Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Novel Cross Protective Vaccine Compositions for Porcine Epidemic Diarrhea
Virus
Field of the Invention
The present invention is directed to novel vaccine compositions that protect
swine
from disease caused by porcine epidemic diarrhea virus (PEDV). The vaccine
compositions
are both safe and efficacious, and are based on novel attenuates of PEDV that
provide cross
protection against a wide variety of PEDV strains, and are safe to use even in
live form.
Backdround of the Invention
Porcine epidemic diarrhea (PED) is highly contagious and is characterized by
dehydration, diarrhea, and high mortality in swine, particularly young
piglets. The causative
agent, porcine epidemic diarrhea virus (PEDV), is a single stranded, positive
sense RNA
virus identified to the Alphacoronoavirus genus of the family Coronaviridae.
PEDV has a
total genome size of approximately 28kb and contains 7 open reading frames.
Symptoms of
PEDV infection are often similar to those caused by transmissible
gastroenteritis virus
(TGEV), also a member of the Coronaviridae. It should be noted that cross
protection
between PEDV and TGEV is not generally observed, the overall viral nucleotide
sequences
being at most about 60% similar.
PED was likely first observed in Europe circa 1970, and the causative virus
was
subsequently characterized (see for example M. Pensaert et al. Arch. Virol, v.
58, pp 243-
247, 1978 and D. Chasey et al., Res. Vet Sci, v. 25, pp 255-256, 1978). PED
disease was
generally considered unknown in North America until 2013, at which point
widespread
outbreaks commenced, and severe economic losses to the swine industry
resulted. These
prototype North American isolates (year 2013 and thereafter) have remained
genetically
closely related (i.e. with overall nucleotide identity generally over 99%),
and are similar to
Asian strains characterized there within a few years prior to the North
American outbreaks.
PEDV generally grows poorly in culture, and there is a need to identify both
particular strains
and culture conditions that are appropriate for the culturing of sufficient
virus for commercial
vaccine preparation. Additionally, there is a need to develop vaccines that
provide effective
cross protection against known isolates of PEDV, and which are expected to
provide
effective cross protection against evolving, non-prototype PEDV strains.
Additionally, additional variant strains of PEDV (termed "variant" or "I NDEL"
strains,
see immediately below) have also been recently identified in North America and
Europe, and
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are among themselves closely related, and are recognizably different from the
aforementioned "prototype" North American strains and classical European
strains. Such
variant strains (based in part on "S" or spike protein sequence) also appeared
earlier in Asia,
and these Asian isolates are again more similar North American or European
variant/IN DEI
strains than to prototype strains. A well known North American prototype
strain, associated
with outbreaks of disease beginning in 2013 is USA/Colorado/2013, whose
sequence is
deposited as GenBank accession No. KF272920, of the NCB! of the United States
National
Institutes of Health. Bethesda, MD). In this regard, see also A. Vlasova et
al. "Distinct
Characteristics and Complex Evolution of PEDV Strains, North America, May 2013-
February
2014", Emerging Infectious Disease, Vol 20, No. 10, 2014. Original reports
from Asia of
variant/ I NDEL strains include D. S. Song et al., Research in Veterinary
Science, v 82, pp.
134-140, 2007; S-J Park et al., Virus Genesõ v 35, pp..55-64, 2007; and
further discussion
thereof by D. Song et al. (Virus Genes (2012) v 44 pp. 167-175) referring to
the DR13 strain,
passaged to level 100, and previously licensed in Korea (see also KR patent
0502008).
Accordingly, there is a need to identity both vaccine strains and appropriate
vaccine
compositions that will be effective against current and emerging worldwide
outbreaks of
PEDV, thus providing needed cross protection.
Further concerning the recent emergence of variant/ I NDEL strains throughout
the
world, a very early report of such a North American variant strain is PEDV-IN
DEL (0H851)
.. first isolated by the Ohio Department of Agriculture (see L. Wang et al.,
Emerg. Infect. Dis.,
2014, v. 20, pp. 917-919, see GenBank KJ399978). 0H851 is reproduced as SEQ ID
NO:7
in the present patent application. Typically, it appears that circulating
variant strains
(whether Asian, North American or European) have, as one feature, insertions
and deletions
in the spike gene (S-INDELS), but such strains nonetheless share about 98-100%
identity at
.. a nucleotide level (spike gene, and the overall genome), but such recent
isolates only
present about 96-97% identity, or lower, at the nucleotide level (spike gene
and overall
genome) with initial (prototype) North American strains (for example
USA/Colorado/2013).
The very first public disclosure of North American S-INDELs may be that of the
Iowa
State University Veterinary Diagnostic Laboratory, on January 30, 2014,
defined such
.. isolates as having having only 93.9-94.6% identity to previously identified
USA prototype
strains, but being nearly identical (99+%) to each other. Useful insertions
and deletions need
not be confined to the spike gene. ORF3 modifications (particularly mutations
causing
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truncation of the encoded protein) have been correlated with adaptation to
cell culture and
reduction of pathogenicity. See S-J. Park etal., Virus Genes, 2008, v 36, pp.
95-104. Others
have commented that classification of PEDVs based on ORF3 may be appropriate
(J. Zhang
et al. Journal of Clinical Microbiology, v. 52(9), pp. 3511-3514, 2014). T.
Oka et al.,
Veterinary Microbiology, 173, pp 258-269 (2014) disclose additional S-INDEL
strains, and
interestingly, a PEDV strain related instead to prototype virulent strains,
but also bearing a
large 197 amino acid deletion from the S protein, possibly resulting from
passaging.
I NDEL-type strains that have recently been reported from Europe include those
described by S. Theuns et al. (2015). "Complete genome sequence of a porcine
epidemic
diarrhea virus from a novel outbreak in Belgium, January 2015." Genome
Announcements
3(3), pp. 1-2, May/June 2015; J. Stadler, et al., "Emergence of porcine
epidemic diarrhea
virus in southern Germany." BMC Veterinary Research, v 11 No.142; pages 1-8,
2015; and.
B. Grasland, et al. "Complete genome sequence of a porcine epidemic diarrhea S-
Gene
Indel strain isolated in France in December 2014." Genome Announcements 3(3),
pp. 1-2,
May/June 2015.
It should be noted that variant and prototype strains are co-circulating in
North
America, and elsewhere, and existing populations of strains may result from
multiple
transmissions across continents or other regions.
Although the so-called variant strains may be less virulent than prototype
strains, at
least as to some age groups of swine, remaining virulence still makes such
viruses unsafe
for use in vaccines, if used in live form. Generally to date, attempts to
passage prototype
strains to avirulence have not been successful, and adequate safety is not
achieved after
well over 100 passages. The present invention is directed to novel mutant
isolates of the
variant European strain Calaf14 (see SEQ ID NO:1 herein, and PCT/US
2015/039475
generally, in regard of the wild type) that have been attenuated so that they
can be safely
administered to swine of all ages, without harm to the animals, and at the
same time, are
highly immunogenic and cross protective against subsequent challenge of the
animals by a
wide variety of PEDV strains, including both prototype and variant strains
from all Continents.
Vaccines from such mutant isolates, whether in killed or live form, are useful
to protect swine
from PEDV on a worldwide basis.
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Summary of the Invention
The present invention is directed to novel mutant isolates of Calf 14 PEDV
virus that
can be safely administered to swine of all ages, without harm to the animals,
and at the same
time, are highly immunogenic and cross protective against subsequent challenge
of the
animals by a wide variety of PEDV strains, including both prototype and
variant strains.
Vaccines from such mutant isolates, whether in killed or live form, are useful
to protect swine
from PEDV on a worldwide basis.
The present invention therefore encompasses an vaccine composition comprising
inactivated mutant Calaf14 PEDV, one or more adjuvants, and optionally one or
more
excipients, in an amount effective to elicit production of neutralizing
antibodies in swine, with
good cross protection against subsequent challenge by both prototype and
variant strain
PEDV strains that circulate in Nature.. The adjuvant preferably provides an
oil-in-water
emulsion with additional components. The vaccine compositions of the invention
protect
swine from infection by PEDV, and are effective in single doses, in two-dose
programs, or in
vaccination programs involving multiple doses, which may be spread apart by at
least a
week, and optionally at greater intervals of time, such as one to several
months.
The present invention similarly provides vaccine compositions comprising the
aforementioned mutant isolates of Calaf14 PEDV, as live vaccines, with our
without
adjuvants, that are also highly effective and provide good cross protection
against
subsequent challenge by both prototype and variant strain PEDV strains that
circulate in
Nature.
It should be noted that depending on the level of epidemic threat in a
particular swine
population, the vaccine dose program of one, two, or multiple doses may be
repeated, from
time to time, as a precautionary measure. Additionally, it should be noted
that vaccinating a
mother sow during pregnancy will provide protection to a young piglet, via
maternal transfer
of antibodies and T-cells in milk, although such protection may need to be
followed up with
additional vaccination doses to the piglet. Vaccination of all swine including
piglets and
adults is contemplated.
Thus, according to the practice of the present invention, there are provided
vaccines
.................................................. against PEDV based on
inactivated virus and modified live virus
Additionally, the immunogenic composition can comprise other swine antigens,
including Escherichia coli and Clostridium perfringens, types A-D, the dosages
of which
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would be equivalent to those found in the commercially-available vaccines,
Gletvax0 and
Litterguarde. The vaccines can contain one or more adjuvants, and optionally
one or more
excipients, in an amount effective to elicit production of neutralizing
antibodies in swine. The
adjuvant preferably provides an oil-in-water emulsion with additional
components. The
5 immunogenic compositions of the invention protect swine from infection by
PEDV are
effective in single doses, in two-dose programs, or in vaccination programs
involving multiple
doses, which may be spread apart by at least a week, and optionally at greater
intervals of
time, such as one to several months.
Preferably, the PEDV vaccines of the present invention are comprise any of the
novel
viruses as disclosed herein, such as those encoded by a polynucleotide
selected from:
(a) the group consisting of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, and
SEQ ID NO:6; or
(b) a PEDV virus that is encoded by a nucleotide sequence that is at least 90%
identical to
one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID
NO:6
at a full length nucleotide level, as long as said claimed encoding sequence
contains a
mutant amino acid residue not found in the virus encoded from SEQ ID NO:1.
More preferably, the PEDV vaccines of the present invention comprise viruses
encoded by a nucleotide sequence that is at least 95%, 96%,97%, 98%, 99% or
99.5%
identical to one or more of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, and
SEQ ID NO:6 at a full length nucleotide level, as long as said novel encoding
sequence
contains a mutant amino acid residue not found in the virus encoded from SEQ
ID NO:l.
