Note: Descriptions are shown in the official language in which they were submitted.
The human parvovirus B19 (for short hereinafter:
B19) was discovered by chance in 1975 in plasma samples
from blood donors (Cossart, Y.E., Field, A.M., Cant, B.,
Widdows, D.: Parvovirus-like particles in human sera.
Lancet I (1975) 72-73j by countercurrent electrophoresis.
In recent years it has been shown that B19 may cause an
aplastic crisis in patients with chronic haemolytic
anaemia, and i~~ the aetiological agent of erythema
infectiosum (EI).
Under the electron microscope, B19 has a size of
about 20 nm. The particles have an icosahedric symmetry.
Besides the virus particles there are also seen to be
"empty" capsids which contain no DNA. The density in
CsCl2 ( sic ) is 1. 36 - 1.40 g/ml. The virus genome consists
of a single-stranded DNA of 5.4kb. The nucleotide
sequence of the genome of a B19 parvovirus has been
derived from a clone which contained virtually the
complete viral genome (RØ Shade et al. Journal of
Virology (1986) p. 921). In each case only one DNA
strand, either of the plus or the minus orientation, is
packaged into each virus particle. B19 is an autonomous
parvovirus, that is to say requires no helper virus
for replication.
The capsid consists of two polypeptides with
molecular weights of 83kDa (VP1) and 58kDa (VP2). In
addition, three non-structural proteins of 52, 63 and
7lkDa can be detected.
CA 02075366 2001-03-14
- 2 -
The DNA codes in the 5' region for the structural
proteins of the caps.id. The coding regions of the struc-
tural proteins are identical apart from an additional N
terminus of VP1. This difference is caused by splicing
processes at the mRNA level, in which in the case of VP2
the translational start for VP1 is taken out and thus
translation can start only with the shorter VP2.
Investigations on various B19 isolates found
world-wide have shown that these differ in part at the
DNA level by the restriction enzyme pattern. These
differences do not, however, correlate with the clinical
spectrum of B19 infection.
It has not been possible to date to find a
permanent cell line :in which B19 can be grown. There has
been just as little success to date in establishing an
experimental animal model for B19. B19 can, however, be
grown in primary bone marrow cells in the presence of
erythropoietin. It has thus been possible to clarify the
mechanism of replication of the virus and show that cells
of erythropoiesis a.re the target cells of this infection.
Inoculation of B19 cells in fetal erythropoietic cells
and erythroblasts of a patient with chronic myeloid
leukaemia has now su~~ceeded.
H19 causes erythema infectiosum (infectious
erythema) which is an infectious disease which usually
has a benign course and mostly occurs between the ages of
childhood and early adulthood. B19 infection may in
addition cause aplastic crises in patients with chronic
haemolytic anaemia ( sickle cell anaemia etc . ) and chronic
bone marrow aplasias in patients with inborn or acquired
immunodefficiency states.
In pregnancy B19 infection may in about 10-15~
result in hydrops fetalis with resulting interuterine
death. Furthermore, B19 is associated with the
occurrer:~s of Schonlein-Henoch purpura.
As a rule, B19 is transmitted by droplet infec-
tion but also by antigen-positive conserved blood and
coagulation products.
CA 02075366 2001-03-14
_
Since no permanent cell line in which B19 can be
obtained in large amounts is yet known, there is thus a
lack of a source for obtaining antigen for diagnostic
tests. To date one has made do with B19 virus discovered
by chance in conserved blond from danors who are just in
the viraemic stage of infection.
The object of the present invention is to provide
immunologically active polypeptides which permit, with
the test systems presented here, detention of B19-
specific antibodies of the IgG and IgM class. This
results in the following possible applicationso
- Serodiagnosis of acute or previous B19 infections in
dermatology, haematology, gynaecology, rheumatology and
paediatrics.
- Determination of the B19 immune status in pregnant
women
- Investigation of conserved blood or donated plasma to
exclude transmission of B19 antigen, since it is highly
probable that transmission of B19 virus is no longer
possible by anti-B19 IgG positive blood or plasma.
- Selection of anti--B19 positive plasma donors fox
production of B19 hyperimmunoglobulin products.
There is a pressing need for the introduction of
test reagents because of 'the broad clinical spectrum of
the diseases caused by B19, and of the risk to B19
seronegative pregnant women.
It has emerged that utilisable immunologically
active palypeptides cannot be prepared directly. Pre-
paration of short peptides by genetic engineering is,
just like that of large polypeptides, possible in a
satisfactory yield only when suitable expression vectors
are used. Although relatively short peptides can be
easily prepared by synthesis, more accurate knowledge of
the immunological activity is necessary.
The invention relates to immunologically active
peptides which have a part of the amino-acid sequence of
the capsid proteins VP 1 or VP 2 of parvovirus B19. These
peptides are characterised in that they are free of
r~ ,."~ 9a t b
r.J~ ti '~ t
impurities which interfere with the detection of anti-
bodies directed against parvovirus 819, This property is
of great importance since it is not possible to utilise
those peptide preparations which contain, by reason of
the preparation, components which .react with the anti-
bodies to be detected. One example of an unwanted
impurity of this type .is protein A, which is able to
react specifically with the Fc portion of IgG antibodies.
A particular advantage of the immunologically actiue
peptides according to the invention is that they can be
prepared in good yield by the preparation process accord-
ing to the invention. This is because, if the antigens
required for a diagnostic test are not synthesised in an
adequate amount in the preparation process, it is not
possible to obtain the required yield after the subse-
quent purification processes.
