U.S. patent application number 15/408485 was filed with the patent office on 2017-09-14 for modified vp1-capsid protein of parvovirus b19.
The applicant listed for this patent is Michael Broker, Torsten Lowin, Markus Mobs, SUSANNE MODROW. Invention is credited to Michael Broker, Torsten Lowin, Markus Mobs, SUSANNE MODROW.
Application Number | 20170260239 15/408485 |
Document ID | / |
Family ID | 35809798 |
Filed Date | 2017-09-14 |
United States Patent
Application |
20170260239 |
Kind Code |
A1 |
MODROW; SUSANNE ; et
al. |
September 14, 2017 |
MODIFIED VP1-CAPSID PROTEIN OF PARVOVIRUS B19
Abstract
The inventions relates to a modified VP1-capsid protein of
parvovirus B19 having reduced phospholipase A2-like enzyme activity
as compared to the wild type VP1-capsid protein of parvovirus B19
having the amino acid sequence of SEQ ID NO: 1.
Inventors: |
MODROW; SUSANNE;
(Altenthann, DE) ; Lowin; Torsten; (Regensburg,
DE) ; Mobs; Markus; (Berlin, DE) ; Broker;
Michael; (Marburg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MODROW; SUSANNE
Lowin; Torsten
Mobs; Markus
Broker; Michael |
Altenthann
Regensburg
Berlin
Marburg |
|
DE
DE
DE
DE |
|
|
Family ID: |
35809798 |
Appl. No.: |
15/408485 |
Filed: |
January 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11575951 |
Mar 23, 2007 |
9573979 |
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PCT/EP2005/054771 |
Sep 23, 2005 |
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15408485 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 14/005 20130101; C12N 2750/14222 20130101; A61K 38/00
20130101; A61K 38/162 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; A61K 38/16 20060101 A61K038/16 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 24, 2004 |
EP |
04450180.7 |
Claims
1.-41. (canceled)
42. A modified VP1-capsid protein of parvovirus B19, wherein the
wild type sequence of the VP-1 capsid protein has been modified to
contain amino acid substitutions at positions corresponding to
histidine 153 of SEQ ID NO:1, tyrosine 157 of SEQ ID NO:1, lysine
162 of SEQ ID NO:1, and tyrosine 168 of SEQ ID NO:1, wherein said
substitutions are histidine 153 to alanine, tyrosine 157 to
phenylalanine, lysine 162 to leucine, and tyrosine 168 to
phenylalanine, and wherein said modified VP1-capsid protein has a
reduced phospholipase A2 enzyme activity as compared to the wild
type VP1-capsid protein.
43. A method of treating parvovirus B19 infection comprising
administering to a human subject a pharmaceutical composition
comprising: (a) an adjuvant, and (b) a modified VP1-capsid protein
of parvovirus B19 wherein the wild type sequence of the VP-1 capsid
protein has been modified to contain amino acid substitutions at
positions corresponding to histidine 153 of SEQ ID NO:1, tyrosine
157 of SEQ ID NO:1, lysine 162 of SEQ ID NO:1, and tyrosine 168 of
SEQ ID NO:1, wherein said substitutions are histidine 153 to
alanine, tyrosine 157 to phenylalanine, lysine 162 to leucine, and
tyrosine 168 to phenylalanine, and wherein said modified VP1-capsid
protein has a reduced phospholipase A2 enzyme activity as compared
to the wild type VP1-capsid protein.
44. The method of claim 43, wherein said adjuvant is an
immunostimulatory substance selected from the group consisting of
an immunostimulatory deoxynucleotide (ODN), a peptide containing at
least two LysLeuLys motifs, a neuroactive compound, alum, Freund's
complete adjuvant and Freund's incomplete adjuvant.
45. The method of claim 43, further comprising a polycationic
peptide.
46. The method of claim 43, further comprising a VP2-capsid
protein.
47. The method of claim 46, wherein the modified VP1-capsid protein
is fused to the VP2-capsid protein.
48. A method of inducing an immune response against parvovirus B19
infection in a human, comprising administering to a human subject a
pharmaceutical composition comprising: (a) an adjuvant, and (b) a
modified VP1-capsid protein of parvovirus B19 wherein the wild type
sequence of the VP-1 capsid protein has been modified to contain
amino acid substitutions at positions corresponding to histidine
153 of SEQ ID NO:1, tyrosine 157 of SEQ ID NO:1, lysine 162 of SEQ
ID NO:1, and tyrosine 168 of SEQ ID NO:1, wherein said
substitutions are histidine 153 to alanine, tyrosine 157 to
phenylalanine, lysine 162 to leucine, and tyrosine 168 to
phenylalanine, and wherein said modified VP1-capsid protein has a
reduced phospholipase A2 enzyme activity as compared to the wild
type VP1-capsid protein.
49. The method of claim 48, wherein said immune response provides
protection against human parvovirus B19 infection.
50. The method of claim 48, wherein said adjuvant is an
immunostimulatory substance selected from the group consisting of
an immunostimulatory deoxynucleotide (ODN), a peptide containing at
least two LysLeuLys motifs, a neuroactive compound, alum, Freund's
complete adjuvant and Freund's incomplete adjuvant.
51. The method of claim 48, further comprising a polycationic
peptide.
52. The method of claim 48, further comprising a VP2-capsid
protein.
53. The method of claim 52, wherein the modified VP1-capsid protein
is fused to the VP2-capsid protein.
Description
[0001] The present invention relates to modified Parvovirus B19
capsid proteins and particles, their uses and the preparation of
medicaments including vaccines for the treatment of parvoviurs B19
associated diseases.
[0002] Parvovirus B19 was discovered in 1975 as cause of systemic
infections of adults who either were asymptomatic or had mild non
specific symptoms such as headache, pyrexia, malaise, fatigue, and
myalgia (1, 2, 3). In 1984 parvovirus B19 was identified as the
etiological agent of erythema infectiosum, also known as fifth
disease (4). The virus is spread worldwide but restricted
exclusively to human hosts. With respect to regional differences
about 40 to 60 percent of the population displays antibodies
against viral proteins at an age of 20 years. The infection is
endemic, however reaches regional outbreaks during late winter and
spring time. The virus is very stable. In general it is transmitted
orally from individuals with acute infection (5). Beside this major
route of infection the virus may be transmitted through
parenterally administered blood and blood derived products and
vertically from mother to fetus (6, 7, 8, 9). Massive productive
replication of parvovirus B19 takes place in erythroid progenitor
cells (10). Therefore during acute infection the virus load is
extremely high and up to 1013 particles and/or virus genomes may be
present per milliliter of the peripheral blood. The virus binds to
erythrocyte precursor cells using the blood group antigen P
(globoside) as the cellular receptor for adsorption (11). After the
infection and elimination from the peripheral blood viral genomes
have been shown to be present in cells of bone marrow and skin, in
synovial cells, in tissues from liver and in the endothelium of the
myocard (12, 13, 14, 15, 16, 17). At present it is not clear if
these cells produce viral proteins and/or infectious B19 particles
and if the virus genome can be reactivated to productive
replication.
[0003] In common parvovirus B19 infection is without clinical
symptoms or may result in a flu like disease. Particularly children
may suffer from erythema infectiosum (fifth disease), a self
limiting rash illness (18, 19, 20). Parvovirus B19 has been further
associated with a wide spectrum of diseases (Table 1): Transient
anemia, leucocytopenia or thrombocytopenia may occur without
requiring any therapy. However, in some patients severe
thrombocytopenia, pure red cell aplasia or pancytopenia were
observed. Besides these hematologic sequelae of acute infection
hepatitis, myocarditis, myositis, acute lung injury and
neurological disease may occur occasionally (12, 21, 22, 23, 24).
In pregnant women spontaneous abortion, non-immune hydrops fetalis
and intrauterine fetal death have been reported as clinical
manifestations (25, 26). In non immune pregnant women with acute
infection a fetal death rate of about 9% has been found (9, 27).
However there are also studies which describe a higher percentage
for the developlment of hydrops fetalis in B19-infected, non-immune
pregnant women (28). Depending on the hematologic status of the
host; e.g. patients with sickle cell anemia or thalassemia, B19
infection results in aplastic crisis. Persistent parvovirus B19
infection has been reported in patients both with and without
underlying immunodeficiencies (29, 30).
