugate of claim 64, wherein the viral antigen is derived
from West Nile Virus.
66. The conjugate of claim 64, wherein the viral antigen is derived
from the envelope protein of the virus.
67. The conjugate of claim 66, wherein the viral antigen is derived
from the E3 domain of said envelope protein.
68. The conjugate of claim 67, wherein said viral antigen comprises
a B cell epitope and a MHC II-restricted epitope.
69. The conjugate of claim 65, wherein the viral antigen has an
amino acid sequence as set forth in any one of SEQ ID NOS:11 and 12
and 21 and analogs, homologs, derivatives and salts thereof.
70. The conjugate of claim 69 having an amino acid sequence as set
forth in any one of SEQ ID NOS: 13-16 and 23-24 and analogs,
derivatives and salts thereof.
71. The conjugate of claim 64, wherein the viral antigen has an
amino acid sequence as set forth in any one of SEQ ID
NOS:25-44.
72. The conjugate of claim 71 having an amino acid sequence as set
forth in any one of SEQ ID NOS:56-64.
73. A conjugate comprising a viral antigen covalently attached to a
synthetic peptide carrier comprising a T cell epitope of HSP60 in
which said synthetic peptide carrier has an amino acid sequence
selected from the group consisting of KKARVEDALHATRAAVEEGV (Ec27;
SEQ ID NO:76) and KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO:86) and
analogs, derivatives and salts thereof.
74. The conjugate of claim 73, wherein the viral antigen is derived
from a virus belonging to a virus family selected from the group
consisting of flaviviridae and herpesviridae.
75. The conjugate of claim 74, wherein the viral antigen is derived
from WNV.
76. The conjugate of claim 74, wherein the viral antigen has an
amino acid sequence as set forth in any one of SEQ ID NOS: 11-12,
25-44 and 21.
77. The conjugate of claim 76 having an amino acid sequence as set
forth in any one of SEQ ID NOS:67-75 and 77-79.
78. An isolated peptide antigen having an amino acid sequence
selected from the group consisting of: LVTVNPFVSVATANS (SEQ ID NO:
11), LVTVNPFVSVATANA (SEQ ID NO:12), YIVVGRGEQQINHHWHK (SEQ ID
NO:21), and SEQ ID NOS:25-44 and analogs, fragments, derivatives
and salts thereof, other than the full length envelope protein of
West Nile Virus or known fragments thereof.
79. An isolated peptide antigen according to claim 78 having an
amino acid sequence as set forth in any one of SEQ ID NOS: 11, 12,
21 and 25-44.
80. A conjugate comprising the peptide antigen of claim 78
covalently attached to a carrier capable of enhancing the
immunogenicity of said peptide.
81. A nucleic acid sequence encoding the peptide of claim 78.
82. The nucleic acid sequence of claim 81, wherein the sequence is
as set forth in any one of SEQ ID NOS:19-20, 22 and 45-55.
83. A vector comprising the nucleic acid molecule of claim 82
operably linked to one or more transcription control sequences.
84. A host cell comprising the vector of claim 83.
85. A vaccine composition comprising a peptide antigen according to
claim 78 further comprising at least one pharmaceutically
acceptable carrier, adjuvant, excipient or diluent.
86. A synthetic peptide having an amino acid sequence as set forth
in any one of SEQ ID NOS:76 and 86, and analogs and derivatives
thereof.
87. A vaccine composition comprising an antigen and a synthetic
peptide adjuvant comprising a T cell epitope of HSP60 in which said
synthetic peptide adjuvant has an amino acid sequence selected from
the group consisting of KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76)
and KKDRVTDALNATRAAVEEGI (Ec27h; SEQ ID NO:86) and analogs,
homologs, derivatives and salts thereof.
88. The composition of claim 87, wherein the antigen is selected
from the group consisting of: a peptide, a peptide derivative, a
protein, a polysaccharide, and an antibody.
89. The composition of claim 87, wherein the antigen is a viral
antigen.
90. The composition of claim 87, wherein the antigen is covalently
attached to said synthetic peptide adjuvant.
91. The composition of claim 87 comprising an admixture of the
antigen and said synthetic peptide adjuvant.
92. The composition of claim 87, wherein said viral antigen has an
amino acid sequence as set forth in any one of SEQ ID NOS:11-12, 21
and 25-44 and analogs, homologs, derivatives and salts thereof.
93. A diagnostic kit comprising at least one peptide antigen
according to claim 78, and means for determining whether the
peptide antigen binds specifically to a biological sample.
94. A method of enhancing the immunogenicity of a viral antigen
comprising covalently conjugating the antigen with a synthetic
peptide carrier selected from the group of peptides consisting of:
(a) NEDQKIGIEIIKRTLKI (p458h) (SEQ ID NO: 1), (b) NEDQKIGIEIIKRALKI
(p458) (SEQ ID NO:2), (c) EGDEATGANIVKVALEA (p458mt) (SEQ ID NO:3),
(d) NEDQNVGIKVALRAMEA (p458e) (SEQ ID NO:4), (e) an analog or
derivative of p458h (SEQ ID NO: 1) that has at least 70% of the
electric and hydrophilicity/hydrophobicity characteristic of human
hsp60 from position 458 to position 474, said peptide, analog or
derivative being capable of increasing substantially the
immunogenicity of the viral antigen when the conjugate is
administered in vivo, wherein the viral antigen is derived from a
virus belonging to a virus family selected from the group
consisting of flaviviridae and herpesviridae.
95. A method of enhancing the immunogenicity of an antigen
comprising covalently conjugating the antigen with a synthetic
peptide carrier having an amino acid sequence as set forth in any
one of SEQ ID NOS:76 and 86 and analogs and derivatives
thereof.
96. A method of immunizing a subject in need thereof against a
viral infection comprising administering to the subject an
effective amount of a vaccine composition comprising a conjugate
according to claim 57.
97. The method of claim 96, wherein the subject is selected from a
group consisting of: humans, non-human mammals and non-mammalian
animals.
98. The method of claim 97, wherein the subject is human.
99. The method according to claim 96 further comprising steps prior
to immunizing the subject comprising: isolating an antigen derived
from a virus comprising at least one epitope selected from: a CTL
epitope, a B cell epitope and a MHC II-restricted epitope;
covalently conjugating the antigen to said synthetic peptide
carrier; and preparing a vaccine composition comprising the
conjugate and a pharmaceutically acceptable carrier, adjuvant,
excipient or diluent.
100. A method for diagnosing exposure of a subject to a flavivirus,
or for diagnosing a flavivirus infection in a subject comprising
the steps of: (a) contacting a suitable biological sample with a
viral antigen according to claim 78 under conditions such that an
immune reaction can occur; (b) determining whether the peptide
antigen binds specifically to the biological sample.
101. A method according to claim 100, wherein step (b) includes
determining the extent of antigen-antibody complex formation,
wherein an antigen-antibody complex formation level significantly
higher than the level obtained for a sample obtained from a
non-infected subject is indicative of exposure of the subject to
the flavivirus.
102. A method according to claim 100 for the differential diagnosis
of a flavivirus infection.
103. The method of claim 95, wherein the antigen is a viral
antigen.
104. A method of immunizing a subject in need thereof against a
viral infection comprising administering to the subject an
effective amount of a vaccine composition comprising a conjugate
according to claim 73.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to vaccines providing enhanced
immunogenicity comprising HSP60 peptides conjugated to or mixed
with a viral antigen. The present invention further identifies
certain novel epitopes, compositions thereof and methods of using
same for vaccination or diagnosis.
BACKGROUND OF THE INVENTION
[0002] Despite remarkable achievements in the development of
vaccines for certain viral infections (i.e., polio and measles),
and the eradication of specific viruses from the human population
(e.g., smallpox), viral diseases remain as important medical and
public health problems. Indeed, viruses are responsible for several
"emerging" (or re-emerging) diseases (e.g., West Nile encephalitis
and Dengue fever), and viral infection is a cause of significant
morbidity and mortality worldwide.
[0003] The presence of adequate T-cell help is important for the
construction of potent vaccines. Vaccines that induce both helper T
cells and CTLs may be more effective that those that induce CTLs
only. Indeed, the importance of cooperation between CD4.sup.+ and
CD8.sup.+ T cells is emphasized in the therapeutic vaccination
against chronic viral infection (Zajac et al., 1998; Matloubian et
al., 1994).
[0004] Classically, vaccines are manufactured by introducing killed
or attenuated organisms into the host along with suitable adjuvants
to initiate the normal immune response to the organisms while,
desirably, avoiding the pathogenic effects of the organism in the
host. The approach suffers from the well known limitations in that
it is rarely possible to avoid the pathogenic response because of
the complexity of the vaccine which includes not only the antigenic
determinant of interest but many related and unrelated deleterious
materials, any number of which may, in some or all individuals,
induce an undesirable reaction in the host.
[0005] For example, vaccines produced in the classical way may
include competing antigens which are detrimental to the desired
immune response, antigens which include unrelated immune responses,
nucleic acids from the organism or culture, endotoxins and
constituents of unknown composition and source. These vaccines,
generated from complex materials, inherently have a relatively high
probability of inducing competing responses even from the antigen
of interest.
[0006] HSP60 belongs to a family of chaperone molecules highly
conserved throughout evolution; a similar HSP60 molecule is present
in all cells, prokaryotes and eukaryotes. The human HSP60 molecule
was formerly designated HSP65, but is now designated HSP60 in view
of more accurate molecular weight information; by either
designation, the protein is the same. Apparently, no cell can exist
without the ability to express HSP60. Mammalian HSP60 is highly
homologous to the bacterial cognates, showing about 50% amino acid
identity (Jindal et al., 1989). Thus, HSP60 is shared by the host
and its parasites, and is immunogenic, cross-reactive, and
universally expressed in inflammation. Furthermore, HSP60 is well
recognized by the immune system (Konen Waisman et al., 1999, Konen
Waisman et al., 1995) and is a part of the set of self-molecules
for which autoimmunity naturally exists; HSP60 is member of the
immunologic homunculus (Cohen, 1992). Heat shock, IFN.gamma.,
bacterial or viral infection, and inflammation, all result in the
presentation of endogenous HSP60 epitopes on MHC class II molecules
leading to the activation of HSP60-specific T cells, even in
healthy individuals (Anderton et al., 1993; Hermann et al., 1991;
Koga et al., 1989).
[0007] European Patent EP 262 710 and U.S. Pat. No. 5,154,923
describe peptides having an amino acid sequence corresponding to
positions 171-240 and 172-192, respectively, of a Mycobacterium
boris BCG 64 kD polypeptide, that are useful as immunogens inducing
resistance to autoimmune arthritis and similar autoimmune
diseases.
[0008] PCT Patent Application No. WO 90/10449 describes a peptide
designated p277 having an amino acid sequence corresponding to
positions 437-460 of the human HSP65 molecule that is useful as
immunogen inducing resistance to insulin dependent diabetes
mellitus (IDDM). A control peptide, designated p278, corresponding
to positions 458-474 of human HSP65, did not induce resistance to
IDDM.
[0009] Lussow et al. (1990) showed that the priming of mice with
live Mycobacterium tuberculosis var. bovis (BCG) and immunization
with the repetitive malaria synthetic peptide (NANP).sub.40
conjugated to purified protein derivative (PPD), led to the
induction of high and long-lasting titers of anti-peptide IgG
antibodies. Later on, Lussow et al. (1991) showed that
mycobacterial heat-shock proteins (HSP) of 65 kDa (GroEL-type) and
70 kDa (DnaK-type) acted as carrier molecules in mice, previously
primed with Mycobacterium tuberculosis var. boris (bacillus
Calmette-Guerin, BCG), for the induction of high and long-lasting
titers of IgG against the repetitive malaria synthetic peptide
(NANP).sub.40. Anti-peptide antibodies were induced when the
malaria peptide, conjugated to the mycobacterial HSP, was given in
the absence of any adjuvants.
[0010] Barrios et al. (1992) have shown that mice immunized with
peptides or oligosaccharides conjugated to the 70 kDa HSP produced
high titers of IgG antibodies in the absence of any previous
priming with BCG. The anti-peptide antibody response persisted for
at least 1 year. This adjuvant-free carrier effect of the 70 kDa
HSP was T cell dependent, since no anti-peptide nor anti-70 kDa IgG
antibodies were induced in athymic nu/nu mice. Previous
immunization of mice with the 65 kDa or 70 kDa HSP did not have any
negative effect on the induction of anti-peptide IgG antibodies
after immunization with HSP-peptide conjugates in the absence of
adjuvants. Furthermore, preimmunization with the 65 kDa HSP could
substitute for BCG in providing effective priming for the induction
of anti-(NANP).sub.40 antibodies. Finally, both the 65 kDa and 70
kDa HSP acted as carrier molecules for the induction of IgG
antibodies to group C meningococcal oligosaccharides, in the
absence of adjuvants, suggesting that the use of HSPs as carriers
in conjugated constructs for the induction of anti-peptide and
anti-oligosaccharide antibodies could be of value in the design of
new vaccines for eventual use in humans.
[0011] U.S. Pat. No. 5,736,146 discloses conjugates of poorly
immunogenic antigens with a synthetic peptide carrier comprising a
T cell epitope derived from the sequence of human heat shock
protein HSP65, or an analog thereof, said peptide or analog being
capable of increasing substantially the immunogenicity of the
poorly immunogenic antigen. The '146 patent discloses conjugates of
a peptide corresponding to positions 458-474 and 437-453 of human
or mouse HSP60 and homologs thereof with a wide variety of antigens
including peptides, proteins and polysaccharides such as bacterial
polysaccharide (e.g. capsular polysaccharide (CPS) Vi of Salmonella
typhi), and antigens derived from HIV virus or from malaria
antigen.
[0012] U.S. Pat. No. 5,869,058 discloses conjugates of poorly
immunogenic antigens, e.g., peptides, proteins and polysaccharides,
with a synthetic peptide carrier comprising a T cell epitope
derived from the sequence of E. coli HSP65 (GroEL), or an analog
thereof, said peptide or analog being capable of increasing
substantially the immunogenicity of the poorly immunogenic antigen.
A suitable peptide according to the invention is Pep278e, which
corresponds to positions 437-453 of the E. coli HSP65 molecule.
[0013] Human cytomegalovirus (HCMV) is a ubiquitous double-stranded
DNA virus from the betaherpesvirus group; it is endemic in all
human populations. In North America, HCMV infects about 50% of the
population outside of urban centers and up to 90% of the population
within cities. HCMV disease presents two major medical problems:
first, it is the most common congenital viral infection, causing
birth defects including mal-development of the central nervous
system; up to 25% of asymptomatic infected infants will develop
neurologic sequelae. Second, HCMV becomes re-activated in
immunocompromised patients.
[0014] A self-limiting acute phase of viral infection, persistent
and latent phases normally characterize the pathogenesis of HCMV
infection in the immunocompetent host. The clinical outcome of HCMV
infection is determined by the ability of infected individuals to
mount protective humoral and T-cell mediated immune responses. In
immunocompromised hosts, including persons with HIV infection,
cancer patients and allograft recipients, primary HCMV infection or
reactivation of a latent virus results in multi-organ HCMV disease,
associated with high rates of morbidity and mortality. These grave
clinical consequences emphasize the need for effective HCMV
vaccines to prevent not only primary infection but also to limit or
prevent reactivation.
[0015] At present there is no protective vaccine available for CMV.
Currently available antiviral drugs which target viral DNA
replication are efficacious but exhibit significant host toxicity
and a high spontaneous resistance rate.
[0016] West Nile virus is a member of the alpha-like Flaviviridae.
The Flavivirus genome is a single-stranded, positive-sense RNA
approximately 11 kb in length, containing a 5'untranslated region
(5'UTR); a coding region encoding the three viral structural
proteins; seven nonstructural proteins, designated NS1, NS2A, NS2B,
NS3, NS4A, NS4B, NS5; and a 3'untranslated region (3'UTR). The
viral structural proteins include the capsid (C),
premembrane/membrane (prM) and envelope (E) proteins. The
structural and nonstructural proteins are translated as a single
polyprotein. The polyprotein is then processed by cellular and
viral proteases.
[0017] West Nile virus affects birds as well as reptiles and
mammals, together with man. The West Nile virus is transmitted to
birds and mammals by the bites of certain mosquitoes (e.g. Culex,
Aedes, Anopheles). Direct transmission may happen from WNV infected
subject to healthy subject by oral transmission (prey and
transmission through colostrum) and blood/organ vectored
transmission. Widespread in Africa, the geographic range of WNV now
also includes Australia, Europe, the Middle East, West Asia and the
USA. West Nile virus can cause a harsh, self-limiting fever, body
aches, brain swelling, coma, paralysis, and death.
[0018] There is no effective treatment for the disease. A number of
different WNV vaccines are now in various stages of development and
testing (Monath, 2001; Pletnev et al., 2003; Tesh et al., 2002;
Hall et al., 2003), but presently a licensed human vaccine is not
available for its prevention. The only currently effective way to
provide immediate resistance to WNV is by passive administration of
protective antibodies (Casadevall, 2002). Mosquito control is
currently considered the practical strategy to combat the spread of
disease, but effective spraying is difficult to perform in urban
areas. Clearly, an effective vaccine is needed to protect at-risk
populations.
[0019] There remains a need for improved vaccines conferring
protection against viral infections, using isolated epitopes.
Furthermore, isolated epitopes are needed for improved diagnostic
tests.
SUMMARY OF THE INVENTION
[0020] The present invention provides compositions and methods
suitable for vaccination against and diagnosis of viral infections.
According to some aspects the present invention provides a vaccine
comprising an isolated viral antigenic peptide and a peptide
comprising a T cell epitope of HSP60, wherein the HSP60 peptide
enhances the immunogenicity of the viral antigenic peptide by at
least two fold compared to the peptide without the HSP60 peptide.
In certain currently preferred embodiments the immunogenicity is
enhanced by at least 4-5 fold. Novel viral peptide antigens useful
in vaccination and diagnosis are also provided.
[0021] In certain embodiments the vaccine compositions comprise a T
cell epitope of HSP60 suitable to enhance the immunogenicity when
used as an adjuvant peptide that is mixed with the viral antigen.
In alternative embodiments the vaccine comprises a T cell epitope
of HSP60 suitable to enhance the immunogenicity of the viral
antigenic peptide when used in conjugates where the HSP60 peptide
is covalently linked to the viral antigenic peptide.
[0022] The enhanced immunogenicity of said viral antigen is
measured by at least one of the following: serum titer of
antibodies directed to said viral antigen; T cell proliferation in
the presence of said viral antigen; cytokine secretion induced by
said viral antigen; specific T cell mediated lysis of
virus-infected cells; and reduction of detectable viral load.
[0023] According to another aspect, the invention provides
conjugates comprising a viral antigen covalently attached to a
synthetic peptide carrier comprising a T cell epitope of HSP60.
According to some embodiments, the synthetic peptide carrier is the
known peptide carrier p458, a Major Histocompatibility Complex
(MHC) class II-restricted peptide derived from murine HSP60 (aa
458-474, also designated previously as p278m), or an analog or
derivative thereof. In other embodiments, the synthetic peptide
carrier is Ec27, a novel peptide derived from E. coli HSP60 (GroEL,
aa 391-410).
[0024] According to the present invention, it is now disclosed that
conjugates comprising a synthetic peptide carrier selected from
p458 and Ec27 covalently attached to a viral antigen are
unexpectedly effective in conferring immunity against viral
infections. It is now demonstrated for the first time that these
conjugates significantly enhance effective immunity against both
DNA and RNA viruses, latent and acute infections, and when combined
with CTL-, B cell- and MHC II-restricted viral epitopes.