It has furthermore been possible within the scope
of the present invention to determine short peptide
segments from VP 1, more accurately from the region of
VP 1 which does not coincide with VP 2, whose epitopes
are suitable for reliable detection of antibodies against
parvovirus B19 in the investigation fluids, especially
sera. This region is called VP 1-VP 2 hereinafter. Fig. 3
shows by way of example the arrangement of some peptides
(PAPEP 1-PAPEP 8) in the region (VP 1-VP 2). Although
these peptides are preferred, it is equally possible to
employ other peptides with 8-50 amino acids, preferably
10 to 32 amino acids, from the VP 1-VP 2 region, This
region approximately corresponds to the polypeptide PAN:L
which is depicted in Fig. 2-1.
In a preferred embodiment of the present inven-
tion, this small, immunodominant and B19-specific region
is employed in the serological test. It is particularly
preferable in this connection to employ a mixture of
synthetic peptides, these pept ides having the amino-acid
sequences PAPEP 1 - PAPEP 8 shown .i.n Exampl.e 3.
In another preferred embodiment of the present
invention, the arnino-acid sequences which are depicted in
- 5 -
Figure 2 of the imrnunologically active peptides PAN-1,
PAN-2, PAN-3, PAN-4,, PCE, PANSE and PAPST prepared
by ge::etic engineering are employed. It is as a rule
sufficient in this case to use one peptide in the test.
It is possible, however, in special cases also to employ
two or more of these peptides.
The peptide's according to the invention can be
prepared either by synthesis or by genetic engineering.
The short peptides, which are explained in detail in
Example 3, are preferably prepared by synthesis. The
longer peptides are, however, preferably prepared by
genetic engineering.
Firstly, they coding regions of the viral DNA were
amplified from the serum of an infected patient by means
of two polymerase chain reactions (PCR) and cloned in
plasmids for further growth in Escherichia (E.) coli.
After further subcloning steps, various regions therefrom
were then expressed by genetic engineering in E. coli,
and the antigens resulting therefrom were investigated
for their use for detecting antibodies against the virus.
Direct preparation of the peptides according to the
invention in expres~;ion vectors is impossible because of
various difficulties. For this reason, according to the
invention, the viral protein segment is fused to a
protein amenable t.o stable expression. This fusion
protein can be employed directly after purification as
antigen for IgG detection. However, the parvovirus
specific portion is preferably cleaved off by suitable
methods, further purified and then employed for sero
logical tests.
The present invention furthermore relates to test
kits for the determination of antibodies which are
directed against parvovirus B19. The immunologically
active peptides according to the invention can in prin-
ciple be used in ai.l diagnostic test kits for detecting
antibodies against parvovirus B19. In a preferred embodi-
ment of the test kits according to the invention, the
solid phase of suitable microtitre plates or polystyrene
CA 02075366 2001-03-14
~~
- 6 -
beads is coated with the immunologically active peptides
according to the invention. After incubation with the
investigation fluid (serum sample) in a suitable
dilution, and after customary washing steps, enzyme- or
radioactively labelled anti-human IgG is added. The
extent of substrate conversion or of the bound radio-
activity then shows whether antibodies directed against
parvovirus B19 are present in the serum sample.
The test kits according to the invention are
IO normally supplied to laboratories of physicians, hospi
tals, investigation facilities etc. They usually contain
all the reagents required for carrying out the test.
Customary test reagents such as buffer solutions etc.
are, however, sometimes not included. As a rule, the test
kits contain microtitre plates or polystyrene beads which
are coated either with one or more peptides according to
the invention or with anti-antibodies. The test kit:a may
furthermore contain, depending on the test principle, one
or more peptides according to the invention. Finally, the
test kits also embrace an indicator component which makes
it possible to quantify 'the test result.
In other preferred test kits, the antigens are
bound to the solid phase of microtitre plates or poly-
styrene beads. After incubation of the test serum, and
suitable washing and saturation steps, a specific enzy-
matically or radioactively labelled antibody against. the
B19 antigens is added and its substrate conversion or the
bound radioactivity is measured. Since this takes the
form of an inhibition test, a small substrate conversion
or low radioactivity indicates 'the presence of specific
antibodies.
It is likewise possible to employ peptides
according to the invention coupled to solid phases far
detecting IgM ant ibadies against B19. In this detection
method, firstly the IgG antibodies are eliminated by
adding beads coated with protein A to the investigation
fluids. Bound antibodies are then detected using an
anti-human IgM antibody which is enzymatically or
~~~ ~t'~~~
_,_
radioacti.vely labelled.
The principle of the so-called ~-capture assay is
used in another preferred test kit. First the IgM from
the investigation fluid (serum) is bound by means of
anti-human IgM antibodies bound to the solid phase. The
immunologically active peptides according to the inven-
tion are then added. The ewtent of 'the binding of the
antigens and thus the amount of anti-B19 IgM present can
be effected by either the antigens being radioactively
labelled or labelled with other substances (digoxigenin,
avidin) and thus being detectable, or by employing a
second labelled antibody against the B19 antigens and
measuring its binding.
Very particularly preferred within the scope of
the present invention are ELISA (enzyme linked immuno
sorbent assay) test kits.
Also provided according to the invention are DNA
sequences cahich can be used for direct detection of the
virus in investigation samples (sera, biopsies, etc.).
Two DNA primers which attach themselves specifically to
DNA regions in VP 1 are preferably used. It is then
possible by means of a commercially available polymerase
chain reaction kit to achieve amplification of the region
lying between them. Amplified DNA which has then been
immobilised in a suitable way is detected by a suitable
DNA sequence. This DNA employed for the hybridisation is
prepared with the aid of a plasmi.d which contains the DNA
region lying between 'the two primers.
It is self-evident that the primer sequences must
not be present in the DNA employed for the hybridisation.
The sequence of the primers used, and the arrangement
with respect to one another, is depicted in F'ig. 1.
Finally, vaccines against parvovirus B19 are also
made available within the scope of the present invention.
This entails the immunologically active peptides accord
ing to the invention being administered, optionally
several times, together with suitable adjuvants to the
people to be protected. The production of antibodies
~i~"~ ~ ~~~
_8_
elicited by this can effect protection from infection
with parvovirus 819.
