Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-05-15
2001-08-14
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S975000
Reexamination Certificate
active
06274307
ABSTRACT:
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) 72-73) by countercurrent electrophoresis. In recent years it has been shown that B19 may cause an aplastic crisis in patients with chronic haemolytic crisis (sic), and is 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 CsCl
2
(sic) is 1.36-1.40 g/ml. The virus genome consists of a single-stranded DNA of 5.4 kb. The nucleotide sequence of the genome of a B19 parvovirus has been derived from a clone which contained virtually the complete viral genome (R. O. 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 parovirus (sic), that is to say requires no helper virus for replication.
The capsid consists of two polypeptides with molecular weights of 83 kDa (VP1) and 58 kDa (VP2). In addition, three non-structural proteins of 52, 63 and 71 kDa can be detected.
The DNA codes in the 5′ region for the structural proteins of the capsid. The coding regions of the structural 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 are 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 succeeded.
B19 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 interuterinal (sic) death. Furthermore, B19 is associated with the occurrence of Schonlein-Henoch purpura.
As a rule, B19 is transmitted by droplet infection but also by antigen-positive conserved blood and coagulation products.
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 blood from donors 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, detection of B19-specific antibodies of the IgG and IgM class. This results in the following possible applications:
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 1319 virus is no longer possible by anti-B19 IgG positive blood or plasma.
Selection of anti-B19 positive plasma donors for 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 polypeptides cannot be prepared directly. Preparation 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 impurities which interfere with the detection of antibodies directed against parvovirus B19. 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 antibodies 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 active peptides according to the invention is that they can be prepared in good yield by the preparation process according 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 subsequent 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 PAN1 which is depicted in
FIG. 2-1
.
In a preferred embodiment of the present invention, 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 peptides having the amino-acid sequences PAPEP 1-PAPEP 8 shown in Example 3.
In another preferred embodiment of the present invention, the amino-acid sequences which are depicted in
FIG. 2
of the immunologically active peptides PAN-1, PAN-2, PAN-3, PAN-4, PCE, PANSE AND (sic) PAPST prepared by genetic 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 peptides 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, the 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 investiga
Motz Manfred
Soutschek Erwin
MIKROGEN molekularbiologische Entwicklungs-GmbH
Mosher Mary E.
Norris & McLaughlin & Marcus
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