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Uh oh, looks like he picked door number 2, the one with the bio weapons.
See you later. Now we see if they actually have an antidote or does
the guy experience like the rest of us. Cuz he's really high up in the network food chain so we'll see.
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Corona Pope
https://www.bitchute.com/video/P9kRvNaAhOVY/
The present invention provides a live, attenuated
coronavirus comprising a variant replicase gene encoding polyproteins
comprising a mutation in one or more of non-structural protein(s)
(nsp)-10, nsp-14, nsp-15 or nsp-16. The coronavirus may be used as a
vaccine for treating and/or preventing a disease, such as infectious
bronchitis, in a subject.
Latest THE PIRBRIGHT INSTITUTE Patents:
Description
FIELD OF THE INVENTION
The
present invention relates to an attenuated coronavirus comprising a
variant replicase gene, which causes the virus to have reduced
pathogenicity. The present invention also relates to the use of such a
coronavirus in a vaccine to prevent and/or treat a disease.
BACKGROUND TO THE INVENTION
Avian
infectious bronchitis virus (IBV), the aetiological agent of infectious
bronchitis (IB), is a highly infectious and contagious pathogen of
domestic fowl that replicates primarily in the respiratory tract but
also in epithelial cells of the gut, kidney and oviduct. IBV is a member
of the Order Nidovirales, Family Coronaviridae, Subfamily Corona
virinae and Genus Gammacoronavirus; genetically very similar coronaviruses cause disease in turkeys, guinea fowl and pheasants.
Clinical
signs of IB include sneezing, tracheal rales, nasal discharge and
wheezing. Meat-type birds have reduced weight gain, whilst egg-laying
birds lay fewer eggs and produce poor quality eggs. The respiratory
infection predisposes chickens to secondary bacterial infections which
can be fatal in chicks. The virus can also cause permanent damage to the
oviduct, especially in chicks, leading to reduced egg production and
quality; and kidney, sometimes leading to kidney disease which can be
fatal.
IBV has been reported to be
responsible for more economic loss to the poultry industry than any
other infectious disease. Although live attenuated vaccines and
inactivated vaccines are universally used in the control of IBV, the
protection gained by use of vaccination can be lost either due to
vaccine breakdown or the introduction of a new IBV serotype that is not
related to the vaccine used, posing a risk to the poultry industry.
Further,
there is a need in the industry to develop vaccines which are suitable
for use in ovo, in order to improve the efficiency and
cost-effectiveness of vaccination programmes. A major challenge
associated with in ovo vaccination is that the virus must be capable of
replicating in the presence of maternally-derived antibodies against the
virus, without being pathogenic to the embryo. Current IBV vaccines are
derived following multiple passage in embryonated eggs, this results in
viruses with reduced pathogenicity for chickens, so that they can be
used as live attenuated vaccines. However such viruses almost always
show an increased virulence to embryos and therefore cannot be used for
in ova vaccination as they cause reduced hatchability. A 70% reduction
in hatchability is seen in some cases.
Attenuation
following multiple passage in embryonated eggs also suffers from other
disadvantages. It is an empirical method, as attenuation of the viruses
is random and will differ every time the virus is passaged, so passage
of the same virus through a different series of eggs for attenuation
purposes will lead to a different set of mutations leading to
attenuation. There are also efficacy problems associated with the
process: some mutations will affect the replication of the virus and
some of the mutations may make the virus too attenuated. Mutations can
also occur in the S gene which may also affect immunogenicity so that
the desired immune response is affected and the potential vaccine may
not protect against the required serotype. In addition there are
problems associated with reversion to virulence and stability of
vaccines.
It is important that new and
safer vaccines are developed for the control of IBV. Thus there is a
need for IBV vaccines which are not associated with these issues, in
particular vaccines which may be used for in ovo vaccination.
SUMMARY OF ASPECTS OF THE INVENTION
The
present inventors have used a reverse genetics approach in order to
rationally attenuate IBV. This approach is much more controllable than
random attenuation following multiple passages in embryonated eggs
because the position of each mutation is known and its effect on the
virus, i.e. the reason for attenuation, can be derived.
Using
their reverse genetics approach, the present inventors have identified
various mutations which cause the virus to have reduced levels of
pathogenicity. The levels of pathogenicity may be reduced such that when
the virus is administered to an embryonated egg, it is capable of
replicating without being pathogenic to the embryo. Such viruses may be
suitable for in ovo vaccination, which is a significant advantage and
has improvement over attenuated IBV vaccines produced following multiple
passage in embryonated eggs.
Thus in a
first aspect, the present invention provides a live, attenuated
coronavirus comprising a variant replicase gene encoding polyproteins
comprising a mutation in one or more of non-structural protein(s)
(nsp)-10, nsp-14, nsp-15 or nsp-16.
The variant replicase gene may encode a protein comprising one or more amino acid mutations selected from the list of:
-
- Pro to Leu at position 85 of SEQ ID NO: 6,
- Val to Leu at position 393 of SEQ ID NO: 7;
- Leu to Ile at position 183 of SEQ ID NO: 8;
- Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may encode a protein comprising the amino acid mutation Pro to Leu at position 85 of SEQ ID NO: 6.
The
replicase gene may encode a protein comprising the amino acid mutations
Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183
of SEQ ID NO: 8; and Val to Ile at position 209 of SEQ ID NO: 9.
The
replicase gene may encodes a protein comprising the amino acid
mutations Pro to Leu at position 85 of SEQ ID NO: 6; Val to Leu at
position 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID NO:8;
and Val to Ile at position 209 of SEQ ID NO: 9.
The replicase gene may comprise one or more nucleotide substitutions selected from the list of:
C to T at nucleotide position 12137;
G to C at nucleotide position 18114;
T to A at nucleotide position 19047; and
G to A at nucleotide position 20139;
compared to the sequence shown as SEQ ID NO: 1.
The coronavirus may be an infectious bronchitis virus (IBV).
The coronavirus may be IBV M41.
