U.S. patent application number 10/457082 was filed with the patent office on 2004-02-19 for flexible vaccine assembly and vaccine delivery platform.
Invention is credited to Lindbo, John A., McCormick, Alison A., Nguyen, Long V., Palmer, Kenneth E., Pogue, Gregory P., Smith, Mark L..
Application Number | 20040033585 10/457082 |
Document ID | / |
Family ID | 29736232 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040033585 |
Kind Code |
A1 |
McCormick, Alison A. ; et
al. |
February 19, 2004 |
Flexible vaccine assembly and vaccine delivery platform
Abstract
Herein described are various methods for making a vaccine that
are made of re-assembled virus like particles (VLP). First, the
VLPs are disassembled into encapsidation intermediate populations.
Each encapsidation intermediate population undergoes, for instance,
chemical conjugation of unique peptide or nucleic moieties to form
separate populations. Thereafter, a predetermined amount of each of
the several (one or more) different encapsidation intermediates
from the different populations is mixed and joined, forming intact
VLPs, surrounding a nucleic acid core, that are composed of
different encapsidation intermediate such that the reassembled VLP
displays more than one peptide or nucleic acid. The nucleic acid
can function either as a scaffold alone or can be engineered for
the expression of an immunomodulatory protein in a eukaryotic
cell.
Inventors: |
McCormick, Alison A.;
(Vacaville, CA) ; Smith, Mark L.; (Davis, CA)
; Palmer, Kenneth E.; (Vacaville, CA) ; Lindbo,
John A.; (Vacaville, CA) ; Nguyen, Long V.;
(Vacaville, CA) ; Pogue, Gregory P.; (Vacaville,
CA) |
Correspondence
Address: |
Douglas W. Schelling, Ph.D.
Waddey & Patterson
414 Union Street, Suite 2020
Bank of America Plaza
Nashville
TN
37219
US
|
Family ID: |
29736232 |
Appl. No.: |
10/457082 |
Filed: |
June 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60386921 |
Jun 7, 2002 |
|
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Current U.S.
Class: |
435/235.1 ;
435/456 |
Current CPC
Class: |
A61K 2039/6075 20130101;
C12N 2770/00022 20130101; C07K 14/005 20130101; C12N 2760/16122
20130101; C12N 2760/18022 20130101; A61K 2039/64 20130101; C12N
2710/20034 20130101; C12N 2710/20022 20130101; C07K 2319/00
20130101; A61P 31/12 20180101; C12N 2770/00023 20130101; C07K
14/4748 20130101; A61K 39/385 20130101; A61K 39/12 20130101; A61K
2039/5258 20130101; A61K 2039/55516 20130101; C12N 7/00 20130101;
A61K 2039/627 20130101 |
Class at
Publication: |
435/235.1 ;
435/456 |
International
Class: |
C12N 015/86; C12N
007/00 |
Goverment Interests
[0002] This invention was made with United States Government
Support under cooperative agreement number 70NANB2H3048 awarded by
the National Institute of Standards and Technology.
Claims
What is claimed is:
1. A method of making a virus-like particle (VLP) containing an RNA
encoding a gene that is capable of expression in a eukaryotic cell,
the method comprising the steps of: a) disassembling TMV virus; b)
forming one or more groups of encapsidation intermediate (20S
disks) populations; c) mixing portions of one or more groups of
encapsidation intermediates (20S disks); d) forming intact VLP of
one or more encapsidation intermediates (20S disks) surrounding RNA
which encodes a gene such that the arrangement of sequences allows
translation and gene expression in a mammalian cell, and
stabilization of the RNA for delivery to mammalian cells or tissues
by VLP structure.
2. A method for making a virus-like particle (VLP) containing
multiple, different in composition, peptides or proteins, said
method comprising the steps of: a) disassembling separate virus or
VLP populations, each displaying a distinct peptide or protein via
genetic fusion; b) forming encapsidation intermediate populations
each displaying a distinct peptide or protein; c) mixing
encapsidation intermediates from different populations; d) forming
intact VLP surrounding a nucleic acid core that is composed of
different encapsidation intermediates such that the VLP displays
one or more peptides or proteins.
3. A method as set forth in claim 2, wherein: said VLP is a tobacco
mosaic virus (TMV) virus-like particle (VLP) containing multiple,
different composition peptides or proteins; in said disassembling
step the separate virus or VLP populations are TMV populations; and
in said first forming step, said mixing, and said second forming
step, the encapsidation intermediate populations are 20S disks.
4. A method for making a virus-like particle (VLP) containing
multiple, different composition peptides, proteins or nucleic acid
moieties, said method comprising the steps of: a) disassembling a
virus or VLP population that has a surface residue for chemical
conjugation, provided by genetic fusion; b) forming encapsidation
intermediate populations; c) effecting chemical conjugation of
unique peptide, protein or nucleic acid moieties to each of several
separate encapsidation intermediate populations; d) mixing
encapsidation intermediates from different populations in the
presence of a nucleic acid scaffold; e) forming intact VLP
surrounding a nucleic acid core that is composed of different
encapsidation intermediates such that the VLP displays more than
one moiety, such as a peptide, protein or nucleic acid, or
combination thereof.
5. A method for making a TMV virus containing one or more,
different composition peptides or proteins displayed, said method
including the steps of: a) constructing a TMV expression vector
with i) a gene for expression in mammalian cells placed downstream,
3', of a internal ribosome initiation sequence (IRES) that lies
either within the TMV 30 kDa movement protein or separately placed
downstream, 3', of the 30 kDa movement protein gene; ii) a coat
protein expressed from a non-native subgenomic promoter downstream
of the gene for expression in mammalian cells; iii) the coat
protein having either a genetic fusion for the expression of a
peptide sequence, protein and/or a surface residue for chemical
conjugation, provided by genetic fusion; b) purifying the TMV
viruses expressing peptide, protein and/or surface residue; c)
effecting chemical conjugation of unique peptide, protein or
nucleic acid moieties to purified TMV virus; d) using TMV virus
with genetic and/or chemical fusion peptides for the stabilization
and delivery of the RNA expression construct into mammalian cells
or tissues.
6. A method for making a TMV virus-like particle (VLP) containing
multiple, different composition peptides, proteins or nucleic
acids, the method comprising the steps of: a) disassembling
separate TMV populations, each having a surface residue for
chemical conjugation provided by genetic fusion; b) forming
encapsidation intermediate, 20S disk, populations from the separate
TMV populations; c) effecting chemical conjugation of unique
peptide, proteins or nucleic acid moieties to separate populations
of the encapsidation intermediate 20S disks; d) mixing at least two
different encapsidation intermediates, 20S disks, from different
populations in the presence of a nucleic acid scaffold; e) forming
intact TMV VLP surrounding a nucleic acid core that is composed of
the at least two different encapsidation intermediates, 20S disks,
such that the VLP displays more than one moiety, be it peptide,
protein or nucleic acid, or some combination thereof.
7. A method as set forth in claim 7, wherein said mixing step a
multiple of differing encapsidation intermediates, each from
different populations, is mixed such that in said forming step, the
VLP displays a multiple number of moieties, be they peptide,
protein or nucleic acid, or some combination of these moieties.
8. A method as set forth in claim 8, wherein in said forming step,
the VLP displays at least three peptides, proteins or nucleic
acids.
9. A method for making a virus-like particle (VLP) containing
multiple, different composition peptides or proteins displayed by a
process comprising the steps of: a) disassembling separate VLP
populations, each displaying a distinct peptide or protein via
genetic fusion; b) disassembling a separate VLP population that has
a surface residue for chemical conjugation, provided by genetic
fusion; c) forming encapsidation intermediate populations such
that: i) each displays a distinct peptide or protein and ii) each
displays a surface residue for chemical conjugation; d) effecting
chemical conjugation of unique peptide, protein or nucleic acid
moieties to separate populations of the encapsidation intermediate
displaying surface residue for chemical conjugation; e) mixing
encapsidation intermediates from different populations displaying
peptides or proteins by genetic fusion or displaying peptides,
proteins or nucleic acids by chemical conjugation; f) forming
intact VLP surrounding a nucleic acid core that is composed of
different encapsidation intermediates such that the VLP displays
more than one moiety, be it peptide, protein or nucleic acid, or
some combination of these moieties.
10. A method as set forth in claim 10, wherein the VLPs are TMV
virus and the encapsidation intermediates are 20S disks.
11. A method as set forth in claim 11, wherein the chemical
conjugation of unique peptide and/or nucleic acid moieties to the
encapsidation intermediates, displaying the residues for chemical
conjugations, is performed following the formation of an intact VLP
surrounding a nucleic acid core.
12. A VLP produced by any one of the methods recited in claims
1-11, wherein multiple different peptides, proteins or nucleic acid
moieties are displayed on said VLP such that said VLP induces an
immune response in two or more organisms.
13. A VLP produced by any one of the methods recited in claims
1-11, wherein multiple different peptides, proteins or nucleic acid
moieties are displayed on said VLP such that said VLP induces in a
host an immune response to one or more epitopes.
14. A VLP produced by any one of the methods recited in claims
1-11, wherein multiple different peptides, proteins or nucleic acid
moieties are displayed on said VLP such that said VLP exhibits an
enhanced cellular uptake in the host.
15. A VLP produced by any one of the methods recited in claims
1-11, wherein multiple different peptides, proteins or nucleic acid
moieties are displayed on said VLP such that said VLP exhibits
immune stimulation or modulation functions thereof in a host.
16. A VLP that contains a nucleic acid moiety that is functional as
a scaffold for building a multivalent VLP generated by the
methodology as set forth in any one of claims 1-11.
17. A VLP made by the methodology as set forth in any one of claims
1-11, said VLP including a nucleic acid moiety that contains a gene
for one or more of the following functions: induction of humoral
immune responses, induction of cellular immune responses, or
stimulation or modulation of host immune responses.
18. A VLP made by the methodology as set forth in any one of claims
1-11, said VLP including a nucleic acid moiety that contains a gene
for one or more of the following: an intact or partial viral
antigen, an intact or partial bacterial antigen, an intact or
partial mycoplasm antigen, an intact or partial eukaryotic pathogen
antigen, a cytokine, a chemokine, and a portion of a chemokine,
cytokine or cellular receptor that could modulate host immune
response.
19. A VLP made by the methodologies as set forth in any one of the
claims 1-11, that by virtue of the moieties displayed on the
surface and/or the functionality associated with the nucleic acid
core, has the properties required to function as a vaccine, an
anti-allergy medication, a diagnostic reagent or a combinatorial
chemistry reagent.
20. A VLP made by any one of the methods set forth in any one of
claims 1-11, comprising: an RNA moiety comprising any one from the
following group: a TMV expression vector containing a gene for
inducing or modulating host immune responses via expression in
mammalian cells; a TMV expression vector containing an internal
ribosome initiation sequence (IRES) upstream of a gene for inducing
or modulating host immune responses via expression in mammalian
cells; a TMV origin of assembly (OAS) and a gene for inducing or
modulating host immune responses via expression in mammalian cells;
a TMV Omega RNA leader, TMV origin of assembly (OAS) and a gene for
inducing or modulating host immune responses via expression in
mammalian cells; an alphavirus replicon a TMV origin of assembly
(OAS) and a gene for inducing or modulating host immune responses
via expression gene in mammalian cells; a rubivirus replicon a TMV
origin of assembly (OAS) and a gene for inducing or modulating host
immune responses via expression gene in mammalian cells; a
nodavirus replicon containing the TMV origin of assembly (OAS) and
a gene for inducing or modulating host immune responses via
expression gene in mammalian cells; and a flavivirus replicon
containing the TMV origin of assembly (OAS) and a gene for inducing
or modulating host immune responses via expression gene in
mammalian cells.
21. A VLP comprising a surface presented, unpaired cysteine residue
on the surface of the tobacco mosaic virus virion, constructed by
genetic expression of a unpaired cysteine residue at the N, C or
surface exposed loop of the TMV coat protein, to augment specific
chemical conjugation reactions.
22. A VLP comprising a surface presented, lysine residue on the
surface of the tobacco mosaic virus virion, constructed by genetic
expression of a lysine residue at the N, C or surface exposed loop
of the TMV coat protein, to augment specific chemical conjugation
reactions.
23. A method for purification of TMV or virus like particles (VLP)
displaying a peptide fusion or whole protein, comprising the steps
of: a) adding PEG to a virus containing S1 or S2 supernatant, at an
empirically determined concentration of 1% to 10%, depending on the
epitope displayed; b) adding salt to 4% w/v if not already present
in the S1 or S2 supernatant; c) chilling the supernatant to
4.degree. C., for 30 minutes to 1 hour; d) recovering the
precipitated virus by centrifugation; and c) re-suspending the
virus and repeating the process to increase purity.
24. A coat protein fusion, comprising: a peptide or protein of
interest genetically fused to a TMV coat protein and flanked by
additional charged amino acids introduced to improve coat fusion
accumulation and/or extraction from an infected plant host.
25. A coat protein fusion as set forth in claim 24 wherein the coat
protein fusion comprises Seq ID No: 22
Description
PRIORITY CLAIM
[0001] This application claims priority to U.S. Provisional
Application No. 60/386,921, filed Jun. 7, 2002, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to a novel vaccine platform that
includes a reassembled virus constructed from one or more subunits,
each subunit containing a different peptide or nucleic acid moiety
added by genetic fusion or in vitro conjugation such that each
subunit incorporates a target therapeutic agent. The invention
further relates to a method for assembling RNA molecules in vitro
for delivery and expression in eukaryotic cells. In particular, the
invention provides for proteins, molecules and nucleic acid
sequences necessary for the packaging of RNA molecules for delivery
and expression in a eukaryotic cell. The packaged RNA molecules of
the invention are capable of delivery to a wide range of eukaryotic
cells. The packaged RNA molecules may also be targeted to specific
eukaryotic cells. The invention further includes a delivery
platform where the above described reassembled viruses or
virus-like particles (VLPs), RNA vaccines are used to induce either
cellular or humoral immunity, or both simultaneously, by the
synergistic action of peptide fusions to the virus or VLP structure
and the encoded proteins of the RNA.
BACKGROUND OF THE INVENTION
[0004] To date, most traditional vaccines have been composed of
live-attenuated or inactivated whole pathogen preparations.
Generation of these sorts of vaccines is limited by the requirement
for long and intensive basic research and development. Reliable
production and scale-up technologies for live-attenuated or
inactivated vaccines would be almost impossible to develop at short
notice. There is, therefore, a need for the development of a safe,
robust and broadly-useful technology that is suitable for the
production of vaccines against unanticipated infectious disease
threats. Vaccines developed from plant-virus-pathogen chimera's may
provide a method to rapidly produce vaccines that can be used to
prevent or treat a number of known or emerging disease threats.
