Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
COMPOSITIONS AND VACCINES CONTAINING ANTIGENS) OF
CRYPTOSPORIDIUMPARTrUMAND OF ANOTHER PATHOGEN
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Patent Application Serial
No. 09/742,512, filed on December 20, 2000, which claims priority from U.S.
Provisional Application Serial No. 60/171,399, filed December 21, 1999. This
application also claims priority from U.S. Provisional Application Serial No.
60/495,045 filed August 14, 2003.
Each of the applications and patents cited in this text, as well as each
document or reference cited in each of the applications and patents (including
during
the prosecution of each issued patent; "application cited documents"), and
each of
the PCT and foreign applications or patents corresponding to and/or claiming
priority from any of these applications and patents, and each of the documents
cited
or referenced in each of the application cited documents, are hereby expressly
incorporated herein by reference. More generally, documents or references are
cited
in this text, either in a Reference List before the claims, or in the text
itself; and,
each of these documents or references ("herein-cited references"), as well as
each
document or reference cited in each of the herein-cited references (including
any
manufacturer's specifications, instructions, etc.), is hereby expressly
incorporated
herein by reference.
FIELD OF THE INVENTION
The invention relates to antigen(s)/epitope(s) of Cryptosporidiunz parvufra
and/or enteric pathogens (such as other enteric pathogens), compositions and
methods comprising or using the same for eliciting an immune response against,
or
for prevention, treatment, or control of Cryptospo~idiurra parwmn and/or
enteric
infections, and uses thereof.
The invention further relates to methods and/or compositions, and/or uses of
such compositions or components thereof in formulating such compositions, for
eliciting an immune response against and/or for the prevention and/or
treatment
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2
and/or control of enteric infections in animals, for instance mammals, such as
bovines, felines, canines or equines or species thereof.
The invention relates also to methods and/or compositions, and/or uses of
such compositions or components thereof in formulating such compositions, for
eliciting an immune response against and/or for the prevention and/or
treatment
and/or control of infection by Cyyptospof°idium pa~vurn.
The invention can also relate to the concurrent use of a monovalent
CryptospoT~idium paf°vunz vaccine with enteric, e.g. bovine enteric
(e.g.,
rota/coronavirus, E, coli) vaccines and/or use of a combination vaccine
containing
Cfyptospo~~idiuyu pamum + rota/coronavirus, E. coli, as well as to preventing,
controlling or treating or eliciting an immune response to reduce exacerbation
of
enteric, e.g., bovine enteric, diseases due to co-infection with
C~yptospo~~idium
pa~~vum. The immunity induced by vaccination against Cryptospo~idium parvurn,
can significantly reduce the severity of the disease induced by herein
mentioned
enteric pathogens. A combination vaccine containing C~yptosponidium pa~~um is
useful for a more complete prevention of multietiological enteric disease in
newborn
animals, such as calves, caused by rota and coronaviruses and E. coli K99 and
F41.
This invention also pertains to the effects of Cryptospof-idum pai~vum co-
infection on other enteric, e.g., bovine enteric, pathogens. Cfyptosporidiuna
parvunz
is commonly found in the feces of newborn animals such as mammals, e.g.,
calves.
Cryptosporidiurn parvum is able to produce clinical signs of enteric disease
by itself,
regardless of the presence or absence of other potentially pathogenic viruses
and
bacteria in the gut. Viruses, such as coronavirus, and bacteria, such as E.
coli e.g.,
F41, that have been recognized in the field as very pathogenic are not able to
cause
important clinical signs of disease in experimental challenge models. Thus,
the
invention can relate to addressing the co-infection of cattle with
Cfyptospor°idiurra
parvum as that co-infection can exacerbate the disease caused by other enteric
pathogens such as coronavirus, rotavirus, and E. coli e.g., F41.
BACKGROUND OF THE INVENTION
Bovine enteric disease is the result of an enteropathogenic intestinal
infection
that most often manifests itself in some form of diarrhea. This disease, also
commonly referred to as neonatal calf diarrhea, is responsible for substantial
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economic loss in the farming industry. The morbidity of the calves, together
with
the need for therapeutic intervention and the possible long term detrimental
effects
on the animals, are the main factors responsible for the economic burden on
the
farmer. One estimate indicates that neonatal calf diarrhea is responsible for
about
75% of the death of dairy calves under 3-weeks of age. Radostits, OM, et al.,
Herd
Health Food Animal Production Medicine, 2"d ed., Sounders, Philadelphia, pp.
184-
213, 1994. The management of neonatal calf diarrhea is difficult for multiple
reasons, some of the most important which include: (1) the involvement of
multiple
agents in the pathogenesis of the disease; (2) the nonspecificity of clinical
signs; (3)
the finding that some infections can be asymptomatic; and, (4) the involvement
of
host factors such as nutrition and endogenous immunity. Moon, HW, et al.,
JAVMA 173 (5): 577 - 583 (1978). Viring, S. et al., Acta Vet. Scand. 34: 271-
279
( 1999).
Developing a strategy to prevent or treat bovine enteric disease has been very
difficult since while it is known that multiple enteropathogens are present
during the
infection, it is not known which pathogen or combination of pathogens is
actually
responsible for the disease. Epidemiological studies in the United States as
well as
in other parts of the world show that the most prevalent enteropathogens
associated
with neonatal calf diarrhea include, but are not limited to, Cryptospo~idium
parvum,
rotavirus, coronavirus and E. coli. While in most cases several of these
enteropathogens are isolated from outbreaks of the disease, the prevalence of
each of
the agents is not consistent within a single diseased population or between
multiple
infected herds.
Traditionally, studies found rotavirus to be the most prevalent
enteropathogen in diarrheic calves. For example, in a study of diarrheic
calves in
Great Britain, rotavirus and Cfyptospo~idium pa~vun2 were detected in 42 and
23%
of the population, respectively. Twenty percent of the calves were infected
with
more than one pathogen. However, more recent reports indicate Cyyptosporidium
pafwuy~a to be the predominant pathogen in enteric bovine infections. In a
recent
study evaluating C~yptospof~idiurn pafwuna and concurrent infections by other
major
enteropathogens in neonatal calves, C~yptospo~~idium pamuryi was the only
enteropathogen found in 52.3% of the population, followed by single infections
with
rotavirus at 42.7%. de la Fuente et al., Preventive Veterinary Medicine 36:
145 -
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4
152 (1998) Concurrent infection with two agents occurred in 21.6% of this
study
group while infection with three and four pathogens was found in 6% and 0.5%,
respectively. The most common mixed infection in this study was a combination
of
C~yptospoTfidium-rotavirus. There is limited information available on the role
of
individual enteric pathogens in neonatal calf diarrhea. Furthermore, combined
mechanisms of viral, bacterial and protozoal pathogenesis underlying the
bovine
enteric disease in neonatal animals are even more poorly understood. However,
irrespective of the lack of understanding of the mechanism of pathogenesis,
infection with more than one pathogen tends to lead to a more severe clinical
outcome than infections caused by a single enteropathogen.
At the present time there is no method of treatment that affords adequate
protection against neonatal calf diarrhea. There is no single drug or
combination of
chemotherapeutic agents useful in the treatment of this disease. While
vaccines are
available which target bovine enteric disease, they have been met with limited
success and acceptance. Presently available are vaccines that contain antigens
to
three enteropathogens found to be associated with the disease, namely
rotavirus,
coronavirus and E. coli. Efficacy of individual components of these
commercially
available bovine enteric vaccines (rota/corona, E. coli) has been shown to
protect in
experimental challenge models. Despite the availability of such vaccines,
under
field conditions neonatal diarrhea, calf scours and winter dysentery continue
to
affect beef,. feedlot and cow calf operations. Producers permanently question
the
efficacy of current enteric vaccines containing E. coli K99, rota and
coronavirus
under field conditions as is reflected by the low usage of the enteric combo
vaccines
in the US market (only 4% of pregnant animals are vaccinated annually with
this
product).
More recently, a monovalent experimental vaccine against Cyptospo~idimn
pa~~vum. has been developed and shown to protect against a CTyptospoi~idimn
pafwuni
experimental challenge. However, the multiple enteropathogens involved in
enteric
disease cannot be overcome by treatment with a C~~yptospo~idiurfa pa~vum
vaccine
alone. Also, enteropathogenic infection appears to be universal; it is found
throughout the world and most vertebrates are susceptible to such infection.
Therefore, a need to combat enteropathogenic infection is not limited to the
bovine
species. Furthermore, enteric disease is difficult to control; it is likely
multifactoral;
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Cryptospor°idiurn parwurrz may be a factor, but heretofore there is no
definitive
showing that Cryptospor~idiurn parwzzrrz indeed enhances enteric disease or
that its
use in a combination immunogenic, immunological or vaccine composition
enhances prevention of enteric disease.
Further, a problem encountered in the preparation and use of combination
vaccines is the phenomenon called "efficacy interference" wherein the efficacy
of
one antigen in the combination is diminished or reduced, believed to be from
dominance by another antigen in the combination vaccine; cf. Paoletti et al.,
U.S.
Patent No. 5,843,456. This phenomenon has been observed with combination
vaccines that employ E. coli antigen or antigens; for instance, single or
multiple
bacterial antigens can interfere with other antigens in combination vaccines.
Thus, it is believed that heretofore the problem of Cryptospor~idiuna
par~vurrz
contributing to enteric infections and symptoms, or the manner in which this
problem is herein addressed, e.g., combination compositions including
Cryptosporidiurn parwurrr antigens) or epitope(s) of interest with at least
one other
antigen or epitope of interest from a pathogen that causes enteric infection
and/or
symptoms and/or recombinants) and/or vectors) and/or plasmid(s) expressing
such
antigens) or epitope(s) of interest and administration of such compositions to
pregnant mammals such as pregnant cows and/or newborn or young mammals such
as calves within the first month of birth, and addressing any potential issue
of
efficacy interference, have not been disclosed or suggested.
OBJECTS AND SUMMARY OF THE INVENTION
An object of the invention can be improved enteric immunological or
vaccine compositions, especially those which can be used in the veterinary
field, for
instance for mammals, such as bovines, canines, felines or equines or species
thereof.
Another object of the invention can be such immunological or vaccine
compositions which can be effectively used to immunize newborn and/or young
animals, such as to passively immunize new-born animals, e.g., mammals, for
instance, bovines, canines, felines or equines or species thereof;
advantageously
bovines.
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6
Still another object of the invention can be improved immunological or
vaccine compositions against Cryptospoi~idium parvum, for instance particular
to be
used in the veterinary field, such as for use with mammals, e.g., for canines,
felines
or equines or species thereof, especially bovines or species thereof.
Yet another object of the invention can be improved methods for immunizing
newborns and/or young animals, such as to passively immunize newborn animals,
e.g., mammals, such as canines, felines or equines or species thereof
especially
bovines or species thereof.
Even further still, objects of the invention can involve methods for eliciting
an immune response against Cfyptosporidium parvum or enteric pathogens
including Cryptospo~~idium paf~vurn or for controlling, preventing and/or
treating
enteric infections and/or symptoms including Cryptosporidium pay~vurn; for
instance,
comprising administering an inventive composition; as well as methods for
preparing such compositions, uses of components of such compositions for
formulating such compositions, inter alia.
Vaccination or immunization against enteric pathogens, such as enteric
pathogens including Cryptosporidium paf~~~um is greatly and unexpectedly
improved
by using an immunological or vaccine composition including a combination of at
least two CryptosporidiunZ parvum antigens or epitopes thereof and/or vectors)
expressing at least two Cryptosporidium. paf~vum. antigens or epitopes
thereof, e.g.,
P21 or an eptitope thereof and/or a vector expressing P21 or an eptitope
thereof or
Cp23 or an epitope thereof and/or a vector expressing Cp23 or an epitope
thereof
and CplS/60 or an epitope thereof and/or a vector expressing CplS/60 (for
instance,
a composition containing at least one epitope of Cp23 and at least one epitope
of
CplS/60; and it is noted that the Cp23 antigen or protein can include P21).