EXAMPLE 1:
Obtaining parvovirus B19 VP 1- and VP 2-encoding
sequences from patient's serum
Viral DNA was isolated from lml of serum from a
patient with acute infection (erythema infectiosum) by
proteinase K digestion in 1~ SDS, phenol extraction and
subsequent alcohol precipitation (this and all the
following steps for obtaining, processing and expressing
DNA, as well as the preparation of recombinant proteins
and fundamental steps for the purification thereof, are
described in detail in: Maniatis, T., Fritsch, E.F.,
Sambrook, J. (1982) Molecular cloning. Cold Spring
Harbor, N.Y.). This DNA was taken up in 501 of TE buffer
and then 1~1 samples were employed for the amplification
by means of the polymerase chain reaction and synthetic
oligodeoxynucleotides. Two pairs of primers were used for
the amplification of the coding regions of the surface
proteins; one of these for obtaining the VP 1 portion,
and the second pair for the complete VP p oligo-
deoxynucleotides used as primers have at each of their 5'
ends sequences which are not homologous with the parvo-
virus sequence, code for restriction enzyme cleavage
sites and are therefore suitable for cloning the DNA
fragments resulting from the PCR into suitable vectors.
The primers identified by O-1 to O-5 in Fig. 1 were used.
In each case five mixtures each containing 1~1 of
isolated parvovirus DNA were amplified with the two pairs
of primers in a volume of 100f~1. The conditions for this
were: 1.5 min denaturation at 94°C, 2 min attachment of
the primers at 45°C, 4 min synthesis at 72°C; total of
50 cycles; buffer, substrates and Taq polymerase were
employed far this as stated by the manufacturer
;Cetus/Perkin-Elmer, LJberlingen, FRG) .
The amplified DNA fragments from the two dif-
ferent mixtures (for VP 1 and VP 2) were in each; case
_ g -
combined, precipitated by alcohol precipitation, washed
with 70% alcohol, dried, dissolved in a volume of 200.1
of TE buffer and digested with the restriction enzymes
EcoRI and HindIII. Fractionation of the fragments by
electrophoresis in a 1.2% agarose gel was then followed
by isolation of the corresponding DNA bands ( 709bp for VP
1, 1704bp for VP 2) and insertion into the EcoRI and
HindIII sites of th.e vector PUC12 (Pharmacia, Sweden).
After transformation of the plasmids into E.coli JM109
(Pharmacia, Sweden), bacterial clones with parvovirus DNA
inserts were characterised by restriction digestion. The
corresponding zones were given the names pUCI2PAN for the
region encoding the VP 1 portion and pUC12VP2 for the VP
2-encoding region.
EXAMPLE 2
Preparation by genetic engineering of VP 1 portion and
VP 2 from E.coli cells
a1 VP1 portion:
1) PAN-1
The VP1-encoding region was isolated from the
plasmi:d pUCI2PAN with BclI and HindIII (see Fig. 1, the
HindIII site originates from the pUC vector) and inserted
behind the 3' end of a truncated p-galactosidase gene of
the vector (for exa~ruple pBD2) into the BamHI and HindLII
restriction cleavage sites. E.coli cells with plasmi.ds
-ssulting therefrom express after induction with IPTG a
~;-gal::VP1 fusion protein (about 67kDA) in large quan-
tity, which reacts very strongly with anti-parvovirus
B19-positive sera in an immunoblot (Western blot.).
Purification of this: protein can be achieved very easily
with conventional mE~thods utilising the insolubility of
the Drotein. After lysis of the cells, the pellet frac-
tion is washed with detergents such as TritonMX100 and
octyl gluco-pyr=~zosi.de, and the fusion protein is subse-
quently dissolved v~i.th 8M urea/1% mercaptoethanol and
CA 02075366 2001-03-14
- 10 -
separated from cellular impurities by DEAF chromatography
with an NaCl gradient.
The VP1 portion can be cleaved off the fusion
protein by BrCN cleavage since the VP1 protein sequence
starts with a methionine, and this amino acid no longer
appears in the fragment itself; by contrast, methionine
occurs relatively often in the bacterial fusion portion
so that this portion is broken up into very small frag-
ments. After cleavage in 35$ formic acid and O.lmg/ml
BrCN at room temperature for 4h, the sample was lyophi-
lised, dissolved in 8M urea, 2mM DTT (dithio-
threitol) and purified by DEAF chromatography in an NaCl
gradient. The VP1 fragment resulting therefrom was called
PAN-1 and can be uses directly for serological determi-
nations. The amino-acid sequence is indicated in
Fig. 2-1.
Further con;~tructs generated were plasmids which
code for fusion proteins consisting of the glutathione S-
transferase from Sclz.istosoma japonicum (Smith, D.B. and
Johnson, K.S.: Sing:Le step purification of polypeptides
expressed in Escherichia coli as fusions with glutathione
S-transferase. Gene:, 67 (1988) 31-40) and the VP1
portion. However, i.t is also possible to use another
fusion partner as long as it does not interfere with the
diagnostic test.
2) PCE:
The B19 DNA fragment was isolated from pUCI2PAN
after BclI/PvuII digestion (618bp) and integrated into
the BamHI and SmaI sites in pGEXl (pGEXIPAN) . The resul.t-
ing 52kDA fusion protein was purified from the super-
natant by means c~f glutathione-coupled agarose and
employed as antigen f:or the serological tests in Example
4 (name:PCE). The amino-acid sequence of this antigen is
shown in Fig. 2-2.
