U.S. patent application number 10/366810 was filed with the patent office on 2003-08-07 for parvovirus capsids.
This patent application is currently assigned to The United States of America, represented by the Secretary, Department of Health and Human Services. Invention is credited to Kajigaya, Sachiko, Takashi, Shimada, Young, Neal S..
Application Number | 20030147919 10/366810 |
Document ID | / |
Family ID | 23029907 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030147919 |
Kind Code |
A1 |
Young, Neal S. ; et
al. |
August 7, 2003 |
Parvovirus capsids
Abstract
The present invention relates to a method of producing
non-infections parvovirus capsids and to diagnostic assays and
vaccines utilizing same. The invention further relates to
recombinant baculoviruses encoding parvovirus structural proteins
and host cells infected therewith. The invention also relates to a
method of packaging and delivering genetic information utilizing
the noninfectious capsids.
Inventors: |
Young, Neal S.; (Washington,
DC) ; Kajigaya, Sachiko; (Rockville, MD) ;
Takashi, Shimada; (Bethesda, MD) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
The United States of America,
represented by the Secretary, Department of Health and Human
Services
|
Family ID: |
23029907 |
Appl. No.: |
10/366810 |
Filed: |
February 13, 2003 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10366810 |
Feb 13, 2003 |
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09611066 |
Jul 6, 2000 |
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6558676 |
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09611066 |
Jul 6, 2000 |
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08407939 |
Mar 21, 1995 |
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6132732 |
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08407939 |
Mar 21, 1995 |
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07612672 |
Nov 14, 1990 |
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5508186 |
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07612672 |
Nov 14, 1990 |
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07270098 |
Nov 14, 1988 |
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Current U.S.
Class: |
424/233.1 ;
424/204.1; 435/6.18; 536/23.72 |
Current CPC
Class: |
C12N 2750/14323
20130101; C07K 16/081 20130101; C12N 2750/14343 20130101; Y10S
977/917 20130101; C12N 2750/14222 20130101; A61K 39/00 20130101;
C12N 2750/14122 20130101; C12N 2750/14243 20130101; C12N 15/86
20130101; Y10S 977/92 20130101; C12N 2750/14322 20130101; Y10S
977/927 20130101; C07K 14/005 20130101; C12N 2710/14043 20130101;
Y10S 977/803 20130101; Y10S 977/802 20130101; Y10S 977/742
20130101; Y10S 977/898 20130101; C12N 2750/14223 20130101; A61P
31/12 20180101; A61P 31/02 20180101 |
Class at
Publication: |
424/233.1 ;
424/204.1; 536/23.72; 435/6 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 039/12; A61K 039/23; A61K 039/235 |
Claims
What is claimed is:
1. 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 nonstructural parvovirus proteins are not included
in the DNA sequence; ii) culturing the cells under conditions such
that said structural proteins are produced and self assemble to
form the capsids; and iii) isolating the capsids.
2. The method according to claim 1 wherein said parvovirus is
B19.
3. The method according to claim 1 wherein said host cell is a
mammalian cell.
4. The method according to claim 3 wherein said mammalian cell is a
dehydrofolate reductase-deficient Chinese hamster ovary cell.
5. A parvovirus antigen consisting essentially of a parvovirus
capsid.
6. The parvovirus antigen according to claim 5 wherein said
parvovirus capsid has a minor structural protein to major
structural protein ratio higher than the ration of the naturally
occurring capsid.
7. The parvovirus antigen according to claim 6 wherein said major
structural protein to minor structural protein ratio is at least
3:1.
8. The parvovirus antigen according to claim 7 wherein said ratio
is between 10:1 to 20:1.
9. The parvovirus antigen according to claim 5 wherein said
parvovirus is B19.
10. A parvovirus antigen consisting essentially of a parvovirus
capsid of major structural proteins free of minor structural
proteins.
11. A diagnostic assay for parvovirus infection comprising: i)
contacting a sample from a patient suspected of being infected with
parvovirus with said parvovirus antigen according to claim 5 or
claim 10, and ii) detecting the formation of a complex between
anti-parvovirus antibodies present in said sample and said
parvovirus antigen.
12. The assay according to claim 11 wherein said parvovirus is
B19.
13. The assay according to claim 11 wherein said sample is a serum
sample.
14. An anti-parvovirus vaccine comprising said parvovirus antigen
according to claim 5 and a pharmaceutically acceptable carrier.
15. The vaccine according to claim 14 wherein said parvovirus is
B19.
16. A method of packaging and transferring genetic information
comprising: i) encapsidating said genetic information in said
parvovirus capsid according to claim 5; and ii) introducing said
encapsidated information into a host cell.
17. The method according to claim 16 wherein said parvovirus is
B19.
18. A diagnostic kit comprising: i) said parvovirus antigen
according to claim 5 or claim 10; and ii) ancillary reagents.
19. The diagnostic kit according to claim 18 wherein said
parvovirus is B19.
20. The diagnostic kit according to claim 18 further comprising a
signal-producing system.
21. A recombinant baculovirus comprising a DNA segment encoding a
minor structural protein of a parvovirus.
22. A recombinant baculovirus comprising a DNA segment encoding a
major structural protein of a parvovirus.
23. A recombinant baculovirus according to claim 21 or claim 22
wherein said baculovirus is Autographa california nuclear
polyhedrosis virus.
24. A recombinant baculovirus according to claim 21 or claim 22
wherein said parvovirus is B19.
