U.S. patent application number 17/460641 was filed with the patent office on 2021-12-23 for recombinant modified vaccinia virus ankara (mva) foot and mouth disease virus (fmdv) vaccine.
This patent application is currently assigned to Bavarian Nordic A/S. The applicant listed for this patent is Bavarian Nordic A/S. Invention is credited to Markus Kalla, Robin Steigerwald.
Application Number | 20210393766 17/460641 |
Document ID | / |
Family ID | 1000005826062 |
Filed Date | 2021-12-23 |
United States Patent
Application |
20210393766 |
Kind Code |
A1 |
Steigerwald; Robin ; et
al. |
December 23, 2021 |
Recombinant Modified Vaccinia Virus Ankara (MVA) Foot and Mouth
Disease Virus (FMDV) Vaccine
Abstract
The present invention relates to modified poxviral vectors and
to methods of making and using the same. In particular, the
invention relates to recombinant modified vaccinia virus
Ankara-based (MVA-based) vaccine against FMDV infection and to
related products, methods and uses. Specifically, the present
invention relates to genetically engineered (recombinant) MVA
vectors comprising at least one heterologous nucleotide sequence
encoding an antigenic determinant of a FMDV protein. The invention
also relates to products, methods and uses thereof, e.g., suitable
to induce a protective immune response in a subject.
Inventors: |
Steigerwald; Robin; (Munich,
DE) ; Kalla; Markus; (Penzberg, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bavarian Nordic A/S |
Hellerup |
|
DK |
|
|
Assignee: |
Bavarian Nordic A/S
Hellerup
DK
|
Family ID: |
1000005826062 |
Appl. No.: |
17/460641 |
Filed: |
August 30, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
15737028 |
Dec 15, 2017 |
11103570 |
|
|
PCT/EP2016/063691 |
Jun 15, 2016 |
|
|
|
17460641 |
|
|
|
|
62175738 |
Jun 15, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2710/24034
20130101; A61K 2039/552 20130101; C12N 2710/24143 20130101; C12N
2770/32134 20130101; A61K 9/0019 20130101; A61K 39/12 20130101 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61K 9/00 20060101 A61K009/00 |
Goverment Interests
[0001] This invention was made with Government support under
HSHQDC-12-C-00051 awarded by U.S. Dept. of Homeland Security Office
of Procurement and Operations. The Government has certain rights in
the invention.
Claims
1. A vaccine that elicits a protective immune response to FMDV when
administered to cattle and that comprises: (a) a recombinant MVA
comprising: (i) a first transcriptional unit that comprises a
nucleotide sequence encoding the foot-and-mouth disease virus
(FMDV) P1 region operably liked to a promoter; and (ii) a second
transcriptional unit that comprises a nucleotide sequence encoding
FMDC 3C protease, wherein the 3C protease comprises the mutation
C142T; and (b) a pharmaceutical or veterinary acceptable carrier,
excipient or vehicle; wherein said protective immune response
prevents the development of FMDV-caused lesions in said cattle.
Description
FIELD OF THE INVENTION
[0002] The present invention relates to an improved FMDV vaccine
comprising a recombinant modified vaccinia virus Ankara-based
(MVA-based) vaccine against FMDV infection and to related products,
methods and uses. Specifically, the present invention relates to
genetically engineered (recombinant) MVA vectors comprising a
heterologous nucleotide sequence encoding an antigenic determinant
of a FMDV protein. The invention also relates to products, methods
and uses thereof, e.g., suitable to induce a protective immune
response in a subject.
BACKGROUND OF THE INVENTION
[0003] Foot-and-mouth disease (FMD) is one of the most virulent and
contagious diseases affecting farm animals. This disease is endemic
in numerous countries in the world, especially in Africa, Asia and
South America. In addition, epidemic outbreaks can occur
periodically. The presence of this disease in a country may have
very severe economic consequences resulting from loss of
productivity, loss of weight and milk production in infected herds,
and from trade embargoes imposed on these countries. The measures
taken against this disease consist of strict application of import
restrictions, hygiene controls and quarantine, slaughtering sick
animals and vaccination programs using inactivated vaccines, either
as a preventive measure at the national or regional level, or
periodically when an epidemic outbreak occurs.
[0004] FMD is characterized by its short incubation period, its
highly contagious nature, the formation of ulcers in the mouth and
on the feet and sometimes, the death of young animals. FMD affects
a number of animal species, in particular cattle, pigs, sheep and
goats. The agent responsible for this disease is a ribonucleic acid
(RNA) virus belonging to the Aphthovirus genus of the
Picornaviridae family (Cooper et al., Intervirology, 1978, 10,
165-180). At present, at least seven types of foot-and-mouth
disease virus (FMDV) are known: the European types (A, O and C),
the African types (SATI, SAT2 and SAT3) and an Asiatic type (Asia
1). Numerous sub-types have also been distinguished (Kleid et al.
Science (1981), 214, 1 125-1 129).
[0005] FMDV is a naked icosahedral virus of about 25 nm in
diameter, containing a single-stranded RNA molecule consisting of
about 8500 nucleotides, with a positive polarity. This RNA molecule
comprises a single open reading frame (ORF), encoding a single
polyprotein containing, inter alia, the capsid precursor also known
as protein P1 or P88. The protein P1 is myristylated at its
amino-terminal end. During the maturation process, the protein P1
is cleaved by the protease 3C into three proteins known as VP0, VP1
and VP3 (or 1AB, 1D and 1C respectively; Belsham G. J., Progress in
Biophysics and Molecular Biology, 1993, 60, 241-261). In the
virion, the protein VP0 is then cleaved into two proteins, VP4 and
VP2 (or 1A and 1B respectively). The mechanism for the conversion
of the proteins VP0 into VP2 and VP4, and for the formation of
mature virions is not known. The proteins VP1, VP2 and VP3 have a
molecular weight of about 26,000 Da, while the protein VP4 is
smaller at about 8,000 Da.
[0006] The simple combination of the capsid proteins forms the
protomer or 5S molecule, which is the elementary constituent of the
FMDV capsid. This protomer is then complexed into a pentamer to
form the 12S molecule. The virion results from the encapsidation of
a genomic RNA molecule by assembly of twelve 12S pentamers, thus
constituting the 146S particles. The viral capsid may also be
formed without the presence of an RNA molecule inside it
(hereinafter "empty capsid"). The empty capsid is also designated
as particle 70S. The formation of empty capsids may occur naturally
during viral replication or may be produced artificially by
chemical treatment.
[0007] Many hypotheses, research routes, and proposals have been
developed in an attempt to design effective vaccines against FMD.
Currently, the only vaccines on the market comprise inactivated
virus. Concerns about safety of the FMDV vaccine exist, as
outbreaks of FMD in Europe have been associated with shortcomings
in vaccine manufacture (King, A. M. Q. et al, (1981) Nature 293:
479-480). The inactivated vaccines do not confer long-term
immunity, thus requiring booster injections given every year, or
more often in the event of epidemic outbreaks. In addition, there
are risks linked to incomplete inactivation and/or to the escape of
virus during the production of inactivated vaccines (King, A. M.
Q., ibid). A goal in the art has been to construct conformationally
correct immunogens lacking the infective FMDV genome to make
effective and safe vaccines.
[0008] Vaccinia virus has been used successfully to immunize
against smallpox, culminating in the worldwide eradication of
smallpox in 1980. Thus, a new role for poxviruses became important,
that of a genetically engineered vector for the expression of
foreign genes (Panicali and Paoletti, 1982; Paoletti et al, 1984).
Genes encoding heterologous antigens have been expressed in
vaccinia, often resulting in protective immunity against challenge
by the corresponding pathogen (reviewed in Tartaglia et al., 1990).
A highly attenuated strain of vaccines, designated MVA, has also
been used as a vector for poxvirus-based vaccines. Use of MVA is
described in U.S. Pat. No. 5,185,146.
[0009] The excellent safety profile of MVA, because of its
replication deficiency in human cells, has been proven in many
clinical trials, including vaccination of immune-compromised
individuals, and during the smallpox eradication campaign in the
1970s, when 120,000 people were vaccinated with MVA (A. Mayr et
al., "The smallpox vaccination strain MVA: marker, genetic
structure, experience gained with the parenteral vaccination and
behavior in organisms with a debilitated defense mechanism,"
Zentralbl. Bakteriol. B 167(5-6):375-390 (1978)). Since then many
different recombinant MVA vaccines have been generated and tested
for the ability to immunize animals and humans against infectious
(e.g., HIV, malaria) and non-infectious (e.g., prostate cancer)
diseases. Its proven safety and good immunogenicity thus make MVA a
prime candidate for a T- and B-cell-inducing vaccine vector.
[0010] Additional vaccine vector systems involve the use of avipox
viruses, which are naturally host-restricted poxviruses. Both
fowlpoxvirus (FPV; Taylor et al. 1988a, b) and canarypoxvirus (CPV;
Taylor et al., 1991 & 1992) have been engineered to express
foreign gene products. Fowlpox virus (FPV) is the prototypic virus
of the Avipox genus of the Poxvirus family. The virus causes an
economically important disease of poultry that has been well
controlled since the 1920's by the use of live attenuated vaccines.
Replication of the avipox viruses is limited to avian species
(Matthews, 1982) and there are no reports in the literature of
avipox virus causing a productive infection in any non-avian
species including man. This host restriction provides an inherent
safety barrier against transmission of the virus to other species
and makes the use of avipox virus based vaccine vectors in
veterinary and human applications an attractive proposition.
[0011] Other attenuated poxvirus vectors have been prepared by
genetic modifications of wild type strains of virus. The NYVAC
vector, derived by deletion of specific virulence and host-range
genes from the Copenhagen strain of vaccinia (Tartaglia et al.,
1992) has proven useful as a recombinant vector in eliciting a
protective immune response against an expressed foreign antigen.
Another engineered poxvirus vector is ALVAC, derived from canarypox
virus (see U.S. Pat. No. 5,756, 103). ALVAC does not productively
replicate in non-avian hosts, a characteristic thought to improve
its safety profile (Taylor et al., 1991 & 1992). ALVAC was
deposited under the terms of the Budapest Treaty with the American
Type Culture Collection under accession number VR-2547. Yet another
engineered poxvirus vector is TROVAC, derived from fowlpox virus
(see U.S. Pat. No. 5,766,599).
[0012] Recombinant poxviruses can be constructed in two steps known
in the art and analogous to the methods for creating synthetic
recombinants of poxviruses such as the vaccinia virus and avipox
virus described in U.S. Pat. Nos. 4,769,330; 4,722,848; 4,603,112;
5,110,587; 5,174,993; 5,494,807; and 5,505,941, the disclosures of
which are incorporated herein by reference. It can thus be
appreciated that provision of an FMDV recombinant poxvirus, and of
compositions and products therefrom, particularly ALVAC or
TROVAC-based FMDV recombinants and compositions and products
therefrom, especially such recombinants containing the P1 genes
and/or 3C protease gene of FMDV, and compositions and products
therefrom, would be a highly desirable advance over the current
state of technology.