[0025] The principles of the invention are exemplified by two model
systems for viral infections. Mouse Cytomegalovirus (MCMV)
infection in mice is an established model system for examining
human infection with Human Cytomegalovirus (HCMV), a DNA virus
which is characterized by a latent infection following a
self-limiting acute phase of viral infection. West Nile virus (WNV)
infection in mice serves as a model for the acute viral infection
of WNV in humans.
[0026] According to a some embodiments, the present invention
provides a conjugate comprising a viral antigen covalently attached
to a synthetic peptide carrier comprising a T cell epitope of HSP60
in which said synthetic peptide carrier is selected from the group
of peptides consisting of:
TABLE-US-00001 (a) NEDQKIGIEIIKRTLKI, (p458h; SEQ ID NO: 1) (b)
NEDQKIGIEIIKRALKI, (p458; SEQ ID NO:2) (c) EGDEATGANIVKVALEA,
(p458mt; SEQ ID NO:3) (d) NEDQNVGIKVALRAMEA, (p458e; SEQ ID
NO:4)
[0027] (e) an analog of p458h (SEQ ID NO: 1) that has at least 70%
of the electric and hydrophilicity/hydrophobicity characteristic of
human HSP60 from position 458 to position 474, said peptide or
analog being capable of increasing substantially the immunogenicity
of the viral antigen when the conjugate is administered in vivo,
and derivatives thereof,
[0028] (f) KKARVEDALHATRAAVEEGV (Ec27; SEQ ID NO:76) and analogs,
fragments and derivatives thereof.
[0029] In one embodiment, the synthetic peptide is an analog of
p458h (SEQ ID NO: 1): .sup.458NEDQKIGIEIIKRTLKI.sup.474 in which
the residue E.sup.459 is either E or D; the residue D.sup.460 is
either D or E; the residue K.sup.462 is either K or R or ornithine
(Orn); the residue I.sup.463 is either I or L, V, M, F, norleucine
(Nle) or norvaline (Nva); the residue I.sup.465 residue is either I
or L, V, M, F, Nle or Nva; the residue E.sup.466 is either E or D;
the residue I.sup.467 is either I or L, V, M, F, Nle or Nva; the
residue I.sup.468 is either I or L, V, M, F, Nle or Nva; the
residue K.sup.469 is either K or R or Orn; the residue R.sup.470 is
either R, K or Orn; the residue L.sup.472 in either L or I, V, M,
F, Nle or Nva; the residue K.sup.473 is either K or R or Orn; and
the residue I.sup.474 is either I or L, V, M, F, Nle or Nva.
[0030] In another aspect, there is provided a novel adjuvant
peptide derived from E. coli HSP60 (GroEL) protein, useful for the
compositions and methods of the invention. The novel adjuvant
peptide, herein designated Ec27, has an amino acid sequence
corresponding to positions 391-410 of GroEL (corresponding to
accession number gi:45686198 without the first methionine residue,
SEQ ID NO:83), as follows: KKARVEDALHATRAAVEEGV (SEQ ID NO:76). It
is to be explicitly understood that the corresponding peptides from
mammalian species are included within the scope of the present
invention. The corresponding human peptide exhibits 80% homology,
having the sequence set forth in SEQ ID NO:86, as follows:
KKDRVTDALNATRAAVEEGI (Ec27h). Ec27 analogs, fragments, derivatives,
conjugates and salts are also contemplated by the present
invention.
[0031] The Ec27 peptide is now demonstrated for the first time to
increase significantly the immunogenicity of a broad array of
antigens, including but not limited to viral antigens, bacterial
antigens and mammalian antigens, e.g., viral peptide antigens,
bacterial polysaccharides and antibodies. Surprisingly Ec27 was
found to increase the immunogenicity of antigens when covalently
conjugated to the antigen, as well as when mixed with the antigen.
Unexpectedly, Ec27 could even further increase the immunogenicity
of antigens of the invention conjugated to the p458 carriers.
[0032] In another embodiment, the invention further provides
vaccine compositions comprising an antigen and a peptide adjuvant
having an amino acid sequence as set forth in SEQ ID NO:76 or an
analog, fragment or derivative thereof. In various embodiments, the
antigen is selected from the group consisting of: a peptide, a
peptide derivative, a protein, a polysaccharide (e.g. a bacterial
polysaccharide), and an antibody. In one embodiment, the vaccine
composition comprises a conjugate of the peptide adjuvant and said
antigen. In alternate embodiments, said vaccine composition
comprises an admixture of said peptide adjuvant and said
antigen.
[0033] In another aspect, the viral antigen used in the conjugates
and compositions of the invention comprises at least one epitope
selected from: a CTL epitope (a MHC I restricted T cell epitope), a
B cell epitope and a MHC II restricted T cell epitope.
[0034] The viral antigen used in the conjugates of the invention
may be derived from any virus of interest. In certain embodiments,
the virus belongs to the herpesviridae family. In other particular
embodiments, the virus belongs to the betaherpesvirus subfamily. In
one particular embodiment, the viral antigen is derived from
immediate early gene 1 (IE-1) protein of a virus belonging to
herpesviridae. In another particular embodiment, the viral antigen
comprises a CTL epitope. In another particular embodiment, the
virus is CMV. In one preferred embodiment, the viral antigen is
derived from immediate early gene 1 (IE-1) protein of CMV. In
another preferred embodiment, the viral antigen comprises a CTL
epitope.
[0035] In other embodiments, the virus belongs to the Flaviviridae
family. In other particular embodiments, the virus belongs to the
flavivirus genus. According to various particular embodiments, the
virus is selected from the group consisting of: West Nile virus
(WNV), Yellow fever virus, St. Louis encephalitis virus, Murray
Valley encephalitis virus, Kunjin virus, Japanese encephalitis
virus, Dengue virus type 1, Dengue virus type 2, Dengue virus type
3 and Dengue virus type 4. In one particular embodiment, the viral
antigen is derived from West Nile Virus (WNV).
[0036] In one preferred embodiment, the viral antigen is derived
from the envelope (E) protein of a virus belonging to the
flaviviridae family. In another preferred embodiment, the viral
antigen is derived from the E3 domain of said protein. In another
preferred embodiment, said viral antigen comprises a B cell epitope
and a MHC II restricted epitope. In another preferred embodiment,
the viral antigen is derived from the WNV envelope (E) protein. In
another preferred embodiment, the viral antigen is derived from the
E3 domain of said protein. In another preferred embodiment, said
viral antigen comprises a B cell epitope and a MHC II restricted
epitope.
[0037] Other embodiments of the present invention are directed to
novel isolated viral peptide antigens that may be used in
conjugation with the carriers of the invention for anti viral
vaccination, as well as for diagnostic purposes, as specified
herein.
[0038] In another aspect, there is provided a novel peptide antigen
derived from WNV E3 domain of E protein, hereby designated p15,
having an amino acid sequence corresponding to positions 355-369 of
the E protein. Depending on the particular strain of WNV, this
novel antigen has an amino acid sequence selected from the group
consisting of: LVTVNPFVSVATANS (SEQ ID NO:11) and LVTVNPFVSVATANA
(SEQ ID NO:12). Other embodiments are directed to analogs,
homologs, fragments and derivatives thereof. In other embodiments,
the invention provides proteins, peptides and conjugates comprising
said antigen. In one particular embodiment, the peptide has an
amino acid sequence as set forth in any one of SEQ ID NOS:34-35
(see Table 1).
[0039] In other embodiments, there is provided a p15 homologous
peptide antigen derived from the E3 domain of the envelope protein
of a flavivirus selected from the group consisting of: West Nile
virus (WNV), Yellow fever virus, St. Louis encephalitis virus,
Murray Valley encephalitis virus, Kunjin virus, Japanese
encephalitis virus, Dengue virus type 1, Dengue virus type 2,
Dengue virus type 3 and Dengue virus type 4. In certain particular
embodiments, the p15 homologous antigen has an amino acid sequence
as set forth in any one of SEQ ID NOS:25-33 and 36-44 (see Table
1), and analogs, homologs, fragments, and derivatives thereof.
[0040] In another aspect, there is provided a second novel WNV
peptide antigen derived from the E protein, herein denoted p17,
having the following amino acid sequence: YIVVGRGEQQINHHWHK (SEQ ID
NO:21). Other embodiments are directed to analogs, homologs,
fragments, and derivatives thereof.
[0041] In other embodiments, the invention provides nucleic acid
molecules encoding said novel peptide antigens, recombinant
constructs comprising these nucleic acid molecules, and vectors and
cells comprising them.
[0042] In another embodiment, there are provided conjugates
comprising a synthetic peptide carrier of the invention and a viral
antigen having an amino acid sequence as set forth in any one of
SEQ ID NOS:11-12 and 34-35 and analogs, homologs, fragments and
derivatives thereof covalently attached to a synthetic peptide
carrier of the invention. In another embodiment, the conjugate has
an amino acid sequence as set forth in any one of SEQ ID NOS:13-16,
65-66 and 77-78. In other embodiments, the conjugates of the
invention comprise a viral antigen having an amino acid sequence as
set forth in any one of SEQ ID NOS:25-33 and 36-44 covalently
attached to a synthetic peptide carrier of the invention. In
another embodiment, the conjugate has an amino acid sequence as set
forth in any one of SEQ ID NOS:56-64, and 67-75. In other
embodiments, the conjugates of the invention comprise a viral
antigen having an amino acid sequence as set forth in SEQ ID NO:21
covalently attached to a synthetic peptide carrier of the
invention. In another embodiment, the conjugate has an amino acid
sequence as set forth in any one of SEQ ID NOS:23-24 and 79 (see
Table 4).
[0043] In another aspect, the invention provides vaccine
compositions comprising the conjugates of the invention and a
pharmaceutically acceptable carrier, adjuvant, excipient or
diluent. In another aspect, the invention provides vaccine
compositions comprising a viral antigen in admixture with Ec27 and
a pharmaceutically acceptable carrier, adjuvant, excipient or
diluent. In another aspect, the invention provides vaccine
compositions comprising the novel isolated viral peptide antigens
of the invention and a pharmaceutically acceptable carrier,
adjuvant, excipient or diluent.
[0044] In yet another aspect, the invention provides methods for
increasing the immunogenicity of a viral antigen which comprises
linking the antigen to a synthetic peptide carrier of the
invention.
[0045] In another aspect, the invention provides methods for
immunizing a subject in need thereof against a viral infection,
comprising administering to the subject an effective amount of a
vaccine composition comprising a conjugate of the invention and a
pharmaceutically acceptable carrier, adjuvant, excipient or
diluent.
[0046] The vaccine composition may be administered to said subject
before the exposure of said subject to the virus or after exposure
of said subject to said virus.
[0047] In another aspect, the invention provides methods
comprising: [0048] (a) isolating a viral antigen, comprising at
least one epitope selected from: a CTL epitope, a B cell epitope
and a MHC II-restricted epitope; [0049] (b) conjugating said viral
antigen to a synthetic peptide carrier of the invention to form a
peptide-carrier conjugate; and [0050] (c) administering to the
subject an effective amount of a vaccine composition comprising the
conjugate and a pharmaceutically acceptable carrier, adjuvant,
excipient or diluent.
[0051] According to various embodiments, the compositions and
methods of the invention are suitable for vaccinating a subject
selected from a group consisting of: humans, non-human mammals and
non-mammalian animals. In a preferred embodiment, the subject is
human.
[0052] Other aspects of the present invention are directed to
diagnostic kits and methods utilizing the novel isolated viral
peptide antigens for determining the exposure of a subject to a
flavivirus.
[0053] In one aspect, there is provided a diagnostic kit comprising
at least one viral peptide antigen of the invention and means for
detecting whether the peptide antigen is bound specifically to a
suitable biological sample.
[0054] In another aspect, the invention provides methods for
diagnosing exposure of a subject to a flavivirus and for diagnosing
a flavivirus infection in a subject, comprising the steps of:
[0055] (a) contacting a suitable biological sample with a viral
antigen having an amino acid sequence as set forth in any one of
SEQ ID NOS:11-12, 25-44 and 21 and analogs, homologs, derivatives
and salts thereof under conditions such that an immune reaction can
occur; [0056] (b) determining the extent of specific antigen
binding to the biological sample, wherein a level significantly
higher than the level obtained for a sample obtained from a
non-infected subject is indicative of exposure of the subject to
the flavivirus.
[0057] In certain embodiments, the kits and diagnostic methods of
the invention are useful for the differential diagnosis of a
flavivirus infection.
[0058] These and other embodiments of the present invention will
become apparent in conjunction with the figures, description and
claims that follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1. Kinetics of MCMV infection in spleen and salivary
gland (SG) of BALB/c mice. Mice were challenged i.p. with
5.times.10.sup.4 pfu of MCMV. A. Infectious virus titers in spleen
and salivary gland were measured at different time points after
infection (calculated as log10 pfu/0.1 g tissue). The data
represent the average of 5 experiments. B. PCR amplification of the
356 bp product of MCMV gB DNA in spleen and salivary gland at
different time points after infection. Results are from 1
representative experiment of 3 performed.
[0060] FIG. 2. The effectiveness of the p458-89pep vaccine. A. The
experimental design. B. Infectious MCMV titers in salivary gland
14, 21 and 28 days after challenge. Data represent the average
titer (.+-.SE) of the salivary glands of 3 individual mice of each
group. In salivary glands of mice immunized with p458-89pep, virus
titers on day 28 (asterisks) were below detection (i.e. <2
log.sub.10 pfu/0.1 gr tissue). C. PCR amplification of the 356 bp
product of MCMV gB. Template DNA was extracted from salivary glands
of immunized mice on day 28 after MCMV challenge. Immunization
with: a. IFA only without challenge; b. IFA only; c. 89pep; d.
p458-89pep; e. control-89pep; f. PCR mix without template DNA
(negative PCR control). Results are from 1 representative
experiment of 2 performed.
[0061] FIG. 3. IFN.gamma. secretion from spleen and salivary gland
(SG) cell cultures of MCMV infected mice. Spleen cell (FIG. 3A) and
fractionated salivary gland mononuclear cell cultures (FIG. 3B)
were prepared on different days after virus infection as described
in methods. IFN.gamma. secretion in supernatant was measured by
ELISA after 3 days culture with (squares) or without (circles)
89pep stimulation (10 .mu.g/ml) in vitro. Data represent the
average (.+-.SE) of 3 experiments.
[0062] FIG. 4. IFN.gamma. levels from spleen cell cultures after
vaccination with p458-89pep. Spleen cell cultures were prepared 10
days after vaccination with the various peptides or after challenge
of naive mice with MCMV. A control group received IFA without any
peptide. IFN.gamma. secretion in supernatants was measured by ELISA
after 3 days stimulation in vitro with p458, 89pep (10 .mu.g/ml),
or without stimulation (No-stim). Data represent the average of 6
experiments (.+-.SE). * p<0.05 compared to p458-89pep,
two-tailed T-test.
[0063] FIG. 5. IFN.gamma.-positive spleen cells after vaccination
with p458-89pep. Spleen-cell cultures were prepared 7 days after
vaccination with various peptides, or after challenge of naive mice
with MCMV. After 5 days of stimulation in vitro with 89pep or
without stimulation (No-stim), the cells were stained for CD4 (FIG.
5A) or CD8 (FIG. 5B) markers and for IFN.gamma.. Numbers (27.43,
9.89 and 0.41) are percentage of IFN.gamma..sup.+ cells in total
CD8.sup.+ cells. Results are from 1 representative experiment of 2
performed.
[0064] FIG. 6. CTL activity in spleen cell cultures after
vaccination with p458-89pep. Spleen-cell cultures were prepared 7
days after immunization with various peptides or after challenge of
naive mice with MCMV. A control group received IFA without any
peptide. CTL activity was measured after 6 days of stimulation in
vitro with 89pep (10 .mu.g/ml). Target cells were P815 pulsed with
the 89pep (1 .mu.g/ml). E:T ratio is 25:1. The data represent the
average (.+-.SE) of 3 different experiments.
[0065] FIG. 7. IFN.gamma.-positive salivary gland mononuclear cells
28 days after MCMV challenge of vaccinated mice. Mice were
vaccinated and then challenged with MCMV. Cells were stained for
CD8 and for IFN.gamma.. No-stimulation (No-stim) or stimulation
with 89pep (89pep) relates to the presence of 89pep during the 8 h
incubation with golgi-stop step in the ICCS protocol for
IFN.gamma.. Results are from 1 representative experiment of 2
performed.
[0066] FIG. 8. Recognition of peptides by IVIG-IL. Wells were
coated with the different peptides (1 .mu.g/well) or with the
WNV-Ag (1:700 dilution). After blocking and washing, IVIG-IL were
added at 1:40 dilution and binding was detected as described in
methods (ELISA). Background of ELISA (no peptide at well, 0.078 OD
to 0.120 OD in different experiments) was subtracted from each
experimental point. Results are the average of 4 independent ELISA
experiments. Bars, .+-.SD.
[0067] FIG. 9. Recognition of peptides by serum from WNV-infected
mice. Wells were coated with the different peptides (1 .mu.g/well)
or with the WNV-Ag (1:700 dilution). After blocking and washing,
naive (gray columns) or WNV-infected (white columns) murine sera
were added at 1:40 dilution and binding was detected as described
in methods (ELISA). Results are from 1 representative experiment of
3 performed. Each experimental point was performed in triplicate.
Bars, .+-.SD.
[0068] FIG. 10. Proliferation of splenocytes from WNV-infected mice
following in vitro stimulation with the different peptides. Mice
were infected with 66 pfu of WNV. Six days after, spleens were
harvested and splenocytes were cultured with the different peptides
(10 .mu.g/ml) or with ConA (5 .mu.g/ml) for 3 days. Proliferation
of cells derived from naive (gray columns) or WNV-infected (white
columns) mice was measured using WST-1 method. Results are the
average of 3 independent proliferation experiments. Bars,
.+-.SD.
[0069] FIG. 11. Anti-WNV Abs in the sera of mice immunized with
different peptides or with WNV Ag. (A) Mice were immunized 3 times
with the different peptides or WNV-Ag at 7 day intervals. Seven
days after the 3.sup.rd immunization mice were bled and sera were
tested as follows. Wells were coated with WNV-Ag (1:700 dilution).
After blocking and washing, the different sera were added at 1:40
dilution and binding was detected as described in methods (ELISA).
Results are the average of 4 independent ELISA experiments. Bars,
.+-.SD. (B) Isotypes of Anti-WNV antibodies.
[0070] FIG. 12. Proliferation of splenocytes from p32-immunized
mice following in vitro stimulation with the different peptides.
Mice were immunized 3 times with p32 at 7 day intervals. Seven days
after the 3.sup.rd immunization, spleens were harvested and
splenocytes were cultured with the different peptides or WNV-Ag (10
.mu.g/ml) or with ConA (5 .mu.g/ml) for 3 days. Cell proliferation
was measured using WST-1 method. Results are the average of 3
independent proliferation experiments. Bars, .+-.SD.
[0071] FIG. 13. IFN.gamma. secretion in the spleens of
p32-vaccinated mice on day 7 after immunization. Mice were
immunized 3 times with p32 at 7 day intervals. Seven days after the
3.sup.rd immunization, spleens were harvested and splenocytes were
cultured with the different peptides or WNV-Ag (10 .mu.g/ml) or
with ConA (5 .mu.g/ml) for 3 days. IFN.gamma. levels in the
supernatants were measured as described in methods. Results are the
average of 4 independent proliferation experiments. Bars,
.+-.SD.