The combination of both antigens (or epitope(s) of interest and/or vectors
expressing the antigens and/or epitope(s)) leads to a synergistic effect with
an
improved or useful production of an immune response, e.g., antibodies,
cellular
responses or both, against Cfyptosporidium payvum and/or enteric infection or
pathogens or symptoms such as a very high production of antibodies against
Cryptosporidiurn parvum. This also allows for the preparation of efficient
immunological or vaccine compositions, useful to protect newborn or young
animals
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7
or mammals, for instance, canines, felines or equines or species thereof;
especially
bovines. For instance, compositions containing antigens and/or epitope(s) of
interest
may be advantageously employed in inoculating dams or pregnant females, e.g.,
to
elicit an immune response that can be passed to the yet born offspring and to
new-
born or young animals via milk or colostrum during weaning, and, compositions
containing vectors) expressing antigens and/or epitope(s) may advantageously
be
employed in inoculating males and females of all ages, e.g., such as those
that are
not pregnant and/or are new-born or young animals, and the inoculation of new-
born
or young animals can be done alone or advantageously in conjunction with the
inoculation of dams or pregnant females, e.g., to allow for immune responses
to be
generated in the young or new-born animals while they also receive antibodies
or
other immunological agents via milk or colostrum during nursing.
Combining in an immunological or vaccine composition antigens) and/or
epitope(s) of interest against Cryptosporidiu~r pai~vum with at least one
other antigen
or epitope of interest against at least one other enteric pathogen of the
animal species
(and advantageously a plurality of antigens) and/or epitope(s) of interest
from a
plurality of pathogen(s), e.g., enteric pathogens) can significantly increase
protection
against enteric pathologies.
An especially advantageous inventive immunological or vaccine composition
can be against C~yptospo~°idiuna parwun~ and can comprise (i) at least
one Cp23
antigen or epitope of interest thereof and/or at least one vector expressing
at least
one Cp23 antigen or epitope of interest thereof or at least one P21 antigen or
epitope
of interest thereof and/or at least one vector expressing at least one P21
antigen or
epitope of interest thereof and (ii) at least one CplS/60 antigen or epitope
of interest
thereof and/or at least one vector expressing at least one CplS/60 The
composition
can advantageously further comprise at least one additional antigen or epitope
of
interest from another enteric pathogen and/or a vector expressing at least one
additional antigen (which can be the same vector that expresses the Cp23 or
P21
antigen or epitope of interest and/or the CplS/60 antigen or epitope of
interest, e.g.,
the composition can comprise a vector that co-expresses the Cp23 or P21
antigen or
epitope of interest and the CplS/60 antigen or epitope of interest, and
optionally the
optional additional antigen or epitope of interest).
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8
Another Cryptospof°idiunz pamunz antigen is the CP41 antigen
described in
Mark C. Jenkins et al., Clinical and Diagnostic Laboratory Immunology, Nov.
1999,
6, 6: 912-920. The immunological or vaccine compositions according to the
invention may comprise this antigen or epitope of interest thereof and/or a
vector
expressing said antigen or epitope thereof, possibly and preferably in
association
with at least one other C~yptospo~idium pa»~um as described herein such as
Cp23,
P21 and CplS/60, e.g. in combination with Cp23 or P21 and/or CplS/60. For
expression of this antigen, one may add a start codon upstream the nucleotide
sequence appearing on Figure 2 of this publication, and a stop codon
downstream
this sequence.
An efficient immunological or vaccine composition against enteritis is also
produced
by using only one of the Cp23 or an epitope thereof or a vector expressing the
antigen or epitope, or P21 or an epitope thereof or a vector expressing the
antigen or
epitope, or CplS/60 or an epitope thereof or a vector expressing the antigen
or
epitope thereof, or CP41 or an epitope thereof or a vector expressing the
antigen or
epitope, as a Cryptospo~idizcm pai~vum antigen or epitope of interest,
advantageously
in combination with at least one other C~yptospo~idiufzz parvum antigen or
epitope
of interest or vector expressing such an antigen or epitope of interest; and,
this
composition can further comprise at least one additional antigen or epitope of
interest from another enteric pathogen and/or a vector expressing the at least
one
additional antigen (and this vector can co-express antigens) and/or
epitope(s)).
The invention further comprehends methods for eliciting an immunological
or protective (vaccine) response against or for controlling, preventing and/or
treating
enteric pathogens or enteric infections or enteric symptoms, including
Gz~yptosporidium. paz~vum; for instance, comprising administering an inventive
composition.
An inventive composition can be administered to a pregnant mammal, such
as a heifer or a cow (hereinafter called cow), dog, cat, or horse during the
gestation
period; for instance, once or twice during the typical gestation period (for a
cow,
typically a 9 month or 170 day gestation period), such as a first
administration about
1 to about 2.5 or about 3 months before calving and a second or sole
administration
close to calving, e.g., in the last 3 weeks before calving, preferably about 3
to about
15 days before calving. In this way, the female can transfer passive immunity
to the
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newborn, e.g., calves after birth via milk or colostrum. Advantageously,
compositions comprising antigens) and/or epitope(s) of interest (as opposed to
compositions comprising vector(s), recombinants) and/or DNA plasmid(s)) are
administered to pregnant mammals as eliciting an antibody response is desired.
And, in contrast, such compositions that comprise vector(s), recombinants)
and/or
DNA plasmid(s) that express the antigens) and/or epitope(s) of interest ifa
vivo are
advantageously administered to a newborn or very young mammal (e.g., a mammal
that is susceptible to enteric disease, such as a bovine during about its
first month of
life and other mammals during analogous periods in their life), as a cellular
and/or
antibody response can be useful to prevent, treat, and/or control enteric
conditions,
infections or symptoms in such newborn and/or very young animals. The newborn
and/or very young animals can receive a booster of an antigenic and/or
epitopic
and/or vector/recombinant/DNA plasmid composition during the period of
susceptibility; and, its mother, optionally and advantageously, can also have
been
vaccinated during pregnancy, as herein described, such that the, newborn
and/or very
young animal can be receiving an immunological response by way of the
administration directly to it and passively.
A particular inventive composition can comprise one or more E. coli
antigens (e.g., inactivated E. coli bearing pili, such as, K99, Y, 31A, and/or
F4land/or these pili in subunit form or recombinantly expressed ifz vivo)
and/or one
or more rotavirus antigens (e.g., advantageously inactivated rotavirus),
and/or one or
more coronavirus antigen (e.g., bovine coronavirus antigen, advantageously
such as
inactivated coronavirus), in combination with one or more Cryptospo~idium
pamum
antigens, such as P21 and/or Cp23 and/or CplS/60. (And, as mentioned
previously,
one or more of these antigens can be an epitope of interest contained within
the
antigen; and, one or more of these antigens or epitopes of interest can be
expressed
i~r vivo by a recombinant or a plasmid.)
Thus, a particular inventive composition can comprise (i) one or more
Cryptosponidium pam~uf~z antigens, such as P21 and/or Cp23 and/or CplS/60
and/or
CP41 and advantageously P21 and/or Cp23 and CplS/60, and (ii) at least one E.
coli
antigen (e.g., at least one or all of of I~99, Y, 31A, F41 and/or other pili
borne by
inactivated E. coli or as subunits or as expressed ifs vivo; K99 and/or F41
are
preferably present and Y and/or 31A are advantageously also present) , and/or
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coronavirus and/or rotavirus antigen; such as one or more C. pa~vuna antigens,
such
as P21 and/or Cp23 and/or CplS/60 and/or CP41 and advantageously P21 and/or
Cp23 and CplS/60 and one or more rotavirus antigen such as inactivated
rotavirus,
or one or more C. pamum antigens, such as P21 and/or Cp23 and/or CplS/60
and/or
5 CP41 and advantageously P21 and/or Cp23 and CplS/60 and one or more
coronavirus antigen such as inactivated coronavirus, e.g., inactivated bovine
coronavirus, or one or more C. pai~vuf~~ antigens, such as P21 and/or Cp23
and/or
CplS/60 and/or CP41 and advantageously P21 and/or Cp23 and CplS/60 and one or
more E. coli antigen such as K99, Y, 31A, F41 and/or other pili borne by
inactivated
10 E. coli or as subunits or as expressed in vivo, e.g., a combination of K99,
Y, 31A
and/or F41. An exemplary E. coli antigen useful in the invention can be pili
as E.
coli pili can avoid efficacy interference. An exemplary composition can
comprise
one or more C. pamum antigens,, such as P21 and/or Cp23 and/or CplS/60 and/or
CP41 and advantageously P21 and/or Cp23 and CplS/60 and at least one E. coli
antigen, and at least one coronavirus antigen, and at least one rotavirus
antigen, e.g.,
P21 and/or Cp23 and/or Cp 15/60 and/or CP41 and advantageously P21 and/or Cp23
and CplS/60 and inactivated rotavirus, and inactivated coronavirus, and at
least one
E coli antigen, advantageously pili or preferably at least one or more of K99,
Y,
31 A, and F41, or a combination of K99, Y, 31A and F41. (And, as mentioned
previously, one or more of these antigens can be an epitope of interest
contained
within the antigen; and, one or more of these antigens or epitopes of interest
can be
expressed isz vivo by a recombinant or a plasmid.) In regard to potential
efficacy
interference by single or multiple bacteria, the inventors have found that by
increasing the amount of other antigens present in a combination vaccine, any
potential efficacy interference is avoided; and, that the use of pili as an E.
coli
antigen also avoids efficacy interference.
In these inventive compositions, a single dose can have the E. coli antigen
(or each E. coli antigen, in the case of multiple E. coli antigens) present in
an
amount usually found in vaccines against enteric pathogens such as an amount
to
obtain a serum titre in guinea pigs of at least 0.9 log 10; the rotavirus
antigen can be
present in an typically found in vaccines against enteric pathogens, such as
an
amount to obtain a serum titre in guinea pigs of at least 2.0 log 10, and the
coranovirus antigen can be present in an amount typically found in vaccines
against
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11
enteric pathogens such as an amount to obtain a serum titre in guinea pigs of
at least
1.5 log 10; and, the inventive compositions can include an adjuvant, such as
aluminum hydroxide, which can be present in a single dose in an amount
typically
found in vaccines such as preferably an amount of about 0.7 to about 0.9 mg.
Accordingly, in an aspect the invention provides combined enteric
immunological, immunogenic or vaccine composition comprising a first antigen
or
epitope of interest from Cnyptospo~idiuyra pamum and/or a first vector that
expresses
the first antigen or epitope of interest, and a second antigen or epitope of
interest
from another enteric pathogen and/or the first vector that expresses the first
antigen
or epitope of interest also expresses the second antigen or epitope of
interest and/or a
second vector that expresses the second antigen or epitope of interest, and a
pharmaceutically acceptable vehicle.
The composition can comprise antigen, which can be from Cfyptospo~idium
pafwum and an antigen from another enteric pathogen. The composition can
comprise an antigen from C~yptospo~idiuffa and an antigen from another enteric
pathogen of a bovine species; or of a canine species; or of a feline species;
or of an
equine species. The antigen from the enteric pathogen can be chosen from the
group
consisting of the antigens from E. coli, rotavirus, coronavirus, Clostridium
spp. and
mixtures thereof. The enteric pathogen can be E. coli. The antigen from E.
coli can
be selected from the group consisting of E. coli bearing K99 antigen, E. coli.
bearing
F41 antigen, E. coli bearing Y antigen, E. coli bearing 31A antigen, K99
antigen,
F41 antigen, Y antigen, 31A antigen, and mixtures thereof.
The enteric pathogen can comprise bovine coronavirus; and/or bovine
rotavirus and/or Clost~°idium pe~f~i~gefzs. The antigen of the enteric
pathogen can
comprise Clostridium pe~fi~ingevrs type C and D toxoids. In certain
embodiments,
the enteric pathogen can comprises E. coli, bovine rotavirus, bovine
coronavirus and
Clostridium perf~ifzgen or E. coli, bovine rotavirus, bovine coronavirus.
Yet further, in certain aspects the invention can comprise a composition
wherein the antigen of the enteric pathogen comprises E. coli antigens
selected from
the group consisting of E. coli bearing K99 antigen, E. coli. bearing F41
antigen, E.
coli bearing Y antigen, E. coli bearing 31A antigen, K99 antigen, F41 antigen,
Y
antigen, 31A antigen, and mixtures thereof; inactivated bovine coronavirus;
inactivated bovine rotavirus and Clostridium per', f~i~rgeas type C and D
toxoids; or E.
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12
coli antigens selected from the group consisting ofE. coli bearing K99
antigen, E.
coli. bearing F41 antigen, E. coli bearing Y antigen, E. coli bearing 31A
antigen,
K99 antigen, F41 antigen, Y antigen, 31A antigen and mixtures thereof;
inactivated
bovine coronavirus; and inactivated bovine rotavirus.
The inventive composition advantageously can comprise sub-unit
Cryptosporidium. parvum antigens selected from the group consisting of P21,
Cp23,
Cp 15/60, CP41 and mixtures thereof, such as Cp23 and Cp 15/60 or P21 and
CplS/60.