[0013] Considering the susceptibility of animals (including humans,
albeit rarely), to FMDV, a method of preventing FMDV infection and
protecting animals is essential. Accordingly, there is a need for
an effective vaccine against FMDV.
BRIEF SUMMARY OF THE INVENTION
[0014] It is determined in the present invention that various
prime-boost combinations of replication deficient and/or
replication incompetent vectors generate effective immune
protection against FMDV infection.
[0015] Accordingly, one aspect of the present invention provides a
recombinant MVA comprising a nucleotide sequence encoding an
antigenic determinant of at least one foot-and-mouth disease virus
(FMDV) antigens. In a preferred embodiment, the MVA is MVA-BN.
[0016] Advantageously, the FMDV antigen(s) can be VP1, VP2, VP3,
VP4, 2A, 2B and 3C. Advantageously, the nucleic acid molecule
encoding one or more foot-and-mouth disease virus (FMDV) antigen(s)
is a cDNA encoding FMDV P1 region and a cDNA encoding FMDV 3C
protease of FMDV. In one embodiment, the FMDV antigens are operably
linked to a promoter sequence, e.g. the PrMVA095R or PO 3.5-long
promoter.
[0017] A further aspect of the invention relates to a composition
comprising the MVA and a pharmaceutical or veterinary acceptable
carrier, excipients, or vehicle.
[0018] A further aspect of the invention relates to a method of
eliciting an immune response to FMDV in a subject, comprising
administering the MVA of the present invention to the subject.
[0019] A further aspect of the invention relates to a method of
treatment and/or prevention of a FMDV caused disease in a
subject.
[0020] In a further aspect the invention relates to a vaccine and
cell comprising the MVA of the present invention.
[0021] A further aspect of the invention relates to a kit
comprising the recombinant MVA of the invention and/or the
composition of the invention in a first vial or container for a
first administration (priming) and in a second vial or container
for a second administration (boosting).
[0022] In yet another aspect of the present invention, a method of
producing a recombinant MVA of the invention or the antigenic
determinant expressed from the genome of said MVA.
[0023] These and other objects of the invention will be described
in further detail in connection with the detailed description of
the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
[0025] FIG. 1 shows schematic presentation of two exemplary
constructs: #7B (MVA-mBN360B) and #8A MVA-mBN361A).
[0026] FIG. 2 shows expression and processing of the P1-2AB antigen
with 3C protease activity.
[0027] FIG. 3 shows co-IP of capsid material with correct
conformation with a conformation specific anti-P1 antibody and
detection of the co-precipitated antigens VP3 and VP0.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The present inventors have determined that a vaccine
comprising a recombinant modified vaccinia virus Ankara (MVA)
comprising a heterologous nucleotide sequence encoding an antigenic
determinant of a FMDV provides a FMDV vaccine capable of inducing
both cellular and humoral responses sufficient to confer protective
immunity to FMDV.
[0029] In one aspect, the present invention relates to a modified
recombinant MVA virus expressing at least one nucleic acid
sequences encoding for one or more FMDV antigens. The viral vector
according to the present invention is preferably an MVA virus, such
as MVA-BN. The modified recombinant vector comprises a heterologous
nucleic acid sequence, which encodes an antigenic protein, e.g.,
derived from FMDV ORFs that are encoded by the P1 (comprising VP1,
VP2, VP3, VP4, and 2A), 2B, and/or 3C regions.
[0030] In another aspect, the present invention relates to a
modified recombinant MVA virus that includes, in a non-essential
region of the virus genome, at least one heterologous nucleic acid
sequence that encodes one or more antigens from FMDV, such as gene
products of the P1 gene (comprising VP1, VP2, VP3, VP4, 2A), 2B,
and/or 3C.
[0031] In a still further aspect, the present invention relates to
methods of eliciting an immune response to FMDV in a subject,
comprising administering the recombinant MVA vector of the present
invention. The present invention also relates to methods of
eliciting an immune response to FMDV in a subject, comprising
administering the recombinant MVA virus of the present
invention.
[0032] In one aspect, the present invention relates to recombinant
MVA viruses containing at least one nucleic acid sequence
expressing one or more antigens from FMDV, advantageously in a
non-essential region of the MVA virus genome. The MVA virus can be
an attenuated MVA virus such as MVA-BN.
[0033] According to the present invention, the recombinant MVA
viral vectors express at least one nucleic acid sequence encoding
one or more FMDV antigens. In particular, any or all genes or open
reading frames (ORFs) encoding FMDV antigens can be isolated,
characterized and inserted into MVA recombinants. The resulting
recombinant MVA virus is used to infect an animal. Expression in
the animal of FMDV antigens results in an immune response in the
animal to FMDV. Thus, the recombinant MVA virus of the present
invention may be used in an immunological composition or vaccine to
provide a means to induce an immune response, which may, but need
not be, protective. The molecular biology techniques used are
described by Sambrook et ah (1969).
[0034] The invention also contemplates FMDV antigens that can be
delivered as a naked DNA plasmid or vector, or DNA vaccine or
immunological or immunogenic compositions comprising nucleic acid
molecules encoding and expressing in vivo an FMDV antigen(s).
[0035] The FMDV antigen of interest can be obtained from FMDV or
can be obtained from in vitro and/or in vivo recombinant expression
of FMDV gene(s) or portions thereof. The FMDV antigen of interest
can also be provided using synthetic FMDV sequences. The FMDV
antigen of interest can be, but are not limited to: U, Lab, P1-2 A
(comprising VP1, VP2, VP3, VP4, and 2A); P2 (comprising 2B and 2C),
and P3 (comprising 3A, 3B, VPg, 3C, and 3D), or portions thereof.
In a preferred embodiment, the FMDV antigens are P1 and 3C. In a
particularly preferred embodiment, the FMDV antigens are P1-2A or
P1-2A, 2B. Reference is made herein to U.S. patent application Ser.
No. 10/327,481, issued as U.S. Pat. No. 7,531,182,relating to
isolation of FMDV genome sequences, the contents of which are
incorporated by reference, herein. An exemplary P1 amino acid
sequence of FMDV strain A10 is set forth in SEQ ID NO:6, and an
exemplary 3C amino acid sequence of FMDV strain A10 is set forth in
SEQ ID NO:7.
[0036] Reference will now be made in detail to exemplary
embodiments of the invention, examples of which are illustrated in
the accompanying drawings.
[0037] In one aspect, the present invention provides a recombinant
modified vaccinia virus Ankara (MVA) comprising a nucleotide
sequence encoding an antigenic determinant of a FMDV. In another
aspect, the present invention provides a recombinant MVA vector
comprising a heterologous nucleotide sequence encoding an antigenic
determinant of a FMDV.
[0038] MVA has been generated by more than 570 serial passages on
chicken embryo fibroblasts of the dermal vaccinia strain Ankara
[Chorioallantois vaccinia virus Ankara virus, CVA; for review see
Mayr et al. (1975), Infection 3, 6-14] that was maintained in the
Vaccination Institute, Ankara, Turkey for many years and used as
the basis for vaccination of humans. However, due to the often
severe post-vaccination complications associated with vaccinia
viruses, there were several attempts to generate a more attenuated,
safer smallpox vaccine.
[0039] During the period of 1960 to 1974, Prof. Anton Mayr
succeeded in attenuating CVA by over 570 continuous passages in CEF
cells [Mayr et al. (1975)]. It was shown in a variety of animal
models that the resulting MVA was avirulent [Mayr, A. & Danner,
K. (1978), Dev. Biol. Stand. 41: 225-234]. As part of the early
development of MVA as a pre-smallpox vaccine, there were clinical
trials using MVA-517 in combination with Lister Elstree [Stickl
(1974), Prev. Med. 3: 97-101; Stickl Hochstein-Mintzel (1971),
Munch. Med. Wochenschr. 113: 1149-1153] in subjects at risk for
adverse reactions from vaccinia. In 1976, MVA derived from MVA-571
seed stock (corresponding to the 571.sup.st passage) was registered
in Germany as the primer vaccine in a two-stage parenteral smallpox
vaccination program. Subsequently, MVA-572 was used in
approximately 120,000 Caucasian individuals, the majority children
between 1 and 3 years of age, with no reported severe side effects,
even though many of the subjects were among the population with
high risk of complications associated with vaccinia (Mayr et al.
(1978), Zentralbl. Bacteriol. (B) 167:375-390). MVA-572 was
deposited at the European Collection of Animal Cell Cultures as
ECACC V94012707.
[0040] As a result of the passaging used to attenuate MVA, there
are a number of different strains or isolates, depending on the
number of passages conducted in CEF cells. For example, MVA-572 was
used in a small dose as a pre-vaccine in Germany during the
smallpox eradication program, and MVA-575 was extensively used as a
veterinary vaccine. MVA as well as MVA-BN lacks approximately 15%
(31 kb from six regions) of the genome compared with ancestral CVA
virus. The deletions affect a number of virulence and host range
genes, as well as the gene for Type A inclusion bodies. MVA-575 was
deposited on Dec. 7, 2000, at the European Collection of Animal
Cell Cultures (ECACC) under Accession No. V00120707. The attenuated
CVA-virus MVA (Modified Vaccinia Virus Ankara) was obtained by
serial propagation (more than 570 passages) of the CVA on primary
chicken embryo fibroblasts.
[0041] Even though Mayr et al. demonstrated during the 1970s that
MVA is highly attenuated and avirulent in humans and mammals,
certain investigators have reported that MVA is not fully
attenuated in mammalian and human cell lines since residual
replication might occur in these cells [Blanchard et al. (1998), J.
Gen. Virol. 79:1159-1167; Carroll & Moss (1997), Virology
238:198-211; U.S. Pat. No. 5,185,146; Ambrosini et al. (1999), J.
Neurosci. Res. 55: 569]. It is assumed that the results reported in
these publications have been obtained with various known strains of
MVA, since the viruses used essentially differ in their properties,
particularly in their growth behaviour in various cell lines. Such
residual replication is undesirable for various reasons, including
safety concerns in connection with use in humans.
[0042] Strains of MVA having enhanced safety profiles for the
development of safer products, such as vaccines or pharmaceuticals,
have been developed by Bavarian Nordic: MVA was further passaged by
Bavarian Nordic and is designated MVA-BN. A representative sample
of MVA-BN was deposited on Aug. 30, 2000 at the European Collection
of Cell Cultures (ECACC), Health Protection Agency, Porton Down,
Salisbury, Wiltshire SP4 0JG, United Kingdom, under Accession No.
V00083008. MVA-BN is further described in WO 02/42480 (US
2003/0206926) and WO 03/048184 (US 2006/0159699), both of which are
incorporated by reference herein.
[0043] MVA-BN can attach to and enter human cells where
virally-encoded genes are expressed very efficiently. MVA-BN is
strongly adapted to primary chicken embryo fibroblast (CEF) cells
and does not replicate in human cells. In human cells, viral genes
are expressed, and no infectious virus is produced. MVA-BN is
classified as Biosafety Level 1 organism according to the Centers
for Disease Control and Prevention in the United States.