Preferably, said mutant amino acid residues are selected from the group
consisting of.
The vaccines of the present invention are capable of protecting swine from
challenge
by both variant and prototype strains of PEDV, and preventing or treating
symptoms
associated with PEDV infection, wherein said protected swine include any of
sows, gilts,
boars, hogs, and piglets, and wherein achievement of protection is determined
by an
endpoint selected from the group consisting of prevention or control of any of
the PEDV
infection symptoms of dehydration, fever, diarrhea, vomiting, poor lactational
performance,
poor reproduction performance, mortality, and prevention or control of weight
loss or failure
to gain weight.
As aforementioned, the novel viruses of the vaccine composition may be live or
killed,
and if killed, a preferred adjuvant is an oil-in-water emulsion, wherein the
adjuvant
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components include Amphigene and aluminum hydroxideõ nmost preferably, wherein
the
final concentration of 20% Amphigen is 25% (v/v).
The invention therefor provides a method of protecting swine from challenge
against
PEDV, comprising administering to the subject a vaccine composition, in an
amount
sufficient to prevent or treat symptoms associated with PEDV infection,
wherein said
protected swine include any of sows, gilts, boars, hogs, and piglets, and
wherein
achievement of protection is determined by an endpoint selected from the group
consisting of
prevention or control of any of the PEDV infection symptoms of dehydration,
fever, diarrhea,
vomiting, poor lactational performance, poor reproduction performance,
mortality, and
prevention or control of weight loss or failure to gain weight.
Vaccine compositions of the invention are effective in piglets that are 1 day
of age or
older, in a single or two dose program. Vaccine compositions of the invention
are also
effective in piglets in a two dose program, wherein the first dose is
administered when the
piglet is about 1-7 days old, and the second dose is administered when the
piglet is 2-5
weeks old. The second does may be optional.
Preferably, the vaccine compositions of the invention have a minimum effective
dose
is between about 10 and about 106 logioTCI D50
In additional aspects of the invention, the vaccination program provides 2
doses
administered to the piglet; and the parent sow, although vaccinated pre-
breeding, is not
vaccinated pre-farrowing. Additionally, the vaccination program provides 1
dose
administered to the piglet; and the parent sow is vaccinated both pre-breeding
and pre-
farrowing. In additional embodiments, in order to prevent disease in a piglet,
there is first
administered the vaccine composition to the sow of said piglet, whether pre-
farrowing or pre-
breeding, following by administering one or more doses of said vaccine
composition to said
piglet after birth. In order to prevent disease in healthy pigs caused by
PEDV, the invention
provides that pigs are first vaccinated, followed by annual or pre-farrowing
administration of
further doses PEDV vaccine.
Brief Description of the Fiqures
Figure 1 shows a comparison of particular encoding nucleotides in I NDEL PEDV
strain
USA/OH 851/2014 (of the Ohio Department of Agriculture, GenBank KJ399978, "OH
851") to
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those found in Calaf14 Passage 0, with any resultant amino acid changes. The
Figure then
further shows evolution of additional nucleotide changes, and resultant amino
acid changes,
as Calaf 14 is subsequently passaged from Passage 0 to Passage 60, and then
individual
clones (A and E) are selected from consensus passage 60. Nucleotides are
numbered
according to the numbered sequence of PEDV USA/0H851/2014. Note that in Figure
4
shows the ORF1a-1b gene sequence for Calaf 14, passage 0 Figure 4 shows the
ORF1a-1b
gene sequence for Calaf 14, passage Othe 5' end, and 5 bp at the 3' end. Thus,
for
example, at line 1 of data herein, position 2929 in 0H851 corresponds to
Calaf14, passage
0, position 2894. In Calaf14, passage 37 (only), there is a further deletion
of "ATA" in the
spike gene, relative to passage 11, and not found in Passage 60, causing
downstream
nucleotides to be renumbered by 3 nucleotides, thus position 21,382, for
example, becomes
21,379.
Figure 2 shows the alignment of encoded ORF3 proteins of Calaf 14 PEDV
Passages 37
(SEQ ID NO:8) and 60 (SEQ ID NO:9).
Figure 3 shows the full nucleotide sequence of ther Passage 60 Calf 14 virus,
again noting
that approximately 35 bases are missing from the 5' end, and approximately 5
bases are
missing from the 3' end, as depicted.
Figure 4 (SEQ ID NO:10) shows the ORF1a-1b gene sequence for Calaf 14, passage
0.
Figure 5 (SEQ ID NO:11) shows the ORF1a-lb gene sequence for Calaf 14, passage
11.
Figure 6 (SEQ ID NO:12) shows the ORF1a-1b gene sequence for Calaf 14, passage
37.
Figure 7 (SEQ ID NO:13) shows the ORF1a-1b gene sequence for Calaf 14, passage
60.
Figure 8 (SEQ ID NO:14) shows the ORF1a-lb gene sequence for Calaf14, passage
60 clnA
Figure 9 (SEQ ID NO:15) shows the ORF1a-lb gene sequence for Calaf14, passage
60 clnE
Figure 10 (SEQ ID NO:16) shows the spike gene sequence for Calaf 14, passage
0.
Figure 11 (SEQ ID NO:17) shows the spike gene sequence for Calaf 14, passage
11.
Figure 12 (SEQ ID NO:18) shows the spike gene sequence for Calaf 14, passage
37.
Figure 13 (SEQ ID NO:19) shows the spike gene sequence for Calaf 14, passage
60.
Figure 14 (SEQ ID NO:20) shows the spike gene sequence for Calaf 14, passage
60, cln A
Figure 15(SEQ ID NO:21) shows the spike gene sequence for Calaf 14, passage
60, cln E
Figure 16 (SEQ ID NO:22) shows the ORF3 gene sequence for Calaf 14, passage 0.
Figure 17 (SEQ ID NO:23) shows the ORF3 gene sequence for Calaf 14, passage
11.
Figure 18 (SEQ ID NO:24) shows the ORF3 gene sequence for Calaf 14, passage
37.
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Figure 19 (SEQ ID NO:25) shows the ORF3 gene sequence for Calaf 14, passage
60.
Figure 20 (SEQ ID NO:26) shows the ORF3 gene sequence for Calaf 14, passage
60, cm n A
Figure 21 (SEQ ID NO:27) shows the ORF3 gene sequence for Calaf 14, passage
60, cm n E
Figure 22 (SEQ ID NO:28) shows the E gene for Passage 60.
Figure 23 (SEQ ID NO:29) shows the M gene for Passage 60.
Figure 24 (SEQ ID NO:30) shows the N gene for Passage 60.
Brief Description of the Sequence Listinq
SEQ ID NO: 1 provides the DNA sequence encoding PEDV strain Calaf 14, wild
type,
passage 0.
SEQ ID NO: 2 provides the DNA sequence encoding PEDV strain Calaf 14, passage
11.
SEQ ID NO: 3 provides the DNA sequence encoding PEDV strain Calaf 14, passage
37.
SEQ ID NO: 4 provides the DNA sequence encoding PEDV strain Calaf 14, passage
60.
SEQ ID NO: 5 provides the DNA sequence encoding PEDV strain Calaf 14, passage
60,
clone A selected therefrom.
SEQ ID NO: 6 provides the DNA sequence encoding PEDV strain Calaf 14, passage
60,
clone E selected therefrom.
SEQ ID NO: 7 provides the DNA sequence encoding PEDV strain 0H851.
SEQ ID NO: 1 provides the DNA sequence encoding PEDV strain Calaf 14, wild
type,
passage 0.
SEQ ID NO: 8 provides the amino acid sequence of the protein encoded from
ORF3,
Calaf14, passage 37.
SEQ ID NO: 9 provides the amino acid sequence of the protein encoded from
ORF3,
Calaf14, passage 60.
Detailed Description of the Invention
The present invention provides novel and efficacious vaccines useful to
preventing
disease caused by PEDV.
Definitions
Vaccines can be made more efficacious by including an appropriate adjuvant in
the
composition. The term "adjuvant" generally refers to any material that
increases the humoral
or cellular immune response to an antigen. Adjuvants are used to accomplish
two objectives:
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They slow the release of antigens from the injection site, and they enhance
stimulation of the
immune system. Traditional vaccines are generally composed of a crude
preparation of
inactivated or killed or modified live pathogenic microorganisms. The
impurities associated
with these cultures of pathological microorganisms may act as an adjuvant to
enhance the
immune response. However, the immunity invoked by vaccines that use
homogeneous
preparations of pathological microorganisms or purified protein subunits as
antigens is often
poor. The addition of certain exogenous materials such as an adjuvant
therefore becomes
necessary. Further, in some cases, synthetic and subunit vaccines may be
expensive to
produce. Also, in some cases, the pathogen cannot be grown on a commercial
scale, and
thus, synthetic/subunit vaccines represent the only viable option. The
addition of an adjuvant
may permit the use of a smaller dose of antigen to stimulate a similar immune
response,
thereby reducing the production cost of the vaccine. Thus, the effectiveness
of some
injectable medicinal agents may be significantly increased when the agent is
combined with
an adjuvant.
Many factors must be taken into consideration in the selection of an adjuvant.
An
adjuvant should cause a relatively slow rate of release and absorption of the
antigen in an
efficient manner with minimum toxic, allergenic, irritating, and other
undesirable effects to the
host. To be desirable, an adjuvant should be non-viricidal, biodegradable,
capable of
consistently creating a high level of immunity, capable of stimulating cross
protection,
compatible with multiple antigens, efficacious in multiple species, non-toxic,
and safe for the
host (eg, no injection site reactions). Other desirable characteristics of an
adjuvant are that it
is capable of micro-dosing, is dose sparing, has excellent shelf stability, is
amenable to
drying, can be made oil-free, can exist as either a solid or a liquid, is
isotonic, is easily
manufactured, and is inexpensive to produce. Finally, it is highly desirable
for an adjuvant to
be configurable so as to induce either a humoral or cellular immune response
or both,
depending on the requirements of the vaccination scenario. However, the number
of
adjuvants that can meet the above requirements is limited. The choice of an
adjuvant
depends upon the needs for the vaccine, whether it be an increase in the
magnitude or
function of the antibody response, an increase in cell mediated immune
response, an
induction of mucosal immunity, or a reduction in antigen dose. A number of
adjuvants have
been proposed, however, none has been shown to be ideally suited for all
vaccines. The first
adjuvant reported in the literature was Freund's Complete Adjuvant (FCA) which
contains a
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water-in-oil emulsion and extracts of mycobacterium. Unfortunately, FCA is
poorly tolerated
and it can cause uncontrolled inflammation. Since the discovery of FCA over 80
years ago
efforts have been made to reduce the unwanted side effects of adjuvants.