In the inventive compositions associating antigens from Cyyptospo~idium
pa~~ufrz. and at least one other enteric pathogen, the Cryptosporidizcm pamum
antigen
may also comprise or be constituted by, inactivated or live attenuated
oocysts, or
sub-units obtained from oocysts.
Inventive compositions can include an adjuvant such as saponin or aluminum
hydroxide; and, inventive compositions can be in the form of an oil-in-water
emulsion.
The invention further envisions an immunological, immunogenic or vaccine
composition against Cfyptospof~idium parvum, which comprises a first antigen
comprising a P21 or Cp23 antigen or an epitope thereof or a first vector that
expresses the first antigen and a second antigen comprising CplS/60 antigen or
epitope thereof or the first vector wherein the first vector expresses both
the first and
second antigens or a second vector that expresses the second antigen, and a
pharmaceutically acceptable vehicle. The composition can comprise Cp23 and
CplS/60 antigens which are in the form of separate fusion proteins. The
composition can comprise a vector expressing Cp23 and CplS/60. The composition
can comprise a first recombinant vector expressing Cp23 and a second
recombinant
vector expressing CplS/60. And, the composition can comprise P21 and CplS/60.
These compositions can further comprise an adjuvant.
Still further, the invention comprehends an immunological, immunogenic or
vaccine composition against Cyptospof°idiunz panvum, which comprises a
first
antigen comprising a P21 or Cp23 or CplS/60 or CP41 antigen or an epitope
thereof
or a first vector that expresses the first antigen and a second antigen
comprising a
second antigen or epitope thereof from C~yptospo~idium parvuna or the first
vector
wherein the first vector expresses both the first and second antigens or a
second
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13
vector that expresses the second antigen, wherein the first and second
antigens are
different from each other, and a pharmaceutically acceptable vehicle.
The invention also comprehends a method of bovine immunization of a
newborn calf against enteric disease comprising administering an inventive
composition to a pregnant female calf before delivering, so that the newborn
calf
receives maternal antibodies against CT~yptospo~idium pa~vunz through
colostrum
and/or mills. The method can further comprise the feeding to the newborn calf
colostrum and/or milk from cowls) which has (have) been administered the
composition during pregnancy. The method can comprise administering the
composition to the newborn calf. The composition administered to the pregnant
female can comprise antigens or epitopes thereof and the composition
administered
to the calf can comprise vectors. Thus, the invention also envisions a method
of
active immunization of adult and newborn calves, comprising administering to
the
calves an inventive composition.
The invention also comprehends a method of bovine immunization of a
newborn calf, comprising feeding to the newborn calf colostrum and/or milk
from
cows that have been administered the composition during pregnancy. Similarly,
in a
broader sense, the invention comprehends a method of immunization of a new-
born
mammal comprising feeding to the new-born colostrum and/milk from a female
mammal which has been administered the composition during pregnancy; and, the
mammal is advantageously, a bovine, a feline, a canine, or an equine. Still
further, the invention can encompass a method for preparing an inventive
composition comprising admixing the antigens or epitopes or vectors and the
carrier.
And, the invention can include a lcit for preparing an inventive composition
comprising the antigens, epitopes or vectors, each in separate container or
containers
(some antigens, epitopes or vectors may be together in one container, such as
the
Cryptosporidium parvuzzz antigens, epitopes or vectors may be together in one
container, and the other antigens, epitopes or vectors in one or more other
containers, or the carrier, diluent and/or adjuvant may be in separate
containers),
optionally packaged together; and further optionally with instructions for
admixture
and/or administration.
In this disclosure, "comprises," "comprising," "containing" and "having" and
the like can have the meaning ascribed to them in U.S. Patent law and can mean
"
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14
includes," "including," and the like; "consisting essentially of or "consists
essentially" likewise has the meaning ascribed in U.S. Patent law and the term
is
open-ended, allowing for the presence of more than that which is recited so
long as
basic or novel characteristics of that which is recited is not changed by the
presence
of more than that which is recited, but excludes prior art embodiments.
Other aspects of the invention are described in or are obvious from (and
within the ambit of the invention) the following disclosure.
BRIEF DESCRIPTION OF THE FIGURES
The following Detailed Description, given by way of example, but not
intended to limit the invention to specific embodiments described, may be
understood in conjunction with the accompanying drawings, incorporated herein
by
reference. Various preferred features and embodiments of the present invention
will
now be described by way of non-limiting example and with reference to the
accompanying dr awings in which:
Figure 1 shows a physical and restriction map of plasmid pJCA155;
Figure 2 shows a physical and restriction map of plasmid pJCA156;
Figure 3 shows a physical and restriction map of plasmid pJCA157;
Figure 4 shows a physical and restriction map of plasmid pJCA158;
Figure 5 shows a physical and restriction map of plasmid pJCA159;
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WO 2005/016383 PCT/US2004/026146
Figure 6 shows a physical and restriction map of plasmid pJCA160;
Figure 7 shows comparative oocysts count in feces in calves challenged with
either
C. parvum, or bovine rotavirus, or both, or non-challenged (example 12);
Figure 8 shows comparative rotavirus excretion in feces in calves according to
5 example 12;
Figure 9 shows comparative animal general condition for calves according to
example 12;
Figure 10 shows comparative animal dehydration status in calves according to
example 12;
10 Figure 11 shows comparative count of liquid feces for calves according to
example
12;
Figure 12 shows comparative anorexia status for calves according to example
12;
and
Figure 13 shows comparative rectal temperature evolution in calves according
to
15 example 12.
Figure 14 depicts average P21 (P21) colostrum antibody levels per vaccine
group.
Figure 15 shows the average CP15/60 colostrum antibody levels per vaccine
group.
Figure 16 shows the average P21 (P21) serum antibody levels per vaccine group.
Figure 17 depicts average CP 15/60 antibody levels per vaccine group.
Figure 18 depicts the hematocrit levels comparing challenged and unchallenged
animals.
Figure 19 illustrates the daily differences in % fecal dry matter by group and
by
daily collection time points.
Figure 20 is a graph showing the results of a P21 indirect ELISA antibody-
detection
assay.
Figure 21 shows the results from a CP15/60 ELISA antibody detection assay.
Figure 22 is a score chart depicting overall sickness of animals for all
vaccines over
time.
Figure 23 is a chart depicting the overall sickness of animals for the GST-
15/60 and
placebo vaccines only.
Figure 24 is a cloud diagram showing the diarrhea score for all vaccines.
Figure 25 is a cloud diagram showing the anorexia score for all vaccines.
Figure 26 is a cloud diagram showing the depression score for all vaccines.
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16
Figure 27 is a cloud diagram showing the fecal dry matter for all vaccines.
Figure 28 depicts oocyst shedding for all vaccines used in this study.
Figure 29 is a graph showing the mean evolution of rectal temperatures.
Figure 30 shows the average local reaction to the first vaccination (crypto +
combo;
combo alone).
Figure 31 shows the average local reaction to the second vaccination (crypto +
combo; combo alone).
Figure 32 is a graph showing the mean ELISA CP15/60 antibody titers.
Figure 33 shows the ELISA antibody titers to bovine coronavirus.
Figure 34 shows the virus neutralizing antibody titers to bovine coronavirus.
Figure 35 illustrates the ELISA antibody titers to bovine rotavirus.
Figure 36 illustrates the virus neutralizing antibody titers to bovine
rotavirus.
Figure 37 depicts the ELISA antibody titers to E. eoli K99 antigen.
Figure 38 depicts the ELISA antibody titers to E. eoli F41 antigen.
DETAILED DESCRIPTION OF THE INVENTION
An aspect of the invention is thus a combined enteric immunological,
immunogenic or vaccine composition comprising at least one an antigen or
epitope
of interest from at least one Cryptosporidiz~~n spp., preferably including
Cryptospo~°idium pamurn, and at least one antigen from at least one
other enteric
pathogen, advantageously a pathogen infecting the animal species to be
protected,
such as canine, feline, equine or bovine species and more advantageously
bovine
species; and/or a vector or vectors and/or a recombinant or recombinants
and/or a
plasmid or plasmids that expresses the C~yptosponidium spp antigen or epitope
of
interest and/or at least one of the antigens) or epitope(s) of interest of the
other
enteric pathogen; and a pharmaceutically acceptable vehicle. Universal
immunological, immunogenic or vaccine compositions are also envisioned as
enteric
pathogens are often infecting several (more than one) animal species.
An immunological composition elicits an immunological response - local or
systemic. An immunogenic composition likewise elicits a local or systemic
immunological response. A vaccine composition elicits a local or systemic
protective response. Accordingly, the terms "immunological composition" and
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17
"immunogenic composition" include a "vaccine composition" (as the two former
terms can be protective compositions).
C~yptospoy~idiusn pafwufn antigens which can be used in this invention
comprise preferably: (1) A protein of 148 amino acids called CplS/60 (See,
e.g.,
U.S. Patent No. 5,591,434. This protein is represented in US-A-5,591,434 in
SEQ
ID N0:2 with 10 further amino acids at the 5' end, upstream the methionine
(Met).
It is within the scope of the present invention to use an antigen comprising
or
consisting essentially of the 148 amino acid sequence of Cp 15/60 or of a
longer
amino acid sequence including these 148 amino acids, e.g. the whole sequence
represented in SEQ ID N0:2 in US-A-5,591,434 or any polypeptide comprising a
fragment of the 148 or 158 amino acid sequences that comprises an epitope
thereof,
advantageously a protection-eliciting epitope or an epitope that has the
immumogenicity of the full length sequence.) and/or (2) Cp23 and/or P21. (Cp23
is
an antigen of about 23 lcDa; see Perryman et al., Molec Biochem Parasitol
80:137-
147 (1996); WO-A-9807320 and L. E. PeiTyman et al., Vaccine 17 (1999) 2142-
2149. The major part of this protein (187 amino acids) is herein termed P21
and has
an amino acid sequence homologous to the amino acid sequence of protein C7,
which is disclosed as SEQ ID NO. 12 in WO-A-98 07320. To be expressed, one or
two or more amino acids can be added at the end of P21, such as, Met-, or Met-
Gly-
or similar amino acids. It is within the scope of the present invention to use
an
antigen comprising or consisting essentially of or consisting of the 187 amino
acid
sequence or a longer amino acid sequence, or a polypeptide comprising a
fragment
of the 187 amino acid sequence that comprises an epitope thereof,
advantageously a
protection-eliciting epitope or an epitope that has the immunogenicity of the
full
length sequence. The whole amino acid sequence of Cp23 and the corresponding
nucleotide sequence is easily obtainable. The P21 protein represents the major
part
and the C-terminal end of Cp23. The P21 nucleotide sequence may be used as a
probe to screen a DNA library, e.g. a library as disclosed in Example 1. This
methodology is well known to the one skilled in the art. On the basis of the
molecular weight of Cp23, it can be asserted that about 25-35 amino acids are
missing at the N-terminal end of P21 to have the complete Gp23 amino acid
sequence. This information gives those sleilled in the art the means to easily
find the
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WO 2005/016383 PCT/US2004/026146
18
start codon and thus the 5' end of the Cp23 nucleotide sequence and the N-
terminal
amino acid sequence.
The antigens or epitopes of interest can be used individually or in
combination in compositions of the invention, e.g., an inventive composition
can
include (1) or (2) or both (1) and (2).
Another possible antigen is the CP41 antigen as disclosed supra.
According to the preferred embodiment, these antigens or epitopes of interest
are incorporated into the composition as proteins or sub-unit antigens. They
can be
produced by chemical synthesis or by expression ifz vitro. The examples
describe
how to obtain the sequences encoding Cp 15/60 and P21 and how to construct
vectors expressing them. These sequences can be cloned into suitable cloning
or
expression vectors. These vectors are then used to transfect suitable host
cells. The
antigens encoded by the nucleotide sequence which is inserted into the vector,
e.g.
Cp23 and/or P21 and/or CplS/60, are produced by growing the host cells
transformed by the expression vectors under conditions whereby the antigen is
produced. This methodology is well known to the one skilled in the art. Host
cells
may be either procaryotic or eucaryotic, e.g. Eschei°ichia coli (E.
coli), yeasts such
as Saechaf~osnyces cenevisiae, animal cells, in particular animal cell lines.
The one
skilled in the art knows the vectors which can be used with a given host cell.
The
vectors may be chosen such that a fusion protein is produced which can be used
then
to easily recover the antigen.
