NZ618885B2 - Rabies virus like particle production in plants - Google Patents
Rabies virus like particle production in plants Download PDFInfo
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- NZ618885B2 NZ618885B2 NZ618885A NZ61888512A NZ618885B2 NZ 618885 B2 NZ618885 B2 NZ 618885B2 NZ 618885 A NZ618885 A NZ 618885A NZ 61888512 A NZ61888512 A NZ 61888512A NZ 618885 B2 NZ618885 B2 NZ 618885B2
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- rabies
- vlp
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Abstract
Disclosed is a method of producing a rabies virus like particle (VLP) in plant comprising: a) introducing s nucleic acid comprising a native rabies glycoprotein into the plant or portion thereof; b) incubating the plant or portion of the plant to permit expression, thereby producing the rabies VLP; c) harvesting the plant, d) extracting the VLPs, wherein the VLPs range in size from 40-300 nm and, wherein the VLP comprises a lipid obtained from a plasma membrane of the plant. Also disclosed are related compositions and methods of inducing immunity to rabies in a subject. c) harvesting the plant, d) extracting the VLPs, wherein the VLPs range in size from 40-300 nm and, wherein the VLP comprises a lipid obtained from a plasma membrane of the plant. Also disclosed are related compositions and methods of inducing immunity to rabies in a subject.
Description
RABIES VIRUS LIKE PARTICLE PRODUCTION IN PLANTS
FIELD OF INVENTION
The present invention relates to producing native viral proteins in plants.
More specifically, the present invention also relates to producing virus-like particles
comprising native rabies virus structural protein in plants.
BACKGROUND OF THE INVENTION
Vaccination provides protection against disease caused by an infectious agent
by inducing a subject to mount a defense prior to infection. Conventionally, this has
been accomplished through the use of live attenuated or whole inactivated forms of
the infectious agents as immunogens. To avoid the danger of using the whole virus
(such as killed or attenuated viruses) as a vaccine, recombinant viral proteins, for
example subunits, have been pursued as vaccines. Both peptide and subunit vaccines
are subject to a number of potential limitations. Subunit vaccines may exhibit poor
immunogenicity, owing to incorrect folding, poor antigen presentation, or differences
in carbohydrate and lipid composition. A major problem is the difficulty of ensuring
that the conformation of the engineered proteins mimics that of the antigens in their
natural environment. Suitable adjuvants and, in the case of peptides, carrier proteins,
must be used to boost the immune response. In addition these vaccines elicit primarily
humoral responses, and thus may fail to evoke effective immunity. Subunit vaccines
are often ineffective for diseases in which whole inactivated virus can be
demonstrated to provide protection.
Virus-like particles (VLPs) are potential candidates for inclusion in
immunogenic compositions. VLPs closely resemble mature virions, but they do not
contain viral genomic material. Therefore, VLPs are nonreplicative in nature, which
make them safe for administration as a vaccine. In addition, VLPs can be engineered
to express viral glycoproteins on the surface of the VLP, which is their most native
physiological configuration. Moreover, since VLPs resemble intact virions and are
multivalent particulate structures, VLPs may be more effective in inducing
neutralizing antibodies to the glycoprotein than soluble envelope protein antigens.
To date, VLPs have been produced for more than 30 different viruses that
infect humans and other animals. One of the most striking features of this group is that
it is extremely diverse in terms of the structure of the individual viruses. It includes
viruses that have a single capsid protein, multiple capsid proteins, and those with and
without lipid envelopes.
The formation of VLPs for viruses with a lipid envelope has a different type of
technical challenge than those produced for viruses with multiple capsids. For these
viruses, the choice of expression system can be important for the efficiency of VLP
formation. For example, Hantaan virus readily forms VLPs when expressed in
mammalian cells from a vaccinia-virus-based vector, but VLP formation is relatively
inefficient in insect cells (Betenbaugh M et al. 1995, Virus Res. 38, 111-124).
The rabies virus (RV) is a member of the family Rhabdoviridae. Like most
members of this family, RV is a non-segmented, negative stranded RNA virus whose
genome codes for five viral proteins: RNA-dependent RNA polymerase (L); a
nucleoprotein (N); a phosphorylated protein (P); a matrix protein (M) located on the
inner side of the viral protein envelope; and an external surface glycoprotein (G).
(Dietzschold B et al., 1991, Crit. Rev. Immunol. 10: 427-439.)
Cell-cultured based vaccines for rabies are limited to growing inactivated
strains of the virus in cell cultures. These vaccines comprise the virus grown in cell
cultures. Current biotechnological approaches aim at expressing the coat protein gene
of the rabies virus to develop a safe recombinant protein that could be deployed as an
active vaccine. Stable expression of rabies virus glycoprotein has been shown in
Chinese Hamster Ovary cells (Burger et al., 1991, J Gen Virol., Feb; 72 ( Pt 2):359-
67). A full length, glycosylated protein of 67 K that co-migrated with the G-protein
isolated from virus-infected cells, was obtained.
Expression of rabies glycoprotein gene by baculoviral vectors in insect cells
gives yields of protein to the extent of 18% of total cellular protein, 48 h post
infection. Prehaud D H et al, (1989, Virology, Dec;173(2):390-9) describe placing a
sequence encoding the G protein of CVS strain under control of the AcNPV
polyhedrin promoter and expressing the construct using a Spodopterafugiperda cell-
line. The insect derived protein exhibited altered electrophoretic mobility compared to
the wild type due to differences in the glycan components.
Rupprecht et al (1993, Vaccine, 11(9):925-8) demonstrate that a glycoprotein
(ERA strain) derived from recombinant baculovirus —infected insect cells was
efficacious as an oral vaccine in raccoons. WO/1993/001833 teaches the production of
a virus like particles (VLPs) in a baculovirus expression system containing an RNA
genome including a 3' domain and a filler domain surrounded by a sheath of rabies N
protein and rabies M protein. The VLP also includes a lipid envelope of rabies G
protein. In view of relatively high costs of the insect and mammalian cell-systems
these are not the systems of choice for G protein expression as a strategy to develop
vaccine against rabies.
McGarvey et al. (1995, Bio/Technology Vol. 13, No. 13, pp. 1484-1487)
describe the transformation of tomato cotyledons using a full length cDNA encoding
glycoprotein G of rabies virus (ERA strain) under the control of the 35S' promoter of
cauliflower mosaic virus. The protein was expressed in tomato and was characterized
as having a molecular weight of 62 and 60 kDa in western blot after
immunoprecipitation, as compared to 66 kDa observed for G protein from virus
grown in BH cells. The difference in molecular weight compared to the natural
glycoprotein was suggested to result from post-translational modification of the
protein (proteolytic cleavage and/or modified glycosylation). The amount of G protein
imrnunoprecipitated was found to be approximately 1-10 ng/mg of soluble protein i.e.
at 0.0001% to 0.001% of soluble protein. The low expression level may have been due
to using a poorly designed gene. For example, a native G protein coding gene was
used along with its native signal peptide.
[001 1] Plant derived immune response against diseases such as mink enteritis and
rabies were reported by expressing viral epitopes on the surface of plant viruses,
followed by infection of susceptible host with the recombinant modified virus
(Modelska et al., 1998, Proc. Natl. Acad. Sci. USA 95: 2481-2485; Yusibov et al.,
2002, Vaccine 20:3 155-3164). The size of the antigen polypeptide expressed on
surface of a vector virus was limited to 37 amino acids, and required epitope mapping
of the antigen. Such thorough knowledge of the antigen is not generally available,
especially with newly discovered diseases where the expression of full-length proteins
may be the only option. I some cases, multiple epitopes may be required to give
acceptable protection against challenge by the pathogenic virus. Furthermore,
containment could be considered as a significant problem at the agricultural level,
especially when environmentally stable plant viruses for example, Tobacco Mosaic
Virus, are used.
[001 2] WO 97/43428 teaches a method for producing the glycoprotein G of the
rabies virus, or a virus related to the rabies virus, in plants. The construct comprised a
sequence encoding a chimeric G protein comprising a mature viral protein G with a
N-terminal signal peptide other than that naturally associated with the viral protein G.
The glycoprotein had a molecular weight of approximately 66 kDa and was highly
insoluble. Detergents, such as SDS, or Triton X-100, were necessary to extract and
solubilize the glycoprotein. The authors concluded that the "insoluble" glycoprotein
was related to the presence of the C-terminal transmembrane domain (the region
located about 40 to 60 amino acids of the carboxy terminus of the glycoprotein) which
is important for a protective response when the glycoprotein is used as vaccine.
Enveloped viruses may obtain their lipid envelope when 'budding' out of the
infected cell and obtain the membrane from the plasma membrane, or from that of an
internal organelle. For example, during the assembly process in rhabdoviruses, the N-P-L
complex encapsulates negative-stranded genomic RNA to form the RNP core. The M
protein forms a capsule, or matrix, around the RNP, and the RNP-M complex migrates to
an area of the plasma membrane containing glycoprotein inserts. The M-protein initiates
coiling, and the M-R P complex binds with the glycoprotein, whereupon the completed
virus buds from the plasma membrane.
[00 1 ] Within the central nervous system (CNS), there is preferential viral budding from
plasma membranes. Conversely, virus in the salivary glands buds primarily from the cell
membrane into the acinar lumen. Viral budding into the salivary gland and virus-induced
aggressive biting-behavior in the host animal maximize chances of viral infection of a
new host. In mammalian or baculovirus cell systems, for example, rabies buds from the
plasma membrane. Only a few enveloped viruses are known to infect plants (for example,
members of the Topoviruses and Rhabdoviruses). Of the known plant enveloped viruses,
they are characterized by budding from internal membranes of the host cell, and not from
the plasma membrane. However, recombinant VLPs have been produced in plant hosts
from the plasma membrane (WO 201 1/035422; which is incorporated herein by
reference).
[00 5] Assembly/budding in rhabdoviruses is driven largely by the matrix (M) protein.
The M protein contains a late budding domain that mediates the recruitment of host
proteins linked to the vacuolar protein sorting pathway of the cell to facilitate virus-cell
separation. Without wishing to be bound by theory, budding of enveloped viruses from
cellular membranes is believed to depend on the presence of transmembrane spike
proteins interacting with cytoplasmic virus components. For example, cells infected with
rabies virus mutants that were deficient for the glycoprotein G, or the G cytoplasmic tail,
released spikeless rhabdovirus particles, demonstrating that the viral surface protein is not
required to drive the budding process. (Mebatsion T. et al., 1996, Cell, Mar
22:84(6):941-51). In contrast, infectious particles produced within M-protein deficient
rabies virus mutants were mainly cell associated, and the yield of cell-free infectious
virus was reduced by as much as 500,000-fold. This demonstrates the significant role of
the M protein in virus budding. Supernatants from cells infected with the M-deficient
rabies virus comprised long, rod-shaped virions, rather than the typical bullet-shaped
rhabdovirus particles, further confirming impairment of the virus formation process.
Complementation with M protein expressed from plasmids rescued rhabdovirus
formation. The M protein therefore appears to play an important role in condensing and
targeting the RNP to the plasma membrane as well as in incorporation of G protein into
budding virions. (Mebatsion T. et al, 1999, J Virol Jan; 73(l):242-50).
SUMMARY OF THE INVENTION
The present invention relates to producing native viral proteins in plants.
More specifically, the present invention also relates to producing virus-like particles
comprising native rabies virus structural protein in plants.
[001 7] According to the present invention there is provided a method (A) of
producing a rabies virus like particle (VLP) in a plant comprising,
a) introducing a first nucleic acid comprising a first regulatory region active
in the plant operatively linked to a nucleotide sequence encoding a native rabies virus
structural protein into the plant, or portion of the plant,
b) incubating the plant or portion of the plant under conditions that permit the
expression of the nucleic acids, thereby producing the rabies VLP.