CA 02075366 2001-03-14
- 11 -
3) PAN-2:
A 458bp fragment was isolated from pUCI2PAN with
BclI/HincII and, after intermediate clonings in other
vectors, inserted into pGEX2 (pGEX2PAN). Insertion of the
fragment in the same reading frame can also be achieved
by using oligodeoxynucleot.ides. At the fusion site of
glutathione S-transferase and the VP1 segment is the
amino-acid sequence which is recognised by thrombin, so
that the B19 portion can be cleaved off the fusion
partner by this enzyme. It is also possible to use any
other fusion partner as long as it has this protease
recognition sequence. The amino-acid sequence of the
antigen, as well as fused-on foreign amino acids ( in bold
print) is indicated in Fig. 2-3.
4) PAN-3:
A 458bp fragment was isolated from pUCI2PAN ceith
BcII/HincTI and, after intermediate clonings in other
vectors, inserted into pGEX3 (pGEX3PAN). Insertion of the.
fragment in the same reading frame can also be achieved
by using synthetic oligodeoxynucleotides. At the fusion
site of glutathione S-transferase and the VP1 segment is
the amino-acid sequence which is recognised by the
protease factor Xa, so that the B19 portion can be
cleaved off the fusion partner by this enzyme. It is also
possible to use any other fusion partner as long as it
has this protease recognition sequence. The amino-acid
sequence of the antigen, as well as fused-on foreign
amino acids (in bold print) is indicated in Fig. 2-4.
5) PAN-4:
The complete B19 DNA insert was obtained .from
pUCI2PAN by BclI and PstI digestion and, after various
intermediate cloning steps, inserted into the vector
pGEX2. This resulted in the plasmid pGEX2PAN. Insertion
of the fragment in the same reading frame can also be
achieved by using synthetic oligodeoxynucleotides. At 'the
fusion site of glutathione S-transferase and the VP1
- 12 -
segment is the amino-acid sequence for the protease
thrombin, so that the B19 portion can be cleaved off the
fusion partner by this enzyme. It is also possible to use
another suitable fusion partner if it has this protease
recognition sequence. The amino-acid sequence of the
antigen, as well as fused-on foreign amino acids (in bold
print) is indicated .in Fig. 2-5.
Purification of the antigens can be achieved by
simple affinity chromatography with a glutathione-coupled
gel matrix.
For further purification of the fusion proteins
based on pGEX2 and _i, the B19 portion was cleaved off by
digestion with thrombin or factor X as stated by the
manufacturer (Boehringer Mannheim). The fragments were
then purified again by affinity chromatography. The
glutathione S-transferase can in this case be selec-
tively fished out, the parvovirus protein portion is to
be found in the flow-through and can be employed after a
final DEAF chromatographic fractionation in serological
tests.
However, as an alternative to this, the protease
can also be added directly to the glutathione-coupled gel
suspension with they fusion protein bound on. After an
incubation time of about 1h, the VPl fragment which
has been cleaved off can be washed out of the gel, while
the glutathione S-transferase portion remains bound to
the gel matrix.
b) VP-2 portion:
1) VP-2
Owing to the: choice of the PCR primers and of the
vect..r, the coding region for VP2 is already in the
correct reading frame in the plasmid pUC12VP2 and can be
purified after induction with IPTG from the insoluble
fraction of the bacterial lysate, in a similar way to
that described for pBD2PAN. The amino-acid sequence of
the recombinant antigen is shown in Fig. 2-6.
CA 02075366 2001-03-14
- 13 -
2) PANSE:
It emerged, surprisingly, that a truncation of
the VP2-encoding sequence is associated with a consider-
able increase in the protein yield, that this truncated
antigen can be stably expressed, is not degraded euen
during purification, and still has the same reactivity
with anti-B19 positive sera too. This expression plasmid
(pUCI9PANSE) was obtained by truncating the 5' region of
VP2 by 355bp as far as an Nsil site. This fragment was
inserted into pCUl9 (Pharmacia, Sweden) which has the
same reading frame in the lacZ peptide as the B19
sequence. Since, because of the PCR primers, a HindIII
site is located at the 3' end, it was necessary also to
produce an EcoRI site by intermediate cloning in order to
be able to insert the required fragment into 'the Pstl and
EcoRI sites of pUCl9.
The antigen with a size of about 38kDa ( PANSE )
can be separated from impurities very simply from the
pellet fraction of the bacterial lysate after dissolving
in 4M urea by DEAE chromatography. The ammo-acid
sequence of the antigen is indicated in Fig. 2-7.
3) PAPST:
A fragment 7I6bp in size which encodes the N-
terminal region of VP-2 was isolated from the plasmid
pUC12VP2 by PstI digestion. After insertion of the
fragment into the vector pUC9 (Pharmacia, Sweden] in the
same orientation of the reading frames as the lacZ of the
vector (characterised by restriction enzyme digestion),
the B19 antigen with a size of about 33kDa is produced in
very large quantity (about 10~ o.f the total E.coli
protein). Purification can take place in a similar way as
for pBDAN from the insoluble constituents by dissolving
in 8M urea and subsequent DEAF chromatography. The amino-
acid sequence is depicted in Fig. 2-8.
- 14 -
c) Complete VP1/VP2:
The plasmid. pUCI2PAN was opened with PstI and
HindIII, and the VP~? encoding region from pUC12VP2 was
inserted after HindIII and partial PstI digestion as
l.7kb fragment (pUC:12VP1/2).
