Note: Descriptions are shown in the official language in which they were submitted.
CA 02414396 2002-12-23
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PRODUCTION OF VACCINES USING TRANSGENIC PLANTS OR MODIFIED
PLANT VIRUSES AS EXPRESSION VECTORS AND TRANSENCAPSIDATED
VIRAL COAT PROTEINS AS EPITOPE PRESENTATION SYSTEMS
BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to methods for production of recombinant peptides in
plants.
More particularly, this invention relates to methods for inserting immunogenic
peptides
into the coat protein of a plant virus for the purpose of generating
quantities of
immunogenic peptides for vaccine and antibody production. Further, the
invention
relates to the recombinant nucleic acid, vectors, and virions used to generate
the
expressed immunogenic peptides and to the transgenic plant expressing the
immunogenic peptides.
_Description of the Relevant Art
1t is now welt established that plants can serve as useful, accessible
resources for
generating large quantities of foreign gene products efficiently and
inexpensively.
"Foreign gene" refers to a gene that is not part of the genome of a particular
plant.
"Foreign gene products" refers to RNAs and proteins that are encoded by the
foreign
gene. Plants may be used to express foreign gene products or to overexpress
endogenous gene products via introduction of a genetically engineered DNA
sequence encoding the foreign or endogenous gene into the genetic material of
a
suitable plant through the use of various biotechnological methods. The term
"introduction" refers to a method which is capable of introducing the
genetically
engineered DNA sequence into the genetic material of a plant cell. Examples of
such
biotechnological methods are Agrobacterium-mediated transfer, plant virus
mediated-transfer, microinjection, microprojectile bombardment,
electroporation,
PEG-mediated transformation and transformation of plant protoplasts with virus-
based
stable vectors, all methods well known and practiced in the art.
In particular, transgenic proteins or viral proteins produced in plant tissues
provide a
system for expression of peptide epitopes useful as immunogenic peptides in
generating vaccines (Usha et al. 1993. Virology 197:366-374; Mason et al.
1993.
Proc. Natl. Acad. Sci. USA 89:11745-11749). Transgenic plant cells express the
genetically introduced foreign peptides which can then be extracted from plant
leaves
and other plant parts.
An additional method for expressing foreign peptides in plants is to infect
plants with
plant viruses which have been genetically engineered to express foreign
peptides as
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antigenic epitopes within modified viral coat protein. The success of such
epitope
presentation strategies depends on a detailed knowledge of virus structure at
the
atomic level. Potato virus X (PVX), bean yellow mosaic virus (BYMV), and
tobacco
mosaic virus (TMV) are examples of well-characterized plant viruses. These
viruses
are characterized by a single positive-sense RNA genome which is encapsidated
by
the capsid made up of approximately 2000 copies of a single type of coat
protein
(CP). For PVX and TMV, proteins which are required for virus replication are
translated directly from the genomic RNA; whereas, CP and movement protein
(MP),
which is involved in cell-to-cell movement, are translated from separate
subgenomic
mRNAs. The amount of mRNA for the viral proteins determines the amount of each
protein produced. The protein produced in the largest amount is the CP, which
is as
much as 5-10% of the total protein made in the infected plant cell. For BYMV
the
genomic RNA is translated into a single polyprotein which is cleaved by three
virus-
encoded proteinases into.the mature viral proteins. For each of these viruses,
the CP
drives the assembly and encapsidation of the viral RNA, which in turn enables
long-
distance movement and thereby systemic spread of the virus within the plant.
Furthermore, the CP is stable, tolerates modification, and exhibits many
characteristics of an ideal antigen system.
Foreign peptides can be fused to viral CP and systemically expressed along
with the
viral CP and in assembled virions. The viruses can be produced at high
concentrations; thus, large quantities of the peptide epitope are generated
and
available for vaccine production. Viruses such as PVX, BYMV, and TMV are
candidates for epitope carriers since they are self assembling viruses which
aggregate
into rod like particles that accumulate in virus infected leaves. The TMV CP,
for
example, has been shown to be immunogenic (Takamatsu et al. 1990. FEBS. Lefts.
269:73-76) and likely to contain helper T-cell epitopes which could function
for
chimeric epitopes. Whereas synthetic and recombinant peptides presented to the
immune system in a soluble form frequently are poor immunogens and thus
require
fusion to carriers and formulation in adjuvants to elicit a vigorous immune
response,
virions carrying repetitive copies of the foreign peptide exposed on the
surface serve
as potent immunogens. A further advantage of epitope presentation is that coat
protein antigens can be isolated and presented in particulate or aggregate
form. The
particulate nature of TMV based antigens, for example, could be advantageous
for
maintaining high local concentrations of antigen in parenteral immunizations
and may
be useful in stimulating mucosal immune responses to orally ingested antigens
(Loor,
F. 1967. Virology 33: 215-220).
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WO 02/00169 PCT/USO1/20272
Particular regions of the CP are known to be exposed on the surface of the
virus
particle and to be highly immunodominant (Shukla et al. 1989. Proc. Natl.
Acad. Sci,
USA 86: 8192-8196). Therefore, sites of insertion for the foreign peptides are
chosen
so that the translated foreign peptides are expressed on the surface of the
coat
protein and thus project outwards on the virus particle. For example,
experiments
where the 12 amino acid angiotensin-I-converting enzyme inhibitor was fused to
tobacco mosaic virus (TMV) coat protein, have suggested that each virus
particle
contained mostly native coat protein interspersed with subunits composed of
the
exposed peptide-fusions projected outward (Sugiyama et al. 1995. FEBS Lest.
359:
247-250). Other examples of successful epitope presentation include malarial
epitopes expressed by TMV coat protein (Turpen et al. 1995. Bio-technology 13:
53-
57) and animal virus epitopes expressed by cowpea mosaic virus (CPMV;
Lomonossoff et al. 1995. Sem. Virol. 6: 257-267). Fernandez-Fernandez et al.
(1998. FEBS Letters 427:229-235) have described an antigen presentation system
based on an infectious clone of plum pox potyvirus. Thus, epitope-expressing
viruses
can be isolated from infected plants and used as epitope presentation vehicles
to
raise specific antibodies against small peptides.
Antigenic determinant II of Newcastle Disease Virus (NDV) is a continuous
epitope
consisting of 17 amino acids spanning from Leu65 to Leu $~ of the fusion
protein Fo
described by Toyoda et al. (1988. J. Virol. 62: 4427-4430). This epitope is
composed
of the amino acids LLPNMPKDKEACAKAPL (SEQ ID N0:12). Monoclonal antibodies
specific to antigenic determinant II have been found to be highly potent at
neutralizing
the infectivity and inhibiting both homolysis and fusion activities of NDV
(Abenes et al.
1986. Arch. Virol. 90: 97-110.) Vaccination of chickens using either a linear
plasmid
DNA vector expressing the entire F protein (553 amino acids) of NDV (Sakaguchi
et
al. 1996. Vaccine 14, 747-752) or a recombinant fowlpox virus expressing the
entire F
protein of NDV (Taylor et al. 1990. J. Virol. 64, 1441-1450) gave efficient
protection
against the disease.
Variability in the human immunodeficiency virus type 1 (HIV-1 ) gp41 epitope
ELDKWA
(SEQ ID N0:8) is limited among characterized isolates of HIV-1 making this
conserved epitope a candidate for use for vaccine production. The ELDKWA
epitope
is recognized by the human monoclonal antibody 2F5 which is able to neutralize
HIV
in vitro. Expression of the ELDKWA epitope from a chimeric influenza virus has
been
shown to induce mucosal immune response in mice, and the antisera induced were
able to neutralize multiple strains of HIV-1 in vitro (Muster et al. 1993. J.
Virol.
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67:6642-6647; Muster et al. 1994. J. Virol. 68:4031-4034; Muster et al. 1995.
J. Virol.
69: 6678-6686).
The development of in vitro expression systems that allow production of
infectious
positive-sense RNAs from cloned full length cDNA genomes (Dawson et al. 1986.
Proc. Natl. Acad. Sci. USA 83:1832-1836; Meshi et al. 1986. Proc. Natl. Acad.
Sci.