The native rabies virus structural protein may be a glycoprotein. If the native rabies
virus structural protein is not an M protein, then the method (A) as described above
may further comprise a step of:
c) introducing a second nucleic acid comprising a second regulatory region
active in the plant and operatively linked to a nucleotide sequence encoding a matrix
protein.
[00 18] The first or second nucleotide sequence or both may further encode,
comprise, or encode and comprise, one or more than one amplification element. The
one or more than one amplification element may be selected from one or more than
one geminivirus amplification element. The one or more than one geminivirus
amplification element may be selected from a Bean Yellow Dwarf Virus long
intergenic region (BeYDV LIR), and a BeYDV short intergenic region (BeYDV SIR).
[00 1 ] The first regulatory region active in the plant, and the second regulatory
region active in the plant may be the same or different.
The method as described above may further comprising a step of:
d) harvesting the plant and extracting the VLPs.
The present invention also includes the method (A) as described above,
wherein the first nucleic acid sequence comprises the regulatory region operatively
linked with a one or more than one comovirus enhancer, and a third nucleic acid
encoding a suppressor of silencing, a geminivirus replicase, or both, are introduced
into the plant or portion of the plant. Alternatively, first the nucleic acid sequence
comprising the regulatory region operatively linked with a one or more than one
comovirus enhancer, the second nucleic acid comprising a second regulatory region
active in the plant and operatively linked to a nucleotide sequence encoding the matrix
protein, and the third nucleic acid encoding a suppressor of silencing, a geminivirus
replicase, or both, may be introduced into the plant or portion of the plant. If the third
nucleic acid only comprises the suppressor of silencing, then a fourth nucleic acid
encoding the geminivirus replicase may be introduced into the plant or portion of the
plant. The one or more than one comovirus enhancer may be a comovirus UTR, for
example, a Cowpea Mosaic Virus hyperanslatable (CPMV-HT) UTR such as the
CPMV-HT 5' and/or 3'UTR.
The present invention also includes the method (A) as described above,
wherein in the step of introducing (step a), the first nucleic acid is transiently
expressed in the plant. Alternatively, in the step of introducing (step a), the first
nucleic acid is stably expressed in the plant.
The present invention also provides a method (B) of producing a rabies virus
like particle (VLP) comprising,
a) providing a plant or portion of the plant comprising a first nucleic acid
comprising a first regulatory region active in the plant operatively linked to a
nucleotide sequence encoding one or more native rabies virus structural protein,
b) incubating the plant or portion of the plant under conditions that permit the
expression of the nucleic acids, thereby producing the native rabies VLP.
The first regulatory region active in the plant, and the second regulatory
region active in the plant may be the same or different.
The native rabies virus structural protein in method (B) may be a
glycoprotein. If the a native rabies virus structural protein is not an M (matrix) protein,
then in method (B) as described above the plant may further comprise a second
nucleic acid comprising a second regulatory region active in the plant and operatively
linked to a nucleotide sequence encoding a matrix protein. Alternatively, the first
nucleic acid might comprise a nucleotide sequence encoding a matrix protein.
In the method as described above (Method B) the first nucleic acid or second
nucleic acid or both may further encode, comprise, or encode and comprise, one or
more than one amplification element. The one or more than one amplification
element may be selected from one or more than one geminivirus amplification
element. The one or more than one geminivirus amplification element may be
selected from a Bean Yellow Dwarf Virus long intergenic region (BeYDV LIR), and a
BeYDV short intergenic region (BeYDV SIR).
In the methods as described above (Methods A or B) the plant or portion of
the plant may further comprise another nucleic acid sequence encoding a suppressor
of silencing, for example HcPro or pi 9, a geminivirus replicase or both. Alternatively,
the plant or portion of the plant may comprise yet another nucleic acid encoding the
geminivirus replicase.
The present invention also includes the method (B) as described above,
wherein the plant or portion of the plant transiently expressed the first nucleic acid.
Alternatively, the first nucleic acid is stably expressed in the plant or portion of the
plant.
The method (B) as described above may further comprising a step of:
d) harvesting the plant and extracting the VLPs.
The present invention provides a VLP produced by the methods (A) and/or
(B) as described above. The VLP may further comprising one or more than one lipid
derived from a plant. The, one or more native rabies virus structural protein
comprises of the VLP may comprise plant-specific N-glycans, or modified N-glycans.
The present invention also provides a polyclonal antibody prepared using the VLP.
[003 1] The present invention includes a composition comprising an effective dose of
the VLP made by the method (A) or (B) as just described, for inducing an immune
response, and a pharmaceutically acceptable carrier.
The present invention also includes a method of inducing immunity to an
rabies virus infection in a subject, comprising administering the VLP as just
described, to the subject. The VLP may be administered to a subject orally,
intradermally, intranasally, intramusclarly, intraperitoneally, intravenously, or
subcutaneously.
The present invention also provides plant matter comprising a VLP produced
by the method (A) and/or (B) described above. The plant matter may be used in
inducing immunity to a rabies virus infection in a subject. The plant matter may also
be admixed as a food supplement.
This summary of the invention does not necessarily describe all features of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the invention will become more apparent from the
following description in which reference is made to the appended drawings wherein:
Figure 1 shows a Western blot analysis of transient rabies G protein
expression in Nicotiana benthamiana. Rabies G protein was expressed under the
control of CPMV-HT with (1091) or without (1071) BeYDV-based DNA
amplification system (see table 2 in the Examples for constructs). Numbers in
parenthesis refer to the amount of Agrobacterium culture, in milliliters, used in the
preparation of the bacterial inoculum. Plants infiltrated with AGLl/1091 were harvest
3 or 4 days post-infiltration (DPI). Leaves of infiltrated plants were harvested and
extracted mechanically. Protein extracts were separated by SDS-PAGE and analyzed
by western blot using anti-rabies G mouse monoclonal antibodies (Santa-Cruz SC-
57995).
Figure 2 shows a comparison of rabies G protein content in protein extracts
from biochemical and mechanical extraction methods for rabies G protein. Protein
extracts were separated by SDS-PAGE and analyzed by western blot using anti-rabies
G mouse monoclonal antibodies (Santa-Cruz SC-57995). Numbers in parenthesis
refer to the amount of Agrobacterium culture, in milliliters, used in the preparation of
the bacterial inoculum (see table 2 for constructs).
Figure 3A shows a western blot analysis of rabies G protein content after
separation, by size exclusion chromatography (SEC), of concentrated protein extracts
from plants infiltrated with AGLl/1091. Elution fractions from SEC were separated
by SDS-PAGE and analyzed by western blot using anti-rabies protein G mouse
monoclonal antibodies (Santa-Cruz SC-57995). Figure 3B shows a western blot
analysis of rabies G protein content after separation, by size exclusion
chromatography (SEC), of concentrated protein extracts from plants infiltrated with
AGLl/1091 +AGL1/1086. Elution fractions from SEC were separated by SDS-PAGE
and analyzed by western blot using anti-rabies protein G mouse monoclonal
antibodies (Santa-Cruz SC-57995).
Figure 4A shows primer IF-RabM-S3.c (SEQ ID NO:l). Figure 4B shows
primer IF-RabM-Sl-4.r (SEQ ID NO: 2). Figure 4C shows synthesized M protein
coding sequence (corresponding to nt 2496-3 04 from Genbank accession number
FJ913470) (SEQ ID NO. 3). Figure 4D shows a schematic representation of construct
number 191. SacII and Stul restriction enzyme sites used for plasmid linearization
are annotated on the representation. Figure 4E shows construct number 1 9 1 from
left to right t-DNA borders (underlined). 2X35S/CPMV-HT/NOS with Plastocyanin-
P 19-Plastocyanin expression cassette (suppressor of silencing) (SEQ ID NO: 4).
Figure 4F shows expression cassette number 1066 from 2X35S promoter to NOS
terminator. The open reading frame of M protein from Rabies virus ERA strains is
underlined (SEQ ID NO: 5). Figure 4G shows the amino acid sequence of M protein
from Rabies virus ERA strain (SEQ ID NO: 6). Figure 4H shows the schematic
representation of construct number 1066.
Figure 5A shows a schematic representation of construct number 193. SacII
and Stul restriction enzyme sites used for plasmid linearization are annotated on the
representation. Figure 5B shows construct number 193 from left to right t-DNA
borders (underlined). 2X3 S/CPMV-HT/NOS into BeYDV+Replicase amplification
system with Plastocyanin-TBSV P19-Plastocyanin expression cassette (suppressor of
silencing) (SEQ ID NO:7). Figure 5C shows expression cassette number 086 from
BeYDV left LIR to BeYDV right LIR. Open reading frame of PDISP/G protein from
Rabies virus ERA strain is underlined. (SEQ ID NO: 8). Figure 5D shows a
schematic representation of construct number 1086.
Figure 6A shows primer IF-RabG-S2+4.c (SEQ ID NO: 9). Figure 6B shows
primer IF-RabG-Sl-4.r (SEQ ID NO:10). Figure 6C shows synthesized Rabies G
protein coding sequence (corresponding to nt 3317-4891 from Genbank accession
number EF206707) (SEQ ID NO: 1). Figure 6D shows a schematic representation of
construct number 1192. Sacll and Stul restriction enzyme sites used for plasmid
linearization are annotated on the representation. Figure 6E shows construct number
1192 from left to right t-DNA borders (underlined). 2X35S/CPMV-HT/PDISP/NOS
with Plastocyanin-P19-Plastocyanin expression cassette (suppressor of silencing)
(SEQ ID NO: 12). Figure 6F shows expression cassette number 1071 from 2X35S
promoter to NOS terminator. Open reading frame of PDISP/G protein from Rabies
virus ERA strain is underlined. (SEQ ID NO: 13). Figure 6G shows amino acid
sequence of PDISP-G protein from Rabies virus ERA strain (SEQ ID NO: 14). Figure
6H shows a schematic representation of construct number 107 .
Figure 7A shows a schematic representation of construct number 1194. Sacll
and Stul restriction enzyme sites used for plasmid linearization are annotated on the
representation. Figure 7B shows construct number 1194 from left to right t-DNA
borders (underlined). 2X35S/CPMV-HT/PDISP/NOS into BeYDV+Replicase
amplification system with Plastocyanin-P19-Plastocyanin expression cassette
(suppressor of silencing) (SEQ ID NO: 15). Figure 7C shows expression cassette
number 1091 from BeYDV left LIR to BeYDV right LIR. Open reading frame of
PDISP/G protein from Rabies virus ERA strain is underlined. (SEQ ID NO: 16).
Figure 7D shows a schematic representation of construct number 1091.
Figure 8A shows a coomassie stained SDS-PAGE (Precast 4-12%,from
BioRad) showing G protein VLP preparation. 1) Rab-VLP preparation, 2) molecular
weight marker. Figure 8B shows responses of neutralizing antibodies (IU/ml) in
groups of mice (5 per group) immunized intramuscularly (i.m.) with three doses (DO,
D7 and D28) of 0.1ml of the NG-VLP (Native G protein VLP) vaccine with or
without adjuvant (Alhydrogel (Alhy)). Blood samples were taken on Day 44, 6 days
after the 3rd doses. Method of testing used: RFFIT test (rapid fluorescent focus
inhibition test): Geometric Mean Titers (GMT) was calculated, using the individual
values obtained for each animal and positive responders are indicated. Bars represent
GMT with 95% CI. Anova was performed between all treatment groups and no
statistically significant differences were reported.
DETAILED DESCRIPTION
The following description is of a preferred embodiment.
The present invention relates to virus-like particles (VLPs) comprising one or
more native rabies virus structural protein, and methods of producing rabies VLPs in
plants. The rabies VLPs may comprise one or more native rabies virus structural
protein, for example a one or more glycoprotein, one or more matrix protein, or both.
The VLP does not comprise virus proteins from a plant virus.