Preparations of MVA-BN and derivatives have been administered to
many types of animals, and to more than 2000 human subjects,
including immune-deficient individuals. All vaccinations have
proven to be generally safe and well tolerated. Despite its high
attenuation and reduced virulence, in preclinical studies MVA-BN
has been shown to elicit both humoral and cellular immune responses
to vaccinia and to heterologous gene products encoded by genes
cloned into the MVA genome [E. Harrer et al. (2005), Antivir. Ther.
10(2):285-300; A. Cosma et al. (2003), Vaccine 22(1):21-9; M. Di
Nicola et al. (2003), Hum. Gene Ther. 14(14):1347-1360; M. Di
Nicola et al. (2004), Clin. Cancer Res., 10(16):5381-5390].
[0044] "Derivatives" or "variants" of MVA refer to viruses
exhibiting essentially the same replication characteristics as MVA
as described herein, but exhibiting differences in one or more
parts of their genomes. MVA-BN as well as a derivative or variant
of MVA-BN fails to reproductively replicate in vivo in humans and
mice, even in severely immune suppressed mice. More specifically,
MVA-BN or a derivative or variant of MVA-BN has preferably also the
capability of reproductive replication in chicken embryo
fibroblasts (CEF), but no capability of reproductive replication in
the human keratinocyte cell line HaCat [Boukamp et al (1988), J.
Cell Biol. 106: 761-771], the human bone osteosarcoma cell line
143B (ECACC Deposit No. 91112502), the human embryo kidney cell
line 293 (ECACC Deposit No. 85120602), and the human cervix
adenocarcinoma cell line HeLa (ATCC Deposit No. CCL-2).
Additionally, a derivative or variant of MVA-BN has a virus
amplification ratio at least two fold less, more preferably
three-fold less than MVA-575 in Hela cells and HaCaT cell lines.
Tests and assay for these properties of MVA variants are described
in WO 02/42480 (US 2003/0206926) and WO 03/048184 (US
2006/0159699).
[0045] The term "not capable of reproductive replication" or "no
capability of reproductive replication" is, for example, described
in WO 02/42480, which also teaches how to obtain MVA having the
desired properties as mentioned above. The term applies to a virus
that has a virus amplification ratio at 4 days after infection of
less than 1 using the assays described in WO 02/42480 or in U.S.
Pat. No. 6,761,893.
[0046] The term "fails to reproductively replicate" refers to a
virus that has a virus amplification ratio at 4 days after
infection of less than 1. Assays described in WO 02/42480 or in
U.S. Pat. No. 6,761,893 are applicable for the determination of the
virus amplification ratio.
[0047] The amplification or replication of a virus is normally
expressed as the ratio of virus produced from an infected cell
(output) to the amount originally used to infect the cell in the
first place (input) referred to as the "amplification ratio". An
amplification ratio of "1" defines an amplification status where
the amount of virus produced from the infected cells is the same as
the amount initially used to infect the cells, meaning that the
infected cells are permissive for virus infection and reproduction.
In contrast, an amplification ratio of less than 1, i.e., a
decrease in output compared to the input level, indicates a lack of
reproductive replication and therefore attenuation of the
virus.
[0048] The advantages of MVA-based vaccine include their safety
profile as well as availability for large scale vaccine production.
Preclinical tests have revealed that MVA-BN demonstrates superior
attenuation and efficacy compared to other MVA strains (WO
02/42480). An additional property of MVA-BN strains is the ability
to induce substantially the same level of immunity in vaccinia
virus prime/vaccinia virus boost regimes when compared to
DNA-prime/vaccinia virus boost regimes.
[0049] The recombinant MVA-BN viruses, the most preferred
embodiment herein, are considered to be safe because of their
distinct replication deficiency in mammalian cells and their
well-established avirulence. Furthermore, in addition to its
efficacy, the feasibility of industrial scale manufacturing can be
beneficial. Additionally, MVA-based vaccines can deliver multiple
heterologous antigens and allow for simultaneous induction of
humoral and cellular immunity.
[0050] MVA vectors useful for the present invention can be prepared
using methods known in the art, such as those described in
WO/2002/042480 and WO/2002/24224, both of which are incorporated by
reference herein.
[0051] In another aspect, an MVA viral strain suitable for
generating the recombinant virus may be strain MVA-572, MVA-575 or
any similarly attenuated MVA strain. Also suitable may be a mutant
MVA, such as the deleted chorioallantois vaccinia virus Ankara
(dCVA). A dCVA comprises del I, del II, del III, del IV, del V, and
del VI deletion sites of the MVA genome. The sites are particularly
useful for the insertion of multiple heterologous sequences. The
dCVA can reproductively replicate (with an amplification ratio of
greater than 10) in a human cell line (such as human 293, 143B, and
MRC-5 cell lines), which then enable the optimization by further
mutation useful for a virus-based vaccination strategy (see WO
2011/092029).
[0052] Antigenic Determinants
[0053] Any DNA of interest or foreign gene can be inserted as a
heterologous nucleotide sequence encoding an antigenic determinant
into the viral vectors described herein. Foreign genes for
insertion into the genome of a virus in expressible form can be
obtained using conventional techniques for isolating a desired
gene. For organisms which contain a DNA genome, the genes encoding
an antigen of interest can be isolated from the genomic DNA; for
organisms with RNA genomes, the desired gene can be isolated from
cDNA copies of the genome. The antigenic determinant can also be
encoded by a recombinant DNA that is modified based on a naturally
occurring sequence, e.g., to optimize the antigenic response, gene
expression, etc.
[0054] The term "antigenic determinant" refers to any molecule that
stimulates a host's immune system to make an antigen-specific
immune response, whether a cellular response or a humoral antibody
response. Antigenic determinants may include proteins,
polypeptides, antigenic protein fragments, antigens, and epitopes
which still elicit an immune response in a host and form part of an
antigen, homologues or variants of proteins, polypeptides, and
antigenic protein fragments, antigens and epitopes including, for
example, glycosylated proteins, polypeptides, antigenic protein
fragments, antigens and epitopes, and nucleotide sequences encoding
such molecules. Thus, proteins, polypeptides, antigenic protein
fragments, antigens and epitopes are not limited to particular
native nucleotide or amino acid sequences but encompass sequences
identical to the native sequence as well as modifications to the
native sequence, such as deletions, additions, insertions and
substitutions.
[0055] The term "epitope" refers to a site on an antigen to which
B- and/or T-cells respond, either alone or in conjunction with
another protein such as, for example, a major histocompatibility
complex ("MHC") protein or a T-cell receptor. Epitopes can be
formed both from contiguous amino acids or noncontiguous amino
acids juxtaposed by secondary and/or tertiary folding of a protein.
Epitopes formed from contiguous amino acids are typically retained
on exposure to denaturing solvents, while epitopes formed by
tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 5, 6, 7, 8, 9, 10
or more amino acids--but generally less than 20 amino acids--in a
unique spatial conformation. Methods of determining spatial
conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
e.g., "Epitope Mapping Protocols" in Methods in Molecular Biology,
Vol. 66, Glenn E. Morris, Ed (1996).
[0056] Preferably, a homologue or variant has at least about 50%,
at least about 60% or 65%, at least about 70% or 75%, at least
about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more
typically, at least about 90%, 91%, 92%, 93%, or 94% and even more
typically at least about 95%, 96%, 97%, 98% or 99%, most typically,
at least about 99% identity with the referenced protein,
polypeptide, antigenic protein fragment, antigen and epitope at the
level of nucleotide or amino acid sequence.
[0057] Techniques for determining sequence identity between nucleic
acids and amino acids are known in the art. Two or more sequences
can be compared by determining their "percent identity." The
percent identity of two sequences, whether nucleic acid or amino
acid sequences, is the number of exact matches between two aligned
sequences divided by the length of the shorter sequences and
multiplied by 100.
[0058] "Percent (%) amino acid sequence identity" with respect to
proteins, polypeptides, antigenic protein fragments, antigens and
epitopes described herein is defined as the percentage of amino
acid residues in a candidate sequence that are identical with the
amino acid residues in the reference sequence (i.e., the protein,
polypeptide, antigenic protein fragment, antigen or epitope from
which it is derived), after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for example,
using publically available computer software such as BLAST, ALIGN,
or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximum alignment over the full
length of the sequences being compared.
[0059] The same applies to "percent (%) nucleotide sequence
identity", mutatis mutandis.
[0060] For example, an appropriate alignment for nucleic acid
sequences is provided by the local homology algorithm of Smith and
Waterman, (1981), Advances in Applied Mathematics 2:482-489. This
algorithm can be applied to amino acid sequences by using the
scoring matrix developed by Dayhoff, Atlas of Protein Sequences and
Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National
Biomedical Research Foundation, Washington, D.C., USA, and
normalized by Gribskov (1986), Nucl. Acids Res. 14(6):6745-6763. An
exemplary implementation of this algorithm to determine percent
identity of a sequence is provided by the Genetics Computer Group
(Madison, Wis.) in the "BestFit" utility application. The default
parameters for this method are described in the Wisconsin Sequence
Analysis Package Program Manual, Version 8 (1995) (available from
Genetics Computer Group, Madison, Wis.). A preferred method of
establishing percent identity in the context of the present
invention is to use the MPSRCH package of programs copyrighted by
the University of Edinburgh, developed by John F. Collins and Shane
S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain
View, Calif.). From this suite of packages the Smith-Waterman
algorithm can be employed where default parameters are used for the
scoring table (for example, gap open penalty of 12, gap extension
penalty of one, and a gap of six). From the data generated the
"Match" value reflects "sequence identity." Other suitable programs
for calculating the percent identity or similarity between
sequences are generally known in the art, for example, another
alignment program is BLAST, used with default parameters. For
example, BLASTN and BLASTP can be used using the following default
parameters: genetic code=standard; filter=none; strand=both;
cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences;
sort by=HIGH SCORE; Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss
protein+Spupdate+PIR. Details of these programs can be found at the
following internet address:
http://www.ncbi.nlm.gov/cgi-bin/BLAST.
[0061] In some embodiments, the heterologous nucleic acid encodes
antigenic domains or antigenic protein fragments rather than the
entire antigenic protein. These fragments can be of any length
sufficient to be antigenic or immunogenic. Fragments can be at
least 8 amino acids long, preferably 10-20 amino acids, but can be
longer, such as, e.g., at least 50, 100, 200, 500, 600, 800, 1000,
1200, 1600, 2000 amino acids long, or any length in between.
[0062] In some embodiments, at least one nucleic acid fragment
encoding an antigenic protein fragment or immunogenic polypeptide
thereof is inserted into the viral vector of the invention. In
another embodiment, about 2-6 different nucleic acids encoding
different antigenic proteins are inserted into one or more of the
viral vectors. In some embodiments, multiple immunogenic fragments
or subunits of various proteins can be used. For example, several
different epitopes from different sites of a single protein or from
different proteins of the same species, or from a protein ortholog
from different species can be expressed from the vectors.