Expression of VP1/2-containing antigens in E.coli:
1) PAV-1-B:
pUC12VP1/2 'was cut with EcoRI and BamHI, and a
DNA band 1466 by in size was isolated and subsequently
inserted into the EcoRI/BamHI sites of the vector
pUCl8stop. pUCl8 stop resembles the abovementioned pUC
vectors; however, it. differs from the latter by contain-
ing between the Pstl and HindIII site a synthetic oligo-
deoxynucleotide which codes for translation stop signals
and for the stop of transcription. The polylinker region
of the vector thus has the following sequence:
ATG ACC ATG ATT AGG ATT TCG AGC TCG GTA CCC GGG GAT CCT
CTA GAG TCG ACC TGC AGT AAT TAA TTA GAT CTC GAG CCC GCC
TAA TGA GCG GGC TTT TTT AAG CTT
(The restriction cleavage sites EcoRI - GAATTC, BamHI -
GGATCC, PstI - CTGCA.G, BglII - AGATCT and HindIII -
AAGCTT are indicated by bold print)
The vector (pUC-V1-B) obtained in this way
encodes the VP-1 stx-uctural protein from the start up to
amino acid 486 followed by some amino acids of the pUC
polylinker and is germinated by the stop codon of the
inserted oligodeoxynucleotide. The expressed antigen
(PAV-1-B) is produced in very good yield after IPTG
induction in the E.coli cells and has a size of 60 kUa.
Its amino-acid sequence is depicted in Fig. 2-9, amino
acids emphasised by bold print are not B19-specific and
are encoded by pUC sequences. The reactivity with anti-
B19-positive sera is very good and efficient
purification can be achieved by removing soluble E.cali
proteins, dissolving in 8M urea and conventional ion
CA 02075366 2001-03-14
- 15 -
exchange chromatography (as described).
2) PAV-1-N:
The vector pUCVP-1-B described above was digested
with EcoRI and Nsil. The band 1137bp in size produced in
this way was inserted into the vector pUCl8stop into the
EcoRI and Pstl sites (see above). The resulting vector
pUCVP-1-N encodes the structural protein from the start
up to amino acid 377; the antigen (PAV--1-N) is produced
after IPTG induction in the E.coli cells somewhat less
well than the antigen PAV-1-B described above> Tt is
45 kDa in size, and the reactivity with awti-B19 sera is
good. The amino-acid sequence is indicated in Fig. 2-10,
amino acids with bold print are encoded by the pUC vector
and are not B19 specific. Purification of the antigen can
be achieved as for PAV-1-B.
3) Expression of the antigens described under c)1) and
c)2) as GST fusion proteins
The two vectors pUCVP-1-H and pUCVP-1-N were
digested with EcoRI/BglII and the resulting bands about
1480bp and 1150bp, respectively, in size were isolated,
with the translation stop signals introduced together
with the pUClBstop being transferred too. (The BglII site
is indicated in the pUCl8stop polylinker sequence indi-
cated above, and the Bcll site (TGATCA) immediately
before the start of translation was introduced with the
primers used for the DNA amplification - see Fig. 1,
0-1). The two fragments which encode the same 5'
sequences but regions of different length of VP-1 were
then inserted into the vector pGEX-1 described above.
Since pGEX-1 has only the Smal and EcoRI restriction
cleavage sites available for insertion of the 3' end of
a fragment, it was initially necessary also to produce a
site compatible for SmaT (blunt end). This was effected
by inserting the two DNA fragments into the EcoRI and
BamHI (compatible with Bgl.II) restriction sites of the
vector pTC20H (The pIC plasmid and phage vectors with
- 16 -
versatile cloning sites for recombinant selection by
insertional inactivation, J.L. Marsh, M. Erfle and
E.J. Wykes, Gene, 32. (1984) 481-485). The two fragments
were then isolated in turn from the two resulting vectors
with BclI and HincI:I (blunt end cleavage) and inserted
into pGEX-1 in BamHI and SmaI. Since HincII also cuts in
the B19 sequence, the two fragments were isolated by a
partial HineII digestion.
The two pGEX vectors now express fusion proteins
consisting of the g7.utathione S-transferase followed by
the two VP-1 segments of different length. The fragment
originating from pUCVP-1-B and now located in pGEXVP-1-B
yields a fusion protein of about 87kDa; the smaller
fragment encoding only up to amino acid 377 a fusion
protein 72kDa in size. The amino-acid sequences are
indicated in Fig. 2--9 and 2-10. The only difference is
that the five N-tex-minal amino acids are omitted and
instead replaced by glutathione S-transferase.
4) Further expression of VP1/VP2:
A 2.4kb fragment was isolated after EcoRI and
HindIII digestion fx:om the plasmid pUC12VP1/2 with the
complete VPl and VP2 encoding region and inserted into
the eukaryotic expression vector pMDIII (Motz, M.,
Deby G., Jilg, W., Wolf, H.: Expression of the Epstein-
Barr virus major membrane proteins in Chinese
hamster ovary cells. Gene, 44 (1986) 353-359. (Obtainab:Le
from ATCC)) after EcoRI and HindIIT digestion. This
plasmid was subsequently linearised again with a Sal:I,
and a 2.4kb SalI fragment with a dihydrofolate reductase
gene (DHFR) and regulatory sequences was also inserted.
The plasmid pMDIIIVPl/2 obtained in this way was trans-
fected into DHFR-negative CHO cells. Colonies resulting
after selection on a:Lpha-minus medium (GIBCO) were
isolated and amplified after washing out with increasing
concentrations of methotrexate (MTX). Particles with
VPl/VP2 can be purified from the culture supernatant from
these cell cultures.
CA 02075366 2001-03-14
- 17 -
Furthermore, the 2.4kb fragment from pUC12VP1/2
was inserted after EcoRI/HindIII digestion into a vector
which has, besides the HindIII site, also a HamHI site.
The parvovirus portion from this intermediate construct
was then isolated as. BclI and BamHI fragment and inserted
into the BamHI site: of a baculovirus expression vector
(various constructs can be used). (The BclI site is
located immediately in front of the translational start
of VP1, it is encoded by the PCR primers, see Fig. 1; the
BamHI digestion must. be carried out partially since there
is also a site of this type still present in the parvo-
virus sequence.). Ai_ter co-transfection of the resulting
plasmid with wild-type baculovirus DNA into an insect
cell culture line, cells which have no so-called
inclusion bodies were isolated. The VP1 which is produced
intracellularly can be purified in large quantity from
those cells in which the baculovirus polyhedrin gene is
replaced by the VP1/2 gene.