USA 83: 5043-5047) has permitted the direct manipulation of the TMV genome at
the
DNA level. Highly infectious RNA transcripts of a full-length infectious cDNA
clone of
the U1 (common) strain of TMV have been produced in vitro using bacteriophage
T7
RNA polymerase (Holt et al. 1991. Virology 181:109-117) Thus, the RNAs of TMV
and other viruses are good candidates as vectors for the expression of foreign
genes
in plants. However, although TMV, PVX, and other viruses have been used as
viral
vectors for expressing foreign gene products, such vectors have not always
been
completely successful. Gene products have been produced via expression from
viral
vectors; however, the usefulness of such vectors can be limited by instability
of
inserted sequences and the failure of the viral vector to replicate
efficiently. Similarly,
there are apparent limitations associated with expression of gene products
from genes
introduced stably into transgenic plant genomes. Low levels of expression of
introduced genes resulting from suppression have been observed and
difficulties in
recovering functional foreign peptide may be encountered. To obtain high
yields of a
foreign gene product wherein the peptide of interest-is stably expressed and
easily
purified, it is sometimes advantageous to utilize a strategy involving
transencapsidation.
Transencapsidation, or phenotypic mixing, describes the coating of the RNA of
one
virus or isolate either partially or completely with the CP of another virus
or isolate
(Rochow 1970. Science 167:875-878). Thus, fusion viral coat proteins, which
comprise foreign antigenic peptides, and which are expressed in a plant either
as a
result of Agrobacterium-mediated transfection or as a result of inoculation
with
infectious viral RNA, can encapsidate the viral genomes of particular related
viruses.
For example, Farinelli et al. (1992. BioTechnology10:1020-1025), Lecoq et al.
(1993.
Mol. Plant Microbe Interact. 6:403-406), and Maiss et al. (1994. pp.129-139
in: Proc.
Int. Symp. Biosafety Results of Field Tests of Genetically Modified Plants and
Microorganisms, 3rd. D.D. Jones, ed. University of California, Division of
Agriculture
and Natural Resources, Oakland) have demonstrated partial transencapsidation
of
potyviruses with heterologous CP in transgenic plants.
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Bean yellow mosaic virus (BYMV) and potato virus Y (PVY) are members of the
potyvirus group. The single genomic RNA is translated into a single
polyprotein which
is then cleaved by three virus-encoded proteases to yield the mature viral
proteins.
The viral coat protein (CP) is the C-terminal product on the viral
polyprotein, and it is
therefore necessary to modify the CP sequence with an initiation codon in a
suitable
context in order to express the CP as a separate gene in transgenic plants
(e.g.
Hammond and Kamo. 1993. Acta Hort 336:171-178; Hammond and Kamo. 1995.
pp.369-389 in: Biotechnology and Plant Protection: Viral Pathogenesis and
Disease
Resistance. D.D. Bills and S.D. Kung, eds. World Scientific, Singapore.) or
from an
engineered viral vector. Potyviruses typically have moderate host ranges, but
many
potyviruses, including both BYMV and PVY, readily infect Nicotiana
benthamiana,
which is readily transformed using Agrobacterium tumefaciens. Potyviruses have
a CP
that is highly conserved at the amino acid level between distinct viruses; the
major
differences between the CP of distinct viruses, and between isolates of a
single virus,
are in the N-terminal domain (Shukla et al. 1994. The Potyviridae. CAB
International,
Wallingford, 516pp). The N-terminal domain varies considerably in length
between
distinct viruses (from about 22 to over 50 amino acids; Hammond, 1992.~Arch.
Virol.
[Suppl.5]:123-128), with natural deletions of up to 15 amino acids reported in
some
isolates.
The similarity of structure between the CP of different potyviruses allows
compatibility
and mixed assembly of subunits derived from two distinct viruses into a single
virion.
Such phenotypic mixing or transcapsidation occurs naturally in mixed
infections, and
can result in complementation leading to aphid transmission of a normally
aphid non-
transmissible isolate (Hobbs and McLaughlin. 1990. Phytopathology 80:268-272;
Bourdin and Lecoq. 1991. Pfiytopathology 81:1459-1464). This is also the basis
for
transcapsidation in potyvirus-infected transgenic plants expressing a
potyviral coat
protein (Lecoq et al. 1993. Mol. Plant-Microbe Interact. 6:403-406; Farinelli
et al.
1992. Bio/Technology 10:1020-1025; Hammond and Dienelt. 1997. Mol. Plant
Microbe Interact. 10:1023-1027).
Virus vector systems for epitope presentation are particularly advantageous
because
such systems ensure that the peptide exposed on the surface can be easily
recognized by the immune system of mammals, the virus can be inoculated
mechanically to large numbers of plants, very large quantities of purified
virus can be
recovered using simple extraction from infected leaves within two to four
weeks after
inoculation, and the resulting viral particles are stable (Scholthof et al.
1996. Annu.
Rev, of Phytopathol. 34: 299-323).
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Thus, systemic infection of plants with a virus vector encoding a foreign
protein can be
an economical means for obtaining unlimited yields of recombinant immunogenic
proteins, which can be recovered from the leaves and somefiimes other parts of
the
plant. However, although there have been advances in the art, the need exists
for
new and improved methods for utilizing positive-sense virus vectors as a means
for
expressing and producing large quantities of immunogenic foreign proteins in
plants
as well as for new and improved methods for ensuring stability, high yields,
and
efficient and economical purification of a functional product.
Accordingly, the present invention provides a method for producing in a plant
a viral
vaccine or an immunogenic peptide, which is very stable, can be economically
purified, and is capable of raising an immune response in a mammal.
SUMMARY OF THE INVENTION
We have discovered a method of reproducibly and economically obtaining large
quantities of foreign peptide by stably overexpressing non-native, foreign
peptides in
plants through the insertion of foreign peptide sequences into the viral coat
protein of
particular plant viruses.
In accordance with this discovery, it is an object of the invention to provide
a plant
systemically infected and producing stable recombinant plant virions
expressing
foreign peptides, as a means of producing and purifying large amounts of
foreign
peptides to be used as genetically engineered vaccines or for antibody
production.
Plants, plant cells, plant parts, and plant progeny are encompassed in the
invention.
In particular, a plant in which NDV or HIV immunogenic peptides are expressed
is
provided.
Another object of the invention is to provide a construct comprising a nucleic
acid
encoding a fusion viral coat protein comprising a foreign peptide and a
truncated bean
yellow mosaic virus coat protein (BYMV-CP).
It is an additional object of the invention to provide. a host cell containing
the nucleic
acid of the invention, wherein said host cell is a bacterial cell, in
particular, an
Escherichia coli cell and an Agrobacterium tumefaciens cell.
Yet another object of the invention is to provide a method of transforming a
plant with
a gene capable of expressing a fusion coat protein comprising a foreign
peptide and
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truncated BYMV-CP. A method of transforming a plant with a gene encoding a
fusion
coat protein comprising an HIV immunogenic peptide and truncated BYMV-CP is
particularly provided.
Still another object of the invention is to provide modified viral vectors
encoding fusion
coat proteins comprising foreign antigenic peptides. Viral vectors which
encode fusion
coat proteins which comprise NDV or HIV immunogenic peptides and BYMV-CP are
particularly provided.
A further object of the invention is to provide a method of capturing, stably
expressing,
and purifying fusion coat proteins comprising foreign antigenic peptides by
utilizing a
transencapsidation strategy. A transencapsidation strategy involving PVY or
BYMV
virions is particularly provided.
A still further object of the invention is to provide transencapsidated
virions
incorporating coat proteins comprising foreign antigenic peptides.
Transencapsidated
virions which encode fusion coat proteins which comprise NDV or HIV
immunogenic
peptides and truncated BYMV-CP are particularly provided.
Other objects and advantages of this invention will become readily apparent
from the
ensuing description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-G are schematic diagrams of methods used to produce the BYMV-
derived
constructs used for expression from PVX and for plant transformation.
Figure 1A shows a linear representation of the BYMV coat protein and the
surface-
located immuno-dominant epitopes replaced with the vaccine epitope for
expression
in a transgenic plant or viral vector.