[0063] Definitions
[0064] Before the present invention is described in detail below,
it is to be understood that this invention is not limited to the
particular methodology, protocols and reagents described herein as
these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Unless
defined otherwise, all technical and scientific terms used herein
have the same meanings as commonly understood by one of ordinary
skill in the art.
[0065] It must be noted that, as used herein, the singular forms
"a", "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "an
antigenic determinant" includes one or more antigenic determinants
and reference to "the method" includes reference to equivalent
steps and methods known to those of ordinary skill in the art that
could be modified or substituted for the methods described
herein.
[0066] Unless otherwise indicated, the term "at least" preceding a
series of elements is to be understood to refer to every element in
the series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention.
[0067] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integer or step. When used herein the term
"comprising" can be substituted with the term "containing" or
"including" or sometimes when used herein with the term "having".
Any of the aforementioned terms (comprising, containing, including,
having), whenever used herein in the context of an aspect or
embodiment of the present invention may be substituted with the
term "consisting of", though less preferred.
[0068] When used herein "consisting of" excludes any element, step,
or ingredient not specified in the claim element. When used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim.
[0069] As used herein, the conjunctive term "and/or" between
multiple recited elements is understood as encompassing both
individual and combined options. For instance, where two elements
are conjoined by "and/or", a first option refers to the
applicability of the first element without the second. A second
option refers to the applicability of the second element without
the first. A third option refers to the applicability of the first
and second elements together. Any one of these options is
understood to fall within the meaning, and therefore satisfy the
requirement of the term "and/or" as used herein. Concurrent
applicability of more than one of the options is also understood to
fall within the meaning, and therefore satisfy the requirement of
the term "and/or."
[0070] As used herein, "affecting an immune response" includes the
development, in a subject, of a humoral and/or a cellular immune
response to a protein and/or polypeptide produced by the
recombinant MVA and/or compositions and/or vaccines comprising the
recombinant MVA of this invention A "humoral" immune response, as
this term is well known in the art, refers to an immune response
comprising antibodies, while the "cellular" immune response, as
this term is well known in the art, refers to an immune response
comprising T-lymphocytes and other white blood cells, especially
the immunogen-specific response by HLA-restricted cytolytic
T-cells, i.e., "CTLs." A cellular immune response occurs when the
processed immunogens, i.e., peptide fragments, are displayed in
conjunction with the major histocompatibility complex.
[0071] The term "substantially similar" in the context of the FMDV
antigenic proteins of the invention indicates that a polypeptide
comprises a sequence with at least 90%, preferably at least 95%
sequence identity to the reference sequence over a comparison
window of 10-20 amino acids. Percentage of sequence identity is
determined by comparing two optimally aligned sequences over a
comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0072] As used herein, the term "operably linked" means that the
components described are in a relationship permitting them to
function in their intended manner.
[0073] By "animal" it is intended mammals, birds, and the like.
Animal or host includes mammals and human. The animal may be
selected from the group consisting of equine (e.g., horse), canine
(e.g., dogs, wolves, foxes, coyotes, jackals), feline (e.g., lions,
tigers, domestic cats, wild cats, other big cats, and other felines
including cheetahs and lynx), ovine (e.g., sheep), bovine (e.g.,
cattle), porcine (e.g., pig), caprine (e.g., goat), avian (e.g.,
chicken, duck, goose, turkey, quail, pheasant, parrot, finches,
hawk, crow, ostrich, emu and cassowary), primate (e.g., prosimian,
tarsier, monkey, gibbon, ape), and fish. The term "animal" also
includes an individual animal in all stages of development,
including embryonic and fetal stages.
[0074] The term "nucleic acid" and "polynucleotide" refers to RNA
or DNA that is linear or branched, single or double stranded, or a
hybrid thereof. The term also encompasses RNA/DNA hybrids. The
following are non-limiting examples of polynucleotides: a gene or
gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,
recombinant polynucleotides, branched polynucleotides, plasmids,
vectors, isolated DNA of any sequence, isolated RNA of any
sequence, nucleic acid probes and primers. A polynucleotide may
comprise modified nucleotides, such as methylated nucleotides and
nucleotide analogs, uracyl, other sugars and linking groups such as
fluororibose and thiolate, and nucleotide branches. The sequence of
nucleotides may be further modified after polymerization, such as
by conjugation, with a labeling component. Other types of
modifications included in this definition are caps, substitution of
one or more of the naturally occurring nucleotides with an analog,
and introduction of means for attaching the polynucleotide to
proteins, metal ions, labeling components, other polynucleotides or
solid support. The polynucleotides can be obtained by chemical
synthesis or derived from a microorganism.
[0075] The term "gene" is used broadly to refer to any segment of
polynucleotide associated with a biological function. Thus, genes
include introns and exons as in genomic sequence, or just the
coding sequences as in cDNAs and/or the regulatory sequences
required for their expression. For example, gene also refers to a
nucleic acid fragment that expresses mRNA or functional RNA, or
encodes a specific protein, and which includes regulatory
sequences.
[0076] As used herein, a "heterologous" gene, nucleic acid,
antigen, or protein is understood to be a nucleic acid or amino
acid sequence which is not present in the wild-type poxviral genome
(e.g., MVA or MVA-BN). The skilled person understands that a
"heterologous gene", when present in a poxvirus such as MVA or
MVA-BN, is to be incorporated into the poxviral genome in such a
way that, following administration of the recombinant poxvirus to a
host cell, it is expressed as the corresponding heterologous gene
product, i.e., as the "heterologous antigen" and\or "heterologous
protein." Expression is normally achieved by operatively linking
the heterologous gene to regulatory elements that allow expression
in the poxvirus-infected cell. Preferably, the regulatory elements
include a natural or synthetic poxvirus promoter.
[0077] The invention further comprises a complementary strand to a
polynucleotide encoding an FMDV antigen, epitope or immunogen. The
complementary strand can be polymeric and of any length, and can
contain deoxyribonucleotides, ribonucleotides, and analogs in any
combination.
[0078] The terms "protein", "peptide", "polypeptide" and
"polypeptide fragment" are used interchangeably herein to refer to
polymers of amino acid residues of any length. The polymer can be
linear or branched, it may comprise modified amino acids or amino
acid analogs, and it may be interrupted by chemical moieties other
than amino acids. The terms also encompass an amino acid polymer
that has been modified naturally or by intervention; for example
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling or bioactive component. The present
invention relates to ovine, bovine, caprine and porcine vaccines or
pharmaceutical or immunological compositions which may comprise an
effective amount of a recombinant FMDV antigens and a
pharmaceutically or veterinary acceptable carrier, excipient, or
vehicle.
[0079] "Pharmaceutically acceptable carriers" are for example
described in Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975). They
describe compositions and formulations using conventional
pharmaceutically acceptable carriers suitable for administration of
the vectors and compositions disclosed herein. Generally the nature
of the carrier used depends on the particular mode of
administration being employed. For example, parenteral formulations
usually comprise injectable fluids that include pharmaceutically
and physiologically acceptable fluids such as water, physiological
saline, balanced salt solutions, aqueous dextrose, glycerol or the
like, as a vehicle. For solid compositions (such as powders, pills,
tablets, or capsules), conventional non-toxic solid carriers
include, for example, pharmaceutical grades of mannitol, lactose,
starch, or magnesium stearate. Pharmaceutical compositions can also
contain minor amounts of non-toxic auxiliary substances such as
wetting or emulsifying agents, preservatives, pH-buffering agents
and the like such as, for example, sodium acetate or sorbitan
monolaurate.
[0080] The term "prime-boost vaccination" refers to a vaccination
strategy using a first, priming injection of a vaccine targeting a
specific antigen followed at intervals by one or more boosting
injections of the same vaccine. Prime-boost vaccination may be
homologous or heterologous. A homologous prime-boost vaccination
uses a vaccine comprising the same immunogen and vector for both
the priming injection and the one or more boosting injections. A
heterologous prime-boost vaccination uses a vaccine comprising the
same immunogen for both the priming injection and the one or more
boosting injections but different vectors for the priming injection
and the one or more boosting injections. For example, a homologous
prime-boost vaccination may use a recombinant MVA vector comprising
the same nucleic acids expressing alphavirus antigens for both the
priming injection and the one or more boosting injections. In
contrast, a heterologous prime-boost vaccination may use a
recombinant MVA vector comprising nucleic acids expressing one
alphavirus protein for the priming injection and another
recombinant MVA vector expressing a second one alphavirus protein
not contained in the priming injection or vice versa. Heterologous
prime-boost vaccination also encompasses various combinations such
as, for example, use of a plasmid encoding an immunogen in the
priming injection and use of a recombinant MVA encoding the same
immunogen in the one or more boosting injections, or use of a
recombinant protein immunogen in the priming injection and use of a
recombinant MVA vector encoding the same protein immunogen in the
one or more boosting injections.
[0081] A "vector" refers to a recombinant DNA or RNA plasmid or
virus that comprises a heterologous polynucleotide to be delivered
to a target cell, either in vitro or in vivo. The heterologous
polynucleotide may comprise a sequence of interest for purposes of
prevention or therapy, and may optionally be in the form of an
expression cassette. As used herein, a vector needs not be capable
of replication in the ultimate target cell or subject. The term
includes cloning vectors and viral vectors.
[0082] The term "recombinant" means a polynucleotide semisynthetic,
or synthetic origin which either does not occur in nature or is
linked to another polynucleotide in an arrangement not found in
nature.
[0083] As used herein, "treat", "treating" or "treatment" of a
disease means the prevention, reduction, amelioration, partial or
complete alleviation, or cure of a disease e.g., an FMDV-caused
disease. It can be one or more of reducing the severity of the
disease, limiting or preventing development of symptoms
characteristic of the disease being treated, inhibiting worsening
of symptoms characteristic of the disease being treated, limiting
or preventing recurrence of the disease in a subject who has
previously had the disease, and limiting or preventing recurrence
of symptoms in subjects.
[0084] Several documents are cited throughout the text of this
specification. Each of the documents cited herein (including all
patents, patent applications, scientific publications,
manufacturer's specifications, instructions, etc.), whether supra
or infra, are hereby incorporated by reference in their entirety.
To the extent the material incorporated by reference contradicts or
is inconsistent with this specification, the specification will
supersede any such material. Nothing herein is to be construed as
an admission that the invention is not entitled to antedate such
disclosure by virtue of prior invention.
[0085] FMDV Proteins
[0086] In one aspect, the present invention provides FMDV
polypeptides from ovine, bovine, caprine, or porcine. In another
aspect, the present invention provides a FMDV polypeptide and
variant or fragment thereof.
[0087] Moreover, homologs of FMDV polypeptides from ovine, bovine,
caprine, or porcine are intended to be within the scope of the
present invention. As used herein, the term "homologs" includes
orthologs, analogs and paralogs. The term "analogs" refers to two
polynucleotides or polypeptides that have the same or similar
function, but that have evolved separately in unrelated organisms.