[0072] FIG. 14. p458-89pep reduces viral load of MCMV-infected
mice. A. The experimental design. B. PCR amplification of the 363
bp product of MCMV IE-1.
[0073] FIG. 15. Viral loads following p32 immunization and WNV
challenge.
[0074] FIG. 16. proliferation and IFN-.gamma. secretion of
splenocytes from mice immunized by Ec27-p15 conjugates following in
vitro stimulation with p15.
[0075] FIG. 17. Specific recognition of p15 (A, B) and p17 (B) by
sera from WNV-infected human patients.
[0076] FIG. 18. Proliferative response of BALB/c lymph node cells
to overlapping GroEL peptides after immunization with E. coli
bacteria (A) or GroEL (B)
[0077] FIG. 19. Proliferative response of BALB/c lymph node cells
to immunization with the Ec27 peptide
[0078] FIG. 20. Proliferative response of different mouse strains
to immunization with the Ec27 peptide. BALB/c (A), BALB/k (B), and
BALB/b (C) and SJL (D) mice were immunized s.c. with 20 mg of the
Ec27 peptide emulsified in IFA. Ten days later lymph node cells
(2.times.10.sup.5 cells per well) were assessed for specific
proliferation to the Ec27 peptide (full circles), the ec35 peptide
(empty circles), or the acetylcholine receptor peptide 259-271
(empty triangles). After 96 hours of incubation, the
.sup.3H-thymidine incorporation was assessed as a measure of
proliferation. Results are shown as mean cpm of quadruplicate
wells. The standard deviations are indicated.
[0079] FIG. 21. Adjuvant effect of the Ec27 peptide. (A)
anti-PAb-246 reactivity; (B) anti-p53 reactivity.
DETAILED DESCRIPTION OF THE INVENTION
[0080] The present invention provides novel conjugates comprising a
viral antigen covalently linked to a synthetic peptide carrier
comprising a T cell epitope of HSP60. The synthetic peptide
carrier, p458, is a MHC class II-restricted peptide derived from
murine HSP60 (aa 458-474, also designated previously as p278m), or
an analog or derivative thereof, which peptide or analog being
capable of increasing substantially the immunogenicity of the viral
antigen. In other embodiments, the carrier is Ec27, a novel peptide
derived from E. coli GroEL (aa 391-410). The invention provides
vaccine compositions comprising the conjugates of the invention,
and methods for their use in vaccinating a subject in need thereof
against a viral infection. The invention further provides novel
viral peptide antigens, conjugates and vaccine compositions thereof
and uses thereof in vaccination and diagnosis.
[0081] The present invention discloses unexpectedly that a vaccine
composition comprising a conjugate of a viral antigen and a peptide
carrier derived from HSP60 p458 or Ec27 is highly efficacious in
conferring protective immunity against a viral infection in vivo.
It is now demonstrated for the first time that p458 and Ec27
enhance effective immunity even for conjugates comprising antigens
that are not poorly immunogenic. The peptide carriers of the
invention were found to enhance the immunogenicity of the viral
antigen by at least two fold compared to the peptide without the
HSP60 peptide.
[0082] The present invention is based, in part, on studies of
p458-viral antigen conjugate vaccination for the treatment of a
chronic (latent) Cytomegalovirus (CMV) infection associated with
persistence of virus in the salivary glands. A conjugate comprising
89pep, an antigen derived from murine CMV (MCMV) immediate early
gene 1 protein (IE-1), fused to p458, was more effective than the
89pep in inducing 89pep-specific IFN.gamma. secretion and specific
CTL activity. The p458-89pep chimeric peptide induced sustained
IFN.gamma. secretion in the salivary gland specific to 89pep and
only this immunization was associated with clearance of virus from
the salivary gland.
[0083] The present invention is also based, in part, on studies of
p458-viral antigen and Ec27-viral antigen conjugate vaccination
against an acute viral infection of West Nile Virus (WNV). A
conjugate comprising p15, a novel antigen derived from WNV envelope
(E) protein, fused to p458 was capable, upon immunization, to
significantly reduce the mortality associated with the infection,
while immunization with p15 alone could only moderately affect the
mortality rate. The conjugate was more effective than the viral
antigen alone in inducing WNV-specific neutralizing antibodies as
well as WNV-specific T cell proliferation and IFN.gamma. secretion.
Ec27-p15 conjugate was also more effective than p15 alone in
inducing p15-specific T cell proliferation and IFN.gamma.
secretion.
[0084] Thus, the conjugates of the invention are herein
demonstrated to be effective against both DNA and RNA viruses,
latent and acute infections, and when combined with CTL-, B cell-
and MHC II-restricted viral epitopes.
[0085] Ec27, a novel adjuvant peptide derived from E. coli HSP60
(GroEL) protein, was found to increase significantly the
immunogenicity of a broad array of antigens, including but not
limited to viral antigens, bacterial antigens and mammalian
antigens, e.g., viral peptide antigens, bacterial polysaccharides
and antibodies. Surprisingly Ec27 was found to increase the
immunogenicity of antigens when covalently conjugated to the
antigen, as well as when mixed with the antigen. Unexpectedly, Ec27
could even further increase the immunogenicity of antigens
conjugated to the p458 carriers. Ec27 has an amino acid sequence
corresponding to positions 391-410 of GroEL (corresponding to
accession number gi:45686198 without the first methionine residue,
SEQ ID NO:83), as follows: KKARVEDALHATRAAVEEGV (SEQ ID NO:76).
[0086] According to a first aspect, the present invention provides
a conjugate comprising a viral antigen covalently attached to a
synthetic peptide carrier comprising a T cell epitope of HSP60 in
which said synthetic peptide carrier is selected from the group of
peptides consisting of: [0087] (a) NEDQKIGIEIIKRTLKI (p458h,
derived from human HSP60; SEQ ID NO: 1), [0088] (b)
NEDQKIGIEIIKRALKI (p458, derived from mouse HSP60; SEQ ID NO:2),
[0089] (c) EGDEATGANIVKVALEA (p458mt, derived from M. tuberculosis
HSP60; SEQ ID NO:3), [0090] (d) NEDQNVGIKVALRAMEA (p458e, derived
from E. coli HSP60; SEQ ID NO:4. It should be noted, that the amino
acid sequence of p458e corresponds to positions 432-448 of SEQ ID
NO:83) [0091] (e) an analog of p458h (SEQ ID NO: 1) that has at
least 70% of the electric and hydrophilicity/hydrophobicity
characteristic of human HSP60 from position 458 to position 474,
said peptide or analog being capable of increasing substantially
the immunogenicity of the viral antigen when the conjugate is
administered in vivo, [0092] (f) KKARVEDALHATRAAVEEGV (Ec27,
derived from E. coli HSP60; SEQ ID NO:76).
[0093] The active peptide carriers according to the invention are
characterized as being highly charged, i.e. of strong electric
properties (7 out of 17 constituent amino acid residues of p458 are
either negatively or positively charged) and highly hydrophobic (6
amino acid residues). The peptide p458h is further characterized as
possessing a polar negatively-charged N-terminal domain, a polar
positively-charged C-terminal domain and a highly hydrophobic core.
These overall features should be maintained in order to preserve
efficacy. Thus, following the above general outline certain amino
acids substitution will lead to active peptides. More specifically,
positions 6, 8, 10, 11, 15 and 17 in the p458 peptide chain
(corresponding to positions 463, 465, 467, 468, 472 and 474 of the
human HSP60 molecule) can be occupied by either I or L or by other
hydrophobic amino acids, natural, such as V, M, or F, or unnatural
amino acids, such as norleucine (Nle) or norvaline (Nva). Positions
5, 12, 13 and 16 in the p458h chain (corresponding to positions
462, 469, 470 and 473 of the human HSP60 molecule) can be occupied
by either K or R or by unnatural positively charged amino acids,
such as ornithine (Orn). Interchange of E and D may also lead to
active derivatives.
[0094] With respect to the peptide carriers of the invention, the
term "analogs" relates to peptides obtained by replacement,
deletion or addition of amino acid residues to the sequence,
optionally including the use of a chemically derivatized residue in
place of a non-derivatized residue, as long as they have the
capability of enhancing substantially the immunogenicity of viral
antigen molecules. Analogs, in the case of p458, are peptides such
that at least 70%, preferably 90-100%, of the electric properties
and of the hydrophobicity of the peptide molecule are conserved.
These peptides can be obtained, without limitation, according to
the instructions in the paragraph hereinbefore. Ec27 analogs are
preferably of at least about 70%, more preferably of at least about
80-90% similarity in their amino acid sequence of Ec27. For
example, the corresponding human peptide, having the sequence set
forth in SEQ ID NO:86 (KKDRVTDALNATRAAVEEGI, Ec27h), exhibits 80%
amino acid identity to Ec27
[0095] The terms "covalently attached" and "conjugated" as used
herein refer to a conjugate comprising an antigen and a synthetic
peptide carrier linked either as a continuous fusion peptide or by
means of chemical conjugation (either directly or through a
spacer), using methods well known in the art.
[0096] By "substantially increasing" the immunogenicity of a viral
antigen molecule it is meant to comprise both the induction of an
increase in the level of antibodies (Abs) against said antigen as
well as the presentation of said antibodies as mainly of the IgG
isotype. Alternatively, the term may represent an increase in
antigen-specific T cell response, as measured either as increased
CTL activity (antigen-dependent lysis) or as increased
antigen-specific T cell proliferation or cytokine secretion (e.g.
Th1 cytokines such as IFN.gamma.). Non-limitative examples for
measuring the level of specific Abs and antigen-specific T cell
response according to the invention are presented in the Examples
hereinbelow.
[0097] In another aspect, the viral antigen comprises at least one
epitope selected from: a CTL epitope (a MHC I restricted T cell
epitope), a B cell epitope and a MHC II restricted T cell epitope.
Methods for identifying suitable candidate epitopes are within the
abilities of those of skill in the art (for example, without
limitation, by using epitope prediction software).
[0098] The viral antigen used in the conjugates of the invention
may be derived from any virus of interest. In certain embodiments,
the virus belongs to the herpesviridae family. This family
includes, but is not limited to, human viruses such as human
herpesvirus 1 (HHV-1, also known as herpes simplex virus 1, HSV1),
HHV-2 (HSV2), HHV-3 (Varicella-zoster virus, VSV), HHV-4
(Epstein-Barr virus, EBV), HHV-5 (cytomegalovirus, CMV), HHV-6,
HHV-7 and HHV-8.
[0099] In other particular embodiments, the virus belongs to the
betaherpesvirus subfamily (e.g. CMV and EBV). In another particular
embodiment, the virus is CMV. In one preferred embodiment, the
viral antigen is derived from immediate early gene 1 (IE-1) protein
of a herpesvirus. In another preferred embodiment, the viral
antigen is derived from immediate early gene 1 (IE-1) protein of a
CMV. In another preferred embodiment, the viral antigen derived
from IE-1 protein comprises a CTL epitope.
[0100] In other embodiments, the virus belongs to the Flaviviridae
family. This family currently contains three genera, the
flaviviruses (e.g. Tick-borne encephalitis viruses, Japanese
encephalitis viruses, Dengue, Yellow fever and viruses such as
Modoc and Uganda virus), the pestiviruses (e.g. bovine viral
diarrhea, Border disease), and the hepatitis C viruses (e.g.
hepatitis C virus, HCV).
[0101] In various embodiments, the virus is selected from the group
consisting of: West Nile virus (WNV), Yellow fever virus, St. Louis
encephalitis virus, Murray Valley encephalitis virus, Kunjin virus,
Japanese encephalitis virus, Dengue virus type 1, Dengue virus type
2, Dengue virus type 3 and Dengue virus type 4. In one particular
embodiment, the viral antigen is derived from West Nile Virus
(WNV). In one preferred embodiment, the viral antigen is derived
from the WNV envelope (E) protein. In another preferred embodiment,
the viral antigen is derived from the E3 domain of said protein. In
another preferred embodiment, said viral antigen comprises a B cell
epitope and a MHC II restricted epitope.
[0102] In other embodiments, there is provided a novel antigen
derived from WNV E3 domain of E protein, hereby designated p15,
corresponding to aa 355-369 of the E protein. In various
embodiments, the antigen has an amino acid sequence as set forth in
any one of SEQ ID NOS: 11 and 12 (LVTVNPFVSVATANS and
LVTVNPFVSVATANA, respectively). In other embodiments, the invention
provides proteins, peptides and conjugates comprising said antigen.
For example, without limitation, said antigen may be conjugated
with a peptide or lipid carrier or adjuvant.
[0103] In another embodiment, the conjugates of the invention
comprise a viral antigen having an amino acid sequence as set forth
in any one of SEQ ID NOS:11 and 12 covalently attached to a
synthetic peptide carrier of the invention. In another embodiment,
the conjugate has an amino acid sequence as set forth in any one of
SEQ ID NOS:13 (NEDQKIGIEIIKRALKILVTVNPFVSVATANS), 14
(NEDQKIGIEIIKRALKILVTVNPFVSVATANA),
(NEDQKIGIEIIKRTLKILVTVNPFVSVATANS), 16
(NEDQKIGIEIIKRTLKILVTVNPFVSVATANA), 77
(KKARVEDALHATRAAVEEGVLVTVNPFVSVATANS), and 78
(KKARVEDALHATRAAVEEGVLVTVNPFVSVATANA).
[0104] Other embodiments are directed to homologs, analogs,
fragments and derivatives of p15, as detailed hereinbelow.
[0105] According to certain embodiments, the invention provides p15
homologs derived from a flavivirus, and active fragments and
extensions thereof, as detailed in Table 1:
TABLE-US-00002 TABLE 1 p15 homologous epitopes from various
flaviviruses, active fragments and extensions thereof, and
nucleotide sequences encoding them. nucleic acid Amino acid
sequence (SEQ ID Virus (SEQ ID NO.) NO.) West Nile virus
LVTVNPFVSVATANS (11) 19 LVTVNPFVSVATANA (12) 20 GRLVTVNPFVSVATANS
(34) 54 GRLVTVNPFVSVATANA (35) 55 Yellow fever LVTVNPIASTNDDEVLIE
(25) 45 virus GILVTVNPIASTNDDEVLIE (36) St. Louis
LVTVNPFISTGGANNKVM (26) 46 encephalitis GRLVTVNPFISTGGANNKVM (37)
virus Murray Valley MYTANPYVASSTANAKVL (27) 47 encephalitis
GRMVTANPYVASSTANAKVL (38) virus Kunjin virus LVTVNPFVSVSTANAKVL
(28) 48 GRLVTVNPFVSVSTANAKVL (39) Japanese LVTVNPFVATSSANSKVL (29)
49 encephalitis GRLVTVNPFVATSSANSKVL (40) virus Dengue virus
LITANPIVTDKEKPVNIE (30) 50 type 1 GRLITANPIVTDKEKPVNIE (41) Dengue
virus LITVNPIVTEKDSPVNIE (31) 51 type 2 GRLITVNPIVTEKDSPVNIE (42)
Dengue virus LITANPVVTKKEEPVNIE (32) 52 type 3 GRLITANPVVTKKEEPVNIE
(43) Dengue virus IISSTPLAENTNSVTNIE (33) 53 type 4
GRIISSTPLAENTNSVTNIE (44)
[0106] However, it should be understood that the amino acid
sequence of these homologous epitopes may be altered in different
variants and strains of these viruses. The present invention is
thus further directed to homologous peptides from different
variants and strains of these viruses.
[0107] With respect to the novel viral peptide antigens of the
invention, the term "analogs" relates to peptides obtained by
replacement, deletion or addition of amino acid residues to the
sequence, optionally including the use of a chemically derivatized
residue in place of a non-derivatized residue, as long as their
ability to confer immunity against a viral infection when
conjugated to the carriers of the invention is retained. The term
also includes homologs corresponding to amino acid sequences which
are significantly related because of an evolutionary relationship,
either between species (ortholog) or within a species (paralog).
Peptide sequences having conserved amino acid sequence domains are
examples of homologs. With respect to the novel viral peptide
antigens of the invention, peptide homologs may have at least about
40% identity in their amino acid sequence, preferably at least 50%,
more preferably at least about 70% and most preferably at least
about 90% identity. These values reflect the short length of the
peptides.
[0108] In another aspect, there is provided a second novel WNV
epitope derived from the E protein, herein denoted p17, having the
following amino acid sequence: YIVVGRGEQQINHHWHK (SEQ ID NO:21).
Other embodiments are directed to analogs, homologs, fragments, and
derivatives thereof.
[0109] In various embodiments, these peptides and homologs may be
used in conjugation with the carriers of the invention. In certain
particular embodiments, the conjugate has an amino acid sequence as
set forth in any one of SEQ ID NOS: 23-24 and 56-75.
[0110] Peptide and Derivative Synthesis
[0111] The polypeptides and peptides of the invention may be
synthesized using any recombinant or synthetic method known in the
art, including, but not limited to, solid phase (e.g. Boc or f-Moc
chemistry) and solution phase synthesis methods. For solid phase
peptide synthesis, a summary of the many techniques may be found
in: Stewart and Young, 1963; and Meienhofer, 1973. For a review of
classical solution synthesis, see Schroder and Lupke, 1965.
[0112] The amino acid residues described herein are preferred to be
in the "L" isomeric form. However, residues in the "D" isomeric
form can be substituted for any L-amino acid residue, as long as
the peptide substantially retains the desired functional property.
Use of "D" amino acids may be used as is known in the art to
increase the stability or half-life of the resultant peptide.
[0113] Whenever p458 and Ec27 conjugates are mentioned in the
invention, also salts and functional derivatives thereof are
contemplated, as long as they are able to substantially enhance the
immunogenicity of the antigen molecules. Thus, the present
invention encompasses polypeptides or peptides containing
non-natural amino acid derivatives or non-protein side chains.
[0114] The term derivative includes any chemical derivative of the
polypeptides or peptides of the invention having one or more
residues chemically derivatized by reaction of side chains or
functional groups. Such derivatized molecules include, for example,
those molecules in which free amino groups have been derivatized to
form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy
groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl
groups. Free carboxyl groups may be derivatized to form salts,
methyl and ethyl esters or other types of esters or hydrazides.
Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl
derivatives. The imidazole nitrogen of histidine may be derivatized
to form N-im-benzylhistidine. Also included as chemical derivatives
are those peptides, which contain one or more naturally occurring
amino acid derivatives of the twenty standard amino acid residues.
For example: 4-hydroxyproline may be substituted for proline;
5-hydroxylysine may be substituted for lysine; 3-methylhistidine
may be substituted for histidine; homoserine may be substituted or
serine; and ornithine may be substituted for lysine.
[0115] In addition, a peptide or conjugate can differ from the
natural sequence of the polypeptides or peptides of the invention
by chemical modifications including, but are not limited to,
terminal-NH.sub.2 acylation, acetylation, or thioglycolic acid
amidation, and by terminal-carboxlyamidation, e.g., with ammonia,
methylamine, and the like. Peptides can be either linear, cyclic or
branched and the like, which conformations can be achieved using
methods well known in the art.
[0116] It is noted that both shorter active fragments derived from
the viral antigens denoted as SEQ ID NOS:11-12, 21 and 25-33 and
longer peptides comprising these sequences are within the scope of
the present invention. Such fragments or peptides may be comprise,
for example, peptides having 1-3 amino acids deleted at either
termini, or addition of 1-3 amino acid residues or more from the
flanking sequences of the viral protein to either termini, as long
as their ability to confer immunity against a viral infection when
conjugated to the carriers of the invention is retained. It is to
be understood that longer peptides, e.g. up to 50 amino acids in
length may also be used for vaccination according to the invention.