The present invention in part provides a method of producing a rabies virus
like particle (VLP) in a plant. The method may comprise introducing a nucleic acid
comprising a regulatory region active in the plant operatively linked to a nucleotide
sequence encoding a native rabies virus structural protein and one or more than one
amplification element, into the plant, or portion of the plant. Followed by incubating
the plant or portion of the plant under conditions that permit the expression of the
nucleic acids, thereby producing the VLP.
The native rabies structural virus protein (also referred to as native rabies
structural viral protein) may refer to all or a portion of an native rabies virus structural
protein sequence isolated from rabies virus, present in any naturally occurring or
variant rabies virus strain or isolate. Thus, the term native rabies virus structural
protein and the like include naturally occurring native rabies virus structural protein
sequence variants produced by mutation during the virus life-cycle or produced in
response to selective pressure (e.g., drug therapy, expansion of host cell tropism or
infectivity, etc.). As one of skill in the art appreciates, such native rabies virus
structural protein sequences and variants thereof may be also produced using
recombinant techniques. The native rabies structural virus protein does not include
chimeric proteins wherein for example a transmembrane domain and/or a cytoplasmic
tail have been replaced with a heterologous transmembrane domain and/or a
cytoplasmic tail with respect to the native rabies structural virus protein.
A non-limiting example of a native rabies virus structural protein is rabies
glycoprotein (G) protein, a fragment of G protein, a matrix (M) protein, a fragment of
M protein, or a combination thereof. Non-limiting examples of G protein, or
fragments of G protein that may be used according to the present invention include
those G protein from rabies ERA strain. An example of a G protein, which is not to
be considered limiting, is set forth in the amino acid sequence of SEQ ID NO: 14.
Furthermore, the native rabies structural virus protein may comprise the sequence set
forth in SEQ ID NO: , or a sequence having at least about 90-100% sequence
similarity thereto, including any percent similarity within these ranges, such as 91,
92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto.
Amino acid sequence similarity or identity may be computed by using the
BLASTP and TBLASTN programs which employ the BLAST (basic local alignment
search tool) 2.0 algorithm. Techniques for computing amino acid sequence similarity
or identity are well known to those skilled in the art, and the use of the BLAST
algorithm is described in ALTSCHUL et al. (1990, J Mol. Biol. 215: 403- 410) and
ALTSCHUL et al. (1997, Nucleic Acids Res. 25: 3389-3402).
The native structural viral protein may exist as a monomer, a dimer, a trimer,
or a combination thereof. A trimer is a macromolecular complex formed by three,
usually non-covalently bound proteins. Without wishing to be bound by theory, the
trimerization domain of a protein may be important for the formation such trimers.
Therefore the viral protein or fragment thereof may comprise a trimerization domain.
By "matrix protein" (also referred to as viral core protein) it is meant a
protein that that organizes and maintains virion structure. Viral matrix proteins
usually interact directly with cellular membranes and can be involved in the budding
process. Viral core proteins are proteins that make up part of the nucelocapsid and
typically are directly associated with the viral nucleic acid. Examples of viral matrix
or core protein are rabies M protein, influenza M 1, RSV M and retrovirus gag
proteins. Examples of Matrix proteins that may be used as described herein include,
but are not limited to rabies M protein. Non-limiting example of sequences that may
be used with the present invention include M protein from rabies virus ERA strain.
An exemplary M protein consists of the amino acid sequence as shown in SEQ ID
NO: 6. Further more, the native rabies structural virus protein may comprise the
sequence set forth in SEQ ID NO: 6 or sequences having at least about 90-100%
sequence similarity thereto, including any percent similarity within these ranges, such
as 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity thereto.
Both vesicular stomatitis virus (a rhabdovirus like rabies) and Herpes
Simplex virus (a herpes virus like varicella-zoster virus) bud in a VSP4-dependent
manner (Taylor et al. J. Virol 8 1:13631-13639, 2007; Crump et al., J. Virol 8 1:7380-
7387, 2007). Since VSP4 interacts with the late domain of the matrix protein, this
suggests that the matrix protein is required for budding and, as a corollary, for VLP
production. However, as described herein, native rabies VLPs may be produced
within plants with or without co-expression of a matrix protein. Therefore, native
rabies VLPs produced in plants from virus derived native structural proteins, in
accordance with the present invention may or may not comprise viral matrix (or viral
core) protein.
The present invention also provides for a method of producing rabies virus
like protein VLPs in a plant, wherein a nucleic acid (a first nucleic acid) encoding a
native rabies virus structural protein, for example a rabies G protein, is co-expressed
with a second nucleic acid encoding a viral matrix protein, for example but not
limited to rabies matrix protein. The nucleic acid, and second nucleic acid, may be
introduced to the plant in the same step, or may be introduced to the plant
sequentially.
As described in more detail below, VLPs may be produced in a plant by
expressing a nucleic acid (a first nucleic acid) encoding one or more native rabies
virus structural protein, for example a rabies G protein. A second nucleic acid
encoding a matrix protein, for example but not limited to rabies matrix protein might
be co-expressed in the plant. The nucleic acid and second nucleic acid may be
introduced to the plant in the same step, or they may be introduced to the plant
sequentially. The nucleic acid and second nucleic acid may be introduced in the
plant in a transient manner, or in a stably manner.
Furthermore, a plant that expresses a first nucleic acid encoding one or more
native rabies virus structural protein, for example a rabies G protein, may be
transformed with a matrix protein, for example but not limited to a rabies matrix
protein, (second nucleic acid) so that both the first and the second nucleic acids are
co-expressed in the plant. Alternatively, a plant that expresses a matrix protein, for
example but not limited to a rabies matrix protein, (second nucleic acid) may be
transformed with a first nucleic acid encoding one or more native rabies virus
structural protein, for example a rabies G protein, so that both the first and the second
nucleic acids are co-expressed in the plant.
Additionally, a first plant expressing the first nucleic acid encoding one or
more native rabies virus structural protein, for example a rabies G protein, may be
crossed with a second plant expressing the second nucleic acid encoding the matrix
protein for example but not limited to rabies matrix protein, to produce a progeny
plant that co-expresses the first and second nucleic acids encoding the native rabies
virus structural protein and the matrix protein, respectively.
The present invention also provides a method of producing rabies virus VLPs
in a plant that involves introducing one or more nucleic acid encoding one or more
native rabies virus structural protein operatively linked to a regulatory region active
in the plant, and one or more than one amplification elements, into the plant or
portion of the plant. The plant or portion of the plant is then incubated under
conditions that permit the expression of the one or more nucleic acid, thereby
producing the rabies virus VLPs. The one or more native rabies virus structural
protein may be one or more rabies G protein, a fragment of the G protein, one or
more M protein, a fragment of the M protein, or a combination thereof.
The present invention further provides for a VLP comprising one or more
native rabies virus structural protein for example but not limited to one or more
rabies virus glycoprotein, one or more matrix protein or both. The VLP may be
produced by the method as provided by the present invention.
The term "virus like particle" (VLP), or "virus-like particles" or "VLPs" refers
to structures that self-assemble and comprise one or more structural proteins such as
for example native rabies virus structural protein for example but not limited to
rabies G protein. VLPs are generally morphologically and antigenically similar to
virions produced in an infection, but lack genetic information sufficient to replicate
and thus are non-infectious. VLPs may be produced in suitable host cells including
plant host cells. Following extraction from the host cell and upon isolation and
further purification under suitable conditions, VLPs may be purified as intact
structures.
The size (i.e. the diameter) of the above-defined VLPs, maybe measures for
example by dynamic light scattering (DLS) or electron microscope (EM) techniques,
is usually between 40 to 300 nm, or any size therebetween. In some embodiments,
the size of the intact VLP structure may range from about 40 r n to about 300 nm, or
any size therebetween, such as 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 10
nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm,
210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm or 280 nm, or any size
therebetween. In an embodiment the size of the intact VLP structure may range
from about 170 nm to about 200 nm or any size therebetween, such as 175 nm, 180
nm,185 nm, 190 nm, 195 nm or any size therebetween.
The present invention further provides a nucleic acid comprising a nucleotide
sequence encoding one or more native rabies virus structural protein operatively
linked to a regulatory region active in a plant. Furthermore one or more native rabies
virus structural protein may be operatively linked to one or more than one
amplification elements. The one or more native rabies virus structural protein may be
for example one or more rabies G protein one or more M protein, or both.
A nucleic acid sequence referred to in the present invention, may be
"substantially homologous" or "substantially similar" to a sequence, or a compliment
of the sequence if the nucleic acid sequence hybridise to one or more than one
nucleotide sequence or a compliment of the nucleic acid sequence as defined herein
under stringent hybridisation conditions. Sequences are "substantially homologous"
"substantially similar" when at least about 70%, or between 70 to 100%, or any
amount therebetween, for example 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,
96, 98, 100%, or any amount therebetween, of the nucleotides match over a defined
length of the nucleotide sequence providing that such homologous sequences exhibit
one or more than one of the properties of the sequence, or the encoded product as
described herein. Correct folding of the protein may be important for stability of the
protein, formation of multimers, formation of VLPs and function. Folding of a
protein may be influenced by one or more factors, including, but not limited to, the
sequence of the protein, the relative abundance of the protein, the degree of
intracellular crowding, the availability of cofactors that may bind or be transiently
associated with the folded, partially folded or unfolded protein.
Such a sequence similarity may be determined using a nucleotide sequence
comparison program, such as that provided within DNASIS (using, for example but
not limited to, the following parameters: GAP penalty 5, #of top diagonals 5, fixed
GAP penalty 10, k-tuple 2, floating gap , and window size 5). However, other
methods of alignment of sequences for comparison are well-known in the art for
example the algorithms of Smith & Waterman (1981, Adv. Appl. Math. 2:482),
Needleman & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc.
Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and BLAST, available through the NIH.), or
by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular
Biology, Ausubel et al., eds. 1995 supplement), or using Southern or Northern
hybridization under stringent conditions (see Maniatis et al., in Molecular Cloning (A
Laboratory Manual), Cold Spring Harbor Laboratory, 1982). Preferably, sequences
that are substantially homologous exhibit at least about 80% and most preferably at
least about 90% sequence similarity over a defined length of the molecule.
An example of one such stringent hybridization conditions may be overnight
(from about -20 hours) hybridization in 4 X SSC at 65°C, followed by washing in
0.1 X SSC at 65°C for an hour, or 2 washes in 0.1 X SSC at 65°C each for 20 or 30
minutes. Alternatively an exemplary stringent hybridization condition could be
overnight (16-20 hours) in 50% formamide, 4 X SSC at 42°C, followed by washing
in 0. X SSC at 65°C for an hour, or 2 washes in 0. X SSC at 65°C each for 20 or
minutes, or overnight (16-20 hours), or hybridization in Church aqueous
phosphate buffer (7% SDS; 0.5M NaP0 buffer pH 7.2; 10 mM EDTA) at 65°C, with
2 washes either at 50°C in 0.1 X SSC, 0.1% SDS for 20 or 30 minutes each, or 2
washes at 65°C in 2 X SSC, 0.1% SDS for 20 or 30 minutes each for unique
sequence regions.
A nucleic acid encoding a native rabies structural polypeptide or a native
rabies virus structural protein may be described as a "rabies nucleic acid", a "rabies
nucleotide sequence", a "native rabies nucleic acid", or a "native rabies nucleotide
sequence". For example, which is not to be considered limiting, a virus-like particle
comprising one or more native rabies virus structural protein or native rabies virus
structural polypeptide, may be described as a "rabies VLP" or "native rabies VLP".