The coronavirus may comprise an S protein at least part of which is from an IBV serotype other than M41.
For example, the S1 subunit or the entire S protein may be from an IBV serotype other than M41.
The
coronavirus according to the first aspect of the invention has reduced
pathogenicity compared to a coronavirus expressing a corresponding
wild-type replicase, such that when the virus is administered to an
embryonated egg, it is capable of replicating without being pathogenic
to the embryo.
In a second aspect, the
present invention provides a variant replicase gene as defined in
connection with the first aspect of the invention.
In
a third aspect, the present invention provides a protein encoded by a
variant coronavirus replicase gene according to the second aspect of the
invention.
In a fourth aspect, the
present invention provides a plasmid comprising a replicase gene
according to the second aspect of the invention.
In
a fifth aspect, the present invention provides a method for making the
coronavirus according to the first aspect of the invention which
comprises the following steps:
-
- (i) transfecting a plasmid according to the fourth aspect of the invention into a host cell;
- (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene;
- (iii)
allowing homologous recombination to occur between the replicase gene
sequences in the plasmid and the corresponding sequences in the
recombining virus genome to produce a modified replicase gene; and
- (iv) selecting for recombining virus comprising the modified replicase gene.
The recombining virus may be a vaccinia virus.
The method may also include the step:
-
- (v)
recovering recombinant coronavirus comprising the modified replicase
gene from the DNA from the recombining virus from step (iv).
In
a sixth aspect, the present invention provides a cell capable of
producing a coronavirus according to the first aspect of the invention.
In
a seventh aspect, the present invention provides a vaccine comprising a
coronavirus according to the first aspect of the invention and a
pharmaceutically acceptable carrier.
In an
eighth aspect, the present invention provides a method for treating
and/or preventing a disease in a subject which comprises the step of
administering a vaccine according to the seventh aspect of the invention
to the subject.
Further aspects of the invention provide:
-
- the vaccine according to the seventh aspect of the invention for use in treating and/or preventing a disease in a subject.
- use
of a coronavirus according to the first aspect of the invention in the
manufacture of a vaccine for treating and/or preventing a disease in a
subject.
The disease may be infectious bronchitis (IB).
The
method of administration of the vaccine may be selected from the group
consisting of; eye drop administration, intranasal administration,
drinking water administration, post-hatch injection and in ovo
injection.
Vaccination may be by in ova vaccination.
The
present invention also provides a method for producing a vaccine
according to the seventh aspect of the invention, which comprises the
step of infecting a cell according to the sixth aspect of the invention
with a coronavirus according to the first aspect of the invention.
DESCRIPTION OF THE FIGURES
FIG. 1—Growth kinetics of M41-R-6 and M41-R-12 compared to M41-CK (M41 EP4) on CK cells
FIG. 2—Clinical
signs, snicking and wheezing, associated with M41-R-6 and M41-R-12
compared to M41-CK (M41 EP4) and Beau-R (Bars show mock, Beau-R, M41-R
6, M41-R 12, M41-CK EP4 from left to right of each timepoint).
FIG. 3—Ciliary
activity of the viruses in tracheal rings isolated from tracheas taken
from infected chicks. 100% ciliary activity indicates no effect by the
virus; apathogenic, 0% activity indicates complete loss of ciliary
activity, complete ciliostasis, indicating the virus is pathogenic (Bars
show mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4 from left to right of
each timepoint).
FIG. 4—Clinical
signs, snicking, associated with M41R-nsp10rep and M41R-nsp14,15,16rep
compared to M41-R-12 and M41-CK (M41 EP5) (Bars show mock, M41-R12;
M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 from left to right of
each timepoint).
FIG. 5—Ciliary
activity of M41R-nsp10rep and M41R-nsp14,15,16rep compared to M41-R-12
and M41-CK in tracheal rings isolated from tracheas taken from infected
chicks (Bars show mock; M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and
M41-CK EP5 from left to right of each timepoint).
FIG. 6—Clinical
signs, snicking, associated with M41R-nsp10, 15rep, M41R-nsp10, 14,
15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared
to M41-CK (Bars show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4;
M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from
left to right of each timepoint).
FIG. 7—Clinical
signs, wheezing, associated with M41R-nsp10, 15rep, M41R-nsp10, 14,
15rep, M41R-nsp10, 14, 16rep, M41R-nsp10, 15, 16rep and M41-K compared
to M41-CK (Bars show mock, M41R-nsp10,15rep1; M14R-nsp10,14,16rep4;
M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from
left to right of each timepoint).
FIG. 8—Ciliary
activity of M41R-nsp10, 15rep, M41R-nsp10, 14, 15rep, M41R-nsp10, 14,
16rep, M41R-nsp10, 15, 16rep and M41-K compared to M41-CK in tracheal
rings isolated from tracheas taken from infected chicks (Bars show mock,
M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8;
M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 from left to right of each
timepoint).
FIG. 9—Growth kinetics of rIBVs compared to M41-CK on CK cells. FIG. 9A shows the results for M41-R and M41-K. FIG. 9B
shows the results for M41-nsp10 rep; M41R-nsp14, 15, 16 rep;
M41R-nsp10, 15 rep; M41R-nsp10, 15, 16 rep; M41R-nsp10, 14, 15 rep; and
M41R-nsp10, 14, 16.
FIG. 10—Position of amino acid mutations in mutated nsp10, nsp14, nsp15 and nsp16 sequences.
FIG. 11—A)
Snicking; B) Respiratory symptoms (wheezing and rales combined) and C)
Ciliary activity of rIBV M41R-nsp 10,14 rep and rIBV M41R-nsp 10,16 rep
compared to M41-CK (Bars show mock, M41R-nsp10,14rep; M41R-nsp10,16rep
and M41-K from left to right of each timepoint).
DETAILED DESCRIPTION
The
present invention provides a coronavirus comprising a variant replicase
gene which, when expressed in the coronavirus, causes the virus to have
reduced pathogenicity compared to a corresponding coronavirus which
comprises the wild-type replicase gene.