However, shorter peptides are preferable, in one embodiment, for
being easier to manufacture. Such extensions of the novel peptide
antigens of the present invention are not intended to include any
known protein of fragment, such as the full length E3 domain of a
flavivirus. The viral antigens, according to the present invention
are preferably 5-50 amino acids in length, more preferably 8-20
amino acids in length. Exemplary fragments and extensions of the
p15 and homologs thereof according to the invention are presented
in Table 1.
[0117] Addition of amino acid residues may be performed at either
terminus of the polypeptides or peptides of the invention for the
purpose of providing a "linker" by which the peptides of this
invention can be conveniently bound to a carrier. Such linkers are
usually of at least one amino acid residue and can be of 40 or more
residues, more often of 1 to 10 residues. Typical amino acid
residues used for linking are tyrosine, cysteine, lysine, glutamic
and aspartic acid, or the like.
[0118] The conjugates of the invention may also be created by means
of chemically conjugating a viral antigen with a p458 or Ec27
synthetic carrier peptide, using methods well known in the art.
[0119] Nucleic Acids
[0120] In another aspect, the invention provides nucleic acid
molecules encoding the peptide antigens of the invention.
[0121] The nucleic acid molecules may include DNA, RNA, or
derivatives of either DNA or RNA. An isolated nucleic acid sequence
encoding a viral antigen or a HSP60 peptide can be obtained from
its natural source, either as an entire (i.e., complete) gene or a
portion thereof. A nucleic acid molecule can also be produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Nucleic acid
sequences include natural nucleic acid sequences and homologs
thereof, including, but not limited to, natural allelic variants
and modified nucleic acid sequences in which nucleotides have been
inserted, deleted, substituted, and/or inverted in such a manner
that such modifications do not substantially interfere with the
nucleic acid molecule's ability to encode a functional peptide of
the present invention.
[0122] A nucleic acid molecule homolog can be produced using a
number of methods known to those skilled in the art (see, for
example, Sambrook et al., 1989). For example, nucleic acid
molecules can be modified using a variety of techniques including,
but not limited to, classic mutagenesis techniques and recombinant
DNA techniques, such as site-directed mutagenesis, chemical
treatment of a nucleic acid molecule to induce mutations,
restriction enzyme cleavage of a nucleic acid fragment, ligation of
nucleic acid fragments, polymerase chain reaction (PCR)
amplification and/or mutagenesis of selected regions of a nucleic
acid sequence, synthesis of oligonucleotide mixtures and ligation
of mixture groups to "build" a mixture of nucleic acid molecules
and combinations thereof. Nucleic acid molecule homologs can be
selected from a mixture of modified nucleic acids by screening for
the function of the protein encoded by the nucleic acid with
respect to the induction of an anti-viral response, for example by
the methods described herein.
[0123] A polynucleotide or oligonucleotide sequence can be deduced
from the genetic code of a protein, however, the degeneracy of the
code must be taken into account. For example, an oligonucleotide
having a nucleic acid sequence:
ctggtgaccgtgaatccatttgtgtctgtggccacagccaactcg (SEQ ID NO:19)
encodes a p15 antigen derived from West Nile Virus E protein:
LVTVNPFVSVATANS (SEQ ID NO:11). However, nucleic acid sequences of
the invention also include sequences, which are degenerate as a
result of the genetic code, which sequences may be readily
determined by those of ordinary skill in the art. In other
particular embodiments, the viral antigens of the invention are
encoded by oligonucleotides having a nucleic acid sequence as set
forth in any one of SEQ ID NOS:20, 22 and 45-55 (see Table 1).
[0124] The oligonucleotides or polynucleotides of the invention may
contain a modified internucleoside phosphate backbone to improve
the bioavailability and hybridization properties of the
oligonucleotide or polynucleotide. Linkages are selected from the
group consisting of phosphodiester, phosphotriester,
methylphosphonate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoroanilidate, phosphoramidate,
phosphorothioate, phosphorodithioate or combinations thereof.
[0125] Additional nuclease linkages include alkylphosphotriester
such as methyl- and ethylphosphotriester, carbonate such as
carboxymethyl ester, carbamate, morpholino carbamate,
3'-thioformacetal, silyl such as dialkyl (C1-C6)- or diphenylsilyl,
sulfamate ester, and the like. Such linkages and methods for
introducing them into oligonucleotides are described in many
references, e.g. reviewed generally by Peyman and Ulmann,
(1990).
[0126] The present invention includes a nucleic acid sequence of
the present invention operably linked to one or more transcription
control sequences to form a recombinant molecule. The phrase
"operably linked" refers to linking a nucleic acid sequence to a
transcription control sequence in a manner such that the molecule
is able to be expressed when transfected (i.e., transformed,
transduced or transfected) into a host cell. Transcription control
sequences are sequences which control the initiation, elongation,
and termination of transcription. Particularly important
transcription control sequences are those which control
transcription initiation, such as promoter, enhancer, operator and
repressor sequences. Suitable transcription control sequences
include any transcription control sequence that can function in at
least one of the recombinant cells of the present invention. A
variety of such transcription control sequences are known to those
skilled in the art. Preferred transcription control sequences
include those which function in animal, bacteria, helminth, insect
cells, and animal cells.
[0127] A nucleic acid molecule of the invention may be inserted
into appropriate expression vector, i.e., a vector which contains
the necessary elements for the transcription and translation of the
inserted coding sequence.
[0128] Vectors can be introduced into cells or tissues by any one
of a variety of known methods within the art, including in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Such methods are generally described in
Sambrook et al., (1989, 1992), in Ausubel et al., Current Protocols
in Molecular Biology, John Wiley and Sons, Baltimore, Md. 1989.
[0129] A recombinant cell of the present invention comprises a cell
transfected with a nucleic acid molecule that encodes a viral
antigen of the invention. A variety of expression vector/host
systems may be utilized to contain and express sequences encoding
the viral antigens of the invention. These include, but are not
limited to, microorganisms such as bacteria transformed with
recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.,
baculovirus); plant cell systems transformed with virus expression
vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed. The expression of the construct
according to the present invention within the host cell may be
transient or it may be stably integrated in the genome thereof.
[0130] Vaccine Compositions and Methods Thereof.
[0131] According to some aspects the present invention provides a
vaccine comprising an isolated viral antigenic peptide and a
peptide comprising a T cell epitope of HSP60, wherein the HSP60
peptide enhances the immunogenicity of the viral antigenic peptide
by at least two fold compared to the peptide without the HSP60
peptide. In certain currently preferred embodiments the
immunogenicity is enhanced by at least 4-5 fold.
[0132] In certain embodiments the vaccine compositions comprise a T
cell epitope of HSP60 suitable to enhance the immunogenicity when
used as an adjuvant peptide that is mixed with the viral antigen.
According to certain particular embodiments, the adjuvant peptide
is selected from Ec27 and analogs and derivatives thereof. In
alternative embodiments the vaccine comprises a T cell epitope of
HSP60 suitable to enhance the immunogenicity of the viral antigenic
peptide when used in conjugates where the HSP60 peptide is
covalently linked to the viral antigenic peptide. In some
particular embodiments, the peptide carrier is selected from p458,
Ec27 and analogs and derivatives thereof. The enhanced
immunogenicity of said viral antigen is measured by at least one of
the following: serum titer of antibodies directed to said viral
antigen; T cell proliferation in the presence of said viral
antigen; cytokine secretion induced by said viral antigen; specific
T cell mediated lysis of virus-infected cells; and reduction of
detectable viral load.
[0133] In another aspect, the invention provides vaccine
compositions comprising the conjugates of the invention and a
pharmaceutically acceptable carrier, adjuvant, excipient or
diluent.
[0134] In another aspect, the invention provides vaccine
compositions comprising a polypeptide or peptide, said polypeptide
or peptide comprising an amino acid sequence as set forth in any
one of SEQ ID NOS:11-12, 21 and 25-44, and a pharmaceutically
acceptable carrier, adjuvant, excipient or diluent.
[0135] In one embodiment of the invention, the composition is
useful for treating or preventing a viral infection in a subject in
need thereof, as described herein.
[0136] The vaccine composition of the invention is administered to
a subject in need thereof in an effective amount. According to the
present invention, an "effective amount" is an amount that when
administered to a subject results in a substantial increase in the
immune response of the subject to said viral antigen, as described
herein.
[0137] According certain embodiments, the subject is selected from
the group consisting of humans, non-human mammals and non-mammalian
animals (e.g. birds). In a preferred embodiment, the subject is
human.
[0138] Pharmaceutical and veterinary compositions for use in
accordance with these embodiments may be formulated in conventional
manner using one or more physiologically acceptable carriers or
excipients (vehicles). The carrier(s) are "acceptable" in the sense
of being compatible with the other ingredients of the composition
and not deleterious to the recipient thereof. The vaccine
composition can be optionally administered in a pharmaceutically or
physiologically acceptable vehicle, such as physiological saline or
ethanol polyols such as glycerol or propylene glycol.
[0139] The polypeptides and peptides of the invention may be
formulated into the vaccine as neutral or salt forms.
Pharmaceutically acceptable salts include the acid addition salts
(formed with free amino groups of the peptide) and which are formed
with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids such as acetic, oxalic,
tartaric and maleic. Salts formed with the free carboxyl groups may
also be derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such
organic bases as isopropylamine, trimethylamine, 2-ethylamino
ethanol, histidine and procaine.
[0140] The vaccine composition may optionally comprise additional
adjuvants such as vegetable oils or emulsions thereof, surface
active substances, e.g., hexadecylamin, octadecyl amino acid
esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium
bromide, N,N-dicoctadecyl-N'-N'bis(2-hydroxyethyl-propane diamine),
methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g.,
pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl
dipeptide, dimethylglycine, tuftsin; immune stimulating complexes;
oil emulsions (including, but not limited to, oil-in-water
emulsions having oil droplets in the submicron range, such as those
disclosed by U.S. Pat. Nos. 5,961,970, 4,073,943 and 4,168,308);
liposaccharides such as MPL.RTM. and mineral gels. The antigens of
this invention can also be incorporated into liposomes, cochleates,
biodegradable polymers such as poly-lactide, poly-glycolide and
poly-lactide-co-glycolides, or ISCOMS (immunostimulating
complexes), and supplementary active ingredients may also be
employed. The protein and peptide antigens of the present invention
can be coupled to albumin or to other carrier molecule in order to
modulate or enhance the immune response, all as are well known to
those of ordinary skill in the vaccine art.
[0141] The vaccines can be administered to a human or animal by a
variety of routes, including but not limited to parenteral,
intradermal, transdermal (such as by the use of slow release
polymers), intramuscular, intraperitoneal, intravenous,
subcutaneous, oral and intranasal routes of administration,
according to protocols well known in the art. The particular dosage
of the conjugate antigen will depend upon the age, weight and
medical condition of the subject to be treated, as well as on the
identity of the antigen and the method of administration. Suitable
doses will be readily determined by the skilled artisan. A
preferred dose for human intramuscular, subcutaneous and oral
vaccination is between about 6 .mu.g to about 70 mg per kg body
weight, preferably between about 15 .mu.g to about 28 mg per kg
body weight, and more preferably between about 40 .mu.g to about 7
mg per kg body weight. Adjustment and manipulation of established
dosage ranges used with traditional carrier antigens for adaptation
to the present vaccine is well within the ability of those skilled
in the art.
[0142] In various embodiments, the vaccine composition s of the
invention may be used in combination with other treatments and
medicaments, e.g. anti-viral drugs. For example, a conjugate
comprising a CTL epitope derived from 1E-1 protein of HCMV and a
peptide carrier of the invention may be administered to HCMV
infected subjects in combination with gancyclovir therapy. Doses
and administration regimes of gancyclovir are known in the art.
[0143] In another aspect, the present invention is directed to the
use of a conjugate of the invention for the preparation of a
vaccine composition useful for conferring anti-vial immunity.
[0144] In yet another aspect, the invention provides methods for
increasing the immunogenicity of a viral antigen which comprises
linking the antigen to a synthetic peptide carrier of the
invention.
[0145] In another aspect, the invention provides methods for
immunizing a subject in need thereof against a viral infection,
comprising administering to the subject an effective amount of a
vaccine composition comprising a conjugate of the invention and a
pharmaceutically acceptable carrier, adjuvant, excipient or
diluent.
[0146] In various embodiments, the vaccine composition may be
administered to said subject before the exposure of said subject to
the virus or after exposure of said subject to said virus.
[0147] In another aspect, the invention provides methods
comprising: [0148] (a) isolating a viral antigen, comprising at
least one epitope selected from: a CTL epitope, a B cell epitope
and a MHC II-restricted epitope; [0149] (b) conjugating said viral
antigen to a synthetic peptide carrier of the invention; and [0150]
(c) administering to the subject an effective amount of a vaccine
composition comprising a conjugate of the invention and a
pharmaceutically acceptable carrier, adjuvant, excipient or
diluent.
[0151] Diagnostic Kits and Methods Thereof.
[0152] Other embodiments of the present invention are directed to
diagnostic compositions and kits and uses thereof for the diagnosis
of flavivirus infection.
[0153] The present invention provides a method for diagnosing the
presence of, or exposure to a flavivirus in a patient, comprising
testing said patient for the presence of anti-flavivirus antibodies
or of a T cells which immunoreact with flavivirus epitopes using a
peptide according to Table 1 or analogs, derivatives and salts
thereof as antigen.
[0154] In one embodiment, the method comprises the steps of: [0155]
(a) contacting a suitable biological specimen with a viral antigen
having an amino acid sequence as set forth in any one of SEQ ID
NOS:11-12, 21 and 25-44 and analogs, homologs, derivatives and
salts thereof under conditions such that an immune reaction can
occur; [0156] (b) quantifying the immune reaction between the
peptide antigen and the biological specimen, [0157] wherein an
immune reaction significantly higher than an immune reaction
obtained for a sample obtained from a non-infected subject is
indicative of exposure to, or, in other embodiments, infection of
the subject with a flavivirus.
[0158] A biological specimen or sample that may be assayed for
flavivirus infection may include, for example, mammalian body
fluids (e.g. serum, tissue extracts, tissue fluids, mucosal
secretions), in vitro cell culture supernatants, cell lysates and
cells or tissue from the subject that have been cultured in cell
culture (e.g. leukocyte samples such as peripheral blood
mononuclear cells). Methods of obtaining a suitable biological
sample from a subject are known to those skilled in the art.
[0159] In certain embodiments, the peptides and peptide
compositions prepared in accordance with the present invention can
be used to detect anti-flavivirus antibodies and diagnose
flavivirus infection by using them as the test reagent in an
enzyme-linked immunoadsorbent assay (ELISA), an enzyme immunodot
assay, a passive hemagglutination assay (e.g., PHA test), an
antibody-peptide-antibody sandwich assay, a
peptide-antibody-peptide sandwich assay, or other well-known
immunoassays. In accordance with the present invention, any
suitable immunoassay can be used with the subject peptides. Such
techniques are well known to the ordinarily skilled artisan and
have been described in many standard immunology manuals and texts.
In one particular embodiment, the immunoassay is an ELISA using a
solid phase coated with the peptide compositions of the present
invention. For example, such a kit for determining the presence of
anti-flavivirus antibodies may contain a solid-phase immobilized
peptide of the invention and a tagged antibody capable of
recognizing the non-variable region of the anti-flavivirus antibody
to be detected, such as tagged anti-human Fab. The kit may also
contain directions for using the kit and containers to hold the
materials of the kit. Any conventional tag or label may be used,
such as a radioisotope, an enzyme, a chromophore or a fluorophore.
A typical radioisotope is iodine-125 or sulfur-35. Typical enzymes
for this purpose include horseradish peroxidase, horseradish
galactosidase and alkaline phosphatase.
[0160] In other embodiments, the presence of T cells immunoreactive
with flavivirus epitopes may be determined, for example, by
determining T cell proliferation or cytokine secretion induced by
the novel viral peptide epitopes of the invention, using methods
well known in the art. Several non-limitative examples of
determining T cell reactivity with peptide antigens are presented
in the Examples herein. For example, a kit for diagnosing
flavivirus exposure or infection by testing for the presence of a T
cell which immunoreacts with flaviviral epitopes, may comprise: an
antigen selected from the peptides of the invention; a suitable
medium for culture of lymphocytes (T cells); and either a labeled
nucleotide for the T cell proliferation test, or a cytokine, e.g.,
interferon-gamma, assay kit, for the cytokine test.
[0161] In various embodiments, the method may comprise the steps
of: [0162] (a) contacting a suitable biological sample with a viral
antigen having an amino acid sequence as set forth in any one of
SEQ ID NOS:11-12, 21 and 25-44 and analogs, homologs, derivatives
and salts thereof under conditions such that an immune reaction can
occur; [0163] (b) determining whether the peptide antigen binds
specifically to the biological sample.
[0164] The term "binds specifically to the biological sample" as
used herein refers to occurrence of an immune reaction between a
component of the biological specimen or sample (e.g. antibodies and
T cells) and the viral peptide antigen having higher affinity or
extent than to another antigen. For example, specific binding may
be measured by determining the extent of antigen-antibody complex
formation, T cell proliferation or cytokine secretion. Thus, for
example, step (b) may include determining the extent of
antigen-antibody complex formation, wherein an antigen-antibody
complex formation level significantly higher than the level
obtained for a sample obtained from a subject not previously
exposed to or infected by a flavivirus is indicative of exposure of
the subject to the flavivirus.
[0165] The kits and methods of the present invention may be used,
in some embodiments, for the differential diagnosis of a flavivirus
infection, enabling the identification of the particular flavivirus
strain infecting the subject or to which the subject was exposed.
For example, a biological specimen may be assayed for the presence
of anti-Dengue antibodies using the peptides having an amino acid
sequence as set forth in SEQ ID NOS:30-33 to determine the strain
of Dengue virus infecting the subject (e.g. to distinguish between
Dengue 1, 2, 3 or 4 infection).
[0166] The following examples are presented in order to more fully
illustrate some embodiments of the invention. They should, in no
way be construed, however, as limiting the broad scope of the
invention.
EXAMPLES
A. CMV Vaccination
[0167] Materials and Methods
[0168] Mice
[0169] BALB/c female mice were purchased from Harlan Olac
(Bicester, UK). Mice were maintained under specific pathogen free
conditions and were allowed to adjust to the facility for 1 week
before any experiments were performed. For the pathogenesis
experiments, mice were used at 6 to 8 weeks of age and for the
immunization experiments, mice were used at 3 weeks of age. The
mouse experiments were approved by and performed according to the
guidelines of the Ben Gurion University Faculty of Health Sciences
Animal Safety Committee.
[0170] MCMV
[0171] The Smith strain of MCMV was obtained from the American Type
Culture Collection (ATCC) (Rockville, Md.). Highly virulent
salivary gland-passaged MCMV stocks were prepared as a 10% (wt/vol)
homogenate of salivary gland from day 14-infected BALB/c in
DMEM-10% FCS. Homogenates were clarified by low speed
centrifugation, DMSO was added to final concentration of 10%, and
virus stocks were stored in aliquots at -70.degree. C. until use
(Palmon et al., 1996).