Figure 1 B shows the approximate location of PCR primers used to accomplish
the
removal of the immunodominant BYMV epitopes and replacement with the HIV
ELDKWA epitope. The designation for each construct is indicated above the
hatched
boxes representing the BYMV and HIV epitope-modified CP open reading frames,
respectively. BYMV CP modified only with a transcriptional start codon has a
predicted molecular mass of 31078 Da. The HIV epitope-modified CP (ELDKWAcp)
has a predicted molecular mass of 29710 Da. H: Hind III; D: Dra I; P: Pst 7;
B: Bam
H7; E: Eco R7.
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Figure 1C depicts the primers JH039, JH040, and JH042, which were used for
introduction of the ELDKWA epitope from HIV gp41 into the BYMV CP, in more
detail:
the forward primer JH039: 5'-
GAAGGAAATCCTAATGAGCTCGATAAGTGGGCAAGT-GTCAGGCAAATAGTACC-
3' (SEQ ID N0:1 ); the reverse primer JH040: 5'-CTTTTT-
CCTTTTATCGAGCTCATTTGACCATGCATTGAGTTGCTCTTGATCTGC-3' (SEQ ID
N0:2) which is the complement of 5'-GCAGATCAAGAGCAACTCAATGCATGGTCA-
AATGAGCTCGATAAAAGGAAAAAG-3' (SEQ ID N0:3); and forward primer JH042:
5'-GATTACGCCAAGCTTTAAAACAATGGCAGATCAAGAGCAACTCAATGC-3' (SEQ
ID N0:4). (1) Upper lines: The N-terminal portion of the BYMV CP sequence (SEQ
ID
N0:6) and translation (one letter code; SEQ ID N0:7). Lower lines: Primer
JH039
sequence (SEQ ID N0:1) and translation (one letter code; SEQ ID NO: 8). Only
those
amino acids contributing to the ELDKWA epitope are shown. (2) Upper lines:
BYMV
sequence (SEQ ID N0:6) and translation (one letter code; SEQ ID N0:7). Lower
lines: Complement (SEQ ID N0:3) of Primer JH040 sequence (SEQ ID N0:2) and
translation (one letter code; SEQ ID NO: 9). Only those amino acids
contributing to
the ELDKWA epitope are shown. (3) Primer JH042 sequence (SEQ ID N0:4) and
translation (one letter code; SEQ ID NO: 5).
Figure 1 D is a diagram indicating the particular amino acids of the highly
hydrophilic
epitopes (BOLDFACE type) which were removed in the native BYMV CP, and
subsequently replaced with the ELDKWA-HIV epitope (BOLDFACE type) to generate
the amino terminal sequence (SEQ ID N0:10) and translation (one letter code;
SEQ
ID NO:11) of the ELDKWA epitope-modified BYMV CP. The nucleic acid sequence
of the N-terminal portion of the BYMV CP sequence, 5'-
ATGGCAGATCAAGAGCAACTCAAT-
G CAG GTGAG GAGAAGAAG GATAAAAG GAAAAAGAATGAAG GAAATCCTAATAAG
GACTCTGAGGGGCAGAGTGTCAGGCAAATAGTACC-3', is identified by SEQ ID
N0:6; the one letter code translation,
MADQEQLNAGEEKKDKRKKNEGNPNKDSEGQSVR-QIV, is identified by SEQ ID
N0:7.
Figure 1 E shows the deliberate primer-dimer of JH042iJH040 used to create the
5'
portion of ELDKWAcp, which was then annealed, extended, and amplified by PCR.
Upper lines: Sequence (SEQ ID N0:4) and translation (one letter code; SEQ ID
NO:
5) of primer JH042; underlined is the Hind III site used in subsequent
subcloning into
pGA643. The M in the one letter code indicates the initiation codon for plant
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expression. Lower lines: Sequence (SEQ ID N0:3) and translation (one letter
code;
SEQ ID N0:9) of the complement of primer JH040; underlined is the Sac I site
used
for assembly with the 3' portion of ELDKWAcp. Only those amino acids
contributing to
the ELDKWA epitope are indicated.
Figure 1 F and 1 G are schematic diagrams of intermediates in the construction
of the
ELDKWA cp. Figure 1 F shows the construction of pCR2.1.JH042/JH040, including
removal of the H:H fragment to remove unwanted Sac1, Bam H1, and Eco R7 sites
from the vector. SP6: SP6 promoter; H: Hind III; S: Sac I; B: BamHl; E: EcoRl;
X: Xba
I.
Figure 1G shows the construction of pCR2.1.JH039/M13F. The deliberate primer-
dimer of JH042/JH040, and the PCR product JH039/M13F were separately cloned
into the vector pCR2.1. The plasmids were screened for the appropriate
inserts,
which were subsequently ligated together at the introduced Sac I site to
create
pCR2.1.ELDKWAcp (See Figure 1 B). SP6: SP6 promoter; H: Hind III; S: Sac I; B:
BamHl; E: EcoRl; X: Xba I.
Figure 2 is a representation of the nucleotide and amino acid sequence of the
entire
modified BYMV CP containing the NDV F protein epitope, LLPNMPKDKEACAKAPL
(SEQ ID N0:12). The locafiion of the forward primer NDV1 (5'-CCCAAGCTTAATTAA-
TACAATGGCAGATCAAGAGCAATTGTTGCC-3'; SEQ ID N0:13), the reverse primer
N DV2 (5'-
TTTGCGCATGCTTCCTTATCCTTTGGCATATTTGGCAACAATTGCTCTTG-3'; SEQ
ID N0:14), the forward primer BYMCP1(5'-GGAAGCATGCGCAAAGGCACC-
ATTGGTCAGGCAAATAGTACCA-3'; SEQ ID N0:15), and the reverse primer
BYMVCP2 (5'-GGAATTCTCGAGCTAAATACGAACACCAAGCA-3'; SEQ ID N0:16)
used in the construction of the BYMVF CP construct, identified by SEQ ID
N0:17, is
shown. The complement of SEQ ID N0:17 is identified by SEQ ID NO:18. SEQ ID
N0:19 identifies the one letter code translation of SEQ ID N0:17.
Figure 3 is a Western analysis of triplicate blots of identical samples
derived from virus
infected plants. Purified virus obtained from infected plants were
electrophoresed on
10% polyacrylamide/SDS gels. The samples lanes contained: PVY, purii~ied
potato
virus Y; BYMV, purified bean yellow mosaic virus; TMVF, potato virus Y
purified from
plants co-infected with the TMV vector expressing the BYMVF CP (NDV-F epitope
on
the BYMV coat protein (CP)); PVXF, potato virus Y purified from plants co-
infected
with the PVX vector expressing the BYMVF CP. ~ The separated protein was
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transferred to Immobilon membranes. Following transfer, the blots were reacted
to
three different types of antisera. The blots were then developed with
NBT/BCIP. The
blots were reacted with the following antisera, as noted on the figure: Figure
3A,
PTY2, Monoclonal antibody that reacts with all potyviruses (here PVY and
BYMV);
Figure 3B, PTY3, Monoclonal antibody that reacts with BYMV and not PVY; Figure
3C, NDV2190, Polyclonal antibody that reacts only with NDV.
Figure 4 shows Western blots of purified potyvirus preparations of BYMV
isolate Ideal
A from non-transgenic plants of Nicotiana benthamiana, and transgenic lines of
N.
benthamiana expressing the HIV epitope-modified ELDKWAcp. Preparations were
diluted to approximately 1 mg/ml, dissociated and electrophoresed as described
(Hammond and Lawson, 1988, J. Virol. Methods 20:203-217), and blotted to
Immobilon PVDF membrane. Following transfer, the blots were reacted with three
different types of antibody: Figure 4A, with a mix of potyvirus cross-reactive
MAbs
(PTY 1, PTY 2, PTY 3, PTY 4, PTY 8, PTY 10, and PTY 21); Figure 4B, with BYMV-
specific MAb PTY 24; and Figure 4C, with HIV ELDKWA epitope-specific human MAb
2F5. In each of Figures 4A, 4B, and 4C, the lanes are: Lane 1, Non-transgenic
(BYMV-Ideal A, purified from non-transgenic plants); Lane 2, ELDKWA 40-7 (BYMV-
Ideal A, purified from transgenic line ELDKWA 40-7, expressing ELDKWAcp); Lane
3,
ELDKWA 42A-7 (BYMV-Ideal A, purified from transgenic line ELDKWA 42A-7); Lane
4, ELDKWA 30-4 (BYMV-Ideal A, purified from transgenic line ELDKWA 30-4); Lane
5, ELDKWA 76B-6 (BYMV-Ideal A purified from transgenic line ELDKWA 76B-6);
Lane 6, ELDKWA 6-4 (BYMV-Ideal A, purified from transgenic line ELDKWA 6-4).