Some other materials that have been used as adjuvants include metallic oxides
(e.g.,
5 aluminum hydroxide), alum, inorganic chelates of salts, gelatins, various
paraffin-type oils,
synthesized resins, alginates, mucoid and polysaccharide compounds,
caseinates, and
blood-derived substances such as fibrin clots. While these materials are
generally efficacious
at stimulating the immune system, none has been found to be entirely
satisfactory due to
adverse effects in the host (e.g., production of sterile abcesses, organ
damage,
10 carcinogenicity, or allergenic responses) or undesirable pharmaceutical
properties (e.g.,
rapid dispersion or poor control of dispersion from the injection site, or
swelling of the
material).
"Cellular immune response" or "cell mediated immune response" is one mediated
by
T-lymphocytes or other white blood cells or both, and includes the production
of cytokines,
chemokines and similar molecules produced by activated T-cells, white blood
cells, or both;
or a T lymphocyte or other immune cell response that kills an infected cell.
The term "emulsifier" is used broadly in the instant disclosure. It includes
substances
generally accepted as emulsifiers, e.g., different products of TWEENO or SPAN
product
lines (fatty acid esters of polyethoxylated sorbitol and fatty-acid-
substituted sorbitan
surfactants, respectively), and different solubility enhancers such as PEG-40
Castor Oil or
another PEGylated hydrogenated oil.
"Humoral immune response" refers to one that is mediated by antibodies.
"Immune response" in a subject refers to the development of a humoral immune
response, a
cellular immune response, or a humoral and a cellular immune response to an
antigen.
Immune responses can usually be determined using standard immunoassays and
neutralization assays, which are known in the art.
"Immunologically protective amount" or "immunologically effective amount" or
"effective amount to produce an immune response" of an antigen is an amount
effective to
induce an immunogenic response in the recipient. The immunogenic response may
be
sufficient for diagnostic purposes or other testing, or may be adequate to
prevent signs or
symptoms of disease, including adverse health effects or complications
thereof, caused by
infection with a disease agent. Either humoral immunity or cell-mediated
immunity or both
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may be induced. The immunogenic response of an animal to an immunogenic
composition
may be evaluated, e.g., indirectly through measurement of antibody titers,
lymphocyte
proliferation assays, or directly through monitoring signs and symptoms after
challenge with
wild type strain, whereas the protective immunity conferred by a vaccine can
be evaluated by
measuring, e.g., reduction in clinical signs such as mortality, morbidity,
temperature number,
overall physical condition, and overall health and performance of the subject.
The immune
response may comprise, without limitation, induction of cellular and/or
humoral immunity.
"Immunogenic" means evoking an immune or antigenic response. Thus an
immunogenic
composition would be any composition that induces an immune response.
"Therapeutically effective amount" refers to an amount of an antigen or
vaccine that
would induce an immune response in a subject receiving the antigen or vaccine
which is
adequate to prevent or reduce signs or symptoms of disease, including adverse
health
effects or complications thereof, caused by infection with a pathogen, such as
a virus or a
bacterium. Humoral immunity or cell-mediated immunity or both humoral and cell-
mediated
immunity may be induced. The immunogenic response of an animal to a vaccine
may be
evaluated, e.g., indirectly through measurement of antibody titers, lymphocyte
proliferation
assays, or directly through monitoring signs and symptoms after challenge with
wild type
strain. The protective immunity conferred by a vaccine can be evaluated by
measuring, e.g.,
reduction in clinical signs such as mortality, morbidity, temperature number,
overall physical
condition, and overall health and performance of the subject. The amount of a
vaccine that is
therapeutically effective may vary depending on the particular adjuvant used,
the particular
antigen used, or the condition of the subject, and can be determined by one
skilled in the art.
"TCID50" refers to "tissue culture infective dose" and is defined as that
dilution of a
virus required to infect 50% of a given batch of inoculated cell cultures.
Various methods may
be used to calculate TCID50, including the Spearman-Karber method which is
utilized
throughout this specification. For a description of the Spearman-Karber
method, see B. W.
Mahy & H. 0. Kangro, Virology Methods Manual, p. 25-46 (1996).
Vaccine & Immunogenic Compositions
The vaccine and immunogenic composition of the present invention induces at
least
one of a number of humoral and cellular immune responses in a subject swine
that has been
administered a vaccine composition of the invention. Generally, the vaccine
compositions of
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the invention may be administered to swine of any age, whether male or female,
irrespective
of reproductive status, and although it is contemplated that a two-dose
regimen will be most
common, single dose and multiple dose vaccine treatments are also effective in
the practice
of the invention. A most preferred virus for use according to all aspects of
the invention
relating to PEDV is that encoded from SEQ ID NOS: 4, 5 and 6.
Such a vaccine protects well against challenge by both prototype and variant
strains,.
such as North American prototype strain USA/Colorado/2013 (whose sequence is
deposited
as GenBank accession No. KF272920, of the NCB! of the United States National
Institutes of
Health. Bethesda, MD), and also numerous other prototype and variant strains,
such as
similarly deposited Chinese strain AH2012, deposited as GenBank accession No.
KC210145; strain 13-019349, deposited as GenBank accession No. KF267450;
strain CH-
ZMDZY-11 deposited as GenBank accession No. KC196276; strain 0H851 (Ohio),
GenBank
KJ399978; European strain CV777 (see R. Kocherhans et al., Virus Genes, vol
23(2), pp
137-144, 2001; strains IA2013-KF452322 and 1N2013-KF452323 (see G. Stevenson
et al. J.
Vet. Diagn. Invest., vol 25, pp.649-654, 2013. Additional strains
representative of those
against which the current vaccines can protect, include, without limitation,
GenBank
Accessions KJ645688 (USA/lowa96/2013); KJ645640 (USA/Oklahoma32/2013);
KJ778615
(NPL-PEDv/2013); KJ645647 (USA/Minnesota41/2013); KJ645637
((USA/Kansas29/2013);
KJ645639 (USA/Texas31/2013); KJ645666 (USA/lowa70/2013); KJ645646
(USA/NorthCarolina40/2013); KM189367 (PEDv ON-018); and KJ645669
(USA/VVisconsin74/2013).
The vaccine compositions of the present invention protect against challenge by
PEDV
generally, including all forms of the virus circulating in Asia, North
America, and Europe.
Such viruses (against which protection is achieved) can also be identified
solely by the amino
acid of nucleotide encoding sequences of surface spike protein S, and thus
additional
isolates against which the present invention is effective include viral coat
sequences reported
in GenBank (US NIH/NCBI) by their spike protein accessions, to include Al
D56757.1;
AHA38139.1; AG058924.1; AHA38125.1; AIM47748.1; Al D56895.1: AID5669.1:
A1120255.1:
AGG34694.1; AIE15986.1; AHG05730.1; AHG05733.1 (all being representative of
those
having above 99% identity to the USA/Colorado/2013 spike sequence), and
further,
A1C82397.1; AFLO2631.1; AHB33810.1; AFQ37598.1; AGG34691.1; AFJ97030.1;
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13
AFR11479.1; and AEW22948.1 (all being representative of those having above 98%
identity
to the USA/Colorado/2013 spike sequence).
GenBank is the recognized US-NI H genetic sequence database, comprising an
annotated collection of publicly available DNA sequences, and which further
incorporates
submissions from the European Molecular Biology Laboratory (EMBL) and the DNA
DataBank of Japan (DDBJ), see Nucleic Acids Research, January 2013,v 41(D1)
D36-42 for
discussion.
Homologous Sequences and Conservative Amino Acid Changes
For purposes of the present invention, the nucleotide sequence of a second
polynucleotide molecule (either RNA or DNA) is "homologous" to the nucleotide
sequence of
a first polynucleotide molecule, or has "identity" to said first
polynucleotide molecule, where
the nucleotide sequence of the second polynucleotide molecule encodes the same
polyaminoacid as the nucleotide sequence of the first polynucleotide molecule
as based on
the degeneracy of the genetic code, or when it encodes a polyaminoacid that is
sufficiently
similar to the polyaminoacid encoded by the nucleotide sequence of the first
polynucleotide
molecule so as to be useful in practicing the present invention. Homologous
polynucleotide
sequences also refers to sense and anti-sense strands, and in all cases to the
complement
of any such strands. For purposes of the present invention, a polynucleotide
molecule is
useful in practicing the present invention, and is therefore homologous or has
identity, where
it can be used as a diagnostic probe to detect the presence of PEDV virus or
viral
polynucleotide in a fluid or tissue sample of an infected pig, e.g. by
standard hybridization or
amplification techniques. Generally, the nucleotide sequence of a second
polynucleotide
molecule is homologous to the nucleotide sequence of a first polynucleotide
molecule if it has
at least about 70% nucleotide sequence identity to the nucleotide sequence of
the first
polynucleotide molecule as based on the BLASTN algorithm (National Center for
Biotechnology Information, otherwise known as NCB!, (Bethesda, Maryland, USA)
of the
United States National Institute of Health). In a specific example for
calculations according to
the practice of the present invention, reference is made to BLASTP 2.2.6
[Tatusova TA and
TL Madden, "BLAST 2 sequences- a new tool for comparing protein and nucleotide
sequences." (1999) FEMS Microbiol Lett. 174:247-2501. Briefly, two amino acid
sequences
are aligned to optimize the alignment scores using a gap opening penalty of
10, a gap
extension penalty of 0.1, and the "b105um62" scoring matrix of Henikoff and
Henikoff (Proc.
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14
Nat. Acad. Sci. USA 89:10915 10919. 1992). The percent identity is then
calculated as:
Total number of identical matches x 100/ divided by the length of the longer
sequence+number of gaps introduced into the longer sequence to align the two
sequences.
Preferably, a homologous nucleotide sequence has at least about 90% nucleotide
sequence
identity, even more preferably at least about 95%, 96%, 97%, 98% and 99%
nucleotide
sequence identity. Since the genetic code is degenerate, a homologous
nucleotide
sequence can include any number of "silent" base changes, i.e. nucleotide
substitutions that
nonetheless encode the same amino acid.