25. A method of producing parvovirus capsids comprising the steps
of: i) infecting an insect cell with said recombinant baculovirus
according to claim 22; ii) culturing said cells under conditions
such that said major structural proteins are produced and self
assemble to form the capsids; and iii) isolating the capsids.
26. The method according to claim 25 wherein said insect is
Spodoptera frugiperda.
27. A method of producing parvovirus capsids comprising the steps
of: i) infecting an insect cell with (a) a first recombinant
baculovirus comprising a DNA segment encoding a minor structural
parvovirus protein and (b) a second recombinant baculovirus
comprising a DNA segment encoding a major structural parvovirus
protein; ii) culturing said cells under conditions such that said
minor structural protein and said major structural protein are
produced and self assemble to form the capsids; and iii) isolating
the capsids.
28. The method according to claim 27 wherein said insect is
Spodoptera frugiperda.
29. 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 said cells under conditions
such that said expressed proteins self assemble to form the
capsids; and iii) isolating the capsids.
30. The method according to claim 29 wherein said nonparvovirus
protein is an antigenic epitope, ligand or enzyme.
31. 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.
Description
BACKGROUND OF THE INVENTION
[0001] This application is a continuation-in-part of application
Ser. No. 07/270,098 filed on Nov. 14, 1988, which is hereby
incorporated in its entirety by reference.
[0002] 1. Technical Field
[0003] 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.
[0004] 2. Background Information
[0005] 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.
[0006] 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.
[0007] 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.
SUMMARY OF THE INVENTION
[0008] It is a general object of the invention to provide a method
of producing large quantities of parvovirus antigens.
[0009] It is a specific object of the invention to provide a method
of effecting the expression of parvovirus structural proteins in
cell culture.
[0010] It is another object of the invention to provide
non-infectious parvovirus capsids.
[0011] It is a further object of the invention to provide a safe
and effective method of producing antibodies against parvovirus
capsid proteins.
[0012] It is a still further object of the invention to provide a
vaccine effective against parvovirus infection.
[0013] It is another object of that invention to provide diagnostic
assays for detecting the presence in biological samples of
parvovirus particles or antibodies thereto.
[0014] 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.
[0015] It is another object of the present invention to provide a
method of presenting antigens, ligands and enzymes utilizing the
parvovirus capsids.
[0016] Further objects will be clear to one skilled in the art from
the following detailed description of the present invention.
[0017] In one embodiment, the present invention relates to a method
of producing parvovirus capsids comprising the steps of:
[0018] i) introducing into a host cell a recombinant DNA molecule
comprising:
[0019] a) an expression vector, and
[0020] 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;
[0021] ii) culturing the cells under conditions such that the
structural proteins are produced and self assemble to form the
capsids; and
[0022] iii) isolating the capsids.
[0023] In another embodiment, the present invention relates to a
parvovirus antigen consisting essentially of a parvovirus
capsid.
[0024] 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.
[0025] In yet another embodiment, the present invention relates to
a diagnostic assay for parvovirus infection comprising:
[0026] i) contacting a sample from a patient suspected of being
infected with parvovirus with the above-described parvovirus
capsid, and
[0027] ii) detecting the formation of a complex between
anti-parvovirus antibodies present in the sample and the parvovirus
capsid.
[0028] In another embodiment, the present invention relates to an
anti-parvovirus vaccine comprising the above-described parvovirus
capsid and a pharmaceutically acceptable carrier.
[0029] In another embodiment, the invention relates to a method of
packaging and transferring genetic information comprising
[0030] i) encapsidating the genetic information in the
above-described parvovirus capsid and
[0031] ii) introducing the encapsidated information into a host
cell.
[0032] In yet another embodiment, the present invention relates to
a diagnostic kit comprising:
[0033] i) the above-described parvovirus capsid; and
[0034] ii) ancillary reagents.
[0035] 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.
[0036] In another embodiment, the present invention relates to a
method of producing parvovirus capsids comprising the steps of:
[0037] 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;
[0038] ii) culturing the cells under conditions such that the major
structural proteins are produced and self assemble to form the
capsids; and
[0039] iii) isolating the capsids.
[0040] In yet a further embodiment, the present invention relates
to a method of producing a protein presenting capsid comprising the
steps of:
[0041] 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;
[0042] ii) culturing the cells under conditions such that the
expressed proteins self assemble to form the capsids; and
[0043] iii) isolating the capsids.
[0044] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1. Human DHFR minigene DM14.
[0046] FIG. 2. Structure of the B19 capsid expression vector.
[0047] FIG. 3. Amplification of B19 capsid genes.
[0048] FIG. 4. Immunoblot of B19 capsid proteins in CHO and bone
marrow cells.
[0049] FIG. 5. Immunofluorescence of a capsid-producing Chinese
hamster ovary (CHO) cell-line: FIG. 2A--control CHO cells, and FIG.
2B transformed CHO cells.
[0050] FIG. 6. Sedimentation of B19 capsids.
[0051] FIG. 7. Electron micrograph of transformed CHO
cells--demonstration of intranuclear viral particles.
[0052] FIG. 8. Growth curves.
[0053] FIG. 9. Plasmid constructions containing the major (VP2) and
minor (VP1) capsid genes of B19 parvovirus. (A) Diagram outlining
relationship of inserts derived from pYT103c, a nearly full-length
molecular clone of parvovirus B19, and the baculovirus vector
pVL941; (B) synthesized regions of DNA used to complete the gene
sequences.