Coronavirus
Gammacoronavirus is
a genus of animal virus belonging to the family Coronaviridae.
Coronaviruses are enveloped viruses with a positive-sense
single-stranded RNA genome and a helical symmetry.
The
genomic size of coronaviruses ranges from approximately 27 to 32
kilobases, which is the longest size for any known RNA virus.
Coronaviruses
primarily infect the upper respiratory or gastrointestinal tract of
mammals and birds. Five to six different currently known strains of
coronaviruses infect humans. The most publicized human coronavirus,
SARS-CoV which causes severe acute respiratory syndrome (SARS), has a
unique pathogenesis because it causes both upper and lower respiratory
tract infections and can also cause gastroenteritis. Middle East
respiratory syndrome coronavirus (MERS-CoV) also causes a lower
respiratory tract infection in humans. Coronaviruses are believed to
cause a significant percentage of all common colds in human adults.
Coronaviruses
also cause a range of diseases in livestock animals and domesticated
pets, some of which can be serious and are a threat to the farming
industry. Economically significant coronaviruses of livestock animals
include infectious bronchitis virus (IBV) which mainly causes
respiratory disease in chickens and seriously affects the poultry
industry worldwide; porcine coronavirus (transmissible gastroenteritis,
TGE) and bovine coronavirus, which both result in diarrhoea in young
animals. Feline coronavirus has two forms, feline enteric coronavirus is
a pathogen of minor clinical significance, but spontaneous mutation of
this virus can result in feline infectious peritonitis (FIP), a disease
associated with high mortality.
There are
also two types of canine coronavirus (CCoV), one that causes mild
gastrointestinal disease and one that has been found to cause
respiratory disease. Mouse hepatitis virus (MHV) is a coronavirus that
causes an epidemic murine illness with high mortality, especially among
colonies of laboratory mice.
Coronaviruses are divided into four groups, as shown below:
-
- Alpha
- Canine coronavirus (CCoV)
- Feline coronavirus (FeCoV)
- Human coronavirus 229E (HCoV-229E)
- Porcine epidemic diarrhoea virus (PEDV)
- Transmissible gastroenteritis virus (TGEV)
- Human Coronavirus NL63 (NL or New Haven)
- Beta
- Bovine coronavirus (BCoV)
- Canine respiratory coronavirus (CRCoV)—Common in SE Asia and Micronesia
- Human coronavirus OC43 (HCoV-OC43)
- Mouse hepatitis virus (MHV)
- Porcine haemagglutinating encephalomyelitis virus (HEV)
- Rat
coronavirus (Roy). Rat Coronavirus is quite prevalent in Eastern
Australia where, as of March/April 2008, it has been found among native
and feral rodent colonies.
- (No common name as of yet) (HCoV-HKU1)
- Severe acute respiratory syndrome coronavirus (SARS-CoV)
- Middle East respiratory syndrome coronavirus (MERS-CoV)
- Gamma
- Infectious bronchitis virus (IBV)
- Turkey coronavirus (Bluecomb disease virus)
- Pheasant coronavirus
- Guinea fowl coronavirus
- Delta
- Bulbul coronavirus (BuCoV)
- Thrush coronavirus (ThCoV)
- Munia coronavirus (MuCoV)
- Porcine coronavirus (PorCov) HKU15
The
variant replicase gene of the coronavirus of the present invention may
be derived from an alphacoronavirus such as TGEV; a betacoronavirus such
as MHV; or a gammacoronavirus such as IBV.
As
used herein the term “derived from” means that the replicase gene
comprises substantially the same nucleotide sequence as the wild-type
replicase gene of the relevant coronavirus. For example, the variant
replicase gene of the present invention may have up to 80%, 85%, 90%,
95%, 98% or 99% identity with the wild type replicase sequence. The
variant coronavirus replicase gene encodes a protein comprising a
mutation in one or more of non-structural protein (nsp)-10, nsp-14,
nsp-15 or nsp-16 when compared to the wild-type sequence of the
non-structural protein.
IBV
Avian
infectious bronchitis (IB) is an acute and highly contagious
respiratory disease of chickens which causes significant economic
losses. The disease is characterized by respiratory signs including
gasping, coughing, sneezing, tracheal rales, and nasal discharge. In
young chickens, severe respiratory distress may occur. In layers,
respiratory distress, nephritis, decrease in egg production, and loss of
internal egg quality and egg shell quality are common.
In
broilers, coughing and rattling are common clinical signs, rapidly
spreading in all the birds of the premises. Morbidity is 100% in
non-vaccinated flocks. Mortality varies depending on age, virus strain,
and secondary infections but may be up to 60% in non-vaccinated flocks.
The
first IBV serotype to be identified was Massachusetts, but in the
United States several serotypes, including Arkansas and Delaware, are
currently circulating, in addition to the originally identified
Massachusetts type.
The IBV strain
Beaudette was derived following at least 150 passages in chick embryos.
IBV Beaudette is no longer pathogenic for hatched chickens but rapidly
kills embryos.
H120 is a commercial live
attenuated IBV Massachusetts serotype vaccine strain, attenuated by
approximately 120 passages in embryonated chicken eggs. H52 is another
Massachusetts vaccine, and represents an earlier and slightly more
pathogenic passage virus (passage 52) during the development of H120.
Vaccines based on H120 are commonly used.
IB
QX is a virulent field isolate of IBV. It is sometimes known as
“Chinese QX” as it was originally isolated following outbreaks of
disease in the Qingdao region in China in the mid 1990s. Since that time
the virus has crept towards Europe. From 2004, severe egg production
issues have been identified with a very similar virus in parts of
Western Europe, predominantly in the Netherlands, but also reported from
Germany, France, Belgium, Denmark and in the UK.
The
virus isolated from the Dutch cases was identified by the Dutch
Research Institute at Deventer as a new strain that they called D388.
The Chinese connection came from further tests which showed that the
virus was 99% similar to the Chinese QX viruses. A live attenuated
QX-like IBV vaccine strain has now been developed.