Each transgenic line represents a separate homozygous line from a distinct
transformation event. MAb 2F5 reacts only with the virus preparations from
transgenic
plants, and with a band (open arrowhead to right) migrating slightly faster
than the
wild-type BYMV-CP (solid arrow head) that forms the major reactive bands in
Figures
4A and B.
Figure 5 shows an immunoelectron micrograph of purified PVY virions from
plants co-
inoculated with PVX-containing the BYMV-NDV coat protein and PVY; the small
black
dots (gold particles) denote a reaction of the NDV antibody to the surface of
the virion,
indicating the presence of the NDV epitope.
Figures 6A-6F show results of an indirect enzyme-linked immunosorbent assay
(ELISA) demonstrating the incorporation of ELDKWAcp expressed in transgenic
plants into virions of BYMV-Ideal A purified from BYMV-inoculated plants.
Serial two-
fold dilutions of virus preparations, starting at 5pg/ml, were coated directly
to ELISA
to
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WO 02/00169 PCT/USO1/20272
plates, and subsequently reacted with a mix of PTY monoclonal antibodies
(cross-
reactive; Figures 6A and 6B), with BYMV-specific PTY 24 (Figures 6C and 6D),
or
with HIV ELDKWA epitope-specific human neutralizing MAb 2F5 (Figures 6E and
6F).
The BYMV preparations from ELDKWA transgenic plants are: o, ELDKWA 40-7; o,
ELDKWA 42A-7; v, ELDKWA 30-4; and o, ELDKWA 76B-6; (o), non-transgenic
plants.
Figure 7 shows the Western blot analysis of NDV epitopes using mouse serum
from
mice injected with purified PVY virions bearing the F epitope of NDV. Purified
recombinant TMV carrying the F epitope on the BYMV CP (Left lane) or
inactivated
NDV were electrophoresed on a 10% SDS polyacrylamide gel. The proteins were
transferred to an Immobilon membrane which was subsequently incubated with
serum
derived from mice that had been injected with purified PVY virions bearing the
NDV F
epitope. The arrow points to the location of the 58,000 Da F protein in
inactivated
NDV isolated from diseased chickens.
Figure 8 shows the bacterial expression of the HIV epitope-modified BYMV CP
(ELDKWAcp), and specific recognition of the ELDKWA epitope by the HIV-specific
neutralizing human monoclonal antibody (MAb) 2F5. Extracts of Escherichia coli
strain
DHSa, and purified BYMV (strain Ideal A) were electrophoresed through a 10%
polyacrylamide gel, and blotted to an Immobilon PVDF membrane. Identical sets
of
lanes were reacted with BYMV isolate GDD- (BYMV-GDD-) specific MAb PTY 43
which reacts with the N-terminal portion of the BYMV-GDD CP, with potyvirus
cross-
reactive MAb PTY 2, reactive with the core region of the CP, and with ELDKWA
epitope-specific MAb 2F5. In each panel, the lanes are as follows: BYMV-Ideal
A
(Purified BYMV, isolate Ideal A); DHSa/pCR2.1 (E, coli strain DHSa carrying
plasmid
vector pCR2.1 - negative control #1 ); DHSa/ELDKWAcp (E. coli strain DHScc
carrying
plasmid pCR2.1/ELDKWAcp, expressing the ELDKWAcp as a IacZ fusion protein);
DH5cx/BYMV-CP (E, coli strain DHSa carrying plasmid pBY9, expressing BYMV-GDD
CP as a IacZ fusion protein); and DHSa (without any plasmid - negative control
#2).
This figure also demonstrates the lack of reactivity of ELDKWAcp with the BYMV
isolate GDD-specific monoclonal antibody PTY 43, as a result of the
replacement of
the N-terminal BYMV epitope (recognized by PTY 43) with the ELDKWA epitope.
Figures 9A-9D show the expression of ELDKWAcp from the PVX vector inoculated
to
non-transgenic plants of Nicotiana benthamiana, as demonstrated by reactivity
with
potyvirus cross-reactive, BYMV-specific, and ELDKWA-specific MAbs in an
indirect
ELISA. ELISA plates were coated with extracts of plants, blocked, and
incubated with
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the appropriate antibody solution (Figure 9A, PVX-specific rabbit polyclonal;
Figure
9B, PTY 1 potyvirus cross-reactive mouse MAb; Figure 9C, PTY 24 BYMV-specific
mouse MAb; and Figure 9D, 2F5 HIV-specific human MAb) . Plates were again
washed prior to addition of goat anti-rabbit (for detection of PVX-specific
polyclonal
antiserum), goat anti-mouse (for PTY 1 and PTY 24), or goat anti-human (for
MAb
2F5) alkaline phosphatase conjugate. Samples of plant extracts were: A,
healthy
Nicotiana benthamiana (negative control); B, BYMV-infected Nicotiana
benthamiana
(BYMV control); C, Nicotiana benthamiana plant 1A infected with PVX carrying
the
ELDKWAcp as an additional gene; and D, Nicotiana benthamiana plant 6A infected
with PVX carrying the ELDKWAcp as an additional gene.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to modified viral vectors encoding fusion
coat
proteins comprising foreign antigenic peptides. The recombinant viral RNA
provides
for systemic infection of a plant and the resulting production of stable
recombinant
plant virions expressing foreign peptides, as a means of producing and
purifying large
amounts of foreign peptides to be used as genetically engineered vaccines or
for
antibody production. Constructs of the invention encode a fusion protein
comprising
foreign peptide and a truncated potyvirus CP. The recombinant construct of the
invention can be used to produce transgenic plants which express the fusion
coat
protein comprising a foreign peptide and a truncated potyvirus CP or to
generate viral
vectors which are genetically engineered to comprise the nucleic acid encoding
the
fusion protein comprising a foreign peptide and a truncated potyvirus CP.
Constructs,
viral vectors, and transgenic plants comprising nucleic acid encoding the
fusion
protein comprising a foreign peptide and a truncated BYMV CP are particularly
provided. The genetically engineered viral vectors of the invention can be
transcribed
and the resultant infectious RNA used to inoculate plants to generate virions
expressing a fusion protein comprising a foreign peptide and a truncated
potyvirus
CP. RNA used to inoculate plants to generate virions expressing a fusion
protein
comprising a foreign peptide and a truncated BYMV CP is particularly provided.
In
plants that have been co-infected with a compatible potyvirus,
transencapsidation can
occur yielding large quantities of virions which stably incorporate a fusion
protein
comprising a foreign peptide and a truncated potyvirus CP. Such virions can be
purified efficiently, economically, and in large quantities. Transencapsidated
virions
which incorporate fusion coat proteins which comprise NDV or HIV immunogenic
peptides and truncated BYMV CP are particularly provided.