A homologous nucleotide sequence can further contain non-silent mutations,
i.e.
base substitutions, deletions, or additions resulting in amino acid
differences in the encoded
polyaminoacid, so long as the sequence remains at least about 90% identical to
the
polyaminoacid encoded by the first nucleotide sequence or otherwise is useful
for practicing
the present invention. In this regard, certain conservative amino acid
substitutions may be
made which are generally recognized not to inactivate overall protein
function: such as in
regard of positively charged amino acids (and vice versa), lysine, arginine
and histidine; in
regard of negatively charged amino acids (and vice versa), aspartic acid and
glutamic acid;
and in regard of certain groups of neutrally charged amino acids (and in all
cases, also vice
versa), (1) alanine and serine, (2) asparagine, glutamine, and histidine, (3)
cysteine and
serine, (4) glycine and proline, (5) isoleucine, leucine and valine, (6)
methionine, leucine and
isoleucine, (7) phenylalanine, methionine, leucine, and tyrosine, (8) serine
and threonine, (9)
tryptophan and tyrosine, (10) and for example tyrosine, tyrptophan and
phenylalanine.
Amino acids can be classified according to physical properties and
contribution to secondary
and tertiary protein structure. A conservative substitution is recognized in
the art as a
substitution of one amino acid for another amino acid that has similar
properties. Exemplary
conservative substitutions may be found in WO 97/09433, page 10, published
Mar. 13. 1997
(PCT/GB96/02197, filed Sep. 6, 1996. Alternatively, conservative amino acids
can be
grouped as described in Lehninger, (Biochemistry, Second Edition; Worth
Publishers, Inc.
NY:NY (1975), pp. 71-77).
Homologous nucleotide sequences can be determined by comparison of nucleotide
sequences, for example by using BLASTN, above. Alternatively, homologous
nucleotide
sequences can be determined by hybridization under selected conditions. For
example, the
nucleotide sequence of a second polynucleotide molecule is homologous to SEQ
ID NO:1 (or
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any other particular polynucleotide sequence) if it hybridizes to the
complement of SEQ ID
NO:1 under moderately stringent conditions, e.g., hybridization to filter-
bound DNA in 0.5 M
NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 C, and washing in
0.2xSSC/0.1 /0 SDS at 42 C (see Ausubel et al editors, Protocols in Molecular
Biology, VViley
5 and Sons, 1994, pp. 6Ø3 to 6.4.10), or conditions which will otherwise
result in hybridization
of sequences that encode a PEDV virus as defined below. Modifications in
hybridization
conditions can be empirically determined or precisely calculated based on the
length and
percentage of guanosine/cytosine (GC) base pairing of the probe. The
hybridization
conditions can be calculated as described in Sambrook, et al., (Eds.),
Molecular Cloning: A
10 Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring
Harbor, New York
(1989), pp. 9.47 to 9.51.
In another embodiment, a second nucleotide sequence is homologous to SEQ ID
NO:1 (or any other sequence of the invention) if it hybridizes to the
complement of SEQ ID
NO:1 under highly stringent conditions, e.g. hybridization to filter-bound DNA
in 0.5 M
15 NaHPO4, 7% SDS, 1 mM EDTA at 65 C, and washing in 0.1xSSC/0.1 /0 SDS at
68 C, as is
known in the art.
It is furthermore to be understood that the isolated polynucleotide molecules
and the
isolated RNA molecules of the present invention include both synthetic
molecules and
molecules obtained through recombinant techniques, such as by in vitro cloning
and
transcription.
Preferred Viral isolates
Referring to Figure 1, it can be seen that important nucleotide changes happen
in
ORF1a/b, spike, and ORF3, causing important amino acid changes in the
resultant proteins,
that provide for the high level of safety achieved for Calaf14 passage 60 (see
Example 5
below). It should be noted that 2 clones (A and E, SEQ ID NOS:5 and 6) were
also
recovered and sequenced from the consensus population (SEQ ID NO:4).
Referring first to spike protein changes, between passages 11 and 37, there is
only
one stable amino acid change that is then retained in passage 60. The
phenylalanine (F) at
amino acid position 1269 in spike changes to leucine (L). Between passages 37
and 60, two
additional amino acid changes in Spike contribute to attenuation. These are
aspartate (D) to
tyrosine (Y) at position 262, and asparagine (N) to aspartate (D) to position
1006. It is thus
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within the practice of the present invention to provide PEDV viruses having
these sequence
modifications (see SEQ ID NOS 4,5 and 6), or conservative amino acid variants
thereof, for
example, position 1269 (or the position that corresponds to residue 1269 as
would be
determined from an appropriate algorithm) could instead be valine, isoleucine,
and the like;
position 262 tyrosine could be replaced by phenyalanine, valineõleucine,
isoleucine and the
like, and asparate at position 1006 could be replaced by by glutamate, for
example.
As can also be seen from Figure 1, there is one amino acid change in ORF1a/1b
of
particular note, that helps explain the increased attenuation between passages
37 and 60
(drop from 10% to 0% mortality). It is the A to G nucleotide change at genome
position
17,171 (0H851) or 17,136 (Calaf14 P37). This results in an E to G (glutamate
to glycine)
change at amino acid position 5627 of the ORF lab polyprotein. This portion of
the
polyprotein is proteolytically cleaved to form nsp14, which is known as the
Exoribonuclease
(ExoN). It is possible that the replacement of a negatively charged amino acid
like glutamate
with a small non-charged glycine residue (or proline or alanine, for example)
could alter the
.. affinity of the enzyme for its RNA substrate. Alternatively, it could alter
the conformation of
the enzyme resulting in reduced activity or stability.
In regard of ORF3, it can be seen that a frameshift inducing truncation "ATTA"
happens between passages 37 and 60, leading to a shortened expression product.
Other
amino acid changes noted in Figure 1 are considered less likely to contribute
substantially to
.. attenuation since they appear in equally attenuated final variants (for
example ORF1a/1b
position 3319 as T or C, where virulent wild type passage 0 is also C), for
example, or where
the mutation already arises prior to passage 11, although this passage is not
yet substantially
attenuated (see, for example, ORF1a/1b at position 5122) Nonetheless, such
amino acid
changes may contribute to the practice of the invention, so that mutant
ORF1a/1b position
5122 (alanine) may be replaced with glycine, and other relatively small non
polar amino
acids, for example.
Culturing of Virus
Isolation and propagation of PEDV has been generally difficult. Initial
studies using
Vero cells for propagation in culture have only been partially effective, and
have required a
trypsin-containing medium, often with excessive cytopathic effect including
cell fusion,
synctia formation, and cell detachment (see, for example K. Kusangi et al., J.
Vet Med Sci,
vol. 54(2), pp.313-318, 1992, and M. Hofmann et al. J. Clinical Microbiology,
vol. 26(11),
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pp2235-2239, 1988). Accordingly, improved passaging methods were developed for
the
practice of the present invention. General and detailed methods are provided
in Examples 1
and 2 below. It should be noted that both USA/Colorado/2013 and Calaf14 can be
cultured
in Vero cells.
Inactivation of virus
Inactivated or killed viral strains are those which have been inactivated by
methods
known to those skilled in the art, including treatment with formalin,
betapropriolactone (BPL),
binary ethyleneimine (BEI), sterilizing radiation, heat, or other such
methods.
Adjuvant component
The vaccine compositions of the invention may or may not include adjuvants. In
particular, as based on an orally infective virus, the modified live vaccines
of the invention
may be used adjuvant free, with a sterile carrier. Adjuvants that may be used
for oral
administration include those based on CT-like immune modulators (rmLT, CT-B,
i.e.
recombinant-mutant heat labile toxin of E. coli, Cholera toxin-B subunit); or
via encapsulation
with polymers and alginates, or with mucoadhesives such as chitosan, or via
liposomes. A
preferred adjuvanted or non adjuvanted vaccine dose at the minimal protective
dose through
vaccine release may provide between approximately 10 and approximately 106
logioTCI D5o
of virus per dose, or higher. Adjuvants, if present, may be provided as
emulsions, more
commonly if non-oral administration is selected, but should not decrease
starting titer by
more than 0.7 logs (80% reduction.
Immunogenic compositions of the present invention can include one or more well
known adjuvants and adjuvant systems. Suitable adjuvants include, but are not
limited to,
the RIBI adjuvant system (Ribi Inc.; Hamilton, MT); alum; aluminum hydroxide
gel; aluminum
phosphate; oil-in water emulsions; water-in-oil emulsions such as Freund's
complete and
incomplete adjuvants; Block copolymer (CytRx; Atlanta, GA); SAF-M (Chiron;
Emeryville,
CA); AMPHIGENO adjuvant; killed Bordetella; saponins such as Stimulon TM QS-21
(Antigenics, Framingham, MA. described in U.S. Patent No. 5,057,540, which is
hereby
incorporated by reference) and particles generated therefrom such as ISCOMS
(immunostimulating complexes), GPI-0100 (Galenica Pharmaceuticals, Inc.;
Birmingham,
AL) or other saponin fractions; monophosphoryl lipid A (MPL-A); avridine;
lipid-amine
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adjuvant; heat-labile enterotoxin from Escherichia coli (recombinant or
otherwise); cholera
toxin; and muramyl dipeptide. Also useful is MPLTM (3-0-deacylated
monophosphoryl lipid
A; Corixa, Hamilton, MT), which is described in U.S. Patent No. 4,912,094, and
is hereby
incorporated by reference.
Also suitable for use as adjuvants are: synthetic lipid A analogs or
aminoalkyl
glucosamine phosphate (AGP) compounds, or derivatives or analogs thereof,
which are
available from Corixa (Hamilton, MT), and which are described in US 6,113,918,
hereby
incorporated by reference; L121/squalene; D-lactide-polylactide/glycoside;
pluronic polyols;
muramyl dipeptide; extracts of Mycobacterium tuberculosis; bacterial
lipopolysaccharides
generally; pertussis toxin (PT); and an E. coli heat-labile toxin (LT),
particularly LT-K63, LT-
R72, PT-K9/G129; see, e.g., International Patent Publication Nos. WO 93/13302
and WO
92/19265, incorporated herein by reference.
Synthetic polynucleotides, such as oligonucleotides containing CpG motifs (US
6,207,646, hereby incorporated by reference), can also be used as adjuvants
for the present
invention. CpG oligonucleotides, such as P-class immunostimulatory
oligonucleotides, are
useful, including E-modified P-class immunostimulatory oligonucleotides.
Sterols can also be useful as adjuvants herein. Those suitable for use can
include
sitosterols, stigmasterol, ergosterol, ergocalciferol, and cholesterol.
The adjuvant compositions useful in the practice of the invention can
generally
.. further include one or more polymers such as, for example, DEAE Dextran,
polyethylene
glycol, polyacrylic acid, and polymethacrylic acid (e.g., CARBOPOLO). The
adjuvant
compositions can also further include one or more Th2 stimulants such as, for
example, Bay
R1005(R) and aluminum.