5 ) Expression of VP~-2
Furthermore, expression of the smaller B19-VP-2
was obtained using recombinant baculoviruses. For this,
the VP-2-encoding plasmid pUCI2VP (see Example 1) was
digested with EcoRI and HindIII, and the resulting l.7kb
fragment was inserted into an abovementioned vector which
has, besides the H:indIII site, also a BamHI site. The
parvovirus portion was then isolated as BclI and
BamFiI fragment from this intermediate construct and
inserted into the BamHI site of a baculovirus expression
vector (various constructs can be used). (The BclI
site is ~:~cated immediately in front of the translational
start of BP2, it is encoded by the PCR primers, see
Fig. 1, 0-3; the BamHI digestion must be carried out
partially since there i~ also a site of this type still
present in the pa:rvovirus sequence). After co-trans-
fection of the resulting plasmid with wild-type baculo-
virus DNA into an insect cell culture line, cells which
have no so-called .inclusion bodies were isolated. VP2 can
CA 02075366 2001-03-14
~'~~r~
be purified in large quantity as particles from those
cells in which the baculovirus polyhedrin gene is
replaced by the VP2 gene . These particles are particu-
larly suitable for use in the ~-capture 'test.
EXAMPLE 3
Synthetic peptides with imrnunodominant epitopes
The reaction patterns of the bacaerial expression
products (especially pGEX::VPl fusion constructs) with
parvovirus-positive sera in a Western blot lead to the
surprising conclusion that a short fragment from the VP1
portion suffices to identify all IgG-positive parvo sera.
The fragment can be covered with the following
peptides which can be produced by synthesis:
PAPEP-1:
Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe
Ala Lys Ala Val Tyr
PAPEP-2:
Leu Lys Asp His Tyr Asn Tle Ser Leu Asp Asn Pro Leu Glu
Asn Pro Ser Ser
PAPEP-3:
Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His
His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala
PAPEP-4:
Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser
Ser G1u Asp Leu His Lys Pro Gly Gln Val
PAPEP-5:
Asn Tyr Val G1y Pro Gly Asn Glu Leu Gln Ala Gly Pro Fro
G1n Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg
'.Cyr Ser Gln Leu
_ 19 _
PAPEP-6:
Pro Tyr Thr Isis Trp Thr Val Ala Asp Glu Glu Leu Leu Lys
Asn Ile Lys Asn Glu Thr G1y Phe
PAPEP-7:
Asn Ala Ser Glu Lys Tyr Pro Sex° Met Thr Ser Val Asn Sex
Ala Glu Ala Ser
PAPEP-8:
Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu
Lys His Ile Lys
These antigens can be prepared in large
quantities by synthesis without problems, purified and
then employed in the ELISA in concentrations between 100-
200 ng per mixture.
Even when good results can be achieved with just
one of the peptides, conjoint use of two or three
peptides is preferred.
It is possible to use for detecting TgG or IgM
antibodies in some cases different peptides or com-
binations thereof.
EXAMPLE 4
a) Determination of serum antibodies against parvovirus
B19
The antigens purified and described in Example 2
were used to test a relatively large quantity of sera for
their reactivity with these antigens. For this, the
various recombinant proteins were added in a
cancentration of 0.5 - 1 ug/rnl in carbonate buffer, pFI
9.5, to the wells of commercially available ELISA plates
for 16h for binding on. After unbound material has been
washed out it is possible then to store these plates in
the dry state at 4°C.
Tncubation with the sera for 2h took plane in a
dilution of 1:100, and the subsequent washing procedures
~~~ ~~,~.~~'
- 20
and the detection of bound antibodies with a peroxidase-
coupled anti-human IgG antibody took place by conven-
tional test procedures.
Various serum panels were tested for anti-B19
IgG:
1. Sera from a patient with acute B19 infection were
investigated consecutively from the appearance of
erythema infectiosum up to 19 weeks after the illness.
Result :
All the sera were recognised as anti-B19 IgG
positive even from the start of the clinical manifes-
tation and remained positive over the observation period
(19 weeks) both with the fusion protein from pGEXIPAN
(PCE, see Example 2) and with a VP-1 region cleaved off
by BrCN (PAN-1, see Example 2) and daith a VP1 portion
cleaved off by thrombin (PAN-4) too as antigens.
2. Serum pairs from pregnant women (n=21) from whom
a serum sample was taken on hospitalisation and four
weeks later were tested for anti-B19 TgG. The same sera
were used for each antigen.
Result:
PCE:
Of the 21 pregnant women, 15 were anti-B19
negative and 6 were anti-B19 IgG positive at the time of
hospitalisation. ~'he serological result on the second
serum sample four weeks later produced an identical
result.
PAN-2:
Of the 21 pregnant women, 14 were anti-B19 2gG
negative and 7 were anti-B19 positive at the time of
hospitalisation. On retesting serum samples taken from
these women four weeks later, IgG was no longer detect-
able in one woman who was previously anti-B19 TgG
positive.
_ 21 _
PAN-4:
Of the 21 pregnant women, 15 were anti-B19
negative and 6 were anti-B19 IgG positive at the time of
hospitalisation. The serological result on the second
serum sample four weeks later produced an identical
result.
b) Testing of a definitely B19 IgG/M~-positive/negative
serum collection (n=13)
The sera used were obtained from clinically
defined cases and had previously been checked in an IgG/M
test which uses purified virus as antigen. Sera 1-6:
anti-B19 negative, 7-9: IgM/IgG-positive, 10-13~ IgM
negative, IgG-positive.
PAN-4 were tested by the procedure desa~:ibed
above. The IgM antibodies were determined by the same
test principle as for the IgG determination but PANSY and
PAV-1-B were bound as antigens to the plates in a 1:1
mixture with a 10-fold higher concentration, furthermore
the serum IgG antibodies were eliminated by pre
adsorption with protein A-coupled beads.