[0172] MCMV titers in these salivary gland suspension (SGS) stocks
were determined by a quantitative plaque assay (Rager Zisman et
al., 1973). Briefly, confluent monolayers of secondary mouse embryo
fibroblasts (MEF) were prepared in 24 well plates. Serial 10-fold
dilutions of SGS containing MCMV were prepared in DMEM supplemented
with 2% FCS. The growth medium from each well in MEF plates was
aspirated, and duplicate wells were inoculated with 0.2 ml of
diluted SGS. After an adsorption period of 1 hour at 37.degree. C.,
monolayers were overlayed with 0.8 ml of growth medium containing
0.75% carboxymethyl cellulose (CMC), incubated for 5 days at
37.degree. C. in a humidified 5% CO.sub.2 incubator, fixed in
PBS-10% formaldehyde and stained with Crystal Violet to visualize
virus plaques. Titers were expressed as log.sub.10 pfu/0.1 gr
tissue. Thorough this study virus stocks containing
1.75.times.10.sup.8 pfu/0.1 g of tissue were used.
[0173] Infection with MCMV and Virus Titers in Target Organs
[0174] To study the course of MCMV infection in naive or immunized
BALB/c mice, mice were inoculated intraperitoneally (i.p.) with
5.times.10.sup.4 pfu of stock virus in 0.2 ml PBS. Mice were
sacrificed at different time points, spleens and salivary glands
(pooled 3 mice per group at each time point) were removed and 10%
(wt/vol) homogenates were prepared as previously described (Palmon
et al., 1996). Samples were stored at -70.degree. C. until
infectious virus titrations were performed on primary cultures of
MEF.
[0175] Preparation of DNA and Amplification by PCR
[0176] DNA was extracted from naive and infected spleens and
salivary gland using QiAmp Tissue Kit (QIAGEN Inc. Chatsworth,
Calif., USA), according to appropriate QiAmp protocols. DNA
oligonucleotide primers were synthesized according to the published
sequence of MCMV gB gene (Rapp et al., 1992). The sequence of gB
sense strand primer was based on the cDNA sequence no. 2416-2443
(5'-AAG-CAG-CAC-ATC-CGC-ACC-CTG-AGC-GCC-3' SEQ ID NO:17) and the
antisense no. 2745-2772 (5'-CCA-GGC-GCT-CCC-GGC-GGC-CCG-CTC-TCG-3'
SEQ ID NO:18). This gB gene primer pair amplifying a 356 bp segment
was found the most sensitive in previous studies (Palmon et al.,
1996). For gene amplification, 1 .mu.g of DNA sample was added to
the reaction mixture containing 200 .mu.M each dNTP, 100 .mu.mol
each primer, 1.0 mM MgSO.sub.4, 10 mM KCl, 10 mM
(NH.sub.4).sub.2SO.sub.4, 20 mM Tris-HCl (pH 8.8), 0.1% Triton
X-100 and 2 U of vent polymerase (Biolabs) in a total reaction
volume of 50 .mu.l each. Samples were amplified for 30 cycles in an
automated thermal cycler (Perkin Elmer Cetus, USA). Each cycle
entailed denaturation at 94.degree. C. for 60 sec, annealing at
68.degree. C. for 90 sec and primer extension at 72.degree. C. for
120 sec. PCR products were electrophoretically separated on 1.5%
agarose gel, stained with ethidium bromide, and photographed. The
lower limit of detection for this method under the experimental
conditions was 5 femtograms of viral DNA corresponding to about 20
copies of the MCMV genome (Palmon et al., 1996).
[0177] Peptides
[0178] Peptides were prepared in the Weizmann Institute of Science
(Rehovot, Israel), and in Albert Einstein College of Medicine
(New-York USA). The purity of the peptides was ascertained by
analytical reversed-phase HPLC and amino acid (aa) analysis. The
sequences of the six peptides synthesized are: 89pep (MCMV pp 89
ie1-CTL epitope, Reddehase et al., 1989)--YPHFHPTNL (SEQ ID NO:5);
p458 (the active peptide derived from mouse HSP60, Konen Waisman et
al., 1999)--NEDQKIGIEIIKRALKI (SEQ ID NO:2); p458-89pep
(combined)--NEDQKIGIEIIKRALKIYPHFHPTNL (SEQ ID NO:6); negative
control for p458 (the p431 peptide of the mycobacterial HSP60,
442val-deleted)--EGDEATGANI-KVALEA (SEQ ID NO:7); control-89pep
(combined)--EGDEATGANI-KVALEAYPHFHPTNL (SEQ ID NO:8); and
TTp30-89pep (combined)--FNNFTVSFWLRVPKVSASHLEYPHFMPTNL (SEQ ID
NO:9). The p30 of TT (aa 947-967) (Panina-Bordignon et al., 1989)
(SEQ ID NO:10) is now being used as a carrier peptide in various
vaccines (Brander et al., 1996; Keitel et al., 1999). The
mycobacterial p431 peptide (442val-deleted) was used as a negative
control peptide since it is homologous in sequence to mammalian
p458, but did not elicit a CD4.sup.+-dependent immune response
against itself or p458.
[0179] Immunization and Challenge of Mice with MCMV
[0180] The immunizing dose of each peptide was equimolar to 15
.mu.g of p458 (Konen Waisman et al., 1999). All peptides were
emulsified in incomplete Freund's adjuvant (IFA), and the volumes
for intra-footpad (i.f.p.) and subcutaneous (s.c.) injections were
50 .mu.l and 100 .mu.l respectively. Two different protocols were
used. To study the immune response of mice to the chimeric peptide
(p458-89pep), groups of 6-week old to 8-week old mice were
immunized once into the hind footpad with peptides emulsified in
IFA. Ten days later several mice were sacrificed, and organs were
harvested for IFN.gamma. and IL-4 assays. To study the protective
efficacy of the combined peptide, groups of 3-week old mice were
immunized and boosted according to the following protocol: mice
were immunized i.f.p. on day (-24), and boosted s.c. two weeks
later on day (-10). Ten days later (day 0), mice were challenged IP
with 5.times.10.sup.4 pfu of MCMV. Mice were sacrificed on days 14,
21, and 28 after challenge, and organs were harvested for virus
titrations, PCR, cytotoxic T cell and cytokine assays.
[0181] Preparation of Spleen and Salivary Gland Mononuclear Cell
Cultures
[0182] Spleen pulp was extruded from the capsule in a non-tissue
culture Petri dish in RPMI-1640 medium supplemented with 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, 2 mM glutamine, 10 mM HEPES
and 5% FCS (base-RPMI). Spleen cell suspensions were passaged
through a cell strainer, washed once, treated 2 min with ACK lysing
buffer (0.15M NH.sub.4Cl, 0.01 KHCO.sub.3; 2 ml/spleen) for
elimination of erythrocytes, and washed twice in base-medium.
Splenocytes were resuspended in RPMI-1640 medium supplemented with
100 U/ml penicillin, 100 .mu.g/ml streptomycin, 2 mM glutamine, 10
mM HEPES, 5.times.10.sup.-5M .beta.-mercaptoethanol and 10% FCS
(complete RPMI) in a final concentration of 5.times.10.sup.6
cells/ml.
[0183] Salivary gland cell suspensions were prepared by initially
cutting the salivary glands into small fragments (<2 mm) in a
non-tissue culture Petri dish. Fragments were treated with
base-medium containing 1 mg of collagenase-dispase (Roche
Diagnostics, Germany)/ml and 50 .mu.g of DNase I (Boehringer
Mannheim, Germany)/ml. After 1 h incubation in 37.degree. C., cells
were resuspended in 45% Percoll (Sigma Chemical Co., Israel),
overlayed on 66% Percoll and centrifuged at 800 g for 25 min.
Mononuclear cells collected at the interphase, were counted and
resuspended in complete-RPMI to a final concentration of
5.times.10.sup.6 cells/ml.
[0184] IFN.gamma. and IL-4 ELISA Assays
[0185] Mononuclear cell cultures from spleens and salivary gland
were prepared as described above. Cell suspensions were divided
into 24 well plates (5.times.10.sup.6 cells/well) and were
stimulated in vitro with either 10 .mu.g/ml of 89pep or p458 or
with 5 .mu.g/ml Concanavalin-A (Con-A). Cells were incubated for 72
h (with or w/o stimulation) at 37.degree. C. in a humidified 5% CO2
incubator. After incubation supernatants were collected, and
IFN.gamma. and IL-4 levels were measured using indirect ELISA
according to Pharmingen cytokine ELISA protocol (Pharmingen, San
Diego, Calif.).
[0186] FACS Analysis of Cell Phenotypes and Intracellular
IFN.gamma.
[0187] For phenotypic analysis, spleen and salivary gland
mononuclear cells of MCMV-infected and naive mice were cultured as
described above and were stained for CD8 and CD4, IFN.gamma. and
IL-5 using directly-labeled antibodies (PharMingen, San Diego,
Calif.). Intracellular cell staining (ICCS) for IFN.gamma., IL-4
and IL-5 was performed using PharMingen's Cytofix/Cytoperm Plus kit
with GolgiPlug (containing Brefeldin A) according to the
manufacturer's instructions. Briefly, GolgiPlug was added to the 8
h-incubated immune cell cultures (established as described above,
with or w/o peptide stimulation). After the additional 6 h of
incubation in the incubator cells (minimum 10.sup.6 per sample)
were harvested, washed in PBS supplemented with 2% FBS and 0.09%
Sodium azide, and incubated in 50 .mu.l of FC blocker, labeled with
anti-CD4 and anti-CD8 surface markers. Then cells were fixed,
permeabilized and treated with anti-IFN.gamma., anti-IL-4 or
anti-IL-5 antibodies for intracellular cytokine detection. Stained
cells were immediately analyzed on a FACSCalibur flow cytometer
(Becton-Dickinson, Mansfield, Mass.) and 50,000 to 100,000
events/sample were acquired and analyzed with CellQuest
software.
[0188] Cytotoxic T Cell Assay
[0189] The cytotoxic activity against the MCMV 89pep was assessed
in a 4-h cytotoxic assay using the CytoTox 96 non-radioactive,
colorimetric-based kit (Promega, Madison, Wis.), according to
manufacturer instructions. This assay is based on the quantitative
measurement of lactate dehydrogenase, a stable cytosolic enzyme
that is released upon cell lysis. Spleen cell suspensions from
immunized mice, prepared as described above, were re-stimulated in
vitro for 6 days with 89pep (10 .mu.g/ml) and rhIL-2 (25 IU/ml)
from day 2. Target cells for the lysis assay were P815 cells
(mastocytoma, H-2.sup.d). P815 were either non-pulsed or pulsed
with 89pep (1 .mu.g/ml) for 2 h and then washed before incubation
with effector cells. In all experiments shown, the spontaneous
release was less than 25% of maximal release. Each point in a lysis
assay represents the average of triplicate values. The range of the
triplicates was within 5% of their mean.
Example 1
Natural History of MCMV Dissemination in Spleen and Salivary
Gland
[0190] MCMV infection is characterized by different kinetics and
viral loads in different organs (Mercer and Spector, 1986). BALB/c
mice, 6-8 weeks old, were injected i.p. with 5.times.10.sup.4 pfu
of MCMV. Mice were sacrificed on days 1, 3, 7, 14 and 28 after
infection, and spleens and salivary glands were assayed for
infectious virus and MCMV DNA. FIG. 1, shows a typical pattern of
MCMV replication in spleen (empty diamonds) and salivary gland
(full squares). Virus replication peaked in the spleen on day 3
after infection, and slowly declined thereafter (FIG. 1A). By day
14, no infectious virus could be recovered from this organ. To
detect MCMV DNA in infected organs, we used a sensitive PCR using a
gB gene primer pair that amplifies a 356 bp segment (Palmon et al.,
1996). Viral DNA was detected in the spleen as early as day 1 after
infection, peaked on day 3 and by day 14 no DNA could be detected
(FIG. 1B).
[0191] In the salivary gland, virus appeared on day 7. Virus
replication in this organ steadily increased, peaking by day 14
(3.times.10.sup.8 pfu/0.1 gr tissue, FIG. 1A). A moderate decline
in virus titers ensued, and at day 28, 1.5.times.10.sup.6 pfu/0.1
gr tissue were still recoverable from the salivary gland. No
infectious virus could be detected in the SG (and in any other
organ) by day 42 post challenge (data not shown and Keitel et al.,
1999) The detection of viral DNA was associated with the presence
of infectious virus. DNA increased from day 7 to 14. Large amounts
of viral DNA could still be detected on day 28 after infection
(FIG. 1B). On this background of viral dissemination, replication,
splenic clearance and salivary gland persistence, we evaluated the
efficacy of immunization with the p458-89pep chimeric peptide. We
also studied MCMV load in lungs after challenge of naive 6-8 week
old mice; MCMV load (pfu) maximized on day 7 and disappeared by day
14 (data not shown). Thus, in our model we concentrated on the
salivary gland because it is considered as the major site for viral
persistence of MCMV in mice (FIG. 1 and Ho, 1991; Koszinowski et
al., 1990, Kirchner, 1983; Mercer and Spector, 1986).
Example 2
Immunization with p458-89pep Suppresses MCMV Persistence in the
Salivary Gland
[0192] 89pep is the H-2L.sup.d-restricted YPHFMPTNL epitope of
MCMV-pp 89 (Reddehase et al., 1989). We synthesized chimeric
p458-89pep and compared its protective efficacy against MCMV to
that induced by 89pep alone or by negative control-89pep. p458 is a
MHC class II-restricted peptide derived from murine HSP60 (aa
458-474) and capable of inducing CD4+ T responses in BALB/c mice
(Amir-Kroll et al., 2003). The mycobacterial HSP60 431-447 aa
peptide (with a val deletion at position 442) did not elicit an
immune response to itself or to p458, and thus served as a negative
control peptide for immunization; control-89pep.
[0193] To investigate whether immunization with the different
peptides would decrease MCMV replication in salivary glands, 3-week
old BALB/c mice were immunized twice with IFA alone, 89pep,
p458-89pep or control-89pep (FIG. 2). Three-week old, female BALB/c
mice were immunized (i.f.p.) with various peptides, and were
boosted (s.c.). Two weeks later, the mice were challenged (i.p.)
with 5.times.10.sup.4 pfu MCMV, day 0. Three mice from each group
were sacrificed on days 14, 21, and 28 after challenge.
[0194] Peptides for vaccination were emulsified in IFA. Ten days
after the last immunization, mice were challenged i.p. with
5.times.10.sup.4 pfu of MCMV (day 0). Mice were sacrificed on days
14, 21, and 28 after challenge, and infectious virus titers and
MCMV-DNA were measured by plaque and PCR assays in the salivary
glands. As shown in FIG. 2B, no effect of immunization with any of
the peptides could be demonstrated on days 14 and 21 after virus
challenge; on day 14, virus titers ranged from 8.1 to 8.8
log.sub.10 pfu/0.1 g, and on day 21 ranged from 7.1 to 8.0
log.sub.10 pfu/0.1 g.
[0195] On day 28, however, MCMV was not detectable in the salivary
glands of the p458-89pep-immunized mice (<2 log.sub.10 pfu/0.1
g). Immunization with 89pep alone showed a marginal advantage
compared to IFA-immunized mice (FIG. 2B); the viral load was 4.5
and 5.1 log.sub.10 pfu/0.1 g, respectively. Immunization with
control-89pep did not affect the viral load. Other experiments were
performed with the same experimental design in which TTp30-89pep
was used; immunization with TTp30-89pep did not affect viral load
on days 14 and 21, reduced viral load on day 28 by two fold, on
average, but failed to eliminate infectious virus on day 28. To
further evaluate the virus suppression induced by the p458-89pep
immunization, we used a sensitive viral gB PCR to detect viral DNA.
We previously showed that 1 pfu is the equivalent of approximately
1500 viral genomes (Palmon et al., 2000). Yet, on day 28, even this
assay failed to reveal any gB PCR product in salivary glands of
mice immunized with p458-89pep (FIG. 2C, lane d). Therefore, only
immunization with the p458-89pep led to the elimination of
detectable MCMV from the salivary gland, on day 28.
Example 3
IFN.gamma. Secretion by 89pep-Specific T Cells Following Infection
and Vaccination
[0196] It is well established that clearance of MCMV during acute
infection depends primarily on Th1 IFN.gamma. secretion and
protective CTL responses (Mercer and Spector, 1986; Reddehase et
al., 1989). We tested whether IFN.gamma. secretion was stimulated
by 89pep from spleen (FIG. 3A) and salivary gland (FIG. 3B) cell
cultures of MCMV-challenged mice. Cell cultures were prepared on
days 1, 3, 7, 14, 21, and 28 after infection, plated for 3 d with
or without 89pep, and IFN.gamma. secretion was measured. In the
absence of 89pep stimulation, secretion of IFN.gamma. was detected
only in spleen cultures from days 1 and 3 after infection. This
result probably reflects NK activity in the early stages of
infection. When 89pep was added to the cultures, IFN.gamma.
secretion in spleen and salivary glands was correlated with the
kinetics of viral replication in these organs (FIGS. 1A and 3). It
is noteworthy that no significant IL-4 secretion was detected in
the culture supernatants; however, the cells were capable of
secreting IL-4 along with other cytokines in response to
stimulation with Con-A (data not shown).
[0197] We investigated whether immunization with p458-89pep induced
89pep-specific IFN.gamma. secretion. Mice received a single
immunization with the following peptides: p458-89pep, 89pep, p458,
control-89pep or TTp30-89pep. TTp30 is a MHC class II-restricted
peptide capable of inducing vigorous CD4+ T responses and
IFN.gamma. production in BALB/c mice and used as a universal
adjuvant (Panina-Bordignon et al., 1989, Amir-Kroll et al., 2003).
In addition, a non-vaccinated group was infected with MCMV. Ten
days after immunization with the different peptides or infection,
mice were sacrificed, and spleen cell and salivary gland cultures
were prepared and stimulated in vitro for 3 d with 89pep or p458.
FIG. 4 shows that spleen cells derived from mice immunized with
p458-89pep and re-stimulated in vitro with 89pep secreted
significantly higher (p<0.05) levels of IFN.gamma. compared to
mice immunized with 89pep, p458, control-89pep or IFA-only.
89pep-restimulated splenocytes from p458-89pep-immunized mice
secreted significantly higher (p<0.05) levels of IFN.gamma.
compared to the same but non-re-stimulated splenocytes (FIG. 4).
Thus, immunization with p458-89pep induced specific and
significantly enhanced IFN.gamma. secretion. In these experiments
we also tested the TTp30-89pep. Immunization with TTp30-89pep
followed by 89pep re-stimulation induced IFN.gamma. levels similar
to those of mice immunized with p458-89pep (FIG. 4). The highest
levels of 89pep-specific IFN.gamma. secretion were obtained in
spleen cell cultures from mice infected with virus (FIG. 4). This
high IFN.gamma. secretion by spleen cells from MCMV-infected mice,
after in vitro stimulation with 89pep, indicates the dominance of
this epitope in the response to MCMV. No 89pep-specific IFN.gamma.
was detected in salivary gland cell cultures after immunization
with the different peptides (data not shown). Thus, infection of
the salivary gland with MCMV appeared to be needed for recruitment
to the organ of 89pep-specific IFN.gamma. producing cells (FIGS. 1A
and 3).
[0198] The response of spleen cell cultures to stimulation with
p458 induced high levels of IFN.gamma. in mice immunized with p458
or p458-89pep, but not in other groups; this indicates that the
responses were immunologically specific (FIG. 4). No significant
IL-4 secretion after either immunization was detected; nonetheless
the cells were capable of secreting IL-4 after stimulation with
Con-A. IL-4 levels measured after Con-A stimulation in vitro were
242 pg/ml, 146 pg/ml, 184 pg/ml and 317 pg/ml for IFA-only, 89pep,
p458, and p458-89pep respectively. Taken together, these results
imply that the protection induced by p458-89pep was associated with
elevation in MCMV-specific IFN.gamma. production.