[0004] Beside these general manifestations parvovirus B19 infection
may induce a broad spectrum of autoimmune phenomena. Autoimmune
cytopenias are well known hematological disorders that may affect
any bone-marrow cell lineage. In the mass majority of cases only
one of the cell lines is affected. However, concurrent
autoimmune-mediated destruction of neutrophil granulocytes and
thrombocytes due to persistent parvovirus B19 infection is known
(31). Parvovirus B19 has been identified as a possible trigger in
some cases of immune thrombocytopenia (32, 33, 34, 35). The
clinical spectrum of autoimmune joint disease ranges from mild
arthralgias to severe necrotizing vasculitis (Table 1). In some
outbreaks of erythema infectiosum, arthralgias and arthritis have
been commonly reported. B19-infection may produce a symmetrical
peripheral polyarthropathy in adults. Symptoms are usually
self-limited but may persist for several months. Occasionally
viremia combined with IgM/IgG-antibodies against structural
proteins VP1 and/or VP2 has been shown in patients with long
lasting polyarthralgia/polyarthritis. Some of them met the criteria
of rheumatoid arthritis (RA) (26, 37). However development of RA
after acute parvovirus infection seems to be rare (38, 39). In
children the relation of the B19-infection with
arthritis-arthralgias is well known. Some of the affected children
may develop chronic arthritis indistinguishable from juvenile
idiopathic arthritis (40, 41, 42, 43). The clinical spectrum
encompasses mono-, oligo- and polyarthritis. Furthermore persisting
viremia is frequently observed in children with systemic juvenile
idiopathic arthritis. In children with various forms of juvenile
arthritis B19-DNA was amplified in 57% of serum and/or synovial
fluid from those with previous parvovirus B19 infection. In many
patients the continuous presence of viral particles and
immunocomplexes could be demonstrated in the peripheral blood as
well as in synovial fluid (44). Despite the development and
presence of B19-specific immunoreaction the children were incapable
of eliminating the virus and showed a prolonged state of viremia or
viral persistence in synovial fluid.
[0005] Parvovirus B19 infection has been associated with various
forms of vasculitis, collagenosis and may mimic systemic lupus
erythematosus (SLE) in children and adults (39, 45, 46). Similar to
the situation in arthritis patients also in SLE-patients parvovirus
B19 infection has been described both as the agent causing or
triggering the rheumatic disease (47, 48, 49, 50, 51). Recently, an
association between persistent parvovirus B19 infection and the
production of anti-phospholipid antibodies in pediatric and adult
patients with rheumatic disease was described (52). The
antiphospholipid syndrome (APS) (53) is, like some parvovirus B19
infections, characterized by a wide variety of hemocytopenic and
vaso-occlusive manifestations. Additionally recurrent fetal loss
and the association with autoantibodies directed against negatively
charged phospholipids and protein co-factors, mainly
.beta.2-glycoprotein-I are important features of the APS. The
hypothesis regarding a common pathogenicity of the
anti-phospholipid syndrome and the autoimmunity features observed
in B19-infection is supported not only by the close association
between the presence of anti-phospholipid antibodies and parvovirus
B19 infection but also by the similarity in the presenting of
clinical symptoms in patients with parvovirus 319 infection and
patients with the APS.
[0006] The icosahedral capsid of parvovirus B19 consist of two
structural proteins, VP1 (83 kDa) and VP2 (58 kDa), which are
identical except for 227 amino acids (aa) at the amino-terminal end
of the VP1 protein (the VP1 unique region). A phospholipase A2
motif is present in the amino acid sequence of the VP1 unique
region spanning positions 130 to 195 (Phospholipase A2 active
center) (54). One of the pathogenic mechanisms involved in
triggering the production of anti-phospholipid antibodies might be
the phospholipase-A2-like activity observed in the VP1-unique
region of the structural protein VP1 of parvovirus B19 (54). This
enzyme activity is present in infectious B19-particles, in
recombinant empty capsids consisting of VP1/VP2-proteins and in
preparations of purified VP1-unique region. It may contribute to
the inflammatory processes induced by the production of
leukotrienes and prostaglandines, but may also lead to the
generation of unusualnatural cleavage products from cellular
phospholipid compounds that may induce aPL-antibodies in
combination with a distinct genetic background.
[0007] Until now a vaccine to prevent parvovirus B19 infection is
not available. With respect to the severe courses that are observed
in association with the infection, the problems associated with the
infection in pregnant women and the virus' potential to induce a
wide variety of autoimmune diseases the development of a safe
vaccine protecting parvovirus B19 infections Purified recombinant
empty capsids consisting of VP1/VP2-proteins expressed by
baculovirus vectors have been used in a phase I trial and showed
the successful induction of B19-neutralizing antibodies in
volunteers (55). The application of this vaccine was, however
combined with a variety of side effects. Besides swelling at the
injections side, more general signs of ill feeling as fever, head
ache and diarrhea were reported by more than 50 percent of the
volunteers. These side effects indicate that the application of the
vaccine results in systemic manifestation of side effects that are
known to be associated with the activation of basic defense
reactions associated with inflammation. During early immune
reactions following e.g. infections these effects are mainly
associated with the activation of cellular and cytosolic
phospholipases A2. These enzymes are responsible for the production
of arachodonic acid as precursor for prostaglandin and leukotriene
production, the induction of various cyto- and chemokines leading
to fever and general ill feeling. The side effects observed by
Ballou and coworkers using VP1/VP2 empty capsids produced by
recombinant baculovirus (55) is likely due to the
phospholipase-A2-like activity which is part of the VP1-unique
region of the viral capsid protein VP1.
[0008] The problem underlining the present inventions is to provide
medicaments such as a parvovirus B19 vaccine for the prevention
and/or treatment of parvovirus B19 associated diseases with minimal
side effects.
[0009] This problem is addressed by the present invention through
advantageously alternating/modifying the VP1-capsid protein of
parvovirus B19 thus reducing the phospholipase-A2-like activity of
the VP1-unique region of the viral capsid protein VP1.
[0010] According to one aspect of the present invention, there is
provided a modified VP1-capsid protein of parvovirus B19 having a
reduced phospholipase A2-like enzyme activity as compared to the
wild type VP1-capsid protein of parvovirus B19 having the amino
acid sequence of Seq.ID No 1.
[0011] According to the present invention, the natural
phospholipase activity is reduced (which includes complete
abolition of the activity). Therefore, regions of the protein which
are involved in reduction of this phospholipase activity can be
altered according to the present invention. These changes compared
to wild type sequence may be introduced by amino acid exchanges,
deletions or insertions. Reduction of phospholipase activity can
easily be assessed by the skilled man in the art e.g. by relying on
the methodology disclosed herein, especially according to the test
described in FIG. 2 of Dorsch et al, 2002 (54). On the other hand,
the changes should not significantly alter the immunological
performance of the vaccine (i.e. in that the immunogenic properties
should not be adversly affected by the mutation).
[0012] According to a preferred embodiment of the present
invention, the amino acid residues in the VP1-unique region of the
VP1-capsid protein (amino acids 100-220, specifically 130 to 195)
are altered. This region is highly conserved in all three genotypes
of parvovirus B19 that have been identified until today (60; FIG.
2). However, although obviously this region is conserved,
differences in the activity of different parvovirus B19 genotypes
can be observed: Genotype 1: 100%, Genotype 2: 70%, genotype 3:
59%. Reference to wild type activity means that the closest
genotype is selected for comparison of wild type mutant (e.g. if a
mutant of genotype 2 is made, phospholipase activity of the mutant
is compared to wild type of genotype 2).
[0013] In general, whole VP1 protein may be subject to the
introduction of mutations for reducing or eliminating phospholipase
A2-like activity of this protein. However, since mutations outside
the VP1 unique region (amino acids 100-220, specifically 130 to
195) have usually a low, if any, reduction potential on the
phospholipase activity, mutations inside this VP1 unique region are
preferred according to the present invention. Nevertheless, also in
the region outside the VP1 unique region mutations according to the
present invention may be introduced, e.g. by amino truncation or by
mutations in the region of amino acid 50-90, especially 59-81.
Whether a mutation fulfills the expected reduction of phospholipase
activity can easily be addressed by the combination of the
expression of the mutant with a functional activity assay. Such
phospholipase activity tests are well available in the art and also
described herein.
[0014] Specifically preferred sites of mutation are around the
Ca.sup.2+ binding amino acids (Tyr(130), Gly(132), Gly(134),
Asp(154)) or around the catalytic network (His(153), Tyr(157),
Tyr(168)), including the phospholipid binding (Lys(162)), i.e.
amino acids 125-140 and 150-175, especially 127-137, 152-160 and
165 to 171 (see also SeqID No. 1 and FIG. 2 for sequences).
[0015] Preferred exchanges are the exchanges usually used in enzyme
activity reducing mutation strategies, e.g. replacing an amino acid
with a functional side chain (His, Lys, Tyr, Asp, Glu, Ser, Thr,
Asn, Gln, etc.) with an amino acid with a non functional side chain
of the same or different size (Ala, Phe, Leu, Pro, Ile, etc.) or
with a different functional side chain (e.g. Asn.fwdarw.Asp,
Gln.fwdarw.Ser, etc.). Sometimes, also a slight difference is
sufficient (Val.fwdarw.Ala, Ser.fwdarw.Thr, etc.).