The adjuvant compositions can additionally or alternatively further include
one or
more immunomodulatory agents, such as quaternary ammonium compounds (e.g.,
DDA),
interleukins, interferons, or other cytokines. A number of cytokines or
lymphokines have
been shown to have immune-modulating activity, and thus may be used as
adjuvants.
These can include, but are not limited to, the interleukins 1-a, 113, 2, 4, 5,
6, 7, 8, 10, 12
(see, e.g., US 5,723,127), 13, 14, 15, 16, 17 and 18 (and its mutant forms),
the interferons-a,
13 and gamma, granulocyte-macrophage colony stimulating factor (see, for
example, US
5,078,996, and ATCC Accession Number 39900), macrophage colony stimulating
factor,
granulocyte colony stimulating factor, GSF, and the tumor necrosis factors a
and 13. Still
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other adjuvants useful in this invention include chemokines, including without
limitation,
MCP-1, MIP-1a, MIP-113, and RANTES. Adhesion molecules, such as a selectin,
e.g., L-
selectin, P-selectin, and E-selectin may also be useful as adjuvants. Still
other useful
adjuvants include, without limitation, a mucin-like molecule, e.g., 0D34,
GlyCAM-1 and
MadCAM-1; a member of the integrin family such as LFA-1, VLA-1, Mac-1 and
p150.95; a
member of the immunoglobulin superfamily such as PECAM, ICAMs (e.g., ICAM-1,
ICAM-2
and ICAM-3), CD2 and LFA-3; co-stimulatory molecules such as CD40 and CD4OL;
growth
factors including vascular growth factor, nerve growth factor, fibroblast
growth factor,
epidermal growth factor, B7.2, PDGF, BL-1, and vascular endothelial growth
factor; receptor
molecules including Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-
3, AIR,
LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, and DR6. Still another
adjuvant
molecule includes Caspase (ICE). See also W098/17799 and W099/43839.
Suitable adjuvants also include, without limitation, M PLTm (3-0-deacylated
monophosphoryl lipid A; Corixa, Hamilton, MT), which is described in U.S.
Patent No.
.. 4,912,094, which is hereby incorporated by reference. Also suitable for use
as adjuvants are
synthetic lipid A analogs or aminoalkyl glucosamine phosphate compounds (AGP),
or
derivatives or analogs thereof, which are available from Corixa (Hamilton,
MT), and which
are described in United States Patent No. 6,113,918, which is hereby
incorporated by
reference. One such AGP is 2-[(R)-3-Tetradecanoyloxytetradecanoylamino] ethyl
2-Deoxy-4-
0-phosphono-3-0-[(R)-3-tetradecanoyoxytetradecanoy1]-2-[(R)-3-
tetradecanoyloxytetradecanoyl-amino]-b-D-glucopyranoside, which is also known
as 529
(formerly known as RC529). The RC529 adjuvant is formulated as an aqueous form
or as a
stable emulsion.
Additional adjuvants useful in the practice of the present invention include
cholera
toxins (CT) and mutants thereof, including those described in published
International Patent
Application number WO 00/18434 (wherein the glutamic acid at amino acid
position 29 is
replaced by another amino acid, other than aspartic acid, preferably a
histidine). Similar CT
toxins or mutants are described in published International Patent Application
number WO
02/098368 (wherein the isoleucine at amino acid position 16 is replaced by
another amino
acid, either alone or in combination with the replacement of the serine at
amino acid position
68 by another amino acid; and/or wherein the valine at amino acid position 72
is replaced by
another amino acid). Other CT toxins are described in published International
Patent
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Application number WO 02/098369 (wherein the arginine at amino acid position
25 is
replaced by another amino acid; and/or an amino acid is inserted at amino acid
position 49;
and/or two amino acids are inserted at amino acid positions 35 and 36). Said
CT toxins or
mutant can be included in the immunogenic compositions either as separate
entities, or as
5 fusion partners for the polypeptides of the present invention.
In one example, adjuvant components are provided from a combination of
lecithin in
light mineral oil, and also an aluminum hydroxide component. Details
concerning the
composition and formulation of Amphigen (as representative lecithin/mineral
oil component)
are as follows.
10 A preferred adjuvanted may be provided as a 2ML dose in a buffered
solution further
comprising about 5% (v/v) Rehydragele (aluminum hydroxide gel) and "20%
Amphigen" 0 at
about 25% final (v/v). Amphigen is generally described in U.S Patent
5,084,269 and
provides de-oiled lecithin (preferably soy) dissolved in a light oil, which is
then dispersed into
an aqueous solution or suspension of the antigen as an oil-in-water emulsion.
Amphigen has
15 been improved according to the protocols of U.S. Patent 6,814,971 (see
columns 8-9
thereof) to provide a so-called "20% Amphigen" component for use in the final
adjuvanted
vaccine compositions of the present invention. Thus, a stock mixture of 10%
lecithin and
90% carrier oil (DRAKEOL , Penreco, Karns City, PA) is diluted 1: 4 with 0.63%
phosphate
buffered saline solution, thereby reducing the lecithin and DRAKEOL components
to 2% and
20 18% respectively (i.e. 20% of their original concentrations). Tween 80
and Span 80
surfactants are added to the composition, with representative and preferable
final amounts
being 5.6% (v/v) Tween 80 and 2.4% (v/v) Span 80, wherein the Span is
originally provided
in the stock DRAKEOL component, and the Tween is originally provided from the
buffered
saline component, so that mixture of the saline and DRAKEOL components results
in the
finally desired surfactant concentrations. Mixture of the DRAKEOL/lecithin and
saline
solutions can be accomplished using an In-Line Slim Emulsifier apparatus,
model 405,
Charles Ross and Son, Hauppauge, NY, USA.
The vaccine composition also includes Rehydragele LV (about 2% aluminum
hydroxide content in the stock material), as additional adjuvant component
(available from
Reheis, NJ, USA, and ChemTrade Logistics, USA). With further dilution using
0.63% PBS,
the final vaccine composition contains the following compositional amounts per
2ML dose;
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5% (v/v) Rehydragele LV; 25% (v/v) of "20% Amphigen", i.e. it is further 4-
fold diluted); and
0.01% (w/v) of merthiolate.
As is understood in the art, the order of addition of components can be varied
to
provide the equivalent final vaccine composition. For example, an appropriate
dilution of
.. virus in buffer can be prepared. An appropriate amount of Rehydragele LV
(about 2%
aluminum hydroxide content) stock solution can then be added, with blending,
in order to
permit the desired 5% (v/v) concentration of Rehydragele LV in the actual
final product.
Once prepared, this intermediate stock material is combined with an
appropriate amount of
"20% Amphigen" stock (as generally described above, and already containing
necessary
amounts of Tween 80 and Span 80) to again achieve a final product having 25%
(v/v) of
"20% Amphigen". An appropriate amount of 10% merthiolate can finally be added.
The vaccinate compositions of the invention permit variation in all of the
ingredients,
such that the total dose of antigen may be varied preferably by a factor of
100 (up or down)
compared to the antigen dose stated above, and most preferably by a factor of
10 or less (up
or down),. Similarly, surfactant concentrations (whether Tween or Span) may be
varied by
up to a factor of 10, independently of each other, or they may be deleted
entirely, with
replacement by appropriate concentrations of similar materials, as is well
understood in the
art.
Rehydragele concentrations in the final product may be varied, first by the
use of
.. equivalent materials available from many other manufacturers (i.e.
Alhydrogele ,Brenntag;
Denmark), or by use of additional variations in the Rehydragele line of
products such as CG,
HPA or HS. Using LV as an example, final useful concentrations thereof
including from 0%
to 20%, with 2-12% being more preferred, and 4-8% being most preferred,
Similarly, the
although the final concentration of Amphigen (expressed as % of "20%
Amphigen") is
preferably 25%, this amount may vary from 5-50%, preferably 20-30% and is most
preferably
about 24-26%.
According to the practice of the invention, the oil used in the adjuvant
formulations of
the instant invention is preferably a mineral oil. As used herein, the term
"mineral oil" refers
to a mixture of liquid hydrocarbons obtained from petrolatum via a
distillation technique. The
term is synonymous with "liquefied paraffin", "liquid petrolatum" and "white
mineral oil." The
term is also intended to include "light mineral oil," i.e., oil which is
similarly obtained by
distillation of petrolatum, but which has a slightly lower specific gravity
than white mineral oil.
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See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pa.:
Mack Publishing
Company, 1990, at pages 788 and 1323). Mineral oil can be obtained from
various
commercial sources, for example, J. T. Baker (Phillipsburg, Pa.), USB
Corporation
(Cleveland, Ohio). Preferred mineral oil is light mineral oil commercially
available under the
name DRAKEOLO.
Typically, the oily phase is present in an amount from 50% to 95% by volume;
preferably, in an amount of greater than 50% to 85%; more preferably, in an
amount from
greater than 50% to 60%, and more preferably in the amount of greater than 50-
52% v/v of
the vaccine composition. The oily phase includes oil and emulsifiers (e.g.,
SPAN 80,
TWEENO 80 etc), if any such emulsifiers are present.
Non-natural, synthetic emulsifiers suitable for use in the adjuvant
formulations of the
present invention also include sorbitan-based non-ionic surfactants, e.g.
fatty-acid-
substituted sorbitan surfactants (commercially available under the name SPAN
or
ARLACELO), fatty acid esters of polyethoxylated sorbitol (TWEENO),
polyethylene glycol
esters of fatty acids from sources such as castor oil (EMULFORO);
polyethoxylated fatty acid
(e.g., stearic acid available under the name SIMULSOLO M-53), polyethoxylated
isooctylphenol/formaldehyde polymer (TYLOXAPOLO), polyoxyethylene fatty
alcohol ethers
(BRIJO); polyoxyethylene nonphenyl ethers (TRITON N), polyoxyethylene
isooctyl phenyl
ethers (TRITON X) . Preferred synthetic surfactants are the surfactants
available under the
name SPAN and TWEENO, such as TWEENO-80 (Polyoxyethylene (20) sorbitan
monooleate) and SPAN -80 (sorbitan monooleate). Generally speaking, the
emulsifier(s)
may be present in the vaccine composition in an amount of 0.01% to 40% by
volume,
preferably, 0.1% to 15%, more preferably 2% to 10%.
In an alternative embodiment of the invention, the final vaccine composition
contains
SP-Oil and Rehydragel LV as adjuvants (or other Rehydragel or Alhydrogele
products),
with preferable amounts being about 5-20% SP-Oil (v/v) and about 5-15%
Rehydragel LV
(v/v), and with 5% and 12%, respectively, being most preferred amounts. In
this regard it is
understood that % Rehydragel refers to percent dilution from the stock
commercial product.