IBV
is an enveloped virus that replicates in the cell cytoplasm and
contains an non-segmented, single-stranded, positive sense RNA genome.
IBV has a 27.6 kb RNA genome and like all coronaviruses contains the
four structural proteins; spike glycoprotein (S), small membrane protein
(E), integral membrane protein (M) and nucleocapsid protein (N) which
interacts with the genomic RNA.
The genome
is organised in the following manner: 5′UTR—polymerase (replicase)
gene—structural protein genes (S-E-M-N)—UTR 3′; where the UTR are
untranslated regions (each ˜500 nucleotides in IBV).
The
lipid envelope contains three membrane proteins: S, M and E. The IBV S
protein is a type I glycoprotein which oligomerizes in the endoplasmic
reticulum and is assembled into homotrimer inserted in the virion
membrane via the transmembrane domain and is associated through
non-covalent interactions with the M protein. Following incorporation
into coronavirus particles, the S protein is responsible for binding to
the target cell receptor and fusion of the viral and cellular membranes.
The S glycoprotein consists of four domains: a signal sequence that is
cleaved during synthesis; the ectodomain, which is present on the
outside of the virion particle; the transmembrane region responsible for
anchoring the S protein into the lipid bilayer of the virion particle;
and the cytoplasmic tail.
All
coronaviruses also encode a set of accessory protein genes of unknown
function that are not required for replication in vitro, but may play a
role in pathogenesis. IBV encodes two accessory genes, genes 3 and 5,
which both express two accessory proteins 3a, 3b and 5a, 5b,
respectively.
The variant replicase gene
of the coronavirus of the present invention may be derived from an IBV.
For example the IBV may be IBV Beaudette, H120, H52, IB QX, D388 or M41.
The
IBV may be IBV M41. M41 is a prototypic Massachusetts serotype that was
isolated in the USA in 1941. It is an isolate used in many labs
throughout the world as a pathogenic lab stain and can be obtained from
ATCC (VR-21™). Attenuated variants are also used by several vaccine
producers as IBV vaccines against Massachusetts serotypes causing
problems in the field. The present inventors chose to use this strain as
they had worked for many years on this virus, and because the sequence
of the complete virus genome is available. The M41 isolate, M41-CK, used
by the present inventors was adapted to grow in primary chick kidney
(CK) cells and was therefore deemed amenable for recovery as an
infectious virus from a cDNA of the complete genome. It is
representative of a pathogenic IBV and therefore can be analysed for
mutations that cause either loss or reduction in pathogenicity.
The genome sequence of IBV M41-CK is provided as SEQ ID NO: 1.
IBV M41-CK Sequence
Reduced Pathogenicity
The
live, attenuated coronavirus of the present invention comprises a
variant replicase gene which causes the virus to have reduced
pathogenicity compared to a coronavirus expressing the corresponding
wild-type gene.
The term “attenuated” as
used herein, refers to a virus that exhibits said reduced pathogenicity
and may be classified as non-virulent. A live, attenuated virus is a
weakened replicating virus still capable of stimulating an immune
response and producing immunity but not causing the actual illness.
The
term “pathogenicity” is used herein according to its normal meaning to
refer to the potential of the virus to cause disease in a subject.
Typically the pathogenicity of a coronavirus is determined by assaying
disease associated symptoms, for example sneezing, snicking and
reduction in tracheal ciliary activity.
The
term “reduced pathogenicity” is used to describe that the level of
pathogenicity of a coronavirus is decreased, lessened or diminished
compared to a corresponding, wild-type coronavirus.
In
one embodiment, the coronavirus of the present invention has a reduced
pathogenicity compared to the parental M41-CK virus from which it was
derived or a control coronavirus. The control coronavirus may be a
coronavirus with a known pathogenicity, for example a coronavirus
expressing the wild-type replicase protein.
The
pathogenicity of a coronavirus may be assessed utilising methods
well-known in the art. Typically, pathogenicity is assessed by assaying
clinical symptoms in a subject challenged with the virus, for example a
chicken.
As an illustration, the chicken
may be challenged at 8-24 days old by nasal or ocular inoculation.
Clinical symptoms, associated with IBV infection, may be assessed 3-10
days post-infection. Clinical symptoms commonly assessed to determine
the pathogenicity of a coronavirus, for example an IBV, include gasping,
coughing, sneezing, snicking, depression, ruffled feathers and loss of
tracheal ciliary activity.
The variant
replicase of the present invention, when expressed in a coronavirus, may
cause a reduced level of clinical symptoms compared to a coronavirus
expressing a wild-type replicase.
For
example a coronavirus expressing the variant replicase may cause a
number of snicks per bird per minute which is less than 90%, less than
80%, less than 70%, less than 60%, less than 50%, less than 40%, less
than 30%, less than 20% or less than 10% of the number of snicks caused
by a virus expressing the wild type replicase.
A
coronavirus expressing a variant replicase according to the present
invention may cause wheezing in less than 70%, less than 60%, less than
50%, less than 40%, less than 30%, less than 20% or less than 10% of the
number of birds in a flock infected with the a virus expressing the
wild type replicase.
A coronavirus
expressing a variant replicase according to the present invention may
result in tracheal ciliary activity which is at least 60%, at least 70%,
at least 80%, at least 90% or at least 95% of the level of tracheal
ciliary activity in uninfected birds.
A
coronavirus expressing a variant replicase according to the present
invention may cause clinical symptoms, as defined in Table 2, at a lower
level than a coronavirus expressing the wild type replicase.
TABLE 2 IBV severity limits based on clinical signs:
The
variant replicase of the present invention, when expressed in a
coronavirus, may cause the virus to replicate at non-pathogenic levels
in ovo.
While developing vaccines to be
administered in ovo to chicken embryos, attention must be paid to two
points: the effect of maternal antibodies on the vaccines and the effect
of the vaccines on the embryo. Maternal antibodies are known to
interfere with active immunization. For example, vaccines with mild
strains do not induce protective antibody levels when administered to
broiler chickens with maternal antibodies as these strains are
neutralized by the maternal antibody pool.