[0016] According to another preferred embodiment of the present
invention, the altered amino acid residues reside in the
phospholipase A2 center of the VP1-unique region of the VP1-capsid
protein.
[0017] According to a further preferred embodiment of the present
invention, one or more of the conserved amino acid residues in the
active centre of the phospholipase A2 are altered. Amino acid
residues at position 153: histidine; 157: tyrosine; 162: lysine;
168: tyrosine; 195: aspartic acid have been described as parts of
the catalytic triad of bovine pancreatic phospholipase A2 and to be
involved in substrate orientation and specificity (58, 59).
Modification of one or more of the residues of 153: histidine, 157:
tyrosine, 162: lysine, 168: tyrosine result in an even more
pronounced reduction of phospholipase A2-like activity, whereas
modifying and alternating the residue of 195: aspartic acid result
in an enhanced phospholipase A2-like activity.
[0018] Thus according to another preferred embodiment of the
present invention, one or more of the amino acid residues are
altered at position 153: histidine; position 157: tyrosine;
position 162: lysine; and/or position 168: tyrosine.
[0019] According to yet another preferred embodiment, histidine 153
is altered into alanine; tyrosine 157 is altered into
phenylalanine; lysine 162 is altered into leucine and/or tyrosine
168 is altered into phenylalanine.
[0020] In general, the mutation according to the present invention
should be as minimal as possible (in order not to alter immunogenic
properties too much): usually a low number of amino acid
substitutions (6, 5, 4, 3, 2 or, preferably, 1) and a short
insertion or deletion (30, 20, 10, 5 or 1 amino acids) should be
sufficient to efficiently reduce or remove phospholipase activity.
However longer insertions, deletion (50, 100, 200 amino acids) or a
higher number of substitutions are not excluded, as long as the
immunogenic properties are not significantly changed. However, the
latter, multiple mutations are also less preferred due to cost
aspects.
[0021] Whether the mutants have similar immunogenic properties or
not can easily be determined e.g. by reacting with specific
antibodies or by determining in vitro or in vivo ability to induce
immune responses. Generally, the mutants should exhibit (with or
without specific adjuvants) about 50% of in vivo immunogenicity as
the wild type (without adjuvants, or preferably, with the same
adjuvant as the mutant), determined by at least one scientifically
applied and (by peer review) accepted method, e.g. antibody
binding, ELISpot assays, etc. (see also: example section of the
present application).
[0022] According to the present invention, the modified VP1-unique
region or the modified VP1-capsid protein of the present invention
results in a reduced phospholipase A2-like enzyme activity as
compared to the wild type VP1-capsid protein. Preferably, the
reduction is at least 30% compared to the wild type (i.e. 70% or
less of wild type activity). Further preferred is a reduction to
50% or less, 30% or less, 20% or less, or 10% or less of the wild
type activity. Reference activity of wild type and mutant are
preferably determined according to a standardized phospholipase
A2-like enzyme activity test, especially according to FIG. 2 of
(54).
[0023] Especially, those mutants which completely lack this
activity (i.e. below 5%, below 1% or below 0.1%, depending on the
detection limits of the method) are most suitable for vaccine use
and safety according to the present invention.
[0024] Preferably, the alternation of the amino acid residues is
made by site-directed mutagenesis.
[0025] According to another aspect of the present invention, there
is provided an isolated nucleic acid molecule encoding the modified
VP1-capsid protein provided by the present invention.
[0026] According to a further aspect of the present invention,
there is provided a recombinant expression vector comprising the
nucleic acid molecules encoding the modified VP1-unique region and
the modified VP1-capsid protein provided by the present
invention.
[0027] Preferably, the recombinant expression vector according to
the present invention further comprises VP2-capsid protein of
parvovirus B19 especially wherein the protein is being expressable
together with the modified VP-1 capsid. However, also the combined
expression of the VP1 and VP2 proteins using two vectors that are
introduced into the cells has its advantages for specific
purposes.
[0028] More preferably, the recombinant expression vector according
to the present invention comprises a fusion product in that the
modified VP1-unique region or the modified VP1-capsid protein is
fused to the VP2-capsid protein.
[0029] According to another aspect of the present invention, there
is provided a host cell comprising the recombinant expression
vector according to the present invention.
[0030] Preferably, the host cell is E. coli, yeast, or an animal
cell.
[0031] Most preferably the host cell is Saccharomyces cerevisiae.
This species of yeast has been used for two decades to produce
recombinant HBsAg-particles that protect against hepatitis B virus
infection. The use of recombinant S. cerevisiae derived HBV
vaccines has been shown to be safe, side effects are observed only
very rarely.
[0032] According to another aspect of the present invention, there
is provided a process of producing the modified VP1-unique region
or the modified VP1-capsid protein or the fusion product of the
modified VP1-capsid protein and VP2-capsid protein by transforming
a host cell, expressing the VP1-capsid (and optionally the
VP2-capsid), recovering the protein(s), optionally as virus-like
particles, using the host cell according to the present
invention.
[0033] Preferably in such process the modified VP1-unique region
and/or the modified VP1-capsid protein and/or the fusion product of
the modified VP1-capsid protein and VP2-capsid protein are isolated
and/or purified.
[0034] According to another aspect of the present invention, there
is provided the modified VP1-unique region, and/or the modified
VP1-capsid protein or the fusion product of the modified VP1-unique
region and VP2-capsid protein obtainable by the process according
to the present invention.
[0035] According to yet another aspect of the present invention,
there is provided the modified VP1-unique region and/or the
modified VP1-protein or the fusion product of the modified
VP1-unique region and/or the modified VP1-protein and VP2-capsid
protein for use as medicaments.
[0036] According to a further aspect of the present invention,
there is provided the use of the modified VP1-unique region, the
modified VP1-protein with or without VP2-capsid protein for the
manufacture of a medicament for the treatment against parvovirus
B19 infection and/or parvovirus B19 associated autoimmune and
rheumatic diseases.
[0037] According to still a further aspect of the present
invention, there is provided the use of the fusion product of the
modified VP1-unique region, the modified VP1-protein and VP2 capsid
protein for the manufacture of a medicament for the treatment
against parvovirus B19 infection and/or parvovirus B19 associated
autoimmune and rheumatic diseases.
[0038] Preferably the treatment is against Arthralgias; Arthritis;
more preferably Monoarthritis, Oligoarthritis, Polyarthritis,
Rheumatoid arthritis and/or Juvenile idiopathic arthritis; Systemic
lupus erythematosus (SLE); Vasculitis, more preferably Leukoclastic
vasculitis, Purpura Henlein-Schoenoch, Papular-purpuric
gloves-and-socks syndrome (PPGSS), Kawasaki disease, Giant cell
arteritis (GCA), Polyarteritis nodosa and/or Wegener's
granulomatosis; Dermatomyositis; Autoimmune neutropenia; Autoimmune
thrombocytpenia; Idiopathic thrombocytopenic purpura (ITP);
Autoimmune hemolytic anemia; and/or Virus-associated hemophagocytic
syndrome (VAHS).
[0039] According to another aspect of the present invention, there
is provided the use of the modified VP1-unique region, the modified
VP1 capsid protein or the fusion product of the modified VP1-unique
region and VP2-capsid protein according to the present invention in
an assay for detecting antibodies directed against the B19 virus
protein VP1 in a sample to be tested.
[0040] According to a further aspect of the present invention,
there is provided the use of the host cells of the present
invention in an assay for detecting antibodies directed against the
B19 virus protein VP1 in a sample to be tested.
[0041] According to another aspect of the present invention, there
is provided the recombinant virus-like particles consisting of the
modified VP1-capsid protein according to the present invention with
or without VP2-capsid protein.
[0042] According to yet another aspect of the present invention,
there is provided the recombinant virus-like particles consisting
of the fusion product of the modified VP1-unique region, the
modified VP1-capsid protein and VP2-capsid protein according to the
present invention.
[0043] According to another aspect of the present invention, those
recombinant virus-like particles can be used for the manufacture of
a medicament for the treatment against parvovirus B19 infection
and/or parvovirus B19 associated autoimmune and rheumatic
diseases.
[0044] The treatment is preferably against Arthralgias; Arthritis;
more preferably Monoarthritis, Oligoarthritis, Polyarthritis,
Rheumatoid arthritis and/or Juvenile idiopathic arthritis; Systemic
lupus erythematosus (SLE); Vasculitis, more preferably Leukoelastic
vasculitis, Purpura Henlein-Schoenoch, Papular-purpuric
gloves-and-socks syndrome (PPGSS), Kawasaki disease, Giant cell
arteritis (GCA), Polyarteritis nodosa and/or Wegener's
granulomatosis; Dermatomyositis; Autoimmune neutropenia; Autoimmune
thrombocytpenia; Idiopathic thrombocytopenic purpura (ITP);
Autoimmune hemolytic anemia; and/or Virus-associated hemophagocytic
syndrome (VAHS).