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The present invention provides a construct encoding a recombinant BYMV fusion
CP
comprising the amino acid sequence for a foreign protein, preferably of
between 5 and
20 amino acids, and a truncated BYMV coat protein. A PVX vector, pP2C2S, was
genetically engineered to contain the construct encoding the fusion protein
comprising
the foreign peptide and the truncated BYMV CP. Specifically, pP2C2S was
engineered to contain the construct FCP encoding a NDV epitope (F) and the
truncated BYMV CP (CP); pP2C2S was separately engineered to contain the
construct ELDKWAcp encoding an HIV epitope "ELDKWA" (SEQ ID N0:8) and the
truncated BYMV CP (ELDKWAcp). The vector pPVX-FCP and pPVX-ELDKWAcp has
two types of promoters. Transcription by T7 polymerise of the cloned cDNA
encompassing the full genome, operably linked to the promoter of the T7
polymerise
gene, produces a full length transcript. This transcript, when inoculated into
suitable
plant species, such as tobacco plants, causes infection and is therefore
referred to
herein as an "infectious clone" of the virus. The PVX vector also contains a
subgenomic promoter which has been duplicated. One copy of the subgenomic
promoter is used to make PVX coat protein. The other copy can be used to make
any
other protein of interest. For example, the vector can be engineered to
promote a
foreign peptide such as a NDV epitope or an HIV epitope. The modified
infectious
cDNA clone so produced encodes a PVX MP and a PVX CP. It also encodes a
modified CP, i.e., the FCP or ELDKWAcp as described above. Both the wild type
PVX coat protein and the coat protein FCP (or ELDKWAcp) are expressed in the
plant; however, only wild-type PVX virions (PVX genome assembled with PVX CP)
will
be assembled.
Thus, as a result of inoculation of the plant with the infectious modified
viral PVX RNA,
modified BYMV coat proteins expressing foreign peptide are expressed
throughout
the plant. Furthermore, in plants that have been previously infected with PVY
or
BYMV virions, transencapsidation will occur as potyvirions are assembled. The
PVY
(or BYMV) coat protein is incorporated into the viral capsid of the
potyvirions along
with the modified CP expressed from the PVX vector, thereby assuring formation
of a
stable capsid that incorporates a substantial proportion of the modified CP
containing
the foreign antigenic peptide. Virions are generated which express the foreign
antigenic peptide, FCP, in the capsid together with PVY (or BYMV) CP. Systemic
infection results in the generation of large numbers of virions which express
the
foreign antigenic peptide. Hence, viral purification yields large quantities
of foreign
antigenic peptide.
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One skilled in the art will appreciate that the modified infectious clone can
also be
further modified to replace the T7 polymerase promoter by any other strong
promoter
for transcription of the cDNA clone in vitro by contact with a polymerase
therefore; or
by replacement with a suitable plant promoter for transcription' in vivo
following
inoculation of plants with plasmid DNA. Such modified clones are also
contemplated
within the scope of this invention.
Any antigenic epitope of from about 5 to 20 amino acids can be used for which
antibodies are known or are discovered that neutralize a virus or other
pathogen, as
shown by routine tests well known in the art. For instance, antigenic
determinant II of
NDV and the antigenic epitope ELDKWA from gp 41 of the HIV virus can be
successfully incorporated into the truncated CP of BYMV, and subsequently into
transencapsidated BYMV or PVY virions. Furthermore, variants of antigenic
epitopes,
including antigenic determinant II of NDV and the antigenic epitope ELDKWA,
can be
used. A "variant" antigenic epitope may have an amino acid sequence that is
different by one or more amino acid "substitutions". The variant may have
"conservative substitutions", wherein a substituted amino acid has similar
structural or
chemical properties, e.g., replacement of leucine with isoleucine. More
rarely, a
variant may have "nonconservative" changes, e.g., replacement of a glycine
with a
tryptophan. Similar minor variations may also include amino acid deletions or
insertions, or both. Guidance in determining which and how many amino acid
residues
may be substituted, inserted or deleted without abolishing biological or
immunological
activity may be found using computer programs well known in the art, for
example,
DNASTAR software. "Immunological activity" defines the capability of the
recombinant
variant epitope, or any oligopeptide thereof, to induce a specific immune
response in
appropriate animals or cells and to bind with specific antibodies. The
recombinant
nucleic acid molecules encoding the variant epitopes are encompassed by the
invention as are virions which are generated to express variants of the
foreign
antigenic peptide.
As used herein a suitable plant host is any variety of plant known to be
subject to
infection by PVX, BYMV, PVY, and TMV. For instance suitable host plants for
TMV
include lettuce, spinach, tomato, potato as well as Nicotiana tabacum, N.
glutinosa, N.
sylvestris, N, benthamiana, Phaseolus vulgaris and Chenopodium amaranticolor.
Since the modified CP is produced under the control of the virus promoter
rather than
under the control of the plant promoter, up to 10 percent of the total protein
in the
plant is the modified CP containing the FCP of this invention.
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Therefore, in one embodiment, transencapsidation involving viral transcripts
of the
CP-modified PVX infectious clone and infectious PVY virions is used to
accomplish
systemic infections of suitable host plants. After construction of the
modified clones,
the PVX DNA constructs are transcribed in vitro by suitable reactions carried
out in the
presence of a polymerase active with the promoter (i.e., T7 polymerase with
the T7
promoter) to generate RNA for inoculation of PVY-infected plants.
In another embodiment, the modified CP comprising the foreign antigenic
peptide is
provided by using a transgenic host plant wherein the transgene encodes a
fusion
protein comprising truncated BYMV CP and the HIV-I gp41 ELDKWA epitope.
Transcription of the transgene and translation yields copies of the modified
CP, i.e.,
the truncated BYMV CP which contains the HIV-I epitope. Infection of this
transgenic
plant with wild type BYMV infectious virions results in viral replication of
the wild type
BYMV within the transgenic host plant. During viral replication and assembly
of wild
type BYMV virions, a portion of the virions isolated from the plants will be
transencapsidated with the transgene CP. Thus, the BYMV virions will contain
foreign
HIV-I epitopes. The plant undergoes systemic infection of the virus, resulting
in
transencapsidation in all infected tissues.
The method of exposure can be by inoculation of the subject with purified
antigen at
appropriately spaced intervals using methods routine in the art or by the
subject
ingesting plants or plant tissue extracts that have been infected with the
virions
produced from the CP-modified clones of this invention into which a nucleotide
sequence encoding a heterologous viral or other antigen has been encoded. The
purified virus or a plant in which the recombinant virus is accumulated is
administered
in an immune response stimulating dose as determined by those skilled in the
art
taking into account, for instance, the body weight and general health of the
subject.
Modified PVY or BYMV virions produced by transencapsidation have the
heterologous
viral or other antigen projecting from the surface of the viral coat, and are
capable of
stimulating producing of antibodies in the subject that neutralize the
antigen.
Alternatively, immunogenic exposure can be by a combination of inoculation and
ingestion of the antigenic epitopes, or by ingestion alone. The virus can be
ingested
directly by intubation or oral inoculation of a subject can be accomplished by
the
subject consuming a sufficient quantity of a plant, such as spinach, that has
been
systemically infected with a CP-modified infectious clone to raise a
neutralizing level
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of antibodies to the heterologous antigenic epitope encoded by the CP-modified
infectious clone.
Virus can also be grown and maintained in protoplasts of plant lines such as
Nicotiana
benthamiana. Generally in vitro transcripts are inoculated into tobacco
protoplasts by
electroporation as described by Watanabe et al. (1987. FEBS Left. 219:65-69).
In
addition, virus can be grown in cultures of infected cells. Such cells can be
maintained
for extended periods of time (Murakishi et al. 1971. Virology 43:62-68).
One skilled in the art will appreciate that antigenic peptides and/or virions
can be
purified from plant leaves using standard methods (Bruening et al. 1976.
Virology 71:
498-517). Generally virus is purified from infected leaf tissues by
homogenizing the
infected leaf tissues in appropriate buffer, removing the leaf debris, and
concentrating
the virus either by a salting out procedure or by ultracentrifugation. The
standard
methods for purification of TMV, for example, can lead to the isolation of 1
to 5 mg of
purified virus per gram fresh weight of tobacco leaves.
EXAMPLES
Having now generally described this invention, the following examples
illustrate the
manner in which the invention can be practiced. It is understood, however,
that the
xamples are included herein only to further illustrate the invention and are
not
intended to limit the scope of the invention as defined by the claims.