The following values for the absorption were
obtained:
IgG determination with PAN-4 ( about 20ng per test
well), IgM with a 1:1 mixture of PANSE and PAV-1-B (about
150ng per test well total protein)
Serum IgG IgM
1 0.09 0.07
2 0.05 0.06
3 0.10 0.08
4 0.07 0.06
5 0.07 0.08
6 0.04 0.07
7 1.82 1.53
8 0.90 0.46
9 0.72 0.56
~'"~~~~f~
- 22
1.10 0.08
11 0.62 0.14
12 0.98 0.11
13 0.87 0.09
5 The results show a clear discrimination of the
positive/negative sera bath for the IgG test and for the
IgM test.
The IgM-positive sera used were obtained from
clinically defined cases and had previously been checked
10 in an IgM test which uses purified virus as antigen. A
test mixture with recombinant antigens from the VP1 and
VP2 regions also recognised all IgM-positive sera. It
emerged that the "PAPST, VP2 but especially PANSE" VP2
portions reacted better in this case than in the IgG
test. Both regions will therefore be represented in a
commercial test kit for IgM.
A further improvement in the sensitivity can be
achieved by selectively binding the serum IgM antibodies
to the solid phase by means of monoclonal antibodies,
adding recombinant antigen (baculovirus-expressed par-
ticulate VP-2) and determining the binding (~c-capture
assay).
These experiments demonstrate the high
reliability of the test carried out using the immunologi
cally active polypeptides according to the invention.
The VP2 region contained in the antigens called
"PANSE, PAPST and VP-2" results in no additional increase
in the sensitivity for the determination of antibodies
from patients with long-passed infection. On the other
hand, a good reaction with these antigens is to be found
in the case of sera within infection only in the recent
past. This antigen is therefore suitable for providing
information about the 'timing of the infection.
In a test kit it is possible to admix one or a
mixture of these antigens either with the VP1 portions
produced by genetic engineering or with the synthetic
peptide, or else to use these in separate mixtures where
the discrimination of the reactivity with these two
- 23 -
regions provides additional information about the timing
of the infection.
A further improvement in the sensitivity can be
achieved by selective binding of the serum IgM antibodies
to the solid phase by means of monoclonal antibodies,
adding recombinant antigen and determining the binding
(~a-capture assay).
EXAMPLE 5
Use of B19-specific DNA primers for direct detection of
pathogen
Any H19 DN.A present were obtained from the
investigation samples (serum, biopsies) by proteinase K
digestion in the presence of 1~ SDS (2h, 37°C), phenol
extraction and precipitation in 70~ ethanol. This, and
the DNA amplification which then followed too, was
carried out in ana:Logy to the procedure described in
Example 1. Primers 0-5 and O-2 (see Fig. 1 for the
sequence and position on the B19 genome) were used; in
the case of B19-positive samples, the amplified fragment
has a size of 319bp. Demonstration of the B19-specificity
of the DNA fragment was carried out after fractionation
of the PCR mixtures by a 1.5$ agarose gel, transfer of
the DNA to a nitrocellulose membrane (Southern blot) and
hybridisation with a piece of DNA which was located
between them and which had been labelled either radio-
actively with P-3c. or with digoxigenin by conven-
tional methods (primer extension). The DNA fragment used
for w:ze hybridisation was obtained in the following way:
a DNA fragment 260 by long was isolated from the plasmid
pUCI2PAN after digestion with HincII and PstI and in
serted into the HincII and Pstl sites in pUCl2. It is now
possible for the B:19~ fragment without the sequences u~~ed
for the amplification to be obtained from the resulting
plasmid (pUCI2PCRDIA) by EcoRI/PstI digestion and be
employed after labe~_bing as hybridisation probe.
CA 02075366 2001-03-14
CA 02075366 2001-11-23
- 23a -
In summary, this invention relates to an
immunologically active peptide or polypeptide which has
part of the amino-acid sequence of the capsid proteins VP1
or VP2 of parvovirus B19, characterised in that it is free
of impurities which may interfere with the detection of
parvovirus Bl9 specific antibodies, and the polypeptide is
a partial sequence of 8 to 50 amino-acid residues, in
particular 10 to 32 amino-acid residues, of the peptide
PAN-1, as depicted in Fig. 2-1, or has one or more amino
acid sequences selected from a group consisting of:
Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys His
Ile Lys;
Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys
Ala Val Tyr;
Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro
Ser Ser;
Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe
Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala;
Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu
Asp Leu His Lys Pro Gly Gln Val;
Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser
Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu;
Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile
Lys Asn Glu Thr Tly Phe;
Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu
Ala Ser;
CA 02075366 2001-11-23
- 23b -
His Met Ser Lys Lys Ser Gly Lys Trp Trp G1u Ser Asp Asp Lys Phe
Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr
Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile
Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val
Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His
His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser
Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser
Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly
Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln
Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln
Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp
Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln
Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Gly Glu Phe Ile Val
Thr Asp;
Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp
Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu
Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu
Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser
Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser
Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp
His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly
Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val
Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu
Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Arg Glu Phe Ile
Val Thr Asp;
Gly Ile Leu Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys
Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe
Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln
Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn
Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys
Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu
Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro
Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly
Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn
Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Leu Glu
Asp Pro Arg Val Pro Ser Ser Asn Ser;
CA 02075366 2001-11-23
- 23c -
Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp
Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu
Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu
Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser
Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser
Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp
His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly
Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val
Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu
Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His
Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr
His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu
Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly
Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro
Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser
Ala Gly Arg Arg Ile Pro Gly Asn Ser Ser;
Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala
Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly
Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser
Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala
Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His
Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser
Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser
Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr
Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser
Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu
Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu
Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val
Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His
Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys
Tyr Pro Ser;
or partial sequences thereof
CA 02075366 2001-11-23
- 23d -
or selected from a group consisting of:
Met Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys Trp
Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val
Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile
Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro
Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn
Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser
Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg
Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln
Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu
Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile
His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr
Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn
Glu Thr Gly Phe Gln Ala G1n Val Val Lys Asp Tyr Phe Thr Leu Lys
Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val
Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn
Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val
Lys Ser Met Trp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr
Cys Thr Phe Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His
Tyr Lys Val Phe Ser Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly
Lys Glu Ala Lys Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr_
Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro
Leu Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp
Ala Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp
Lys Thr Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu
Cys Met Leu Val Asp His Glu Tyr Lys Tyr Pro Tyr Val Leu Gly Gln
Gly Gln Asp Thr Leu Ala Pro Glu Leu Pro Ile Trp Val Tyr Phe Pro
Pro Gln Tyr Ala Tyr Leu Thr Val Gly Asp Val Asn Thr Gln Gly Ile
Ser Gly Asp Ser Lys Lys Leu Ala Ser Glu Glu Ser Ala Phe Tyr Val
Leu Glu His Ser Ser Phe Gln Leu Leu Gly Thr Gly Gly Thr Ala Ser
Met Ser Tyr Lys Phe Pro Pro Val Pro Pro G1u Asn Leu Glu Gly Cys
Ser Gln His Phe Tyr Glu Met Tyr Asn Pro Leu Tyr Gly Ser Ser Arg
Val Asp Leu Gln;
CA 02075366 2001-11-23
- 23e -
Met Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys Trp
Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val
Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile
Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro
Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn
Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser
Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg
Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln
Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu
Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile
His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr
Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn
Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys
Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val
Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn
Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val
Lys Ser Met Trp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr
Cys Thr Phe Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His
Tyr Lys Val Phe Ser Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly
Lys Glu Ala Lys Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr
Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro
Leu Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp
Ala Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp
Lys Thr Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu
Cys Ser Asn
This invention further relates to a test kit for
the detection of antibodies against human parvovirus B19,
characterised in that it has at least one immunologically
active peptide or polypeptide according which is able to
react with the antibodies present in the investigation
fluids, and in that it has at least one indicator component
which makes it possible to detect complexes of
immunologically active peptide and antibody.
CA 02075366 2001-11-23
- 23f -
This invention further relates to a process for
the purification of immunologically active peptides or
polypep.tides characterised in that it comprises the
dissolving of unpurified peptide or polypeptide in 8M urea,
and fractionation by a DEAF SephacelTM column with an NaCl
gladient.
This invention further relates to the use of at
least one DNA sequence selected from 0-1:
GTG AAT TCT GAT CAT ATG AGT AAA AAA AGT GGC AAA TGG
0-2:
C TTC GGT CGT GAC CAC GTC CTC CCC
0-3:
G AGG AAT TCT CTG ATC ATG ACT TCA GTT AAT TCT GCA GAA GCC
0-4:
GAG GGG TGG CAC GGG ACT CGG TCC TTC GAA GAG
0-5:
G CTA CAA GCT GGG CCC CCG CAA AG
for the direct detection of parvovirus B19 by means of DNA
amplification, especially by means of polymerase chain
reaction.
This invention further relates to the use of
immunologically active peptides as vaccines against
infections with parvovirus B19.
- 24 -°
DESCRTPTION OF THE FIGURES
Fig. 1: Diagrammatic representation of the VP1/2 encoding
region of parvovirus B19 with the primer sequences used
for the amplification.
The structure of the single-stranded 819 genome
with the inverse regions at the ends (double strand) and
with coding regions is depicted diagrammatically in the
upper part. The coding region for the non-structural
proteins (NS) which are synthesised as polypeptide and
then processed is in the left region. The right region
codes for the surface proteins of the viral capsid
(VP1/2), with VP1 being, apart from an additional N-
terminal region (shaded bar), identical to VP2 (black
bar). Underneath this are indicated the regions of
oligodeoxynucleotides 0-1 to O-4 which were used as
primers for the amplification (PCR) of the H19 sequences
located between them (0-1 and O-2 for the VP1 region, and
O-3 and O-4 for VP-2).
The DNA sequences of the corresponding B19
regions as well as of the oligodeoxynucleotides are
indicated in the lower part of the figure. The oligo
deoxynucleotide sequences are identified by bold print,
non-homologous regions, that is to say sequences which do
not hybridise with B19, are contrasted by a line spacing.
These non-hybridising sequences represent restriction
enzymes sites for EcoRI (GAATTC) and BclI (TGATCA) in the
case of O-l, for EcoRI, BclI and BspHII (TC-ATGA) in the
case of 0-3, and for HindITI (AAGCT-T) in 'the case of
0-4. The amplified VP2 encoding fragment (O-3 and O-4)
was digested with EcoRI and HindIII before insertion in
pUC vectors, the VP1 encoding fragment with EcoRI and
PstI, the PstI cleavage site being located in the B19 DNA
(from position no. 4 in the indicated sequence for 0-2,
C'PGCAG ) .
- 25 -
Fig. 2: Amino-acid sequences of the antigens described in
Example 2 and produced by recombination in E.coli cells.
Owing to cloning steps, in each case some non
B19-authentic foreign amino acids are also contained at
the N-termini and at. the C-termini (apart from PANSE and
VP-2) and are emphasised by bold print.
The amino-acid sequences of the antigens
described in Example 2 are described:
Fig. 2-1: PAN-1.
Fig. 2-2: PCE
Fig. 2-3: PAN-2:
Fig. c-4: PAN-3~
Fig. 2-5: PAN-9
Fig. 2-6: VP2
Fig. 2-7: PANSE~
Fig. 2-8: PAPST
Fig. 2-9: PAV-1B
Fig. 2-10: PAV-1N
Fig. 3: Diagrammatic' representation of the arrangement of
some peptides with respect to one another
CA 02075366 2001-03-14