[0045] According to another aspect of the present invention, there
is provided the use of the recombinant virus-like particles
according to the present invention in an assay for detecting
antibodies directed against the B19 virus protein VP1 in a sample
to be tested.
[0046] According to a further aspect of the present invention,
there is provided a pharmaceutical composition, especially a
vaccine preparation for inducing an immune response which provides
protection against the human parvovirus B19, comprising the
modified VP1-unique region, the modified VP1-capsid protein
according to the present invention, with or without the VP2-capsid
protein.
[0047] According to another aspect of the present invention, there
is provided a pharmaceutical composition, especially a vaccine
preparation for inducing an immune response which provides
protection against the human parvovirus B19, comprising the fusion
product of the modified VP1-capsid protein according to the present
invention.
[0048] According to still another aspect of the present invention,
there is provided a pharmaceutical composition, especially a
vaccine preparation for inducing an immune response which provides
protection against the human parvovirus B19, comprising the
recombinant virus-like particles according to the present
invention.
[0049] Preferably, the pharmaceutical compositions according to the
present invention are further comprising one or more carriers
and/or adjuvants suitable for vaccination purposes.
[0050] Therefore, the compositions provided by the present
invention, especially in the form of a vaccine, may further
comprise an immunostimulatory substance, preferably selected from
the group comprising a polycationic compound, preferably a
polycationic polymer, more preferably a polycationic peptide,
especially polyarginine, polylysine or an antimicrobial peptide.
polymers; immunostimulatory deoxynucleotides (ODNs); peptides
containing at least two LysLeuLys motifs; neuroactive compounds,
especially human growth hormone; alumn, Freund's complete or
incomplete adjuvants or combinations thereof.
[0051] The present vaccine preferably comprises [0052] an modified
VP1 capsid protein according to the present invention as antigen
[0053] a peptide comprising a sequence
R.sub.1--XZXZ.sub.NXZX--R.sub.2, whereby N is a whole number
between 3 and 7, preferably 5, X is a positively charged natural
and/or non-natural amino acid residue, Z is an amino acid residue
selected from the group consisting of L, V, I, F and/or W, and
R.sub.1 and R.sub.2 are selected independently one from the other
from the group consisting of --H, --NH.sub.2, --COCH.sub.3, --COH,
a peptide with up to 20 amino acid residues or a peptide reactive
group or a peptide linker with or without a peptide; X--R.sub.2 may
be an amide, ester or thioester of the C-terminal amino acid
residue of the peptide (in the following also referred to as
"Peptide A") and/or [0054] an immunostimulatory oligodeoxynucleic
acid molecule (ODN) having the structure according to the formula
(I)
##STR00001##
[0054] wherein R1 is selected from hypoxanthine and uracile,
any X is O or S,
[0055] any NMP is a 2' deoxynucleoside monophosphate or
monothiophosphate, selected from the group consisting of
deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-,
deoxyuridine-, deoxythymidine-, 2-methyl-deoxyinosine-,
5-methyl-deoxycytosine, deoxypseudouridine-, deoxyribosepurine-,
2-amino-deoxyribosepurine-, 6-S-deoxyguanine-,
2-dimethyldeoxyguanosine- or
N-isopentenyl-deoxyadenosine-monophosphate or -monothiophosphate,
NUC is a 2' deoxynucleoside, selected from the group consisting of
deoxyadenosine-, deoxyguanosine-, deoxyinosine-, deoxycytosine-,
deoxyinosine-, deoxythymidine-, 2-methyl-deoxyuridine-,
5-methyl-deoxycytosine-, deoxypseudouridine-, deoxyribosepurine,
2-amino-deoxyribosepurine-, 6-S-deoxyguanine-,
2-dimethyldeoxyguanosine- or N-isopentenyl-deoxyadenosine, a and b
are integers from 0 to 100 with the proviso that a+b is between 4
and 150, and B and E are common groups for 5' or 3' ends of nucleic
acid molecules (in the following also referred to as
"I-/U-ODN").
[0056] Of course, the present vaccine may further contain other
substances, e.g. suitable pharmaceutically acceptable diluents or
carrier, buffer or stabilising substances, etc.
[0057] The vaccine according to the present invention may also
contain other or additional adjuvants, especially an Al(OH).sub.3
adjuvant (Alum).
[0058] Alum, as meant herein includes all forms of Al.sup.3+ based
adjuvants used in human and animal medicine and research.
Especially, it includes all forms of aluminum hydroxide as defined
in Rompp, 10th Ed. pages 139/140, gel forms thereof, aluminum
phosphate, etc.
[0059] The polycationic peptides or compound to be used according
to the present invention may be any polycationic compound which
shows the characteristic effect according to the WO 97/30721.
Preferred polycationic compounds are selected from basic
polypeptides, organic polycations, basic polyaminoacids or mixtures
thereof. These polyaminoacids should have a chain length of at
least 4 amino acid residues. Especially preferred are substances
containing peptidic bounds, like polylysine, polyarginine and
polypeptides containing more than 20%, especially more than 50% of
basic amino acids in a range of more than 8, especially more than
20, amino acid residues or mixtures thereof. Other preferred
polycations and their pharmaceutical compositions are described in
WO 97/30721 (e.g. polyethyleneimine) and WO 99/38528. Preferably
these polypeptides contain between 20 and 500 amino acid residues,
especially between 30 and 200 residues.
[0060] These polycationic compounds may be produced chemically or
recombinantly or may be derived from natural sources.
[0061] Cationic (poly)peptides may also be polycationic
anti-bacterial microbial peptides. These (poly)peptides may be of
prokaryotic or eukaryotic origin or may be produced chemically or
recombinantly. Peptides may also belong to the class naturally
occurring antimicrobial peptides. Such host defense peptides or
defensives are also a preferred form of the polycationic polymer
according to the present invention. Generally, a compound allowing
as an end product activation (or down-regulation) of the adaptive
immune system, preferably mediated by APCs (including dendritic
cells) is used as polycationic polymer.
[0062] Furthermore, also neuroactive compounds, such as (human)
growth hormone (as described e.g. in WO01/24822) may be used as
immunostimulants (Immunizers).
[0063] Polycationic compounds derived from natural sources include
HIV-REV or HIV-TAT (derived cationic peptides, antennapedia
peptides, chitosan or other derivatives of chitin) or other
peptides derived from these peptides or proteins by biochemical or
recombinant production. Other preferred polycationic compounds are
cathelin or related or derived substances from cathelicidin,
especially mouse, bovine or especially human cathelicidins and/or
cathelicidins. Related or derived cathelicidin substances contain
the whole or parts of the cathelicidin sequence with at least 15-20
amino acid residues. Derivations may include the substitution or
modification of the natural amino acids by amino acids which are
not among the 20 standard amino acids. Moreover, further cationic
residues may be introduced into such cathelicidin molecules. These
cathelicidin molecules are preferred to be combined with the
antigen/vaccine composition according to the present invention.
However, these cathelin molecules surprisingly have turned out to
be also effective as an adjuvant for a antigen without the addition
of further adjuvants. It is therefore possible to use such
cathelicidin molecules as efficient adjuvants in vaccine
formulations with or without further immunactivating
substances.
[0064] The vaccine according to the present invention preferably
contains as Peptide A KLKL.sub.5KLK and/or as I-/U-ODN oligo
d(IC).sub.13 (The combination of Peptide A and Oligo-d(IC).sub.13
is also referred as IC31). These two substances are specifically
advantageous according to the present invention.
[0065] The vaccine according to the present invention may contain
an oligodeoxynucleotide containing a CpG-motif as immunomodulating
nucleic acids. The immunomodulating nucleic acids to be used
according to the present invention can be of synthetic, prokaryotic
and eukaryotic origin. In the case of eukaryotic origin, DNA should
be derived from, based on the phylogenetic tree, less developed
species (e.g. insects, but also others). In a preferred embodiment
of the invention the immunogenic oligodeoxynucleotide (ODN) is a
synthetically produced DNA-molecule or mixtures of such molecules.
Derivates or modifications of ODNs such as thiophosphate
substituted analogues (thiophosphate residues substitute for
phosphate) as for example described in U.S. Pat. No. 5,723,335 and
U.S. Pat. No. 5,663,153, and other derivatives and modifications,
which preferably stabilize the immunostimulatory composition(s) but
do not change their immunological properties, are also included. A
preferred sequence motif is a six base DNA motif containing an
(unmethylated) CpG dinucleotide flanked by two 5' purines and two
3' pyrimidines (5'-Pur-Pur-C-G-Pyr-Pyr-3'). The CpG motifs
contained in the ODNs according to the present invention are more
common in microbial than higher vertebrate DNA and display
differences in the pattern of methylation. Surprisingly, sequences
stimulating mouse APCs are not very efficient for human cells.