Thus
a viral particle must be sufficiently efficient at replicating and
propagating to ensure that it is not neutralized by the
maternally-derived antibodies against the virus. Maternally-derived
antibodies are a finite pool of effective antibodies, which decrease as
the chicken ages, and neutralization of the virus in this manner does
not equate to the establishment of long-term immunity for the
embryo/chick. In order to develop long-term immunity against the virus,
the embryo and hatched chicken must develop an appropriate protective
immune response which is distinct to the effect of the
maternally-derived antibodies.
To be
useful for in ovo vaccination, the virus must also not replicate and
propagate at a level which causes it to be pathogenic to the embryo.
Reduced
pathogenicity in terms of the embryo may mean that the coronavirus
causes less reduction in hatchability compared to a corresponding,
wild-type control coronavirus. Thus the term “without being pathogenic
to the embryo” in the context of the present invention may mean “without
causing reduced hatchability” when compared to a control coronavirus.
A
suitable variant replicase may be identified using methods which are
known in the art. For example comparative challenge experiments
following in ovo vaccination of embryos with or without
maternally-derived antibodies may be performed (i.e. wherein the layer
has or has not been vaccinated against IBV).
If
the variant replicase enables the virus to propagate at a level which
is too high, the embryo will not hatch or will not be viable following
hatching (i.e. the virus is pathogenic to the embryo). A virus which is
pathogenic to the embryo may kill the embryo.
If
the variant replicase causes a reduction in viral replication and
propagation which is too great, the virus will be neutralised by the
maternally-derived antibodies. Subsequent challenge of the chick with
IBV will therefore result in the development of clinical symptoms (for
example wheezing, snicking, loss of ciliary activity) and the onset of
disease in the challenged chick; as it will have failed to develop
effective immunity against the virus.
Variant
As
used herein, the term ‘variant’ is synonymous with ‘mutant’ and refers
to a nucleic acid or amino acid sequence which differs in comparison to
the corresponding wild-type sequence.
A
variant/mutant sequence may arise naturally, or may be created
artificially (for example by site-directed mutagenesis). The mutant may
have at least 70, 80, 90, 95, 98 or 99% sequence identity with the
corresponding portion of the wild type sequence. The mutant may have
less than 20, 10, 5, 4, 3, 2 or 1 mutation(s) over the corresponding
portion of the wild-type sequence.
The
term “wild type” is used to mean a gene or protein having a nucleotide
or amino acid sequence which is identical with the native gene or
protein respectively (i.e. the viral gene or protein).
Identity
comparisons can be conducted by eye, or more usually, with the aid of
readily available sequence comparison programs. These commercially
available computer programs can calculate % identity between two or more
sequences. A suitable computer program for carrying out such an
alignment is the GCG Wisconsin Bestfit package (University of Wisconsin,
U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples
of other software that can perform sequence comparisons include, but
are not limited to, the BLAST package (see Ausubel et al., 1999
ibid—Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410)
and the GENEWORKS suite of comparison tools, ClustalX (see Larkin et al.
(2007) Clustal W and Clustal X version 2.0. Bioinformatics,
23:2947-2948). Both BLAST and FASTA are available for offline and online
searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However,
for some applications, it is preferred to use the GCG Bestf it program.
A new tool, called BLAST 2 Sequences is also available for comparing
protein and nucleotide sequence (see FEMS Microbiol Lett 1999 174(2):
247-50; FEMS Microbiol Lett 1999 177(1): 187-8 and
tatiana@ncbi.nlm.nih.gov).
The sequence
may have one or more deletions, insertions or substitutions of amino
acid residues which produce a silent change and result in a functionally
equivalent molecule. Deliberate amino acid substitutions may be made on
the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the
residues as long as the activity is retained. For example, negatively
charged amino acids include aspartic acid and glutamic acid; positively
charged amino acids include lysine and arginine; and amino acids with
uncharged polar head groups having similar hydrophilicity values include
leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine,
serine, threonine, phenylalanine, and tyrosine.
Conservative
substitutions may be made, for example according to the Table below.
Amino acids in the same block in the second column and preferably in the
same line in the third column may be substituted for each other:
ALIPHATIC Non-polar G A P I L V Polar- uncharged C S T M N Q Polar- charged D E K R AROMATIC H F W Y
The
coronavirus of the present invention may comprise a variant replicase
gene which encodes a protein which comprises a mutation compared to any
one of SEQ ID NO: 6, 7, 8 or 9 which, when expressed in a coronavirus,
causes the virus to have reduced pathogenicity compared to a coronavirus
expressing the corresponding wild-type replicase.
The
variant replicase gene may encode a protein which comprises at least
one or more amino acid mutations in any combination of nsp-10, nsp-14,
nsp-15 and nsp-16.
The variant replicase
gene of the coronavirus of the present invention may encode a protein
comprising a mutation as defined in the M41 mod sequences presented in FIG. 10.
The
variant replicase gene of the coronavirus of the present invention may
encode a protein which comprises one or more amino acid mutations
selected from the list of:
-
- Pro to Leu at position 85 of SEQ ID NO: 6,
- Val to Leu at position 393 of SEQ ID NO: 7;
- Leu to Ile at position 183 of SEQ ID NO: 8;
- Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene of the coronavirus of the present invention may
encode a protein which does not comprise a mutation in nsp-2, nsp-3,
nsp-6 or nsp-13.
The variant replicase
gene of the coronavirus of the present invention may encode a protein
which does not comprise a mutation in nsp10 which corresponds to the
threonine to isoleucine mutation caused by a mutation at nucleotide
position 12,008 in the gene reported by Ammayappan et al. (Arch Virol
(2009) 154:495-499).