The term "orthologs" refers to two polynucleotides or polypeptides
from different species, but that have evolved from a common
ancestral gene by speciation. Normally, orthologs encode
polypeptides having the same or similar functions. The term
"paralogs" refers to two polynucleotides or polypeptides that are
related by duplication within a genome. Paralogs usually have
different functions, but these functions may be related. Analogs,
orthologs, and paralogs of a wild-type FMDV polypeptide can differ
from the wild-type FMDV polypeptide by post-translational
modifications, by amino acid sequence differences, or by both. In
particular, homologs of the invention will generally exhibit at
least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, 99% sequence
identity, with all or part of the wild-type FMDV or polynucleotide
sequences, and will exhibit a similar function. Variants include
allelic variants. The term "allelic variant" refers to a
polynucleotide or a polypeptide containing polymorphisms that lead
to changes in the amino acid sequences of a protein and that exist
within a natural population (e.g., a virus species or variety).
Such natural allelic variations can typically result in 1-5%
variance in a polynucleotide or a polypeptide. Allelic variants can
be identified by sequencing the nucleic acid sequence of interest
in a number of different species, which can be readily carried out
by using hybridization probes to identify the same gene genetic
locus in those species. Any and all such nucleic acid variations
and resulting amino acid polymorphisms or variations that are the
result of natural allelic variation and that do not alter the
functional activity of gene of interest, are intended to be within
the scope of the invention.
[0088] As used herein, the term "derivative" or "variant" refers to
a polypeptide, or a nucleic acid encoding a polypeptide, that has
one or more conservative amino acid variations or other minor
modifications such that (1) the corresponding polypeptide has
substantially equivalent function when compared to the wild type
polypeptide or (2) an antibody raised against the polypeptide is
immunoreactive with the wild-type polypeptide. These variants or
derivatives include polypeptides having minor modifications of the
FMDV polypeptide primary amino acid sequences that may result in
peptides which have substantially equivalent activity as compared
to the unmodified counterpart polypeptide. Such modifications may
be deliberate, as by site-directed mutagenesis, or may be
spontaneous. The term "variant" further contemplates deletions,
additions and substitutions to the sequence, so long as the
polypeptide functions to produce an immunological response as
defined herein.
[0089] The term "conservative variation" denotes the replacement of
an amino acid residue by another biologically similar residue, or
the replacement of a nucleotide in a nucleic acid sequence such
that the encoded amino acid residue does not change or is another
biologically similar residue. In this regard, particularly
preferred substitutions will generally be conservative in nature,
as described above.
[0090] The polynucleotides of the disclosure include sequences that
are degenerate as a result of the genetic code, e.g., optimized
codon usage for a specific host. As used herein, "optimized" refers
to a polynucleotide that is genetically engineered to increase its
expression in a given species. To provide optimized polynucleotides
coding for FMDV polypeptides, the DNA sequence of the FMDV protein
gene can be modified to 1) comprise codons preferred by highly
expressed genes in a particular species; 2) comprise an A+T or G+C
content in nucleotide base composition to that substantially found
in said species; 3) form an initiation sequence of said species; or
4) eliminate sequences that cause destabilization, inappropriate
polyadenylation, degradation and termination of RNA, or that form
secondary structure hairpins or RNA splice sites. Increased
expression of FMDV protein in said species can be achieved by
utilizing the distribution frequency of codon usage in eukaryotes
and prokaryotes, or in a particular species. The term "frequency of
preferred codon usage" refers to the preference exhibited by a
specific host cell in usage of nucleotide codons to specify a given
amino acid. There are 20 natural amino acids, most of which are
specified by more than one codon. Therefore, all degenerate
nucleotide sequences are included in the disclosure as long as the
amino acid sequence of the FMDV polypeptide encoded by the
nucleotide sequence is functionally unchanged.
[0091] 3C Protease
[0092] During the maturation process, the FMDV protein P1 is
cleaved by the protease 3C into three proteins known as VP0, VP1
and VP3 (or 1AB, 1D and 1C respectively; Belsham G. J., Progress in
Biophysics and Molecular Biology, 1993, 60, 241-261). In the
virion, the protein VP0 is then cleaved into two proteins, VP4 and
VP2 (or 1A and 1B respectively).
[0093] High level expression of the 3C protease may lead to
toxicity in the cells. The residual activity of 3C in the virus in
eukaryotic cells is expected to be low although it is wt-3C, as the
HIV frame shift upstream of the 3C coding sequence is expected to
decrease translation by 20-fold, meaning equally lower amounts of
3C protein. In order to overcome the toxicity of high level
expression of the 3C protease, the inventors of the present
invention have found the following strategies. One strategy is to
make mutations in the cDNA encoding FMDV 3C protease such that the
expression levels of the 3C protease is decreased compared with
expression levels of the 3C protease when un-mutated. Another
strategy is to make mutations in the cDNA encoding FMDV 3C protease
such that the activity level of the 3C protease is changed compared
with expression level of the 3C protease when un-mutated.
[0094] According to a preferred embodiment, the cDNA encoding the
3C protease comprise the following mutations: C147T, C142L, and/or
C31A/L47A.
[0095] Still another strategy is to clone the expression cassettes
into a low copy-number-plasmid (pACYC177; e.g. commercially
available at New England Biolabs). Still another strategy is to
carefully choose the promoter responsible for lowering the
expression level of the 3C protease. According to a preferred
embodiment the expression level of the 3C protease is lowered by
applying a weak promoter. The term "weak promoter" refers a
promoter that weakens the expression level of the genes of
interest.
[0096] Recombinant MVA
[0097] Provided herein are recombinant poxviruses (e.g., MVA or
MVA-BN) comprising heterologous or foreign nucleic acid sequences
derived from FMDV incorporated in a variety of insertion sites in
the poxviral (e.g., MVA or MVA-BN) genome. The heterologous nucleic
acids can encode one or more foreign proteins and/or foreign
antigens including, for example, viral antigens.
[0098] Generally, a "recombinant" MVA as described herein refers to
MVAs that are produced by standard genetic engineering methods,
i.e., MVAs of the present invention are thus genetically engineered
or genetically modified MVAs. The term "recombinant MVA" thus
includes MVAs which have stably integrated recombinant nucleic
acid, preferably in the form of a transcriptional unit, in their
genome. A transcriptional unit may include a promoter, enhancer,
terminator and/or silencer. Recombinant MVAs of the present
invention may express heterologous antigenic determinants,
polypeptides or proteins (antigens) upon induction of the
regulatory elements.
[0099] As used herein, a "heterologous" gene, nucleic acid,
antigen, or protein is understood to be a nucleic acid or amino
acid sequence which is not present in the wild-type poxviral genome
(e.g., MVA or MVA-BN). The skilled person understands that a
"heterologous gene", when present in a poxvirus such as MVA or
MVA-BN, is to be incorporated into the poxviral genome in such a
way that, following administration of the recombinant poxvirus to a
host cell, it is expressed as the corresponding heterologous gene
product, i.e., as the "heterologous antigen" and\or "heterologous
protein." Expression is normally achieved by operatively linking
the heterologous gene to regulatory elements that allow expression
in the poxvirus-infected cell. Preferably, the regulatory elements
include a natural or synthetic poxviral promoter.
[0100] In one aspect, the present invention comprises a recombinant
MVA vector comprising a heterologous nucleotide sequence encoding
an antigenic determinant of a FMDV.
[0101] For the embodiments as described herein the FMDV may be
derived from a virulent strain of FMDV, advantageously the FMDV 01
Manisa, 01 BFS or Campos, A24 Cruzeiro, Asia 1 Shamir, A Iran '96,
A22 Iraq, SAT2 Saudi Arabia strains.
[0102] Still other strains may include FMDV strains A 10-61, A5, A
12, A24/Cruzeiro, C3/Indaial, 01, CI-Santa Pau, C1-05,
A22/550/Azerbaij an/65, SAT1-SAT3, A, A/TNC/71/94, A/IND/2/68,
A/IND/3/77, A/IND/5/68, A/IND/7/82, A/IND/16/82, A/IND/17/77,
A/IND/17/82, A/IND/19/76, A/TND/20/82, A/IND/22/82, A/IND/25/81,
A/IND/26/82, A/IND/54/79, A/IND/57/79, A/TND/73/79, A/IND/85/79,
A/IND/86/79, A/APA/25/84, A/APN/41/84, A/APS/44/05, A/APS/50/05,
A/APS/55/05, A/APS/66/05, A/APS/68/05, A/BIM/46/95, A/GUM/33/84,
A/ORS/66/84, A/ORS/75/88, A/TNAn/60/947/Asia/I, A/IRN/05,
Asia/IRN/05, O/HK/2001, O/UKG/3952/2001, O/UKG/4141/2001, Asia
I/HNK/CHA/05 (GenBank accession number EF149010, herein
incorporated by reference), Asia I/XJ (Li, ZhiYong et al. Chin Sci
Bull, 2007), HK/70 (Chin Sci Bull, 2006, 51(17): 2072-2078),
O/UKG/7039/2001, O/UKG/9161/2001, O/UKG/7299/2001, O/UKG/4014/2001,
O/UKG/4998/2001, O/UKG/9443/2001, O/UKG/5470/2001, O/UKG/5681/2001,
O/ES/2001, HKN/2002, 05India, O/BKF/2/92, K/37/84/A, KEN/1/76/A,
GAM/51/98/A, A10/Holland, O/KEN/1/91, O/IND49/97, O/IND65/98,
O/IND64/98, O/IND48/98, O/IND47/98, O/IND82/97, O/IND81/99,
O/IND81/98, O/IND79/97, O/IND78/97, O/IND75/97, O/IND74/97,
O/IND70/97, O/IND66/98, O/IND63/97, O/IND61/97, O/IND57/98,
O/IND56/98, O/IND55/98, O/IND54/98, O/IND469/98, O/IND465/97,
O/IND464/97, O/IND424/97, O/IND423/97, O/IND420/97, O/IND414/97,
O/IND411/97, O/IND410/97, O/IND409/97, O/IND407/97, O/IND399/97,
O/IND39/97, O/IND391/97, O/IND38/97, O/IND384/97, O/IND380/97,
O/IND37/97, O/IND352/97, O/IND33/97, O/IND31/97, O/IND296/97,
O/IND23/99, O/IND463/97, O/IND461/97, O/IND427/98, O/IND28/97,
O/IND287/99, O/IND285/99, O/IND282/99, O/IND281/97, O/IND27/97,
O/IND278/97, O/IND256/99, O/IND249/99, O/IND210/99, O/IND208/99,
O/IND207/99, O/IND205/99, O/IND185/99, O/IND175/99, O/IND170/97,
O/IND164/99, O/IND160/99, O/IND153/99, O/IND148/99, O/IND146/99,
O/SKR 2000, A22/India/17/77.