The native rabies virus structural protein or polypeptide may include a signal
peptide that is the same as, or heterologous with, the remainder of the polypeptide or
protein. The term "signal peptide" is well known in the art and refers generally to a
short (about 5-30 amino acids) sequence of amino acids, found generally at the N-
terminus of a polypeptide that may direct translocation of the newly-translated
polypeptide to a particular organelle, or aid in positioning of specific domains of the
polypeptide chain relative to others. As a non-limiting example, the signal peptide
may target the translocation of the protein into the endoplasmic reticulum and/or aid
in positioning of the N-terminus proximal domain relative to a membrane-anchor
domain of the nascent polypeptide to aid in cleavage and folding of the mature
protein, for example which is not to be considered limiting, a native rabies virus
structural protein.
A signal peptide (SP) may be native to the protein or virus protein, or a signal
peptide may be heterologous with respect to the primary sequence of the protein or
virus protein being expressed. For example the native signal peptide of native rabies
G protein or native rabies M protein may be used to express the native rabies virus
structural protein in a plant system.
A signal peptide may also be non-native, for example, from a protein, viral
protein or native structural protein of a virus other than virus protein, or from a plant,
animal or bacterial polypeptide. A non limiting exemple of a signal peptide that may
be used is that of alfalfa protein disulfide isomerase (PDI SP; nucleotides 32-103 of
Accession No. Z 1499).
The present invention therefore provides for a native rabies virus structural
protein comprising a native, or a non-native signal peptide, and nucleic acids
encoding such rabies virus structural proteins.
Amplification Element
The native rabies virus structural protein or polypeptide may be expressed in
an expression system comprising a viral based, DNA or RNA, expression system, for
example but not limited to, a comovirus-based expression cassette and geminivirus-
based amplification element.
The expression system as described herein may comprise an expression
cassette based on a bipartite virus, or a virus with a bipartite genome. For example,
the bipartite viruses may be of the Comoviridae family. Genera of the Comoviridae
family include Comovirus, Nepovirus, Fabavirus, Cheravirus and Sadwavirus.
Comoviruses include Cowpea mosaic virus (CPMV), Cowpea severe mosaic virus
(CPSMV), Squash mosaic virus (SqMV), Red clover mottle virus (RCMV), Bean
pod mottle virus (BPMV), Turnip ringspot virus (TuRSV), Broad bean true mosaic
virus (BBtMV), Broad bean stain virus (BBSV), Radish mosaic virus (RaMV).
Examples of comoviruse RNA-2 sequences comprising enhancer elements that may
be useful for various aspects of the invention include, but are not limited to: CPMV
RNA-2 (GenBank Accession No. NC_003550), RCMV RNA-2 (GenBank Accession
No. NC 003738), BPMV RNA-2 (GenBank Accession No. NC_003495), CPSMV
RNA-2 (GenBank Accession No.NC _003544), SqMV RNA-2 (GenBank Accession
No.NC_003800), TuRSV RNA-2 (GenBank Accession No. NC 013219.1). BBtMV
RNA-2 (GenBank Accession No. GU8 10904), BBSV RNA2 (GenBank Accession
No. FJ028650), RaMV (GenBank Accession No. NC 003800).
Segments of the bipartite comoviral RNA genome are referred to as RNA-1
and RNA-2. RNA-1 encodes the proteins involved in replication while RNA-2
encodes the proteins necessary for cell-to-cell movement and the two capsid proteins.
Any suitable comovirus-based cassette may be used including CPMV, CPSMV,
SqMV, RCMV, or BPMV, for example, the expression cassette may be based on
CPMV.
"Expression cassette" refers to a nucleotide sequence comprising a nucleic
acid of interest under the control of, and operably (or operatively) linked to, an
appropriate promoter or other regulatory elements for transcription of the nucleic acid
of interest in a host cell.
The expression systems may also comprise amplification elements from a
geminivirus for example, an amplification element from the bean yellow dwarf virus
(BeYDV). BeYDV belongs to the Mastreviruses genus adapted to dicotyledonous
plants. BeYDV is monopartite having a single-strand circular DNA genome and can
replicate to very high copy numbers by a rolling circle mechanism. BeYDV-derived
DNA replicon vector systems have been used for rapid high-yield protein production
in plants.
As used herein, the phrase "amplification elements" refers to a nucleic acid
segment comprising at least a portion of one ore more long intergenic regions or long
intergenic repeat (LIR) of a geminivirus genome. As used herein, "long intergenic
region" or "long intergenic repeat" refers to a region of a long intergenic region that
contains a rep binding site capable of mediating excision and replication by a
geminivirus Rep protein. In some aspects, the nucleic acid segment comprising one
or more LIRs, may further comprises a short intergenic region or small intergenic
region (SIR) of a geminivirus genome. As used herein, "short intergenic region" or
"small intergenic region" refers to the complementary strand (the short IR (SIR) of a
Mastreviruses). Any suitable geminivirus-derived amplification element may be
used herein. See, for example, WO2000/20557; WO2010/025285; Zhang X. et al.
(2005, Biotechnology and Bioengineering, Vol. 93, 271-279), Huang Z. et al. (2009,
Biotechnology and Bioengineering, Vol. 103, 706-714), Huang Z. et al.(2009,
Biotechnology and Bioengineering, Vol. 106, 9-17); which are herein incorporated
by reference). If more than one LIR is used in the construct, for example two LIRs,
then the promoter, CMPV-HT regions and the nucleic acid sequence of interest and
the terminator are bracketed by each of the two LIRs. Furthermore, the amplification
element might for example originate from the sequence as disclosed in Halley-Stott
et al. (2007) Archives of Virology 152: 1237-1240, deposited under Gen Bank
accession number DQ458791, which are herein incorporated by reference. The
nucleic acid segment comprising LIRs are joined nucleotides 2401 to 2566 and 1 to
128. The nucleic acid segment comprising SIRs are nucleotides 154 to 1212.
As described herein, co-delivery of bean yellow dwarf virus (BeYDV)-
derived vector and a Rep/RepA-supplying vector, by agroinfiltration of Nicotiana
benthamiana leaves results in efficient replicon amplification and robust protein
production.
A comovirus-based expression cassette and a geminivirus-derived
amplification element may be comprised on separate vectors, or the component parts
may be included in one vector. If two vectors are used, the first and second vectors
may be introduced into a plant cell simultaneously, or separately.
A viral replicase may also be included in the expression system as described
herein to increase expression of the nucleic acid of interest. An non-limiting example
of a replicase is a BeYDV replicase (pREPl 10) encoding BeYDV Rep and RepA
(C2/C1; Huang et al., 2009, Biotechnol. Bioeng. 103, 706-714; which is incorporated
herein by reference). Another non-limiting example of a replicase is disclosed in
Halley-Stott et al. (2007) Archives of Virology 152: 1237-1240, deposited under Gen
Bank accession number DQ458791, which are herein incorporated by reference. The
nucleic acid segment comprising C1:C2 gene are nucleotides 1310 to 2400.
By "co-expressed" it is meant that two or more than two nucleotide sequences
are expressed at about the same time within the plant, and within the same tissue of
the plant. However, the nucleotide sequences need not be expressed at exactly the
same time. Rather, the two or more nucleotide sequences are expressed in a manner
such that the encoded products have a chance to interact. The two or more than two
nucleotide sequences can be co-expressed using a transient expression system, where
the two or more sequences are introduced within the plant at about the same time
under conditions that both sequences are expressed. Alternatively, a platform plant
comprising one of the nucleotide sequences may be transformed in a stable manner,
with an additional sequence encoding the protein of interest for example the native
rabies virus structural protein, introduced into the platform plant in a transient manner.
Chaperon
Correct folding of the expressed native rabies virus structural protein may be
important for stability of the protein, formation of multimers, formation of VLPs,
function of the native rabies virus structural protein and recognition of the native
rabies virus structural protein by an antibody, among other characteristics. Folding
and accumulation of a protein may be influenced by one or more factors, including,
but not limited to, the sequence of the protein, the relative abundance of the protein,
the degree of intracellular crowding, the pH in a cell compartment, the availability of
cofactors that may bind or be transiently associated with the folded, partially folded or
unfolded protein, the presence of one or more chaperone proteins, or the like.
Heat shock proteins (Hsp) or stress proteins are examples of chaperone
proteins, which may participate in various cellular processes including protein
synthesis, intracellular trafficking, prevention of misfolding, prevention of protein
aggregation, assembly and disassembly of protein complexes, protein folding, and
protein disaggregation. Examples of such chaperone proteins include, but are not
limited to, Hsp60, Hsp65, Hsp 70, Hsp90, HsplOO, Hsp20-30, HsplO, HsplOO-200,
Hsp 100, Hsp90, Lon, TF55, FKBPs, cyclophilins, ClpP, GrpE, ubiquitin, calnexin,
and protein disulfide isomerases (see, for example, Macario, A.J.L., Cold Spring
Harbor Laboratory Res. 25:59-70. 1995; Parsell, D.A. & Lindquist, S. Ann. Rev.
Genet. 27:437-496 (1993); U.S. Patent No. 5,232,833). As described herein,
chaperone proteins, for example but not limited to Hsp40 and Hsp70 may be used to
ensure folding of a rabies virus protein.
Examples of Hsp70 include Hsp72 and Hsc73 from mammalian cells, DnaK
from bacteria, particularly mycobacteria such as Mycobacterium leprae,
Mycobacterium tuberculosis, and Mycobacterium bovis (such as Bacille-Calmette
Guerin: referred to herein as Hsp71). DnaK from Escherichia coli, yeast and other
prokaryotes, and BiP and Grp78 from eukaryotes, such as A. thaliana (Lin et al. 2001
(Cell Stress and Chaperones 6:201-208). A particular example of an Hsp70 is A.
thaliana Hsp70 (encoded by Genbank ref: AY 20747.1). Hsp70 is capable of
specifically binding ATP as well as unfolded polypeptides and peptides, thereby
participating in protein folding and unfolding as well as in the assembly and
disassembly of protein complexes.
Examples of Hsp40 include DnaJ from prokaryotes such as E. coli and
mycobacteria and HSJ1, HDJ1 and Hsp40 from eukaryotes, such as alfalfa (Frugis et
al., 1999. Plant Molecular Biology 40:397-408). A particular example of an Hsp40 is
M . sativa MsJl (Genbank ref: AJ000995.1). Hsp40 plays a role as a molecular
chaperone in protein folding, thermotolerance and DNA replication, among other
cellular activities.
Among Hsps, Hsp70 and its co-chaperone, Hsp40, are involved in the
stabilization of translating and newly synthesized polypeptides before the synthesis is
complete. Without wishing to be bound by theory, Hsp40 binds to the hydrophobic
patches of unfolded (nascent or newly transferred) polypeptides, thus facilitating the
interaction of Hsp70-ATP complex with the polypeptide. ATP hydrolysis leads to the
formation of a stable complex between the polypeptide, Hsp70 and ADP, and release
of Hsp40. The association of Hsp70-ADP complex with the hydrophobic patches of
the polypeptide prevents their interaction with other hydrophobic patches, preventing
the incorrect folding and the formation of aggregates with other proteins (reviewed in
Hartl, FU. 1996. Nature 381 :571-579).
Native chaperone proteins may be able to facilitate correct folding of low
levels of recombinant protein, but as the expression levels increase, the abundance of
native chaperones may become a limiting factor. High levels of expression of native
rabies virus structural protein in the agroinfiltrated leaves may lead to the
accumulation of native rabies virus structural protein in the cytosol, and co-expression
of one or more than one chaperone proteins such as Hsp70, Hsp40 or both Hsp70 and
Hsp40 may reduce the level of misfolded or aggregated proteins, and increase the
number of proteins exhibiting tertiary and quaternary structural characteristics that
allow for formation of virus-like particles.
Therefore, the present invention also provides for a method of producing rabies VLPs
in a plant, wherein a first nucleic acid encoding a native rabies virus structural protein
is co-expressed with a second nucleic acid encoding a chaperone. The first and
second nucleic acids may be introduced to the plant in the same step, or may be
introduced to the plant sequentially.