[0054] FIG. 10. Expression of B19 parvovirus proteins in insect
cells infected with recombinant baculoviruses. (A)
Immunofluorescence of Sf9 cells infected with pVP1/941 (a),
pVP2/941 (b), and wild type baculovirus (c) after staining with
convalescent phase antiserum to B19 parvovirus (.times.1500). (B)
Immunoblot of lysates from cells infected with pVP1/941 (a),
pVP2/941 (b) both pVP1/941 and pVP2/941 (c), and wild type
baculovirus after development with convalescent phase antiserum
followed by .sup.125I-labeled protein A. (C) Coomassie blue
dye-stained polyacrylamide gel of Sf9 cell lysates after infection
with recombinant baculovirus pVP1/941 (a), pVP2/941 (b), both
pVP1/941 and pVP2/941 (c), and wild type baculovirus (d).
[0055] FIG. 11. Electron micrographs of empty capsids. After
infection with either pVP1/941 plus pVP2/941 or pVP2/941, cell
lysates were subjected to equilibrium density gradient
sedimentation and examined by transmission electron microscopy
after negative staining (.times.171,000).
[0056] FIG. 12. Capture immunoassay comparing antigen derived from
serum of infected patients with Sf9 c311 lysate after in vitro
infection with pVP2/941.
[0057] FIG. 13. Shows supernormal amounts of VP1 were present in
the recombinant capsids when the relative multiplicity of infection
for VP1 and VP2 baculoviruses was increased from 1:1 (7% VP1) to
100:1 (30% VP1). These results have two significant implications.
First, because VP1 is immunogenic and probably contains the
receptor binding site, a capsid enriched for VP1 compared to virion
may be particularly effective as a vaccine reagent because it
increases the amount of desirable antigen presented to the immune
system. Second, the unique region of the VP1 plasmid could be
replaced with other epitopes and other recombinant baculoviruses
generated. In this way, the basic capsid structure could be used to
present multiple or different antigens to the host (that is,
tetanus, gp120 of HIV) in the context a stable, highly immunogenic
particulate structure.
[0058] FIG. 14. Neutralization of B19 parvovirus infectivity for
human erythroid progenitors. Sera from six rabbits immunized with
partially purified capsids composed of VP2 alone or VP2 and VP1
were tested for their ability to abrogate the toxic effect of B19
parvovirus in bone marrow cultures.
[0059] FIG. 15. Schematic representation of a protein presenting
capsid.
DETAILED DESCRIPTION OF THE INVENTION
[0060] The present invention relates to a method of producing
parvovirus structural proteins utilizing recombinant DNA
techniques, to expression vectors containing DNA sequences encoding
the structural proteins, and to cells transformed with such
recombinant molecules. The present invention also relates to
recombinant baculoviruses encoding parvovirus structural proteins
and to insect cells infected with such recombinant viruses. The
invention further relates to diagnostic assays utilizing the
recombinantly produced parvovirus protein products, or antibodies
to such proteins. The invention also relates to a vaccine effective
against parvoviral infection comprising the recombinantly produced
viral protein product. The invention further relates to methods of
treating diseases amenable to genetic therapy, i.e.,
hemoglobinopathies and enzyme deficiency states, utilizing the
recombinantly produced parvovirus protein products, specifically
parvoviral capsids, in cell transfections. The present invention
further relates to methods of producing protein presenting capsid
vehicles.
[0061] The present invention developed from Applicants' discovery
that empty, and thus non-infectious, parvovirus capsids can be
produced from the major and minor parvovirus capsid protein species
or from the major parvovirus capsid protein species alone, without
the non-structural proteins. (The minor structural protein alone
can not form a capsid.) The elimination of the noncapsid proteins
allows for the production of parvoviral particles, microscopically
indistinguishable from infectious particles, which are incapable of
killing the host cell.
[0062] In one embodiment, the present invention relates to a method
of producing parvovirus structural proteins, for example, B19
structural proteins, utilizing recombinant DNA techniques.
Advantageously, the structural proteins self assemble in the host
cell (eucaryotic or procaryotic) to form an empty, but intact,
parvoviral capsid. Quantities of parvovirus capsids equal to or
greater than those present in infected bone marrow cells, can be
produced by the method of the invention.
[0063] In a preferred embodiment, eucaryotic cells are transfected
with a recombinant DNA molecule comprising an expression vector and
the coding sequences of either the major capsid protein or the
major and minor capsid proteins of a parvovirus, under control of a
promoter. For selection, cells carrying a marker that alters the
phenotype of the cell are used as the host. The recombinant DNA
molecule containing the capsid-encoding sequences is cotransfected
with the sequence encoding the marker gene (i.e., a gene encoding
an enzyme deficient in the untransfected cell). Transformants
having the appropriate phenotype are readily selected by growing
the cells in a selective medium. (Cells can be selected positively
or negatively; negatively by the presence of a gene conferring
resistance in selective medium and positively by the expression of
a detectable marker allowing for identification and isolation of
positive cells.) Such transformants are then screened, using known
techniques, to determine which contain the capsid proteins. The
capsid proteins are isolated in substantially pure form using
protocols known in the art.
[0064] In a most preferred embodiment, CHO cells deficient in
dihydrofolate reductase (DHFR) are cotransfected with: i) a
recombinant DNA molecule comprising an expression vector and a DNA
sequence encoding the two B19 parvovirus capsid proteins driven by
the strong single B19 promoter, and ii) a human DHFR minigene
driven by the SV40 early promoter enhancer unit. Transformants
bearing the DHFR+ phenotype are selected by growing the cells in a
medium lacking nucleosides; colonies are screened by RNA Northern
analysis for expression of B19 genes. Coamplification of the
integrated B19 capsid-encoding sequence and the DHFR sequence can
be accomplished by treating the cells with increasing
concentrations of methotrexate; coamplification results in
detectable levels of protein expression.
