Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1995-06-05
2002-04-30
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
Reexamination Certificate
active
06379885
ABSTRACT:
The invention relates both to the field of genetic manipulation by means of the recombinant DNA technology for the production of certain proteins and/or particles that consist of one or more of these proteins, and to the fields of diagnostics and vaccine preparation. The invention concerns certain viral proteins, which may or may not be in the form of virus-like particles, which proteins or particles can for instance be used in assays for detecting antibodies directed against these proteins, or can be used to obtain such antibodies, or can be used to accomplish protection against the virus, or can be used for the incorporation therein of epitopes of proteins of other pathogenes to accomplish protection against these other pathogenes (and thus offers various possibilities of use for vaccination purposes).
More particularly, the invention relates to the coat proteins VP1 and VP2 of the human parvovirus B19 and to virus like particles that consist of VP2 or of VP1 and VP2. The invention further comprises genetic information in the form of recombinant expression vectors which contain the genes coding for these proteins, and organisms that have acquired the ability to produce the proteins and/or particles in question owing to genetic manipulation using such vectors.
The human parvovirus B19 was serendipitously discovered in 1975 in serum samples of some healthy blood donors. Since that time it has been found that the virus causes erythema infectiosium 13 also known as “fifth disease”—and of the so-called “aplastic crisis” in patients with chronic hemolytic anemia. The B19 virus is further associated with abortion and fetal death, with arthritis and with chronic anemia in immuno-deficient patients. Infections may also occur under other syndromes or occur entirely asymptomatically.
Infections with this virus, which is found throughout the world, usually occur in epidemics which take place about every 3-6 years, but may occur sporadically in intervening years. Today, fourteen years after the discovery of the B19 virus, the diagnostics for infection with the virus are still performed in only a limited number of laboratories in the world. Because the virus cannot be demonstrated anymore in the patients at the time when the symptoms arise (viremia and virus excretion precede the symptoms), diagnostics must focus on demonstrating B19-specific (IgMr)-antibodies.
To this end (and also for the preparation of suitable vaccines, for example) it is necessary to have a sufficient supply of B19-antigen for setting up the tests. What is lacking, however, is a suitable in vitro cell culture system for propagating the virus, with which sufficient antigen can be obtained.
The existing parvovirus B19 diagnostics are performed with virus antigen which becomes available more or less by chance (screening blood donors offers an estimated chance of 1 in 50,000 that viremic blood is found).
For these reasons there is a great need for antigen which is produced using recombinant DNA techniques. Accordingly, various proposals in this direction have already been made, but none of them has proved really useful for the construction of a diagnostic test.
The present invention is based on the use of an expression vector system that was developed fairly recently, viz. the “Baculovirus Expression Vector System”. In this system use is made of a recombinant virus vector of the baculo-virus
Autoaraphica californica
nuclear polyhedrosis virus (AcNPV) to express the B19 virus proteins in insect cells:
Spodoptera frugiperda
(Sf9). This system offers many advantages over the current systems of expression vectors:
a) In view of the use for diagnostic and possibly therapeutic (vaccination) purposes, no cross reactivity is to be expected against proteins of the baculovirus or the insect cells (in proteins which are expressed in
E.coli
, this cannot always be precluded).
b) The virus proteins can be produced in large amounts (1-500 mg/1) up to even 50-75% of the total protein, detected on SDS-polyacrylamide gel (Summers and Smith, 1986, a manual of methods for baculovirus vector and insect cell culture procedures; Yong Kang, 1988; Adv. in Virus Res. 35, 177-192). These are considerably larger amounts than those produced in prokaryotic expression systems or in Chinese hamster ovary cells, as described by Kajigaya et al. (Blood 75(5), suppl.1, 44a, abstr. 86; 1988).
c) The proteins can be produced as non-fusion proteins, in contrast to for instance the B19 protein, which has been produced as a fusion protein in
E.coli
by Sisk and Berman (Biotechnology 5, 1077-1080, 1987). This recombinant &bgr;-galactosidase-B19 fusion protein goes into solution only in the presence of sodium dodecylsulphate (SDS). The proteins VP1 and VP2 expressed in insect cells in accordance with the invention, by contrast, can easily be dissolved by sonification of the cells in a buffer which contains 25 mM NaHCO
3
and 20 mg/l NaN
3
(pH 9.5). In such a treatment 95% of the cellular proteins go over into the soluble supernatant fraction.
d) The proteins can be produced in an insect cell line which is easy to culture, as opposed to the production of virus proteins in human erythroid bone marrow cells (Ozawa et al., 1987; Blood 70, 384-391) or human foetal erythroid liver cells (Yaegashi et al., 1989; J. Virol.
63, 2422-2426).
e) Because in the baculovirus expression vector system pre- and post-translation modifications occur, such as phosphorylation, glycosylation, signal peptide split-off and the removal of introns by splicing, the system is potentially very suitable for the production of biologically active proteins with a (virtually) native structure (Yong Kang, 1988; Adv. in Virus Res. 35, 177-192). In this system VP1 and VP2 of B19 can be expressed both separately and collectively. Moreover, the possibility exists that virus-like particles are spontaneously formed from one or more of these proteins.
f) An additional advantage of the baculovirus is that it does not multiply in mammalian cells and hence is not pathogenic for humans, which makes it much safer to work with and utilize this system.
According to the invention it has actually been accomplished to produce in a high yield the coat proteins VP1 and VP2 of the human parvovirus B19 in an antigenically active form as non-fusion proteins, as virus-like particles or not, using the baculovirus expression system in insect cells (
Spodoptera frugiperda
). Further, it has been accomplished to develop, using the B19 virus proteins producing insect cells, a specific and sensitive immunofluorescence-assay (IFA) and a specific and sensitive Enzyme-Linked-Immuno-Sorbent-Assay (ELISA) for the detection of antibodies directed against the B19 virus proteins. However, on the basis of the B19 virus proteins and virus-like particles produced in insect cells in conformity with the invention, other diagnostic assays can be developed as well, such as a Radio-Immuno-Assay (RIA) or an agglutination test.
The invention is primarily embodied in recombinant VP1 and VP2 protein of the human parvovirus B19, formed in
Spodoptera frugiperda
cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of the B19 virus protein VP1 and/or VP2. The invention further comprises recombinant virus-like particles which consist of VP2 protein or of VP1 and VP2 protein of the human parvovirus B19, formed in
Spodoptera frugiperda
cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information necessary for expression of VP2 protein or of VP1 and VP2 protein.
Further, the invention is embodied in
Spodoptera frugiperda
cells which, by means of a baculovirus expression vector system, have been equipped with the genetic information which is necessary for expression of VP1 and/or VP2 protein of the human parvovirus B19.
The invention further provides a method of producing VP1 and/or VP2 protein of the human parvovirus B19 (optionally in the-form of virus-like particles which are composed of VP2 protein or
Greenberg & Traurig, LLP
Heath David L.
Mosher Mary E.
Rijksuniversiteit te Leiden
Rzucidlo Eugene C.
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