Regulatory Element
The use of the terms "regulatory region", "regulatory element" or "promoter"
in the present application is meant to reflect a portion of nucleic acid typically, but not
always, upstream of the protein coding region of a gene, which may be comprised of
either DNA or RNA, or both DNA and RNA. When a regulatory region is active, and
in operative association, or operatively linked, with a gene of interest, this may result
in expression of the gene of interest. A regulatory element may be capable of
mediating organ specificity, or controlling developmental or temporal gene activation.
A "regulatory region" may includes promoter elements, core promoter elements
exhibiting a basal promoter activity, elements that are inducible in response to an
external stimulus, elements that mediate promoter activity such as negative regulatory
elements or transcriptional enhancers. "Regulatory region", as used herein, may also
includes elements that are active following transcription, for example, regulatory
elements that modulate gene expression such as translational and transcriptional
enhancers, translational and transcriptional repressors, upstream activating sequences,
and mR A instability determinants. Several of these latter elements may be located
proximal to the coding region.
In the context of this disclosure, the term "regulatory element" or "regulatory
region" typically refers to a sequence of DNA, usually, but not always, upstream (5')
to the coding sequence of a structural gene, which controls the expression of the
coding region by providing the recognition for RNA polymerase and/or other factors
required for transcription to start at a particular site. However, it is to be understood
that other nucleotide sequences, located within introns, or 3' of the sequence may also
contribute to the regulation of expression of a coding region of interest. An example
of a regulatory element that provides for the recognition for RNA polymerase or other
transcriptional factors to ensure initiation at a particular site is a promoter element.
Most, but not all, eukaryotic promoter elements contain a TATA box, a conserved
nucleic acid sequence comprised of adenosine and thymidine nucleotide base pairs
usually situated approximately 25 base pairs upstream of a transcriptional start site. A
promoter element comprises a basal promoter element, responsible for the initiation of
transcription, as well as other regulatory elements (as listed above) that modify gene
expression.
There are several types of regulatory regions, including those that are
developmentally regulated, inducible or constitutive. A regulatory region that is
developmentally regulated, or controls the differential expression of a gene under its
control, is activated within certain organs or tissues of an organ at specific times
during the development of that organ or tissue. However, some regulatory regions that
are developmentally regulated may preferentially be active within certain organs or
tissues at specific developmental stages, they may also be active in a developmentally
regulated manner, or at a basal level in other organs or tissues within the plant as well.
Examples of tissue-specific regulatory regions, for example see-specific a regulatory
region, include the napin promoter, and the cruciferin promoter (Rask et al., 1998, J.
Plant Physiol. 152: 595-599; Bilodeau et al, 1994, Plant Cell 14: 125-130). An
example of a leaf-specific promoter includes the plastocyanin promoter (see US
7,125,978, which is incorporated herein by reference).
An inducible regulatory region is one that is capable of directly or indirectly
activating transcription of one or more DNA sequences or genes in response to an
inducer. In the absence of an inducer the DNA sequences or genes will not be
transcribed. Typically the protein factor that binds specifically to an inducible
regulatory region to activate transcription may be present in an inactive form, which is
then directly or indirectly converted to the active form by the inducer. However, the
protein factor may also be absent. The inducer can be a chemical agent such as a
protein, metabolite, growth regulator, herbicide or phenolic compound or a
physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly
through the action of a pathogen or disease agent such as a virus. A plant cell
containing an inducible regulatory region may be exposed to an inducer by externally
applying the inducer to the cell or plant such as by spraying, watering, heating or
similar methods. Inducible regulatory elements may be derived from either plant or
non-plant genes (e.g. Gatz, C. and LenJk, LR.P., 1998, Trends Plant Sci. 3, 352-358;
which is incorporated by reference). Examples, of potential inducible promoters
include, but not limited to, tetracycline-inducible promoter (Gatz, C.,1997, Ann. Rev.
Plant Physiol. Plant Mol. Biol. 48,89-108; which is incorporated by reference), steroid
inducible promoter (Aoyama. T. and Chua, N.H.,1997, Plant 1. 2, 397-404; which is
incorporated by reference) and ethanol-inducible promoter (Salter, M.G., et al, 1998,
Plant lOurnal 16, 127-132; Caddick, M.X., et al,1998, Nature Biotech. 16, 177-180,
which are incorporated by reference) cytokinin inducible IB6 and CKI 1 genes
(Brandstatter, . and K.ieber, 1.1.,1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996,
Science 274,982-985; which are incorporated by reference) and the auxin inducible
element, D 5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which is
incorporated by reference).
A constitutive regulatory region directs the expression of a gene throughout
the various parts of a plant and continuously throughout plant development. Examples
of known constitutive regulatory elements include promoters associated with the
CaMV 35S transcript (Odell et al., 1985, Nature, 313: 810-812), the rice actin 1
(Zhang et al, 1991, Plant Cell, 3: 1155-1 165), actin 2 (An et al, 1996, Plant J., 10:
107-121), or tins 2 (U.S. 5,428,147, which is incorporated herein by reference), and
triosephosphate isomerase 1 (Xu et. al., 1994, Plant Physiol. 106: 459-467) genes, the
maize ubiquitin 1 gene (Cornejo et ai, 1993, Plant Mol. Biol. 29: 637-646), the
Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al, 1995, Plant Mol. Biol. 29: 637-
646), and the tobacco translational initiation factor 4A gene (Mandel et al, 1995, Plant
Mol. Biol. 29: 995-1004).
The term "constitutive" as used herein does not necessarily indicate that a gene
under control of the constitutive regulatory region is expressed at the same level in all
cell types, but that the gene is expressed in a wide range of cell types even though
variation in abundance is often observed. Constitutive regulatory elements may be
coupled with other sequences to further enhance the transcription and/or translation of
the nucleotide sequence to which they are operatively linked. For example, the
CPMV-HT system is derived from the untranslated regions of the Cowpea mosaic
virus (CPMV) and demonstrates enhanced translation of the associated coding
sequence. By "native" it is meant that the nucleic acid or amino acid sequence is
naturally occurring, or "wild type". By "operatively linked" it is meant that the
particular sequences, for example a regulatory element and a coding region of interest,
interact either directly or indirectly to carry out an intended function, such as
mediation or modulation of gene expression. The interaction of operatively linked
sequences may, for example, be mediated by proteins that interact with the operatively
linked sequences.
The invention also provides VLPs that obtain a lipid envelope from the plasma
membrane of the cell in which the VLPs are expressed. For example, if the one or
more native rabies virus structural protein is expressed in a plant-based system, the
resulting VLP may obtain a lipid envelope from the plasma membrane of the plant
cell.
Generally, the term "lipid" refers to a fat-soluble (lipophilic), naturally-
occurring molecule. A VLP produced in a plant according to some aspects of the
invention may be complexed with plant-derived lipids. The plant-derived lipids may
be in the form of a lipid bilayer, and may further comprise an envelope surrounding
the VLP. The plant-derived lipids may comprise lipid components of the plasma
membrane of the plant where the VLP is produced, including phospholipids, tri-, di-
and monoglycerides, as well as fat-soluble sterol or metabolites comprising sterols.
Examples include phosphatidylcholine (PC), phosphatidylethanolamine (PE),
phosphatidylinositol, phosphatidylserine, glycosphingolipids, phytosterols or a
combination thereof. A plant-derived lipid may alternately be referred to as a 'plant
lipid'. Examples of phytosterols include campesterol, stigmasterol, ergosterol,
brassicasterol, deltastigmasterol, deltaavenasterol, daunosterol, sitosterol, 24-
methylcholesterol, cholesterol or beta-sitosterol - see, for example, Mongrand et ai.,
2004. As one of skill in the art would understand, the lipid composition of the plasma
membrane of a cell may vary with the culture or growth conditions of the cell or
organism, or species, from which the cell is obtained. Generally, beta-sitosterol is the
most abundant phytosterol.
Cell membranes generally comprise lipid bilayers, as well as proteins for
various functions. Localized concentrations of particular lipids may be found in the
lipid bilayer, referred to as 'lipid rafts'. These lipid raft microdomains may be enriched
in sphingolipids and sterols. Without wishing to be bound by theory, lipid rafts may
have significant roles in endo and exocytosis, entry or egress of viruses or other
infectious agents, inter-cell signal transduction, interaction with other structural
components of the cell or organism, such as intracellular and extracellular matrices.
The VLP produced within a plant may induce a native rabies virus structural
protein comprising plant-specific N-glycans. Therefore, this invention also provides
for a VLP comprising native rabies virus structural protein having plant specific N-
glycans.
Furthermore, modification of N-glycan in plants is known (see for example
U.S. 60/944,344; which is incorporated herein by reference) and native rabies virus
structural proteins having modified N-glycans may be produced. Native rabies virus
structural proteins comprising a modified glycosylation pattern, for example with
reduced fucosylated, xylosylated, or both, fucosylated and xylosylated, N-glycans may
be obtained, or native rabies virus structural proteins having a modified glycosylation
pattern may be obtained, wherein the protein lacks fucosylation, xylosylation, or both,
and comprises increased galatosylation. Furthermore, modulation of post-
translational modifications, for example, the addition of terminal galactose may result
in a reduction of fucosylation and xylosylation of the expressed native rabies virus
structural proteins when compared to a wild-type plant expressing native rabies virus
structural proteins.
For example, which is not to be considered limiting, the synthesis of native
rabies virus structural proteins having a modified glycosylation pattern may be
achieved by co-expressing the native rabies virus structural protein along with a
nucleotide sequence encoding beta- 1.4 galactosyltransferase (GalT), for example, but
not limited to mammalian GalT, or human GalT however GalT from another sources
may also be used. The catalytic domain of GalT may also be fused to a CTS domain
(i.e. the cytoplasmic tail, transmembrane domain, stem region) of N-
acetylglucosaminyl transferase (GNT1), to produce a GNTl-GalT hybrid enzyme, and
the hybrid enzyme may be co-expressed with native rabies virus structural protein.
The native rabies virus structural protein may also be co-expressed along with a
nucleotide sequence encoding N-acetylglucosaminyltrasnferase III (GnT-III), for
example but not limited to mammalian GnT-III or human GnT-III, GnT-III from other
sources may also be used. Additionally, a GNTl-GnT-III hybrid enzyme, comprising
the CTS of GNT1 fused to GnT-IIl may also be used .
Therefore the present invention also provides VLP's comprising native rabies
virus structural protein having modified N-glycans.
Without wishing to be bound by theory, the presence of plant N-glycans on
native rabies virus structural protein may stimulate the immune response by
promoting the binding of native rabies virus structural protein by antigen presenting
cells. Stimulation of the immune response using plant N glycan has been proposed by
Saint-Jore-Dupas et al. (2007). Furthermore, the conformation of the VLP may be
advantageous for the presentation of the antigen, and enhance the adjuvant effect of
VLP when complexed with a plant derived lipid layer.
[001 00] The occurrence of VLPs may be detected using any suitable method for
example, sucrose gradients, or size exclusion chromatography. VLPs may be
assessed for structure and size by, for example electron microscopy, or by size
exclusion chromatography.
For size exclusion chromatography, total soluble proteins may be
extracted from plant tissue by homogenizing (Polytron) sample of frozen-crushed
plant material in extraction buffer, and insoluble material removed by centrifugation.
Precipitation with ice cold acetone or PEG may also be of benefit. The soluble
protein is quantified, and the extract passed through a Sephacryl™ column, for
example a Sephacryl™ S500 column. Blue Dextran 2000 may be used as a
calibration standard. Following chromatography, fractions may be further analyzed by
immunoblot to determine the protein complement of the fraction.