Preferred palindromic or non-palindromic ODNs to be used according
to the present invention are disclosed e.g. in Austrian Patent
applications A 1973/2000, A 805/2001, EP 0 468 520 A2, WO 96/02555,
WO 98/16247, WO 98/18810, WO 98/37919, WO 98/40100, WO 98/52581, WO
98/52962, WO 99/51259 and WO 99/56755 all incorporated herein by
reference. ODNs/DNAs may be produced chemically or recombinantly or
may be derived from natural sources. Preferred natural sources are
insects.
[0066] The vaccine according to the present invention may
preferably contain a polycationic peptide and an
oligodeoxynucleotide containing a CpG-motif in combination. The
combination of CpG-ODN and polycationic peptide has improvement
effects in vaccine compositions, which are comparable to the
effects of the combination of Peptide A and I-/U-ODNs and cannot
only be combined with Peptide A and I-/U-ODNs but even be used
instead of them. Of course, also mixtures of different
immunostimulatory nucleic acids (I-/U-ODNs, CpG-ODNs, . . . ) and
Peptide A variants (as well as other Immunizers) may be used
according to the present invention.
[0067] It has been shown previously (WO 02/13857) that naturally
occurring, cathelicidin-derived antimicrobial peptides or
derivatives thereof have an immune response stimulating activity
and therefore constitute highly effective type 1 inducing adjuvants
(Immunizers). Main sources of antimicrobial peptides are granules
of neutrophils and epithelial cells lining the respiratory,
gastro-intestinal and genitourinary tracts. In general they are
found at those anatomical sites most exposed to microbial invasion,
are secreted into internal body fluids or stored in cytoplasmic
granules of professional phagocytes (neutrophils).
[0068] In the WO 02/32451 a type 1 inducing adjuvant (Immunizer)
that is able to strongly enhance the immune response to a specific
co-administered antigen and therefore constitutes a highly
effective adjuvant is disclosed, Peptide A comprising a sequence
R.sub.1--XZXZ.sub.NXZX--R.sub.2. A specifically preferred peptide
is KLKLLLLLKLK. Besides naturally occurring antimicrobial peptides,
synthetic antimicrobial peptides have been produced and
investigated. The synthetic antimicrobial peptide
KLKLLLLLKLK-NH.sub.2 was shown to have significant chemotherapeutic
activity in Staphylococcus aureus-infected mice; human neutrophils
were activated to produce the superoxide anion (O.sub.2.sup.-) via
cell surface calreticulin. The exact number and position of K and L
was found to be critical for the antimicrobial activity of the
synthetic peptide.
[0069] The present invention is especially beneficial if the
vaccine is administered subcutaneously, intramusculary,
intradermally or transdermally. However, other application forms,
such as parenteral, intravenously, intranasally, oral or topical
application, are also suitable for the present invention.
[0070] The Parvovirus antigen according to the present invention
may be mixed with an adjuvant (Immunizer) (composition) or
otherwise specifically formulated e.g. as liposome, retard
formulation, etc.
[0071] The vaccines according to the present invention may be
administered to an individual in effective amounts known to the
skilled man in the art. Optimisation of antigen amount and
Immunizer amount can be started from established amounts and using
available methods.
[0072] According to another aspect of the present invention, there
is provided a diagnostic kit comprising a modified VP1-unique
region, a modified VP1-capsid protein according to the present
invention and ancillary reagents.
[0073] According to still another aspect of the present invention,
there is provided a diagnostic kit comprising a fusion product of
the modified VP1-capsid protein according to the present invention
and ancillary reagents.
[0074] According to a further aspect of the present invention,
there is provided a diagnostic kit comprising a recombinant
virus-like particles according to the present invention and
ancillary reagents.
[0075] According to another aspect of the present invention, there
is provided the use of the modified VP1-unique region, the modified
VP1-capsid protein according to the present invention with or
without VP2-capsid protein as an agent to modify the activity of
host phospholipase A2 activity, e.g. by gene therapy on using
antisense KNA or RNAi.
[0076] According to another aspect of the present invention, there
is provided the use of the fusion product of the modified
VP1-capsid protein according to the present invention as an agent
to modify the activity of host phospholipase A2 activity.
[0077] According to a further aspect of the present invention,
there is provided the use of the recombinant virus-like particles
according to the present invention as an agent to modify the
activity of host phospholipase A2 activity.
[0078] The present invention is further illustrated by the
following examples and the figures, from which further features,
embodiments and advantages may be taken. It is to be understood
that the present examples are given by way of illustration only and
not by way of limitation of the disclosure.
[0079] FIG. 1 shows an immunological comparison of wt and mutant
protein
[0080] FIG. 2 shows an alignment of Parvovirus VP1 protein.
EXAMPLE 1
[0081] The production of VP1/VP2 antigens without enzymatic
activity can be achieved by alteration of the residues that are
part of the active centre by site-directed mutagenesis. Despite the
fact that the overall size of cellular enzymes with
Ca.sup.2+-dependent phospholipase A2 activity and the viral
VP1-unique region are different, alignments comparing the amino
acid sequences revealed a number of conserved residues in the
region that represents the active centre of the enzyme. Conserved
amino acids were observed at the following positions:
[0082] Residue 153: histidine; residue 157: tyrosine; residue 162:
lysine; residue 168: tyrosine; residue 195: aspartic acid. The
respective amino acids have been described as parts of the
catalytic triad of bovine pancreatic phospholipase A2 and to be
involved in substrate orientation and specificity. Therefore the
alteration of these residues in the VP1-unique region by
site-directed mutagenesis was performed using polymerase chain
reaction with mutated primer and overlap extension as initially
described by Ho and coworkers (56). As shown in table 2 the
phospholipase A2-like activity of the VP1-unique region could be
reduced by exchanging both the tyrosine 157 and 168 to
phenylalanine and by the alteration of lysine 162 to leucine.
However the exchange of aspartic acid 195 to alanine led to an
unexpected enhancement of the activity of the viral enzyme
indicating distinct differences between viral and cellular
phospholipase A2 enzymes. An almost total destruction of the
enzymatic activity could only be achieved by the alteration of
histidine 153 to alanine. It can be concluded that this amino acid
residue is part of the active centre and most important for the
enzymatic activity of the VP1-unique region. Its alteration is
associated with the complete destruction of the viral phospholipase
A2-like activity.
TABLE-US-00001 TABLE 1 Autoimmune diseases that are reported in
association to parvovirus B19 infection. Involved organs Disease
Joints Arthralgias Arthritis Monoarthritis Oligoarthritis
Polyarthritis Rheumatoid arthritis Juvenile idiopathic arthritis
Connective Systemic lupus erythematosus (SLE) tissue/vessels
Vasculitis Leukoclastic vasculitis Purpura Henlein-Schoenoch
Papular-purpuric gloves-and-socks syndrome (PPGSS) Kawasaki
disease? Giant cell arteritis (GCA) Polyarteritis nodosa Wegener's
granulomatosis Dermatomyositis Blood cells Autoimmune neutropenia
Autoimmune thrombocytpenia Idiopathic thrombocytopenic purpura
(ITP) Autoimmune hemolytic anemia Virus-associated hemophagocytic
syndrome (VAHS)
TABLE-US-00002 TABLE 2 Enzmyatic phopholipase A2-like activity in
the VP1-unique region of parvovirus B19 and variants constructed by
site-directed mutagenesis. Position altered by site-directed
mutagenesis enzyme acitivty (%) wildtype, genotype 1 100 active
center mutants histidine 153 .fwdarw. alanine 0 tyrosine 157
.fwdarw. phenylalanine 10 lysine 162 .fwdarw. leucine 61 tyrosine
168 .fwdarw. phenylalanine 54 aspartic acid 195 .fwdarw. alanine
204 not-active center mutants leucine 76 .fwdarw. glutamine,
phenylalanine 81 .fwdarw. alanine 80 isoleucine 66 .fwdarw.
leucine, leucine 70 .fwdarw. glutamine 144 leucine 59 .fwdarw.
glutamine, leucine 62 .fwdarw. glutamine 143 variations in
non-conserved regions parvovirus B19, VP1-unique region genotype
2/strain Berlin 70 ala18.fwdarw.asp, gln21.fwdarw.lys,
asn68.fwdarw.ser, asn72.fwdarw.asp, ser73.fwdarw.thr,
ser96.fwdarw.pro, ala101.fwdarw.thr, val123.fwdarw.ile,
val192.fwdarw.ala parvovirus B19, VP1-unique region, genotype
3/strain V9 59 lys4.fwdarw.thr, ser5.fwdarw.thr, gly6.fwdarw.asn,
asp12.fwdarw.ser, lys17.fwdarw.gln, ala18.fwdarw.asp,
gln21.fwdarw.1ys, glu25.fwdarw.gln, val30.fwdarw.ala,
asn68.fwdarw.ser, ser98.fwdarw.asn, his100.fwdarw.ser ,
val123.fwdarw.ile, ser144.fwdarw.asn, val192.fwdarw.ala
EXAMPLE 2
[0083] Comparison Study for the Immunogenicity between wild type
and mutant of VP1 proteins.