(SP-Oil 0 is a fluidized oil emulsion with includes a polyoxyethylene-
polyoxypropylene block
copolymer (Pluronice L121, BASF Corporation, squalene, polyoxyethylene
sorbitan
monooleate (Tween080, ICI Americas) and a buffered salt solution.)
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It should be noted that the present invention may also be successfully
practiced using
wherein the adjuvant component is only Amphigene.
In another embodiment of the invention, the final vaccine composition contains
TXO
as an adjuvant; TXO is generally described in WO 2015/042369. All TXO
compositions
disclosed therein are useful in the preparation of vaccines of the invention.
In TXO, the
immunostimulatory oligonucleotide ("T"), preferably an ODN, preferably
containing a
palindromic sequence, and optionally with a modified backbone, is present in
the amount of
0.1 to 5 ug per 50 ul of the vaccine composition (e.g., 0.5 ¨ 3 ug per 50 ul
of the composition,
or more preferably 0.09-0.11 ug per 50 ul of the composition). A preferred
species thereof is
SEQ ID NO: 8 as listed (page 17) in the W02015/042369 publication
(PCT/U52014/056512).
The polycationic carrier ("X") is present in the amount of 1-20 ug per 50 ul
(e.g., 3-10 ug per
50 ul, or about 5 ug per 50 u1). Light mineral oil ("0") is also a component
of the TXO
adjuvant.
In certain embodiments, TXO adjuvants are prepared as follows:
a) Sorbitan monooleate, MPL-A and cholesterol are dissolved in light
mineral oil. The
resulting oil solution is sterile filtered;
b) The immunostimulatory oligonucleotide, Dextran DEAE and Polyoxyethylene
(20)
sorbitan monooleate are dissolved in aqueous phase, thus forming the aqueous
solution; and
c) The aqueous solution is added to the oil solution under continuous
homogenization
thus forming the adjuvant formulation TXO.
All the adjuvant compositions of the invention can be used with any of the
PEDV
strains and isolates covered by the present Specification.
Additional adjuvants useful in the practice of the invention include Prezent-A
(see
generally United States published patent application U520070298053; and
"QCDCRT" or
"QCDC"-type adjuvants (see generally United States published patent
application
U520090324641.
All the adjuvant compositions of the invention can be used with any of the
PEDV
strains and isolates covered by the present Specification.
Excipients
The immunogenic and vaccine compositions of the invention can further comprise
pharmaceutically acceptable carriers, excipients and/or stabilizers (see e.g.
Remington: The
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Science and practice of Pharmacy (2005) Lippincott VVilliams), in the form of
lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients, or
stabilizers are
nontoxic to recipients at the dosages and concentrations, and may comprise
buffers such as
phosphate, citrate, and other organic acids; antioxidants including ascorbic
acid and
.. methionine; preservatives (such as Mercury((o-carboxyphenyl)thio)ethyl
sodium salt
(THIOMERSAL), octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol;
alkyl
parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol;
and m-cresol); proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic
polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine,
histidine, arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates
including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars
such as
sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal
complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as
polyethylene
glycol (PEG), TWEEN or PLURONICS.
Dosing
A preferred clinical indication is for treatment, control and prevention in
both breeding
sows and gilts pre-farrowing, followed by vaccination of piglets. In a
representative example
(applicable to both sows and gilts), two 2-ML doses of vaccine will be used,
although of
course, actual volume of the dose is a function of how the vaccine is
formulated, with actual
dosing amounts ranging from 0.1 to 5ML, taking also into account the size of
the animals.
Single dose vaccination is also appropriate.
The first dose may be administered as early as pre-breeding to 5-weeks pre-
farrowing, with the second dose administered preferably at about 1-3 weeks pre-
farrowing.
Doses vaccine preferably provide an amount of viral material that corresponds
to a TCI D50
(tissue culture infective dose) of between about 106 and 108, more preferably
between about
107 and 1075, and can be further varied, as is recognized in the art. Booster
doses can be
given two to four weeks prior to any subsequent farrowings. Intramuscular
vaccination (all
doses) is preferred, although one or more of the doses could be given
subcutaneously. Oral
administration is also preferred. Vaccination may also be effective in naïve
animals, and
non-naïve animals as accomplished by planned or natural infections.
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In a further preferred example, the sow or gilt is vaccinated intramuscularly
or orally at
5-weeks pre-farrowing and then 2-weeks pre-farrowing. Under these conditions,
a protective
immune response can be demonstrated in PEDV-negative vaccinated sows in that
they
developed antibodies (measured via fluorescent focal neutralization titer from
serum
5 samples) with neutralizing activity, and these antibodies were passively
transferred to their
piglets. The protocols of the invention are also applicable to the treatment
of already
seropositive sows and gilts, and also piglets and boars. Booster vaccinations
can also be
given and these may be be via a different route of administration. Although it
is preferred to
re-vaccinate a mother sow prior to any subsequent farrowings, the vaccine
compositions of
10 the invention nonetheless can still provide protection to piglets via
ongoing passive transfer
of antibodies, even if the mother sow was only vaccinated in association with
a previous
farrowing.
It should be noted that piglets may then be vaccinated as early as Day 1 of
life. For
example, piglets can be vaccinated at Day 1, with or without a booster dose at
3 weeks of
15 age, particularly if the parent sow, although vaccinated pre-breeding,
was not vaccinated pre-
farrowing. Piglet vaccination may also be effective if the parent sow was
previously not
naïve either due to natural or planned infection. Vaccination of piglets when
the mother has
neither been previously exposed to the virus, nor vaccinated pre-farrowing may
also
effective. Boars (typically kept for breeding purposes) should be vaccinated
once every 6
20 months. Variation of the dose amounts is well within the practice of the
art. It should be
noted that the vaccines of the present invention are safe for usein pregnant
animals (all
trimesters) and neonatal swine. The vaccines of the invention are attenuated
to a level of
safety (i.e. no mortality, only transient mild clinical signs or signs normal
to neonatal swine)
that is acceptable for even the most sensitive animals again including
neonatal pigs.
25 It should also be noted that animals vaccinated with the vaccines of the
invention are
also immediately safe for human consumption, without any significant slaughter
withhold,
such as 21 days or less.
When provided therapeutically, the vaccine is provided in an effective amount
upon
the detection of a sign of actual infection. Suitable dose amounts for
treatment of an existing
infection include between about 10 and about 106 logioTCI D5o , or higher, of
virus per dose
(minimum immunizing dose to vaccine release). A composition is said to be
"pharmacologically acceptable" if its administration can be tolerated by a
recipient. Such a
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composition is said to be administered in a "therapeutically or
prophylactically effective
amount" if the amount administered is physiologically significant.
At least one vaccine or immunogenic composition of the present invention can
be
administered by any means that achieve the intended purpose, using a
pharmaceutical
.. composition as described herein. For example, route of administration of
such a composition
can be by parenteral, oral, oronasal, intranasal, intratracheal, topical,
subcutaneous,
intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and
intravenous
administration. In one embodiment of the present invention, the composition is
administered
by intramuscularly. Parenteral administration can be by bolus injection or by
gradual
.. perfusion over time. Any suitable device may be used to administer the
compositions,
including syringes, droppers, needleless injection devices, patches, and the
like. The route
and device selected for use will depend on the composition of the adjuvant,
the antigen, and
the subject, and such are well known to the skilled artisan. Administration
that is oral, or
alternatively, subcutaneous, is preferred. Oral administration may be direct,
via water, or via
.. feed (solid or liquid feed). When provided in liquid form, the vaccine may
be lyophilized with
reconstitution, pr provided as a paste, for direct addition to feed (mix in or
top dress) or
otherwise added to water or liquid feed.
Variation of the dose amounts is well within the practice of the art.
Generation of Vero Cells Suitable for Large Scale Virus Production
Viruses of the invention can be conveniently grown in Vero cell stocks that
are
approved for vaccine production. The following provides a representative
method to
generate safe and approved cell stock, a vial of Vero cells was subject to
additional
passaging.. The cells were passed four times in PMEM w/wheat to produce Master
Cell
Stock (MCS) Lot "1834430". The MCS was tested in accordance with 9CFR & EP
requirements. The MCS tested satisfactory for sterility, freedom from
mycoplasmas, and
extraneous agents. Therefore, PF-Vero MCS lot "1834430", is deemed eligible
for
submission to the Center for Veterinary Biologics Laboratories (CVB-L) for
confirmatory
testing.
Seed Origin and Passage History is as follows. A Pre-master Cell stock of
global Vero
cells was previously frozen. For production of the cell stock, the cells were
grown in PMEM
containing 1% bovine serum (item # 00-0710-00, BSE compliant) and 3 mM L-
glutamine.
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They were derived from Vero WCS Pass # 136, Lot #071700 MCS+3, 28-Jul-00. The
new
Pre-master cell stock was frozen at pass # 166, which is MCS+33 from the
original global
Vero master cell stock. MCS "1833440" was produced from a pre-master Lot All
cultures
were grown in PMEM w/wheat, 1.0% L-glutamine and 1.0% Bovine Calf serum. Cells
were
planted (passage # 167) in 150 cm2 T-Flasks on August 14, 2008. The flasks
were
incubated in 5.0% CO2 at 36 1 liC for 7 days then expanded. (passage # 168)
After
flasks reached 100% confluency 4 days later, the cultures were passed (#169)
into 850 cm2
roller bottles. Rollers were incubated at 36 +/- 1 degree C at 0.125 ¨ 0.250
rpm without CO2.
The final passage of rollers (#170) was done 4 days later. Cryopreservation
was completed
by adding 10.0% bovine calf serum and 10.0% dimethyl sulfoxide (DMSO) to the
condensed
cell suspension.. Vials were labeled as passage level #170. A total of 231
containers
containing 4.2 ml were placed into a controlled rate freezer then transferred
into liquid
nitrogen tank for long term storage at vapor phase. The MCS was produced
without the use
of antibiotics.
Mycoplasma Testing and Extraneous Testing were accomplished as follows. The
MCS was tested as per 9CFR (028-PUO) and EP 2.6.7. The MCS was found to be
free of
any Mycoplasma contamination. Extraneous testing was completed as per 9CFR
113.52
using NL-BT-2 (Bovine), Vero, NL-ED-5 (Equine), NL-ST-1 (Porcine), NL-DK
(Canine),NL-FK
(Feline) cells, . The MCS was negative for MGG, CPE and HAd and tested
negative by FA
.. for BVD, BRSV, BPV, BAV-1, BAV-5, Rabies, Reo, BTV, ERV, Equine arteritis,
PPV, TGE,
PAV, HEV, CD, CPV, FPL and Fl P. The MCS was tested by ELISA for FIV and was
found to
be satisfactory.