[0005] Controlling immune responses to pathogens and tumor cells
has been the focus of immunology, cell biology and pharmaceutical
development for several decades. Much has been learned about the
complexity of immune cells and the patterns and effect of cytokine
expression in response to pathogen challenge, and vaccine
administration. One key aspect of this work has been the
identification of two major arms of the immune response, the Th1
response, which is largely cellular, and the Th2 response, which is
predominantly humoral. The two types of immune responses are
mounted in response to how foreign antigens are presented to the
immune system, what cytokines are expressed by presenting cells and
what types of immune cells are activated. Th1 responses result in
cytotoxic immune cell function and production of neutralizing
antibodies of a different subtype than observed with Th2 responses.
While some pathogens can be susceptible to Th2 responses, the Th1
response is key to mounting an effective response to both pathogen
and tumor cells. However, both pathogens and tumor cells have
developed strategies to avoid immune surveillance, bypassing
mechanisms that are essential to Th1 immunity.
[0006] A key goal in vaccine development is to direct Th1 type
immunity, in addition to Th2 humoral responses, upon vaccine
administration to the host. By using an attenuated cowpox virus,
Jenner unknowingly took advantage of the powerful activation of Th1
pathway to prevent smallpox infections. Since his time, most
pathogen vaccines have been killed or attenuated, which have
generally shown good success in controlling pathogen morbidity and
viral spread. However, two aspects of recent vaccine development
have led to growing concerns for live or attenuated viral vaccines.
The use of an attenuated or killed virus to treat human
immunodeficiency virus (HIV) is impractical for several reasons.
Occupational safety concerns, low yield of attenuated virus, and
the threat of viral mutation or escape are serious drawback to both
vaccine development and public acceptance. In other cases, as
observed with measles virus and respiratory syncytial virus (RSV),
unpredictable and severe adverse events are associated with whole
virus immunization. Therefore, much research has focused on
"subunit" vaccines, which are composed of pathogen protein(s) or
peptides that are generally targeted by the host immune response
for protective immunity (Vaccines, 3.sup.rd ed 1999, Plotkin and
Orenstein, Philadelphia Pa., Saunders Co). Unfortunately, protein
subunit vaccines don't often elicit strong Th1 responses by
themselves, and DNA subunit vaccines often fail to elicit
antibodies. In most cases both antibodies and CTL responses are
necessary in controlling pathogenesis or disease progression.
[0007] Two new types of vaccines have been created to overcome the
deficiencies of current subunit vaccines. Non-pathogenic viruses
have been genetically modified to encode immunogenic subunit
proteins of a pathogen, thus taking advantage of the Th1 immune
response to viral antigen presentation. Strong Th1 type immune
responses have been demonstrated for many pathogen and
self-antigens using adenovirus, vaccinia, fowlpox and alphavirus
delivery systems (Walther and Stein. 2000 Drugs 60, 249). However,
these "first generation" viral delivery systems encountered
problems due to the vector immunogenicity, which precluded their
subsequent use in booster immunizations. Viral priming followed by
either protein or DNA boosting has been successful, but this
approach requires the manufacture of at least two agents for a
single vaccine. The large-scale manufacture of DNA and/or protein
for these vaccines has encountered both technical and financial
challenges.
[0008] A second strategy takes advantage of the self-assembly of
viral coat proteins into virus like particles (VLPs), which by
themselves stimulate strong Th1 antigen responses (Schiller and
Lowy. 2001 Expert Opin Biol Ther. 1, 571). VLPs constructed from
arrayed viral coat have been shown to be effective in stimulating
both neutralizing antibody and cytotoxic T lymphocyte (CTL)
responses. Viral coat proteins are also effective carriers of
antigens through fusion to the external solvent-exposed residues,
usually by genetic fusion (Pogue et al. 2002 Ann Rev Phyto Path 40,
3; Da Silva. 1999 Curr Opin Mol Ther 1, 82). Though promising, VLP
technology also has drawbacks. Production is again limiting, and
often fusion of a heterologous antigen to the coat reduces VLP
yield, solubility, or prevents self-assembly. In addition, immune
clearance, the same mechanism that limits whole virus boosting,
also limits the use of VLPs. Clearly, there is a need for a cost
effective viral coat antigen delivery system that overcomes the
limitations of both whole virus and VLP technology for vaccine
delivery. The properties of this system would include all the
benefits of boosting Th1 responses via a virus-like antigen
presentation to the immune system without pathogenicity,
flexibility to rotate the VLP backbone to which the antigen is
fused, generation of and control of immunogenicity, high yield and
low cost.
[0009] Applicant and others have shown that coat proteins from
plant viruses have all the immunologic presentation properties of
mammalian virus coat, but without pathogenicity. A large number of
positive (+) strand RNA plant viruses, including Tobacco Mosaic
Virus (TMV), type member of the tobamovirus family, have been
cloned and manipulated in vitro to express heterologous gene
products in plants as well as to display biologically relevant
peptides on its virion surface. A unique property of TMV virions is
their ability to be disassociated to form monomers and self
assemble into VLPs using a RNA scaffold. Plant coat proteins,
including TMV, engineered to display foreign epitopes have been
shown to promote functional immunity to both self-antigens
(Savelyeva N 2001 Nat Biotechnol 19 760) and various pathogens
(Pogue et al. 2002 Ann Rev Phyto Path 40, 3).
[0010] Essential for the encapsidation of the viral genomic RNA
molecule into an infectious particle is the presence of a sequence
element referred to as the origin of assembly (OAS). The TMV OAS is
located approximately 1 Kb from the 3' end of the viral genome and
consists of a 440 nucleotide sequence that is predicted to form
three hairpin stem-loop structures (Turner and Butler, 1986). The
viral coat protein disks initially bind to loop 1 during viral
assembly. In vitro packaging assays using mutual assembly origin
transcripts have defined the 75 nucleotides comprising loop 1 as
necessary and sufficient for encapsidation of foreign or viral RNA
sequences (Turner et al., 1988). In vitro reconstitution studies
have shown that preparations of purified coat protein, derived from
virions from infected plant cells, are able to assemble into
helical structures with TMV RNA at pH 7.0, resulting in assembly of
TMV-like viral particles containing RNA (Fraenkel-Conrat and
Williams, 1955). Furthermore, it has been shown that foreign
chimeric RNA molecules containing OAS sequences, transcribed in
vitro using SP6 or T7 RNA polymerase, may be assembled in vitro
into pseudovirus particles (Sleat et al., 1986).
[0011] The cloning and sequencing of the viral coat proteins
responsible for encapsidation has led to the insertion of these
genes into bacterial expression vectors in, for example, E. coli
(Shire et al., 1990). However, in vitro assembly with recombinant
E. coli viral coat proteins results in a decreased reconstitution
rate relative to native coat protein produced in plants (Shire et
al., 1990). U.S. Pat. No. 5,443,969 attempts to overcome this
deficiency in E. coli by packaging RNA sequences containing a
TMV-OAS in vivo in E. coli, instead of in vitro. However,
introduction of the encapsidated viral vectors into hosts outside
of plants is problematic. The lack of acetylation of the TMV coat
protein in E. coli results in poorly efficient encapsidation of
non-capped RNAs. These RNAs are poorly translated in eukaryotic
cells due to the lack of the cap structure. Further, the yields of
recombinant TMV products in E. coli are very poor and not
commercially feasible.
[0012] The process of intracellular delivery of genetic material
for therapeutic purposes by either correcting an existing
abnormality or providing cells with a new function is the basis
behind gene therapy (Drew and Martin, 1999), and for DNA
immunizations. Practically speaking, nucleic acid immunization
technologies present an attractive front-line defense against new
pathogens: there is probably no other system that can compete as
the first line in a rapid-response subunit vaccine strategy.
However, conventional DNA vaccines suffer from a number of
significant drawbacks that makes reliance on this technology alone
unwise. Most significantly, the dose of DNA required to stimulate
an effective immune responses is very high, with the implication
that production of significant quantities for large scale
immunization will be challenging. DNA and RNA vaccines are
generally capable of promoting good Th1 type cytotoxic T cell
responses, which are essential for elimination of non-cytopathic
pathogens. However, with few exceptions, the antibody response
induced by DNA vaccines is poor. Hence, although nucleic acid
vaccines are attractive from the prospective that production can be
very rapid, ideally an initial DNA or RNA vaccination should be
followed by a booster vaccination, preferably with protein, to
induce efficient antibody production and more complete protection
against pathogen challenge. The current invention addresses the
issues raised above by introducing a novel and flexible vaccine
delivery platform
SUMMARY OF THE INVENTION
[0013] The present invention includes several unique solutions that
address current limitations of VLP technology, while retaining all
the positive characteristics of a successful VLP antigen scaffold.
Applicant presents a method for generating VLP vaccines in
adaptable, predictable, stable and scaleable manners. This work is
highly innovative, and there is continuing development. The method
includes generating muli-valent vaccines where different vaccine
protein moieties are fused to the surface of a single VLP structure
conferring a multi-functional effect--the availability of immune
peptides (protein elements stimulating protective immunity) and
peptides that either modulate the host immune response or
facilitate efficient immune cell recognition or processing. The
proposed vaccines will be also bi-functional, where the protein
elements of the VLP, with or without a peptide fusion or series of
fusions, encapsidate a modified RNA moiety. The modified RNA can
carry an mRNA of interest and that protected RNA can then be used
to carry nucleic acid content, along with protein, into an immune
cell that takes up the vaccine. The RNA constituents works
synergistically to generate strong, lasting immunological responses
by encoding either an intact pathogen or oncology antigen, proteins
that stimulate host immune responses or proteins that modulate
either a type Th1 or Th2 immune response to the vaccine. The method
alleviates problems associated with other VLP systems by having
robust production potential, improved cellular uptake, and
multi-epitope valency. A selection of structurally similar, yet
immunologically distinct VLP carriers allows rotation of the coat
backbone for prime-boost strategies that have proven unworkable in
other VLP systems.
[0014] Vaccination with bi-functional RNAs presents an alternative
to DNA vaccination, with some distinct advantages. In the first
instance, there is little concern that an RNA-based vaccine could
cause oncogenesis because it cannot incorporate into or transform
the genome. Secondly, there is good evidence that one could deliver
an RNA vaccine derived from an RNA virus (such as an alphavirus) as
a safe self-amplifying vaccine vector. Alphavirus replicons are
cytolytic for cells, and thus the replicating RNA vaccine is
intrinsically transient and self-eliminating. Alphavirus "replicon"
vaccines cause powerful immune responses--both antibody and
cell-mediated--associated with both increases in the amount of
antigen produced as well as the production of inflammatory
cytokines induced by intracellular accumulation of the viral dsRNA
replicative intermediate. These features indicate that the dosage
of replicative RNA required for induction of effective immune
responses would be orders of magnitude lower than that required by
DNA immunization. However, the major drawback associated with naked
RNA vaccines is the notoriously labile nature of the nucleic acid:
this severely limits the application of RNA vaccines for mass
immunizations.
[0015] Alphavirus replicon vaccines are currently delivered either
as naked RNA transcribed in vitro, packaged in alphavirus-like
particles (replicon particles), or as plasmids containing
infectious cDNAs, driven by the cytomegalovirus immediate early
promoter (CMV promoter). Replicon particles are very efficient as
vehicles for carrying the replicon RNAs into cells, but production
is complicated, inefficient and unreliable. An efficient packaging
and RNA stabilization technology is therefore required to protect
alphavirus-based RNA vaccines from degradation. Two viable options
present themselves: (1) to deliver recombinant alphavirus
constructs as infectious cDNA plasmids; (2) to package alphavirus
RNA transcribed in vitro such that it is protected from nucleases
and has good stability and storage properties. An approach for the
latter option is presented below.
[0016] The inventors employ as a VLP carrier the well-characterized
plant virus, tobacco mosaic virus (TMV), and exploit its unique
abilities to reconstitute VLP structures in vitro onto various
heterologous RNA sequences.
[0017] By introducing a cysteine in the solvent exposed sequences
of TMV coat, we can introduce and fuse foreign antigen epitopes
ex-vivo. Epitope sequences that are not amenable to in vitro
synthesis will be fused in-frame genetically to the TMV coat
protein. TMV VLPs will be reassembled in vitro decorated with a
single epitope (monovalent), or with a collection of different
epitopes (multivalent), derived from in vitro conjugation or
expressed from a genetic fusion.
[0018] As a scaffold for reassembly, the present invention includes
using an RNA that encodes a protein that will enhance vaccine
potency, thereby creating a bi-functional antigen delivery system
that derives its activity from both protein and nucleic acid. The
RNA can also incorporate an alphavirus replicon to augment
translation. Essential for the encapsidation of the RNA molecule by
the TMV coat protein, to generate an RNA-containing VLP, is the
presence of the 75 nucleotide sequence comprising loop 1 of the
origin of assembly (OAS). By combining this 75 nucleotide sequence
with foreign sequences encoding protein(s) or peptide(s) of
therapeutic interest, the RNA molecule can function as an effective
scaffold for the generation of a TMV-like VLP. The RNA can encode
any number of immunomodulating factors (e.g. IL4, IL1.beta. or
IFN.gamma.) that ensure a highly successful immune response to the
vaccine, and help generate either protective or therapeutic
immunity to the pathogen, or deliver inhibitory RNA signal (RNAi)
for targeted gene inhibition. This VLP strategy can be applied to
effectively target immune cells and stimulate Th1 type
responses.
[0019] An important requirement to inducing a Th1 type immune
response is getting VLPs into cells for processing and antigen
presentation. Peptides with known cell targeting have been
identified (Samuel O, Shai, Y., 2001 Bichem. 40, 1340; Magnusson et
al. 2001 J. Virol. 75 7280; Bushkin-Harav et al. 1998 FEBS L. 424
243) and can be tested in vitro by direct examination of cell
entry, and in vivo for augmented antigen presentation by examining
the type and speed of immune response to target antigens. Targeting
and fusion peptides will be tested for their ability to augment
cellular uptake of TMV, as well as their ability to deliver
encapsidated RNA in vitro and in vivo.
[0020] A common method to improving vaccination is to co-administer
an adjuvant or a specific T-helper peptide to stimulate T-cell
help. CpG DNA has been shown to be an easily administered adjuvant
that improves Th1 type immune responses when co-administered with
an appropriate vaccine (Krieg. 2000 Vaccine 19, 618). Most CpG DNA
adjuvants have been given mixed with the vaccine and administered
subcutaneously (s.c.), although the single strand thiolated DNA can
also be fused to a protein carrier through SPDP conjugation
chemistry. Also, several universal T-helper peptides have been
identified (Kulkarni, A. B., et al., 1995 J. Virol.69,1261;
Panina-Bordignon, 1989 Eu.J.Imm.19, 2237; Boraschi, 1988 J Exp Med.