Inoculation of Mice.
[0084] Groups of 5 female Balc/C mice were inoculated with 50 ug of
purified preparations of the VP1-unique region/wildtype and the
VP1-unique region/H153A in emulsion with complete's Freund's
Adjuvans. Retrobulbar blood samples were taken at days 0, 3, 7, 10,
14, 18 and 28 after inoculation. The sera were tested for the
presence of IgG antibodies against the VP1-unique region/wildtype
in ELISA assays.
Protein Production.
[0085] The sequences encoding the VP1-unique region/wildtype and
the VP1-unique region/H153A were cloned into the T7-expression
vector pET21a_int in fusion with an intein and a chitin-binding
domain as described previously (Dorsch et al., 2001, Dorsch et al.,
2002). The constructs were introduced into the E. coli strain BL21.
Bacteria were inoculated with LB (luria broth) medium containing
100 .mu.g/ml ampicillin and incubated at 37.degree. C. Expression
of the recombinant protein was induced by addition of 1 mM IPTG for
at least 3 h of culture. The bacteria were harvested by
centrifugation, resuspended in 30 ml 20 mM HEPES, 1 mM EDTA, 100 mM
NaCl, pH8.5 and lysed by the use of a French Press. The debris was
pelleted at 10000 g. The supernatant was loaded on a chitin column
(NEB) using FPLC-system (Pharmacia Biosystems, Freiburg). The
column was washed with 2 volumes of 20 mM HEPES, 1 mM EDTA, 100 mM
NaCl, pH8.5, 8 volumes of 20 mM HEPES, 1 mM EDTA, 2 mM NaCl, pH8.5
and 2 volumes of 20 mM HEPES, 1 mM EDTA, 100 mM NaCl, pH8.5.
Afterwards the protein was eluted using 3 volumes of 50 mM DTT in
buffer 20 mM HEPES, 1 mM EDTA, 100 mM NaCl, pH8.5. Fractions were
tested for the recombinant proteins by SDS-PAGE and silver
staining. Positive fractions were unified and concentrated by using
a Centriplus concentrator (3 kD exclusion volume; Amicon, Beverly,
USA). The protein concentration was determined after dialysis
against PBS (0.9 mM KH.sub.2PO.sub.4, 8.0 mM
Na.sub.2HPO.sub.2.times.12H.sub.2O, 2.7 mM KCl, 137 mM NaCl) using
a Bradford assay (Bio Rad Laboratories, Hercules, USA).
ELISA-Assay.
[0086] Microtiter plates (Maxisorb, Nunc, Wiesbaden, FRG) were
coated over night with purified protein (VP1-unique
region/wildtype, 100 ng/well) in 0.2 M NaCO.sub.3 buffer, pH 9.2
containing 0.15 M NaCl. Sera were used in dilutions of 1:100 in
PBS/0.5% Tween-20 and IgG-antibodies were detected using
HRP-coupled polyclonal rabbit anti-mouse IgG as second antibodies
(ditution 1:5000 in PBS/0.5% Tween-20, Dako, Hamburg FRG) and TMB
as substrate, the optical density was determined at 450 nm.
Results
[0087] Starting from day 7 after inoculation IgG antibodies
directed against the VP1-unique region/wildtype were detectable in
mice that had been inoculated both with purified preparations of
the VP1-unique region/wildtype and the VP1-unique region/H153A
(FIG. 1). The amounts of antibodies continually increased until day
28 after inoculation. Differences in the reactivity of mice
inoculated with either the VP1-unique region/wildtype or the
VP1-unique region/H153A could not be observed. These results
indicate that both proteins are highly antigenic. Antibodies
induced against the variant VP1-unique region/H153A have the
capacity to bind to the VP1-unique region/wildtype which had used
as antigen in the ELISA indicating an high degree of cross
reactivity. Mice that were inoculated with PBS in emulsion with
complete Freund's adjuvans did not develop any significant amounts
of VP1-specific antibodies.
[0088] The wildtype VP1-unique region antigen and the mutant
antigen (His153A1a) were inoculated in mice. VP1-specific antibody
production was analysed by ELISA. No differences were observed in
the capacity of both antigens to elicit VP1-specific antibody
production. Antibodies against the mutant antigen His153Ala were
similarly active to bind to the wildtype VP1-unique region and vice
versa. This indicates that the mutant His153Ala variant of the
VP1-unique region of parvovirus B19 has a comparable capacity to
elicit the production of antibodies as the wildtype protein domain.
Since the main neutralizing epitopes are known to be located in
protein parts different from the active centre of the viral
phospholipase A2-like enzyme and were not affects by any of the
introduced mutations effects on the protein's immunogenicity are
unlikely (57).
[0089] The combination of both approaches--the production
VP1/VP2-capsids in recombinant S. cerevisiae and the destruction of
the phospholipase A2-like activity--has the potential to produce a
vaccine that allows the prevention of parvovirus B19 infection
without a reduced number of side effects due to elevated
leukotriene and prostaglandin production and without the dangerous
potential to induce autoimmune reactions that may result in
life-long rheumatic disease.
Legend
[0090] FIG. 1. The development of IgG-antibodies against the
VP1-unique region/wildtype. Groups of 5 mice were inoculated with
either the VP1-unique region/wildtype, the VP1-unique region/H153A
or PBS. Serum samples taken at days after inoculation as indicated
were tested in ELISA using the VP1-unique region as antigen.
Average values obtained from testing the individual samples of 5
mice were determined in each case.
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76, 2014-2018.