[0103] Further details of these FMDV strains may be found on the
European Bioinformatics Information (EMBL-EBI) web pages, and all
of the associated nucleotide sequences are herein incorporated by
reference. The inventors contemplate that all FMDV strains, both
herein listed, and those yet to be identified, could be expressed
according to the teachings of the present disclosure to produce,
for example, effective vaccine compositions. Both homologous and
heterologous strains are used for challenge to test the efficacy of
the vaccines. The animal may be challenged intradermally,
subcutaneously, spray, intra-nasally, intra-ocularly,
intra-tracheally, and/or orally.
[0104] In another aspect, the present invention comprises a
recombinant MVA vector comprising a heterologous nucleotide
sequence encoding an antigenic determinant of a FMDV as described
above, and further comprises heterologous nucleotide sequences
encoding additional proteins required to form virus-like particles
(VLPs).
[0105] Integration Sites into MVA
[0106] Heterologous nucleotide sequences encoding antigenic
determinants of a FMDV may be inserted into one or more intergenic
regions (IGR) of the MVA. In certain embodiments, the IGR is
selected from IGR07/08, IGR 44/45, IGR 64/65, IGR 88/89, IGR
136/137, and IGR 148/149. In certain embodiments, less than 5, 4,
3, or 2 IGRs of the recombinant MVA comprise heterologous
nucleotide sequences encoding antigenic determinants of a FMDV. The
heterologous nucleotide sequences may, additionally or
alternatively, be inserted into one or more of the naturally
occurring deletion sites, in particular into the main deletion
sites I, II, Ill, IV, V, or VI of the MVA genome. In certain
embodiments, less than 5, 4, 3, or 2 of the naturally occurring
deletion sites of the recombinant MVA comprise heterologous
nucleotide sequences encoding antigenic determinants of a FMDV.
[0107] The number of insertion sites of MVA comprising heterologous
nucleotide sequences encoding antigenic determinants of a FMDV
protein can be 1, 2, 3, 4, 5, 6, 7, or more. In certain
embodiments, the heterologous nucleotide sequences are inserted
into 4, 3, 2, or fewer insertion sites. Preferably, two insertion
sites are used. In certain embodiments, three insertion sites are
used. Preferably, the recombinant MVA comprises at least 2, 3, 4,
5, 6, or 7 genes inserted into 2 or 3 insertion sites.
[0108] The recombinant MVA viruses provided herein can be generated
by routine methods known in the art. Methods to obtain recombinant
poxviruses or to insert exogenous coding sequences into a poxviral
genome are well known to the person skilled in the art. For
example, methods for standard molecular biology techniques such as
cloning of DNA, DNA and RNA isolation, Western blot analysis,
RT-PCR and PCR amplification techniques are described in Molecular
Cloning, A laboratory Manual (2nd Ed.) [J. Sambrook et al., Cold
Spring Harbor Laboratory Press (1989)], and techniques for the
handling and manipulation of viruses are described in Virology
Methods Manual [B. W. J. Mahy et al. (eds.), Academic Press
(1996)]. Similarly, techniques and know-how for the handling,
manipulation and genetic engineering of MVA are described in
Molecular Virology: A Practical Approach [A. J. Davison & R. M.
Elliott (Eds.), The Practical Approach Series, IRL Press at Oxford
University Press, Oxford, UK (1993) (see, e.g., Chapter 9:
Expression of genes by Vaccinia virus vectors)] and Current
Protocols in Molecular Biology [John Wiley & Son, Inc. (1998)
(see, e.g., Chapter 16, Section IV: Expression of proteins in
mammalian cells using vaccinia viral vector)].
[0109] For the generation of the various recombinant MVAs disclosed
herein, different methods may be applicable. The DNA sequence to be
inserted into the virus can be placed into an E. coli plasmid
construct into which DNA homologous to a section of DNA of the MVA
has been inserted. Separately, the DNA sequence to be inserted can
be ligated to a promoter. The promoter-gene linkage can be
positioned in the plasmid construct so that the promoter-gene
linkage is flanked on both ends by DNA homologous to a DNA sequence
flanking a region of MVA DNA containing a non-essential locus. The
resulting plasmid construct can be amplified by propagation within
E. coli bacteria and isolated. The isolated plasmid containing the
DNA gene sequence to be inserted can be transfected into a cell
culture, e.g., of chicken embryo fibroblasts (CEFs), at the same
time the culture is infected with MVA. Recombination between
homologous MVA DNA in the plasmid and the viral genome,
respectively, can generate an MVA modified by the presence of
foreign DNA sequences.
[0110] According to a preferred embodiment, a cell of a suitable
cell culture as, e.g., CEF cells, can be infected with a poxvirus.
The infected cell can be, subsequently, transfected with a first
plasmid vector comprising a foreign or heterologous gene or genes,
preferably under the transcriptional control of a poxvirus
expression control element. As explained above, the plasmid vector
also comprises sequences capable of directing the insertion of the
exogenous sequence into a selected part of the poxviral genome.
Optionally, the plasmid vector also contains a cassette comprising
a marker and/or selection gene operably linked to a poxviral
promoter. Suitable marker or selection genes are, e.g., the genes
encoding the green fluorescent protein, .beta.-galactosidase,
neomycin-phosphoribosyltransferase or other markers. The use of
selection or marker cassettes simplifies the identification and
isolation of the generated recombinant poxvirus. However, a
recombinant poxvirus can also be identified by PCR technology.
Subsequently, a further cell can be infected with the recombinant
poxvirus obtained as described above and transfected with a second
vector comprising a second foreign or heterologous gene or genes.
In case, this gene shall be introduced into a different insertion
site of the poxviral genome, the second vector also differs in the
poxvirus-homologous sequences directing the integration of the
second foreign gene or genes into the genome of the poxvirus. After
homologous recombination has occurred, the recombinant virus
comprising two or more foreign or heterologous genes can be
isolated. For introducing additional foreign genes into the
recombinant virus, the steps of infection and transfection can be
repeated by using the recombinant virus isolated in previous steps
for infection and by using a further vector comprising a further
foreign gene or genes for transfection.
[0111] Alternatively, the steps of infection and transfection as
described above are interchangeable, i.e., a suitable cell can at
first be transfected by the plasmid vector comprising the foreign
gene and, then, infected with the poxvirus. As a further
alternative, it is also possible to introduce each foreign gene
into different viruses, co-infect a cell with all the obtained
recombinant viruses and screen for a recombinant including all
foreign genes. A third alternative is ligation of DNA genome and
foreign sequences in vitro and reconstitution of the recombined
vaccinia virus DNA genome using a helper virus. A fourth
alternative is homologous recombination in E. coli or another
bacterial species between a vaccinia virus genome cloned as a
bacterial artificial chromosome (BAC) and a linear foreign sequence
flanked with DNA sequences homologous to sequences flanking the
desired site of integration in the vaccinia virus genome.
[0112] Expression of Heterologous FMDV Genes
[0113] In certain embodiments, expression of one, more, or all of
the heterologous nucleotide sequences encoding antigenic
determinants of a FMDV protein is under the control of one or more
poxvirus promoters. In certain embodiments, the poxvirus promoter
is a Pr7.5 promoter, a hybrid early/late promoter, a PrS promoter,
a PrS5E promoter, a synthetic or natural early or late promoter, or
a cowpox virus ATI promoter. In certain embodiments, the poxvirus
promoter is selected from the group consisting of the PrS promoter
(SEQ ID NO:1), the PrS5E promoter (SEQ ID NO:2), the Pr7.5 (SEQ ID
NO:3), the PrLE1 promoter (SEQ ID NO:4), the Pr13.5 long promoter
(SEQ ID NO:5) and the PrMVA095R promoter. Suitable promoters are
further described in WO 2010/060632, WO 2010/102822, WO 2013/189611
and WO 2014/063832 incorporated fully by reference herewith.
[0114] A heterologous nucleotide sequence encoding an antigenic
determinant of a FMDV protein can be expressed as a single
transcriptional unit. For example, a heterologous nucleotide
sequence encoding an antigenic determinant of a FMDV protein can be
operably linked to a vaccinia virus promoter and/or linked to a
vaccinia virus transcriptional terminator.
[0115] In certain embodiments, the "transcriptional unit" is
inserted by itself into an insertion site in the MVA genome. In
certain embodiments, the "transcriptional unit" is inserted with
other transcriptional unit(s) into an insertion site in the MVA
genome. The "transcriptional unit" is not naturally occurring
(i.e., it is heterologous, exogenous or foreign) in the MVA genome
and is capable of transcription in infected cells.
[0116] Preferably, the recombinant MVA comprises 1, 2, 3, 4, 5, or
more transcriptional units inserted into the MVA genome. In certain
embodiments, the recombinant MVA stably expresses heterologous
nucleotide sequences encoding antigenic determinants of a filovirus
protein encoded by 1, 2, 3, 4, 5, or more transcriptional units. In
certain embodiments, the recombinant MVA comprises 2, 3, 4, 5, or
more transcriptional units inserted into the MVA genome at 1, 2, 3,
or more insertion sites in the MVA genome.
[0117] FMDV Vaccines and Pharmaceutical/Veterinary Compositions
[0118] Since the recombinant MVA viruses described herein are
highly replication restricted and, thus, highly attenuated, they
are ideal candidates for the treatment of a wide range of mammals
including humans and even immune-compromised humans. Hence,
provided herein are pharmaceutical/veterinary compositions and
vaccines for inducing an immune response in a living animal body,
including a human. Additionally provided is a recombinant MVA
vector comprising a nucleotide sequence encoding an antigenic
determinant of a FMDV protein for use in the treatment and/or
prevention of a FMDV-caused disease.
[0119] The vaccine preferably comprises any of the recombinant MVA
viruses described herein formulated in solution in a concentration
range of 10.sup.4 to 10.sup.9 TCID.sub.50/ml, 10.sup.5 to
5.times.10.sup.8 TCID.sub.50/ml, 10.sup.6 to 10.sup.8
TCID.sub.50/ml, or 10.sup.7 to 10.sup.8 TCID.sub.50/ml. A preferred
vaccination dose for humans comprises between 10.sup.6 to 10.sup.9
TCID.sub.50, including a dose of 10.sup.6 TCID.sub.50, 10.sup.7
TCID.sub.50, or 10.sup.8 TCID.sub.50.
[0120] The pharmaceutical/veterinary compositions provided herein
may generally include one or more pharmaceutically/veterinary
acceptable and/or approved carriers, additives, antibiotics,
preservatives, adjuvants, diluents and/or stabilizers. Such
auxiliary substances can be water, saline, glycerol, ethanol,
wetting or emulsifying agents, pH buffering substances, or the
like. Suitable carriers are typically large, slowly metabolized
molecules such as proteins, polysaccharides, polylactic acids,
polyglycolic acids, polymeric amino acids, amino acid copolymers,
lipid aggregates, or the like.
[0121] For the preparation of vaccines, the recombinant MVA viruses
provided herein can be converted into a physiologically acceptable
form. This can be done based on experience in the preparation of
poxvirus vaccines used for vaccination against smallpox as
described by H. Stickl et al., Dtsch. med. Wschr. 99:2386-2392
(1974).
[0122] For example, purified viruses can be stored at -80.degree.