Furthermore, with respect to sequences, nucleic acid sequences useful for
expressing the C. ~amurra antigen or epitope of interest can include nucleic
acid
sequences that are capable of hybridizing under high stringency conditions or
those
having a high homology with nucleic acid molecules employed in the invention
(e.g., nucleic acid molecules in documents mentioned herein); and,
''hybridizing
under high stringency conditions"can be synonymous with "stringent
hybridization
conditions", a term which is well known in the art; see, for example, Sambrook
et
al., "Molecular Cloning, A I;aboratory Manual" second ed., CSH Press, Cold
Spring
Harbor, 1989; "Nucleic Acid Hybridisation, A Practical Approach", Hames and
Higgins eds., IRL Press, Oxford, 1985; both incorporated herein by reference.
With respect to nucleic acid molecules and polypeptides which can be used
in the practice of the invention, the nucleic acid molecules and polypeptides
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WO 2005/016383 PCT/US2004/026146
19
advantageously have at least about 75% or greater homology or identity,
advantageously 80% or greater homology or identity, more advantageously 85% or
greater homology or identity, such as at least about 85% or about 86% or about
87%
or about 88% or about 89% homology or identity, for instance at least about
90% or
homology or identity or greater, such as at least about 91 %, or about 92%, or
about
93%, or about 94% identity or homology, more advantageously at least about 95%
to 99% homology or identity or greater, such as at least about 95% homology or
identity or greater e.g., at least about 96%, or about 97%, or about 98%, or
about
99%, or even about 100% identity or homology, or from about 75%,
advantageously
from about 85% to about 100% or from about 90% to about 99% or about 100% or
from about 95% to about 99% or about 100% identity or homology, with respect
to
sequences set forth in herein cited documents (including subsequences thereof
discussed herein); and thus, the invention comprehends a vector encoding an
epitope
or epitopic region of a C. parwuna isolate or a composition comprising such an
epitope, compositions comprising an epitope or epitopic region of a C. paywuni
isolate, and methods for making and using such vectors and compositions, e.g.,
the
invention also comprehends that these nucleic acid molecules and polypeptides
can
be used in the same fashion as the herein mentioned nucleic acid molecules,
fragments thereof and polypeptides.
Nucleotide sequence homology can be determined using the "Align"
program of Myers and Miller, ("Optimal Alignments in Linear Space", CABIOS 4,
11-17, 1988, incorporated herein by reference) and available at NCBI.
Alternatively
or additionally, the term "homology" or "identity", for instance, with respect
to a
nucleotide or amino acid sequence, can indicate a quantitative measure of
homology
between two sequences. The percent sequence homology can be calculated as
(N,.ef
- Ndf)* 100/N,.ef, wherein Ndlf is the total number of non-identical residues
in the
two sequences when aligned and wherein N,,ef is the number of residues in one
of
the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence
similarity of 75% with the sequence AATCAATC (N,.ef = 8; Nd,~-2).
Alternatively or additionally, "homology" or "identity" with respect to
sequences can refer to the number of positions with identical nucleotides or
amino
acids divided by the number of nucleotides or amino acids in the shorter of
the two
sequences wherein alignment of the two sequences can be determined in
accordance
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WO 2005/016383 PCT/US2004/026146
with the Wilbur and Lipman algorithm (Wilbur and Lipman, 1983 PNAS USA
80:726, incorporated herein by reference), for instance, using a window size
of 20
nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and
computer-
assisted analysis and interpretation of the sequence data including alignment
can be
5 conveniently performed using commercially available programs (e.g.,
Intelligenetics
TM Suite, Intelligenetics Inc. CA). When RNA sequences are said to be similar,
or
have a degree of sequence identity or homology with DNA sequences, thymidine
(T)
in the DNA sequence is considered equal to uracil (U) in the RNA sequence. RNA
sequences within the scope of the invention can be derived from DNA sequences,
by
10 thymidine (T) in the DNA sequence being considered equal to uracil (U) in
RNA
sequences.
Additionally or alternatively, amino acid sequence similarity or identity or
homology can be determined using~the BlastP program (Altschul et al., Nucl.
Acids
Res. 25, 3389-3402, incorporated herein by reference) and available at NCBI
(used
15 in determining sequence homology, as shown in Appendix I; see also the
Examples). The following references (each incorporated herein by reference)
also
provide algorithms for comparing the relative identity or homology of amino
acid
residues of two proteins, and additionally or alternatively with respect to
the
foregoing, the teachings in these references can be used for determining
percent
20 homology or identity: Needleman SB and Wunsch CD, "A general method
applicable to the search for similarities in the amino acid sequences of two
proteins,"
J. Mol. Biol. 48:444-453 (1970); Smith TF and Waterman MS, "Comparison of Bio-
sequences," Advances in Applied Mathematics 2:482-489 (1981); Smith TF,
Waterman MS and Sadler JR, "Statistical characterization of nucleic acid
sequence
functional domains," Nucleic Acids Res., 11:2205-2220 (1983); Feng DF and
Dolittle RF, "Progressive sequence alignment as a prerequisite to correct
phylogenetic trees," J. of Molec. Evol., 25:351-360 (1987); Higgins DG and
Sharp
PM, "Fast and sensitive multiple sequence alignment on a microcomputer,"
CABIOS, 5: 151-153 (1989); Thompson JD, Higgins DG and Gibson TJ,
"ClusterW: improving the sensitivity of progressive multiple sequence
alignment
through sequence weighing, positions-specific gap penalties and weight matrix
choice, Nucleic Acid Res., 22:4673-480 (1994); and, Devereux J, Haeberlie P
and
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WO 2005/016383 PCT/US2004/026146
21
Smithies O, "A comprehensive set of sequence analysis program for the VAX,"
Nucl. Acids Res., 12: 387-395 (1984).
Furthermore, as to nucleic acid molecules used in this invention (e.g., as in
herein cited documents), the invention comprehends the use of codon equivalent
nucleic acid molecules. For instance, if the invention comprehends "X" protein
(e.g., P21 and/or Cp23 and/or CplS/60 and/or CP41) having amino acid sequence
"A" and encoded by nucleic acid molecule "N", the invention comprehends
nucleic
acid molecules that also encode protein X via one or more different codons
than in
nucleic acid molecule N.
The antigen or epitope of interest used in the practice of the invention can
be
obtained from the particular pathogen(s), e.g., C. pa~vufn, E. coli,
rotovirus,
coronavirus, and the like or can be obtained from in. vita°o and/or i~r
vivo recombinant
expression of genes) or portions thereof. Methods for malting and/or using
vectors
(or recombinants) for expression can be by or analogous to the methods
disclosed in:
U.S. Patent Nos. 4,603,112, 4,769,330, 5,174,993, 5,505,941, 5,338,683,
5,494,807,
4,722,848, 5,942,235, PCT publications WO 94/16716, WO 96/39491, Paoletti,
"Applications of pox virus vectors to vaccination: An update," PNAS USA
93:11349-11353, October 1996, Moss, "Genetically engineered poxviruses for
recombinant gene expression, vaccination, and safety," PNAS USA 93:11341-
11348, October 1996, Smith et al., U.S. Patent No. 4,745,051 (recombinant
baculovirus), Richardson, C.D. (Editor), Methods in Molecular Biolo~v 39,
"Baculovirus Expression Protocols" (1995 Humana Press Inc.), Smith et al.,
"Production of Huma Beta Interferon in Insect Cells Infected with a
Baculovirus
Expression Vector," Molecular and Cellular Biology, Dec., 1983, Vol. 3, No.
12, p.
2156-2165; Pennoclc et al., "Strong and Regulated Expression of
Esche~°iclaia coli B-
Galactosidase in Infect Cells with a Baculovirus vector," Molecular and
Cellular
Biology Mar. 1984, Vol. 4, No. 3, p. 399-406; EPA 0 370 573, U.S. application
Serial No. 920,197, filed October 16, 1986, EP Patent publication No. 265785,
U.S.
Patent No. 4,769,331 (recombinant herpesvirus), Roizman, "The function of
herpes
simplex virus genes: A primer for genetic engineering of novel vectors," PNAS
USA 93:11307-11312, October 1996, Andrearislcy et al., "The application of
genetically engineered herpes simplex viruses to the treatment of experimental
brain
tumors," PNAS USA 93:11313-11318, October 1996, Robertson et al. "Epstein-Barr
CA 02535618 2006-02-13
WO 2005/016383 PCT/US2004/026146
22
virus vectors for gene delivery to B lymphocytes," PNAS USA 93:11334-11340,
October 1996, Frolov et al., "Alphavirus=based expression vectors: Strategies
and
applications," PNAS USA 93:11371-11377, October 1996, I~itson et al., J.
Virol. 65,
3068-3075, 1991; U.S. Patent Nos. 5,591,439, 5,552,143, allowed U.S.
applications
Serial Nos. 08/675,556 and 08/675,566, filed July 3, 1996 (recombinant
adenovirus),
Grunhaus et al., 1992, "Adenovirus as cloning vectors," Seminars in Virology
(Vol.
3) p. 237-52, 1993, Ballay et al. EMBO Journal, vol. 4, p. 3861-65, Graham,
Tibtech
8, 85-87, April, 1990, Prevec et al., J. Gen Virol. 70, 429-434, PCT
W091/11525,
Felgner et al. (1994), J. Biol. Chem. 269, 2550-2561, Science, 259:1745-49,
1993
and McClements et al., "Immunization with DNA vaccines encoding glycoprotein D
or glycoprotein B, alone or in combination, induces protective immunity in
animal
models of herpes simplex virus-2 disease," PNAS USA 93:11414-11420, October
1996, and U.S. Patents Nos 5,591,639, 5,589,466, and 5,580,859 relating to DNA
expression vectors, irrte~ alia. See also WO 98/33510; Ju et al.,
Diabetologia,
41:736-739, 1998 (lentiviral expression system); Sanford et al., U.S. Patent
No.
4,945,050; Fischbach et al. (Intracel), WO 90/01543; Robinson et al., seminars
in
IMMUNOLOGY, vol. 9, pp.271-283 (1997) (DNA vector systems); Szoka et al.,
U.S. Patent No. 4,394,448 (method of inserting DNA into living cells);
McCormick
et al., U.S. Patent No. 5,677,178 (use of cytopathic viruses); U.S. Patent No.
5,928,913 (vectors for gene delivery), and Tartaglia et al. U.S. Patent No.
5,990,091
(vectors having enhanced expression), as well as other documents cited herein.
A
viral vector, for instance, selected from herpes viruses, adenoviruses,
poxviruses,
especially vaccinia virus, avipox virus, canarypox virus, as well as DNA
vectors
(DNA plasmids) are advantageously employed in the practice of the invention,
especially for i~r vivo expression (whereas bacterial and yeast systems are
advantageously employed for is vitro expression).
If the host-vector combination leads to the production of antigen without
excretion, for the convenience of their production, and their recovering,
these
antigens are preferably under the form of fusion proteins (e.g., a HIS tag).
In other
words, the antigen can comprise the antigen peg se and foreign amino acids.
Techniques for protein purification and/or isolation from this disclosure and
documents cited herein, ifzte~ alia, and thus within the ambit of the skilled
artisan,
can be used, without undue experimentation, to purify and/or isolate
recombinant or
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23
vector expression products and/or antigen(s), in the practice of the
invention, and
such techniques, in general, can include: precipitation by taking advantage of
the
solubility of the protein of interest at varying salt concentrations,
precipitation with
organic solvents, polymers and other materials, affinity precipitation and
selective
denaturation; column chromatography, including high performance liquid
chromatography (HPLC), ion-exchange, affinity, immunoaffinity or dye-ligand
chromatography; immunoprecipitation and the use of gel filtration,
electrophoretic
methods, ultl~afiltration and isoelectric focusing, inter alia.
As mentioned herein, according to another aspect, the invention
comprehends that the antigens and/or epitopes of interest are not incorporated
as
subunits in the composition, but rather that they are expressed ifz vivo;
e.g., the
invention comprehends that the composition comprises recombinant vectors)
expressing the antigens ifz vivo when administered to the animal. The vector
can
comprise a DNA vector plasmid, a herpesvirus, an adenovirus, a poxvirus,
including
a vaccinia virus, an avipox virus, a canarypox virus, and a swinepox virus,
and the
like. The vector-based compositions can comprise a vector that contains and
expresses a nucleotide sequence of the antigen to be expressed, e.g., CplS/60
and/or
Cp23 for C~yptosporidiuyn parvu~r.
The word plasmid is intended to include any DNA transcription unit in the
form of a polynucleotide sequence comprising the sequence to be expressed.
Advantageously, the plasmid includes elements necessary for its expression;
for
instance, expression irr vivo. The circular plasmid form, supercoiled or
otherwise, is
advantageous; and, the linear form is also included within the scope of the
invention.