EXAMPLE 1
BYMV-Foreign Epitope Constructs
The immunodominant surface epitopes of bean yellow mosaic virus coat protein
were
replaced with a vaccine epitope for subsequent expression in a transgenic
plant or
viral vector (Figure 1A). The forward primer JH039 (5'-
GAAGGAAATCCTAATGAGCTCGAT-AAGTGGGCAAGTGTCAGGCAAATAGTACC-
3') (SEQ ID NO:1 ), the reverse primer JH040, 5'-
CTTTTTCCTTTTATCGAGCTCATTTGACCATGCATTGAGTTGCTCTTG-ATCTGC-3'
(SEQ ID N0:2) which is the complement of 5'-GCAGATCAAGAGCAACTC-
AATGCATGGTCAAATGAGCTCGATAAAAGGAAAAAG-3' (SEQ ID N0:3), and
forward primer JH042, 5'-
GATTACGCCAAGCTTTAAAACAATGGCAGATCAAGAGCAACTC-AATGC-3' (SEQ
ID N0:4) were used to accomplish the removal of the immunodominant BYMV
epitopes and replacement with the HIV ELDKWA epitope (Figures 1 B). BYMV CP
modified only with a transcriptional start codon has a predicted molecular
mass of
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31078 Da. The HIV epitope-modified CP (ELDKWAcp) has a predicted molecular
mass of 29710 Da (Figure 1 B). The C-terminal portion of ELDKWAcp is identical
to
that of the FCP construct shown in Figure 2. The primers are described in more
detail
in Figure 1 C. The particular amino acids of the highly hydrophilic epitopes
which were
removed in the native BYMV CP, and subsequently replaced with the ELDKWA-HIV
epitope are shown in Figure 1 D . A deliberate primer-dimer of JH042/JH040 was
used
to create the 5' portion of ELDKWAcp which was then annealed, extended, and
amplified by PCR (Figure 1 E). The deliberate primer-dimer of JH042/JH040, and
the
PCR product JH039/M13F were separately cloned into the vector pCR2.1. The
plasmids were screened for the appropriate inserts, which were subsequently
ligated
together at the introduced Sac I site. Figure 1 F and Figure 1 G are schematic
diagrams of intermediates in the construction of the ELDKWAcp; Figure 1 B
shows the
construction of pCR2.1 ELDKWAcp containing the ELDKWA peptide at the 6'
terminus
as a fusion protein.
EXAMPLE 2
Plasmid Construction to Produce Chimeric Virus Vectors
Newcastle Disease Virus (NDV)
Antigenic determinant II of Newcastle Disease Virus (NDV) fusion protein (F)
is a
continuous epitope which consists of 17 amino acids spanning from Leu65 to
Leus~ of
Fo (Toyoda et al. 1988. J. Virol. 62: 4427-4430). A DNA fragment encoding the
major
portion of the epitope was PCR amplified with a pair of partially
complementary
oligonucleotide primers NDV1 and NDV2. The location of the forward primer NDV1
(5'-CCCAAGCTTAATTAATACAATGGCAGATCAAGAGCAATTGTTGCC-3'; SEQ ID
N0:13) and the reverse primer NDV2 (5'-
TTTGCGCATGCTTCCTTATCCTTTGGCATA-TTTGGCAACAATTGCTCTTG-3'; SEQ
ID N0:14) are shown in Figure 2. Specific sequence tags were engineered into
the
primers so that the amplified epitope-encoding fragment contains a Hind III
recognition
site as well as a translation initiation codon at the 5' end and a Sphl
recognition site at
the 3' end. A truncated BYMV CP gene lacking the first 40 codons was amplified
from
plasmid pBY9 (Hammond and Hammond. 1989. J. Gen. Virol. 70: 1961-1974) with
primer pair BYMVCPIIBYMVCP2. The location of the forward primer BYMCP1 (5'-
GGAAGCATGCGCAAAGGCACCATTGGTCAGG-CAAATAGTACCA-3'; SEQ ID
N0:15) and the reverse primer BYMVCP2 (5'-GGAATT-
CTCGAGCTAAATACGAACACCAAGCA-3'; SEQ ID N0:16) are also shown in Figure
2. Primer BYMVCP1 was tagged with an Sphl recognition site and a sequence
encoding the last 5 amino acids of the F epitope, overlapping primer NDV2.
EcoRl
and Xhol recognition sites were engineered at the end of primer BYMVCP2. The
amplified epitope-encoding fragment and the truncated BYMV-CP gene were
digested
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with Sphl and ligated to form an NDV-F/BYMV-CP chimera. This chimera was in
turn
inserted into plasmid vector pUC19 at Hindlll/EcoRl sites giving rise to pFCP.
The
resulting construct is shown in Figure 2 and is identified by SEQ ID N0:17.
To create pPVX-FCP, a PVX (potato virus X)-based vector pP2C2S was digested
with
EcoRV and dephosphorylated with CIP. The NDV-FIBYMV-CP insert was prepared
from pFCP by Hindlll/EcoRl double digestion followed by Klenow fragment
filling-in.
After ligation of the insert into the pP2C2S vector, a recombinant with the
desired
orientation was selected.
To create pTMV-FCP, a TMV-based vector p30BRz was digested with PinAl, filled-
in
with Klenow fragment of E.coli DNA polymerase I, and digested with Xhol. The
NDV-
F/BYMV-CP insert was prepared from pFCP by Hindlll digestion and Klenow
fragment
filling-in followed by Xhol digestion. The above-treated vector and insert
were ligated
with T4 DNA ligase.
Human Immunodeficiency Virus Type I (HIV)
Two approaches were used to express a unique HIV epitope as part of a plant
viral
capsid protein. The first involved the transformation of Nicotiana benthamiana
with
Agrobacterium tumefaciens containing a binary plasmid in which a bean yellow
mosaic virus (BYMV) coat protein gene where 22 amino acids were replaced by 9
amino acid residues of the HIV gp41 epitope (amino acids ELDKWA) was
engineered
(Figures 1 B, 1 F, and 1 G). The ELDKWAcp construct was transferred as a
HindllllBamH1 fragment into pGA643 digested with Hindlll and Bgl II, creating
pGA/ELDKWAcp. The pGA/ELDKWAcp was transformed into Agrobacterium
tumefaciens strain C58C1, and used to transform leaf pieces of N, benthamiana.
Plants were transformed and selected essentially as described by Hammond and
Kamo (1995. Mol. Plant-Microbe Interact. 8:674-682). Transformed plants
expressing
detectable ELDKWAcp were identified by antigen-coated plate indirect ELISA
with
potyvirus cross-reactive MAbs (Jordan and Hammond. 1991. J. Gen. Virol. 72:25-
36)
and ELDKWA-specific human MAb 2F5 (Muster et al. 1993. J. Virol. 67:6642-
6647).
The plants expressing the modified CP were then inoculated with wild type BYMV
and
a portion of the virions isolated from infected plants were transencapsidated
with the
ELDKWAcp (See Example 4, below).
The second approach was similar to that described above for the NDV epitope.
The
engineered CP gene was excised from pCR2.1 (ELDKWAcp) with Dra I, which
cleaves
immediately 5' to the initiation codon, and 3' to the termination codon of the
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ELDKWAcp (Figure 1 B), and inserted at the Eco RV site of pSKAS (immediately
downstream of the PVX subgenomic promoter), creating pSKAS/ELDKWAcp. The
Apa 1/Spe 1 fragment from pSKAS/ELDKWAcp was then inserted into the equivalent
sites of the full-length PVX viral-based vector pP2C2S and infectious
transcripts were
prepared and delivered to plants as outlined below for the NDV-epitope
containing
CP.
EXAMPLE 3
Preparation and Delivery of Infectious Transcripts
Both the PVX-based vector pP2C2S and the TMV-based vector p30BRz contain a
bacteriophage T7 promoter prior to the respective viral template, facilitating
the
production of infectious viral transcripts in vitro using T7 RNA polymerase-
driven
transcription. For the PVX-based construct pPVX-FCP, the plasmid DNA was
linearized with Spel prior to transcription. For TMV-based construct pTMV-FCP,
since
the vector contains an engineered cis-acting ribozyme at the 3' terminus of
the viral
template, no template linearization is necessary before transcription.
Approximately
one pg of each DNA template was used in a 20 p1 reaction for synthesis of
capped
transcripts using T7 Message Machine kit (Ambion) at 37°C. Considering
the size of
the expected transcripts, 1 p1 of 30 mM GTP was supplemented 15 min after the
start
of the reaction. The transcription reaction was continued at 37°C for
90 min. An
aliquot of the product (2 p1) was fractionated on a 1.0% agarose gel to assess
the
quantity and the integrity of the transcripts. The transcripts were diluted to
0.5 pg/pl
with 50 mM potassium phosphate (pH 7.0) for inoculation.