The separated fraction may be for example a supernatant (if
centrifuged, sedimented, or precipitated), or a filtrate (if filtered), and is enriched for
proteins, or suprastructure proteins, such as for example rosette-like structures or
higher-order, higher molecular weight, particles such as VLPs. The separated fraction
may be further processed to isolate, purify, concentrate or a combination thereof, the
proteins, or suprastructure proteins, by, for example, additional centrifugation steps,
precipitation, chromatographic steps (e.g. size exclusion, ion exchange, affinity
chromatography), tangential flow filtration, or a combination thereof. The presence of
purified proteins, or suprastructure proteins, may be confirmed by, for example, native
or SDS-PAGE, Western analysis using an appropriate detection antibody, capillary
electrophoresis, electron microscopy, or any other method as would be evident to one
of skill in the art.
Figures 3A and 3B, show an example of an elution profile of a size
exclusion chromatography analysis of a plant extract comprising VLPs. In this case,
VLPs comprising native rabies virus structural proteins elute in fractions 8 to approx.
11, rosettes and high molecular weight structures elute from about fractions 12 to
about 14, and lower molecular weight, or soluble form of the native rabies virus
structural proteins elute in fractions from about 15 to about 17.
The VLPs may be purified or extracted using any suitable method for
example chemical or biochemical extraction. VLPs are relatively sensitive to
desiccation, heat, pH, surfactants and detergents. Therefore it may be useful to use
methods that maximize yields, minimize contamination of the VLP fraction with
cellular proteins, maintain the integrity of the proteins, or VLPs, and, where required,
the associated lipid envelope or membrane, methods of loosening the cell wall to
release the proteins, or VLP. For example, methods that produce protoplast and/or
spheroplasts may be used (see for example WO 201 1/035422, which is incorporated
herein by reference) to obtain VLPs as described herein. Minimizing or eliminating
the use of detergence or surfactants such for example SDS or Triton X-100 may be
beneficial for improving the yield of VLP extraction. VLPs may be then assessed for
structure and size by, for example, electron microscopy, or by size exclusion
chromatography as mentioned above.
The one or more than one or more genetic constructs of the present
invention may be expressed in any suitable plant host that is transformed by the
nucleotide sequence, or constructs, or vectors of the present invention. Examples of
suitable hosts include, but are not limited to, agricultural crops including alfalfa,
canola, Brassica spp., maize, Nicotiana spp., alfalfa, potato, ginseng, pea, oat, rice,
soybean, wheat, barley, sunflower, cotton and the like.
The one or more genetic constructs of the present invention can further
comprise a 3' untranslated region. A 3' untranslated region refers to that portion of a
gene comprising a DNA segment that contains a polyadenylation signal and any other
regulatory signals capable of effecting mRNA processing or gene expression. The
polyadenylation signal is usually characterized by effecting the addition of
polyadenylic acid tracks to the 3' end of the mRNA precursor. Polyadenylation signals
are commonly recognized by the presence of homology to the canonical form 5'
AATAAA-3' although variations are not uncommon. Non-limiting examples of
suitable 3' regions are the 3' transcribed nontranslated regions containing a
polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as
the nopaline synthase (NOS) gene, plant genes such as the soybean storage protein
genes, the small subunit of the ribulose-I, 5-bisphosphate carboxylase gene
(ssRUBISCO; US 4,962,028; which is incorporated herein by reference), the promoter
used in regulating plastocyanin expression, described in US 7,125,978 (which is
incorporated herein by reference).
One or more of the genetic constructs of the present invention may also
include further enhancers, either translation or transcription enhancers, as may be
required. Enhancers may be located 5' or 3' to the sequence being transcribed.
Enhancer regions are well known to persons skilled in the art, and may include an
ATG initiation codon, adjacent sequences or the like. The initiation codon, if present,
may be in phase with the reading frame ("in frame") of the coding sequence to provide
for correct translation of the transcribed sequence.
The constructs of the present invention can be introduced into plant
cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation,
micro-injection, electroporation, etc. For reviews of such techniques see for example
Weissbach and Weissbach, Methodsfor Plant Molecular Biology, Academy Press,
New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology,
2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant
Metabolism, 2d Ed. DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison
Wesly, Langmans Ltd. London, pp. 561-579 (1997). Other methods include direct
DNA uptake, the use of liposomes, electroporation, for example using protoplasts,
micro-injection, microprojectiles or whiskers, and vacuum infiltration. See, for
example, Bilang, et al. (Gene 100: 247-250 (1991), Scheid et al. (Mol. Gen. Genet.
228: 104-1 12, 1991), Guerche et al. (Plant Science 52: 1 1-1 16, 1987), Neuhause et
al. (Theor. Appl Genet. 75: 30-36, 1987), Klein et al., Nature 327: 70-73 (1987);
Howell et al. (Science 208: 1265, 1980), Horsch et al. (Science 227: 1229-1231,
1985), DeBlock et al, Plant Physiology 91: 694-701, 1989), Methods for Plant
Molecular Biology (Weissbach and Weissbach, eds., Academic Press Inc., 1988),
Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press
Inc., 1989), Liu and Lomonossoff (J Virol Meth, 105:343-348, 2002,), U.S. Pat. Nos.
4,945,050; 5,036,006; and 5,100,792, U.S. patent application Ser. Nos. 08/438,666,
filed May 10, 1995, and 07/951,715, filed Sep. 25, 1992, (all of which are hereby
incorporated by reference).
As described below, transient expression methods may be used to
express the constructs of the present invention (see Liu and Lomonossoff, 2002,
Journal of Virological Methods, 105:343-348; which is incorporated herein by
reference). Alternatively, a vacuum-based transient expression method, as described
by Kapila et al., 1997, which is incorporated herein by reference) may be used. These
methods may include, for example, but are not limited to, a method of Agro-
inoculation or Agro-infiltration, syringe infiltration, however, other transient methods
may also be used as noted above. With Agro-inoculation, Agro-infiltration, or syringe
infiltration, a mixture of Agrobacteria comprising the desired nucleic acid enter the
intercellular spaces of a tissue, for example the leaves, aerial portion of the plant
(including stem, leaves and flower), other portion of the plant (stem, root, flower), or
the whole plant. After crossing the epidermis the Agrobacteria infect and transfer t-
DNA copies into the cells. The t-DNA is episomally transcribed and the mRNA
translated, leading to the production of the protein of interest in infected cells,
however, the passage of t-DNA inside the nucleus is transient.
[001 10] To aid in identification of transformed plant cells, the constructs of this
invention may be further manipulated to include plant selectable markers. Useful
selectable markers include enzymes that provide for resistance to chemicals such as an
antibiotic for example, gentamycin, hygromycin, kanamycin, or herbicides such as
phosphinothrycin, glyphosate, chlorosulfuron, and the like. Similarly, enzymes
providing for production of a compound identifiable by colour change such as GUS
(beta-glucuronidase), or luminescence, such as luciferase or GFP, may be used.
[001 1 ] Also considered part of this invention are transgenic plants, plant cells
or seeds containing the gene construct of the present invention. Methods of
regenerating whole plants from plant cells are also known in the art. In general,
transformed plant cells are cultured in an appropriate medium, which may contain
selective agents such as antibiotics, where selectable markers are used to facilitate
identification of transformed plant cells. Once callus forms, shoot formation can be
encouraged by employing the appropriate plant hormones in accordance with known
methods and the shoots transferred to rooting medium for regeneration of plants. The
plants may then be used to establish repetitive generations, either from seeds or using
vegetative propagation techniques. Transgenic plants can also be generated without
using tissue cultures.
[001 12]
The present invention includes nucleotide sequences:
Table 1. List of Sequence Identification numbers.
SEQ ID Description Table/Figure
1 IF-RabM-S3.c Figure 4A
2 IF-RabM-Sl-4.r
Figure 4B
3 Synthesized M protein coding sequence (corresponding to Figure 4C
nt2496-3 104 from Genbank accession number FJ9 13470)
4 Construct number 1191 from left to right t-DNA borders
Figure 4E
(underlined).2X35S/CPMV-HT/NOS with Plastocyanin-
P19-Plastocyanin expression cassette (suppressor of
silencing)
Expression cassette number 066 from 2X35S promoter to Figure 4F
NOS terminator.Open reading frame of M protein from
Rabies virus ERA strainis underlined.
Amino acid sequence of M protein from Rabies virus ERA Figure 4G
strain
7 Construct number 1193 from left to right t-DNA borders
Figure 5B
(underlined). 2X35S/CPMV-HT/NOS into
BeYDV+Replicase amplification system with Plastocyanin-
TBSV P19-Plastocyanin expression cassette(suppressor of
Table/Figure
SEQ ID Description
silencing)
Expression cassette number 086 from BeYDV left LIR to Figure 5C
BeYDV right LIR. Open reading frame ofPDISP/G protein
from Rabies virus ERA strain is underlined.
Figure 6A
9 IF-RabG-S2+4.c
Figure 6B
IF-RabG-Sl-4.r
11 Synthesized Rabies G protein coding sequence Figure 6C
(corresponding to nt 3317-4891 from Genbank accession
number EF206707)
12 Construct number 1192 from left to right t-DNA borders Figure 6E
(underlined).2X35S/CPMV-HT/PDISP/NOS with
Plastocyanin-PI9-Plastocyanin expression cassette
(suppressor of silencing)
13 Expression cassette number 1071 from 2X3 5S promoter to Figure 6F
NOS terminator. Open reading frame ofPDISP/Gprotein
from Rabies virus ERA strain is underlined.
14 Amino acid sequence of PDISP-G protein from Rabies Figure 6G
virus ERA strain
Construct number 1194 from left to right t-DNA borders
Figure 7B
(underlined).2X35S/CPMV-HT/PDISP/NOS into
BeYDV+Replicase amplification system with Plastocyanin-
P19-Plastocyanin expression cassette (suppressor of
silencing)
Expression cassette number 1091 from BeYDV left LIR to Figure 7C
BeYDV right LIR. Open reading frame ofPDISP/G protein
from Rabies virus ERA strain is underlined.
[001 13] The present invention will be further illustrated in the following examples.
Examples
Constructs
Table 2. Constructs comprising sequences encoding native rabies virus structural
protein
Expression Amplification Signal Structural
Construct number
system system peptide Protein
CPMV HT - -
Rabies M 1066
- Rabies M
CPMV HT BeYDV+rep 1086
- Sp PDI Rabies G 1071
CPMV HT
CPMV HT BeYDV+rep Sp PDI Rabies G 1091
Example 1: Assembly of expression cassettes with rabies prot
A-2X35S/CPMV-HT/Rabies M/NOS (Construct number 1066)
[001 14] A sequence encoding M protein from Rabies virus ERA strain was cloned
into 2X35S/CPMV-HT/NOS expression system in a plasmid containing
Plasto_pro/P19/Plasto_terexpression cassette using the following PCR-based method
A fragment containing the complete M protein coding sequence was amplified using
primers IF-RabM-S3.c (Figure 4A, SEQ ID NO:l) and IF-RabM-Sl-4.r (Figure 4B,
SEQ ID NO: 2) using synthesized M gene (corresponding to nt 2496-3104 from
Genbank accession number FJ913470) (Figure 4C, SEQ ID NO: 3) as template. The
PCR product was cloned in 2X35S/CPMV-HT/NOS expression system using In-
Fusion cloning system (Clontech,Mountain View, CA). Construct 1191(Figure 4D)
was digested with SacII and Stul restriction enzyme and the linearized plasmid was
used for the In-Fusion assembly reaction. Construct number 1191 is an acceptor
plasmid intended for "In Fusion" cloning of genes of interest in a CPMV-HT-based
expression cassette. It also incorporates a gene construct for the co-expression of the
TBSV P 9 suppressor of silencing under the alfalfa Plastocyanin gene promoter and
terminator. The backbone of construct number 1191 is a pCAMBIA binary plasmid
and the sequence from left to right t-DNA borders is presented in Figure 4E (SEQ ID
NO:4). The resulting construct was given number 1066 (Figure 4F, SEQ ID NO: 5).