The plasmid can be either naked plasmid or plasmid formulated, for example,
inside
lipids or liposomes, e.g., cationic liposomes (see, e.g., WO-A-90 11082; WO-A-
92
19183; WO-A-96 21797; WO-A-95 20660). The plasmid immunological or vaccine
composition can be administered by way of a gene gun, or intramuscularly, or
nasally, or by any other means that allows for expression in vivo, and
advantageously an immunological or protective response. Reference is also made
to
U.S. applications Serial Nos. 09/232,278, 09/232,468, 09/232,477, 09/232,279,
09/232,478, and 09/232,469, each filed January 15, 1999 (and incorporated
herein
by reference), and to U.S. applications Serial Nos. 60/138,352 and 60/138,478,
each
filed June 10, 1999 (and incorporated herein by reference), as these
applications
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WO 2005/016383 PCT/US2004/026146
24
involve DNA and/or vector vaccines or immunogenic or immunological
compositions for felines, canines, bovines, and equines, and inventive
compositions
can include DNA and/or vector vaccines or immunogenic or immunological
compositions from these applications and/or inventive compositions can be
prepared
and/or formulated and/or administered in a fashion analogous to the
compositions of
these applications:
Compositions for use in the invention can be prepared in accordance with
standard techniques well known to those skilled in the veterinary or
pharmaceutical
or medical arts. Such compositions can be administered in dosages and by
techniques well known to those skilled in the veterinary arts taking into
consideration such factors as the age, sex, weight, condition and particular
treatment
of the animal, and the route of administration. The components of the
inventive
compositions can be administered alone, or can be co-administered or
sequentially
administered with other compositions (e.g., the C. paf~~u~rz antigens) and/or
epitope(s) can be administered alone, and followed by the administration
sequentially of antigens) and/or epitope(s) of other enteric pathogens, or
compositions comprising a enteric antigens) or epitope(s) can include vectors
or
recombinants or plasmids that also express enteric antigens) or epitope(s) of
the
same or different pathogens) or with other prophylactic or therapeutic
compositions
(e.g., other immunogenic, immunological or vaccine compositions). Thus, the
invention provides multivalent or "cocktail" or combination compositions and
methods employing them. The ingredients and manner (sequential, e.g., as part
of a
prime-boost regimen, or as part of a booster program wherein immunogenic,
immunological or vaccine composition is administered periodically during the
life of
the animal such as an annual, seasonal, biannual and the like booster program;
or co
administration) of administration, as well as dosages, can be.determined,
taking into
consideration such factors as the age, sex, weight, condition and particular
treatment
of the animal, e.g., cow, and, the route of administration. In this regard,
reference is
made to U.S. Patent No. 5,843,456, incorporated herein by reference, and
directed to
rabies compositions and combination compositions and uses thereof.
Compositions of the invention may be used for parenteral or mucosal
administration, preferably by intradermal, subcutaneous or intramuscular
routes.
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When mucosal administration is used, it is possible to use oral, nasal, or
vaginal
routes. .
In such compositions, the vector(s), or antigens) or epitope(s) of interests)
may be in admixture with a suitable carrier, diluent, or excipient such as
sterile
water, physiological saline, glucose or the like. The compositions can also be
lyophilized. The compositions can contain auxiliary substances such as pH
buffering
agents, adjuvants, preservatives, polymer excipients used for mucosal routes,
and the
like, depending upon the route of administration and the preparation desired.
Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE",
10 17th edition, 1985, incorporated herein by reference, may be consulted to
prepare
suitable preparations, without undue experimentation. Suitable dosages can
also be
based upon the text herein and documents cited herein.
Adjuvants are substances that enhance the immune response to antigens.
Adjuvants, can include aluminum hydroxide and aluminum phosphate, saponins
15 e.g., Quil A, mineral oil emulsions, pluronic polymers with mineral or
metabolizable
oil emulsion, the water-in-oil adjuvant, the oil-in-water adjuvant, synthetic
polymers
(e.g., homo- and copolymers of lactic and glycolic acid, which have been used
to
produce microspheres that encapsulate antigens, see Eldridge et al., Mol.
Immunol.
28:287-294 (1993), e.g., biodegradable microspheres), nonionic block
copolymers,
20 low molecular weight copolymers in oil-based emulsions (see Hunter et al.,
The
Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D.E.S.). John
Wiley and Sons, NY, pp51-94 (1995)), high molecular weight copolymers in
aqueous formulations (Todd et al., Vaccine 15:564-570 (1997)), cytolcines such
as
IL-2 and IL-12 (see, e.g., U.S. Patent No. 5,334,379), and GM-CSF (granulocyte
25 macrophage-colony stimulating factor; see, genes°ally, U.S. Patents
Nos. 4,999,291
and 5,461,663, see also Clarlc et al., Science 1987, 230:1229; Grant et al.,
Drugs,
1992, 53:516), advantageously GM-CSF from the animal species to be vaccinated,
Intel- alia. Certain adjuvants can be expressed ifz vioo with antigens) and/or
epitope(s); e.g., cytolcines, GM-CSF (see, e.g., C. R. Maliszewslci et al.
Molec
Immunol 25(9): 843-50 (1988); S.R. Leong, Vet Immunol and Immunopath 21:261-
78 (1989) concerning bovine GM-CSF. A plasmid encoding GM-CSF can be
modified to contain and express DNA encoding an antigen from a bovine pathogen
according to the instant invention and/or an epitope thereof optionally also
with
CA 02535618 2006-02-13
WO 2005/016383 PCT/US2004/026146
26
DNA encoding an antigen and/or epitope of another bovine pathogen, or can be
used
in conjunction with such a plasmid)
A further instance of an adjuvant is a compound chosen from the polymers of
acrylic or methacrylic acid and the copolymers of malefic anhydride and
alkenyl
derivative. Advantageous adjuvant compounds are the polymers of acrylic or
methacrylic acid, which are cross-linked, especially with polyalkenyl ethers
of
sugars or polyalcohols. These compounds are known by the term carbomer
(Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also
refer to
U.S. Patent No. 2,909,462 (incorporated herein by reference) which describes
such
acrylic polymers cross-linked with a polyhydroxylated compound having at least
3
hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least
three
hydroxyls being replaced by unsaturated aliphatic radicals having at least 2
carbon
atoms. The preferred radicals are those containing from 2 to 4 carbon atoms,
e.g.
vinyls, allyls and other ethylenically unsaturated groups. The unsaturated
radicals
may themselves contain other substituents, such as methyl. The products sold
under
the name Carbopol~ (BF Goodrich, Ohio, USA) are particularly appropriate. They
are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among
then,
there may be mentioned Carbopol~ 974P, 934P and 971P. Among the copolymers
of malefic anhydride and alkenyl derivative, the copolymer s EMA~ (Monsanto),
which are copolymers of malefic anhydride and ethylene, linear or cross-
linked, for
example cross-linked with divinyl ether, are preferred. Reference may be made
to J.
Fields et al., Nature, 186: 778-780, 4 June 1960, incorporated herein by
reference.
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WO 2005/016383 PCT/US2004/026146
27
From the point of view of their structure, the polymers of acrylic or
methaciylic acid and the copolymers EMA~ are preferably formed of basic units
of
the following formula:
Z
1
- ---~ -( ),~-----~ (c~2) y ---
'
in which:
Rl and RZ, which are identical or different, represent H or CH3;
x = 0 or l, preferably x = l; and
y=lor2,withx+y=2.
For the copolymers EMAO, x = 0 and y = 2. For the carbomers, x = y =1.
The dissolution of these polymers in water leads to an acid solution that will
be neutralized, preferably to physiological pH, in order to give the adjuvant
solution
into which the immunogenic, immunological or vaccine composition itself will
be
incorporated. The carboxyl groups of the polymer are then partly in COO- form.
Preferably, a solution of adjuvant according to the invention, especially of
carbomer, is prepared in distilled water, preferably in the presence of sodium
chloride, the solution obtained being at acidic pH. This stock solution is
diluted by
adding it to the desired quantity (for obtaining the desired final
concentration), or a
substantial part thereof, of water charged with NaCI, preferably physiological
saline
(NaCI 9 g/1) all at once in several portions with concomitant or subsequent
neutralization (pH 7.3 to 7.4), preferably with NaOH. This solution at
physiological
pH will be used as it is for mixing with the vaccine, which may be especially
stored
in freeze-dried, liquid or frozen form.
The polymer concentration in the final vaccine composition can be 0.01% to
2% w/v, e.g., 0.06 to 1% w/v, such as 0.1 to 0.6% w/v.
Adjuvanting immunogenic and vaccine compositions according to the
invention may also be made with formulating them in the form of emulsions, in
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28
particular oil-in-water emulsions, e.g. an emulsion such as the SPT emulsion
described p 147 in " Vaccine Design, The Subunit and Adjuvant Approach "
edited
by M. Powell, M. Newman, Plenum Press 1995,.or the emulsion MF59 described
p183 in the same book. In particular, the oil-in-water emulsion may be based
on
light liquid paraffin oil (according to European Pharmacopoeia); isoprenoid
oil, such
as squalane, squalene ; oil obtained by oligomerisation of alkenes, in
particular of
isobutylene or of decene ; acid or alcohol esters with linear alkyl groups,
particularly
vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate),
glycerol
tri(caprylate / caprate), propylene glycol dioleate; esters of branched fatty
acids or
alcohols, in particular esters of isostearic acid. The oil is used in
combination with
emulsifiers to form the emulsion. Emulsifiers are preferably non-ionic
surfactants, in
particular sorbitan esters, mannide esters, glycerol esters, polyglycerol
esters,
propylene glycol esters or esters of oleic acid, of isostearic acid, of
ricinoleic acid, of
hydroxystearic acid, possibly ethoxylated, block-copolymers such as
polyoxypropylene-polyoxyethylene, in particular the products called Pluronic,
namely Pluronic L121.
From this disclosure and the knowledge in the art, the skilled artisan can
select a suitable adjuvant, if desired, and the amount thereof to employ in an
immunological, immunogenic or vaccine composition according to the invention,
without undue experimentation.
The immunological, immunogenic or vaccine compositions according to the
invention may be associated to at least one live attenuated, inactivated, or
sub-unit
vaccine, or recombinant vaccine (e.g. poxvirus as vector or DNA plasmid)
expressing at least one immunogen, antigen or epitope of interest from another
pathogen.
Compositions in forms for various administration routes are envisioned by
the invention. And again, the effective dosage and route of administration are
determined by known factors, such as age, weight. Dosages of each active agent
e.g., of each C. pamur~a antigen or epitope of interest and/or of each antigen
or
epitope from each enteric pathogen can be as in herein cited documents or as
otherwise mentioned herein and/or can range from one or a few to a few hundred
or
thousand micrograms, e.g., 1 ~g to lmg, for a subunit immunogenic,
immunological
or vaccine composition; and, 104 to 101° TCIDS° advantageously
106 to 108 TCIDS°,
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29
before inactivation, for an inactivated immunogenic, immunological or vaccine
composition.
Recombinants or vectors can be administered in a suitable amount to obtain
ih vivo expression corresponding to the dosages described herein and/or in
herein
cited documents. For instance, suitable ranges for viral suspensions can be
determined empirically. The viral vector or recombinant in the invention can
be
administered to the animal or infected or transfected into cells in an amount
of about
at least 103 pfu; more preferably about 10~ pfu to about 101° pfu,
e.g., about 105 pfu
to about 109 pfu, for instance about 106 pfu to about 10$ pfu, with doses
generally
ranging from about 106 to about 101°, preferably about 101°
pfu/dose, and
advantageously about 108 pfu per dose of about 1 ml to about 5 ml,
advantageously
about 2 ml. And, if more than one gene product is expressed by more than one
recombinant, each recombinant can be administered in these amounts; or, each
recombinant can be administered such that there is, in combination, a sum of
recombinants comprising these amounts. In plasmid compositions employed in the
invention, dosages can be as described in documents cited herein or as
described
herein. Advantageously, the dosage should be a sufficient amount of plasmid to
elicit a response analogous to compositions wherein the antigens) or
epitope(s) of
interest are directly present; or to have expression analogous to dosages in
such
compositions; or to have expression analogous to expression obtained ih vivo
by
recombinant compositions. For instance, suitable quantities of each plasmid
DNA
in plasmid compositions can be 1 ~g to 2 mg, preferably 50 ~g to lmg.
Documents
cited herein regarding DNA plasmid vectors may be consulted by the skilled
auisan
to ascertain other suitable dosages for DNA plasmid vector compositions of the
invention, without undue experimentation.