C. with a titer of 5.times.10.sup.8 TCID.sub.50/ml formulated in
about 10 mM Tris, 140 mM NaCl pH 7.4. For the preparation of
vaccine shots, e.g., 10.sup.2-10.sup.8 or 10.sup.2-10.sup.9
particles of the virus can be lyophilized in 100 ml of
phosphate-buffered saline (PBS) in the presence of 2% peptone and
1% human albumin in an ampoule, preferably a glass ampoule.
Alternatively, the vaccine shots can be produced by stepwise
freeze-drying of the virus in a formulation. This formulation can
contain additional additives such as mannitol, dextran, sugar,
glycine, lactose or polyvinylpyrrolidone or other aids such as
antioxidants or inert gas, stabilizers or recombinant proteins
(e.g., human serum albumin) suitable for in vivo administration.
The glass ampoule is then sealed and can be stored between
4.degree. C. and room temperature for several months. However, as
long as no need exists, the ampoule is stored preferably at
temperatures below -20.degree. C.
[0123] For vaccination or therapy, the lyophilisate can be
dissolved in an aqueous solution, preferably physiological saline
or Tris buffer, and administered either systemically or locally,
i.e., parenteral, subcutaneous, intravenous, intramuscular,
intranasal, or any other path of administration known to the
skilled practitioner. The mode of administration, the dose and the
number of administrations can be optimized by those skilled in the
art in a known manner.
[0124] Vaccines Using Homologous/Heterologous Prime-Boost
Regimens
[0125] The vaccines and methods described herein may also be used
as part of a homologous prime-boost regimen. In the homologous
prime-boost, a first priming vaccination is given followed by one
or more subsequent boosting vaccinations. The boosting vaccinations
are configured to boost the immune response generated in the first
vaccination by administration of the same recombinant poxvirus that
was used in the first vaccination.
[0126] In one exemplary embodiment a homologous prime-boost regimen
may be employed wherein a MVA viral vector as defined herein is
administered in a first dosage. One or more subsequent
administrations of an MVA viral vector as defined herein can be
given to boost the immune response provided in the first
administration. Preferably, the one or more antigenic determinants
are the same or similar to those of the first administration
[0127] The MVA recombinant viral vectors according to the present
invention may also be used in heterologous prime-boost regimens in
combination with another poxviral vector in which one or more of
the initial prime vaccinations are done with either the MVA or the
other poxviral vector as defined herein and one or more subsequent
boosting vaccinations are done with the poxviral vector not used in
the prime vaccination, e.g., if a MVA vector defined herein is
given in a prime boost, then subsequent boosting vaccinations would
be with the other poxviral vectors and vice versa.
[0128] Vaccines and Kits Comprising Recombinant MVA Viruses
[0129] Also provided herein are vaccines and kits comprising any
one or more of the recombinant MVAs described herein. The kit can
comprise one or multiple containers or vials of the recombinant
MVA, together with instructions for the administration of the
recombinant MVA to a subject at risk of FMDV infection. In certain
embodiments, the instructions indicate that the recombinant MVA is
administered to the subject in a single dose, or in multiple (i.e.,
2, 3, 4, etc.) doses. In certain embodiments, the instructions
indicate that the recombinant MVA virus is administered in a first
(priming) and second (boosting) administration to naive or
non-naive subjects. Preferably, a kit comprises at least two vials
for prime/boost immunization comprising the recombinant MVAs as
described herein for a first inoculation ("priming inoculation") in
a first vial/container and for an at least second and/or third
and/or further inoculation ("boosting inoculation") in a second
and/or further vial/container.
[0130] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
appended claims.
EXAMPLES
[0131] The detailed examples which follow are intended to
contribute to a better understanding of the present invention.
However, the invention is not limited by the examples. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein.
Example 1: Construction of Recombinant MVA
[0132] The following sections describe construction of two
recombinant MVAs comprising one or more heterologous nucleic acids
expressing an antigenic determinant of a FMDV protein. All other
constructs described herein are made using similar methods.
1.1 Cloning and Generation of Two Recombinant MVA-BN.RTM.-FMDV
Constructs
[0133] For the insertion of foreign genes into the MVA-BN.RTM.
genome, BN has constructed a set of plasmids. These basic plasmids
contain specific regions of the MVA-BN.RTM. genome covering
deletion sites or intergenic regions of the MVA virus backbone,
promoters and different selection cassettes. In order to clone a
recombinant MVA-BN.RTM.-FMDV vaccine candidate, the transgenes were
inserted into one of these basic plasmids, resulting in a final
recombination plasmid which was used to promote the insertion of
the transgenes into a specific site within the MVA genome via
homologous recombination. To allow for homologous recombination
between the plasmid and the MVA genome, primary CEF cells were
infected with MVA-BN.RTM. and subsequently transfected with the
respective recombination plasmids. During homologous recombination,
flanking sequences of the plasmids recombine with the homologous
sequences of the insertion sites in the MVA-BN.RTM. virus genome
and target the plasmid sequences into the respective site. The
presence of the selection cassettes within the inserted sequence
allows for positive selection of recombinant MVA-BN.RTM. viruses.
After initial amplification and 3-4 plaque purification steps under
selective conditions, the recombinant product containing the
FMDV-derived transgenes and the selection cassette was obtained. By
further amplification and plaque purification steps under
non-selective conditions, the selection cassette was excised and
the final vaccine candidate was isolated. Final plaque purified
clones were selected and amplified in 2-3 T175 tissue culture
flasks to generate a Pre-Master virus stock which will be
extensively characterized as described in section 1.2.
[0134] Two recombinant MVA-BN.RTM.-FMDV candidates in which the 3C
protease was expressed in trans using separate promoters. 7
transgenes was inserted into one basic plasmid supporting
homologous recombination into one of the well-established insertion
sites of MVA-BN.RTM. described above.
[0135] Codon optimization of the nucleotide sequences in the
proposed constructs involves the identification and removal of
homologous sequences which could affect the stability of the
construct and the optimization of the codon usage for the optimal
expression of the transgenes in the respective host. This was
performed in collaboration with a highly experienced CRO (GeneArt
AG, Regensburg, Germany).
1.2 Genetic Analysis of Recombinant MVA-BN.RTM.-FMDV
[0136] Following generation of recombinant MVA-BN.RTM.-FMDV
constructs by homologous recombination and plaque purifications,
final clones were selected and amplified in T175 tissue culture
flasks to generate a Pre-Master virus stock. The presence of the
recombinant inserts, correct insertion into the targeted genome
sites and absence of parental MVA-BN.RTM. virus in the Pre-Master
virus stock was confirmed by Polymerase Chain Reaction (PCR)
analysis. The correct sequence of the recombinant inserts was
confirmed by sequence analysis. Sequencing was performed for the
recombinant inserts including the flanking regions (more than 600
bp each, covering the sites of homologous recombination as
contained in the recombination plasmids). A nested PCR was
performed to verify the absence of the selection cassettes used
during homologous recombination.
1.3 Analysis of Expression and Processing of Inserted FMDV
Proteins
[0137] Following the generation of two viable MVA-BN.RTM.-FMDV
viruses, their functionality were proven by analysis of their
expressed recombinant proteins. The expression and processing of
the FMDV proteins is essential for robust induction of immune
response. BN will analyse expression and processing by Western blot
and the ability of processed proteins to interact by
co-immunoprecipitation assays. Further analysis of VLP formation
was assessed by PIADC using electron microscopy. Based upon these
analyses,
1.3.1 Expression and Processing of Antigens: Western Blot
[0138] Western blotting for analysis of recombinant proteins
expressed by MVA-BN.RTM. in various cell types was used for the
detection of FMDV proteins with respect to their size and also for
the relative estimation of expression levels from the respective
recombinant MVA-BN.RTM. vectors. Depending on the nature of the
FMDV-specific antibody, expression of native or denatured FMDV
proteins can be detected. As antibodies specific for non-structural
proteins as well as for virulent FMDV are commercially available
only for serotype O1, BN recommends to use FMDV-antibodies
(serotype A24) provided by PIADC. Concerning practical application,
cells will be infected with MVA-BN.RTM.-FMDV at a defined MOI and
harvested after 24 h. Cell extracts will be prepared and analysed
by SDS-PAGE. The result of the assay will confirm expression of
recombinant FMDV proteins and their correct size (indicative of
correct processing).
1.3.2 Interaction of Proteins: IP-Western Blot
[0139] The interaction of structural proteins during the infectious
cycle of FMDV is essential for the formation of infectious, highly
immunogenic virus particles. Thus, the formation of non-infectious
virus-like particles (VLPs) from MVA-BN.RTM. expressed FMDV
proteins is desirable and requires specific protein Construction
and Evaluation of Recombinant MVA-BN.RTM. FMDV Candidates Revised
Statement of Work
Example 2: Construction of Two Recombinant MVA-BN-FMDV Constructs
(MVA-mBN360B and mBN361A)
[0140] The two constructs shown in FIG. 1 were generated as
candidates for animal experiments. Construct #7B was selected as a
candidate and production of MVB and FDP were performed. The
recombinant MVA-BN constructs were generated as disclosed under
heading 1.1 above.
[0141] A Master Virus Bank of construct #7B was produced in three
roller bottles according to the SOPs at Bavarian Nordic. Cells were
lysed and the product was aliquoted and stored for later use at
-80.degree. C. Genetic analysis for identity, purity and absence of
empty vector and selection cassette was confirmed by PCR based
methods and sequencing. Further a sterility test and a PCR based
test for absence of mycoplasma were performed. The MVB of
MVA-mBN360B (#7B) passed all tests. The titer of the MVB-material
was determined to be 8.25.times.10.sup.6 TCID.sub.50/ml, which is
regarded sufficient to go into BDS production.
[0142] The quality tests on the FDP material were finalized,
including expression analysis (FIG. 2), co-IP (FIG. 3) titration,
the later resulting in a titer of 1.47.times.10.sup.9
TCID.sub.50/ml.
[0143] The 3C protease and the P1-2AB are expressed by MVA-mBN360B
(construct #7) in HeLa cells and lysates were applied to western
blotting. The VP2 specific western shows, that P1 is processed to
VP0, which is indicative for 3C protease activity, and the VP3
specific WB shows, that VP3 is efficiently released from the P1
precursor by 3C (FIG. 2).
[0144] FDP material of MVA-BN360B construct #7 was applied to
co-precipitation with a P1 conformation specific antibody. A VP3
specific band was detected with the VP3 antibody, which indicates
interaction of VP3 single protein in a `good` conformational
structure of a capsid. The detection of a VP0 specific band with
the VP2 antibody indicates interaction of P0 premature protein in a
`good` conformational structure (FIG. 3).
Example 3: MVA-BN-FMDV (MVA-mBN360B) in Cattle
[0145] Cattle were immunized on day 0 and on day 21 with doses of
10.sup.9 TCID.sub.50 MVA-mBN360B. On day 4, animals were challenged
with 10.sup.4 pfu of strain A24 Cruzeiro and analysed for signs of
infection (tongue) and general disease as scored by the number of
infected feet per animal.