168,675; Weiner, G. et al., 1997 Proc.Nat. Acad. Sci 94 10833).
Immunostimulatory peptides, usually fragments of cytokines, have
also been identified that direct Th1 type immunity after
vaccination in combination with pathogen or self-antigen peptides
or subunit vaccines (IL1.beta., Boraschi, 1988 J Exp Med. 168,675).
Coat fusions containing T-helper or adjuvant peptides or CpG DNA
oligo will be used to augment the immunogenicity of co-expressed
peptides, or encapsidated RNA.
[0021] Lastly, it is well established that cytokines play an
important role in determining which arm of the immune system is
activated after vaccine delivery. Interleukin 4 (IL4) has been
implicated in directing Th2 type immune responses and interferon
gamma (IFN.gamma.) is an important contributor to Th1 responses
(Spellberg and Edwards.2001 Clin Infect Dis 32, 76). By introducing
IL4 and IFN.gamma. RNA into cells by encapsidation into a TMV VLP,
we may be able to influence the type of immune response that is
generated. Applicant can test both antibody isotype responses to
antigen, which are a reflection of Th1 or Th2 antigen presentation,
as well as assess CTL responses that are primarily a consequence of
Th1 immunity.
[0022] Cell fusion peptides, T-help, adjuvants, pathogen antigens,
tumor antigens and encapsidated cytokine RNA will be tested
systematically in combination with antigens from Papillomavirus and
melanoma murine disease models. Immunogencity and challenge models
will establish incremental improvements over vaccination with
single peptides, and define the best peptide/RNA combinations for
generating Th1 or Th2 immune responses.
[0023] The availability of such a flexible and effective vaccine
platform provides opportunities to apply non-live vaccines for
humans and livestock thus reducing side effects and increasing
effectiveness. New vistas of medical practice, including
applications for breaking self-tolerance and driving immune
responses against weak antigens, may be opened by the synergistic
and high specific-activity of the disclosed vaccine platform.
[0024] The invention relates to a method where a specified virus,
such as a tobacco mosaic virus (TMV), is disrupted into a plurality
of subunits. Each subunit contains a genetically fused peptide or
is subjected to a conjugation reaction in order to attach a
predetermined epitope, peptide or nucleotide thereto. A plurality
of subunits are processed in this manner to produce a plurality of
subunit groups, where one subunit group has attached thereto a
predetermined peptide; another subunit group has a second peptide;
another subunit has a predetermined epitope attached there to; and
another subunit group has a nucleotide attached thereto, and so on,
for as many subunit groups necessary to provide the building blocks
for a plurality of virus vaccines.
[0025] An alternative strategy is to employ TMV RNA modified to
initiate internal ribosomal entry by introducing specific sequences
known to cause such an effect. These internal ribosomal entry sites
(IRES) are effective in causing internal translation products from
a polycystronic RNA in mammalian cells (Yang et al., J Virol 1989
63(4):1651-60). Introduction of an IRES into a TMV genome in frame
with an RNA encoding either a full length gene product or
immunostimulatory cytokine or other kind of immunmodulatory protein
allows for translation of that protein. Because these IRES are
introduced into non-replicating RNA, the amount of TMV and
proportional transcript taken up by a cell after vaccination is
conceivably lower than with a self replicating RNA such as encoded
by an alphavirus replicon, but the level of translation product
should be sufficient to induce the correct response.
[0026] The present invention includes research and development of
technological solutions to help the USA to produce and supply
effective vaccine reagents in response to unanticipated pathogen
threats. Applicant specifically addresses issues that limit
bio-defense application of nucleic acid vaccines: poor
environmental stability and high dosage requirements. In addressing
these issues, we will draw upon the core of knowledge that the
inventors possesses in the field of positive stranded RNA viruses
and their applications in biotechnology to develop a set of
molecular tools to improve nucleic acid vaccines. Applicant will
also demonstrate our capacity to produce protein subunit vaccines
that will provide effective antibody responses. Production of
protein subunit vaccines is inherently slower than nucleic acid
vaccines and so, practically, will only be available within a
delayed period following encounter with a new pathogen threat.
However, the inventor's non-transgenic plant-based vaccine
expression platform (GENEWARE.RTM.) has the capability to express a
variety of proteins, including virus-like particles (VLP)--known to
be potent inducers of antibodies in vaccinated
individuals--rapidly. Applicant has recently used a modified TMV
expression vector to produce 16 different human therapeutic
vaccines in tobacco plants, and have shown excellent safety in a
Phase I clinical trial (BB-IND #9283). Unlike other competing
technologies, GENEWARE.RTM. does not require specialized
fermentation facilities, and uses the efficient, rapid protein
production strategy of the plant virus TMV to harness plant protein
production machinery to produce vaccine proteins. A typical harvest
time, post inoculation is less than 21 days. Since the same virus
is used from pilot testing to large-scale manufacturing, there is
little or no transition time between validation and manufacturing
scale up. Most of the delay in delivery of vaccines via
GENEWARE.RTM. technology would be in the growth of plants, and
establishment of antigen-specific purification protocols. These
aspects of the technology result in a low cost of production for
plant-derived VLP vaccines.
LIST OF FIGURES
[0027] FIG. 1 is a flow diagram outlining the standard methods for
the generation of multivalent vaccines via chemical fusions
[0028] FIG. 2 is a flow diagram outlining methods to generate
multivalent TMV-based vaccines via chemical fusions that can be
bifunctional through the use of a translatable RNA species as a
scaffold
[0029] FIG. 3 is a flow diagram outlining the standard methods for
the generation of multivalent vaccines via genetic fusions
[0030] FIG. 4 is a flow diagram outlining methods to generate
multivalent TMV-based vaccines via genetic fusions that can be
bifunctional through the use of a translatable RNA species as a
scaffold
[0031] FIG. 5 is a rendering of TMV virion disassembly and in vitro
virion reassembly, showing from left to right: an electron
micrograph of a single TMV virion; space filling models of an
individual TMV coat protein, with schematic placement of surface
exposed N- (N) and C- (C) terminal domains and surface exposed loop
(SL); space filling models of 20S disk subunits; and a reassembled
VLP surrounding RNA.
[0032] FIG. 6 is a schematic of in vitro conjugation, or molecular
fusion, of heterologous peptides of various biological
functionalities to modified TMV 20S subunits and reassembly of
heteropolymeric (multiple peptide display) VLP surrounding
bioactive RNA.
[0033] FIG. 7 shows the expression levels of TMV-HA peptide fusions
at different insertion sites in TMV U1 coat protein. N. Benthamiana
plants (21 days post sow) were inoculated with encapsidated RNA
with a mild abrasive and approximately 200 .mu.g tissue was
harvested 9 to 10 days post infection. Samples were ground in 300
.mu.l acetate buffer pH 5, and insoluble material was pelleted by
centrifugation. Total plant proteins were harvested by grinding 100
.mu.g tissue in 100 .mu.l SDS-PAGE buffer. The soluble supernatant
was removed and then the pellet was resuspended in 200 .mu.l Tris
buffer pH 7.5 for a final pH extraction at pH7. 10 .mu.l of each
sample was then separated by 10-20% SDS-PAGE, stained in Coomassie
brilliant blue and destained before photographing. HA N accumulates
as a pH5 insoluble pH7 soluble coat fusion at approximately 19 kD
(arrow). HA Loop is expressed, but insoluble (present in total SDS
grind but not soluble fractions 5 or 7). HA GPAT is expressed and
soluble at pH5 but is cleaved, and only partially cleaved at pH7.
Ha C is expressed and is insoluble at pH5 and soluble at pH7 with
minor cleavage products visible. 5: Acetate buffer pH5; 7: Tris
buffer pH 7; S: SDS PAGE buffer total tissue grind.
[0034] FIG. 8 shows TMV proteins that were harvested from plants
infected with p15eTMV or p15e DE TMV after signs of infection were
evident. 20 mg leaf discs were then processed in Acetate buffer A:
50 mM Na-acetate (pH 5.0)/5 mM EDTA, then the insoluble material
was resuspended in tris buffer T: 50 mM TRIS (pH 7.5)/10 mM EDTA,
and material was compared to processing in SDS page buffer S: 78 mM
TRIS (7.0)/10% (w/v) sodium dodecyl sulfate/0.05% bromophenyl
blue/6.25% Glycerol/10% .beta.-mercaptoethanol, for total protein
analysis. Materials were then separated by SDS-PAGE, and visualized
by Coomassie staining. The control was U1: wild type coat protein
of tobacco mosaic virus strain U1, M: protein molecular weight
standard.
[0035] FIG. 9A (1) shows the nucleic acid and amino acid
composition for N terminal Cysteine TMV U1 (Seq ID No: 19).
Alternatively the cysteine can be incorporated into other
tobamovirus coats and at other positions within the coat protein,
e.g., 60s loop, C terminus, read through position. (2) Composition
for N terminal Lysine TMV U1 (Seq ID No: 20). Alternatively the
lysine can be incorporated into other tobamovirus coats and at
other positions within the coat protein, e.g., 60s loop, C
terminus, read through position.
[0036] FIG. 9B shows chemical conjugation to cysteine containing
TMV coat protein by glutaraldehyde. 1.0 mg of peptide was mixed
with 1.0 mg of Cyst-N TMV (C-N), in a volume of 1 ml, and a 20
.mu.l sample was removed for T=0. This sample was added to 20 .mu.l
of water and 40 .mu.l of 2.times.PAGE buffer, and immediately
boiled. (The water in T=0 equalizes its concentration with that of
T=4 which has added glutaraldehyde.) Glutaraldehyde was added to
the reaction to a final concentration of 1%, in a final volume of 2
ml. The reaction was allowed to proceed for 4 hours at room
temperature, with constant rotation. After 4 hours, a sample was
removed for a T=4 time point, added to an equal volume of
2.times.PAGE buffer and immediately boiled. 8 .mu.l (2 .mu.g
peptide & 2 .mu.g carrier) of each time point was loaded on a
gel for Western transfer to nitrocellulose, and 16 .mu.l (4 .mu.g
peptide & 4 .mu.g carrier) of each time point was loaded on a
gel for Coomassie staining.
[0037] FIG. 10 shows transmission electron micrograph (TEM) images
of TMV wild-type and myc or V5 N terminal fusion virus. TMV,
TMV-myc-N or TMV-V5-N were coated onto 400-mesh carbon-coated
copper grids at 20 to 80 g/ml. Samples were then negatively stained
with 1% phosphotungstic acid, dried and stored at RT until
visualized using a Philips CM120 TEM, at
37,000.times.magnification. The bar represents 130 nm.
[0038] FIG. 11 (A) shows a flow diagram for the purification of TMV
U1 virus from infected plant material. (B) SDS-PAGE analysis
(10-20% tris-glycine gel) for the isolation of TMV U1 from infected
N tabacum MD609 plants. Since the majority of the virus partitioned
into the S1 supernatant the S2 supernatant was not processed. GJ,
green juice; S1, supernatant S1; S1 PEG1, resuspended virus from
the first PEG precipitation; S1 PEG2, resuspended virus from the
second PEG precipitation.
[0039] FIG. 12 shows an SDS gel (10-20% tris glycine) illustrating
the effect of salt on the virus partitioning between the S1 and S2
process streams for the Cysteine N coat protein fusion. GJ, initial
green juice; S1, S1 process stream; S2, S2 process stream.
[0040] FIG. 13 shows a flow diagram for the precipitation of TMV
virus in the presence of polyethylene glycol (PEG) and sodium
chloride (NaCl).
[0041] FIG. 14 shows a flow diagram for the generation of free coat
protein from TMV virus.
[0042] FIG. 15 (A) shows the ultraviolet absorption spectrum for
TMV U1 coat protein at pH 8.0. (B to D) Treatment of Myc N coat
protein with DEAE Sepharose to remove contaminating residual RNA.
(B) and (C) Comparison of the ultraviolet absorbance spectrum
before and after DEAE resin treatment. (D) Agarose gel
electrophoresis to track contaminating RNA. Following binding of
the starting coat (L) to the DEAE resin, the coat protein was
eluted with 50 mM NaCl (E50) yielding a preparation free from RNA.
500 mM NaCl was required to elute the RNA from the resin (E500). FT
represents the resin flow through.
[0043] FIG. 16 shows the change in the chromatogram for TMV U1 coat
protein, analyzed by size exclusion chromatography, before and
after incubation at room temperature. (A) Chromatogram profile for
coat protein stored at 4.degree. C. (B) Chromatogram profile for
coat protein following storage for 16 hours at room temperature. A
YMC-Pack Diol-300 column (5 .mu.m bead; pore size, 30 nm) was
employed and the flow rate was 0.5 ml/min. The buffer employed was
0.1 M phosphate, pH 7.0 at either 4.degree. C. or room temperature,
based on the temperature of the sample injected.
[0044] FIG. 17 (A) Shows the kinetics of virion reconstitution from
viral RNA plus the U1 coat protein, which were followed by the
increase in solution turbidity at 310 nm. This is approximately
proportional to the average rod length. TMV virus, at a molarity
equivalent to that of the starting RNA, was employed to indicate
the optical density of a fully reconstituted in vitro
encapsidation. (1) Standard IVE conditions; 0.1 M sodium phosphate,
pH 7.2. (2) 0.1 M phosphate pH 7.2 with RNasin at 0.4 U/.mu.l. (3)
0.1 M sodium pyrophosphate, pH 7.2. (B) Agarose gel electrophoresis
of final reassembly reactions to assess RNA integrity. RNA Cntrl,
RNA lacking coat protein; PO4, phosphate buffered reassembly
reaction; Pyro PO4, pyrophosphate buffered reassembly reaction; PO4
RNasin, phosphate buffered reassembly reaction containing
RNasin.
[0045] FIG. 18. (A) shows the A310 nm kinetic profile for
reassembly reactions (IVE) with the ELDKWAS coat protein fusion, in
the presence and absence of RNasin. The ELDKWAS virus control was
present at the same molar concentration as the RNA in the
reassembly reactions. (B) Agarose gel electrophoresis of reassembly
reactions 5 hours after initiation. Reassembly reactions were
performed in the presence (+) or absence (-) of RNasin and the coat
protein employed is indicated. RNA alone, at the same concentration
as in the reassembly reactions, was run as a control.
[0046] FIG. 19 shows images and data analysis for reassembly
reactions viewed by transmission electron microscopy (TEM). The
samples were negatively stained with 1% phosphotungstic acid, dried
and stored at RT until visualized using a Philips CM120 TEM, at
37,000.times.magnification- . (A) Coat protein control sample (no
RNA present) (B) reassembly reaction with the same coat protein
concentration as in (A) but with TMV RNA present at 50 .mu.g/ml.