However, the dosage of the composition(s), concentration of components
therein and timing of administering the composition(s), which elicit a
suitable
immunological response, can be determined by methods such as by antibody
titrations of sera, e.g., by ELISA and/or seroneutralization and/or
seroprotection
assay analysis. Such determinations do not require undue experimentation from
the
lrnowledge of the skilled artisan, this disclosure and the documents cited
herein.
And, the time for sequential administrations can be likewise ascertained with
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WO 2005/016383 PCT/US2004/026146
methods ascertainable from this disclosure, and the knowledge in the art,
without
undue experimentation.
Preferably, the combined enteric immunological, immunogenic or vaccine
composition comprises both Cryptospo~~idium parvunz antigens as defined above.
5 Antigens or epitopes of enteric pathogens advantageously combined with
Cryptospo~~idium antigens) or epitope(s) (advantageously P21 and/or Cp23
and/or
CplS/60 and/or CP41 such as P21 or Cp23 and CplS/60, or epitope(s) thereof)
comprise preferably one or more antigen or epitope' of interest from E. cola,
and/or
rotavirus, and/or coronavirus, and/or Clost~~idiurn spp., such as Cl.
perf~ingens; for
10 instance, at least one antigen or epitope of interest from E. coli,
rotavirus, and
coronavirus. Antigens from E. coli include preferably one, preferably several
(more
than one), more preferably all, of the antigens called K99, F41, Y and 31A
and/or
epitopes therefrom. Preferred antigens are K99 and F41. A composition thus
advantageously comprises one of K99 and F41, and preferably both. It is also
15 preferred for a composition to comprise also Y and/or 31A, advantageously Y
and
31A. For instance, these antigens may be incorporated as subunits or can be
borne
by E. coli bacteria. Preferably the compositions according to the invention
comprise
at least one antigen chosen from the group consisting of E. eoli bearing K99
antigen,
E. coli bearing F41 antigen, E. coli bearing Y antigen, E. eoli bearing 31A
antigen,
20 K99 antigen, F41 antigen, Y antigen, 31A antigen and any mixtures thereof.
As mentioned herein, E. coli may be used to produce C~~yptosporidiuni
pa~~vuna antigens or epitopes. The Cryptospo~~idium pa~~vurrz antigens or
epitopes can
be expressed in an E. coli strain expressing at least one of the E. coli
antigens so that
simultaneous expression of E. coli and Cf-yptosporidiufn pa~~vuna antigens is
25 performed. For in vita~o expression, the cells may then be disrupted as
usual and the
E. coli and C~ptospor~idiuna pamum antigens or epitopes recovered;
advantageously, if there is internal or non-surface expression of the antigens
or
epitopes, the antigens or epitopes are expressed as fusion proteins or with
tags, e.g.
HIS tags. For in vivo expression, advantageously the nucleic acid molecules
30 encoding the antigens or epitopes are linked to a signal sequence so that
there is
extracellular expression of the antigens or epitopes; and, advantageously, the
E. coli
is non-pathogenic. Thus, E. coli can, in certain embodiments, be the vector
and the
antigen or epitope of interest.
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31
Antigens from Clostridium perfifi~gehs are preferably type C and/or D
toxoids, more preferably type C and D toxoids.
A particular aspect of the invention is a combined enteric immunological,
immunogenic or vaccine composition for bovine species, comprising at least one
antigen or epitope from at least one C~yptospof°idiu~ra spp.,
preferably including
Cfyptosporidiufn pan~un7, advantageously P21 and/or Cp23 and/or CplS/60 and/or
CP41 such as P21 or Cp23 and Cp 15/60 and/or an epitope of interest thereof,
and at
least one antigen or epitope from at least one additional bovine enteric
pathogen
such as E. coli, bovine rotavirus, bovine coronavirus and Clost~idiufn
penfi~ihgens, or
combinations thereof, and preferably including at least one antigen or epitope
from
each of these pathogens or at least one antigen or epitope from E. coli,
rotavirus, and
coronavirus. With respect to an epitope of interest of a desired antigen and
how to
determine what portion of an antigen is an epitope of interest, reference is
made to
U.S. Patent No. x,990,091 and U.S. applications Serial Nos. 08/675,566 and
08/675,556, as well as other documents cited herein. From the disclosure
herein and'
the knowledge in the art, such as in herein cited documents, there is no undue
experimentation needed to ascertain an epitope of interest, or to formulate a
composition within the invention comprising antigens) and/or epitope(s) and/or
vectors) expressing antigens) and/or epitope(s).
According to a preferred embodiment, the invention provides a bovine
enteric immunological, immunogenic or vaccine composition comprising E. coli
antigens as discussed herein such as antigens K99, F41, Y and 31A, as well as
inactivated bovine coronavirus, inactivated bovine rotavirus. This composition
can
further include Clostridiu~a perfringens type C and D toxoids. Preferably the
E. coli
valency comprises either inactivated E. coli bearing K99 antigen, inactivated
E coli.
bearing F41 antigen, inactivated E. coli bearing Y antigen and inactivated E.
coli
bearing 31A antigen, or, K99 antigen, F41 antigen, Y antigen and 31A antigen.
Another aspect of the present invention is an immunological, immunogenic
or vaccine composition against C~yptosporidiurri pa~vum, which comprises Cp23
or
P21 and CplS/60 antigens or epitopes thereof, and a pharmaceutically
acceptable
vehicle.
According to an advantageous embodiment, these antigens are incorporated
in the composition as proteins or sub-unit antigens. They can be produced by
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32
chemical synthesis or by expression in vitro. For the convenience of
production by
expression in a suitable host, and their recovery, these antigens are
preferably under
the form of fusion protein (e.g., with HIS tag). In other words, the antigen
can
comprise the antigen per se and foreign amino acids.
According to another embodiment, these antigens are not incorporated as
subunits in the composition, but the composition comprises either a
recombinant
vector expressing Cp23 or P21 and CplS/60 or an epitope thereof or a
recombinant
vector expressing Gp23 or P21 or an epitope thereof and a recombinant vector
expressing Cp 15/60 or an epitope thereof, wherein these vectors express the
antigens) or epitope(s) in vivo when administered to the animal. The
composition
can contain an antigen or epitope and a vector expressing the other antigen or
epitope.
A still further aspect of the present invention is the methods of vaccination
wherein one administers to a target animal a combined enteric immunological or
vaccine composition or an immunological or vaccine composition against
Cryptosporidium parvun2 according to the invention. The invention can concern
a
method of immunization of a newborn calf against enteric disease, comprising
administering an immunological or vaccine composition comprising Cp23 or P21
and CplS/60 Cfyptospof°idium parvum antigens or epitopes thereof and a
pharmaceutically acceptable vehicle, to the pregnant cow or pregnant heifer
before
delivering, so that the newborn calf has maternal antibodies against
Cr~ptosporidium
pammn. Preferably, the method comprises the feeding of the newborn calf with
colostrum and/or mills coming from a cow, e.g. the mother, which has been so
vaccinated. For vaccination or immunization against enteric disease, one may
not
only use a combined vaccine, immunogenic or immunological composition,
containing the various valencies, but also separate vaccine, immunogenic or
immunological compositions which can be administered separately, e.g.,
sequentially, or which can be mixed before use.
Antigens and epitopes of interest useful in inventive compositions and
methods may be produced using any method available to the one sleilled in the
art
and for instance using the methods in US-A-5,591,434 and WO-A-9807320.
Further, one can obtain antigens of other enteric pathogens from commercially
available sources, such as TRIVACTON~6; for instance, Cp23 and/or P21 and/or
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WO 2005/016383 PCT/US2004/026146
33
CplS/60 or an epitope thereof, e.g., P21 or Cp23 and CplS/60 or an epitope
thereof,
or a vector expressing these antigens) or epitope(s) can be added to
TRIVACTON~6, in herein specified amounts. Clostridium peg, fiirzgens toXOids C
and D may advantageously be added to TRIVACTON~6. Also, the inactivated E.
coli bearing pili may be replaced in TRIVACTON~6 by the isolated pili. Such a
vaccine, immunogenic or immunological composition (with inactivated E. coli or
isolated pili) to which C. pa~vum antigens) or epitope(s) and/or
Clostf°idiuyn
peg, fi~ihgens antigens) or epitope(s) is/are added and methods of making and
using
such a composition and kits therefor are also within the invention.
Furthermore, as to the E. coli valency and/or antigens) and/or epitope(s)
useful in the practice of the invention, reference is made to EP-A-80,412, EP-
A-
60,129, GB-A-2,094,314, and U.S. Patents Nos. 4,298,597, 5,804,198, 4,788,056,
3,975,517, 4,237,115, 3,907.987, 4,338,298, 4,443,547, 4,343,792, 4,788,056,
and
4,311,797. As to rotavirus antigens) and/or epitope(s), reference is made to
P.S.
Paul and Y.S. Lyoo, Vet Microb 37:299-317 (1993) and U.S. Patents Nos.
3,914,408
and 5,620,896. With respect to coronavirus antigens) and/or epitope(s),
reference is
made to WO-A-98 40097, WO-A-96 41874, and U.S. Patents Nos. 3,914,408 and
3,919,413. For Clostridiuf~i, e.g., Cl. pef, fi~i~gerzs, antigens) and/or
epitope(s),
reference is made to WO-A-94 22476, EP-A-734,731, WO-A-98 27964, GB-A-
2,050,830, GB-A-1,128,325, D. Calmels and Ph. Desmettre, IV Symposium ofthe
Commission for the study of animal diseases caused by anaerobes, Paris, Nov.
16-
18, 1982, U.S. Patents Nos. 5,178,860, 4,981,684, and 4,292,307; and, to
IMOTOXAN~ (MERIAL, Lyon, France) (containing types B, C, D, Cl.
peg, fi~i~rgens, toxoids from Cl. septicurrr, Cl. ~covyi, Cl. tetar~i and
culture of Cl.
chauvoei). And, in addition to TRIVACTON~6, one may use other commercial
combined vaccines to which C. pa~wuyrz valency can be added, in accordance
with
this invention; for instance, SCOURGUARD 3 (K)/C~ (SmithKline Beecham)
containing inactivated bovine rotavirus and coronavirus, K99 E. coli bacterin
and Cl.
pe~frihgens type C toxoid.
A preferred method to obtain antigens or epitopes of interest is to clone the
DNA sequence encoding the antigen or epitope of interest into a fusion or non-
fusion plasmid and to have its expression in E. coli. Fusion plasmids (e.g.,
that
express the antigens) or epitope(s) with a tag such as a His tag) are
preferred as they
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WO 2005/016383 PCT/US2004/026146
34
allow one to recover easily the produced antigen. Suitable plasmids are
described in
the examples. Production of antigens by chemical synthesis is also within the
scope
of the invention.
The invention further comprehends methods for using herein discussed
antigens or epitopes or vectors expressing such antigens or epitopes for the
preparation of a vaccine, immunological, or immunogenic composition, e.g.,
against
C. parvum or against enteric disease; for instance, by admixing the antigens,
epitopes or vectors with a suitable or acceptable carrier or diluent and
optionally also
with an adjuvant. The compositions may be lyophilized for reconstitution. The
invention further comprehends a kit for the preparation of an inventive
composition.
The kit can comprise the antigen(s), epitope(s) and/or vector(s), carrier
and/or
diluent and optionally adjuvant; the ingredients can be in separate
containers. The
containers containing the ingredients can be within one or more than one
package;
and, the kit can include instructions for admixture of ingredients and/or
administration of the vaccine, immunogenic or immunological composition
composition.
Another aspect of the invention is the production of hyperimmune colostrum
and/or milk; for instance, by hyperimmunization of the pregnant female mammal
(such as a cow) by at least 1, advantageously at least 2, and more
advantageously at
least 3, administrations of inventive compositions) (e.g., C. paf~~um
composition or
combined enteric composition according to the invention). Optionally, but
advantageously, the colostrum and/or milk so produced can then be treated to
concentrate the immunoglobulins and to eliminate components of the colostrum
or
mills that do not contribute to the desired immunological, immunogenic and/or
vaccine response or to the nutritional value of the colostrum or milk. That
treatment
can advantageously comprise coagulation of the colostrum or milk, e.g., with
rennet,
and the liquid phase containing the immunoglobins recovered. The invention
also
comprehends the hyperimmune colostrum or milk or mixture thereof and/or
compositions comprising the hyperimmune colostrum or mills or mixture thereof.
Further, the invention envisions the use of the hyperimmune colostrum or mills
or
mixture thereof or composition comprising the same to prevent or treat G. paf-
vum
and/or enteric infection in a young animal, such as a newborn; for instance, a
calf.