Ammayappan et al (as
above) reports the identification of sequence changes responsible for
the attenuation of IBV strain Arkansas DPI. The study identified 17
amino acid changes in a variety of IBV proteins following multiple
passages, approx. 100, of the virus in embryonated eggs. It was not
investigated whether the attenuated virus (Ark DPI 101) is capable of
replicating in the presence of maternally-derived antibodies against the
virus in ovo, without being pathogenic to the embryo. Given that this
virus was produced by multiple passage in SPF embryonated eggs, similar
methodology for classical IBV vaccines, it is likely that this virus is
pathogenic for embryos. The virus may also be sensitive to
maternally-derived antibodies if the hens were vaccinated with a similar
serotype.
The variant replicase gene of
the coronavirus of the present invention may encode a protein which
comprises any combination of one or more amino acid mutations provided
in the list above.
The variant replicase
gene may encode a protein which comprises the amino acid mutation Pro to
Leu at position 85 of SEQ ID NO: 6.
The
variant replicase gene may encode a protein which comprises the amino
acid mutation Val to Leu at position 393 of SEQ ID NO: 7.
The
variant replicase gene may encode a protein which comprises the amino
acid mutation Leu to Ile at position 183 of SEQ ID NO: 8.
The
variant replicase gene may encode a protein which comprises the amino
acid mutation Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the amino
acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, and Val to Leu
at position 393 of SEQ ID NO: 7.
The
variant replicase gene may encode a protein which comprises the amino
acid mutations Pro to Leu at position 85 of SEQ ID NO: 6 Leu to Ile at
position 183 of SEQ ID NO: 8.
The variant
replicase gene may encode a protein which comprises the amino acid
mutations Pro to Leu at position 85 of SEQ ID NO: 6 and Val to Ile at
position 209 of SEQ ID NO: 9.
The variant
replicase gene may encode a protein which comprises the amino acid
mutations Val to Leu at position 393 of SEQ ID NO: 7 and Leu to Ile at
position 183 of SEQ ID NO: 8.
The variant
replicase gene may encode a protein which comprises the amino acid
mutations Val to Leu at position 393 of SEQ ID NO: 7 and Val to Ile at
position 209 of SEQ ID NO: 9.
The variant
replicase gene may encode a protein which comprises the amino acid
mutations Leu to Ile at position 183 of SEQ ID NO: 8 and Val to Ile at
position 209 of SEQ ID NO: 9.
The variant
replicase gene may encode a protein which comprises the amino acid
mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at
position 393 of SEQ ID NO: 7 and Leu to Ile at position 183 of SEQ ID
NO: 8.
The variant replicase gene may
encode a protein which comprises the amino acid mutations Pro to Leu at
position 85 of SEQ ID NO: 6 Leu to Ile at position 183 of SEQ ID NO: 8
and Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the amino
acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at
position 393 of SEQ ID NO: 7 and Val to Ile at position 209 of SEQ ID
NO: 9.
The variant replicase gene may
encode a protein which comprises the amino acid mutations Val to Leu at
position 393 of SEQ ID NO: 7, Leu to Ile at position 183 of SEQ ID NO: 8
and Val to Ile at position 209 of SEQ ID NO: 9.
The
variant replicase gene may encode a protein which comprises the amino
acid mutations Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at
position 393 of SEQ ID NO: 7, Leu to Ile at position 183 of SEQ ID NO: 8
and Val to Ile at position 209 of SEQ ID NO: 9.
The variant replicase gene may also be defined at the nucleotide level.
For
example the nucleotide sequence of the variant replicase gene of the
coronavirus of the present invention may comprise one or more nucleotide
substitutions within the regions selected from the list of:
11884-12318, 16938-18500, 18501-19514 and 19515-20423 of SEQ ID NO:1.
For
example the nucleotide sequence of the variant replicase gene of the
coronavirus of the present invention may comprise one or more nucleotide
substitutions selected from the list of:
-
- C to Tat nucleotide position 12137;
- G to C at nucleotide position 18114;
- T to A at nucleotide position 19047; and
- G to A at nucleotide position 20139;
compared to the sequence shown as SEQ ID NO: 1.
As
used herein, the term “substitution” is synonymous with the term
mutation and means that the nucleotide at the identified position
differs to that of the wild-type nucleotide sequence.
The nucleotide sequence may comprise any combination of the nucleotide substitutions selected from the list of:
-
- C to Tat nucleotide position 12137;
- G to Cat nucleotide position 18114;
- T to A at nucleotide position 19047; and
- G to A at nucleotide position 20139;
compared to the sequence shown as SEQ ID NO: 1.
The nucleotide sequence may comprise the substitution C12137T.
The nucleotide sequence may comprise substitution G18114C.
The nucleotide sequence may comprise the substitution T19047A.
The nucleotide sequence may comprise the substitution G20139A.
The nucleotide sequence may comprise the substitutions C12137T and G18114C.
The nucleotide sequence may comprise the substitutions C12137T and T19047A.
The nucleotide sequence may comprise the substitutions C12137T and G20139A.
The nucleotide sequence may comprise the substitutions G18114C and T19047A.
The nucleotide sequence may comprise the substitutions G18114C and G20139A.
The nucleotide sequence may comprise the substitutions T19047A and G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C and T19047A.
The nucleotide sequence may comprise the substitutions C12137T, T19047A and G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C and G20139A.
The nucleotide sequence may comprise the substitutions G18114C, T19047A and G20139A.
The nucleotide sequence may comprise the substitutions C12137T, G18114C, T19047A and G20139A.
The
nucleotide sequence may not comprise a substitution which corresponds
to the C12008T substitution reported by Ammayappan et al. (as above).
The
nucleotide sequence may be natural, synthetic or recombinant. It may be
double or single stranded, it may be DNA or RNA or combinations
thereof. It may, for example, be cDNA, PCR product, genomic sequence or
mRNA.
The nucleotide sequence may be codon optimised for production in the host/host cell of choice.
It may be isolated, or as part of a plasmid, virus or host cell.
Plasmid
A
plasmid is an extra-chromosomal DNA molecule separate from the
chromosomal DNA which is capable of replicating independently of the
chromosomal DNA. They are usually circular and double-stranded.