Image is for reassembly reaction performed in the presence of
RNasin. The scale bar represents 200 nm. (C) Comparison of the
normalized particle size distribution for reassembly reactions with
the ELDKWAS coat protein fusion, performed in the presence and
absence of RNasin. n indicates the number of rods counted in the
electron microscopy images.
[0047] FIG. 20 (A) shows the A310 nm kinetic profile for separate
reassembly reactions (IVE) with the ELDKWAS, Myc and HPV ep2 coat
protein fusions, all performed in the presence of RNasin. Wild type
TMV RNA was employed as a scaffold. The ELDKWAS virus control was
present at the same molar concentration as the RNA in the
reassembly reactions. The RNA alone control is also shown, however,
the coat protein alone and HPV ep2 and Myc virus controls are
omitted for clarity. (B) shows the A310 nm kinetic profile for
bivalent reassembly reactions (IVE) with the ELDKWAS, Myc and HPV
ep2 coat protein fusions taken in pair wise combinations. All
reassembly reactions were performed in the presence of RNasin and
wild type TMV RNA was employed as a scaffold. The ELDKWAS virus
control was present at the same molar concentration as the RNA in
the reassembly reactions. The RNA alone control is also shown,
however, the coat protein alone and HPV ep2 and Myc virus controls
are omitted for clarity.
[0048] FIG. 21 shows MALDI and SDS-PAGE data for the CRPV 2.1 coat
protein fusion. (A) MALDI TOF trace showing the spectrum for
purified CRPV 2.1 coat protein fusion. The predicted sequence
weight for the protein, with the Met cleaved is 19320 Da, in
excellent agreement with the observed molecular weight. (B)
SDS-PAGE gel for the CRPV 2.1 coat protein fusion, showing a
protein purity of greater than 97% for the final virus
preparation.
[0049] FIG. 22 shows various RNA constructs which may be used as a
scaffold for the reassembly of multivalent TMV-based vaccines and
which impart bifunctionality to the reconstituted virion, by virtue
of the gene (s) that they encode. (A) TMV RNA containing a
structural or non-structural gene. (B) TMV RNA containing IRES
structural or non-structural gene. (C) Alphavirus replicon
containing TMV OAS and structural or non-structural gene. (D)
Chimeric mRNA containing TMV OAS and Omega with structural,
non-structural or immune modulatory gene. (E) Chimeric mRNA
containing TMV OAS with structural, non-structural or immune
modulatory gene. For illustrative purposes the Figure shows the
CRPV L1 or CRPV E7 genes in the RNA constructs. However, melanoma
associated gene e.g. p15e, GP100 or any other structural or
non-structural gene can replace these CRPV-associated genes.
[0050] FIG. 23 shows humoral responses to TMV coat fusion vaccines
as measured by ELISA against the peptide. Sera were collected 10
days post vaccine 3 (pV3), serially diluted onto ELISA plates
coated with either a c-myc-BSA conjugate or a foreign antigen (FNR)
V5 fusion. Plates were then reacted with anti-mouse HRP and
positives were visualized using a colorimetric substrate, and
quantitated using statistical software. Commercially available
positive controls were used as standards.
[0051] FIG. 24 shows the cellular response to TMV ova G vaccination
as measured by fluorescent assisted cell sorting (FACS). Spleens
from animals vaccinated with TMV ova G were harvested, and then
cultured with 1 g/ml ova peptide for 5 hours in the presence of
Brefeldin A. Cells were then fixed, incubated with anti-CD4-FITC or
anti-CD8-FITC antibodies, and then refixed and permeabilized. Cells
were then incubated with anti-IFN-PE or anti-TNF-PE, and then
visualized for PE and FITC staining by flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
[0052] Definitions and Abbreviations
[0053] In order to facilitate understanding of the invention,
certain terms used throughout are herein defined:
[0054] "GM-CSF" means Granulocyte-Macrophage Colony Stimulating
Factor. GM-CSF may increase the immunogenicity of antigens by
stimulating antibody production mechanisms.
[0055] "Non-native" means not derived or obtained from the same
species.
[0056] "Native" means derived or obtained from the same
species.
[0057] "IgG" means immunoglobulin-G.
[0058] "Intergenic sequences" means the non-coding DNA sequences,
wherein the viral origin of replication is situated, that are
located between open reading frames of viruses.
[0059] "OAS" means origin of assembly sequence. The origin of
assembly sequence is necessary for assembling the RNA molecule with
viral coat proteins into a viral particle.
[0060] "Reconstituted protein" means the isolated and hydrated form
of protein from a complex protein mixture
[0061] "IL4" means interleukin 4, a cytokine that activates immune
cells, especially B cells
[0062] "IL1b" means Interleukin 1, beta subtype, a cytokine that
activates immune cells
[0063] "IL1b peptide" means a 9 amino acid section of IL1b that can
stimulate T cells
[0064] "IFN.gamma." means interferon, gamma subtype, a cytokine
that activates immune cells, especially T cells
[0065] "TMV" means tobacco mosaic virus
[0066] "VLP" means virus like particle
[0067] "Th1" means T-helper type one immune response, which is
characterized by both antibody and cellular immunity
[0068] "Th2" means T-helper type two immune response, which is
characterized by primarily an antibody response
[0069] "IVE" means in vitro encapsidation
[0070] "RNA" means ribonucleic acid
[0071] "DNA" means deoxyribonucleic acid
[0072] "HA" means a peptide sequence derived from influenza
hemaglutinin
[0073] "V5" means a peptide sequence derived from simian virus
5
[0074] "myc or Myc" means the peptide derived from the myc
oncogene
[0075] "N" position means the position the peptide or modification
is inserted, at the N terminal location of coat protein
[0076] "L" position means the position the peptide or modification
is inserted, at the extracellular loop location of coat protein
[0077] "G or GPAT" means the position the peptide or modification
is inserted, at four amino acids from the C terminal location of
coat protein
[0078] "C" position means the position the peptide or modification
is inserted, at the C terminal location of coat protein
[0079] "Cys" means the amino acid Cysteine
[0080] "20S" subunit describes the sedimentation profile of the 34
subunit coat protein disk in a density gradient
[0081] "4S" subunit describes the sedimentation profile of the 4
subunit coat in a density gradient, which is an intermediate to the
formation of a 20S disk
[0082] "kDa" means kiloDalton, which refers to the molecular weight
or mass of the protein
[0083] "TEM" means transmission electron microscopy
[0084] "RT" means room temperature
[0085] "4C" means 4 degrees Celsius, or near zero Fahrenheit
[0086] "PAGE" means polyacrylamide agarose gel electrophoresis
[0087] "SDS" means sodium dodecyl sulfate, a detergent
[0088] "PEG" means poly ethylene glycol (molecular weight
6000-8000) "NaCl" means sodium chloride, or salt
[0089] "DEAE" mean diethyl aminoethyl, a molecule used on anion
exchange resins
[0090] "PO4" means phosphate
[0091] "pyro PO4" means pyrophosphate
[0092] "SU" mean subunits
[0093] "CRPV" means cottontail rabbit papillomavirus
[0094] "ROPV" means rabbit oral papillomavirus
[0095] "HPV" means human papillomavirus
[0096] "OVA" means ovalbumin
[0097] "GJ" means green juice, or total plant homogenate
[0098] "S1" means clarified plant extract supernatant
[0099] "S2" means supernatant derived from the S1 insoluble
material by resuspension at pH 7
[0100] "BSA" means bovine serum albumin
[0101] "MW MALDI" means molecular weight mass determination by
Matrix Assisted Laser Desorption lonisation mass spectrometry
[0102] "w/v" means weight per volume
[0103] "OD" means optical density
[0104] "DDT" means Dithiothreitol
[0105] "RNAse" is an ubiquitous cellular enzyme that degrades
RNA
[0106] "RNAsin" is a commercially available RNase inhibitor
[0107] "DEPC" is diethyl pyro carbonate, a chemical inhibitor of
RNAse activity
[0108] "Nab" means neutralizing antibody
[0109] "L1" means papillomavirus capsid protein L1
[0110] "L2" means papillomavirus capsid protein L2
[0111] "E1, 2, 4, 6, 7, and E8" are papillomavirus early gene
products
[0112] "CTL" means cytotoxic T lymphocyte
[0113] "SFV" means semliki forest virus
[0114] "IRES" means internal ribosomal entry site, which allows for
the initiation of translation in the middle (or anywhere that is
not at the first ATG) of the RNA
[0115] "ORF" means open reading frame, the functional unit of RNA,
which when translated encodes a protein
[0116] "B16" means the mouse melanoma tumor cell line named B16
[0117] "SPDP" N-succinimidyl-3-(2-pyridyldithio) propionate
[0118] "BCA assay" Protein assay based on bicinchoninic acid
[0119] The present invention relates to a novel method for for the
colorimetric detection and quantitation of total protein.
[0120] The present invention relates to a novel method for
construction of a plurality of vaccines and pharmaceuticals using
viruses, such as the tobacco mosaic virus (TMV). In broad terms,
the invention is practiced in a manner depicted generically in
FIGS. 1-6, as described below.
[0121] The description of the present invention is first provided
in general terms, followed by a more detailed description that
includes many bio-chemical procedures.
[0122] Standard methodologies can produce a pseudo-multivalent
vaccine product by a chemical conjugation process outlined in FIG.
1. VLP particles are produced (S1) and isolated (S2). Individual
peptides are individually chemically conjugated to the surface of
independent lots of VLPs (S3) to produce distinct populations of
VLPs, each displaying a unique peptide adduct. It is possible to
conceive that multiple peptides could be simultaneously conjugated
on the surface of the same population of VLPs to produce VLPs with
a random distribution of unique peptides. The distinct populations
of immune particles are then mixed (S4) to produce a population of
VLP particles with distinct peptides covalently attached to the
surface (P1). The resulting product will display distinct peptides
in its mixture, but each will be independently taken up by immune
cells and independently used to stimulate the immune system. There
will be a lack of synergy between the fused peptides since there is
no special connection between different peptides; each functions
independently.
[0123] The described invention exploits the unique properties of
the tobacco mosaic virus (TMV) that is amenable to the procedures
outlined in FIG. 1, but also new methods (FIG. 2) with significant
advantages. As represented by the box S5 in FIG. 1, a TMV virion is
constructed with a surface associated amino acid allowing for
improved chemical conjugation. This can be the presence of a
unique, surface associated cysteine or lysine residue, although
other methods can be employed. Large quantities of TMV are produced
(S6) using, for instance, tobacco plants that are infected with the
desired strain of TMV, then processed as described in co-pending
patent application Ser. No. 09/962,527 filed Sep. 24, 2001,
entitled PROCESS FOR ISOLATING AND PURIFYING VITAMINES AND SUGARS
FROM PLANT SOURCES, and related U.S. Pat. Nos. 6,303,779, 6,033,895
and 6,037,456 all commonly assigned to Large Scale Biology
Corporation, Vacaville, Calif., all of which are incorporated
herein by reference in their entirety. Once large quantities of TMV
are available, a process that is described in greater detail below
disrupts the TMV in order to produce a large number of subunits
(SU) or 20S disks, as represented by the box S7 and S8. The
subunits are then separated at step S9 to form a plurality of
subunits, each to be processed separately, as is described in
greater detail below. As represented by step S9, each individual
subunit group is subjected to a conjugation reaction in order to
add predetermined components, such as a functional peptide,
epitope, proteins or nucleic acid sequence to the subunits in that
subunit group, in a manner that is described in greater detail
below. As represented at step S10, pluralities of groups of
subunits are now constructed into a single VLP structure where each
subunit having specific epitopes, peptides, proteins or nucleotides
attached thereto. TMV 20S disks naturally reassociate to form a
rod-shaped virion surrounding an RNA molecule containing a unique
sequence termed the TMV ori, or origin of assembly (OAS). This
produces a multivalent vaccine (P2) that is not equivalent to a
simple mixing reaction. Multifunctional peptide or nucleic acid
adducts are linked physically to one another allowing each to
synergistically enhance the cellular uptake of the VLP vaccine,
immune processing, number of immune peptides presented to the
immune system and the nature of the stimulated immune response. The
simultaneous presentation of each peptide or nucleic acid component
on the same VLP, rather than on distinct, unlinked VLP populations,
is predicted to enhance the effectiveness of the VLP vaccine and
lower the lower dose.
[0124] Further basic steps in the method of the present invention
are depicted in FIG. 2. Specifically, at step S10 a specific
recombinant RNA sequence is selected to be the scaffold for
assembly of the TMV VLPs. The specific VLP subunits selected in
step S10 are combined with the RNA selected to form a reassembled
TMV via a process that is described in greater detail below. The
RNA can act only as a structural scaffold and could represent only
the TMV RNA itself, not offering any augmented function other than
a building block of the new VLP vaccine. However, recombinant RNAs
can be constructed containing the TMV ori (S10) that also encode
proteins. Once the VLP is taken up in immune cells, the TMV virion
has unique function. It is preferentially bound by ribosomes and
disassembled by a co-translational mechanism (Mundry et al., J Gen
Virol. 1991 April;72 (Pt 4):769-77.). This would allow the
efficient translation of this RNA so that the encoded protein is
produced within the host immune cells. The encoded protein can
either be an intact antigen to stimulate humoral or cellular immune
responses against the targeted pathogen or cancer. Conversely, the
RNA could encode immune stimulatory proteins (enhancing the
amplitude of immune response) or modulatory proteins (insuring the
direction, Th1 or Th2, of the immune response). This combination of
protein elements that stimulate the immune response, as well as
promoting the efficiency and effectiveness of the response,--in
combination with an encoded nucleic acid component that is
functional for augmenting the immune response, makes this vaccine
truly bifunctional.
[0125] It should be noted that RNA is inherently unstable as a
`naked` element, or one not coated with a protective protein
coating. However, it has an advantage over DNA in nucleic acid
vaccines since it promotes translation of the desired product
within immune cells, but is degraded and does not risk the
immunized host with DNA recombination and the associated oncologic
events. `Naked` or uncoated nucleic acid vaccines of RNA or DNA
types are very inefficient, where milligram (mg) quantities of DNA
are required for any immune response in humans. Out of the mg of
vaccine administered, picograms or less are taken up by immune
cells. This results in expensive manufacturing and formulation
costs, and very inefficient unpredictable immune responses. This
invention allows the `naked` RNA encoding important antigens or
immune enhancing proteins to be coated and protected within the VLP
structure of TMV. Such coating enhances the stability of the RNA
and improves the delivery efficiency.
