1. Technical Field
The present invention relates, in general, to a method of producing parvovirus antigens, and in particular, to a method of producing empty, and thus non-infectious, parvovirus capsids, and to diagnostic assays and vaccines utilizing same. The invention also relates to a method of packaging and delivering genetic information using the empty parvovirus capsids. The invention further relates to a method of packing and delivering nonparvovirus proteins, such as other antigens, ligands and enzymes, using empty parvovirus capsids.
2. Background Information
Parvoviruses are common agents of animal disease. The first strong link between parvovirus infection and human disease came from the serendipitous discovery in 1975 of parvovirus-like particles in the sera of normal human blood donors (one of the samples having been designated B19). Since that time, B19 parvovirus has been identified as the causative agent of: i) transient aplastic crisis (TAC) of hemolytic disease, ii) the common childhood exanthem called fifth disease; iii) a polyarthralgia syndrome in normal adults that may be chronic and resembles in its clinical features, rheumatoid arthritis; iv) some cases of chronic anemia and/or neutropenia; and v) some cases of hydrops fetalis. The entire spectrum of human illness caused by parvoviruses, however, is not yet clear due, in large part, to the fact that an appropriate assay is not widely available.
Parvoviruses require replicating cells for propagation, and parvovirus infection, therefore, results in pathologic changes in mitotically active host tissue. In infected children and adults, B19 parvovirus replicates in the bone marrow; in the fetus, B19 parvovirus replicates in the liver, there a hematopoietic organ. Erythroid progenitor cells are the only cell type known to be subject to infection by this virus.
The limited host and tissue range of B19 parvovirus has hampered the development of assays specific for the virus. Since the discovery of the virus, the quantity of B19 antigen available as a reagent has been limited to that obtainable from sera fortuitously obtained from infected patients. The virus has an extraordinary tropism for human erythroid progenitor cells and has only been propagated in human bone marrow cell cultures (Ozawa et al. Science 233:883 (1986)), fetal liver (Yaegashi et al. J. Virol. 63:2422 (1989)) and, to a much lesser degree, in erythroleukemia cells (Takahashi et al. J. Inf. Dis. 160:548 (1989)). The bone marrow cultures, however, require explanted bone marrow cells and, therefore, are not practical for virus propagation. The development of and availability of clinical assays continue to be limited by the availability of the antigen. The production of stable transformants capable of producing B19 protein products has been prevented by the fact that some of these products are lethal to transfected cells.
It is a general object of the invention to provide a method of producing large quantities of parvovirus antigens.
It is a specific object of the invention to provide a method of effecting the expression of parvovirus structural proteins in cell culture.
It is another object of the invention to provide non-infectious parvovirus capsids.
It is a further object of the invention to provide a safe and effective method of producing antibodies against parvovirus capsid proteins.
It is a still further object of the invention to provide a vaccine effective against parvovirus infection.
It is another object of that invention to provide diagnostic assays for detecting the presence in biological samples of parvovirus particles or antibodies thereto.
It is a further object of the invention to provide a method of treating hemoglobinopathies, enzyme deficiency states and other diseases that may be amenable to genetic therapy.
It is another object of the present invention to provide a method of presenting antigens, ligands and enzymes utilizing the parvovirus capsids.
Further objects will be clear to one skilled in the art from the following detailed description of the present invention.
In one embodiment, the present invention relates to a method of producing parvovirus capsids comprising the steps of:
i) introducing into a host cell a recombinant DNA molecule comprising:
a) an expression vector, and
b) a DNA sequence encoding the structural proteins of a parvovirus, with the proviso that genes encoding non-structural parvovirus protein are not included in the DNA sequence;
ii) culturing the cells under conditions such that the structural proteins are produced and self assemble to form the capsids; and
iii) isolating the capsids.
In another embodiment, the present invention relates to a parvovirus antigen consisting essentially of a parvovirus capsid.
In a further embodiment, the present invention relates to a parvovirus antigen consisting essentially of a parvovirus capsid of major structural proteins free of minor structural proteins.
In yet another embodiment, the present invention relates to a diagnostic assay for parvovirus infection comprising:
i) contacting a sample from a patient suspected of being infected with parvovirus with the above-described parvovirus capsid, and
ii) detecting the formation of a complex between anti-parvovirus antibodies present in the sample and the parvovirus capsid.
In another embodiment, the present invention relates to an anti-parvovirus vaccine comprising the above-described parvovirus capsid and a pharmaceutically acceptable carrier.
In another embodiment, the invention relates to a method of packaging and transferring genetic information comprising
i) encapsidating the genetic information in the above-described parvovirus capsid and
ii) introducing the encapsidated information into a host cell.
In yet another embodiment, the present invention relates to a diagnostic kit comprising:
i) the above-described parvovirus capsid; and
ii) ancillary reagents.
In a further embodiment, the present invention relates to a recombinant baculovirus comprising a DNA segment encoding a minor structural protein of a parvovirus and to a recombinant baculovirus comprising a DNA segment encoding a major structural protein of a parvovirus.
In another embodiment, the present invention relates to a method of producing parvovirus capsids comprising the steps of:
i) infecting an insect cell with the recombinant baculovirus encoding the major structural protein or co-infecting an insect cell with both of the above-described recombinant baculoviruses;
ii) culturing the cells under conditions such that the major structural proteins are produced and self assemble to form the capsids; and
iii) isolating the capsids.
In yet a further embodiment, the present invention relates to a method of producing a protein presenting capsid comprising the steps of:
i) coinfecting an insect cell with (a) a first recombinant baculovirus encoding a major structural parvovirus protein and (b) a second recombinant baculovirus encoding the nonunique region of a minor structural parvovirus protein and a nonparvovirus protein;
ii) culturing the cells under conditions such that the expressed proteins self assemble to form the capsids; and
iii) isolating the capsids.
In another embodiment, the present invention relates to a protein presenting capsid comprising a major structural parvovirus protein and a nonunique region of a minor structural parvovirus protein joined to a nonparvovirus protein.
USPTO Backgrounds
USPTO Backgrounds