Example 4
Immunization with p458-89pep Induces 89Pep-Specific
IFN.gamma..sup.+CD8.sup.+ T Cells and CTL Activity
[0199] We characterized the nature of cells secreting the
IFN.gamma. by flow cytometry. Mice were immunized once with the
different peptides and an additional group was infected with MCMV.
Seven days later, spleens were removed and cell suspensions were
cultured for 5 days with or without 89pep. Immunization with
p458-89pep followed by 5 days of re-stimulation with 89pep induced
IFN.gamma..sup.+CD8.sup.+T cells and no IFN.gamma..sup.+CD4.sup.+ T
cells (FIG. 5); very few IFN.gamma..sup.+CD8.sup.+ T cells were
detected after immunization and re-stimulation with 89pep alone
(FIG. 5B). Infection with MCMV and re-stimulation with 89pep
induced the highest percentage of IFN.gamma..sup.+CD8.sup.+ T cells
(FIG. 5B). Staining was specific to IFN.gamma. since no
CD8.sup.+IL-4.sup.+ or CD8.sup.+IL-5.sup.+ cells were observed.
[0200] We also investigated whether the CD8.sup.+ IFN.gamma..sup.+
cells induced by the p458-89pep were able to lyse specifically
89pep-loaded target cells. Mice were immunized once and 7 d later,
spleens were harvested and re-stimulated with 89pep. Six days
later, lytic activity was assayed on P815 (H-2.sup.d) loaded with
the 89pep. No lytic activity was observed from the cultures of
89pep-immunized mice, but CTLs induced by p458-89pep lysed the
target cells (FIG. 6). Similar to our results with IFN.gamma.
production by CD8.sup.+ T cells, the 89pep-specific lytic activity
induced by MCMV infection was higher than that induced by
p458-89pep immunization.
Example 5
Salivary Gland-Specific Response after Immunization and Virus
Challenge
[0201] We found, above, that IFN.gamma. secretion in the salivary
gland was virus-specific and depended on MCMV infection (FIGS. 1
and 3). In the present experiment, we monitored IFN.gamma.
production in immunized mice 28 days after virus challenge.
Staining of mononuclear cells for IFN.gamma.-production was
performed immediately after excision of the salivary gland, and
stimulation with 89pep for 8 hr. The salivary glands of mice
immunized with IFA, 89pep or TTp30-89pep and challenged with virus
contained infectious virus on day 28 post challenge (FIG. 2).
Likewise, CD8.sup.+ IFN.gamma..sup.+ cells were observed in these
day 28-infected salivary glands. In contrast, mice immunized with
p458-89pep showed no infectious MCMV in the salivary gland 28 days
after infection (FIG. 2). Nevertheless, the number of CD8.sup.+
IFN.gamma..sup.+ cells was larger than that of the other groups
(FIG. 7). This indicates that vaccination with p458-89pep induced a
large reservoir of 89pep-specific CD8.sup.+ T cells along with
termination of salivary gland infection.
Example 6
p458-89pep Reduces Viral Load of MCMV-Infected Mice
[0202] 3-week old BALB/c mice were challenged i.p. with
5.times.10.sup.4 pfu of MCMV (day 0). On day 6, mice were immunized
once with IFA alone, 89pep or p458-89pep emulsified in IFA, as
described above. Mice were treated with 100 .mu.g of the anti viral
medication Gancyclovir (GCV, Roche, Basel, Switzerland) i.p. on
days 1, 2, 3, 4, 8, 9, 10 and 11 after challenge. Mice were
sacrificed on day 30 after challenge and MCMV-DNA was measured by a
PCR assay in the salivary glands, as specified above (FIG.
14A).
[0203] As can be seen in FIG. 14B, immunization with p458-89pep was
able to suppress CMV load at the salivary gland even when applied
after CMV challenge and in combination with GCV. GCV treatment
alone did not suffice for therapy; also GCV treatment combined with
one immunization with the non-conjugated 89pep did not affect CMV
load. Only GCV treatment combined with one p458-89pep immunization
reduced viral load to undetectable levels.
B. WNV Vaccination
[0204] Materials and Methods
[0205] Mice
[0206] BALB/c female mice were purchased from Harlan Olac
(Jerusalem, Ill.) at the age of 14 days (10-12 g body weight). Mice
were maintained under specific pathogen free conditions and were
allowed to adjust to the facility for 1 week before experiments
were performed. Mice were used at the age of 3-6 weeks unless
otherwise stated. Age- and sex-matched animals were used as
controls. Mice were maintained in isolation cages and were fed and
watered ad libitum. The mouse experiments were approved and
performed according to the guidelines of the Ben Gurion University,
Faculty of Health Sciences, Animal Safety Committee.
[0207] Cell Cultures
[0208] The Vero cell line was derived from African Green Monkey
(ATCC.RTM. number: CCL-81). The cells were grown in DMEM
supplemented with 10% FCS, 1% nonessential amino acids and
antibiotics. The cells were maintained in a humidified atmosphere
at 37.degree. C. in 5% CO.sub.2 and were used for growing virus
stocks, virus titration and neutralization assays.
[0209] Virus, Virus Titrations and WNV Antigen
[0210] The strain of West Nile Virus (WNV) was isolated from a
human case of WNV infection (Goldblum et al., 1954). Signature
amino acid motifs indicate that this strain belongs to lineage I.
Virus plaque assays were performed on Vero cell monolayers in 24
well plates as previously described (Ben-Nathan et al., 1996).
Virus stock titers were expressed as plaque-forming units (pfu) per
ml. A single virus stock containing 5.times.10.sup.7 pfu/ml was
prepared in Vero cells, stored in aliquots at -70.degree. C., and
was used throughout this study. WNV antigen (WNV Ag) was prepared
as previously described (Ben-Nathan et al., 2003).
[0211] Peptides
[0212] Peptides were synthesized at Sigma-Aldrich (Rehovot,
Israel). Peptide purity was ascertained by analytical
reversed-phase HPLC and amino acid (aa) analysis and was assessed
on >95% purity. The sequences of the six peptides synthesized
are: Ep15 (derived from the E3 domain of WNV)--LVTVNPFVSVATANS, SEQ
ID NO:11; p458 (the active peptide derived from mouse
HSP60)--NEDQKIGIEIIKRALKI, SEQ ID NO:2; p32 (p458-Ep15
combined)--NEDQKIGIEIIKRALKILVTVNPFVSVATANS, SEQ ID NO:13; pmock, a
negative control for p458 (the p431 peptide of the mycobacterial
HSP60, 442val-deleted)--EGDEATGANIKVALEA, SEQ ID NO:7; p458-89pep
(combined p458 and 89pep, 89pep is a nonapeptide, YPHFHPTNL SEQ ID
NO:5, which consists of a MCMV pp 89 ie1-CTL
epitope)--NEDQKIGIEIIKRALKIYPHFHPTNL, SEQ ID NO:6. The
mycobacterial p431 peptide (442-val-deleted) was used as a negative
control peptide since it is homologous in sequence to mammalian
p458, but did not elicit a CD4.sup.+-dependent immune response or
antibodies against itself or p458.
Antibodies and Sera
[0213] Human intravenous immunoglobulin-IL (IVIG-IL): the IgG
preparation from Israeli donors (IVIG-IL; OMRIGAM 5% intravenous
IgG) containing 50 mg/ml IgG (total protein 5% w/v) was a gift from
Omrix Biopharmaceuticals Ltd, Israel. This product has an anti-WNV
antibody titer of 1:1600 by ELISA and of >1:80 by
plaque-reduction neutralization testing (PRNT) (Ben-Nathan et al.,
2003). Mouse WNV antiserum was prepared by intraperitoneal (IP)
injection of 5-week old BALB/c mice with 1.times.10.sup.4 pfu of
WNV per mouse. Two weeks later, surviving mice were boosted with
1.times.10.sup.4 pfu and bled 7 days later. Blood was centrifuged
(4000 rpm for 7 min), and serum was collected and stored at
-20.degree. C. The antibody titer, measured by ELISA, was 1:2400.
Serum from mock-injected naive mice was used as a negative
control.
[0214] Recognition of WNV-Ag and Peptides by IVIG and Mouse
Sera
[0215] ELISA tests were performed according to the method described
by Martin et al (Martin et al, 2000) with slight modifications.
Briefly, microtiter plates were coated and incubated overnight at
4.degree. C. with 100 .mu.l of the different peptides (1
.mu.g/well) or WNV antigen diluted 1:700 in coating buffer
(NaHCO.sub.3, pH=9.6). After incubation, the coating buffer was
decanted and the plates were washed twice in PBS containing 0.05%
Tween 20 and 0.2% sodium azide (washing buffer). After blocking for
1 h with a 200 .mu.l/well of PBS containing 0.05% Tween 20 and 2.5%
nonfat dry milk, the plates were washed 4 times in washing buffer
and 100 .mu.l of IVIG-IL or mouse sera at 1:40 dilution were added
to each well (2-4 wells per sample). Negative and positive controls
of human or mouse sera were tested in each plate. After incubation
for 1 h at 37.degree. C. in a humidified atmosphere, the plates
were washed 5 times, and 100 .mu.l of 1:1000 diluted
HRP-Streptavidin-conjugated anti-human IgG (Sigma-Aldrich) or
1:1000 diluted HRP-Streptavidin-conjugated anti-mouse IgG
(SouthernBiotech, Birmingham, Ala.) respectively, was added to each
well. After incubation at 37.degree. C. for 1 h, the plates were
washed 5 times and 100 .mu.l of TMB substrate (DAKO Carpinteria,
Calif.) was added to each well and incubated at room temperature
for 30 min. The color intensity was measured by ELISA-reader
(Dynatec MR 5000) at the absorbance of 405 nm.
[0216] Lymphocyte Proliferation Assay
[0217] Splenocytes from immunized or naive mice were prepared as
described above, resuspended in complete RPMI in a final
concentration of 2.times.10.sup.5 cells per well in 96-well plates
and stimulated in vitro with either 10 .mu.g/ml of different
peptides, 10 .mu.l/ml of WNV-Ag, or 5 .mu.g/ml of Concanavalin-A
(Con-A). Cell cultures were incubated at 37.degree. C. in 5%
CO.sub.2 for 5 days. At the end of incubation, 1 .mu.Ci of
.sup.3H-Thymidine (Amersham Biosciences, Buckinghamshire, England)
was added to each well for 12 h. Radioactive counting was performed
on a .beta.-counter (WALLAC 1409).
[0218] Immunization of Mice with Peptides and WNV Challenge
[0219] The immunizing dose of each peptide was equimolar to 15
.mu.g of p458 (Konen Waisman et al., 1999). All peptides were
emulsified in incomplete Freund's adjuvant (IFA), and injected at
50 .mu.l/mouse intrafootpad (IFP). Mice were immunized with the
different peptides 2-3 times according to the experimental
protocol. One week after the last immunization, mice were bled and
sacrificed. Spleens were harvested, splenocyte cultures prepared
and tested for T cell proliferation and cytokine secretion. Blood
samples were centrifuged (4000 rpm for 7 min), and then sera were
collected and tested for anti-WNV antibodies by ELISA and
neutralization assays.
[0220] To study the immunogenicity and protective efficacy of the
peptides, groups of 3-week old mice were immunized and boosted
according to the following protocol: mice were immunized IFP and
boosted once or twice, 1 week apart. One week after the last boost,
the mice were challenged IP with 1.times.10.sup.6 pfu of WNV.
Mortality was recorded for the next 21 days. For virology studies,
surviving and moribund mice were sacrificed on day 7 after the
challenge, and organs were harvested for virus titrations and
RT-PCR.
[0221] Virus Load in Brain Tissue of Infected Mice
[0222] Brain tissues were removed from infected or immunized and
challenged mice, and 10% (wt/vol) homogenates were prepared in
DMEM-10% DMSO. The homogenates were then aliquoted and stored at
-70.degree. C. until further analysis. Virus levels were determined
by plaque titration on Vero cell monolayers as previously described
(Ben-Nathan et al., 1996), and expressed as pfu/0.1 gr brain
tissue.
[0223] RNA Extraction and RT-PCR
[0224] RNA was extracted from brain tissues from mice using RNEasy
Midi Kit (QIAGEN, Hilden, Germany), and RT-PCR was performed on RNA
extracts using Endo-Free Reverse Transcriptase (Ambion, Huntingdon,
UK) and Biomix-Red (Bioline, London, UK). WNV E protein primers
(5'-ACGAAGTGGCCATTTTTGTC-3', SEQ ID
NO:81/5'-TTGATGCAGAGCTCCCTCTT-3', SEQ ID NO:82) were chosen using
Primer3 program (Whitehead Institute for Biomedical Research). PCR
products were electrophoretically separated on 1.5% agarose gel,
stained with ethidium bromide and imaged using a CCD camera
(Imagechem 5500).
[0225] Preparation of Spleen Cell Cultures and Cytokine
Analysis
[0226] Spleens of immunized mice were harvested, and spleen pulp
was extruded from the capsule in RPMI-1640 medium supplemented with
100 U/ml penicillin, 100 .mu.g/ml streptomycin, 2 mM glutamine, 10
mM HEPES and 5% FCS (base-RPMI). Spleen cell suspensions were
passed through a cell strainer, washed, treated for 2 min with ACK
lysing buffer (0.15M NH.sub.4Cl, 0.01 KHCO.sub.3) for elimination
of erythrocytes, and washed twice in base-medium. Splenocytes were
resuspended in RPMI-1640 medium supplemented with 100 U/ml
penicillin, 100 .mu.g/ml streptomycin, 2 mM glutamine, 10 mM HEPES,
5.times.10.sup.-5M .beta.-mercaptoethanol and 10% FCS (complete
RPMI) in a final concentration of 5.times.10.sup.6 cells/ml in a
24-well plates. Cell suspensions were stimulated in vitro with
either 10 .mu.g/ml of different peptides, 10 .mu.l/ml of WNV-Ag, or
5 .mu.g/ml of Con-A. The cells were then incubated at 37.degree. C.
in 5% CO.sub.2 for 72 h. Supernatants were collected, diluted by 2,
and IFN.gamma. and IL-4 concentrations were measured by indirect
ELISA method using commercial kits (PharMingen, San Diego, Calif.)
according to manufacturer instructions.
[0227] Virus Plaque Reduction Neutralization Testing (PRNT)
[0228] The titer of neutralizing antibodies was determined using a
modified plaque reduction test (PRNT). Briefly, serial 2 fold
dilutions (1:4 to 1:512) of mouse sera were prepared in 96-well
flat-bottom microtiter plates and 10.sup.4 pfu of WNV in equal
volumes was added to duplicate wells of each dilution. After 30 min
of incubation at room temperature, 5.times.10.sup.4 of Vero cells
were added to each well of the sera-virus mixtures. The plates were
incubated for 72 h in a humidified atmosphere at 37.degree. C. and
5% CO.sub.2 and plaques were counted. Plaque-reduction neutralizing
antibody titers were expressed as the reciprocal of the highest
dilution that gave 50% plaque reduction (PRNT.sub.50).
[0229] Isotypes of Anti-WNV Antibodies
[0230] Plates were coated with the WNV-Ag diluted 1:700 in coating
buffer. After overnight incubation at 4.degree. C., blocking and
washing, various dilutions of mouse sera were added in triplicates.
Negative and positive controls of mouse sera were tested in each
plate. Total IgG assessment was performed using
HRP-Streptavidin-conjugated anti-mouse IgG (SouthernBiotech). For
antibody isotype identification, incubation with biotin-conjugated
anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgM antibodies
(PharMingen, San Diego, Calif.) was followed by incubation with
1:1000 diluted HRP-Streptavidin (Jackson Laboratories, West Grove,
Pa.). After substrate addition the color intensity was measured by
ELISA-reader at 405 nm.
Example 7
Identification of a WNV Epitope
[0231] Based on the antigenic propensity method (Kolaskar et al.,
1990), calculated using a free B-cell epitope program (BcePred
Server, http://www.imtech.res.in/raghava/bcepred/index.html), and
based on a free MHC epitope program ProPred server,
http://www.imtech.res.in/raghava/propred), we have identified a p15
peptide from different WNV-E proteins (from the immunogenic E3
domain, aa LVTVNPFVSVATANS or aa LVTVNPFVSVATANA; SEQ ID NOS:11 and
12, respectively) as a candidate B-cell continuous epitope and
MHC-II-restricted epitope of the WNV (recognized by different human
and murine MHC-II molecules).
[0232] Next, it was examined whether the candidate epitope is
recognized by a serum immunoglobulin pool taken from Israeli donors
(IVIG-IL), which contains anti-WNV Abs. Wells were coated with the
different peptides (1 .mu.g/well) or with the WNV-Ag (1:700
dilution). After blocking and washing, IVIG-IL was added at 1:40
dilution and binding was detected. The results demonstrate for the
first time that the p15 peptide is recognized by IVIG-IL (FIG. 8).
Subsequently, mice were challenged IP with a sublethal dose of WNV
(10.sup.4 pfu) WNV and 14 days later surviving mice were
re-infected with the same dose of WNV. Sera were collected 7 days
after the second challenge, and ELISA assays were performed. As can
be seen in FIG. 9, serum from WNV-infected mice specifically
recognized p15, and, to a greater extent, the p15 conjugate p32. In
addition, T cells from these mice proliferated in response to in
vitro stimulation with the p15 conjugate p32 (FIG. 10). Thus, the
phenotype of p15 as WNV-B-cell epitope and WNV-MHC II-restricted
peptide was hereby demonstrated.
Example 8
Immunization with P458-p15 Elicits WNV Protection
[0233] We showed that HSP60-p458 (NEDQKIGIEIIKRALKI SEQ ID NO:2)
used as a carrier peptide for a MHC-I epitope of CMV enhanced
immune response to CMV and immunization with the chimeric peptide,
p458-CMV epitope, cleared MCMV from salivary glands of mice
challenged with the virus. In accordance we tested the
WNV-protective efficacy of p458-p15
(NEDQKIGIEIIKRALKILVTVNPFVSVATANS) chimeric peptide (termed as p32,
SEQ ID NO:13). Table 2 summarizes 5 different experiments;
immunization with p15 alone showed slight protective efficacy while
immunization with a mixture of p15 and p458 did not protect mice.
Nonetheless, when mice were immunized with p32, the chimeric
p458-p15 peptide, a high degree of protection against a fatal
challenge with virulent WNV was achieved.
TABLE-US-00003 TABLE 2 In vivo protective efficacy of p32
immunization against WNV challenge Treatment - Deaths/Total (%
deaths) No p458 p15 + treatment p15 (x3) p32 (x3) (x3) p458 (x3)
Mortality 22/32 8/15 2/21 4/7 5/7 (dead of total) (69%) (53%) (10%)
(57%) (71%)
[0234] Mice were immunized 3 times with the different peptides or a
peptide mix at 7 day intervals. Seven days after the 3rd
immunization mice were challenged with 10.sup.6 pfu of WNV and
survival was monitored for 21 days post challenge. Results are the
summary of 5 different experiments, performed in the same
conditions.
[0235] To investigate whether immunization with p32 resulted in
clearance of WNV, virus levels in the brain, which is the prime
target organ of this neurotropic virus, were examined.
[0236] Mice were immunized 3 times with p32 at 7 day intervals.