The production of vaccines in our system is based on the observation that
potyvirus
capsid proteins are capable of encapsidating heterologous potyvirus RNAs and
that
progeny virions in some cases may contain up to 25% of the second coat protein
(Hammond and Dienelt. 1997. Mol. Plant Microbe Interact. 10: 1023-1027). Young
hPalthv Nirntiana tabacum seedlings were first inoculated with potato virus Y
(PVYI
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tissue in 1 % K~HP04 at approximately 1 g leaf/1 Oml with a mortar and pestle
in the
presence of a small amount of Celite, and manually inoculating three to four
leaves of
plants with a finger dipped in the inoculum. The virus purification procedure
was
essentially the same as described by Hammond and Lawson (1988. J. Virol. Meth.
20:
203-217). Briefly, leaves were homogenized in 5 volumes (w/v) of 0.5M
potassium
phosphate buffer (pH 8.4) supplemented with 0.5% Na2S03. The homogenate was
filtered through two layers of cheesecloth and the filtrate was centrifuged at
4500 rpm
(Sorvall GSA) for 10 min at 4°C. Crude virus particles were
precipitated from the
supernatant with the addition of PEG 8000 to a final concentration of 4% in
the
presence of 0.1 M NaCI. After centrifugation at 7000 rpm (Sorvall GSA,
4°C, 10 min),
the pellet was collected and resuspended in 0.1 M borate/KCI buffer (8.0). The
virus
particles were dispersed and concentrated by centrifugation through a 30%
sucrose
pad (27,000 rpm, 2.5 hrs, Beckman R30), resuspended again in 0.1 M borate/KCI
buffer (8.0) and finally purified through CsCI gradient centrifugation at
40,000 rpm
(Beckman R65) for 16 hrs at 10°C, followed by dialysis to remove CsCI.
EXAMPLE 5
Immunoblot Analysis of Transencapsidated Virions
For the NDV epitope experiments, three Ng of viral proteins as determined by
Bradford
assay were separated by 12% SDS-PAGE and transferred to an Immobilon-P
membrane (Millipore). The transferred proteins were probed with mouse anti-
potyvirus CP monoclonal antibodies PTY 2, PTY 3, or PTY 24, or a mix of
potyvirus
cross-reactive MAbs (PTY 1, PTY 2, PTY 3, PTY 4, PTY 8, PTY 10, and PTY 21 )
and
separately with the chicken anti-NDV polyclonal antibody '2-1-90' (NDV2190;
obtained
from Jack King, USDA-ARS) followed by alkaline phosphatase (AP)-conjugated
goat
anti-mouse, goat anti-chicken, or goat anti-human antibodies (Kirkegaard &
Parry
Laboratories, Inc.) as appropriate. The immunoreactions were visualized by the
addition of AP substrate BCIP-NBT.
The samples lanes of the experiment shown in Figure 3 contain: PVY, purified
potato
virus Y; BYMV, purified bean yellow mosaic virus; TMVF, potato virus Y
purified from
plants co-infected with the TMV vector expressing the BYMVF CP (NDV-F epitope
on
the BYMV coat protein (CP)); PVXF, potato virus Y purified from plants co-
infected
with the PVX vector expressing the BYMVF CP. The blots were reacted with the
following antisera, as noted on the figure: PTY2, monoclonal antibody that
reacts with
all potyviruses (here PVY and BYMV); PTY3, monoclonal antibody that reacts
with
BYMV and not PVY; NDV2190, polyclonal antibody that reacts only with NDV.
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The results are as follows. In Figure 3A, all samples react with PTY2,
indicating that
all contain potyvirus coat proteins, as expected, for the TMV and PVX samples
are
from plants co-infected with PVY and the BYMV CP should be also expressed. In
Figure 3B, only three samples react with PTY3, which is specific for BYMV. The
results demonstrate that BYMV CP is being encapsidated on the purified PVY
particles in TMVF and PVXF. In Figure 3C, NDV2190 only reacts with TMVF and
PVXF, showing that the BYMVF CP that is expressed from both of these vectors
is
encapsidated in the PVY particle and the F epitope of NDV is expressed on the
particle in such a way that it is recognized by the antiserum to NDV.
For the HIV epitope experiments, 15 u1 of approximately 1 mg/ml purified virus
preparations as determined by UV absorption were separated by 10% SDS-PAGE.
Purified virus preparations of BYMV isolate Ideal A from non-transgenic plants
of
Nicotiana benthamiana, and transgenic lines of N. benthamiana expressing the
HIV
epitope-modified ELDKWAcp were diluted to approximately 1 mg/ml, dissociated
and
electrophoresed as described (Hammond and Lawson. 1988. J. Virol. Mefihods
20:203-217), and blotted to Immobilon PVDF membrane (Millipore). The purified
viral
preparations were: BYMV-Ideal A, purified from non-transgenic plants; ELDKWA
40-7
- BYMV-Ideal A, purified from transgenic line ELDKWA 40-7, expressing
ELDKWAcp;
ELDKWA 42A-7 - BYMV-Ideal A, purified from transgenic line ELDKWA 42A-7;
ELDKWA 30-4 - BYMV-Ideal A, purified from transgenic line ELDKWA 30-4;
ELDKWA 76B-6 - BYMV-Ideal A, purified from transgenic line ELDKWA 76B-6;
ELDKWA 6-4 - BYMV-Ideal A, purified from transgenic line ELDKWA 6-4. Each
transgenic line represents a separate homozygous line from a distinct
transformation
event. The transferred proteins were probed with mouse anti-potyvirus CP
monoclonal antibodies: PTY 24 , a mix of potyvirus cross-reactive MAbs (PTY 1,
PTY
2, PTY 3, PTY 4, PTY 8, PTY 10, and PTY 21; Jordan and Hammond, supra ), or
with
human monoclonal antibody 2F5 specific for the ELDKWA epitope (obtained from
H.
Katinger, Austria; Muster et al, 1993, supra) followed by alkaline phosphatase
(AP)-
conjugated goat anti-mouse, goat anti-chicken, or goat anti-human antibodies
(Kirkegaard & Parry Laboratories, Inc.) as appropriate. The immunoreactions
were
visualized by the addition of AP substrate BCIP-NBT.
ELDKWAcp, expressed from the transgenic plants, is incorporated into potyvirus
virions formed in plants challenged with BYMV (transcapsidation) as indicated
by
reactivity of HIV epitope ELDKWA-specific MAb 2F5 with virus preparations
purified
from transgenic plants expressing the epitope-modified ELDKWAcp (Figure 4C).
All
virus preparations reacted with the mix of potyvirus cross-reactive MAbs
(Figure 4A)
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and with the BYMV-specific MAb PTY 24 (Figure 4B). MAb ZF5 reacted only with
the
virus preparations from transgenic plants, and with a band (open arrowhead to
right)
migrating slightly faster than the wild-type BYMV-CP (solid arrow head) that
forms the
major reactive bands in Figures 4A and 4B (Figure 4C). A faint band
corresponding to
ELDKWAcp is reactive with the "PTY mix" (Figure 4A) and PTY 24 (Figure 4B).
EXAMPLE 6
Immunoelectron Microscopy
Purified virions were analyzed by electron microscopy following labeling with
antibodies specific for potyviruses, NDV, and/or ELDKWA, and by ELISA with
appropriate antibodies, essentially as reported (Hammond and Dienelt. 1997.
Mol.
Plant Microbe Interact. 10; 1023-1027). The results provide evidence for the
transencapsidation fo the chimeric BYMV coat proteins into infectious virus
particles of
PVY (Figure 5).