[0126] VLP vaccines are not dependent only on chemical conjugation
to add immune peptides to their surface. The art describes methods
for generating VLP vaccine through the genetic fusion of
immunologically relevant peptides to the surface of VLPs. This
process is described in FIG. 3. In this case, individual (S11) or
multiple (S14) peptides are fused to the surface of the VLP protein
through recombinant DNA procedures where the protein coding
sequence for the immune peptide is fused to that of the VLP
structure. Each individual or multi-peptide displayed VLP structure
is purified (S11) and then qualified for its properties (S12). A
multivalent vaccine is constructed by mixing either individual VLP
populations displaying one or more peptides by genetic fusion (S13)
or simply using a single population of VLP that is displaying more
than one peptide by genetic means (S14). These procedures produce a
multivalent VLP immunogen composed of multiple separate VLP
populations, each displaying a unique immune peptide (P3). This
approach suffers from the same limitations of the vaccines produced
in FIG. 1 where little to no synergistic activity can be predicted
by the simple mixture of non-linked peptides. Further, the VLP
vaccines lack a nucleic acid component and are simply single
functional vaccines--only providing a protein-based signal to the
immune system.
[0127] This invention overcomes these difficulties by allowing
truly multi-valent and multi-functional vaccines to be derived. TMV
is amenable to the same procedures described in FIG. 3 to produce
mixtures of VLPs each with unique genetic fusions. However, its
unique properties permit the procedure described in FIG. 4.
Individual TMV virions can be prepared with single or multiple
peptides by genetic means (S15). Each individual virion is isolated
(S15) and qualified. Each TMV virion is separately disassembled
(S16) and SU are prepared (S17) composed of 20S disks displaying a
unique array of immune peptides. This plurality of SU are then
reassembled surrounding a RNA containing the TMV ori to produce TMV
VLP (S18). The final product is indeed a VLP vaccine that displays
multiple immune peptides simultaneously on the surface of each VLP
(P4) and contains RNA that functions both as a scaffold for VLP
assembly and as a separate immune stimulus. The advantages of this
approach are the same as described above in that the particle is
multi-functional in terms of the plurality of immune, immune
modulatory, immune stimulatory or cell uptake facilitating peptides
simultaneously displayed on the surface of the VLP. This allows
more efficient cellular uptake, processing and immune stimulation
resulting in reduced dose and improved immune protection. The RNA
again contributes essential functions beyond a scaffolding device.
It can encode intact antigens, immune modulatory, immune
stimulatory proteins to further augment the immune response. The
RNA is protected within the VLP and is delivered efficiently to the
cellular translation apparatus by the natural functions of TMV
VLPs.
[0128] It should be understood that the above description is only a
basic framework of steps upon which the present invention
functions, and a basic understanding of the platform for
constructing vaccines and pharmaceutical products in accordance
with the present invention. The steps outlined in the flow diagrams
in FIG. 2 and FIG. 4 are illustrated visually in FIGS. 5 and 6. Two
further points should be noted with regard to the basic frameworks
outlined in FIGS. 1 to 4. Firstly these figures indicate that
various vaccine compositions contain 3 unique epitopes either
displayed on separate VLPs or virions or all reassembled onto one
VLP or virion. The number three was chosen purely for illustrative
purposes and it should be understood that any number of epitopes
can be recombined to form a multivalent vaccine. Secondly the
entity displayed on the surface of the VLP or virion need not be
limited to a peptide epitope as indicated in FIGS. 3 and 4. The
displayed entity can also be a nucleotide, introduced by chemical
fusion, or a complete protein, introduced by either chemical or
genetic fusion. Furthermore all possible combinations of
nucleotide, peptide epitope and complete protein, in terms of both
number and ratio, can be envisioned for multivalent vaccine
reassembly. For example peptide 1, nucleotide A and complete
protein X, each displayed on separate virions or VLPs can be
combined to yield a multivalent VLP vaccine similar to P3 in FIG.
3. Alternatively separate pools of 20S disks each displaying
peptide 1, nucleotide A and complete protein X can be reassembled
in vitro to generate a multivalent vaccine similar to P4 in FIG. 4,
where all entities reside on a single VLP or virion.
[0129] Following are a series of detailed examples, which
illustrate the general flow diagrams described on the preceding
pages.
EXAMPLE 1
[0130] Peptide Fusions and Solubility as a Function of pH
[0131] The current industry standard for success with peptide
fusions is 40-50%. To improve on this a series of fusions were
tested at multiple insertion locations on the TMV U1 coat protein
and each fusion was extracted under multiple conditions, to
determine the influence of fusion position on virus solubility.
This example describes the influence of genetic fusion position on
the isolation of recombinant TMV viruses (step S15, FIG. 4).
[0132] FIG. 7 illustrates results for the fusion HA, inserted at
four different locations on the U1 coat protein; the N terminal, C
terminal, surface loop (L) and 4 amino acids from the C terminus
(GPAT). Clear differences in the extent of cleavage and virus
solubility were evident. Approximately 100% HA GPAT was cleaved
back to wild type U1 protein molecular weight when extracted at pH
5. Re-extraction at pH 7 improved full-length yield to 50%. Tissue
extraction in SDS PAGE buffer yielded full-length coat fusion
product, suggesting that cleavage was occurring during processing.
This also occurred at the C terminal fusion location, although to a
lesser extent. The processing of coat fusions appears to be site
specific, as locating the epitope at the N-terminus yielded a
full-length product. No virus was recovered at pH 5 or pH 7 with
the HA epitope at the loop position; the SDS-PAGE buffer grind
indicated that loop insertion was expressed but resulted in an
insoluble product. For fusions that show cleavage during extraction
e.g. HA GPAT, protease inhibitor cocktails can be incorporated to
reduce or eliminate cleavage. Alternatively, other strains of N.
tobaccum can be screened to identify hosts with reduced protease
activity.
[0133] Table 1 summarizes the influence of epitope location on the
solubility and relative recoveries for HA and two additional model
epitope fusions, V5 and Myc. The V5, HA and myc epitope TMV fusion
proteins were tested for reactivity to peptide specific antibodies
by Western analysis, to confirm the identity and integrity of each
fusion peptide (data not shown).
1TABLE 1 Expression levels by insertin site for three antibody
binding epitopes. Position of insert and Extraction buffer pH N L
GPAT C Fusion name pH 5 pH 7 pH 5 pH 7 pH 5 pH 7 pH 5 pH 7 V5 +++
++ + + ++ + ++ + HA - ++ - - - ++ - ++ Myc +++ ++ - +/- ++ +++ ++
++
[0134] Following the confirmation of expression with the three
model fusions the list of fusions was expanded to include
clinically relevant epitopes of papillomavirus and melanoma as well
as immuostimulatory and cell fusion epitopes aimed at incorporating
biological functionality to reassembled fusion products. Table 2
summarizes the solubility results for all the epitope fusions. Of
the 18 target epitopes attempted 15 were successfully expressed as
soluble products, an 83% success rate. This represented a doubling
of the previous industry standard of 40% expression/solubility.
This improvement is due to the rotation of the insert position,
performed in parallel with the extraction with two different pH
buffers.
2TABLE 2 15 of 18 peptides have been expressed in frame with TMV U1
coat at either the N-terminus (N) the GPAT position (G) or at the C
terminal location (C). Those fusions that were soluble in either pH
5 or 7 extraction buffer from leaf punch grinds (.about.200 .mu.g
leaf tissue) are indicated in the Solubility column. Those fusions
that were also successfully scaled up (>500 grams leaf tissue)
are also indicated. Peptides Name Solubility Scalability
GKPIPNPLLGLDSTK (Seq ID No: 1) V5 N, G, C N, G, C YPYDVPDYAK (Seq
ID No: 2) HA N, G, C G, C EQKLISEEDLK (Seq ID No: 3) c-myc N, G, C
N, G, C Papillomavirus VGPLDIVPEVADPGGPTLV (Seq ID No: 4) CRPV 2.1
N, G G PGGPTLVSLHELPAETPY (Seq ID No: 5) CRPV 2.2 N, G G
VGPLEVIPEAVDPAGSSIV (Seq ID No: 6) ROPV 2.1 N, G G
PAGSSIVPLEEYPAEIPT (Seq ID No: 7) ROPV 2.2 N, G G AALQAJELM (Seq ID
No: 8) HPV16 ep2 N N Melanoma SVYDFFVWL (Seq ID No: 9) TRP-2181-188
-- KSPWFTTL (Seq ID No: 10) p15E 604-611 -- SIINFEKL (Seq ID No:
11) OVA N, G, C N, G HIV ELDKWAS (Seq ID No: 12) ELDKWAS N N
Immunostimulatory CEYNVFHNKTFELPRA (Seq ID No: 13) Th SH 45-60 G, C
QYIKANSKFIGITELKK (Seq ID No: 14) P2 TT 830-846 -- VQGEESNDK(Seq ID
No: 15) IL1.beta. N, G, C N Cell fusion FAGVVLAGAALGVATAAQI F1
Measles L, G (Seq ID No: 16) SGRGDSG (Seq ID No: 17) integrin N, G,
C N GYIGSR (Seq ID No: 18) laminin N, G, C N
EXAMPLE 2
[0135] Improving Solubility and Accumulation by Modifying the
Linker Amino Acids
[0136] Molecular fusion of epitopes to TMV sometimes fail to
accumulate when aromatic (for example W) or hydrophobic amino acids
are present in the peptide. For example, p15e, a mouse melanoma
antigen, contains the aromatic amino acid tryptophan (W). This
peptide, when introduced onto the N or C-terminal positions on U1
coat, caused virus instability and no TMV systemic infection was
observed. Applicant reasoned that to create a more favorable
environment for peptide solubility, flanking amino acids could be
added to increase hydrophilic interactions, counteracting the
negative effects on virus assembly or stability when amino acids
like W are introduced onto the solvent exposed surface of coat
protein. Aspartic Acid (D) and Glutamic Acid (E) are amino acids
that are charged, and were used to show that such a method will
rescue the insoluble fusion of p15e to TMV coat (FIG. 8). Before
addition of DE adjacent to the p15e peptide, no accumulation of
product was observed (*). After addition DE to p15e, product
accumulation is clearly visible (arrow). Other amino acids could
also be used to alleviate negative effects of peptide composition
on TMV accumulation, such as Asparagine (N), Glutamine (Q),
Histidine (H), Lysine (K), Serine (S) or Threonine (T). The number
and type of flanking amino acids that are sufficient to overcome
negative effects on TMV expression levels or assembly may be
fusion-peptide specific, and may need to be tested empirically for
each peptide. This example illustrates the use of mitigating
sequences to permit isolation of genetic fusions (S15, FIG. 4)
EXAMPLE 3
[0137] Chemically Conjugated Epitope Fusions to TMV U1
[0138] Only a percentage (70-80%; see Example 1) of genetic fusions
are capable of functional VLP formation for many plant viruses.
Many fusions fail to accumulate while others are simply insoluble.
The present invention includes construction of coat protein fusions
containing cysteine (Cys) residues as either N-terminal or surface
loop fusions. The initial fusions to TMV U1, and to other
tobamovirus coat proteins showing good expression in the U1 vector,
are composed of glycine-cysteine-glycin- e (GCG) or GGCGG as N- and
surface loop fusions (FIG. 9A (1)). Previous LSBC experiences have
indicated that cysteine residues are tolerated on the virion
surface and that under the reducing conditions of the plant
cytosol, no disulfide bridges are formed between coat protein
subunits or host proteins. The production of coat protein with
surface exposed Cys residues allows peptide conjugation to the TMV
virions through conjugation using heterobifunctional chemical
cross-linking reagents, e.g. N-succinimidyl-3-(2-pyridyldithio)
propionate (SPDP). SPDP allows coupling of free sulfhydryl group
with a free arnine group, such as that found on lysine (K), at
neutral pH under mild reaction conditions. SPDP fused
immuno-conjugates have been used extensively in in vivo
administrations. Peptides used for initial studies and comparative
biochemical response of the various tobamovirus coat proteins (CPs)
are the c-myc tag (EQKLISEEDLK), the HA tag (YPYDVPDYAK) and the V5
tag (GKPIPNPLLGLDSTK). Each is synthesized (Sigma chemical) to
contain a C-terminal lysine for conjugation to the sulfhydryl
group. In addition to peptides, SPDP could be used to fuse the
immune stimulatory single stranded DNA CpG polynucleotide using a
thiolated 3' terminus to the TMV virions as well. An alternative
approach is to introduce a different reactive amino acid, such as
lysine, into the region of solvent exposed residues of TMV coat
protein (FIG. 9A (2)), and synthesize peptides with a C terminal or
N terminal cysteine for conjugation.
[0139] Initially, SPDP conjugations are tested for reactivity to
cysteine containing TMV that is not disassembled. Cross-linking
reactions are carried out using short chain, long chain and
sulfo-NHS forms of SPDP as described (Hermanson, G. Bioconjugate
Techniques 1996 Rockford Ill., Academic Press, and references
therein). Peptide-SPDP adducts are mixed with cysteine TMV U1 virus
and then analyzed 16 hours later for a size shift that represents
physical association of the peptide with the virus. The procedure
is then extended to 20S disks. An alternative approach was to use a
less specific chemical conjugation strategy employing
glutaraldehyde. The HA peptide was mixed with either TMV or N
terminal cysteine TMV in the presence of glutaraldehyde. After a
four hour incubation with glutaraldehyde, a HA peptide-TMV cysteine
conjugate was formed and was visible as an increase in mass by
Coomassie, as well as by an increase in apparent molecular weight
by Western analysis (FIG. 9B). No such conjugate was present if
wild type TMV (with no solvent exposed cysteine) was used in the
conjugation reaction (data not shown). Conjugation by non-specific
cross-linking agents, such as glutaraldehyde, leads to higher
molecular weight aggregates as is clearly visible in the Western
blot. Other conjugation reagents with more specific chemistry, such
as SPDP, EDC or other heterobifunctional linkers, generate one to
one or directional coat to fusion peptide chemistry, and result in
more controlled conjugation reactions.
[0140] An alternative strategy is to assemble N cysteine coat into
20S discs, reassemble these discs with other discs that carry
functional epitopes (ie, by molecular fusion) onto an RNA, and
incubate the fully reassembled mixture with SPDP-associated peptide
or moiety in order to add a new functionality. This is especially
useful if the SPDP conjugation renders 20S discs chemically inert
and unable to reassemble with other discs, or if the peptide that
is carried interferes sterically with reassembly. As well, the
ability to add a variety of agents after reassembling a monomer or
a multimer has great utility. For example, SPDP conjugation of
ssDNA such as CpG oligonucleotides may allow for the augmentation
of immune modulation, which is greater than simply mixing the CpG
with the vaccine. This could lead to better efficacy and or the
potential to reduce the dose. This example illustrates the steps S3
(FIG. 1) and S9 (FIG. 2) as well as providing alternative routes to
combine chemically and genetically attached epitopes.