Seven days after the last immunization, immunized and non-immunized
mice were challenged IP with 10.sup.6 pfu of WNV. Seven days after
challenge, mice were sacrifice and different organs were extracted
(brain, lungs, heart, liver and spleen). Organs were taken also
from control naive mice (non-immunized and non-infected matched
mice). Viral loads were determined by RT-PCR for viral genome and
plaque assay for infectious virus titers (pfu/0.1 gr tissue).
RT-PCR results are representative from one experimental mouse while
infectious titers are the average for all experimental groups. *
Detection level of the plaque assay is <10.sup.1.
[0237] Virus levels were determined in the brain by RT-PCR and
plaque assay on day 7 after challenge (FIG. 15). Virus titers were
up to 4.times.108 pfu/ml in the non-immunized and challenged mice,
while in the naive and in the immunized and challenged group no
virus was detected (FIG. 15). These findings confirm that
vaccination of mice with the p32 peptide protects them against an
otherwise fatal WNV infection.
Example 9
Immunization with p458-p15 Induces WNV-Neutralizing Abs
[0238] We next investigated the immune responses elicited following
immunization with p32. Immunization with p32 induced high titer of
WNV-specific Abs as compared to immunization with p15 alone (FIG.
11A). The Ab generated following immunization with p32 exhibited
WNV-neutralizing capacity, essential for the protective effect of
the p32 vaccine (Table 3).
TABLE-US-00004 TABLE 3 Titers of anti-WNV neutralizing Abs in the
sera of p32-immunized or WNV-infected mice on day 7 after treatment
Source of serum Titers of anti-WNV neutralizing Abs Naive mice
<1:10 p32-immunized mice 1:80 WNV-infected mice 1:320
[0239] Mice were either immunized 3 times with p32 or challenged
with WNV. Seven days after the 3rd immunization or WNV challenge,
respectively, mice were bled and titers of WNV-neutralizing Abs in
sera were tested (PRNT.sub.50). Results are the average of 4
independent ELISA experiments.
[0240] Next, it was examined whether p32 immunization and sublethal
infection with WNV induce similar isotypes of anti-WNV antibodies.
WNV-Ag was used as the antigen in the ELISA plates. FIG. 1B shows
that levels of anti-WNV IgG1 and IgG2a isotypes were similarly high
in sera from p32-immunized and WNV-infected mice. WNV-specific
IgG2b levels were higher in sera from WNV-infected mice while
WNV-specific IgM were higher in p32-immunized mice. Neither IgA nor
IgG3 specific for WNV were detected in both types of sera (FIG.
11B).
Example 10
Immunization with p458-p15 Induces WNV-Specific T Cell Response and
IFN-.gamma. Secretion
[0241] WNV-specific T-cell responses were examined as follows:
Six-week old BALB/c mice were infected with a sublethal dose of WNV
(0.66.times.10.sup.2 pfu), and spleens were harvested 6 days later.
Spleen cell suspensions were prepared, cultured in 96 wells plates
and incubated for 3 days with the 10 .mu.g/ml of EP15, p32, p458 or
Con-A (5 .mu.g/ml) for 3 days. Cell proliferation was measured by
the WST-1 method and the results are the average of 3 independent
experiments. As shown in FIG. 12, splenocytes from mice immunized
with p32 proliferated in response to in vitro stimulation with WNV
antigen. This stimulation also induced secretion of high levels of
IFN.gamma., essential for TH1 response that characterizes
virus-protective immune responses (FIG. 13).
Example 11
Immunization with Conjugates of p458 and Ec27 with Viral
Antigens
[0242] Conjugates of p458 and Ec27 with the novel viral antigens of
the invention, having amino acid sequences as set forth in SEQ ID
NOS:13-14, 23, 56-75 and 77-79 (see Table 4 below), are synthesized
as described above.
TABLE-US-00005 Ec27 conjugate Virus P458 conjugate (SEQ ID NO):
(SEQ ID NO): WNV NEDQKIGIEIIKRALKILVTVNPFVSVATANS (13)
KKARVEDALHATRAAVEE NEDQKIGIEIIKRALKILVTVNPFVSVATANA (14)
GVGRLVTVNPFISTGGAN NEDQKIGIEIIKRALKIYIVVGRGEQQINHHWHK (23) NKVM
(77) NEDQKIGIEIIKRALKIGRLVTVNPFVSVATANS (65) KKARVEDALHATRAAVEE
NEDQKIGIEIIKRALKIGRLVTVNPFVSVATANA (66) GVLVTVNPFVSVATANA (78)
KKARVEDALHATRAAVEE GVYIVVGRGEQQINHHWH K (79) Yellow
NEDQKIGIEIIKRALKIGILVTVNPIASTNDDEVLIE (56) KKARVEDALHATRAAVEE fever
GVGILVTVNPIASTNDDE virus VLIE (67) St. Louis
NEDQKIGIEIIKRALKIGRLVTVNPFISTGGANNKVM (57) KKARVEDALHATRAAVEE
encephalitis GVGRLVTVNPFISTGGAN virus NKVM (68) Murray
NEDQKIGIEIIKRALKIGRMVTANPYVASSTANAKVL (58) KKARVEDALHATRAAVEE
Valley GVGRMVTANPYVASSTAN encephalitis AKVL (69) virus Kunjin
NEDQKIGIEIIKRALKIGRLVTVNPFVSVSTANAKVL (59) KKARVEDALHATRAAVEE virus
GVGRLVTVNPFVSVSTAN AKVL (70) Japanese
NEDQKIGIEIIKRALKIGRLVTVNPFVATSSANSKVL (60) KKARVEDALHATRAAVEE
encephalitis GVGRLVTVNPFVATSSAN virus SKVL (71) Dengue
NEDQKIGIEIIKRALKIGRLITANPIVTDKEKPVNIE (61) KKARVEDALHATRAAVEE virus
GVGRLITANPIVTDKEKP type 1 VNIE (72) Dengue
NEDQKIGIEIIKRALKIGRLITVNPIVTEKDSPVNIE (62) KKARVEDALHATRAAVEE virus
GVGRLTTVNPIVTEKDSP type 2 VNIE (73) Dengue
NEDQKIGIEIIKRALKIGRLITANPVVTKKEEPVNIE (63) KKARVEDALHATRAAVEE virus
GVGRLTTANPVVTKKEEP type 3 VNIE (74) Dengue
NEDQKIGIEIIKRALKIGRIISSTPLAENTNSVTNIE (64) KKARVEDALHATRAAVEE virus
GVGRIISSTPLAENTNSV type 4 TNIE (75)
[0243] Mice are immunized 3 times with the different peptides or
with control peptides (corresponding non-conjugated viral antigens
and with the carrier peptides alone) at 7 day intervals, as
described above. Seven days after the 3rd immunization mice are
challenged with 10.sup.6 pfu of the corresponding flavivirus and
survival is monitored for 21 days post challenge.
[0244] In other experiments, the immunogenicity of the conjugates
is determined by the following assays: in some experiments, mice
are immunized and assayed for serum titers of virus-specific and
viral peptide-specific antibodies by ELISA essays, as described
above. In other experiments, LNC from immunized mice are examined
in the presence of the corresponding viral antigen in proliferation
assays and IFN-.gamma. secretion assays, as described above.
Example 12
Use of Ec27-Antigen Conjugates for Increasing the Immunogenicity of
a Viral Antigen
[0245] Mice were immunized with the following peptides as described
in Example 8 using the following peptides: p15 (SEQ ID NO:11), p32
(SEQ ID NO:13), Ec27-p15 (KKARVEDALHATRAAVEEGVLVTVNPFVSVATANS, SEQ
ID NO:77), and Mock-p15 (EGDEATGANIKVALEALVTVNPFVSVATANS, SEQ ID
NO:80--the p431 peptide fused to p15).
[0246] Spleens were harvested 10 days after the immunization and
splenocytes were cultured with p15 (25 .mu.g/ml) for 3 days. Cell
proliferation and IFN.gamma. levels in the supernatants of spleen
cell cultures were measured. Results are the average of 3
independent proliferation experiments. Ec27-Ep15* and Ec27-Ep15**
are two identical but independent groups in the same
experiment.
[0247] As shown in FIG. 16, p458 and Ec27 were both able to
increase the immunogenicity of p15. Vaccination with conjugates
comprising either p458 or Ec27 fused to the N-terminus of p15
resulted in enhanced proliferation and IFN-.gamma. secretion of
spleen cells in the presence of p15, compared to those of spleen
cells derived from animals vaccinated with p15 conjugated to the
control peptide. Spleen cells from mice immunized by IFA alone or
unconjugated Ec27 did not induce significant proliferation and
IFN-.gamma. secretion when incubated with p15.
Example 13
Specific Recognition of the WNV Peptides by Sera Derived from
WNV-Exposed Subjects
[0248] Sera from WNV-exposed and non-exposed human subjects was
assayed for antibodies recognizing the novel WNV peptide antigens,
p15 (SEQ ID NO:11) and p17 (SEQ ID NO:21). FIG. 17 presents the
results of an ELISA test in which the p15 and p17 served as the
antigen, human sera were used as the primary antibody and
HRP-conjugated anti-human Abs were used as the secondary antibody.
RLU are the relative luminescence units produced by HRP activity on
the luminal substrate.
[0249] Sera S2 and S6 are from human that were not infected with
WNV while sera S1, S3-S5 and S7 are from human that had history of
WNV infection. IVIG is pool of plasmas from Israeli citizens that
was shown to contain antibodies against WNV.
[0250] As can be seen, both p15 and p17 were specifically
recognized by WNV-exposed sera and not by sera from unexposed
subjects. Human sera from Dengue-infected humans also did not
recognize the p15 (data not shown). Thus, p15 and p17 are suitable
diagnostic peptides useful for determining WNV exposure or
infection.
Example 14
Identification of Ec27, an Immunodominant Peptide Derived from E.
coli GroEL
[0251] To find dominant T helper cell epitopes derived from the
sequence of the E. coli variant of HSP60 (GroEL), BALB/c mice were
inoculated with heat-inactivated E. coli bacteria and the
proliferative responses of draining LNCs to a set of overlapping
GroEL peptides (Table 5), and the whole GroEL molecule (Purchased
from Sigma), were analyzed.
TABLE-US-00006 TABLE 5 Overlapping peptides of the E. coli HSP60
molecule (GroEL); amino acid designation is corresponding to
accession number gi: 45686198 without the first methionine residue,
SEQ ID NO: 83. Peptide Number Position 1 1-20 2 16-35 3 31-50 4
46-65 5 61-80 6 76-95 7 91-110 8 106-125 9 121-140 10 136-155 11
151-170 12 166-185 13 181-200 14 196-215 15 211-230 16 226-245 17
241-260 18 256-275 19 271-290 20 286-305 21 301-320 22 316-335 23
331-350 24 346-365 25 361-380 26 376-395 27 (Ec27) 391-410 28
406-425 29 421-440 30 436-455 31 451-470 32 466-485 33 481-500 34
496-515 35 511-530 36 526-545 37 526-547
[0252] BALB/c mice were immunized s.c. with 10.sup.7
glutaraldehyde-attenuated E. coli bacteria (FIG. 18A) in PBS, or
with 30 .mu.g GroEL in PBS (FIG. 18B). Ten days later lymph node
cells (2.times.10.sup.5 cells per well) were assessed for specific
proliferation to the indicated overlapping GroEL peptides (20
.mu.g/ml). After 96 hours of incubation, the .sup.3H-thymidine
incorporation was assessed as a measure of proliferation. Results
are shown as mean cpm of quadruplicate wells. The standard
deviations are indicated.
[0253] T-cell proliferation assays were performed as follows:
Draining lymph node cells (LNC) of immunized mice were cultured
(2.times.10.sup.5/well) in 200 .mu.l RPMI 1640 medium supplemented
with 2 mM glutamine, non-essential amino acids, 1 mM sodium
pyruvate, 100 U/ml penicillin, 100 .mu.g/ml streptomycin (BioLab,
Jerusalem, Israel), 5.times.10.sup.-5M .beta.-mercaptoethanol
(Fluka A G, Buchs, Switzerland), 10 mM HEPES buffer (Sigma), and 1%
syngeneic normal mouse serum. After four days of incubation,
[.sup.3H]-thymidine (0.5 .mu.Ci of 5 Ci/mmol, Nuclear Research
Center, Negev, Israel) was added for additional sixteen hours, and
the thymidine incorporation was measured. Results are expressed as
the mean cpm, or the stimulation index (SI), i.e. the mean cpm of
test cultures (with antigen) divided by the mean cpm of control
cultures (without antigen).
[0254] As can be seen in FIG. 18A, a peptide corresponding to amino
acid residues 391-410 of GroEL was highly immunogenic. Immunization
of BALB/c mice with the GroEL molecule instead of the whole E. coli
bacteria led to a similar proliferative response of draining LNC
(FIG. 18B) to the same GroEL peptides and the whole GroEL molecule
as used for FIG. 18A: both immunogens gave rise to an immune
response to the GroEL molecule and, predominantly, to the Ec27
peptide of GroEL.
[0255] It was also tested whether the Ec27 peptide itself is
immunogenic by immunization of BALB/c mice with 20 .mu.g of the
Ec27 peptide in IFA.
[0256] BALB/c mice were immunized s.c. with 20 .mu.g Ec27 peptide
emulsified in IFA. Ten days later lymph node cells were taken and
assessed for specific proliferation of 2.times.10.sup.5 cells in
the presence of the Ec27 peptide, the acetylcholine receptor
peptide 259-271 (AcR 259-271, VIVELIPSTSSAV SEQ ID NO:84), or GroEL
at the concentrations 10 .mu.g/ml, 2 .mu.g/ml, or without antigen
(BG). Results are shown as mean cpm of quadruplicate wells. The
standard deviations are indicated.
[0257] FIG. 19 shows that the lymph node cells of immunized mice
responded the Ec27 peptide and to the whole GroEL molecule, but not
to the control peptide AcR 259-271.
[0258] To learn whether the immunogenicity of the Ec27 peptide is
restricted to a certain MHC haplotype, we compared four strains
mice with different MHC haplotypes in their response to the Ec27
peptide. The immunized mice were of the BALB/c (H-2.sup.d), BALB/k
(H-2.sup.k), BALB/b (H-2.sup.b), or SJL (H-2.sup.s) strain. As can
be seen in FIG. 20, all four different mouse strains responded
specifically to the Ec27 immunogen peptide. Thus, the Ec27 peptide
could replace the GroEL, or the whole bacteria in priming for a
GroEL-specific immune response, due to its immuno-dominance.
Example 15
Use of the Ec27 Peptide as an Adjuvant for Antibody Responses
[0259] Because the Ec27 peptide was found to be an immunodominant
peptide of the GroEL molecule, which is the major immunogen of
bacteria, it was interesting to learn whether the Ec27 peptide can
be used as an adjuvant, as can the bacteria. Therefore, BALB/c and
C57BL/6 mice were immunized subcutaneously with 20 .mu.g of the
monoclonal anti-p53 antibody PAb-246 (described in Yewdell et al.,
1986) in different immunogenic compositions: the antibody was
either emulsified in Complete Freund's Adjuvant (246 in CFA), in
Incomplete Freund's Adjuvant (246 in IFA), or together with 50
.mu.g the Ec27 peptide (as a mixture) in IFA (246 in Ec27). Three
weeks later, all mice received a boost of the PAb-246 antibody
subcutaneously in IFA. Ten days after the boost, mice were bled and
their antibody responses to the PAb-246 immunogen were compared by
ELISA (FIG. 21A). Immunization of the PAb-246 antibody in IFA alone
did not result in a significant antibody response. In contrast,
immunization with the antibody in IFA mixed with the Ec27 peptide
resulted in an effective antibody response in both strains. The
adjuvant effect of the Ec27 peptide was comparable to the effect of
CFA, which is one of the most potent adjuvant materials known. FIG.
21B shows the induction of anti-p53 antibodies in the immunized
mice by ways of an idiotypic network, i.e. these anti-p53
antibodies are anti-idiotypic to the anti-PAb-246 antibodies.
Again, the use of the Ec27 peptide as an adjuvant was at least as
effective as the use of CFA.
[0260] Dots represent individual sera, bars indicate the median of
each group.
Example 16
Use of the ec27 Peptide as an Adjuvant for P458-polysacharride
Conjugates
[0261] S. pneumoniae serotype 4 capsular polysaccharide (PnTy4) was
obtained from the American Type Culture Collection (ATCC;
Rockville, Md., USA). PnTy4 was coupled to carrier peptides by
using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(CDI; Aldrich, Wis., USA) using standard procedure.
[0262] The peptide carriers used in this example are: p458 (SEQ ID
NO:2), p458s (SEQ ID NO:87, EGDIETGVNIVLKALTA), MOG (SEQ ID NO: 85,
MEVGWYRSPFSRVVHLYRNGK).
[0263] BALB/c mice were immunized with the indicated
peptide-polysaccharide conjugate in IFA or IFA mixed with 50 .mu.g
Ec27 as described above, in the WNV vaccination example. LNC were
collected and assayed for nitric oxide (NO) production and
proliferation.
[0264] Table 6 shows the levels of Nitric Oxide (NO) Production
(nM) in lymph node cells (LNC) derived from mice immunized with
peptide-sugar conjugates in IFA or in IFA+Ec27. The cells were
cultured for 72 h with either Concanavalin A (Con A) or the
peptide-sugar immunogen in the presence or absence of a macrophage
cell line (J774). NO production was assayed using a Nitrate/Nitrile
colorimetric assay according to the manufacturer's
instructions.
TABLE-US-00007 LNC LNC + J774 Immunogen BG Con A P458-Ty4 BG Con A
P458-Ty4 P458-Ty4/IFA 0.13 4.73 0.21 0.55 6.24 10.29 P458-Ty4/ec27
0.18 3.32 2.36 0.66 4.33 34.97 LNC LNC + J774 Immunogen BG Con A
P458s-Ty4 BG Con A P458s-Ty4 P458.sub.S-Ty4/IFA 0.13 9.91 0.55 0.93
6.35 0.66 P458.sub.S-Ty4/ 0.18 8.53 2.41 0.45 5.23 27.73 ec27
[0265] The proliferation of lymph node cells in response to
immunogen was performed as described above (Example 14).
[0266] Table 7 shows the proliferation of LNC derived from mice
immunized with peptide-sugar conjugates in IFA or in IFA+ec27. The
cells were cultured for 72 h with Concanavalin A (Con A), the
peptide-sugar immunogen (Ty4), the peptide immunogen without the
sugar, or the sugar conjugated to a control peptide (MOG-Ty4). The
proliferative response is given as the stimulation index (SI).
TABLE-US-00008 TABLE 7 proliferation of LNC derived from mice
immunized with peptide-sugar conjugates in IFA or in IFA + ec27.
Immunogen BG Con A P458-Ty4 P458 MOG-Ty4 P458-Ty4/IFA 1.0 2.3 4.7
2.9 2.7 P458-Ty4/ec27 1.0 6.5 15.4 3.1 4.0 Immunogen BG Con A
P458.sub.S-Ty4 P458.sub.S MOG-Ty4 P458.sub.S-Ty4/IFA 1.0 12.8 2.9
2.2 1.3 P458.sub.S-Ty4/ec27 1.0 15.3 8.7 2.1 6.5
[0267] In the tables, BG refers to background values; ConA refers
to concanavalin A; P458-Ty4 is a conjugate between the p458 peptide
and the S. pneumoniae serotype 4 capsular polysaccharide; P458s-Ty4
is a conjugate between the p458s peptide and the S. pneumoniae
serotype 4 capsular polysaccharide.