[0065] Empty B19 parvovirus capsids are found in the nuclei and
cytosol of the CHO cells transfected and cultivated as described
above. Large quantities of capsids are not released into the
culture supernatants. The expression of the empty capsids does not
affect growth of the CHO cells.
[0066] In another embodiment, the present invention relates to a
method of producing parvovirus capsids utilizing baculoviruses, for
example, Autographa california nuclear polyhedrosis virus. Capsids
composed of the major and the minor structural proteins and capsids
composed of the major structural protein alone are produced. The
resulting capsids are suitable reagents for human use and are easy
to produce and purify.
[0067] In a preferred embodiment, recombinant baculoviruses are
produced which encode the major structural parvovirus protein, the
minor structural parvovirus protein, or a fusion protein. To form
an empty, but intact parvovirus capsid containing the major and the
minor structural proteins, host insect cells (for example,
Spodoptera frugiperda cells) are either infected with recombinant
baculoviruses encoding both structural proteins or are coinfected
with recombinant baculoviruses encoding the major protein species
and recombinant baculoviruses encoding the minor protein species.
Preferably, the cells are infected with viruses at a multiplicity
of infections ranging from 10 to 50. The infected cells are
cultured and the capsids self assemble. The assembled capsids are
isolated therefrom. Capsids composed of only the major structural
protein are produced by infecting insect cells with recombinant
baculoviruses encoding the major protein.
[0068] That capsids, morphologically indistinguishable from natural
empty capsids, assemble from the major parvovirus structural
protein, VP2, alone was unexpected. In computer modeling, VP1, the
minor parvovirus structural protein species, has been proposed as
an internal protein that stabilized the capsid structure
(Parvoviridae, In Animal Virus Structure ed. Nermut and Steven,
Elsevier, New York 325-334 (1987)). The present data indicate
instead that the minor structural protein is not required for
capsid assembly; in fact, the minor structural protein alone is
incapable of forming a capsid structure. Several pieces of data are
consistent with the presence of VP1 on the surface of the capsid
structure. Only VP1-containing capsids allow production of
neutralizing antibodies in rabbits. Individual human convalescent
phase antisera and pooled immunoglobin recognize mainly VP1 rather
than VP2, even though the main acid sequence of VP2 is
entirely-contained within VP1; antisera that recognize
predominantly VP1 are equivalent to antisera that recognize VP2 in
their ability to neutralize virus (J. Clin. Invest. 84:1114-1123
(1989)). VP1's unique region of 228 amino acids contains at least
one neutralizing epitope by peptide mapping.
[0069] The minor structural protein appears to be more immunogenic
than the major protein. For example, most convalescent sera, that
is sera from people who have been exposed to the virus previously,
recognize predominantly the minor structural protein. The minor
structural protein may be the binding site for the receptor. Empty
capsids having a higher minor structural protein to major
structural protein ratio are useful for immunization as the number
of neutralizing sites is increased.
[0070] In a preferred embodiment, capsids enriched with the minor
structural protein are produced by altering the relative amounts of
VP1 and VP2 containing baculoviruses, so that the ratio of VP1 to
VP2 is between 10:1 to 100:1.
[0071] In another embodiment, the present invention relates to a
safe and effective method of producing antibodies against
parvovirus capsids. The method comprises immunizing a mammal with
the non-infectious, empty parvoviral capsids described above
comprising the major and the minor structural proteins, using
protocols known in the art, and isolating the antibodies produced.
Monoclonal antibodies specific for the parvoviral capsid can also
be produced and isolated using known techniques. In a preferred
embodiment, the antibodies, or useful binding fragments thereof,
are specific for an epitope present on the B19 capsid.
[0072] In another embodiment, the present invention relates to a
vaccine effective against parvoviral infection. The vaccine
includes the empty, non-infectious capsids described above (or an
immunogenically effective portion thereof), purified so as to be
essentially free of other proteins (that is, so as to be safe for
use as a vaccine). The capsids are preferably, composed of both the
minor and the major structural proteins since neutralizing
antibodies are elicited by capsids containing the minor structural
protein and are not elicited by capsids containing only, the major
structural protein. In a preferred embodiment, the capsids are B19
capsids. Naturally VP1 accounts for 3-5% of the protein in virion.
VP1 also accounts for about 3-5% of the protein in capsids produced
in host cells coinfected at a ratio of 1:1 with baculoviruses
encoding VP1 and baculoviruses encoding VP2. When host cells are
coinfected at a ratio of between 10:1 and 100:1 with baculoviruses
encoding VP1 and baculoviruses encoding VP2, the amount of VP1 is
increased to 25-30% of the capsid proteins (see FIG. 13).
[0073] The invention also relates to diagnostic assays and kits
based thereon for detecting the presence in a biological sample of
either parvoviral antigens or antibodies thereto. When parvoviral
antigens are sought to be detected, antibodies specific for same,
produced as described above, can be used according to known
protocols to effect antigen detection. When antibodies are sought
to be detected, the above-described empty, non-infectious
parvoviral capsids (or portions thereof recognized by the
antibody), can be used as the antigen, in accordance with known
techniques. Capsids containing the minor and the major structural
proteins as well as capsids containing only the major structural
protein can be used as the antigen. It is contemplated that
immunodeficient individuals incapable of producing antibodies
against parvovirus can be detected by challenging such individuals
with the empty, non-infectious capsid containing the minor
structural protein described above and determining whether antibody
is produced in response to the challenge.