EXAMPLE 4
[0141] Electron Microscopy of TMV Coat Protein Fusions
[0142] To determine the influence of the fusions on virus
structure, transmission electron microscopy (TEM) was performed
(FIG. 10). Wild type TMV rods have the dimensions 18-20
nm.times.300 nm. The N terminal epitope fusions of the model
peptides V5 and Myc were visually similar the wild type U1 virus,
as were the rod dimensions. This indicates that the fusion does not
hinder normal coat protein reassembly in vivo and that the fusions
constitute good candidates for in vitro reassembly.
EXAMPLE 5
[0143] Extraction and Partitioning of Wild Type TMV U1
[0144] The extraction and processing of TMV U1 has been extensively
discussed in the above mention commonly assigned U.S. Pat. Nos.
6,303,779, 6,033,895 and 6,037,456, which are incorporated herein
by reference in their entirety. The processing is summarized in
FIG. 11A. Briefly, a weighed mass of infected tissue is combined
with two volumes of chilled water, containing 0.04% w/v sodium
metabisulfite and grinding is preformed in a Waring blender. The
homogenate is passed through 4 layers of cheesecloth to remove the
fiber, leaving the green juice (GJ). The pH of the GJ is adjusted
to 5.0. followed to heating to 47.degree. C. for 5 minutes. After
chilling the GJ is spun to precipitate insolubles, yielding a first
supernatant. In cases where the virus partitions into the remaining
pellet P1, the pellet is resuspended in water and adjusted to pH
7.0. Following a centrifuge spin the virus is recovered in a second
supernatant and the final pellet P2 is discarded. To purify and
concentrate the virus, two serial selective precipitations are
performed on the first and second supernatants processing streams.
Precipitation of the virus is achieved by adjusting the
supernatants to 4% w/v polyethyleneglycol (PEG) and 4% w/v NaCl,
and chilling for 30-60 minutes. Following a centrifuge spin the
virus is recovered as a pellet and contaminating proteins remain in
the supernatant, which is discarded.
[0145] FIG. 11B and Table 3 show representative results for wild
type TMV U1isolated from N. tabacum MD609. The SDS gel clearly
demonstrates that the process yields a final virus preparation of
high purity. Using BSA as a standard the coat protein bands were
quantified densitometrically and a material balance for the process
performed to determine recovery (Table 3). From the data it is
clear that the majority of the virus partitioned into the S1
process stream and with minimal losses during the PEG precipitation
a total process recovery of 76% was achieved.
3TABLE 3 Material balance for the isolation of TMV U1 from infected
N tabacum MD609 plants. Data was generated from the densitometric
analysis of the gel in FIG. 11, using a BSA standard curve. Losses
Losses Mg virus Total mg virus/ % in during S1 during S1 recovered
process g FW % in S1 S2/P1 PEG1 PEG2 /g FW recovery 1.7 86% 14% 9%
2% 1.3 76%
EXAMPLE 6
[0146] Influence of Epitope Fusion on Virus Extraction and
Partitioning
[0147] The process outlined in Example 5 was employed for a
selection of the coat protein fusions listed in Table 2. Material
balances were performed to determine the partitioning of the virus
between the S1 and S2 process streams, in addition to the total
process recovery. The identity of each fusion was confirmed by MW
MALDI. The results for these purifications are summarized in Table
4. From the table it is clear that the processing characteristics
are epitope fusion and location dependent. A material balance on
the extraction gave initial recoveries (S1+S2 process streams) from
90-100% (e.g. HPV ep2 N) to lower than 10% (e.g. V5 N).
Partitioning between the S1 and S2 streams also varied
substantially. Overall recoveries also ranged from 0.5% to 79%.
Based on this data the cysteine N, Myc N and V5N coat protein
fusions were carried forward to optimization studies to determine
conditions which would improve overall process recoveries. This
optimization is detailed in Examples 7 and 8 and illustrates
process modifications that can be employed in order to isolate TMV
virus displaying genetic fusions (step S15, FIG. 4)
4TABLE 4 Virus partitioning and overall process recovery for
various coat protein fusion epitopes. Fusion location designation;
N, N terminus; C, C terminus; GPAT, N terminal to GPAT sequence. #
indicates the purification run number for fusions isolated more
than once. Overall process Fusion % in S1 % in S2 Streams processed
recovery Cysteine N 10% 44% S2 0.5% Myc #1 N 38% 26% S1 and S2 13%
Myc #2 N .about.20% 24% S1 and S2 6% Myc C N/A N/A S1 and S2 13% V5
#1 N N/A N/A S1 2% V5 #2 N .about.5% .about.5% S1 and S2 3% HPV ep2
N 60% 40% S1 51% OVA N 26% 50% S1 and S2 79%
EXAMPLE 7
[0148] Influence of Sodium Chloride on Virus Extraction and
Partitioning
[0149] The incorporation of sodium chloride into the extraction
buffer was tested as a means to improve virus recovery and alter
virus partitioning. GENEWARE-infected N benthamiana plants were
harvested and the biomass split, to perform a head to head
comparison of extraction in the presence and absence of salt. One
half of the plant material was extracted in chilled water
containing 0.04% sodium metabisulfite and the remaining biomass was
extracted in a 50 mM acetate buffer, pH 5.0, containing 4% w/v NaCl
and 0.04% sodium metabisulfite. Processing was performed following
the procedure outlined in Example 5. A comparison of the S1 and S2
fractions by SDS-PAGE, for the Cysteine N TMV fusions (FIG. 12),
clearly illustrates that the presence of salt forces the virus to
partition to the S1 fraction. This is favorable as the virus
obtained from this stream is typically less contaminated by plant
pigments and impurities. Also, from FIG. 11A it is clear that S1
partitioning is preferential to S2 partitioning as it reduces the
number of processing steps.
[0150] A material balance for extractions in the presence and
absence of salt is given in Table 5. From the data for Cysteine N,
it is clear that the overall process recovery was improved
substantially with the addition of salt; although the total virus
extracted in both cases was identical, the virus loss in the
absence of salt was 44% (remained associated with the P2 pellet)
compared to only 7% with 4% w/v sodium chloride. Table 5 also has
data for recovery and virus partitioning of the Myc N and V5N coat
protein fusions during extraction. The benefits of sodium chloride
are again evident, indicating that this process modification has
general applicability.
5TABLE 5 Material balance for the isolation of viruses displaying
multiple epitopes from infected N benthamiana plants. Data was
generated from the densitometric analysis of the SDS gels, using a
BSA standard curve. Losses mg virus/g during Fusion Buffer FW % in
S1 % in S2 extraction Cysteine N No NaCl 1.9 10% 46% 44% 4% w/v
NaCl 1.9 88% 6% 7% Myc N No NaCl 3.3 38% 26% 36% 4% w/v NaCl 2.9
90% 8% 2% V5 N No NaCl 1.2 .about.5% .about.5% 90% 4% w/v NaCl 1.2
63% 0% 37%
EXAMPLE 8
[0151] Influence of Salt and PEG Concentration of Virus
Precipitation
[0152] As illustrated in FIG. 11 the virus in either the S1 or S2
processing streams is further purified and concentrated by a series
of two PEG precipitations. The steps involved in the first PEG
precipitation are outlined in the flow diagram below (FIG. 13). The
S1 (or S2) supernatant is adjusted to 4% w/v polyethylene glycol
and 4% w/c NaCl. If the supernatant already contains NaCl only
solid PEG is added, dissolved with agitation and the sample chilled
on ice. The precipitated virus is pelleted by centrifugation and
the supernatant discarded. The virus-containing pellet is then
resuspended in a low ionic strength buffer and a low speed
clarification spin performed. This will pellet any residual pigment
and aggregated contaminating plant proteins, leaving the virus in
solution. This solution is then resubmitted to a second PEG
precipitation by adjusting to 4% w/v PEG and 4% w/v NaCl and
repeating the process.
[0153] Table 6 compares the recoveries obtained from the two-step
PEG precipitation for wild-type TMV U1 and two coat protein
fusions, Myc N and V5N. The standard procedure outlined in FIG. 13
resulted in poor recoveries for both coat protein fusions compared
to the wild type U1. From the flow diagram the losses can result
from incomplete precipitation of the virus by the PEG, or pelleting
of the virus during the clarification step. A material balance
around each step in the PEG precipitation indicated that for Myc N
4% w/v PEG was insufficient to pellet the virus and the majority
remained in the supernatant. In this case an increase in the PEG
concentration, to 8% w/v, was required and this modification
improved recovery from 6% to 60%. For the V5N virus complete
precipitation was achieved with 4% w/v PEG, however, the virus
failed to remain in solution during the clarification spin. By
resuspending the virus-containing pellet in 10 mM Na K PO4
containing 4% w/v NaCl, the virus remained soluble and recovery was
increased from <1% to 95%. These two examples illustrate how the
fusion can influence the virus properties and provide methods to
maintain virus solubility during processing.
6TABLE 6 Optimization of PEG precipitation steps for TMV coat
protein fusions Stream Losses during Losses during Recovery PEG
Fusion processed Conditions S1 PEG1 S1 PEG2 precipitation steps
Wild type S1 Standard 9% 2% 89% U1 Myc N S1 Standard 80% 63% 6% V5N
S1 Standard .about.95% .about.95% <1% Myc N 1 8% w/v PEG 25% 20%
60% Resuspend in 4% w/v NaCl V5 N 1 Resuspend in 5% 1% 94% 4% w/v
NaCl
EXAMPLE 9
[0154] Generation of Free Coat Protein and 20S Disks
[0155] This example illustrates in greater detail steps S7 and S8
(FIG. 2) and steps S16 and S17 (FIG. 4). Coat protein was generated
from purified virus using a modified version of the protocol
developed by Fraenkel Conrat (Virology 1957, 4, 1-4), which is
summarized in FIG. 14. Briefly the virus was combined with 2
volumes of glacial acetic acid and incubated for 1 hour at
4.degree. C., resulting in disassociation of the virus and
degradation/precipitation of the RNA. Following centrifugation to
remove the degraded RNA, the acetic acid was removed by dialysis.
Alternatively an ultrafiltration/diafiltration can be employed to
remove the majority of the acetic acid, prior to dialysis. With
dialysis the coat protein precipitates at its isoelectric point.
The precipitated coat protein was isolated by centrifugation and
resuspended in water. By adjusting the pH to 8, the coat protein
was resolubilized and subjected to a final spin to remove any
remaining aggregated species.
[0156] This process was employed to generate a number of free coat
protein fusions from purified virus. Table 7 summarizes the process
recoveries for a selection of the epitope fusions for which coat
protein was generated.
7TABLE 7 Free coat protein generation for a selection of epitope
fusions. Fusion location designation; N, N terminus; C, C terminus.
Acetic Acid removal Overall process Fusion UF/DF Dialysis recovery
HPV ep2 N + 49% ELDKWAS N + 68% Myc C + 50% Myc N + 38% V5 N +
34%
[0157] The quality of the coat protein was assessed by its
ultraviolet absorption spectrum (Durham, J Mol Biol, 1972, 67:
289). The spectrum should have an absorbance maximum at 282 nm, an
absorbance minimum at 251 nm and a maximum to minimum ratio between
2.0 and 2.5. A lower ratio indicates residual RNA contamination of
the coat protein preparation. FIG. 15A shows the typical absorption
spectrum for wild type TMV U1 coat protein. Table 8 summarizes the
absorbance ratio for free coat protein preparations displaying
various epitope fusions. In cases where the maximum to minimum
ratio was lower than expected, e.g. Myc N, the coat protein
preparation was treated with an anion exchange resin, such as DEAE
Sepharose. The contaminating RNA associates strongly with the
positively charged resin, while the coat protein's association will
be lower, permitting selective elution of the coat protein at low
chloride ion concentrations. This approach was successful at
separating Myc N coat protein from contaminating residual RNA by a
50 mM NaCl elution, to yield a coat protein preparation with a
maximum to minimum absorbance ratio greater then two (FIG. 15B to
D)
8TABLE 8 Ratio of absorbance maximum (282 nm) to absorbance minimum
(251 nm) for free coat protein displaying various epitope fusions.
Fusion location designation; N, N terminus; C, C terminus. Fusion
OD Ratio HPV ep2 N 2.1 ELDKWAS N 2.2 Myc C 2 Myc N 1.22
[0158] Prior to use in reassembly reactions, the coat protein
preparation is converted from 4 S subunits, consisting of 3 to 4
coat proteins, to 20 S disks (see FIG. 5). This is accomplished by
incubating the coat protein preparation at room temperature for 24
to 48 hours prior to use, under the correct pH and ionic strength
conditions. For example, TMV U1 coat protein, in 0.1 M phosphate
buffer, pH 7.0 was allowed to equilibrate to room temperature
(20-22.degree. C.), from 4.degree. C., over 16 hours and the
initial and equilibrated coat preparation was analyzed by size
exclusion chromatography. As seen in FIG. 16, room temperature
incubation results in a bimodal distribution, resulting from the
formation of 20 S disks.
EXAMPLE 10
[0159] Reassembly of Wild Type TMV Virions from 20S Disks
[0160] This Example, together with Examples 11-13, illustrate the
methods for the generation of multivalent and bifunctional vaccines
i.e. step S18 (FIG. 4) to yield P4 (FIG. 4). The standard
conditions for TMV reassembly have been outlined for wild type U1
coat protein and a wild type TMV RNA scaffold (Fraenkel-Conrat, H
and Singer, B (1959) Biochim Biophys Acta, 33, 359-370). Typically
a 0.1 M phosphate or pyrophosphate buffer at a pH of 7.0 to 7.5 is
employed with a mass ratio of coat protein to RNA of 22:1.
[0161] TMV U1 coat protein was generated from wild type virus
isolated from N. tabacum var. MD609 plants, as described in Example
9. Wild type RNA was isolated from the same virus with the RNeasy
Plant Mini Kit (Qiagen, Valencia, Calif.). Reassembly reactions
were performed in 200 .mu.l volumes, at a coat protein
concentration of 1100 .mu.g/ml and a RNA concentration of 50
.mu.g/ml, in a 96 well plate format. The reactions were buffered
with 0.1 M phosphate or pyrophosphate, pH 7.2 and the coat protein
preincubated for two days at room temperature prior to use. This
preincubation results in the formation of 20S disks from the 4 S
subunits (FIG. 16). In addition to the standard conditions, the
addition of the ribonuclease inhibitor RNasin to the 0.1 M
phosphate buffered reaction was also tested. The reassembly
reactions were followed by measuring the change in absorbance at
310 nm over time, which corresponds to the increase in the average
length of the reassembly products.
[0162] FIG. 17A shows the A310 nm profiles for the reassembly
reactions. The wild type virus control was such that the molar RNA
concentration was equivalent to that of the reassembly reactions.