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"Solid Phase Peptide Synthesis," W. H. Freeman Co. (San Francisco).
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John Wiley and Sons, Baltimore, Md. 1989.
Sequence CWU 1
1
87117PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 1Asn Glu Asp Gln Lys
Ile Gly Ile Glu Ile Ile Lys Arg Thr Leu Lys1 5 10
15Ile217PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 2Asn Glu Asp Gln
Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10
15Ile317PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 3Glu Gly Asp Glu
Ala Thr Gly Ala Asn Ile Val Lys Val Ala Leu Glu1 5 10
15Ala417PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 4Asn Glu Asp Gln
Asn Val Gly Ile Lys Val Ala Leu Arg Ala Met Glu1 5 10
15Ala59PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 5Tyr Pro His Phe His
Pro Thr Asn Leu1 5626PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 6Asn
Glu Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10
15Ile Tyr Pro His Phe His Pro Thr Asn Leu 20 25716PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 7Glu Gly Asp Glu Ala Thr Gly Ala Asn Ile
Lys Val Ala Leu Glu Ala1 5 10 15825PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 8Glu Gly Asp Glu Ala Thr Gly Ala Asn Ile Lys Val Ala Leu
Glu Ala1 5 10 15Tyr Pro His Phe His Pro Thr Asn Leu 20
25930PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 9Phe Asn Asn Phe Thr
Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser1 5 10 15Ala Ser His Leu
Glu Tyr Pro His Phe Met Pro Thr Asn Leu 20 25 301021PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 10Phe Asn Asn Phe Thr Val Ser Phe Trp Leu
Arg Val Pro Lys Val Ser1 5 10 15Ala Ser His Leu Glu
201115PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 11Leu Val Thr Val Asn
Pro Phe Val Ser Val Ala Thr Ala Asn Ser1 5 10 151215PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 12Leu Val Thr Val Asn Pro Phe Val Ser Val
Ala Thr Ala Asn Ala1 5 10 151332PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 13Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala
Leu Lys1 5 10 15Ile Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr
Ala Asn Ser 20 25 301432PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE
14Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1
5 10 15Ile Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn
Ala 20 25 301532PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 15Asn Glu
Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Thr Leu Lys1 5 10 15Ile
Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ser 20 25
301632PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 16Asn Glu Asp Gln Lys
Ile Gly Ile Glu Ile Ile Lys Arg Thr Leu Lys1 5 10 15Ile Leu Val Thr
Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ala 20 25
301727DNAARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 17aagcagcaca
tccgcaccct gagcgcc 271827DNAARTIFICIAL SEQUENCESYNTHETIC PEPTIDE
18ccaggcgctc ccggcggccc gctctcg 271945DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 19ctggtgaccg tgaatccatt tgtgtctgtg
gccacagcca actcg 452045PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE
20Thr Thr Gly Gly Thr Cys Ala Cys Thr Gly Thr Cys Ala Ala Cys Cys1
5 10 15Cys Thr Thr Thr Thr Gly Thr Thr Thr Cys Ala Gly Thr Gly Gly
Cys 20 25 30Cys Ala Cys Gly Gly Cys Cys Ala Ala Cys Gly Cys Thr 35
40 452117PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 21Tyr Ile Val Val
Gly Arg Gly Glu Gln Gln Ile Asn His His Trp His1 5 10
15Lys2251DNAARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 22tatattgtgg
tgggccgcgg cgaacagcag attaaccatc attggcataa a 512334PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 23Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile
Ile Lys Arg Ala Leu Lys1 5 10 15Ile Tyr Ile Val Val Gly Arg Gly Glu
Gln Gln Ile Asn His His Trp 20 25 30His Lys2434PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 24Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile
Ile Lys Arg Thr Leu Lys1 5 10 15Ile Tyr Ile Val Val Gly Arg Gly Glu
Gln Gln Ile Asn His His Trp 20 25 30His Lys2518PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 25Leu Val Thr Val Asn Pro Ile Ala Ser Thr
Asn Asp Asp Glu Val Leu1 5 10 15Ile Glu2618PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 26Leu Val Thr Val Asn Pro Phe Ile Ser Thr
Gly Gly Ala Asn Asn Lys1 5 10 15Val Met2718PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 27Met Val Thr Ala Asn Pro Tyr Val Ala Ser
Ser Thr Ala Asn Ala Lys1 5 10 15Val Leu2818PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 28Leu Val Thr Val Asn Pro Phe Val Ser Val
Ser Thr Ala Asn Ala Lys1 5 10 15Val Leu2918PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 29Leu Val Thr Val Asn Pro Phe Val Ala Thr
Ser Ser Ala Asn Ser Lys1 5 10 15Val Leu3018PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 30Leu Ile Thr Ala Asn Pro Ile Val Thr Asp
Lys Glu Lys Pro Val Asn1 5 10 15Ile Glu3118PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 31Leu Ile Thr Val Asn Pro Ile Val Thr Glu
Lys Asp Ser Pro Val Asn1 5 10 15Ile Glu3218PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 32Leu Ile Thr Ala Asn Pro Val Val Thr Lys
Lys Glu Glu Pro Val Asn1 5 10 15Ile Glu3318PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 33Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn
Thr Asn Ser Val Thr Asn1 5 10 15Ile Glu3417PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 34Gly Arg Leu Val Thr Val Asn Pro Phe Val
Ser Val Ala Thr Ala Asn1 5 10 15Ser3517PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 35Gly Arg Leu Val Thr Val Asn Pro Phe Val
Ser Val Ala Thr Ala Asn1 5 10 15Ala3620PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 36Gly Ile Leu Val Thr Val Asn Pro Ile Ala
Ser Thr Asn Asp Asp Glu1 5 10 15Val Leu Ile Glu 203720PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 37Gly Arg Leu Val Thr Val Asn Pro Phe Ile
Ser Thr Gly Gly Ala Asn1 5 10 15Asn Lys Val Met 203820PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 38Gly Arg Met Val Thr Ala Asn Pro Tyr Val
Ala Ser Ser Thr Ala Asn1 5 10 15Ala Lys Val Leu 203920PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 39Gly Arg Leu Val Thr Val Asn Pro Phe Val
Ser Val Ser Thr Ala Asn1 5 10 15Ala Lys Val Leu 204020PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 40Gly Arg Leu Val Thr Val Asn Pro Phe Val
Ala Thr Ser Ser Ala Asn1 5 10 15Ser Lys Val Leu 204120PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 41Gly Arg Leu Ile Thr Ala Asn Pro Ile Val
Thr Asp Lys Glu Lys Pro1 5 10 15Val Asn Ile Glu 204220PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 42Gly Arg Leu Ile Thr Val Asn Pro Ile Val
Thr Glu Lys Asp Ser Pro1 5 10 15Val Asn Ile Glu 204320PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 43Gly Arg Leu Ile Thr Ala Asn Pro Val Val
Thr Lys Lys Glu Glu Pro1 5 10 15Val Asn Ile Glu 204420PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 44Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala
Glu Asn Thr Asn Ser Val1 5 10 15Thr Asn Ile Glu 204560DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 45ggcattctgg tgaccgtgaa cccgattgcg
agcaccaacg atgatgaagt gctgattgaa 604660DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 46ggccgcctgg tgaccgtgaa cccgtttatt
agcaccggcg gcgcgaacaa caaagtgatg 604760DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 47ggccgcatgg tgaccgcgaa cccgtatgtg
gcgagcagca ccgcgaacgc gaaagtgctg 604860DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 48ggccgcctgg tgaccgtgaa cccgtttgtg
agcgtgagca ccgcgaacgc gaaagtgctg 604960DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 49ggccgcctgg tgaccgtgaa cccgtttgtg
gcgaccagca gcgcgaacag caaagtgctg 605060DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 50ggccgcctga ttaccgcgaa cccgattgtg
accgataaag aaaaaccggt gaacattgaa 605160DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 51ggccgcctga ttaccgtgaa cccgattgtg
accgaaaaag atagcccggt gaacattgaa 605260DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 52ggccgcctga ttaccgcgaa cccggtggtg
accaaaaaag aagaaccggt gaacattgaa 605360DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 53ggccgcatta ttagcagcac cccgctggcg
gaaaacacca acagcgtgac caacattgaa 605451DNAARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 54ggcagactgg tgaccgtgaa tccatttgtg
tctgtggcca cagccaactc g 515551DNAARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 55ggcagattgg tcactgtcaa cccttttgtt tcagtggcca cggccaacgc t
515637PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 56Asn Glu Asp Gln Lys
Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10 15Ile Gly Ile Leu
Val Thr Val Asn Pro Ile Ala Ser Thr Asn Asp Asp 20 25 30Glu Val Leu
Ile Glu 355737PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 57Asn Glu Asp
Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10 15Ile Gly
Arg Leu Val Thr Val Asn Pro Phe Ile Ser Thr Gly Gly Ala 20 25 30Asn
Asn Lys Val Met 355837PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 58Asn
Glu Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10
15Ile Gly Arg Met Val Thr Ala Asn Pro Tyr Val Ala Ser Ser Thr Ala
20 25 30Asn Ala Lys Val Leu 355937PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 59Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala
Leu Lys1 5 10 15Ile Gly Arg Leu Val Thr Val Asn Pro Phe Val Ser Val
Ser Thr Ala 20 25 30Asn Ala Lys Val Leu 356037PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 60Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile
Ile Lys Arg Ala Leu Lys1 5 10 15Ile Gly Arg Leu Val Thr Val Asn Pro
Phe Val Ala Thr Ser Ser Ala 20 25 30Asn Ser Lys Val Leu
356137PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 61Asn Glu Asp Gln Lys
Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10 15Ile Gly Arg Leu
Ile Thr Ala Asn Pro Ile Val Thr Asp Lys Glu Lys 20 25 30Pro Val Asn
Ile Glu 356237PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 62Asn Glu Asp
Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10 15Ile Gly
Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu Lys Asp Ser 20 25 30Pro
Val Asn Ile Glu 356337PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 63Asn
Glu Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala Leu Lys1 5 10
15Ile Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu Glu
20 25 30Pro Val Asn Ile Glu 356437PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 64Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile Ile Lys Arg Ala
Leu Lys1 5 10 15Ile Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn
Thr Asn Ser 20 25 30Val Thr Asn Ile Glu 356534PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 65Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile
Ile Lys Arg Ala Leu Lys1 5 10 15Ile Gly Arg Leu Val Thr Val Asn Pro
Phe Val Ser Val Ala Thr Ala 20 25 30Asn Ser6634PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 66Asn Glu Asp Gln Lys Ile Gly Ile Glu Ile
Ile Lys Arg Ala Leu Lys1 5 10 15Ile Gly Arg Leu Val Thr Val Asn Pro
Phe Val Ser Val Ala Thr Ala 20 25 30Asn Ala6740PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 67Lys Lys Ala Arg Val Glu Asp Ala Leu His
Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val Gly Ile Leu Val Thr
Val Asn Pro Ile Ala Ser Thr 20 25 30Asn Asp Asp Glu Val Leu Ile Glu
35 406840PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 68Lys Lys Ala Arg
Val Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly
Val Gly Arg Leu Val Thr Val Asn Pro Phe Ile Ser Thr 20 25 30Gly Gly
Ala Asn Asn Lys Val Met 35 406940PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 69Lys Lys Ala Arg Val Glu Asp Ala Leu His Ala Thr Arg Ala
Ala Val1 5 10 15Glu Glu Gly Val Gly Arg Met Val Thr Ala Asn Pro Tyr
Val Ala Ser 20 25 30Ser Thr Ala Asn Ala Lys Val Leu 35
407040PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 70Lys Lys Ala Arg Val
Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val
Gly Arg Leu Val Thr Val Asn Pro Phe Val Ser Val 20 25 30Ser Thr Ala
Asn Ala Lys Val Leu 35 407140PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 71Lys Lys Ala Arg Val Glu Asp Ala Leu His Ala Thr Arg Ala
Ala Val1 5 10 15Glu Glu Gly Val Gly Arg Leu Val Thr Val Asn Pro Phe
Val Ala Thr 20 25 30Ser Ser Ala Asn Ser Lys Val Leu 35
407240PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 72Lys Lys Ala Arg Val
Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val
Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp 20 25 30Lys Glu Lys
Pro Val Asn Ile Glu 35 407340PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 73Lys Lys Ala Arg Val Glu Asp Ala Leu His Ala Thr Arg Ala
Ala Val1 5 10 15Glu Glu Gly Val Gly Arg Leu Ile Thr Val Asn Pro Ile
Val Thr Glu 20 25 30Lys Asp Ser Pro Val Asn Ile Glu 35
407440PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 74Lys Lys Ala Arg Val
Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val
Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys 20 25 30Lys Glu Glu
Pro Val Asn Ile Glu 35 407540PRTARTIFICIAL SEQUENCESYNTHETIC
PEPTIDE 75Lys Lys Ala Arg Val Glu Asp Ala Leu His Ala Thr Arg Ala
Ala Val1 5 10 15Glu Glu Gly Val Gly Arg Ile Ile Ser Ser Thr Pro Leu
Ala Glu Asn 20 25 30Thr Asn Ser Val Thr Asn Ile Glu 35
407620PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 76Lys Lys Ala Arg Val
Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val
207735PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 77Lys Lys Ala Arg Val
Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val
Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr 20 25 30Ala Asn Ser
357835PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 78Lys Lys Ala Arg Val
Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val
Leu Val Thr Val Asn Pro Phe Val Ser Val Ala Thr 20 25 30Ala Asn Ala
357937PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 79Lys Lys Ala Arg Val
Glu Asp Ala Leu His Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Val
Tyr Ile Val Val Gly Arg Gly Glu Gln Gln Ile Asn 20 25 30His His Trp
His Lys 358031PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 80Glu Gly Asp
Glu Ala Thr Gly Ala Asn Ile Lys Val Ala Leu Glu Ala1 5 10 15Leu Val
Thr Val Asn Pro Phe Val Ser Val Ala Thr Ala Asn Ser 20 25
308120PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 81Ala Cys Gly Ala Ala
Gly Thr Gly Gly Cys Cys
Ala Thr Thr Thr Thr1 5 10 15Thr Gly Thr Cys 208220PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 82Thr Thr Gly Ala Thr Gly Cys Ala Gly Ala
Gly Cys Thr Cys Cys Cys1 5 10 15Thr Cys Thr Thr
2083547PRTEscherichia coli 83Ala Ala Lys Asp Val Lys Phe Gly Asn
Asp Ala Arg Val Lys Met Leu1 5 10 15Arg Gly Val Asn Val Leu Ala Asp
Ala Val Lys Val Thr Leu Gly Pro 20 25 30Lys Gly Arg Asn Val Val Leu
Asp Lys Ser Phe Gly Ala Pro Thr Ile 35 40 45Thr Lys Asp Gly Val Ser
Val Ala Arg Glu Ile Glu Leu Glu Asp Lys 50 55 60Phe Glu Asn Met Gly
Ala Gln Met Val Lys Glu Val Ala Ser Lys Ala65 70 75 80Asn Asp Ala
Ala Gly Asp Gly Thr Thr Thr Ala Thr Val Leu Ala Gln 85 90 95Ala Ile
Ile Thr Glu Gly Leu Lys Ala Val Ala Ala Gly Met Asn Pro 100 105
110Met Asp Leu Lys Arg Gly Ile Asp Lys Ala Val Thr Val Ala Val Glu
115 120 125Glu Leu Lys Ala Leu Ser Val Pro Cys Ser Asp Ser Lys Ala
Ile Ala 130 135 140Gln Val Gly Thr Ile Ser Ala Asn Ser Asp Glu Thr
Val Gly Lys Leu145 150 155 160Ile Ala Glu Ala Met Asp Lys Val Gly
Lys Glu Gly Val Ile Thr Val 165 170 175Glu Asp Gly Thr Gly Leu Gln
Asp Glu Leu Asp Val Val Glu Gly Met 180 185 190Gln Phe Asp Arg Gly
Tyr Leu Ser Pro Tyr Phe Ile Asn Lys Pro Glu 195 200 205Thr Gly Ala
Val Glu Leu Glu Ser Pro Phe Ile Leu Leu Ala Asp Lys 210 215 220Lys
Ile Ser Asn Ile Arg Glu Met Leu Pro Val Leu Glu Ala Val Ala225 230
235 240Lys Ala Gly Lys Pro Leu Leu Ile Ile Ala Glu Asp Val Glu Gly
Glu 245 250 255Ala Leu Ala Thr Leu Val Val Asn Thr Met Arg Gly Ile
Val Lys Val 260 265 270Ala Ala Val Lys Ala Pro Gly Phe Gly Asp Arg
Arg Lys Ala Met Leu 275 280 285Gln Asp Ile Ala Thr Leu Thr Gly Gly
Thr Val Ile Ser Glu Glu Ile 290 295 300Gly Met Glu Leu Glu Lys Ala
Thr Leu Glu Asp Leu Gly Gln Ala Lys305 310 315 320Arg Val Val Ile
Asn Lys Asp Thr Thr Thr Ile Ile Asp Gly Val Gly 325 330 335Glu Glu
Ala Ala Ile Gln Gly Arg Val Ala Gln Ile Arg Gln Gln Ile 340 345
350Glu Glu Ala Thr Ser Asp Tyr Asp Arg Glu Lys Leu Gln Glu Arg Val
355 360 365Ala Lys Leu Ala Gly Gly Val Ala Val Ile Lys Val Gly Ala
Ala Thr 370 375 380Glu Val Glu Met Lys Glu Lys Lys Ala Arg Val Glu
Asp Ala Leu His385 390 395 400Ala Thr Arg Ala Ala Val Glu Glu Gly
Val Val Ala Gly Gly Gly Val 405 410 415Ala Leu Ile Arg Val Ala Ser
Lys Leu Ala Asp Leu Arg Gly Gln Asn 420 425 430Glu Asp Gln Asn Val
Gly Ile Lys Val Ala Leu Arg Ala Met Glu Ala 435 440 445Pro Leu Arg
Gln Ile Val Leu Asn Cys Gly Glu Glu Pro Ser Val Val 450 455 460Ala
Asn Thr Val Lys Gly Gly Asp Gly Asn Tyr Gly Tyr Asn Ala Ala465 470
475 480Thr Glu Glu Tyr Gly Asn Met Ile Asp Met Gly Ile Leu Asp Pro
Thr 485 490 495Lys Val Thr Arg Ser Ala Leu Gln Tyr Ala Ala Ser Val
Ala Gly Leu 500 505 510Met Ile Thr Thr Glu Cys Met Val Thr Asp Leu
Pro Lys Asn Asp Ala 515 520 525Ala Asp Leu Gly Ala Ala Gly Gly Met
Gly Gly Met Gly Gly Met Gly 530 535 540Gly Met
Met5458413PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 84Val Ile Val Glu
Leu Ile Pro Ser Thr Ser Ser Ala Val1 5 108521PRTARTIFICIAL
SEQUENCESYNTHETIC PEPTIDE 85Met Glu Val Gly Trp Tyr Arg Ser Pro Phe
Ser Arg Val Val His Leu1 5 10 15Tyr Arg Asn Gly Lys
208620PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 86Lys Lys Asp Arg Val
Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val1 5 10 15Glu Glu Gly Ile
208717PRTARTIFICIAL SEQUENCESYNTHETIC PEPTIDE 87Glu Gly Asp Ile Glu
Thr Gly Val Asn Ile Val Leu Lys Ala Leu Thr1 5 10 15Ala
* * * * *
References