[0074] The diagnostic kits of the invention comprise the
above-described antibodies (or binding fragments) and/or capsid
antigens and reagents, such as ancillary agents, for example,
buffering agents. Where necessary, the kit can further include
members of a signal-producing system, numerous examples of which
are known in the art.
[0075] In another embodiment, the present invention relates to
methods for packaging and delivering genetic material to the genome
of a cell. The method comprises encapsidating the genetic material
sought to transferred into the empty, non-infectious parvoviral
capsid described above containing the minor and the major
structural proteins, and introducing the capsid into a host cell
under conditions such that, once inside the cell, the genetic
material is released from the capsid and expressed. In a preferred
embodiment, adenoassociated virus DNA is used as that vector
system. (See Lebkowski et al. Mol. Cell. Biol. 8:3988 (1988) and
McLaughlin at al. J. Virol. 62:1963 (1988)).
[0076] Genetic material suitable for use in such a method includes
genes encoding proteins useful in the treatment of genetic defects,
for example, hemoglobinopathies and enzyme deficiency states. Host
cells include, for example, mammalian stem cells.
[0077] In another embodiment, the present invention relates to a
method of producing a protein presenting capsid (see FIG. 14).
Protein presenting capsid can be made by substituting nonparvovirus
proteins, such as, antigenic epitopes, ligands, enzymes or peptide
sequences, for the unique region of the minor structural protein
(e.g., VP1). (The unique region of VP1 contains amino acids 1-226.)
Recombinant baculoviruses encoding the modified minor structural
protein can be produced using methods known in the art. The
recombinant baculovirus can then be used to coinfect an insect cell
together with a recombinant baculovirus encoding the major
structural protein to effect self-assembly of a parvovirus capsid
having nonparvovirus proteins expressed on the surface thereof.
Such capsids can be used for example, to present antigenic epitopes
for vaccination purposes.
[0078] Epitopes which can be substituted for the unique region of
VP1 include, for example, vaccine epitopes, such as diphtheria or
pertussis epitopes. Further, capsids expressing multiple epitopes
(for example, pertussis and B19 and diphtheria), can be generated
using multiple recombinant minor structural protein genes. The use
of such capsids in vaccines eliminates the use live vaccine and
therefore related complications.
[0079] In addition, the unique region of the minor structural
protein can be replaced with a ligand for a cell surface receptor
or an enzyme. Capsids including ligand proteins can be targeted to
certain cells. For example, capsids expressing a portion of a
growth factor molecule would only bind to cells that had a receptor
for that molecule. Capsids of the present invention can also be
used to deliver enzymes to the circulation system to treat
diseases. As it is contemplated that different proteins can be
expressed on a single capsid surface, enzymes that attack, label or
destroy cells (for example, performs which poke holes in cells),
can be combined with ligands that target the capsid to a cell to
effect efficient cell killing or labeling. Such capsids can be used
as general delivery system for proteins.
[0080] The protein presenting capsids of the present invention can
also be used in vitro as well as for therapeutic treatment. For
example, the protein presenting capsids can be used in assays, such
as immunoassays for the detection of antibodies to various
proteins.
[0081] The following non-limiting Examples describe the invention
in more detail.
EXAMPLE I
Preparation of Recombinant DNA Molecules and Transfection of CHO
Cells
[0082] The DHFR minigene employed consisted of the entire encoding
region of the DHFR gene and included the first intron; this
construct was derived by restriction enzyme digestion and ligation
from that original DHFR minigene, DM-1 (Molec. Cell. Biol. 7:2830,
1987). The promoter-enhancer and polyadenylation signals were
derived from the SV40 virus. For transfection, the DHFR minigene
was cloned in pUC19 (see FIG. 1).
[0083] To prepare the B19 capsid expression vector, the nearly
full-length B19 genomic clone pYT103c was digested with the enzymes
EcoRl and Aat and subcloned into the standard vector pLTN-1. The
nonstructural region was deleted by digestion with Xbal and Smal
enzymes and recircularized (see FIG. 2).
[0084] CHO cells were cotransfected with DNA from two plasmid
constructs, one containing the DHFR minigene and the other
containing the B19 capsid genes. Transformants bearing the DHFR+
phenotype were selected by growing the cells in medium lacking
nucleosides and colonies were screened by RNA Northern analysis for
expression of B19 genes. Coamplification of the integrated B19
capsid encoding sequence and the DHFR sequence was accomplished by
treating the cells with increasing concentrations of methotrexate.
3-11-5 is a cell line established as described above which
expresses the B19 capsid.
EXAMPLE II
DNA and RNA Analysis
[0085] DNA was prepared by:conventional phenol-chloroform
extraction and proteinase K digestion and RNA by the conventional
guanidinium sulfate method from 3-11-5 cells before and after
culture in increasing concentrations of methotrexate (final
concentration=10 .mu.M). DNA was analyzed by Southern and RNA by
Northern hybridization using pYT103c, a B19 specific labeled DNA
probe (Science 233:883 (1986)). The migration on agarose gel
electrophoresis of the B19 DNA from 3-11-5 cells is consistent with
the size of the transfected DNA insert and that of the RNA with the
transcripts expected from the right side of the virus genome (J.
Virol. 61:2395 (1987)) (see FIG. 3).