EP extraneous testing was as per 5.2.4 (52-2002). Extraneous testing using
Bovine
NL-BT-2 and EBK (Primary), Vero, NL-ED-5 (Equine), NL-ST-1 (Porcine), MARC MA
104,
NL-DK (Canine) NL-FK (Feline) cells were negative for MGG, CPE, HAd and tested
negative by FA for BVD, BPV, BAV-1, BAV-5, Bovine corona, Bovine rotavirus,
BHV-3, P13,
IBR, BRSV and BEV-1, Reo, BTV, ERV, Equine arteritis, PPV, PRV, TGE, HEV, PAV,
P.
rota Al, rota A2, PRRSV, CD, CPI, CAV-2, Measles, C. rota, Rabies, CCV, FP,
FCV, FVR,
FIP and FeLV.
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Methods of Use
The invention encompasses methods of preventing PEDV virus infection
comprising
administering the immunogenic and vaccine compositions of the invention in a
swine subject
of any age.
When provided therapeutically, the vaccine is provided in an effective amount
upon
the detection of a symptom of actual infection. A composition is said to be
"pharmacologically acceptable" if its administration can be tolerated by a
recipient. Such a
composition is said to be administered in a "therapeutically or
prophylactically effective
amount" if the amount administered is physiologically significant.
At least one vaccine or immunogenic composition of the present invention can
be
administered by any means that achieve the intended purpose, using a
pharmaceutical
composition as described herein. For example, route of administration of such
a composition
can be by parenteral, oral, oronasal, intranasal, intratracheal, topical,
subcutaneous,
intramuscular, transcutaneous, intradermal, intraperitoneal, intraocular, and
intravenous
.. administration. In one embodiment of the present invention, the composition
is administered
by intramuscularly. Parenteral administration can be by bolus injection or by
gradual
perfusion over time. Any suitable device may be used to administer the
compositions,
including syringes, droppers, needleless injection devices, patches, and the
like. The route
and device selected for use will depend on the composition of the adjuvant,
the antigen, and
.. the subject, and such are well known to the skilled artisan.
According to the present invention, an "effective amount" of a vaccine or
immunogenic composition is one which is sufficient to achieve a desired
biological effect, in
this case at least one of cellular or humoral immune response to one or more
strains of
PEDV. It is understood that the effective dosage will be dependent upon the
age, sex, health,
and weight of the subject, kind of concurrent treatment, if any, frequency of
treatment, and
the nature of the effect desired. The ranges of effective doses provided below
are not
intended to limit the invention and represent examples of dose ranges which
may be suitable
for administering compositions of the present invention. However, the dosage
may be
tailored to the individual subject, as is understood and determinable by one
of skill in the art,
without undue experimentation.
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Examples
The following examples illustrate only certain and not all embodiments of the
invention, and thus, should not be viewed as limiting the scope of the
invention.
Example 1: General representative procedures applicable to collection of PEDV
virus from
tissue samples
Approximately 1 cm of tissue was used for extraction of PEDV virus. The tissue
was
chopped into fine pieces using a sterile scalpel and sterile scissors in a
sterile Petri dish.
Work was done in a Bio-safety cabinet to ensure aseptic conditions. 2 ml of
sterile PBS was
added to the Petri dish to collect tissue and material was transfer to a 15 ml
conical tube.
Tissue was homogenized using a Qiagen TissueRuptor at 80% of maximum by
pulsing for a
total of 30 seconds . Homogenization was performed in an ice bucket to lessen
the effect of
heat on the PEDV virus. The homogenized material was filtered through a 0.45
uM filter and
60 ul of material was used for RNA isolation and PEDV Q-PCR to confirm the
presence of
the PEDV virus. The filtered material containing the PEDV virus was further
diluted 1:10 in
sterile PBS and then filtered through a 0.20 uM filter.
The sterile-filtered PEDV homogenate was used to infect confluent mono-layers
of
Vero 76 cells by transferring 1 ml of filtered material to a T-25 flask
containing 2.8E+06 cells
planted 3 to 4 days prior. The T-25 flasks of confluent Vero 76 cells were
washed 2X with
sterile PBS and 1X with DMEM media containing 10% TPB, 20ug/mIgeneticin and
4ug/m1
TPCK trypsin (equivalent to 18.8 USP units/m1). Cells were infected for 1 hour
at 37 C and
5%CO2in an incubator with gentle swirling every 15 minutes to ensure virus was
evenly
distributed to all cells. 5 ml of DMEM media containing 10% TPB,
20ug/mIgeneticin and
4ug/m1TPCK trypsin (equivalent to 18.8 USP units/m1) was added to flasks and
flask were
allowed to incubate 2 days. After 2 days, flasks were frozen at -80 C and
thawed at 37 C.
This material is considered as Passage 1 of the virus. One milliliter of the
total volume from
the flask was then used for Passage 2 of the virus. The 1 ml of Passage 1
material is used
to infect a T-25 flask containing 2.8E+06 cells seeded 3 to 4 days prior.
Cells were first
washed 2X with sterile PBS and 1X with DM EM media containing 10% TPB, 20ug/m1
geneticin and 4ug/m1TPCK trypsin (equivalent to 18.8 USP units/m1). Cells were
infected for
1 hour at 37 C and 5%CO2 in an incubator with gentle swirling every 15 minutes
to ensure
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virus was evenly distributed to cells. 5 ml of DMEM media containing 10% TPB,
20ug/m1
geneticin and 4ug/m1TPCK trypsin (equivalent to 18.8 USP units/m1) was added
to flasks
and flask were allowed to incubate for 2 days. This material is Passage 2 of
the PEDV virus.
Passages are repeated every 2 days until the cells show signs of infection
indicated by
5 clusters of cells surrounded by a filmy layer of material and/or a bubble
effect on the
clustered cells (see Figures 1-3). The appearance of PEDV infected cells was
confirmed by
a decrease in Ct value in the PEDV Taqman assay. The PEDV-infected cells have
a
rounded up appearance with a layer of shiny film surrounding the rounded up
cells.
10 Example 2: Propagation and Harvest
Plastic flasks or roller bottles are used for growing and expanding cell
cultures. Roller
bottles or bioreactors will be used for virus propagation. Cells may be
washed, to remove
serum, prior to inoculation with virus. The virus may be diluted in virus
production medium
and added directly to the cell monolayer. When bioreactors are used for virus
propagation,
15 trypsinized cells will be removed from the roller bottles and a final
cell passage grown in
uninoculated cell growth medium. Microcarriers for the bioreactors are
prepared. The seed
virus is diluted to an appropriate volume within a multiplicity of infection
(M01) range of
0.0001 to 10.0
The PED virus causes observable cytopathic effect (CPE). Virus is harvested
when
20 viral-induced CPE has reached 50-100% and infected cells have begun
sloughing off into the
medium (cell monolayer loss exceeding 50%). The roller bottle vessels are
removed from
the incubator and inspected microscopically for both CPE and evidence of
microbial
contamination. Following the examination, the antigen fluid is harvested into
appropriate
sterile containers in an aseptic manner. Bioreactor fluids are examined
microscopically for
25 evidence of microbial contamination and for the presence of desired
cytopathic effects
(CPE).
Following examination, the viral fluids are passed through a 100 micron filter
or
stainless steel mesh screen to remove microcarriers and harvested into
appropriate sterile
containers in an aseptic manner. Fluids may be stored at 2 C - 7 C for a
maximum of 24
30 hours until inactivation. The harvested fluids may be used for seed if
it is at the proper
passage level and has an acceptable infectivity titer.
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In the case of Calaf14 isolation, the following specific steps were used. PEDV
Calaf14 isolate was obtained from a PEDV positive case detected in a Spanish
farm in 2014.
Small intestines from 4-day-old piglets displaying clinical features
associated with PED,
including watery diarrhea and severe dehydration, were collected at necropsy.
Individual
intestine samples were processed to obtain a clarified intestine homogenate.
For that
purpose 59 g of intestine sample were suspended in 90 ml PBS (supplemented
with
Penicillin, Streptomycin and Gentamicin), disrupted using a tissue homogenizer
and
subsequently frozen at -80 10 C. Homogenate was thawed at 37 2 C and clarified
by
centrifugation at 2000rpm for 10 minutes at 4 C. Supernatant was collected,
aliquoted and
stored frozen at -80 10 C for further uses. Clarified intestine homogenate was
found to be
PEDV positive and TGEV negative by real-time RT-PCR analysis (Zoetis PEDV N
gene-
based real-time RT-PCR assay and PEDV-TEGV RT-PCR Rgt kit QIAGEN#283615).
Isolate
was identified as PEDV Calaf14 and considered passage 0 (PO).
For cell culture and isolation, intestine clarified homogenate was filtered
through a
0.45-pm filter. 1 ml of filtered material was diluted in 10m1 of PBS and then
filtered again
through a 0.22-pm filter and used as inoculum for virus isolation. Virus
isolation was
attempted using previously described PEDV cell culture conditions (Pan, Tian
et al. 2012,
VVicht, Li et al. 2014). Confluent Vero cells in 25-cm2 tissue culture flask
were washed twice
with PBS and one with the maintenance media-E (MM-E). Maintenance media-E
consisted of
Eagle's Minimum Essential medium Alpha Modification (MEMa, Gibco) supplemented
with
0.3% tryptose phosphate broth (TPB, Gibco), 20 mM HEPES (Gibco) and 15 g/ml
trypsin
(Dyfco). For the initial infection of cells, 1m1 of inoculum was adsorbed at
37 2 C with + 4-7%
CO2 for 1 hour. Flasks were replenished with 4m1 of maintenance media-E and
incubated up
to 2 days before being frozen at -80 10 C, thawed, and passaged as as
described above.
Blind passages were performed from P1 to P4 using 1m1 of previous passage as
inoculum
and maintenance media-E. Viral replication was verified by real-time RT-PCR
analysis each
passage. From passage 3 onward, a decrease of Ct value, indicative of an
increase of the
viral load, demonstrated a productive virus replication.