The use of pyrophosphate in place of phosphate improved the initial
rate of reassembly and the OD maximum corresponded to that of the
TMV virus control. For the phosphate buffered reassembly reaction
the maximum OD was lower than the virus trace (0.12 OD vs. 0.14
OD). Assessing the RNA integrity in the final reassembly reaction
by agarose gel electrophoresis (FIG. 17B) indicated that RNA
degradation was occurring in both the pyrophosphate and phosphate
samples, and to a greater extent in the latter. The addition of
different ribonuclease inhibitors to the coat protein was therefore
tested. The ribonuclease inhibitor (either RNasin (Promega,
Madison, Wis.) with and without additional DTT, or SUPERase
(Ambion, Austin, Tex.)) was added to the coat protein preparation
30 minutes prior to RNA addition (0.2-4 U/ul). SUPERase at all
concentrations tested was ineffective whereas the RNasin reduced
RNA degradation substantially (FIG. 17B). The presence also
improved the maximum OD 310 nm attained for the reassembly reaction
(FIG. 17A).
[0163] To determine the functional significance of the different
buffer combinations, aliquots of the reassembly reactions were
analyzed by the local lesion host assay (Table 9). The reassembly
reactions, naked RNA and virus controls were serially diluted and
applied to the leaves of N tobacum `Xanthi` NN plants, with
carborundum employed as an abrasive. Five days post inoculation the
lesion numbers were counted and provided a semi-quantitative
measure of the titer of functional virus in the reassembly
reactions.
9TABLE 9 Local lesion host assay data for reassembly reactions with
wild type U1 coat protein and TMV RNA. PO4, 0.1 M phosphate
buffered; pyro PO4, 0.1 M pyrophosphate buffered; PO4 RNasin, 0.1 M
phosphate buffered with 0.4 U/.mu.l RNasin ribonuclease inhibitor.
Virus Free RNA Reassembly Reassembly Reassembly Dilution control
control PO4 pyro PO4 PO4 RNasin 10-2 45 .+-. 13 10-3 4 .+-. 1 10-4
123 .+-. 41 1 .+-. 1 25 .+-. 7 95 .+-. 31 122 .+-. 44 10-5 14 .+-.
10 6 .+-. 4 3 .+-. 1 19 .+-. 7
[0164] Comparing the infectivity of free RNA to the reassembly
reactions, which contained an equivalent molar concentration of
RNA, clearly illustrates the improvement in infectivity with RNA
encapsidation. Within the reassembly reactions, a marked
improvement in infectivity was evident for the phosphate buffer
when RNasin was present, which correlated with the improvement in
RNA integrity and A310 nm OD maximum. The observed infectivity with
RNasin was comparable to that of the virus control. The
pyrophosphate buffer also improved infectivity due to the
accelerated reassembly, which aided in the protection of the
RNA.
EXAMPLE 11
[0165] Reassembly of Coat Protein Fusions onto TMV RNA
[0166] A central aim of this work is the generation of a
multifunctional TMV-based reassembly product, which displays
epitopes with different functionalities e.g. a cell targeting or
immunomodulation sequence together with an antibody or CTL target.
As a first step, the ability of various coat protein fusions to
reassemble onto TMV RNA was examined. The fusions chosen were
ELDKWAS and HPV ep2 at the N terminus and Myc at the C terminus.
The reassembly reactions were performed in 200 .mu.l volumes, at a
coat protein concentration of 1100 .mu.g/ml and a RNA concentration
of 50 .mu.g/ml, in a 96 well plate format. The reactions were
buffered with 0.1 M phosphate, pH 7.0 and the coat protein
preincubated for two days at room temperature prior to use. In a
subset of the reactions the ribonuclease inhibitor RNasin was
incorporated. The reassembly reactions were followed by measuring
the change in absorbance at 310 nm over time, which corresponds to
the increase in the average length of the reassembly products.
[0167] FIG. 18A shows the A310 nm profiles for the reassembly
reactions involving the ELDKWAS coat protein fusion. The presence
of RNasin in the reaction mixture clearly resulted in an improved
absorbance profile with a higher final OD. The RNA integrity of the
reassembly reactions was assessed by agarose gel electrophoresis
(FIG. 18B). Although the extent of degradation was substantially
higher than for the reassembly reactions involving U1 coat protein,
the presence of RNasin did reduce the extent of RNA degradation in
the ELDKWAS coat protein reassemblies.
[0168] The A310 nm kinetics together with the RNA profile suggest
that RNasin increases the proportion of full-length rods formed
during reassembly. To confirm this, samples were analyzed by
electron microscopy (FIG. 19). Comparing the images for coat
protein in the presence and absence of RNA shows that reassembly of
the ELDKWAS coat protein fusion onto the TMV RNA scaffold occurred.
To assess the influence of RNasin, the normalized particle size
distribution, obtained from the electron microscopy images was
determined. With RNasin present there was a reduction in the 0-100
nm length rods with a concurrent increase, from 5% to 20% of full
length (>275 nm) rods, which correlates with the A310 nm
absorbance data.
[0169] The reduction in full-length rods presumably results from
the reduced pool of full length RNA. This would be expected to
reduce the number of functional i.e. infectious reassembly
products. Analysis of the reassembly products by the local lesion
host assay confirmed this reduction; omission of RNasin reduced the
average number of lesions observed by a factor of 9 (Table 10).
10TABLE 10 Local lesion host assay data for reassembly reactions
with multiple coat protein fusion and TMV RNA. PO4, 0.1 M phosphate
buffered; PO4 RNasin, 0.1 M phosphate buffered with 0.4 U/.mu.l
RNasin ribonuclease inhibitor. All dilutions were at 10.sup.-3. At
this dilution no lesions were detected for free wild type RNA. N, N
terminal fusion; C, C terminal fusion. Reassembly Reassembly PO4
Coat protein 1 Coat protein 2 PO4 RNasin ELDKWAS (N) -- 4 .+-. 6 37
.+-. 16 Myc (C) -- 2 .+-. 2 31 .+-. 17 HPV ep2 (N) -- 2 .+-. 1 11
.+-. 6 ELDKWAS (N) HPV ep2 (N) 6 .+-. 4 ELDKWAS (N) Myc (C) 21 .+-.
13 HPV ep2 (N) Myc (C) 50 .+-. 43
[0170] Reassembly reactions were also performed with the HPV ep2
and the Myc coat protein fusions, in the presence or absence of
RNasin. Similar to the ELDKWAS coat protein fusion, the presence of
RNasin during the reassembly resulted in A310 nm profiles with a
higher final OD and improved RNA integrity. From a functional
standpoint the reassembly products generated in the presence of
RNasin showed greater activity by the local lesion host assay
(Table 10). For Myc and HPV ep2 the average number of lesions were
15 and 6 fold higher respectively when RNasin was present. These
infectivity studies clearly illustrate the ability of a TMV coat
protein carrying a solvent exposed epitope to reassemble and
encapsidate a functional RNA.
[0171] The coat protein preparations do have a plant-derived
ribonuclease activity associated with them, which can be partially
mitigated by the inclusion of RNasin in the reassembly reaction.
Alternative approaches can also be used to reduce the ribonuclease
activity associated with the starting virion preparations, from
which the coat protein preparations are generated. The virus
preparation can be treated with bentonite, which inhibits
ribonuclease activity (Jacoli, G., Ronald, W., and Lavkulich, L.:
Inhibition of Ribonuclease Activity by Bentonite, Can J Biochem 51,
1558, 1973). Alternatively the virus preparation can be treated
with diethylpyrocarbonate (DEPC) at 0.05% -0.1% v/v, which
inactivates RNases by reacting specifically with the histidine
residues in the enzymatic site. Residual DEPC is removed by
dialyzing the treated virus extensively against any buffer
containing a primary amine group, e.g. Tris
(2-amino-2-hydroxymethyl-1,3-propanediol), with which DEPC
reacts.
[0172] Reassembly reactions to generate a multivalent TMV-based
vaccine were performed using a TMV RNA scaffold. The ELDKWAS, Myc
and HPV ep2 coat protein fusions were combined pair wise at a 1 to
1 ratio. FIG. 20 compares the A310 nm reassembly kinetics for the
bivalent encapsidations to those for the coat protein fusions used
individually. The bivalent reactions showed a similar rise in
absorbance over time indicating that reassembly was occurring
efficiently in the presence of two independent coat protein
fusions. To test for the generation of functional bivalent
reassembled virions, local lesion host assays were performed (Table
10). Lesion numbers comparable to the monovalent assemblies were
obtained, confirming the presence of functional reassembly
products.
EXAMPLE 12
[0173] Multivalent Papillomavirus Prophylactic Vaccine
Introduction
[0174] Animals may be protected against infection with
papillomaviruses by vaccination with either or both papillomavirus
structural proteins, L1 and L2 (Da Silva D M et al., 2001, Journal
of Cellular Physiology 186:169-182; Koutsky L A et al., 2002, New
England Journal of Medicine 347:1645-51). Protection against
papillomavirus infection primarily requires a specific humoral
response, which results in production of virus neutralizing
antibodies (Nab) directed at epitopes in the structural proteins. A
cellular immune response directed against the structural proteins
may also contribute to vaccine-induced immunity. Live recombinant
virus and DNA vaccine vectors carrying L1, or one or more of the
non-structural genes E1, E2, E4, E6, E7 and E8, can induce
protective immunity in vaccinated animals; in these cases both
cellular and humoral immune responses are detected (Sundaram P et
al., 1997, Vaccine 15:664-71; Moore R A et al. J Gen Virol
20:2299-301). It is well established that a humoral response
directed against papillomavirus structural proteins is both
necessary and sufficient for protective immunity against
papillomavirus infection (Embers et al., 2002 Journal of Virology
76:9798-9805). A cellular immune response against virus-encoded
proteins will enhance the level and robustness of the protective
immune response, but will not prevent initial infection (Tobery T W
et al., 2003, Vaccine 21: 1539-47).
[0175] Bivalent or Multivalent Reassembled Vaccines
[0176] The most important papillomavirus Nabs bind conformational
epitopes in L1, and recognize only intact virus, or correctly
assembled virus-like particles (VLP). These Nabs recognize epitopes
in hypervariable loops on the capsid surface, and generally will
only neutralize closely related papillomavirus types. Antibodies
that bind linear epitopes in the N-terminal region of L2 may also
neutralize virus infectivity. Most importantly, Nabs directed
against L2 epitopes show the ability to cross-neutralize distinct
viral strains (Embers M E et al., 2002; Journal of Virology
76:9798-9805; Kawana Y et al., 2001, Journal of Virology 75:
2331-2336; Kawana K et al., 1999, Journal of Virology 73:6188-6190;
Kawana K et al. 2001, 1496-1502; Roden R B S et al., Virology
270:254-257). Embers et al. (2002; Journal of Virology
76:9798-9805) demonstrated that peptides that represent linear
epitopes in the L2 proteins of the rabbit papillomaviruses rabbit
oral papillomavirus (ROPV) and cottontail rabbit papillomavirus
(CRPV) could induce good protective immunity against challenge with
the homologous virus, but not against the heterologous virus.
[0177] Recombinant TMV U1 that display the linear, neutralizing
rabbit papillomavirus epitopes CRPV L2.1; CRPV L2.2; ROPV L2.1 and
ROPV L2.2 (Embers M E et al., 2002 Journal of Virology were
constructed (Table 2). Each recombinant virus will induce
neutralizing antibodies that will protect animals against challenge
with high titer of homologous virus. However, each vaccine may not
induce sufficient titer of Nabs to neutralize the heterologous
virus.
[0178] Assembling at least two different coat proteins, each of
which displays a different peptide, on a structural RNA that
contains the TMV OAS, can make a multivalent recombinant vaccine
that will induce protective immunity against both CRPV and ROPV.
For example, the methods described in Example 9 may be used to
isolate free coat protein from recombinant TMV virions that display
the CRPV L2.1 peptide at the "GPAT" position proximal to the C
terminus of TMV U1. Likewise, free coat protein may be isolated
from recombinant TMV virions that display the ROPV L2.1 peptide at
the "GPAT" position proximal to the C terminus of TMV U1. Wild type
TMV RNA, or a recombinant RNA that contains that TMV U1 origin of
assembly sequence (OAS) may be used as the scaffold on which the
reassembled bivalent vaccine is built, according to methods
described in Example 10 and 11. Similarly, additional recombinant
U1 coat proteins that display peptides with the ability to induce
Nabs in vaccinated animals may be incorporated into the reassembly
reaction to generate a multivalent vaccine virus or virus-like
particle. Animals that are vaccinated with bivalent or multivalent
vaccines will produce antibodies that recognize the various peptide
antigens fused to the recombinant vaccine molecule; these
antibodies are capable of neutralizing both CRPV and ROPV. New
Zealand white rabbits will thus be protected against infection
against two distinct virus species after vaccination with a single
vaccine moiety.
[0179] Multifunctional Vaccine: Induction of Humoral and Cellular
Immunity
[0180] The sequences of human papillomavirus type 16 L2 that are
homologous with the CRPV L2.1, ROPV L2.1; CRPV L2.2 and CRPV L2.2
peptides are capable of binding to specific receptors, and on
binding to the cell surface are able to mediate cellular entry of
proteins fused to these sequences by receptor-mediated mechanisms
(Kawana Y et al., 2001 Journal of Virology 75: 2331-2336; Yang et
al. 2003, Journal of Virology 77:3531-3541). It is thus expected
that virions and reassembled virus-like structures that display
these sequences will be able to bind to the surface of rabbit
cells, and mediate entry of the reassembled virus structure into
the cell. The additional cell fusion function of reassembled
particles with one or more of the CRPV L2.1, ROPV L2.1; CRPV L2.2
and CRPV L2.2 peptides displayed by the assembled virus or
virus-like particles allows delivery of a functional RNA payload to
the cytoplasm of transduced cells.
[0181] To augment the protective antibody mediated immunity induced
by the L2 peptides displayed on the surface of the reassembled
viral structure, the RNA scaffold will have additional biological
activity. For example, the scaffold RNA is a recombinant RNA
molecule that encodes the Semliki forest alphavirus (SFV) RNA
sequences that are required for autonomous replication, with the
CRPV L1 gene that may be expressed under the control of the 26S RNA
promoter from SFV, and the TMV U1 OAS inserted downstream of the
CRPV L1 gene. This construct is shown in FIG. 22. Animals are
immunized with reassembled virus structures that contain the capped
SFV::CRPVL1::OAS RNA molecule as a scaffold, protected by
recombinant TMV coat proteins that display one or more of the CRPV
L2.1, ROPV L2.1; CRPV L2.2 and CRPV L2.2 epitopes assembled on the
scaffold RNA. The recombinant TMV coat proteins perform several
important functions: (1) they protect the recombinant SFV RNA
molecule from nuclease digestion; (2) they form a particulate,
quasicrystalline structure, such as are preferentially recognized
and engulfed by macrophages, dendritic cells, and other antigen
presenting cells; (3) through specific cell-binding activity, they
deliver the recombinant particles to the cytoplasm.