EXAMPLE III
Comparison of B19 Capsid Accumulation by Immunoblot
[0086] 3-11-5 cells were compared to normal or erythroid bone
marrow cells inoculated with virus and harvested at 48 hours (the
peak of virus production; Blood 70:384 (1987)). Capsid protein was
detected by Western blot using convalescent phase antiserum
containing high titer anti-B19 capsid protein IgG (J. Virol.
61:2627 (1987)) (see FIG. 4). The amount of 58 kd and 83 kd protein
in 3-11-5 cells was intermediate between that harvested from
cultures of normal and erythroid bone marrow. From comparison to
known standard plasma preparations, it has been estimated that each
3-11-5 cell contains between 1000-20000 capsids.
EXAMPLE IV
Immunofluorescence
[0087] 3-11-5 and control CHO cells ware fixed with acetone and
stained with human convalescent phase serum containing anti-B19
capsid antibodies followed by fluorescein-conjugated anti-human IgG
(J. Clin. Invest. 74:2024 (1984)). All 3-11-5 cells show a pattern
of strong and specific immunofluorescence in both cytoplasm and
nuclei (see FIG. 5).
EXAMPLE V
Sedimentation Analysis of Capsids from 3-11-5 Cells
[0088] Capsids from CHO 3-11-5 cells were compared to viral
particles from human bone marrow culture Blood 70:385 (1987)).
Proteins were labeled by exposure of cultures to 35S-methionine,
the cells were lysed, and the particulate fraction obtained by
centrifugation over a 40% sucrose cushion (J. Virol. 61:2627
(1987)). After suspension of the particulate fraction in a small
volume of buffer, radioactively labeled capsids or virions were
applied to sucrose (J. Clin. Invest. 73:224 (1984)) or cesium
chloride (Science 233:883 (1986)) gradients (see FIG. 6). On
sucrose gradient sedimentation, empty capsids were clearly
distinguished from intact virions, and isopycnic sedimentation in
cesium showed a density consistent with empty capsids.
EXAMPLE VI
Electron Microscopy of 3-11-5 Cells
[0089] Cells were fixed and prepared for transmission EM as
described (J. Clin. Invest. 74:2024 (1984)). Characteristic
clusters of 20 nm particles were observed in the nuclei of 3-11-5
cells only (see FIG. 7).
EXAMPLE VII
Growth Curves of 3-11-5 Cells Compared to Other CHO Cells
[0090] Cells were serially harvested from microtiter wells and
manually counted. Empty capsid production does not adversely affect
cell proliferation of 3-11-5 (see FIG. 8).
EXAMPLE VIII
Preparation of Recombinant Baculoviruses, Transfection of Sf9 Cells
and Expression of Capsids
[0091] Cell culture and virus stocks were prepared as follows.
Recombinant plasmid was used to generate recombinant baculoviruses.
Autographa california nuclear polyhedrosis virus (AcMNPV) and
recombinant polyhedrosis viruses were grown in monolayers of Sf9
cells. The Sf9 cell line (American Type Culture Collection,
Rockville Md.), which is derived from Spodoptera frugiperda (fall
army worm) ovary, was maintained in Grace's insect tissue culture
medium containing 10% heat inactivated fetal bovine serum, 2.5
.mu.g/ml fungizone, 50 .mu.g/ml gentamicin, 3.33 mg/ml lactalbumin
hydrolysate, and 3.33 mg/ml yeastolate (provided complete by Gibco
BRL Life Technologies, Gaithersburg Md.) at 100% room air, 95%
humidity, at 27.degree. C.
[0092] Recombinant plasmids and recombinant baculoviruses were
constructed as follows. Two plasmids were constructed, one
containing the full length major capsid protein gene (VP2), the
other the full length minor capsid protein gene (VP1). To construct
plasmid pVP1/941, a cDNA encoding the VP1 gene was excised from
pYT103c, a nearly full length molecular clone of B19 parvovirus
(Cotmore et al. Science 226:1161 (1984); Ozawa et al. J. Virol.
62:2884) 1988)), by digestion with the restriction enzymes Hind III
(which cuts at map unit 45) and EcoRI (which cuts at map unit 95)
followed by treatment with mung bean nuclease to complement single
stranded ends.
[0093] The resultant DNA fragment was inserted into the BamHI site
(made blunt ended with the Klenow fragment of DNA polymerase) of
the baculovirus transfer vector pVL941, a vector derived by
deletion of the polyhedrin gene of AcMNPV followed by cloning into
the pUC8 plasmid (Summers et al. Tex. Agric. Exp. Stn. 1555
(1987)). Construction of pVP2/941 was performed by the insertion of
a PstI-EcoRI digestion fragment of pYT103c (map units 58-95; the
EcoRI site was blunt-ended) and a synthetic DNA fragment of 20
nucleotides corresponding to the SstI-PstI region (again with the
SstI site blunt-ended) into the BamHI site of pVL941 (FIG. 9).
[0094] Recombinant plasmids were used to generate recombinant
baculoviruses. Eight .mu.g of each of the recombinant plasmids was
cotransfected into Sf9 cells with 2 .mu.g of wild type AcMNPV,
using calcium phosphate-mediated precipitation. Six days after
transfection, progeny virus was harvested and replaqued onto fresh
Sf9 cells. Recombinant viruses were recognized visually by the
absence of occlusion bodies in the nucleus of cells (the
occlusion-positive phenotype is the result of synthesis of large
quantities of the polyhedrin protein). Recombinant viruses were
subjected to three cycles of plaque purification before large scale
virus stocks were prepared.