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The following examples are put forth so as to provide those of ordinary skill
in the art with a complete disclosure and description of how to make and use
the
cells of the invention, and are not intended to limit the scope of what the
inventors
regard as their invention.
5
EXAMPLES
List of sequences:
SEQ ID NO: 1 oligonucleotide JCA295
SEQ ID NO: 2 oligonucleotide JCA296
10 SEQ ID NO: 3 oligonucleotide JCA297
SEQ ID NO: 4 oligonucleotide JCA298
SEQ ID NO: 5 oligonucleotide JCA299
SEQ ID NO: 6 oligonucleotide JCA300
SEQ ID NO: 7 oligonucleotide JCA301
15 SEQ ID NO: 8 oligonucleotide JCA302
SEQ ID NO: 9 oligonucleotide JCA303
SEQ ID NO: 10 oligonucleotide JCA304
All plasmid constructs have been done using standard molecular biology
techniques (cloning, restriction digestion, polymerase chain reaction (PCR))
as
20 described in Sambroolc J. et al. (Molecular Clo~i~rg: A Laboy~atory Manual.
2"d
Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. New Yorlc. 1989).
All
DNA restriction fragments generated and used for the present invention, as
well as
PCR fragments, have been isolated and purified using the "Geneclean~" lcit
(BIO101 Inc. La Jolla, CA).
25 Examine 1: Cloning ofthe C. paT~vur~a P21 and CplS/60 enes
Oocysts of Cfyptospof°idiuoa pa~vuf~2 are isolated from an infected
calf and
are purified from bovine fecal samples as described by Sagodira S. et al.
(Vaccine.
1999. 17. 2346-2355). Purified oocysts are then stored in distilled water at
+4°C. For
use as a template for PCR reactions, genomic DNA is released from the purified
30 oocysts as described by Iochmann S. et al. (Microbial Pathogenesis 1999.
26. 307-
315).
An alternative source for C. pamum DNA is constituted by the EcoRI
genomic libraries for the Cfyptosporidiu~rz pafvuyn Iowa (A), Iowa (I), I~SU-1
and
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36
KSU-2 isolates available from the American Tissue Culture Collection (ATCC
numbers 87667, 87668, 87439 and 87664 respectively). The specific P2land
Cp 15/60 genes are isolated as follows:
The sequence encoding the P21 protein is amplified by a polymerase chain
reaction (PCR) using C. pa~vu~r2 DNA and the following primers:
oligonucleotide JCA295 (35 mer) SEQ ID NO: 1
5' TTT TTT CCA TGG GGC TCG AGT TTT CGC TTG TGT TG 3'
and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2
5' TTT TTT GAA TTC TTA GGC ATC AGC TGG CTT GTC 3'
This PCR generates a fragment of about 585 by PCR fragment. This PCR
fragment is then digested with NcoI and EcoRI restriction enzymes to isolate,
after
agarose gel electrophoresis and recovery with the GeneClean lcit (BIO101
Inc.), a
575 by NcoI-EcoRI restriction fragment (= fragment A). The sequence of this
fragment encodes a protein homologous to the sequence described as SEQ ID NO:
12 in patent application WO 98/07320 (PCT/US97/14834).
A second PCR is run to amplify the sequence encoding the Cp 15/60 protein
and to add convenient restriction sites in 5' and 3' for further cloning. The
PCR is
done using C. pa~~vufn DNA and the following primers:
oligonucleotide JCA297 (35 mer) SEQ ID NO: 3
5' TTT TTT CTC GAG ATG GGT AAC TTG AAA TCC TGT TG 3'
and oligonucleotide JCA298 (42 mer) SEQ ID NO: 4
5' TTT TTT GAA TTC TTA GTT AAA GTT TGG TTT GAA TTT GTT TGC 3'
This PCR generates a fragment of about 465 bp. This fragment is purified
and then digested with XhoI and EcoRI in order to get, after agarose gel
electrophoresis and recovery with the GeneClean lcit (BIO101 Inc.), the 453 by
XhoI-EcoRI fragment (= fragment B). The amplified sequence is homologous to be
similar to the sequence defined from nucleotide #31 to #528 of SEQ ID NO: 1 in
US
Patent # 5,591,434 and to the sequences deposited in GenBanlc under Accession
Numbers U22892 and AAG47447.
Example 2: Construction of plasmid pJCA155 (GST-P21 fusion protein in vector
pBAD/HisA)
The sequences required to express the GST-P21 fusion protein are amplified
by PCR in order to generate 2 fragments that can be cloned easily into the
pBAD/HisA expression plasmid vector (Cat # V430-O1 InVitrogen Corp., Carlsbad,
CA 02535618 2006-02-13
WO 2005/016383 PCT/US2004/026146
37
CA 92008, USA). The first PCR is done using the pGEX-2TK plasmid (Cat # 27-
4587-01 Amersham-Pharmacia Biotech) and the following primers:
oligonucleotide JCA299 (35 mer) SEQ ID NO: 5
5' TTT TTT CCA TGG GGT CCC CTA TAC TAG GTT ATT GG 3'
and oligonucleotide JCA300 (45 mer) SEQ ID NO: 6
5' TTT TTT CTC GAG CCT GCA GCC CGG GGA TCC AAC AGA TGC ACG
ACG 3'
This PCR generates a fragment of about 720 by encoding the GST moiety
with the addition of a NcoI restriction site at the 5' end for cloning
purposes into
pBAD/HisA; this modification adds a Glycine codon to the GST-P21 fusion
protein). This PCR fragment is then digested with NcoI and XhoI in order to
get,
after agarose gel electrophoresis and recovery with the GeneClean kit (BIO101
Inc.),
the 710 by NcoI-XhoI fragment (= fragment C).
The second PCR is done using C. pa~vuyrz DNA and the following primers:
oligonucleotide JCA301 (33 mer) SEQ ID NO: 7
5' TTT TTT CTC GAG TTT TCG CTT GTG TTG TAC AGC 3'
and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2
This PCR generates a fragment of about 580 by encoding the P21 moiety
with the addition of XhoI and EcoRI restriction sites at the 5' and 3' ends
respectively. This PCR fragment is then digested with XhoI and EcoRI in order
to
get, after agarose gel electrophoresis and recovery with the GeneClean kit
(BIO101
Inc.), the 572 by XhoI-EcoRI fragment (= fragment D).
The pBAD/HisA plasmid (Cat # V430-O1, InVitrogen Corp.) is digested with
NcoI and EcoRI. The digested fragments are separated by agarose gel
electrophoresis in order to recover (GeneClean kit, BIO101 Inc.) the # 3960 by
NcoI-EcoRI restriction fragment (= fragment E).
Fragments C, D and E are then ligated together to generate plasmid
pJCA155. This plasmid has a total size of 5243 by (Figure 1) and encodes a 425
amino acids GST-P21 fusion protein.
Example 3: Construction of plasmid pJCA156 (His6-P21 fusion protein in vector
pBAD/HisA)
The pBAD/HisA vector (Cat # V430-O1, InVitrogen) is digested with NcoI
and EcoRI and the # 3960 by NcoI-EcoRI restriction fragment (= fragment E) is
recovered and isolated as described in Example 2.
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A PCR is done to amplify the sequence encoding the His6-P21 fusion and to
add the NcoI and EcoRI restriction sites respectively in 5' and 3' in order to
subclone this PCR fragment into the pBAD/HisA plasmid vector.
The PCR is done using G. parvuJrz DNA and the following primers:
oligonucleotide JCA302 (65 mer) SEQ ID NO: 8
5' TTT TTT CCA TGG GGG GTT CTC ATG ATC ATC ATC ATC ATG GTC
TCG AGT TTT CGC TTG TGT TGT AC 3'
and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2
This PCR generates a fragment of about 610 bp. This fragment is purified,
and then digested with NcoI and EcoRI in order to isolate, after agarose gel
electrophoresis and recovery with the GeneClean lcit (BIO101 Inc.), the 600 by
NcoI-EcoRI fragment (= fragment F).
Fragments E and F are ligated together to generate plasmid pJCA156. This
plasmid has a total size of 4562 by (Figure 2) and encodes a 199 amino acids
His-
6/P21 fusion protein.
Example 4: Construction of plasmid pJCA157 (P21 protein alone in pBAD/HisA
vector
The pBAD/HisA vector (Cat # V430-O1, InVitrogen Corp.) is digested with
NcoI and EcoRI and the # 3960 by NcoI-EcoRI restriction fragment (= fragment
E)
is recovered and isolated as described in Example 3.
A PCR is done to amplify the sequence encoding the P21 protein and to add
the NcoI and EcoRI restriction sites respectively in 5' and 3' in order to
subclone
this PCR fragment into the pBAD/HisA plasmid vector. The PCR is done using C.
pamnu DNA and the following primers:
oligonucleotide JCA295 (35 mer) SEQ ID NO: 1
and oligonucleotide JCA296 (33 mer) SEQ ID NO: 2
to get, as described in Example 1, a 575 by NcoI-EcoRI fragment (fragment A).
Fragments E and A are ligated together in order to generate plasmid
pJCA157. This plasmid has a total size of 4535 by (Figure 3) and encodes 189
amino acids including the P21 protein.
Example 5: Construction of plasmid pJGA158~GST-CplS/60 fusion protein in
pBAD/HisA vector)
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A PCR is done to amplify the sequence encoding the GST protein and to add
convenient restriction sites in 5' and 3' in order to subclone the PCR
fragment into
the final pBAD/HisA plasmid vector. The PCR uses the DNA of plasmid pGEX-
2TK (Cat # 27-4587-O1, Amersham-Pharmacia Biotech) as a template and the
following primers:
oligonucleotide JCA299 (35 mer) SEQ ID NO: 5
and oligonucleotide JCA300 (45 mer) SEQ ID NO: 6
to get, as described in example 2, a 710 by NcoI-XhoI fragment (= fragment C).
The pBAD/HisA vector (Cat # V430-O 1, InVitrogen) is digested with NcoI
and EcoRI and the # 3960 by NcoI-EcoRI restriction fragment (= fragment E) is
recovered and isolated as described in Example 2.
Fragments C, E and B (Example 1) are ligated together in order to generate
plasmid pJCA158. This plasmid has a total size of 5132 by (Figure 4) and
expresses
a 388 amino acids GST-CplS/60 fusion protein.
Exam~ale 6: Construction of plasmid pJCA159 (His6-CplS/60 fusion protein in
pBAD/HisA vector)
The pBAD/HisA vector (Cat # V430-O1, InVitrogen Corp.) is digested with
NcoI and EcoRI and the # 3960 by NcoI-EcoRI restriction fragment (= fragment
E)
is recovered and isolated as described in Example 2.
A PCR is run to amplify the sequence encoding the His6-CplS/60 fusion and
to add convenient restriction sites in 5' and 3' in order to subclone this PCR
fragment into the pBAD/HisA plasmid vector. The PCR is done using either C.
~af~~~um DNA and the following primers:
oligonucleotide JCA303 (64 mer) SEQ ID NO: 9
5' TTT TTT CCA TGG GGG GTT CTC ATC ATC ATC ATC ATC ATG GTA
TGG GTA ACT TGA AAT CCT GTT G 3'
and oligonucleotide JCA298 (42 mer) SEQ ID NO: 4
This PCR generates a fragment of about 495 bp. This fragment is purified
and then digested with NcoI and EcoRI in order to get, after agarose gel
electrophoresis and recovery with the GeneClean kit (BI0101 Inc.), the 483 by
NcoI-EcoRI fragment (= fragment G).
Fragments E and G are ligated together in order to generate plasmid
pJCA159. This plasmid has a total size of 4445 by (Figure 5) and expresses a
159
amino acids His-6/CplS/60 fusion protein.
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Example 7: Construction of lasmid pJCA160 (C lka 5/60 protein alone in
pBAD/HisA vector)
The pBAD/HisA vector (Cat # V430-O1, InVitrogen Corp.) is digested with
NcoI and EcoRI and the # 3960 by NcoI-EcoRI restriction fragment (= fragment
E)
5 is recovered and isolated as described in Example 2.
A PCR is run to amplify the sequence encoding the CplS/60 protein and to
add convenient restriction sites in 5' and 3' in order to subclone this PCR
fragment
into the pBAD/HisA plasmid vector.
The PCR is done using C. pa~vmn DNA and the following primers:
10 oligonucleotide JCA304 (31 mer) SEQ ID NO: 10
5' TTT TTT CCA TGG GTA ACT TGA AAT CCT GTT G 3'
and oligonucleotide JCA298 (42 mer) SEQ ID NO: 4
This PCR generates a fragment of about 460 bp. This fragment is purified
and then digested with NcoI and EcoRI in order to get, after agarose gel
15 electrophoresis and recovery with the GeneClean kit (BIO101 Inc.), the 450
by
NcoI-EcoRI fragment (= fragment H).