Plasmids,
or vectors (as they are sometimes known), may be used to express a
protein in a host cell. For example a bacterial host cell may be
transfected with a plasmid capable of encoding a particular protein, in
order to express that protein. The term also includes yeast artificial
chromosomes and bacterial artificial chromosomes which are capable of
accommodating longer portions of DNA.
The
plasmid of the present invention comprises a nucleotide sequence capable
of encoding a defined region of the replicase protein. It may also
comprise one or more additional coronavirus nucleotide sequence(s), or
nucleotide sequence(s) capable of encoding one or more other coronavirus
proteins such as the S gene and/or gene 3.
The plasmid may also comprise a resistance marker, such as the guanine xanthine phosphoribosyltransferase gene (gpt) from Escherichia coli,
which confers resistance to mycophenolic acid (MPA) in the presence of
xanthine and hypoxanthine and is controlled by the vaccinia virus P7.5
early/late promoter.
Recombinant Vaccinia Virus
The present invention also relates to a recombinant vaccinia virus (rVV) comprising a variant replicase gene as defined herein.
The recombinant vaccinia virus (rVV) may be made using a vaccinia-virus based reverse genetics system.
In this respect, the present invention also provides a method for making a viral particle by:
-
- (i) transfecting a plasmid as described in the previous section into a host cell;
- (ii) infecting the host cell with a recombining virus comprising the genome of a coronavirus strain with a replicase gene;
- (iii)
allowing homologous recombination to occur between the replicase gene
sequences in the plasmid and the corresponding sequences in the
recombining virus genome to produce a modified replicase gene;
- (iv) selecting for recombining virus comprising the modified replicase gene.
The
term ‘modified replicase gene’ refers to a replicase gene which
comprises a variant replicase gene as described in connection with the
first aspect of the present invention. Specifically, the term refers to a
gene which is derived from a wild-type replicase gene but comprises a
nucleotide sequence which causes it to encode a variant replicase
protein as defined herein.
The
recombination may involve all or part of the replicase gene. For example
the recombination may involve a nucleotide sequence encoding for any
combination of nsp-10, nsp-14, nsp-15 and/or nsp-16. The recombination
may involve a nucleotide sequence which encodes for an amino acid
mutation or comprises a nucleotide substitution as defined above.
The
genome of the coronavirus strain may lack the part of the replicase
protein corresponding to the part provided by the plasmid, so that a
modified protein is formed through insertion of the nucleotide sequence
provided by the plasmid.
The recombining
virus is one suitable to allow homologous recombination between its
genome and the plasmid. The vaccinia virus is particularly suitable as
homologous recombination is routinely used to insert and delete
sequences for the vaccinia virus genome.
The above method optionally includes the step:
-
- (v)
recovery of recombinant coronavirus comprising the modified replicase
gene from the DNA from the recombining virus from step (iv).
Methods
for recovering recombinant coronavirus, such as recombinant IBV, are
known in the art (See Britton et al (2005) see page 24; and
PCT/GB2010/001293).
For example, the DNA
from the recombining virus from step (iv) may be inserted into a plasmid
and used to transfect cells which express cytoplasmic T7 RNA
polymerase. The cells may, for example be pre-infected with a fowlpox
virus expressing T7 RNA polymerase. Recombinant coronavirus may then be
isolated, for example, from the growth medium.
When
the plasmid is inserted into the vaccinia virus genome, an unstable
intermediate is formed. Recombinants comprising the plasmid may be
selected for e.g. using a resistance marker on the plasmid.
Positive recombinants may then be verified to contain the modified replicase gene by, for example, PCR and sequencing.
Large
stocks of the recombining virus including the modified replicase gene
(e.g. recombinant vaccinia virus, (rVV) may be grown up and the DNA
extracted in order to carry out step (v)).
Suitable
reverse genetics systems are known in the art (Casais et al (2001) J.
Virol 75:12359-12369; Casais et al (2003) J. Virol. 77:9084-9089;
Britton et al (2005) J. Virological Methods 123:203-211; Armesto et al
(2008) Methods in Molecular Biology 454:255-273).
Cell
The coronavirus may be used to infect a cell.
Coronavirus particles may be harvested, for example from the supernatant, by methods known in the art, and optionally purified.
The cell may be used to produce the coronavirus particle.
Thus the present invention also provides a method for producing a coronavirus which comprises the following steps:
(i) infection of a cell with a coronavirus according to the invention;
(ii) allowing the virus to replicate in the cell; and
(iii) harvesting the progeny virus.
The
present invention also provides a cell capable of producing a
coronavirus according to the invention using a reverse genetics system.
For example, the cell may comprise a recombining virus genome comprising
a nucleotide sequence capable of encoding the replicase gene of the
present invention.
The cell may be able to produce recombinant recombining virus (e.g. vaccinia virus) containing the replicase gene.
Alternatively
the cell may be capable of producing recombinant coronavirus by a
reverse genetics system. The cell may express or be induced to express
T7 polymerase in order to rescue the recombinant viral particle.
Vaccine
The
coronavirus may be used to produce a vaccine. The vaccine may by a live
attenuated form of the coronavirus of the present invention and may
further comprise a pharmaceutically acceptable carrier. As defined
herein, “pharmaceutically acceptable carriers” suitable for use in the
invention are well known to those of skill in the art. Such carriers
include, without limitation, water, saline, buffered saline, phosphate
buffer, alcohol/aqueous solutions, emulsions or suspensions. Other
conventionally employed diluents and excipients may be added in
accordance with conventional techniques. Such carriers can include
ethanol, polyols, and suitable mixtures thereof, vegetable oils, and
injectable organic esters. Buffers and pH adjusting agents may also be
employed. Buffers include, without limitation, salts prepared from an
organic acid or base. Representative buffers include, without
limitation, organic acid salts, such as salts of citric acid, e.g.,
citrates, ascorbic acid, gluconic acid, histidine-Hel, carbonic acid,
tartaric acid, succinic acid, acetic acid, or phthalic acid, Iris,
trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers
can include sodium chloride solution, Ringer's dextrose, dextrose,
trehalose, sucrose, and sodium chloride, lactated Ringer's or fixed
oils. Intravenous carriers can include fluid and nutrient replenishers,
electrolyte replenishers, such as those based on Ringer's dextrose and
the like. Preservatives and other additives such as, for example,
antimicrobials, antioxidants, chelating agents (e.g., EDTA), inert gases
and the like may also be provided in the pharmaceutical carriers. The
present invention is not limited by the selection of the carrier. The
preparation of these pharmaceutically acceptable compositions, from the
above-described components, having appropriate pH isotonicity, stability
and other conventional characteristics is within the skill of the art.