The amino acid sequence of M protein from Rabies virus ERA strain is presented in
Figure 4G (SEQ ID NO: 6). A representation of plasmid 1066 is presented in Figure
B-2X35S/CPMV-HT/Rabies M/NOS intoBeYDV+Replicase amplification system
(Construct number 1086)
[001 15] A sequence encoding M protein from Rabies virus ERA strain was cloned
into 2X35S/CPMV-HT/NOS expression system comprising the BeYDV+replicase
amplification system in a plasmid containing Plasto_pro/P19/Plasto ter expression
cassette using the following PCR-based method. A fragment containing the complete
M protein coding sequence was amplified using primers IF-RabM-S3.c (Figure 4A,
SEQ ID NO: 1) and IF-RabM-Sl-4.r (Figure 4B, SEQ ID NO: 2) using synthesized M
gene (corresponding to nt 2496-3104 from Genbank accession number FJ9 13470)
(Figure 4C, SEQ ID NO: 3) as template. The PCR product was cloned in
2X35S/CPMV-HT/NOS expression system using In-Fusion cloning system (Clontech,
Mountain View, CA). Construct 193 (Figure 5A, SEQ ID NO: Bl) was digested with
SacII and Stul restriction enzyme and the linearized plasmid was used for the In-
Fusion assembly reaction. Construct number 1193 is an acceptor plasmid intended for
"In Fusion" cloning of genes of interest in a CPMV-HT-based expression cassette into
the BeYDV amplification system. It also incorporates a gene construct for the co-
expression of the TBSV P 9 suppressor of silencing under the alfalfa Plastocyanin
gene promoter and terminator. The backbone of construct number 1193 is a
pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is
presented in Figure 5B (SEQ ID NO: 7). The resulting construct was given number
1086 (Figure 5C, SEQ ID NO: 8). The amino acid sequence of M protein from Rabies
virus ERA strain is presented in Figure 4G (SEQ ID NO: 6). A representation of
plasmid 1086 is presented in Figure 5D.
C-2X35S/CPMV-HT/PDISP/Rabies G NOS (Construct number 1071)
[001 16] A sequence encoding G protein from Rabies virus ERA strain was cloned
into 2X35S-CPMV-HT-PDISP-NOS expression system in a plasmid containing
Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method.
A fragment containing the G protein coding sequence without its wild type signal
peptide was amplified using primersIF-RabG-S2+4.c (Figure 6A, SEQ ID NO:9) and
IF-RabG-Sl-4.r (Figure 6B, SEQ ID NO: 10), using synthesized G gene
(Corresponding to nt 3317-4891 from Genbank accession number EF206707) (Figure
6C, SEQ ID NO: 11) as template. The PCR product was cloned in-frame with alfalfa
PDI signal peptide in 2X35S/CPMV-HT/NOS expression system using In-Fusion
cloning system (Clontech, Mountain View, CA). Construct 1192 (Figure 6D) was
digested with SacII and Stul restriction enzyme and the linearized plasmid was used
for the In-Fusion assembly reaction. Construct number 192 is an acceptor plasmid
intended for "In Fusion" cloning of genes of interest in frame with an alfalfa PDI
signal peptide in a CPMV-HT-based expression cassette. It also incorporates a gene
construct for the co-expression of the TBSV P 9 suppressor of silencing under the
alfalfa Plastocyanin gene promoter and terminator. The backbone of construct 1192 is
a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is
presented in Figure 6E (SEQ ID NO: 12). The resulting construct was given number
1071 (Figure 6F, SEQ ID NO: 13). The amino acid sequence of PDISP/G protein from
Rabies virus ERA strain is presented in Figure 6G (SEQ ID NO: 1 ). A representation
of plasmid 1071 is presented in Figure 6H.
D-2X35S/CPMV-HT/PDISP/Rabies G/NOSintoBe YDV+Replicase amplification
system (Construct number 1091)
[001 17] A sequence encoding G protein from Rabies virus ERA strain was cloned
into 2X35S/CPMV-HT/PDISP/NOS comprising the BeYDV+replicase amplification
system in a plasmid containing Plastojpro/P19/Plasto_ter expression cassette using
the following PCR-based method. A fragment containing Rabies G protein coding
sequence without his wild type signal peptide was amplified using primers IF-RabG-
S2+4 (Figure 6A, SEQ ID NO: 9) and IF-RabG-Sl-4.r (Figure 6B, SEQ ID NO: 10),
using synthesized G gene (Corresponding to nt 3317-4891 from Genbank accession
number EF206707) (Figure 6C, SEQ ID NO: 1) as template. The PGR product was
cloned in-frame with alfalfa PDI signal peptide in 2X35S/CPMV-HT/NOS expression
cassette into the BeYDV amplification system using In-Fusion cloning system
(Clontech, Mountain View, CA). Construct number 1194 (Figure 7A) was digested
with SacII and Stul restriction enzyme and the linearized plasmid was used for the In-
Fusion assembly reaction. Construct number 1194 is an acceptor plasmid intended for
"In Fusion" cloning of genes of interest in frame with an alfalfa PDI signal peptide in
a CPMV-HT-based expression cassette into the BeYDV amplification system. It also
incorporates a gene construct for the co-expression of the TBSV P I9 suppressor of
silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone
of construct number 1194 is a pCAMBIA binary plasmid and the sequence from left
to right t-DNA borders is presented in Figure 7B (SEQ ID NO: 5). The resulting
construct was given number 1091 (Figure 7C, SEQ ID NO: 16). The amino acid
sequence of Influenza PDISP/G protein from Rabies virus ERA strain is presented in
Figure 6G (SEQ ID NO: 14). A representation of plasmid 1091 is presented in Figure
Example 2 : Preparation of plant biomass, inoculum and agro infiltration
[001 18] The terms "biomass" and "plant matter" as used herein are meant to reflect
any material derived from a plant. Biomass or plant matter may comprise an entire
plant, tissue, cells, or any fraction thereof. Further, biomass or plant matter may
comprise intracellular plant components, extracellular plant components, liquid or
solid extracts of plants, or a combination thereof. Further, biomass or plant matter
may comprise plants, plant cells, tissue, a liquid extract, or a combination thereof,
from plant leaves, stems, fruit, roots or a combination thereof. A portion of a plant
may comprise plant matter or biomass.
[001 19] Nicotiana benthamiana plants were grown from seeds in flats filled with a
commercial peat moss substrate. The plants were allowed to grow in the greenhouse
under a 16/8 photoperiod and a temperature regime of 25°C day/20°C night. Three
weeks after seeding, individual plantlets were picked out, transplanted in pots and left
to grow in the greenhouse for three additional weeks under the same environmental
conditions.
Agrobacteria transfected with each construct were grown in a YEB medium
supplemented with 0 mM acid (MES), 20 m M
acetosyringone, 50 g l kanamycin and 25 m g/ml of carbenicillin pH5.6 until they
reached an OD oo between 0.6 and 1.6. Agrobacterium suspensions were centrifuged
before use and resuspended in infiltration medium (10 mM MgCl and 10 mM MES
pH 5.6) and stored overnight at 4°C. On the day of infiltration, culture batches were
diluted in 2.5 culture volumes and allowed to warm before use. Whole plants of N .
benthamiana were placed upside down in the bacterial suspension in an air-tight
stainless steel tank under a vacuum of 20-40 Torr for 2-min. Plants were returned to
the greenhouse for a 2-6 day incubation period until harvest.
A . tumefaciens strains comprising the various constructs as described herein
are referred to by using an "AGLfprefix. For example A. tumefaciens comprising
construct number 1091 is termed "AGL1/1091".
Leaf harvest and total protein extraction (mechanical extraction)
Following incubation, the aerial part of plants was harvested, frozen at -80°C
and crushed into pieces. Total soluble proteins were extracted by homogenizing
(Polytron) each sample of frozen-crushed plant material in 3 volumes of cold 50 mM
Tris pH 8.0, 150 mM NaCl, 0.1% Triton X-100 and 1 mM phenylmethanesulfonyl
fluoride. After homogenization, the slurries were centrifuged at 10,000 g for 0 min at
4°C and these clarified crude extracts (supernatant) kept for analysis.
Leaf harvest and total protein extraction (biochemical extraction)
Following incubation, the aerial part of plants was harvested and cut into
small pieces (3 mm square) with a rolling cutter and special care was taken not to
crush the leaves. Cut biomass was incubated 15-17 hours in 2.5 volumes of 200 mM
mannitol, 125 mM citrate, 75 mM NaP0 , 500 mM NaCl, 25 mM
ethylenediaminetetraacetic acid, 1% (v/v) Multifect CX CG (Genencor, Cat. No.
A03140G190), 1% (v/v) Multifect CX B (Genencor, Cat. No. A03042G190) and 1%
(v/v) Multifect Pectinase FE (Genencor, Cat. No. A02080G190) pH 6.5 at 21°C under
agitation at 100-200 rpm. Digested biomass was then filtered on Miracloth
(Calbiochem, Cat 475855) and centrifuged at 10,000 g for 10 min at 4°C. The
supernatant was transferred to a clean tube and centrifuged again under the same
conditions and the supernatant was kept for analysis.
Protein analysis and immunoblotting
[00 124] The total protein content of clarified crude extracts was determined by the
Bradford assay (Bio-Rad, Hercules, CA) using bovine serum albumin as the reference
standard. Proteins were separated by SDS-PAGE and electrotransferred onto
polyvinylene difluoride (PVDF) membranes (Roche Diagnostics Corporation,
Indianapolis, IN) for immunodetection. Prior to immunoblotting, the membranes were
blocked with 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS-T) for
16-18h at 4°C. Immunoblotting was performed by incubation with 0.25 m g l of a
Santa Cruz SC-57995 primary antibody in 2% skim milk in TBS-Tween 20 0.1%.
Chemiluminescence detection was carried on after incubation with peroxidase-
conjugated goat anti mouse, (JIR, 15146) secondary antibody diluted :10,000
in 2% skim milk in TBS-Tween 20 0.1%. Immunoreactive complexes were detected
by chemiluminescence using luminol as the substrate (Roche Diagnostics
Corporation).
Size exclusion chromatography of protein extract
For size exclusion chromatography (SEC), supernatant from biochemically
extracted biomass was concentrated by centrifugation at 70,000 g for 20 min at 4°C
and the resulting pellet was resuspended in 1/50 volume of cold 50 mM Tris pH 8.0,
150 mM NaCl. Size exclusion chromatography columns of 32 ml Sephacryl™ S-500
high resolution beads (S-500 HR : GE Healthcare, Uppsala, Sweden, Cat. No. 17-
0613-10) were packed and equilibrated with equilibration/elution buffer (50 mM Tris
pH8, 150 mM NaCl). One and a half millilitre of concentrated extract was loaded onto
the column followed by an elution step with 45 mL of equilibration/elution buffer.
The elution was collected in fractions of .5 mL relative protein content of eluted
fractions was monitored by mixing 10 of the fraction with 200 m of diluted Bio-
Rad protein dye reagent (Bio-Rad, Hercules, CA). Two hundred microliters from each
fraction were precipitated by addition of 5 volumes of ice cold acetone followed by
freezing overnight at -20°C. Precipitated proteins were pelleted by centrifugation at
20000 g for 10 min. (4°C) and resuspended in 50 m ΐ of hot SDS-PAGE sample
loading buffer. The column was washed with 2 column volumes of 0.2N NaOH
followed by 10 column volumes of 50 mM Tris pH8, 150 mM NaCl, 20% ethanol.
Each separation was followed by a calibration of the column with Blue Dextran 2000
(GE Healthcare Bio-Science Corp., Piscataway, NJ, USA). Elution profiles of Blue
Dextran 2000 and host soluble proteins were compared between each separation to
ensure uniformity of the elution profiles between the columns used.