EXAMPLE 7
Enzyme-linked Immunosorbent Assay (ELISA)
Purified virions were analyzed by ELISA following labeling with antibodies
specific for
potyviruses, NDV, and/or ELDKWA, essentially as reported (Jordan and Hammond,
supra). Serial two-fold dilutions of virus preparations, starting at 5pg/ml,
were coated
directly to ELISA plates. Plates were washed and blocked with 1 % BSA in PBS
prior
to incubation with the appropriate antibody solution, and subsequently reacted
with a
mix of PTY monoclonal antibodies (cross-reactive; Figures 6A and 6B), with
BYMV-
specific PTY 24 (Figures 6C and 6D), or with HIV ELDKWA epitope-specific human
neutralizing MAb 2F5 (Figures 6E and 6F). Plates were again washed prior to
addition of goat anti-mouse (for mix of PTY monoclonal antibodies and for PTY
24), or
goat anti-human (for MAb 2F5) alkaline phosphatase conjugate.
The relative reactions of the BYMV preparations from ELDKWA transgenic plants
with
the PTY mix and PTY 24 are less than the reaction of a control BYMV
preparation
from non-transgenic plants (Figures 6A-6D). In contrast, the reactions of
virus from
ELDKWA transgenic plants with HIV-specific MAb 2F5 are significantly higher
than the
background level reaction of the BYMV from non-transgenic control plants, thus
demonstrating the presence of the ELDKWAcp in virions purified from the
transgenic
plants.
EXAMPLE 8
Stimulation of Epitope-specific Antibodies in Mice Injected with Purified
Virions
Mice were injected with purified PVY virions bearing fihe F epitope of NDV
isolated as
described previously. Immunizations contained 200 ug of purified virus in 200
u1 of
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Tris-buffered saline (TBS) emulsified with 240 u1 Hunters's TiterMaxGold
adjuvant and
100 u1 injected in each of four mice. The mice were injected a minimum of 4
times
over a 2-3 month time frame with injections 10-14 days apart with a minimum 21
day
rest after the third injection. Tail bleeds were 10 - 14 days after the 4t"
injection. All
injections were done intraperitoneally.
Purified recombinant TMV carrying the F epitope on the BYMV CP or inactivated
NDV
were elecfirophoresed on a 10% SDS polyacrylamide gel. The proteins were
transferred to an Immobilon membrane which was subsequently incubated with the
serum derived from mice that had been injected as described above.
The results provide evidence that the NDV F protein epitope presented at the
surface
of the potyvirus CP could elicit the production of antibodies that react with
the F
protein of NDV (Figure 7). The positive reaction in the left lane indicates
the location
of the BYMV-F CP that is translated from the TMV construct in infected plants.
The
arrow points to the location of the 58,000 Da F protein in inactivated NDV
isolated
from diseased chickens.
Mice that were injected with the BYMV virions carrying HIV ELDKWA epitope-
containing CPs have not been bled.
EXAMPLE 9
Bacterial expression of ELDI~CWAcp
The HIV epitope/BYMV CP fusion protein ELDKWAcp was also expressed as an in-
frame fusion with the IacZ a peptide in Escherichia coli from the vector
pCR2.1. This
was used to confirm the reactivity of the ELDKWAcp protein with the HIV-
specific MAb
2F5, and replacement of the BYMV-specific immunodominant epitopes by the
ELDKWA epitope. Expression of bacterially-expressed potyvirus CP (wild-type
BYMV
CP and ELDKWAcp) was enhanced by IPTG induction as previously reported
(Hammond and Hammond. 1989. J. Gen. Virol. 70:1961-1974). Fifteen p1 of
extracts
of Escherichia coli strain DH5cx and purified BYMV (strain Ideal A) were
electrophoresed through a 10% polyacrylamide gel, and blotted to an Immobilon
PVDF membrane. The extracts analyzed were: BYMV-Ideal A (Purified BYMV,
isolate
Ideal A); DHSa/pCR2.1 (E. coli strain DHSa carrying plasmid vector pCR2.1 -
negative
control #1 ); DHSa/ELDKWAcp (E. coli strain DHSa carrying plasmid
pCR2.1 /ELDKWAcp, expressing the ELDKWAcp as a lacZ fusion protein);
DHSa/BYMV-CP (E. coli strain DHSa carrying plasmid pBY9, expressing BYMV-GDD
CP as a IacZ fusion protein); and DH5oc (without any plasmid - negative
control #2).
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Membranes were probed with (a) MAb PTY 43 (specific for BYMV isolate GDD;
Jordan and Hammond, supra) which reacts with the N-terminal portion of the
BYMV-
GDD CP (Jordan, 1992, Arch. Virol. [Suppl.5]81-95); (b) potyvirus cross-
reactive MAb
PTY 2 (Jordan and Hammond, supra); and (c) HIV-specific MAb 2F5 (Muster et al.
1993. J. Virol. 67:6642-6647).
MAb PTY 43 reacts only with BYMV-GDD CP, expressed from E. coli, and not BYMV-
Ideal A. MAb 2F5 reacts only with ELDKWAcp. In contrast, MAb PTY 2 reacts with
BYMV-Ideal A, and both bacterially-expressed BYMV-GDD CP and ELDKWAcp.
There is also an apparent non-specific activity, with one band present in all
DHSa
extracts, and one or more bands apparently derived from pCR2.1. The ELDKWAcp
band has lower apparent molecular mass than BYMV-CP, as predicted. These
experiments demonstrate that the BYMV-GDD specific epitope recognized by MAb
PTY 43 had been replaced with the ELDKWA epitope recognized by MAb 2F5, and
that the HIV epitope was serologically active (Figure 8).
EXAMPLE 10
Expression of ELDKWAcp from the PVX vector
The ELDKWAcp construct was also expressed from the infectious PVX vector in N.
benthamiana and N. tabacum plants. T7 transcripts were inoculated to young
plants,
and sap from the systemically-infected plants used to infect additional
plants. Indirect,
antigen-coated plate ELISA was performed essentially as described by Jordan
and
Hammond, 1991 (supra). Extracts of infected leaves (1:100, w/v) of plants were
prepared in coating buffer plus polyvinylpyrrolidone (PVP) plus
diethyldithiocarbamic
(DIECA) and coated directly to ELISA plates. Following incubation the plates
were
washed and blocked with 1 % BSA in PBS prior to incubation with the
appropriate
antibody solution (Figure 9A, PVX-specific rabbit polyclonal; Figure 9B, PTY 1
potyvirus cross-reactive mouse MAb; Figure 9C, PTY 24 BYMV-specific mouse MAb;
and Figure 9D, 2F5 HIV-ELDKWA-specific human MAb). Plates were again washed
prior to addition of goat anti-rabbit (for detection of PVX-specific
polyclonai antiserum),
goat anti-mouse (for PTY 1 and PTY 24), or goat anti-human (for MAb 2F5)
alkaline
phosphatase conjugate. Samples were: A, healthy Nicotiana benthamiana
(negative
control); B, BYMV-infected Nicotiana benfihamiana (BYMV control); C, Nicotiana
benthamiana plant 1A infected with PVX carrying the ELDKWAcp as an additional
gene; and D, Nicotiana benthamiana plant 6A infected with PVX carrying the
ELDKWAcp as an additional gene.
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The results demonstrate the expression of ELDKWAcp from the PVX vector
inoculated to non-transgenic plants of Nicotiana benthamiana (Figures 9A-9D).
Figure 9A (PVX polyclonal) shows that PVX is detected only in the PVX/ELDKWA-
inoculated plants. Figure 9C (lower left) shows that the PVX/ELDKWA-inoculated
plants are not infected with BYMV, thus confirming that the reactions of
samples C
and D with MAbs PTY 1 and 2F5 are due to the ELDKWAcp expressed from the PVX
vector.
All publications and patents mentioned in this specification are herein
incorporated by
reference to the same extent as if each individual publication or patent was
specifically and individually indicated to be incorporated by reference.
It is understood that the foregoing detailed description is given merely by
way of
illustration and that modifications and variations may be made therein without
departing from the spirit and scope of the invention. Accordingly, the
following
claims are intended to be interpreted to embrace all such modifications.
2s
CA 02414396 2002-12-23
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SEQUENCE LISTING
<110> Hammond, Rosemarie
Zhao, Yan
Hammond, John
<120> Production of Vaccines Using Transgenic Plants or
Modified Plant Viruses as Expression Vectors and
Transencapsidated Viral Coat Proteins as Epitope
Presentation Systems
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3
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4
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<2l0> 19
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260 265