See, e.g., texts such as Remington: The Science and Practice of
Pharmacy, 20th ed, Lippincott Williams & Wilkins, pub!., 2000; and
The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C.
Rowe et al, APhA Publications, 2003.
The
vaccine of the invention will be administered in a “therapeutically
effective amount”, which refers to an amount of an active ingredient,
e.g., an agent according to the invention, sufficient to effect
beneficial or desired results when administered to a subject or patient.
An effective amount can be administered in one or more administrations,
applications or dosages. A therapeutically effective amount of a
composition according to the invention may be readily determined by one
of ordinary skill in the art. In the context of this invention, a
“therapeutically effective amount” is one that produces an objectively
measured change in one or more parameters associated Infectious
Bronchitis condition sufficient to effect beneficial or desired results.
An effective amount can be administered in one or more administrations.
For purposes of this invention, an effective amount of drug, compound,
or pharmaceutical composition is an amount sufficient to reduce the
incidence of Infectious Bronchitis. As used herein, the term
“therapeutic” encompasses the full spectrum of treatments for a disease,
condition or disorder. A “therapeutic” agent of the invention may act
in a manner that is prophylactic or preventive, including those that
incorporate procedures designed to target animals that can be identified
as being at risk (pharmacogenetics); or in a manner that is
ameliorative or curative in nature; or may act to slow the rate or
extent of the progression of at least one symptom of a disease or
disorder being treated.
The present
invention also relates to a method for producing such a vaccine which
comprises the step of infecting cells, for example Vero cells, with a
viral particle comprising a replicase protein as defined in connection
with the first aspect of the invention.
Vaccination Method
The coronavirus of the present invention may be used to treat and/or prevent a disease.
To
“treat” means to administer the vaccine to a subject having an existing
disease in order to lessen, reduce or improve at least one symptom
associated with the disease and/or to slow down, reduce or block the
progression of the disease.
To “prevent”
means to administer the vaccine to a subject who has not yet contracted
the disease and/or who is not showing any symptoms of the disease to
prevent or impair the cause of the disease (e.g. infection) or to reduce
or prevent development of at least one symptom associated with the
disease.
The disease may be any disease
caused by a coronavirus, such as a respiratory disease and and/or
gastroenteritis in humans and hepatitis, gastroenteritis, encephalitis,
or a respiratory disease in other animals.
The
disease may be infectious bronchitis (IB); Porcine epidemic diarrhoea;
Transmissible gastroenteritis; Mouse hepatitis virus; Porcine
haemagglutinating encephalomyelitis; Severe acute respiratory syndrome
(SARS); or Bluecomb disease.
The disease may be infectious bronchitis.
The
vaccine may be administered to hatched chicks or chickens, for example
by eye drop or intranasal administration. Although accurate, these
methods can be expensive e.g. for large broiler flocks. Alternatives
include spray inoculation of administration to drinking water but it can
be difficult to ensure uniform vaccine application using such methods.
The vaccine may be provided in a form suitable for its administration, such as an eye-dropper for intra-ocular use.
The
vaccine may be administered by in ovo inoculation, for example by
injection of embryonated eggs. In ovo vaccination has the advantage that
it provides an early stage resistance to the disease. It also
facilitates the administration of a uniform dose per subject, unlike
spray inoculation and administration via drinking water.
The
vaccine may be administered to any suitable compartment of the egg,
including allantoic fluid, yolk sac, amnion, air cell or embryo. It may
be administered below the shell (aircell) membrane and chorioallantoic
membrane.
Usually the vaccine is injected
into embryonated eggs during late stages of embryonic development,
generally during the final quarter of the incubation period, such as 3-4
days prior to hatch. In chickens, the vaccine may be administered
between day 15-19 of the 21-day incubation period, for example at day 17
or 18.
The process can be automated using a robotic injection process, such as those described in WO 2004/078203.
The
vaccine may be administered together with one or more other vaccines,
for example, vaccines for other diseases, such as Newcastle disease
virus (NDV). The present invention also provides a vaccine composition
comprising a vaccine according to the invention together with one or
more other vaccine(s). The present invention also provides a kit
comprising a vaccine according to the invention together with one or
more other vaccine(s) for separate, sequential or simultaneous
administration.
The vaccine or vaccine
composition of the invention may be used to treat a human, animal or
avian subject. For example, the subject may be a chick, chicken or mouse
(such as a laboratory mouse, e.g. transgenic mouse).
Typically,
a physician or veterinarian will determine the actual dosage which will
be most suitable for an individual subject or group of subjects and it
will vary with the age, weight and response of the particular
subject(s).
The composition may optionally
comprise a pharmaceutically acceptable carrier, diluent, excipient or
adjuvant. The choice of pharmaceutical carrier, excipient or diluent can
be selected with regard to the intended route of administration and
standard pharmaceutical practice. The pharmaceutical compositions may
comprise as (or in addition to) the carrier, excipient or diluent, any
suitable binder(s), lubricant(s), suspending agent(s), coating agent(s),
solubilising agent(s), and other carrier agents that may aid or
increase the delivery or immunogenicity of the virus.
The
invention will now be further described by way of Examples, which are
meant to serve to assist one of ordinary skill in the art in carrying
out the invention and are not intended in any way to limit the scope of
the invention.
https://patents.justia.com/patent/10130701