For subsequent attenuation of Calaf14, Subsequent passages (from P5 onwards)
were performed using maintenance media-C (MM-C): Dulbecco's modified Eagle's
medium
(DMEM, Gibco), supplemented with 0.3% TPB and 10 pg/ml trypsin (DMEMs + 0.30%
TPB +
10pg/m1 trypsin) as previously described (Pan, Tian et al. 2012, VVicht, Li et
al. 2014). From
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P5 to P42, 25-cm2 tissue culture tissue culture flasks of confluent Vero cells
were washed
twice with MM-C media and 0.01-1m1 of inoculum (previous passage material) was
adsorbed
at 37 2 C with + 4-7% CO2 for 1 hour. Flasks were replenished with MM-C media
and
incubated again for a period 1-3 days at 37 2 C with + 4-7% CO2. Flasks were
frozen,
thawed and harvested. Flasks were observed with a light microscope and
cytopatic effect
(CPE) with syncitia formation started to be evident at passage 6. From P43 to
P60 infection
was performed as follows: 25-cm2 tissue culture flasks of confluent Vero cells
were washed
once with MM-C media. Then flasks were filled in with the appropriate volume
(5-10 ml) of
MM-C media, inoculated with the appropriate volume (0.01-1mI) of the viral
suspension from
.. previous passage without adsorption and incubated for a period 1-3 days at
37 2 C with + 4-
7% CO2. When needed, relevant passages (P11, P37 and P60) viral cultures were
scaled
up in 150 cm2 or 75 cm2 tissue culture flasks to assure enough volume for
attenuation
assessment studies. In all the passages, one T-flask was incubated as a cell
control culture.
For cloning associated with Passage 60, PEDV P60 was cloned three times by end-
point dilution method. The aim is to dilute the virus in order to generate a
pure virus stock
starting from a single infectious unit. Before virus inoculation 96-well plate
of confluent vero
cells were washed once with MM-C media. Tenfold serial dilutions of the PEDV
in MM-C
without serum were performed from 10-1 to 10-8. Each dilution was seeded
eleven times
(50 1/well of virus dilutions) in a 96-well plate flat bottom. One column of
the plate was kept
as cell growth control. Plates were incubated at 37 C 2 C, 4-7% CO2 during 4-5
days. Each
well was observed for virus replication under an inverted microscope. All
wells with signs of
infection (CPE) were scored as positive. From the highest dilution at which
virus replication
was detected, the medium from one infected well was harvested. This inoculum
was purified
twice times more following the same procedure described above. Two clones were
finally
.. selected and identify as ClonA and ClonE. From the clones, a 24-well plate
of confluent vero
cells were infected in order to amplify the virus.
For next generation sequencing, viral RNA from samples PO, P11, P37, P60,
ClonA
and ClonE was extracted using the Biosprint technology (QIAGEN) according to
the
manufacturer's instructions. Cellular DNA was removed by using the Rnase free
Dnase set
(QIAGEN) and RNA cleanup by using the Rneasy Mini Kit (QIAGEN) following
manufacturer's instructions. Purified RNA was used to obtain the complete
genome for each
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33
sample by next-generation sequencing (NGS). Samples were sequenced using the
IIlumina
MySeq system at the Service of Genomic and Bioinformatic laboratory in the
University
Autonoma de Barcelona. Tehcniques generally applicable herein are found in
Pan, Y., X.
Tian, W. Li, Q. Zhou, D. Wang, Y. Bi, F. Chen and Y. Song (2012). "Isolation
and
characterization of a variant porcine epidemic diarrhea virus in China." Virol
J 9: 195, and
VVicht, 0., W. Li, L. VVillems, T. J. Meuleman, R. W. Wubbolts, F. J. van
Kuppeveld, P. J.
Rottier and B. J. Bosch (2014). "Proteolytic activation of the porcine
epidemic diarrhea
coronavirus spike fusion protein by trypsin in cell culture." J Virol 88(14):
7952-7961.
Example 3: Inactivation and Neutralization
Acceptable harvested antigen production fluids will be pooled into suitable
inactivation containers and inactivated using a 5mM binary ethylenimine (BEI)
solution. The
mixture is cyclized for 60-80 minutes at 36 2 C. Following the addition of
inactivant, the
antigen will be thoroughly mixed and transferred to an inactivation vessel for
the duration of
the process (4.8 hours, with agitation). Neutralization of the inactivated
antigen fluids will be
facilitated through the addition of sterile 1M Sodium Thiosulfate to a final
concentration of
approximately 20 mM ¨ 25 mM. Post-inactivated/neutralized antigen production
fluids will be
tested for sterility and completeness of inactivation and stored at 2-7 C for
future use in
vaccine serial formulation. Genatamicin can then be used as preservative. This
antibiotic
will be added at the lot stage. The concentration of gentamicin in the final
product will be
pg/mL.6.
Example 4: Specific Adjuvant Compositions and Formulations
A preferred adjuvanted vaccine composition was assembled as follows. The
killed
vaccine may provide about 7.8 logioTCI D5o of killed Calaf14, Passage 60, SEQ
ID NO:4, 5 or
25 6 virus per 2ML dose in a buffered solution further comprising about 5%
(v/v) Rehydragele
(aluminum hydroxide gel) and "20% Amphigen" 0 at about 25% final (v/v). Doses
down to
7.0 logioTCI D5o of killed virus are also preferred.
Amphigen is generally described in U.S Patent 5,084,269 and provides de-oiled
lecithin (preferably soy) dissolved in a light oil, which is then dispersed
into an aqueous
30 solution or suspension of the antigen as an oil-in-water emulsion.
Amphigen has been
improved according to the protocols of U.S. Patent 6,814,971 (see columns 8-9
thereof) to
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34
provide a so-called "20% Amphigen" component for use in the final adjuvanted
vaccine
compositions of the present invention. Thus, a stock mixture of 10% lecithin
and 90% carrier
oil (DRAKEOL , Penreco, Karns City, PA) is diluted 1: 4 with 0.63% phosphate
buffered
saline solution, thereby reducing the lecithin and DRAKEOL components to 2%
and 18%
respectively (i.e. 20% of their original concentrations). Tween 80 and Span 80
surfactants
are added to the composition, with representative and preferable final amounts
being 5.6%
(v/v) Tween 80 and 2.4% (v/v) Span 80, wherein the Span is originally provided
in the stock
DRAKEOL component, and the Tween is originally provided from the buffered
saline
component, so that mixture of the saline and DRAKEOL components results in the
finally
desired surfactant concentrations. Mixture of the DRAKEOL/lecithin and saline
solutions was
accomplished using an In-Line Slim Emulsifier apparatus, model 405, Charles
Ross and Son,
Hauppauge, NY, USA.
The vaccine composition also includes Rehydragele LV (about 2% aluminum
hydroxide content in the stock material), as additional adjuvant component
(available from
Reheis, NJ, USA, and ChemTrade Logistics, USA). With further dilution using
0.63% PBS,
the final vaccine composition contains the following compositional amounts:
7.8 logioTCI D5o
of killed virus per 2ML dose; 5% (v/v) Rehydragele LV; 25% (v/v) of "20%
Amphigen", i.e. it
is further 4-fold diluted); and 0.01% (w/v) of merthiolate.
As is understood in the art, the order of addition of components can be varied
to
provide the equivalent final vaccine composition. For example, an appropriate
dilution of
killed virus in buffer can be prepared. An appropriate amount of Rehydragele
LV (about 2%
aluminum hydroxide content) stock solution can then be added, with blending,
in order to
permit the desired 5% (v/v) concentration of Rehydragele LV in the actual
final product.
Once prepared, this intermediate stock material is combined with an
appropriate amount of
"20% Amphigen" stock (as generally described above, and already containing
necessary
amounts of Tween 80 and Span 80) to again achieve a final product having 25%
(v/v) of
"20% Amphigen". An appropriate amount of 10% merthiolate can finally be added.
The vaccinate compositions of the invention permit variation in all of the
ingredients,
such that the total dose of antigen may be varied preferably by a factor of
100 (up or down)
compared to the antigen dose stated above, and most preferably by a factor of
10 or less (up
or down),. Similarly, surfactant concentrations (whether Tween or Span) may be
varied by
up to a factor of 10, independently of each other, or they may be deleted
entirely, with
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replacement by appropriate concentrations of similar materials, as is well
understood in the
art.
Rehydragele concentrations in the final product may be varied, first by the
use of
equivalent materials available from many other manufacturers (i.e. Alhydrogele
,Brenntag;
5 Denmark), or by use of additional variations in the Rehydragele line of
products such as CG,
HPA or HS. Using LV as an example, final useful concentrations thereof
including from 0%
to 20%, with 2-12% being more preferred, and 4-8% being most preferred,
Similarly, the
although the final concentration of Amphigen (expressed as % of "20%
Amphigen") is
preferably 25%, this amount may vary from 5-50%, preferably 20-30% and is most
preferably
10 about 24-26%.
Other embodiments and uses of the invention will be apparent to those skilled
in the
art from consideration of the specification and practice of the invention
disclosed herein. All
references cited herein, including all publications, U.S. and foreign patents
and patent
applications, are specifically and entirely incorporated by reference. It is
intended that the
15 specification and examples be considered exemplary only with the true
scope and spirit of
the invention indicated by the following claims.
Example 5: Safety Studies with Live Calaf14 virus, Passages 0, 11, 37, and 60
A safety study was conducted to confirm the safety of Calaf 14 subsequent
passages,
20 by challenge of 4-day old, mixed sex pigs, thus Day 0 of challenge is
day 4 of life 4 =1- 1 day.
Route of infection/ was by esophageal gavage (2ML) using an appropriate tube
feed
catheter.challenge, of an undiluted cell culture supernatant. Piglets were
suckled on their
mothers until day 4, and during the study were fed an artificial milk diet,
milk having been
wiothdrawn for 1-2 hours prior to challenge.
25 Mortality was determined based on actual deaths and also piglets that
must be
euthanized per regulatory requirements. Depression, anorexia, and digestive
disorders were
scored as follows. Depression was scored as normal (active), slightly
inactive, pronounced
inactivity or severe depression (moribund). Piglets showing any level of
depression are
quantified below. Anorexia was scored as normal, moderate or severe. Piglets
showing any
30 level of anorexia are scored below. Digestive disorders were quantified
as low (mild
diarrhea), moderate (vomiting, abdominal pain or marked watery diarrhea) and
severe
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36
(fibrinous or hemorrhagic diarrhea). Piglets showing any level of digestive
disorder are
scored below. It can thus be seen that Passage 60 substantially achieves
safety criteria.
Table 1
Challenge Digestive First
First
dose
Passage Mortality Depression Anorexia
disorders detected Shedding detected
2.2
P11 10 TCID50 69% 77% 92% 92% 1 days p.i.
100% 1 days p.i.
3.4
P37 10 TCID50 10% 50% 50% 100% 2 days p.i.
100% 2 days p.i.
3.0
P60 10 TCID50 0% 0% 0% 0% NA 60%
6 days p.i.