TABLE-US-00001 TABLE 1 Disease scoring of cattle immunized or not
with MVA-mBN360B vaccine. Generalized Disease (days post
challenge).sup.b TG Vaccine ID 3 7 10 14 01 none 1 2 4 4 4 2 2 3 3
3 02 MVA- .sup. 3.sup.b 0 0 0 0 mBN360B 4 0 0 0 0 5 0 0 0 0
[0146] All vaccinated animals were fully protected from a FMDV
challenge, while none of the non-vaccinated animals were protected.
.sup.a Animal without tongue lesion .sup.b Generalized Disease is
given as the number of feet infected with FMDV (maximum
score=4).
DESCRIPTION OF THE SEQUENCE LISTING
[0147] SEQ ID NO:1 [DNA sequence of PrS promoter]
[0148] SEQ ID NO:2 [DNA sequence of PrS5E promoter: 1x (PrS)+5x
(Pr7.5e)]
[0149] SEQ ID NO:3 [DNA sequence of Pr7.5 promoter]
[0150] SEQ ID NO:4 [DNA sequence of PrLE1
promoter--5X-ATI+Pr7.5e]
[0151] SEQ ID NO:5 [Pr13.5 promoter sequence]
[0152] SEQ ID NO:6 [P1 amino acid sequence of FMDV strain A10, from
U.S. Pat. No. 7,531,182]
[0153] SEQ ID NO:7 [3C amino acid sequence of FMDV strain A10, from
U.S. Pat. No. 7,531,182]
Sequence CWU 1
1
7140DNAArtificial sequenceSynthetic promoter 1aaaaattgaa attttatttt
ttttttttgg aatataaata 402234DNAArtificial sequenceSynthetic
promoter 2aaaaattgaa attttatttt ttttttttgg aatataaata aaaaattgaa
aaactattct 60aatttattgc acggtccggt aaaaattgaa aaactattct aatttattgc
acggtccggt 120aaaaattgaa aaactattct aatttattgc acggtccggt
aaaaattgaa aaactattct 180aatttattgc acggtccggt aaaaattgaa
aaactattct aatttattgc acgg 2343104DNAArtificial sequenceSynthetic
promoter 3tccaaaccca cccgcttttt atagtaagtt tttcacccat aaataataaa
tacaataatt 60aatttctcgt aaaagtagaa aatatattct aatttattgc acgg
1044227DNAArtificial sequenceSynthetic promoter 4gttttgaaaa
tttttttata ataaatatcc ggtaaaaatt gaaaaactat tctaatttat 60tgcacggtcc
ggtaaaaatt gaaaaactat tctaatttat tgcacggtcc ggtaaaaatt
120gaaaaactat tctaatttat tgcacggtcc ggtaaaaatt gaaaaactat
tctaatttat 180tgcacggtcc ggtaaaaatt gaaaaactat tctaatttat tgcacgg
2275124DNAArtificial sequenceSynthetic promoter 5taaaaataga
aactataatc atataatagt gtaggttggt agtattgctc ttgtgactag 60agactttagt
taaggtactg taaaaataga aactataatc atataatagt gtaggttggt 120agta
1246737PRTFoot-and-mouth disease virus 6Met Gly Ala Gly Gln Ser Ser
Pro Ala Thr Gly Ser Gln Asn Gln Ser1 5 10 15Gly Asn Thr Gly Ser Ile
Ile Asn Asn Tyr Tyr Met Gln Gln Tyr Gln 20 25 30Asn Ser Met Ser Thr
Gln Leu Gly Asp Asn Thr Ile Ser Gly Gly Ser 35 40 45Asn Glu Gly Ser
Thr Asp Thr Thr Ser Thr His Thr Thr Asn Thr Gln 50 55 60Asn Asn Asp
Trp Phe Ser Lys Leu Ala Ser Ser Ala Phe Thr Gly Leu65 70 75 80Phe
Gly Ala Leu Leu Ala Asp Lys Lys Thr Glu Glu Thr Thr Leu Leu 85 90
95Glu Asp Arg Ile Leu Thr Thr Arg Asn Gly His Thr Thr Ser Thr Thr
100 105 110Gln Ser Ser Val Gly Val Thr Tyr Gly Tyr Ser Thr Glu Glu
Asp His 115 120 125Val Ala Gly Pro Asn Thr Ser Gly Leu Glu Thr Arg
Val Val Gln Ala 130 135 140Glu Arg Phe Phe Lys Lys Phe Leu Phe Asp
Trp Thr Thr Asp Lys Pro145 150 155 160Phe Gly Tyr Leu Thr Lys Leu
Glu Leu Pro Thr Asp His His Gly Val 165 170 175Phe Gly His Leu Val
Asp Ser Tyr Ala Tyr Met Arg Asn Gly Trp Asp 180 185 190Val Glu Val
Ser Ala Val Gly Asn Gln Phe Asn Gly Gly Cys Leu Leu 195 200 205Val
Ala Met Val Pro Glu Trp Lys Ala Phe Asp Thr Arg Glu Lys Tyr 210 215
220Gln Leu Thr Leu Phe Pro His Gln Phe Ile Ser Pro Arg Thr Asn
Met225 230 235 240Thr Ala His Ile Thr Val Pro Tyr Leu Gly Val Asn
Arg Tyr Asp Gln 245 250 255Tyr Lys Lys His Lys Pro Trp Thr Leu Val
Val Met Val Leu Ser Pro 260 265 270Leu Thr Val Ser Asn Thr Ala Ala
Pro Gln Ile Lys Val Tyr Ala Asn 275 280 285Ile Ala Pro Thr Tyr Val
His Val Ala Gly Glu Leu Pro Ser Lys Glu 290 295 300Gly Ile Phe Pro
Val Ala Cys Ala Asp Gly Tyr Gly Gly Leu Val Thr305 310 315 320Thr
Asp Pro Lys Thr Ala Asp Pro Val Tyr Gly Lys Val Tyr Asn Pro 325 330
335Pro Lys Thr Asn Tyr Pro Gly Arg Phe Thr Asn Leu Leu Asp Val Ala
340 345 350Glu Ala Cys Pro Thr Phe Leu Arg Phe Asp Asp Gly Lys Pro
Tyr Val 355 360 365Val Thr Arg Ala Asp Asp Thr Arg Leu Leu Ala Lys
Phe Asp Val Ser 370 375 380Leu Ala Ala Lys His Met Ser Asn Thr Tyr
Leu Ser Gly Ile Ala Gln385 390 395 400Tyr Tyr Thr Gln Tyr Ser Gly
Thr Ile Asn Leu His Phe Met Phe Thr 405 410 415Gly Ser Thr Asp Ser
Lys Ala Arg Tyr Met Val Ala Tyr Ile Pro Pro 420 425 430Gly Val Glu
Thr Pro Pro Asp Thr Pro Glu Glu Ala Ala His Cys Ile 435 440 445His
Ala Glu Trp Asp Thr Gly Leu Asn Ser Lys Phe Thr Phe Ser Ile 450 455
460Pro Tyr Val Ser Ala Ala Asp Tyr Ala Tyr Thr Ala Ser Asp Thr
Ala465 470 475 480Glu Thr Thr Asn Val Gln Gly Trp Val Cys Val Tyr
Gln Ile Thr His 485 490 495Gly Lys Ala Glu Asn Asp Thr Leu Leu Val
Ser Ala Ser Ala Gly Lys 500 505 510Asp Phe Glu Leu Arg Leu Pro Ile
Asp Pro Arg Thr Gln Thr Thr Thr 515 520 525Thr Gly Glu Ser Ala Asp
Pro Val Thr Thr Thr Val Glu Asn Tyr Gly 530 535 540Gly Asp Thr Gln
Val Gln Arg Arg His His Thr Asp Val Gly Phe Ile545 550 555 560Met
Asp Arg Phe Val Lys Ile Asn Ser Leu Ser Pro Thr His Val Ile 565 570
575Asp Leu Met Gln Thr His Lys His Gly Ile Val Gly Ala Leu Leu Arg
580 585 590Ala Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Ile Val Val Arg
His Asp 595 600 605Gly Asn Leu Thr Trp Val Pro Asn Gly Ala Pro Glu
Ala Ala Leu Ser 610 615 620Asn Thr Ser Asn Pro Thr Ala Tyr Asn Lys
Ala Pro Phe Thr Arg Leu625 630 635 640Ala Leu Pro Tyr Thr Ala Pro
His Arg Val Leu Ala Thr Val Tyr Asp 645 650 655Gly Thr Asn Lys Tyr
Ser Ala Ser Asp Ser Arg Ser Gly Asp Leu Gly 660 665 670Ser Ile Ala
Ala Arg Val Ala Thr Gln Leu Pro Ala Ser Phe Asn Tyr 675 680 685Gly
Ala Ile Gln Ala Gln Ala Ile His Glu Leu Leu Val Arg Met Lys 690 695
700Arg Ala Glu Leu Tyr Cys Pro Arg Pro Leu Leu Ala Ile Lys Val
Thr705 710 715 720Ser Gln Asp Arg Tyr Lys Gln Lys Ile Ile Ala Pro
Ala Lys Gln Leu 725 730 735Leu7214PRTFoot-and-mouth disease virus
7Ser Gly Ala Pro Pro Thr Asp Leu Gln Lys Met Val Met Gly Asn Thr1 5
10 15Lys Pro Val Glu Leu Asn Leu Asp Gly Lys Thr Val Ala Ile Cys
Cys 20 25 30Ala Thr Gly Val Phe Gly Thr Ala Tyr Leu Val Pro Arg His
Leu Phe 35 40 45Ala Glu Lys Tyr Asp Lys Ile Met Leu Asp Gly Arg Ala
Met Thr Asp 50 55 60Ser Asp Tyr Arg Val Phe Glu Phe Glu Ile Lys Val
Lys Arg Thr Gly65 70 75 80His Ala Leu Arg Arg Gly Thr His Trp Leu
Leu His Arg Gly Asn Cys 85 90 95Val Arg Asp Ile Thr Lys His Phe Arg
Asp Thr Ala Arg Met Lys Lys 100 105 110Gly Thr Pro Val Val Gly Val
Val Asn Asn Ala Asp Val Gly Arg Leu 115 120 125Ile Phe Ser Gly Glu
Ala Leu Thr Tyr Lys Asp Ile Val Val Cys Met 130 135 140Asp Gly Asp
Thr Met Pro Gly Leu Phe Ala Tyr Lys Ala Ala Thr Arg145 150 155
160Ala Gly Tyr Cys Gly Gly Ala Val Leu Ala Lys Asp Gly Ala Asp Thr
165 170 175Phe Ile Val Gly Thr His Ser Ala Gly Gly Asn Gly Val Gly
Tyr Cys 180 185 190Ser Cys Val Ser Arg Ser Met Leu Gln Lys Met Lys
Ala His Val Asp 195 200 205Pro Glu Pro His His Glu 210
* * * * *
References