[0095] For analysis of protein expression and capsid structure, Sf9
cells were infected with recombinant viruses at multiplicity of
infections (m.o.i.) ranging from 10 to 50. Cells were harvested and
examined for expression of VP1 and VP2 at variable times after
infection; four days post-infection was judged optimal for
recombinant protein expression. Cytocentrifuge preparations
(approximately 1.times.10.sup.5 cells/slide well) of recombinant
(VP1-VP2 baculovirus) or wild type virus-infected cells were fixed
in acetone at -20.degree. C. for 30 seconds, washed twice in
phosphate buffered saline (PBS) containing 0.5% bovine serum
albumin, and blotted dry. Cells were stained with convalescent
phase human anti-B19 parvovirus antiserum (diluted 1:20), followed
by application of fluorescein isothiocyanate-conjugated goat
antihuman IgG (diluted 1:50: Kierkegaard and Perry, Gaithersburg,
Md.).
[0096] All cells stained specifically with the human convalescent
phase human antiserum, with bright fluorescence observed over
cytoplasm and nuclei of fixed cells (FIG. 10); the fluorescent
signal was maximal 3-4 days after infection and had faded after one
week of culture, at which time most of the cells were no longer
viable.
[0097] For analysis of proteins by gel electrophoresis, lysates
from 4 day old cultures were prepared by heat disruption at
100.degree. C. for 3 minutes in 100 .mu.l of Laemmli sample buffer
(Nature 227:680-685 (1970)). Aliquots of each sample were applied
to 8% polyacrylamide gels (10 .mu.l/lane) in the presence of sodium
dodecyl sulfate as described by Laemmli. Proteins were directly
visualized by staining with visualized by staining with 0.25%
Coomassie brilliant blue dye. For immunoblotting, proteins were
transferred by eletroblotting onto nitrocellulose membranes
(Hoeffer Scientific, San Francisco Calif.). Specific proteins were
detected by sequential application of convalescent phase human
antiserum (diluted I:300) and .sup.125I-labeled protein A
(Amersham, Arlington Heights Ill.) by the BLOTTO method (GeneAnal.
Tech. 1:3-8 (1984)).
[0098] Bands of the appropriate molecular weight were detected
after infection with the VP1-baculovirus (FIG. 10B, lane a),
VP2-baculovirus (FIG. 10B, lane b), or after coinfection with both
recombinant viruses (FIG. 10B, lane c). Large enough quantities of
parvovirus structural proteins were produced to be visible after
dye staining of polyacrylamide gels of lysates (FIG. 10C);
parvovirus protein was estimated by densitometry to constitute 2-3%
of total cell protein present.
[0099] Capsids were examined by electron microscopy after
equilibrium density gradient sedimentation. Sf9 cells were
harvested 4 days after inoculation with recombinant baculoviruses
(VP1 alone, VP2 alone, or VP1 plus VP2). Lysates were centrifuged
at 100,000.times.g over 40% (wt/vol) sucrose in Hank's balanced
salt solution. Precipitates were mixed with CsCl in 50 mM Tris-HCl,
pH 8.7, 5 mM EDTA, and 0.1% sarcosyl at an initial density of 1.31
gr/ml, centrifuged at 100,00.times.g in an SW41 rotor for 35 hrs at
18.degree. C. Transmission electron microscopy was performed after
three such banding procedures.
[0100] Banding of parvovirus proteins (determined by immunoblot and
immunoprecipitation) was detected at 1.31 gr/ml, the appropriate
density for empty capsids, for cells infected with VP1-baculovirus
and cells coinfected with VP2 and VP1-baculoviruses. No parvovirus
protein was detected in cell lysates from VP1-baculovirus infected
cells.
[0101] Direct electron microscopy was done on pellets after
ultracentrifugation of 50 .mu.l of the sample in 3.5 ml Dulbecco A
PBS. Immune electron microscopy was performed by incubating 50
.mu.l of human serum containing IgG antibody to B19 parvovirus for
45 minutes at 20.degree. C. prior to dilution in PBS and
ultracentrifugation. Pellets after centrifugation were resuspended
in 50 .mu.l of distilled water and negatively stained using 3%
phosphotungstic acid, pH 6.5. Grids were examined at
60,000.times.magnification in Jeol 1200EX electron microscope.
Magnifications were calibrated with catase.
[0102] Immune electron microscopy showed typical empty parvovirus
capsids, aggregated by the B19 antibody, in samples from cultures
coinfected with VP1 and VP2-containing baculoviruses and also in
cultures after infection only with VP2-baculoviruses (FIG. 11). No
virus particles were seen in lysates of cells infected with
VP1-containing baculovirus alone. Direct electron microscopy of
harvests from cultures coinfected with VP1 and VP2-containing
baculoviruses and from cultures infected with VP2-containing virus
only revealed numerous typical parvovirus-like particles that were
not coated with antibody. A minority of the particles were electron
dense, the majority were less dense, and some particles had
intermediate density. Particles tended to cluster together.
[0103] The capture immunoassay was adapted from a previously
published Elisa immunoassay procedure(J. Clin. Microbiol.
24:522-526 (1986)). Capture antibody, either goat anti-human IgG or
IgM antibodies (Tago, Burlingame Calif. was added to 96
2311-microtiter plates (Immunolon, Dynatech, Alexandria Va.) and
incubated for 1 hour at 37.degree. C.; the plates were washed and
human serum specimens (diluted 1:100) were added for 1.5 hours at
37.degree. C. After washing, positive and control antigens were
added overnight at room temperature; antigens included pooled sera
from viremic human specimens and baculovirus-expressed antigen.