Sequence CWU 1
1
91781PRTParvovirus B19 1Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser
Asp Asp Lys Phe Ala 1 5 10 15 Lys Ala Val Tyr Gln Gln Phe Val Glu
Phe Tyr Glu Lys Val Thr Gly 20 25 30 Thr Asp Leu Glu Leu Ile Gln
Ile Leu Lys Asp His Tyr Asn Ile Ser 35 40 45 Leu Asp Asn Pro Leu
Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala 50 55 60 Arg Ile Lys
Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His 65 70 75 80 Phe
Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser 85 90
95 Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser
100 105 110 Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro
Gly Thr 115 120 125 Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly
Pro Pro Gln Ser 130 135 140 Ala Val Asp Ser Ala Ala Arg Ile His Asp
Phe Arg Tyr Ser Gln Leu 145 150 155 160 Ala Lys Leu Gly Ile Asn Pro
Tyr Thr His Trp Thr Val Ala Asp Glu 165 170 175 Glu Leu Leu Lys Asn
Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val 180 185 190 Val Lys Asp
Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His 195 200 205 Phe
Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys 210 215
220 Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala
225 230 235 240 Gly Gly Gly Gly Ser Asn Ser Val Lys Ser Met Trp Ser
Glu Gly Ala 245 250 255 Thr Phe Ser Ala Asn Ser Val Thr Cys Thr Phe
Ser Arg Gln Phe Leu 260 265 270 Ile Pro Tyr Asp Pro Glu His His Tyr
Lys Val Phe Ser Pro Ala Ala 275 280 285 Ser Ser Cys His Asn Ala Ser
Gly Lys Glu Ala Lys Val Cys Thr Ile 290 295 300 Ser Pro Ile Met Gly
Tyr Ser Thr Pro Trp Arg Tyr Leu Asp Phe Asn 305 310 315 320 Ala Leu
Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln His Leu Ile Glu 325 330 335
Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu Thr Val Thr Ile Ser Glu 340
345 350 Ile Ala Val Lys Asp Val Thr Asp Lys Thr Gly Gly Gly Val Gln
Val 355 360 365 Thr Asp Ser Thr Thr Gly Arg Leu Cys Met Leu Val Asp
His Glu Tyr 370 375 380 Lys Tyr Pro Tyr Val Leu Gly Gln Gly Gln Asp
Thr Leu Ala Pro Glu 385 390 395 400 Leu Pro Ile Trp Val Tyr Phe Pro
Pro Gln Tyr Ala Tyr Leu Thr Val 405 410 415 Gly Asp Val Asn Thr Gln
Gly Ile Ser Gly Asp Ser Lys Lys Leu Ala 420 425 430 Ser Glu Glu Ser
Ala Phe Tyr Val Leu Glu His Ser Ser Phe Gln Leu 435 440 445 Leu Gly
Thr Gly Gly Thr Ala Ser Met Ser Tyr Lys Phe Pro Pro Val 450 455 460
Pro Pro Glu Asn Leu Glu Gly Cys Ser Gln His Phe Tyr Glu Met Tyr 465
470 475 480 Asn Pro Leu Tyr Gly Ser Arg Leu Gly Val Pro Asp Thr Leu
Gly Gly 485 490 495 Asp Pro Lys Phe Arg Ser Leu Thr His Glu Asp His
Ala Ile Gln Pro 500 505 510 Gln Asn Phe Met Pro Gly Pro Leu Val Asn
Ser Val Ser Thr Lys Glu 515 520 525 Gly Asp Ser Ser Asn Thr Gly Ala
Gly Lys Ala Leu Thr Gly Leu Ser 530 535 540 Thr Gly Thr Ser Gln Asn
Thr Arg Ile Ser Leu Arg Pro Gly Pro Val 545 550 555 560 Ser Gln Pro
Tyr His His Trp Asp Thr Asp Lys Tyr Val Thr Gly Ile 565 570 575 Asn
Ala Ile Ser His Gly Gln Thr Thr Tyr Gly Asn Ala Glu Asp Lys 580 585
590 Glu Tyr Gln Gln Gly Val Gly Arg Phe Pro Asn Glu Lys Glu Gln Leu
595 600 605 Lys Gln Leu Gln Gly Leu Asn Met His Thr Tyr Phe Pro Asn
Lys Gly 610 615 620 Thr Gln Gln Tyr Thr Asp Gln Ile Glu Arg Pro Leu
Met Val Gly Ser 625 630 635 640 Val Trp Asn Arg Arg Ala Leu His Tyr
Glu Ser Gln Leu Trp Ser Lys 645 650 655 Ile Pro Asn Leu Asp Asp Ser
Phe Lys Thr Gln Phe Ala Ala Leu Gly 660 665 670 Gly Trp Gly Leu His
Gln Pro Pro Pro Gln Ile Phe Leu Lys Ile Leu 675 680 685 Pro Gln Ser
Gly Pro Ile Gly Gly Ile Lys Ser Met Gly Ile Thr Thr 690 695 700 Leu
Val Gln Tyr Ala Val Gly Ile Met Thr Val Thr Met Thr Phe Lys 705 710
715 720 Leu Gly Pro Arg Lys Ala Thr Gly Arg Trp Asn Pro Gln Pro Gly
Val 725 730 735 Tyr Pro Pro His Ala Ala Gly His Leu Pro Tyr Val Leu
Tyr Asp Pro 740 745 750 Thr Ala Thr Asp Ala Lys Gln His His Arg His
Gly Tyr Glu Lys Pro 755 760 765 Glu Glu Leu Trp Thr Ala Lys Ser Arg
Val His Pro Leu 770 775 780 2781PRTParvovirus B19 2Met Ser Lys Lys
Ser Asp Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala 1 5 10 15 Lys Asp
Val Tyr Lys Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Glu 20 25 30
Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser 35
40 45 Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val
Ala 50 55 60 Arg Ile Lys Ser Asn Leu Lys Asp Thr Pro Asp Leu Tyr
Ser His His 65 70 75 80 Phe Gln Ser His Gly Gln Leu Phe Asp His Pro
His Ala Leu Ser Pro 85 90 95 Ser Ser Ser His Thr Glu Pro Arg Gly
Glu Asp Ala Val Leu Ser Ser 100 105 110 Glu Asp Leu His Lys Pro Gly
Gln Val Ser Ile Gln Leu Pro Gly Thr 115 120 125 Asn Tyr Ile Gly Pro
Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser 130 135 140 Ala Val Asp
Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu 145 150 155 160
Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu 165
170 175 Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln
Ala 180 185 190 Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro
Val Ala His 195 200 205 Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr
Asn Ala Ser Glu Lys 210 215 220 Tyr Pro Ser Met Thr Ser Val Asn Ser
Ala Glu Ala Ser Thr Gly Ala 225 230 235 240 Gly Gly Gly Gly Ser Asn
Pro Val Lys Ser Met Trp Ser Glu Gly Ala 245 250 255 Thr Phe Thr Ala
Asn Ser Val Thr Cys Thr Phe Ser Arg Gln Phe Leu 260 265 270 Ile Pro
Tyr Glu Pro Glu His Arg Tyr Lys Val Phe Ser Pro Ala Ala 275 280 285
Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys Val Cys Thr Ile 290
295 300 Ser Pro Ile Met Gly Tyr Ser Thr Pro Trp Arg Tyr Leu Asp Phe
Asn 305 310 315 320 Ala Leu Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln
His Leu Ile Glu 325 330 335 Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu
Thr Val Thr Ile Ser Glu 340 345 350 Ile Ala Val Lys Asp Val Thr Asp
Lys Thr Gly Gly Gly Val Gln Val 355 360 365 Thr Asp Ser Thr Thr Gly
Arg Leu Cys Met Leu Val Asp His Glu Tyr 370 375 380 Lys Tyr Pro Tyr
Val Leu Gly Gln Gly Gln Asp Thr Leu Ala Pro Glu 385 390 395 400 Leu
Pro Ile Trp Val Tyr Phe Pro Pro Gln Tyr Ala Tyr Leu Thr Ala 405 410
415 Gly Asp Val Asn Thr Gln Gly Ile Ser Gly Asp Ser Lys Lys Leu Ala
420 425 430 Ser Glu Glu Ser Ala Phe Tyr Val Leu Glu His Ser Ser Phe
Glu Leu 435 440 445 Leu Gly Thr Gly Gly Ser Ala Thr Met Ser Tyr Lys
Phe Pro Pro Val 450 455 460 Pro Pro Glu Asn Leu Glu Gly Cys Ser Gln
His Phe Tyr Glu Met Tyr 465 470 475 480 Asn Pro Leu Tyr Gly Ser Arg
Leu Gly Val Pro Asp Thr Leu Gly Gly 485 490 495 Asp Pro Lys Phe Arg
Ser Leu Thr His Glu Asp His Ala Ile Gln Pro 500 505 510 Gln Asn Phe
Met Pro Gly Pro Leu Val Asn Ser Val Ser Thr Lys Glu 515 520 525 Gly
Asp Thr Ser Asn Thr Gly Ala Gly Lys Ala Leu Thr Gly Leu Ser 530 535
540 Thr Gly Thr Ser Gln Ser Thr Arg Ile Ser Leu Arg Pro Gly Pro Val
545 550 555 560 Ser Gln Pro Tyr His His Trp Asp Thr Asp Lys Tyr Val
Thr Gly Ile 565 570 575 Asn Ala Ile Ser His Gly Gln Thr Thr Tyr Gly
Asn Ala Glu Asp Lys 580 585 590 Glu Tyr Gln Gln Gly Val Gly Arg Phe
Pro Asn Glu Lys Glu Gln Leu 595 600 605 Lys Gln Leu Gln Gly Leu Asn
Ile His Thr Tyr Phe Pro Asn Lys Gly 610 615 620 Thr Gln Gln Tyr Thr
Asp Gln Ile Glu Arg Pro Leu Met Val Gly Ser 625 630 635 640 Val Trp
Asn Arg Arg Ala Leu His Tyr Glu Ser Gln Leu Trp Ser Lys 645 650 655
Ile Pro Asn Leu Asp Asp Ser Phe Lys Thr Gln Phe Ala Ala Leu Gly 660
665 670 Gly Trp Gly Leu His Gln Pro Pro Pro Gln Ile Phe Leu Lys Ile
Leu 675 680 685 Pro Gln Ser Gly Pro Ile Gly Gly Ile Lys Ser Met Gly
Ile Thr Thr 690 695 700 Leu Val Gln Tyr Ala Val Gly Ile Met Thr Val
Thr Met Thr Phe Lys 705 710 715 720 Leu Gly Pro Arg Lys Ala Thr Gly
Arg Trp Asn Pro Gln Pro Gly Val 725 730 735 Tyr Pro Pro His Ala Ala