Example 3: Evaluation of rabies G protein expression and co-expression
strategies.
Nicotiana benthamiana plants were agro-infiltrated with AGL1/1071 (with
or without AGL1/1066) or AGL1/1091 (with or without AGL1/1086) inoculums at
different concentration and leaves were harvested after 5 days post infiltration (DPI)
for 1071- and 1071 +1066-infiltrated plants or 3 to 4 DPI for 1091- and 1091+1086-
infiltrated plants. Western blot analysis of leaf protein extracts from transformed
plants showed that the BeYDV elements were required to reach a detectable G protein
accumulation level (compare 1071- and 1091 -infiltrated plants in figure 1). Maximum
accumulation level was reached at 3 DPI for plants infiltrated with AGL1/1091 with
or without co-expression of M protein (AGL1/1086) (Figure 1).
The biochemical extraction method was compared to the mechanical
extraction for its capacity to release rabies G protein from the leaves of transformed
plants. Western blot analysis of extracts obtained by biochemical extraction and
mechanical extraction on 1091- and 091+1 086-infiltrated plants showed that the
protein extracts from biochemical extraction contained significantly more G protein
than the extract from mechanical extraction (Figure 2).
Protein extracts from 1091- and 109 1+1 086-infiltrated plants were subjected
to size exclusion chromatography to assess their assembly into high molecular weight
structures. Elution fractions were collected and an aliquot of each fraction was
concentrated by acetone precipitation and analyzed for protein G content by western
blot. As shown in figure 3A, protein G content peaked in SEC elution fractions 8 to
, corresponding to the void volume of the column where enveloped VLPs are
expected to be found. A similar elution profile was obtained when separating, by SEC,
protein extracts from plants expressing rabies protein G with protein M (1091+1 086-
infiltrated plants, figure 3B).
Example 4 : Animal studies
Rapidpurification of Rab- VLPfrom plants
N . benthamiana plants were agroinfiltrated with AGL1/1091 as described for
example in WO/201 1/035422 which is incorporated herein by reference. Extraction of
Rab-VLP (also referred to as NG-VLP, Native G protein VLP, G-VLP or G protein VLP)
was undertaken as described above. Briefly, leaves were collected on day 4 post-
infiltration, cut into ~ 1 cm pieces and digested for 1 h at room temperature in an orbital
shaker. The digestion buffer contained 1.0% (v/v) Multifect Pectinase FE, 1.0% (v/v)
Multifect CX CG and/or 1.0% (v/v) Multifect CX B (all from Genencor), each in a
solution of 200 mM Mannitol, 75 mM Citrate, 0.04%, 500 mM NaCl sodium bisulfite pH
6.0 buffer using a biomass : digestion buffer ratio of 1:2.5 (w/v).
Following digestion, the apoplastic fraction was filtered through a 400 m
nylon filter to remove coarse undigested vegetal tissue (<5% of starting biomass). The
filtered extract was then centrifuged at room temperature for 15 min at 5000xg to
remove protoplasts and intracellular contaminants (proteins, DNA, membranes,
vesicles, pigments, etc). Next, the supernatant was depth-filtered (for clarification)
using a .2 m glass fiber filter (Sartorius Stedim) and a 0.45/0.2 mpi filter (Sartorium
Stedim), before being subjected to chromatography.
[00 13 ] In order to prepare for chromatography, the extract may be concentrated by
suitable methods known in the art, for example the extract may be centrifuge and the
pellet resuspended in appropriate buffer and volume or the extract may be
concentrated and diafiltrated on tangential flow filtration (TFF) systems, equipped
with appropriate membrane prior to loading on chromatographic media. Membranes
may be flat-sheet or hollow fibres, pore size may range between 100, 300, 500, 750
kDa or 1 m molecular weight cut-off. Lumen size may be chosen according to supplier
line of product, usually 0.75 to 1 mm lumen size is preferable. Shear rates may be
adjusted between 2000 s to 0 000 s with appropriate retentate and permeate flow
rates.
[001 32] The clarified apoplastic fraction was loaded over a cation exchange column
(Poros HS Applied Biosystems) equilibrated with an equilibration/elution buffer (50
mM NaPC-4, 100 mM NaCl, 0.005% Tween 80 pH 6.0). Once the UV was back to
zero, the extract was step-eluted with the equilibration/elution buffer containing
increasing concentrations of NaCl (500 mM). Where necessary, the chromatographic
fractions were concentrated 0 times using Amicon™ devices equipped with 10 kDa
MWCO. Protein analysis was performed as described in previous examples.
Finally, a last formulation approach may be employ to buffer exchange the
final candidate vaccine, by methods known in the art. For example a centrifugation or
a TFF step may be used for such purpose, as described above.
[001 34] Under conditions described as above, purity of 75% or greater for Rab-VLP
vaccines preparation with sufficient quality for immunogenicity study may be
obtained. As illustrated in figure 8A, the G protein migrates to the expected molecular
weight of ca. 55 kDa. The size of the Rab-VLP is estimated to be between 175-190
n by dymamic light scattering (Zeta sizer 90, Malvern instrument), a particle size
similar to that described for the Rabies virus.
Immunization studies in mice
[001 35] Immunogenicity of the Rab-VLP was evaluated in the Balb/c mouse model.
Briefly, groups of female BALB/c mice (8-10 weeks old; Charles River Laboratories,
USA) were vaccinated with a total of three injections (0.05 mL/site) on each
immunization day (Study Days 0, 7 and 28) in the left and right hind limb musculature
intramuscularly (i.m.) for a total of 0.1 ml volume injected that contained various
doses of Rab-VLP vaccine ( 1 or 5 - with or without adjuvants). The doses were
based upon G protein concentrations determined bybicinchoninic acid (BCA) assay
combined with purity assessment by densitometry. Alhydrogel® at final
concemtration of 1% was used as the adjuvant. The placebo group was immunized by
the same route and regimen as the candidate vaccine. Fifteen mice per group were
used to provide adequate statistical power to the study. Serum samples were collected
prior vaccination (pre-immune sera) and on day 7, 2 1 and 44. Five animals per group
were sacrificed on day 7, 2 1 and 44 in order to perform rapid fluorescent focus
inhibition test (RFFIT) to evaluate the protective antibody in the sera. The protective
dose established by the World Health Organization (WHO) for Rabies vaccine is 0.5
international unit (IU) per ml. As shown in figure 8b, the non-adjuvanted Rab-VLP
vaccine, with doses as low as 1 g, allows for achieving higher titers than the standard
titers established by the WHO.
All citations are hereby incorporated by reference.
[001 37] The present invention has been described with regard to one or more
embodiments. However, it will be apparent to persons skilled in the art that a number
of variations and modifications can be made without departing from the scope of the
invention as defined in the claims.
Claims (30)
1 A method of producing a rabies virus like particle (VLP) in a plant comprising, a) introducing a nucleic acid comprising a regulatory region active in the plant operatively linked to a nucleotide sequence encoding a native rabies glycoprotein (G), into the plant, or portion of the plant, b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the rabies VLP, c) harvesting the plant, and d) extracting the VLPs, wherein the VLPs range in size from 40-300 nm and, wherein the VLP comprises a lipid obtained from a plasma membrane of the plant.
2. A method of producing a rabies virus like particle (VLP) in a plant comprising, a) providing a plant or portion of the plant comprising a nucleic acid comprising a regulatory region active in the plant operatively linked to a nucleotide sequence encoding one or more native rabies glycoprotein (G), b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the rabies VLP, c) harvesting the plant, and d) extracting the VLPs, wherein the VLPs range in size from 40-300 nm, and wherein the VLP comprises a lipid obtained from a plasma membrane of the plant.
3. The method of claim 1 or 2, wherein the nucleotide sequence further encodes, comprises or both encodes and comprises, one or more than one amplification element.
4. The method of claim 3, wherein the one or more than one amplification element is a geminivirus amplification element.
5. The method of claim 4, wherein the one or more than one geminivirus amplification element is selected from a Bean Yellow Dwarf Virus long intergenic region (BeYDV LIR), and a BeYDV short intergenic region (BeYDV SIR).
6. The method of claim 1, wherein a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding a rabies matrix protein (M) or fragment thereof is introduced into the plant, or portion of the plant and is expressed when incubating the plant or portion of the plant in step b).
7. The method of claim 2, wherein the plant or portion of the plant further comprises a second nucleic acid comprising a second regulatory region active in the plant and operatively linked to a second nucleotide sequence encoding a rabies matrix protein (M) or fragment thereof and is expressed when incubating the plant or portion of the plant in step b).
8. The method of claim 1, wherein in the step of introducing (step a), the nucleic acid is transiently expressed in the plant.
9. The method of claim 1, wherein, in the step of introducing (step a), the nucleic acid is stably expressed in the plant.
10. The method of any one of claims 1-9, wherein the VLP is extracted by biochemical extraction.
11. A plant-derived VLP comprising one or more native rabies glycoprotein (G) wherein the VLP comprises a lipid obtained from the plasma membrane of the plant.
12. A VLP produced by the method of any one of claims 1-10.
13. The VLP of claim 12, wherein the lipid comprises one or more than one lipid derived from the plant.
14. The VLP of claim 12, wherein one or more glycoprotein (G), comprises plant-specific N- glycans, or modified N-glycans.
15. A composition comprising an effective dose of the VLP of any one of claims 11-14 for inducing an immune response in a subject, and a pharmaceutically acceptable carrier.
16. Use of the composition of claim 15 for manufacture of a medicament for inducing immunity to a rabies virus infection in a subject
17. The use of claim 16, wherein the medicament is adapted for oral, intradermal, intranasal, intramuscular, intraperitoneal, intravenous, or subcutaneous administration.
18. The VLP of any one of claims 11-14 adapted for use in inducing immunity to a rabies virus infection in a subject.
19. A food supplement comprising the VLP of any one of claims 11-14.
20. The method of claim 1 or 6, further comprising introducing an additional nucleic acid sequence, the additional nucleic acid sequence encoding a suppressor of silencing, a geminivirus replicase or both.
21. The method of claim 20, wherein the suppressor of silencing and the geminivirus replicase are encoded by two different additional nucleic acid sequences that are introduced into the plant, or portion of the plant.
22. The method of claim 20 wherein the suppressor of silencing is selected from the group HcPro and p19.
23. The method of any one of claims 1, 2, 6 and 7, wherein the native rabies glycoprotein (G) has at least 90% sequence identity to SEQ ID NO: 14.
24. The method of any one of claims 1, 2, 6 and 7, wherein the native rabies glycoprotein (G) has the amino acid sequence shown as in SEQ ID NO:14.
25. The method of claim 6 or 7, wherein the rabies matrix protein (M) has at least 90% sequence identity to SEQ ID NO: 6.
26. The method of claim 6 or 7, wherein the rabies matrix protein (M) has the amino acid sequence shown as in SEQ ID NO: 6.
27. The method of any one of claims 1, 2, 6 and 7, wherein the nucleic acid sequence comprising the regulatory region is further operatively linked with one or more than one comovirus enhancer.
28. The method of claim 27, wherein the one or more than one comovirus enhancer is a comovirus UTR.
29. The method of claim 28, wherein the comovirus UTR is a Cowpea Mosaic Virus (CPMV) UTR.
30. The method of claim 1 or 2, wherein the VLP does not contain a viral matrix or a core protein.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161496371P | 2011-06-13 | 2011-06-13 | |
US61/496,371 | 2011-06-13 | ||
US201161578787P | 2011-12-21 | 2011-12-21 | |
US61/578,787 | 2011-12-21 | ||
PCT/CA2012/000581 WO2012171104A1 (en) | 2011-06-13 | 2012-06-13 | Rabies virus like particle production in plants |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ618885A NZ618885A (en) | 2015-12-24 |
NZ618885B2 true NZ618885B2 (en) | 2016-03-30 |
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