[0182] Once the particles are in the cytoplasm, the recombinant RNA
molecule is translated, and the RNA undergoes one or more cycles of
replication mediated by the SFV non-structural proteins (NSP)
replicase activity. The subgenomic RNA encoding the CRPV L1 RNA and
TMV OAS is transcribed and the CRPV L1 RNA translated. The
intracellularly expressed L1 protein is then available for
processing and presentation via MHC Class I to T-cells, thereby
priming a cellular immune response against the L1 protein.
Replication of the recombinant SFV RNA delivered to the cytoplasm
of transduced cells induces the innate immune response, via
pathogen surveillance signaling molecules such as the dsRNA-induced
protein kinase (PKR), resulting in secretion of inflammatory
cytokines such as interferon gamma. This augments the specific
cellular immune response induced against the L1 ORF. Thus, a broad,
robust immune response against both structural proteins (L1 and L2)
is induced. Rabbits vaccinated with these multifunctional vaccines
are protected against challenge with both CRPV and ROPV
viruses.
[0183] An alternative method to generate a functional RNA is to
insert an IRES and coding sequence for L1 into TMV RNA, which also
expresses a molecular fusion of L2 peptide epitope onto coat
protein. This method has the advantage of encapsidating the RNA in
vivo, and does not rely on reencapsidation to protect the RNA from
degradation until after cellular uptake mechanisms allow for
transcription of the gene. In a third strategy, the coat protein
also carries an N terminal cysteine for conjugation of a T-helper
epitope, a cell fusion epitope, an adjuvant, or the full-length
gene product of a non-structural protein such as E7.
[0184] Papillomavirus nonstructural proteins, including E1, E2, E4,
E6, E7 and E8 are known to mediate protective immunity, or lesion
regression and clearance in vaccinated animals (Han R et al., 2002,
Cancer Detect Prev 26:458-67; Han R et al., 2000. Journal of
Virology 74: 9712-6). In the same manner as described above, mRNAs
or autonomously replicating RNAs encoding other papillomavirus
proteins which are known to mediate protective immunity, and which
can induce regression or cure of virus infection, may be
encapsidated within virus structures (FIG. 22).
EXAMPLE 13
[0185] Multivalent Melanoma Vaccine
[0186] Melanoma antigens that stimulate good protection against
tumor growth are typically characterized as CTL epitopes. CTL
responses are highly dependant upon the context for antigen
presentation, including immunostimulation during vaccine
presentation to the immune system. This is characterized by a need
for either immunostimulatory cytokines, such as GM-CSF or
IFN.gamma., adjuvants that specifically activate T cells, such as
CpG oligo, or immunomodulatory peptides or proteins, such as Il1B
or MIP1a or IP10, to be delivered along with the vaccine, or fused
directly to the vaccine product. Melanoma CTL epitope fusions,
either molecular or chemical conjugates, are reassembled onto wild
type TMV RNA and tested for appropriate stimulation of peptide
specific CTL responses. The same melanoma CTL epitope fusions are
then reassembled onto an RNA that contains both a TMV origin of
assembly, and a mammalian translatable codon for IFNg, GM-CSF,
MIP1a, or IP 10. After vaccination with epitope TMV or epitope
TMV/IFN.gamma. (for example), the level of CTL response is measured
and compared. Translation of the functional RNA produces a protein
that results in immune activation, thereby increasing the CTL
response.
[0187] Alternatively, the RNA encodes a second full-length antigen
that primes the cellular or humoral immune response for broader
immune coverage. For example, melanoma tumors express several
specific antigens that generate both CTL and antibody responses in
challenged individuals. In murine tumors, such antigens include
p15e, tryrosinase and GP100. Several CTL epitopes, as well as
antibody stimulating domains, exist for each tumor specific
antigen. Defined CTL epitopes, e.g. the p15e CTL epitope, are fused
to the surface of TMV, and the encapsidated RNA encodes the
entirety of gp100, or tyrosinase coding sequences. CTL reactivity
to the p15e epitopes is measured, and further cellular or humoral
reactivity to the gp100 or tyrosinase epitopes encoded by the RNA
demonstrate RNA expression and activity of the resulting gene
product.
[0188] Cellular or humoral assays indicate the level at which the
vaccine is stimulating an immune response. Another way to show
immune reactivity is by challenging animals with the tumor encoding
those antigens and monitoring the rate of tumor growth, or the
morbidity that that tumor causes. Such models exist for melanoma,
and are widely used to prototype the effectiveness of melanoma
vaccines. The B16 melanoma model expresses p15e, tryosinase, and
gp100, and requires an effective CTL response after vaccination to
reduce or eliminate the rate of tumor growth. Animals vaccinated
with CTL epitope fusion vaccines are challenged with tumor, and an
effective immune response will decrease the rate of tumor growth or
morbidity compared to controls. If either an immunostimulatory RNA
or full-length gene product encapsidated by a TMV coat or a TMV
coat fusion is effective, then the rate of tumor growth should
decrease compared to a protein vaccine alone, or the overall
morbidity should decrease. These finding will corroborate the
cellular and humoral response data, considering that these
responses are essential to reducing or eliminating tumor.
[0189] As described above for papillomavirus applications, the
functional encapsidated RNA can be self-replicating, such as an
engineered alphavirus containing a TMV origin of assembly, or can
contain an IRES, to stimulate translation from an internal site in
TMV RNA (see FIG. 22 for examples). Also as described above, the
combination of epitope fusions can be made either by molecular or
chemical conjugation methods, and need not be limited to peptides.
DNA sequences and whole proteins may also be added to reassembled
TMV, or to TMV coat fusions that also encode an N-terminal
cysteine.
EXAMPLE 14
[0190] Immunogenicity of TMV Coat Protein Fusions Immunogenicity to
V5 and Myc U1 Coat Fusions: Responses to Antibody Epitopes
[0191] To verify that coat fusion peptides can stimulate
appropriate immunity, we tested myc and V5 U1 peptide fusions, with
known antibody binding properties, as vaccines in mice, and then
looked for anti-myc and anti-V5 antibody responses. V5 and myc TMV
U1 coat fusions were prepared by extraction methods, optimized for
the recovery of the fusion of interest. Material was quantitated by
the BCA protein assay, evaluated for peptide integrity by MALDI-TOF
and for purity by SDS-PAGE. 10 g of TMV protein was then injected
into Balb-C mice three times, every two weeks. After the second and
third vaccines, animals were bled and sera was collected and
analyzed for peptide specific reactivity by ELISA. The results of
serum titers after the third vaccination are show in FIG. 23, and
are boosted from levels observed after two vaccines.
[0192] Results from this study indicate that at all three
positions, V5 and myc peptide fusions to TMV U1 coat can elicit the
appropriate anti-peptide antibody response even when given without
adjuvant. Varied response levels in individual mice are typical of
subunit vaccines, and have been observed for other antigen
vaccines. Overall, the average response in each vaccine group
tested was not significantly different by position of the peptide
fusion, even though the maximum response levels differed
significantly in each group. Interestingly, responses to the TMV
carrier were generally lower in magnitude than the anti-peptide
response (data not shown). Of note, these vaccines were
administered without adjuvant, and the high levels of responses in
each group show that the viral carrier can provide humoral immune
stimulation that is antigen specific.
[0193] CTL Response Assay Development for Ova Peptide U1 Coat
Fusions
[0194] In addition to testing the ability of antibody-target
peptides to stimulate appropriate humoral responses in vaccinated
mice, we also tested the ability of a CTL epitope, derived from the
chicken ovalbumin protein, to stimulate appropriate cellular
immunity in appropriately MHC restricted mice. 20 g Ova-N or Ova-G
TMV fusions were administered 4 times every two weeks without
adjuvant to mice, and then spleens were harvested from vaccinated
animals five days after the final vaccine. Cells were isolated,
cultured with either media or media plus ova peptide for 5 hours in
the presence of the Golgi transport inhibitor Brefeldin A, and then
cells were fluorescently stained with FITC conjugated antibodies
against surface expression of CD4 and CD8 T cell receptors, in
conjunction with PE staining of the intracellular cytokines IFN
gamma or TNF alpha. Stimulation with ova peptide should upregulate
these cytokines in T cells that are specific for the peptide, and
be measured by an increase in cell number by Fluorescence Activated
Cell Sorting (FACS). 5.times.10.sup.5 events were collected, about
20% of which are T cells.
[0195] Both CD4 and CD8 cells were monitored for increased
intracellular expression of IFN .gamma. (gamma) and TNF .alpha.
(alpha). As shown in FIG. 24, after a five-hour peptide
stimulation, intracellular IFN gamma levels rose in CD4 positive
cells (from 0.08% of gated events to 0.17% or 10 to 22 cells), and
in CD8 positive cells (from 0.08% of gated events to 0.13% or 11 to
17 cells; data not shown), which represent statistically
significant increases. TNF alpha levels rose significantly in
CD4+cells (0.08 to 0.13%) but did not change in CD8+ cells (0.12%
to 0.10%; data not shown).
[0196] Considering that no adjuvant was administered with the
vaccine, these modest increases in cytokine levels suggest that the
vaccine is stimulating an appropriate cellular response.
Administration of an adjuvant with the vaccine, or the fusion of
immunostimulatory peptides to the TMV vaccine, is expected to
increase the percentage of activated T cells. For example, the T
cell activating adjuvant, single stranded CpG DNA oligo 1758,
specifically augments cellular responses in ova and other CTL
systems. For our system the nucleotides are either mixed with the
vaccine, or fused directly to TMV U1. In other systems, the IL1b
peptide has been shown to augment both antibody and CTL responses
but only if the IL1b peptide is physically linked to the ova
peptide vaccine, such as in a multivalent vaccine.
Sequence CWU 1
1
22 1 15 PRT Simian Virus 5 1 Gly Lys Pro Ile Pro Asn Pro Leu Leu
Gly Leu Asp Ser Thr Lys 1 5 10 15 2 10 PRT Influenza virus 2 Tyr
Pro Tyr Asp Val Pro Asp Tyr Ala Lys 1 5 10 3 11 PRT human antibody
3 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Lys 1 5 10 4 19 PRT
cottontail rabit papillomavirus 4 Val Gly Pro Leu Asp Ile Val Pro
Glu Val Ala Asp Pro Gly Gly Pro 1 5 10 15 Thr Leu Val 5 18 PRT
cottontail rabit papillomavirus 5 Pro Gly Gly Pro Thr Leu Val Ser
Leu His Glu Leu Pro Ala Glu Thr 1 5 10 15 Pro Tyr 6 19 PRT rabbit
oral papillomavirus 6 Val Gly Pro Leu Glu Val Ile Pro Glu Ala Val
Asp Pro Ala Gly Ser 1 5 10 15 Ser Ile Val 7 18 PRT rabbit oral
papillomavirus 7 Pro Ala Gly Ser Ser Ile Val Pro Leu Glu Glu Tyr
Pro Ala Glu Ile 1 5 10 15 Pro Thr 8 9 PRT Human papillomavirus 8
Ala Ala Leu Gln Ala Ile Glu Leu Met 1 5 9 9 PRT mouse tyrosinase 9
Ser Val Tyr Asp Phe Phe Val Trp Leu 1 5 10 8 PRT mouse 10 Lys Ser
Pro Trp Phe Thr Thr Leu 1 5 11 8 PRT chicken 11 Ser Ile Ile Asn Phe
Glu Lys Leu 1 5 12 7 PRT Human immunodeficiency virus 12 Glu Leu
Asp Lys Trp Ala Ser 1 5 13 16 PRT respiratory syncytial virus 13
Cys Glu Tyr Asn Val Phe His Asn Lys Thr Phe Glu Leu Pro Arg Ala 1 5
10 15 14 17 PRT tetanus 14 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile
Gly Ile Thr Glu Leu Lys 1 5 10 15 Lys 15 9 PRT Human IL1beta 15 Val
Gln Gly Glu Glu Ser Asn Asp Lys 1 5 16 19 PRT Measles virus 16 Phe
Ala Gly Val Val Leu Ala Gly Ala Ala Leu Gly Val Ala Thr Ala 1 5 10
15 Ala Gln Ile 17 7 PRT human integrin 17 Ser Gly Arg Gly Asp Ser
Gly 1 5 18 6 PRT human laminin 18 Gly Tyr Ile Gly Ser Arg 1 5 19
489 DNA Tobacco mosaic virus 19 atgggatgtg gatcttacag tatcactact
ccatctcagt tcgtgttctt gtcatcagcg 60 tgggccgacc caatagagtt
aattaattta tgtactaatg ccttaggaaa tcagtttcaa 120 acacaacaag
ctcgaactgt cgttcaaaga caattcagtg aggtgtggaa accttcacca 180
caagtaactg ttaggttccc tgacagtgac tttaaggtgt acaggtacaa tgcggtatta
240 gacccgctag tcacagcact gttaggtgca ttcgacacta gaaatagaat
aatagaagtt 300 gaaaatcagg cgaaccccac gactgccgaa acgttagatg
ctactcgtag agtagacgac 360 gcaacggtgg ccataaggag cgcgataaat
aatttaatag tagaattgat cagaggaacc 420 ggatcttata atcggagctc
tttcgagagc tcttctggtt tggtttggac ctctggtcct 480 gcaacttga 489 20
489 DNA Tobacco mosaic virus 20 atgggaaaag gatcttacag tatcactact
ccatctcagt tcgtgttctt gtcatcagcg 60 tgggccgacc caatagagtt
aattaattta tgtactaatg ccttaggaaa tcagtttcaa 120 acacaacaag
ctcgaactgt cgttcaaaga caattcagtg aggtgtggaa accttcacca 180
caagtaactg ttaggttccc tgacagtgac tttaaggtgt acaggtacaa tgcggtatta
240 gacccgctag tcacagcact gttaggtgca ttcgacacta gaaatagaat
aatagaagtt 300 gaaaatcagg cgaaccccac gactgccgaa acgttagatg
ctactcgtag agtagacgac 360 gcaacggtgg ccataaggag cgcgataaat
aatttaatag tagaattgat cagaggaacc 420 ggatcttata atcggagctc
tttcgagagc tcttctggtt tggtttggac ctctggtcct 480 gcaacttga 489 21 15
PRT Artificial Sequence Created from Seq ID No. 10 inserted into
TMV 21 Ala Met Lys Ser Pro Trp Phe Thr Thr Leu Ala Gly Pro Ala Thr
1 5 10 15 22 17 PRT Artificial Sequence Created from Seq ID No 10
inserted into TMV 22 Ala Met Asp Glu Lys Ser Pro Trp Phe Thr Thr
Leu Ala Gly Pro Ala 1 5 10 15 Thr
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