Further washing of the plates to remove antigen was followed by
addition of biotinylated monoclonal antibody (MAb 521-5d, diluted
1:2000) for 1 hour at 37.degree. C., another wash step, and
addition of peroxidase-conjugated streptavidin (plc, Amersham
International, United Kingdom) for 10 minutes at room temperature.
Substrate for the enzyme (0.1 mg/ml of
3,3'5,5'-tetramethyl-benzidine and 0.005% H.sub.2O.sub.2 in
dimethyl sulfoxide and acetate-citrate buffer, pH 5.5) was added to
the plates after further washing for 15 minutes at room
temperature; the reaction was stopped with 2 M H.sub.2SO.sub.4 and
the absorbance at A.sub.450 determined. Capture antibody was
diluted in 0.01 M carbonate buffer, pH 9.6; other reagents were
diluted in phosphate buffered saline (pH 7.2) with 0.5% gelatin and
0.15% Tween-20; plates were washed with phosphate buffered saline,
0.15% Tween-20.
[0104] Each serum specimen was tested in duplicate against
baculovirus antigen and negative control antigen at 1:2000 dilution
and against a human serum pool of viremic blood at a dilution of
1:200. A serum specimen was considered positive in the baculovirus
IgG immunoassay if the P-N was >0.35 and the P/N ration was
>2.0, in the baculovirus IgM immunoassays if P-N was >3.0 and
P/N was >2.0, and in the human serum IgG and IgM immunoassays if
P-N was >3.0 and P/N was >2.5. P is the mean absorbance for
the serum specimen reacted against the B19 viral antigen less mean
absorbance due to nonspecific binding of B19 viral antigen
(negative serum or diluent reacted against positive antigen minus
negative serum reacted against control antigen). N is the mean
absorbance for the same serum specimen reacted against the
respective negative control antigen. These values of P-N and P/N
are .gtoreq.3 standard deviations above the mean values for
specimens previously determined to be antibody-negative.
[0105] For the IgG assay, 23 specimens were negative in both
immunoassays, 45 were positive in both assays, and none was
discordant. For the IgM assay, 25 specimens were negative in both
assays, and none was discordant. The assays based on the two
different sources of antigen also gave comparable qualitative
results (FIG. 12). The correlation coefficients for P-N absorbance
values for serum antigen versus baculovirus antigen wa 0.95 for the
IgG immunoassays and 0.91 for the IgM assay.
[0106] For the production of antisera, rabbits were immunized with
partially purified empty capsids obtained after coinfection of
insect cells with either VP1 and VP2-containing baculovirus or with
only VP2-containing baculovirus. After lysis, capsids were
subjected to sedimentation over sucrose and in cesium chloride, as
described above. Animals were inoculated with either 20 or 200
.mu.g of capsid protein by subcutaneous injection, initially in
complete Freund's adjuvant and with booster injections in
incomplete Freund's adjuvant at 2-4 week intervals. Rabbit sera
were analyzed by immunoblot and in neutralization assays.
[0107] To determine neutralizing activity, sera were heated to
56.degree. C. for 30 minutes to destroy complement activity and
incubated at varying concentrations with quantities of B19
parvovirus known to inhibit erythropoiesis in vitro. The inhibitory
activity of virus treated with antiserum was compared to virus
alone in conventional assays of late erythroid progenitors (CFU-E),
cultured in 0.8% methylcellulose containing 30% fetal calf serum,
1% bovine serum albumin, 10.sup.-1 beta-mercaptoethanol, and 1
.mu./ml recombinant erythropoietin (Amgen, Thousand Oaks Calif. at
37.degree. C., 95% humidity for 6-7 days. Control experiments
included assay of preimmune rabbit sera and similarly diluted
normal human sera that had been obtained from patients in the
convalescent phase of parvovirus infection; these sera contained
antibody to B19 parvovirus, as determined in the capture
immunoassay.
[0108] None of the animals inoculated with low doses of antigen (20
.mu.g/injection) produced neutralizing antisera. However, in 3/3
animals immunized with larger quantities of empty capsids (200
.mu.g/injection), composed of both VP1 and VP2, obtained after
coinfection of insect cells with the two individual recombinant
viruses, neutralizing antisera was produced. The titers of
neutralizing activity in two animals were comparable to those
observed in convalescent phase human sera. (FIG. 14 shows the
production of neutralizing antisera in response to capsid
containing both VP1 and VP2).
[0109] In contrast, none of three sera from animals immunized with
VP2-containing capsids produced neutralizing antibody. Ouchterlony
analysis was used to determine if precipitating antibodies were
made by these animals, using empty capsids made in mammalian cells
or VP2-only capsids produced in baculovirus as antigens: sera from
the animals which had produced neutralizing antibodies after
immunization with VP1 and VP2 also contained precipitating
antibodies, and sera from the animals immunized with VP2-capsule
also demonstrated precipitating antibodies.
[0110] The foregoing invention has been described in some detail by
way of examples for purposes of clarity and understanding. It will
be obvious to those skilled in the art from a reading of the
disclosure that site-directed mutagenesis can be used to alter the
amino acid sequence of the above described capsids and thereby
alter the tissue specificity of the virus. Furthermore, it will be
clear that the DHFR-deficient CHO cells can be used to study the
effect of nonstructural parvoviral proteins on cell replication. It
will also be apparent that various combinations in form and detail
can be made without departing from the scope of the invention.
[0111] The entire contents of all published articles cited herein
are hereby incorporated herein by reference.
* * * * *