Gly His Leu Pro Tyr Val Leu Tyr Asp Pro 740 745 750 Thr Ala Thr Asp
Ala Lys Gln His His Arg His Gly Tyr Glu Lys Pro 755 760 765 Glu Glu
Leu Trp Thr Ala Lys Ser Arg Val His Pro Leu 770 775 780
3781PRTParvovirus B19 3Met Ser Lys Thr Thr Asn Lys Trp Trp Glu Ser
Ser Asp Lys Phe Ala 1 5 10 15 Gln Asp Val Tyr Lys Gln Phe Val Gln
Phe Tyr Glu Lys Ala Thr Gly 20 25 30 Thr Asp Leu Glu Leu Ile Gln
Ile Leu Lys Asp His Tyr Asn Ile Ser 35 40 45 Leu Asp Asn Pro Leu
Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala 50 55 60 Arg Ile Lys
Ser Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His 65 70 75 80 Phe
Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser 85 90
95 Ser Asn Ser Ser Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser
100 105 110 Glu Asp Leu His Lys Pro Gly Gln Val Ser Ile Gln Leu Pro
Gly Thr 115 120 125 Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly
Pro Pro Gln Asn 130 135 140 Ala Val Asp Ser Ala Ala Arg Ile His Asp
Phe Arg Tyr Ser Gln Leu 145 150 155 160 Ala Lys Leu Gly Ile Asn Pro
Tyr Thr His Trp Thr Val Ala Asp Glu 165 170 175 Glu Leu Leu Lys Asn
Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Ala 180 185 190 Val Lys Asp
Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His 195 200 205 Phe
Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys 210 215
220 Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala
225 230 235 240 Gly Gly Gly Gly Ser Asn Pro Thr Lys Ser Met Trp Ser
Glu Gly Ala 245 250 255 Thr Phe Thr Ala Asn Ser Val Thr Cys Thr Phe
Ser Arg Gln Phe Leu 260 265 270 Ile Pro Tyr Asp Pro Glu His His Tyr
Lys Val Phe Ser Pro Ala Ala 275 280 285 Ser Ser Cys His Asn Ala Ser
Gly Lys Glu Ala Lys Val Cys Thr Ile 290 295 300 Ser Pro Ile Met Gly
Tyr Ser Thr Pro Trp Arg Tyr Leu Asp Phe Asn 305 310 315 320 Ala Leu
Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln His Leu Ile Glu 325 330 335
Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu Thr Val Thr Ile Ser Glu 340
345 350 Ile Ala Val Lys Asp Val Thr Asp Lys Thr Gly Gly Gly Val Gln
Val 355 360 365 Thr Asp Ser Thr Thr Gly Arg Leu Cys Met Leu Val Asp
His Glu Tyr 370 375 380 Lys Tyr Pro Tyr Val Leu Gly Gln Gly Gln Asp
Thr Leu Ala Pro Glu 385 390 395 400 Leu Pro Ile Trp Val Tyr Phe Pro
Pro Gln Tyr Ala Tyr Leu Thr Val 405 410 415 Gly Glu Val Asn Thr Gln
Gly Ile Ser Gly Asp Ser Lys Lys Leu Ala 420 425 430 Ser Glu Glu Ser
Ala Phe Tyr Val Leu Glu His Ser Ser Phe Glu Leu 435 440 445 Leu Gly
Thr Gly Gly Ser Ala Thr Met Ser Tyr Lys Phe Pro Ala Val 450 455 460
Pro Pro Glu Asn Leu Glu Gly Cys Ser Gln His Phe Tyr Glu Met Tyr 465
470 475 480 Asn Pro Leu Tyr Gly Ser Arg Leu Gly Val Pro Asp Thr Leu
Gly Gly 485 490 495 Asp Pro Lys Phe Arg Ser Leu Thr His Glu Asp His
Ala Ile Gln Pro 500 505 510 Gln Asn Phe Met Pro Gly Pro Leu Ile Asn
Ser Val Ser Thr Lys Glu 515 520 525 Gly Asp Asn Ser Asn Thr Gly Ala
Gly Lys Ala Leu Thr Gly Leu Ser 530 535 540 Thr Gly Thr Ser Gln Asn
Thr Arg Ile Ser Leu Arg Pro Gly Pro Val 545 550 555 560 Ser Gln Pro
Tyr His His Trp Asp Thr Asp Lys Tyr Val Thr Gly Ile 565 570 575 Asn
Ala Ile Ser His Gly Gln Thr Thr Tyr Gly Asn Ala Glu Asp Lys 580 585
590 Glu Tyr Gln Gln Gly Val Gly Arg Phe Pro Asn Glu Lys Glu Gln Leu
595 600 605 Lys Gln Leu Gln Gly Leu Asn Met His Thr Tyr Phe Pro Asn
Lys Gly 610 615 620 Thr Gln Gln Tyr Thr Asp Gln Ile Glu Arg Pro Leu
Met Val Gly Ser 625 630 635 640 Val Trp Asn Arg Arg Ala Leu His Tyr
Glu Ser Gln Leu Trp Ser Lys 645 650 655 Ile Pro Asn Leu Asp Asp Ser
Phe Lys Thr Gln Phe Ala Ala Leu Gly 660 665 670 Gly Trp Gly Leu His
Gln Pro Pro Pro Gln Ile Phe Leu Lys Ile Leu 675 680 685 Pro Gln Ser
Gly Pro Ile Gly Gly Ile Lys Ser Met Gly Ile Thr Thr 690 695 700 Leu
Val Gln Tyr Ala Val Gly Ile Met Thr Val Thr Met Thr Phe Lys 705 710
715 720 Leu Gly Pro Arg Lys Ala Thr Gly Arg Trp Asn Pro Gln Pro Gly
Val 725 730 735 Tyr Pro Pro
His Ala Ala Gly His Leu Pro Tyr Val Leu Tyr Asp Pro 740 745 750 Thr
Ala Thr Asp Ala Lys Gln His His Arg His Gly Tyr Glu Lys Pro 755 760
765 Glu Glu Leu Trp Thr Ala Lys Ser Arg Val His Pro Leu 770 775 780
411PRTArtificial SequenceSynthetic Polypeptide 4Lys Leu Lys Leu Leu
Leu Leu Leu Lys Leu Lys 1 5 10 59PRTArtificial SequenceSynthetic
PolypeptideMOD_RES(1)..(1)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(1)..(1)X is any positively charged natural and/or
non-natural amino acidMISC_FEATURE(2)..(2)X can be L, V, I, F,
and/or WMISC_FEATURE(3)..(3)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(4)..(6)X can be L, V, I,
F, and/or WMISC_FEATURE(7)..(7)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(8)..(8)X can be L, V, I,
F, and/or WMOD_RES(9)..(9)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(9)..(9)X is any positively charged natural and/or
non-natural amino acid 5Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
610PRTArtificial SequenceSynthetic
PolypeptideMOD_RES(1)..(1)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(1)..(1)X is any positively charged natural and/or
non-natural amino acidMISC_FEATURE(2)..(2)X can be L, V, I, F,
and/or WMISC_FEATURE(3)..(3)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(4)..(7)X can be L, V, I,
F, and/or WMISC_FEATURE(8)..(8)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(9)..(9)X can be L, V, I,
F, and/or WMOD_RES(10)..(10)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(10)..(10)X is any positively charged natural and/or
non-natural amino acid 6Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 711PRTArtificial SequenceSynthetic
PolypeptideMOD_RES(1)..(1)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(1)..(1)X is any positively charged natural and/or
non-natural amino acidMISC_FEATURE(2)..(2)X can be L, V, I, F,
and/or WMISC_FEATURE(3)..(3)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(4)..(8)X can be L, V, I,
F, and/or WMISC_FEATURE(9)..(9)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(10)..(10)X can be L, V,
I, F, and/or WMOD_RES(11)..(11)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(11)..(11)X is any positively charged natural and/or
non-natural amino acid 7Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 812PRTArtificial SequenceSynthetic
PolypeptideMOD_RES(1)..(1)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(1)..(1)X is any positively charged natural and/or
non-natural amino acidMISC_FEATURE(2)..(2)X can be L, V, I, F,
and/or WMISC_FEATURE(3)..(3)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(4)..(9)X can be L, V, I,
F, and/or WMISC_FEATURE(10)..(10)X is any positively charged
natural and/or non-natural amino acidMISC_FEATURE(11)..(11)X can be
L, V, I, F, and/or WMOD_RES(12)..(12)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(12)..(12)X is any positively charged natural and/or
non-natural amino acid 8Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 913PRTArtificial SequenceSynthetic
PolypeptideMOD_RES(1)..(1)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(1)..(1)X is any positively charged natural and/or
non-natural amino acidMISC_FEATURE(2)..(2)X can be L, V, I, F,
and/or WMISC_FEATURE(3)..(3)X is any positively charged natural
and/or non-natural amino acidMISC_FEATURE(4)..(10)X can be L, V, I,
F, and/or WMISC_FEATURE(11)..(11)X is any positively charged
natural and/or non-natural amino acidMISC_FEATURE(12)..(12)X can be
L, V, I, F, and/or WMOD_RES(13)..(13)Modified by H, NH2, COCH3, or
COHMISC_FEATURE(13)..(13)X is any positively charged natural and/or
non-natural amino acid 9Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10
* * * * *