Fragments E and H are ligated together in order to generate plasmid
pJCA160. This plasmid has a total size of 4412 by (Figure 6) and expresses a
148
amino acids CplS/60 protein.
Example 8: Culture of E. coli recombinant clones and induction of recombinant
roteins
Plasmid DNA (Examples 2 to 7) is transformed into Esclzerichia coli DHSa
(or any other suitable E. coli K12 strain well known to those skilled in the
art, such
as E. coli TOP10 (Cat # C4040-03 InVitrogen Corp.)) and grown on Luria-Bertani
(LB) medium agar plates with SOp.g/ml of ampicillin. One colony is picked for
each
plasmid transformed E. coli population and placed in 10 ml of LB medium with
ampicillin (or other appropriate antibiotic) for overnight growth. One ml from
the
overnight culture is added to one liter of LB medium and grown at +30°C
until
OD6oo nm reaches approximately 3Ø
Protein production is induced with different final concentrations of DL-
arabinose (Cat# A9524, Sigma, St Louis, MO) (range of 0.002% to 0.2% for
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41
determining the concentration for optimal yield) added to the culture and
incubated
at +30°C for 4-6 hours.
Example 9: Extraction and purification of the recombinant fusion proteins
At the end of the induction (Example 8), cells are harvested by centrifugation
(3000 g, 10 minutes, +4°C) and resuspended in lysis buffer (50 mM Tris
pH 8.0, 1
mM EDTA, 1 p.M PMSF, 1 mg/ml lysozyme) and sonicated 25 times for 30 seconds
bursts with 1-minute pauses between bursts. Triton X-100 is added to a final
concentration of 0.1%. Debris is removed by centrifugation.
If necessary, alternative techniques (known to those of skill in the art) may
be used for the lysis of bacterial cells.
9.1. GST-fusion recombinant proteins:
Recombinant GST-fusion proteins (produced by E. coli transformed with
plasmids pJCA155 or pJCA158) were affinity purified from the bacterial
lysates,
prepared as described in Example 8, using a glutathione-agarose (Cat# 64510,
Sigma) or glutathione-Sepharose 4B (Cat# 17-0756-O1, Amersham-Pharmacia
Biotech). Bacterial lysates and the glutathione-agarose were incubated for 4
hours at
+4°C. GST-fusion proteins were then eluted from the agarose in a batch
format with
10 mM reduced form glutathione (Cat# 64705, Sigma) under mild conditions (K.
Johnson and D. Smith Gene. 1988. 67. 31-40). (Reference: Anonymous. GST gene
fusion system : technical manual. 3rd edition. Arlington Heights, IL: Amersham-
Pharmacia Biotech, 1997). Anyone skilled in the art can achieve scaling up of
this
process for purifying large quantities of GST-fusion proteins, from this
disclosure
and the knowledge in the art, without undue experimentation.
9.2. His6-fusion recombinant proteins:
Recombinant His6-fusion proteins have all been prepared and purified using
the ProBondTM Nickel-Chelating resin (Cat# 8801-15, InVitrogen Corp.)
following
the manufacturer's instructions.
Preparation of native E. coli cell lysate (soluble recombinant protein) : the
bacterial cells from a 1 liter culture of E. coli (transformed with plasmids
pJCA156
or pJCA159) are harvested by centrifugation (3000 g for 5 minutes). The pellet
is
resuspended in 200 ml ofNative Binding Buffer (20 mM phosphate, 500 mM NaCI,
pH 7.8). The resuspended pellet is then incubated with egg lysozyme at a final
concentration of 100 ~g/ml, for 15 minutes on ice. This mixture is then
sonicated
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42
with 2-3 10-second bursts at medium intensity while holding the suspension on
ice.
The mixture is then submitted to a series of freezing/thawing cycles for
completing
the lysis and the insoluble debris are finally removed by centrifugation at
3000 g for
15 minutes. The lysate is cleared by passage through a 0.8 pm filter and
stored on
ice or at -20°C until purification.
The soluble recombinant His6-fusion protein present in the clear lysate is
batch bound to a 50 ml pre-equilibrated ProBondTM resin column (Cat # 8640-50
and 8801-15, InVitrogen Corp.) with two 100 ml lysate aliquots. The column is
gently rocked for 10 minutes to keep the resin resuspended and allow the
polyhistidine-tagged protein to fully bind. The resin is settled by gravity or
low
speed centrifugation (800 g) and the supernatant is carefully aspirated. An
identical
cycle is repeated with the second aliquot.
Column washing and elution
4 successive steps are done according to the manufacturer's instructions
(Anonymous. XpressTM System Protein Purification - A Manual of Methods for
Purification of Polyhistidine - Containing Recombinant Proteins. InVitrogen
Corp.
Editor. Version D. 1998)
1. The column is washed with 100 ml ofNative Binding Buffer pH 7.8, by
resuspending the resin, rocking for 2 minutes and then separating the resin
from the
supernatant by gravity or centrifugation. This procedure is repeated 2 more
times
(total of 3 washes)
2. The column is washed with 100 ml of Native Wash Buffer pH 6.0 by
resuspending the resin, rocking for 2 minutes and then separating the resin
from the
supernatant by gravity or centrifugation. This procedure is repeated at least
3 more
times until ODZBO is less than 0.01.
3. The column is washed with 100 ml of Native Wash Buffer pH 5.5 by
resuspending the resin, rocking for 2 minutes and then separating the resin
from the
supernatant by gravity or centrifugation. This procedure is repeated once
(total of 2
washes).
4. The column is then clamped in vertical position and the cap is snapped off
on the
lower end. The recombinant protein is eluted with 150 ml of the Native pH
Elution
Buffer. 10 ml fractions are collected. Elution is monitored by taking OD~BO
readings
of the fractions.
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43
If needed, the eluted recombinant protein can be concentrated either by
dialysis, or
by precipitation with ammonium sulfate.
Final concentration of the recombinant protein batch is measured by ODZBo
readings.
Anyone skilled in the art can achieve scaling up of this process for purifying
large quantities of His6-fusion proteins, from this disclosure and the
knowledge in
the art, without undue experimentation.
Example 10: Extraction and purification of the C. pa~~uwr P21 and Cp 15
recombinant non-fusion proteins
The bacterial cells of E. eoli (transformed with plasmids pJCA157 or
pJCA160) are cultured in 4 liters of the M9 minimum medium (supplemented with
the appropriate amino acids) (Sambrook J. et al. (Molecular Clohircg: A
Laboratory
Manual. 2"d Edition. Cold Spring Harbor Laboratory. Cold Spring Harbor. New
York. 1989) at 30°C until OD6oo nm reaches approximately 3.0 and are
induced as
described in Example 8. The bacterial cells are then disrupted by passing
through a
high pressure RANNIE homogeneizer Mini-Lab type 8.30 H with a maximum flow
of 10 liters per hour and working pressure between 0 and 1000 bars. The lysate
is
cleared by filtration through a CUNO filter Zeta plus, LP type, and then
concentrated 50 times on an ultrafilter PALL Filtron (reference OSOlOG01) OF
10
kDa. The protein suspension concentrate is loaded on a size-exclusion
chromatography column with High Resolution Sephacryl S-100 gel under a volume
corresponding to 2-3% of the column volume. Elution is done with a PBS buffer.
The collected fractions corresponding to the expected molecular weight for the
subunit vaccine proteins are concentrated 10 times on a hollow fibers
cartridge A/G
Technology type Midgee cartridge model UFP-10-B-MBOl (or model UFP-10-C-
MBO1 or model UFP-10-E-MBO1). The concentrated samples are then stored at -
70°C until use. The specific C. pafwufn recombinant proteins can be
then mixed in
the appropriate proportions to the final associated vaccine (see Example 11).
Example 11: Formulation of vaccines vaccination of ~reQnant cows' passive
immunization and challenge experiment in newborn calves
Product (adjuvanted or not) is administered intramuscular (IM),
subcutaneous (SQ) or intradennal (ID) to elicit serum antibody responses
against C.
pamur~a. When administered twice to pregnant animals it elicits a serum
antibody
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44
response that will be passively transferred to the newborn via colostrum and
milk.
Vaccination protocol for pregnant animals can comprise 2 doses given between
when pregnancy is diagnosed and calving, such as about 1 month before calving
and
about 3 to 5 days before calving; or, 2 months prior to calving (which
coincides with
dry-off in dairy cows) and a boost prior to calving (e.g., anywhere from 3
weeks to 1
week prior to calving), depending on management practices (however, these
schedules favor maximum efficacy) . Current management practices favor that
are
products administered in the last trimester. Volume of the product can be from
1 ml
to 5 ml, such as 2 ml. Combination vaccines can have a lyophylized and a
liquid
portion that can be mixed prior to injection. To afford maximum protection
under
field conditions the Cnyptospo~~idium antigen can be added as a component of
an E.
coli/Rota/Corona combination vaccine.
The following studies are conducted:
Study A: C. pas°vu~n enhances the pathogenicity of enteric virus and/or
bacteria
Experimental challenge utilizing 3 newborn calves per group as follow:
1. Coronavirus only
2. Coronavirus plus C. paf~vum
3. E. coli F41 only
4. E. eoli F41 plus C. pafwum
5. C. paJ~vun7 only '
6. Unchallenged controls
Calves are challenged within 24 hours of being born, by the oral route. The
amount of challenge material used is that which is necessary to produce
clinical
signs (depression, diarrhea, dehydration) and may depend on the type of animal
(gnotobiotic artificially raised or conventional calve nursing its dam).
Common
clinical signs (temperature, demeanor, hydration, diarrhea scores, etc.) are
collected.
Additional serological and shedding information is collected.
Outcome
Coronavirus or E. coli F41 monovalent experimental challenges do not
produce clinical signs of enteric disease in newborn calves. Dual challenge
with
coronavirus or E. coli F41 with C. parvum, at a C. parvum dose that normally
does
not cause clinical disease, will produce significant clinical signs of enteric
disease.
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Study B: A combo vaccine (E. coli K99/)N'41, rota and coronavirus) containing
C. patrunz provides enhanced protection against enteric disease cause by
concurrent infection of multiple enteric virus and/or bacteria in newborn
calves.
5 Treatment groups are 30 pregnant cows vaccinated with:
1. Combo (rota and coronavirus, E. coli K99 and F41), 8 animals;
2. Combo plus C~ypto, 8 animals;
3. Unvaccinated controls, 14 animals.
Experimental challenge as follow:
10 1. Multiple challenge (coronavirus and F41 plus C. pa~~vum at subclinical
level);
2. Sentinel animals
3. unchallenged.
Calves receive colostrum (manually fed or allowing the calve to nurse from
the dam) and those that are challenged are challenged within 24 hours of being
born,
15 by the oral route. The amount of challenge material is an amount necessary
to
produce clinical signs (e.g., as determined in Study A, and as mentioned under
Study
A, can vary depending upon the type of animal used (e.g., gnotobiotic
artificially
raised or conventional calves nursing their dams). Common clinical signs
(temperature, demeanor, diarrhea scores) are collected. Additional serological
and
20 shedding information is collected.
Design:
6 calves born from vaccinated (combo and combo plus Cf-ypto) or control cows
are
challenged with a challenge containing 3 components (coronavirus and F41 plus
C.
pamum), and 3 calves (from unvaccinated control cows) remain as sentinels.
25 Outcome
Use of a combo vaccine containing C. pa~vurra produces a better protection
than a combo vaccine alone under a multiple challenge situation (coronavirus
and E.
coli F41 with C. pa~~um at a subclinical dose).
30 Example 12 : Effect of dual infection with C. pasw~tf~z and bovine
rotavirus in an
experimental challen,~e model in newborn calves
This study is designed to compare the severity of clinical signs and fecal
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46
excretion in calves after monovalent challenge with C. paswunz or bovine
rotavirus
and after a dual challenge with bovine rotavirus plus C. pa~vurn.
Four groups of six calves are used in order to yield sufficient data to be
able
to detect differences in incidence of clinical signs between .groups.
Cows are individually housed in pens or paddocks. Newborn calves are
separated from their dams as soon as possible after birth, inspected to
eliminate
feces or dirt on the calf and their ombilical cord dipped in approximate 7%
iodine
solution. They are then immediately transferred to containment accomodations
and
housed individually in metabolic crates. Calves are challenged within 6 hours
after
birth.
Calves are fed 1 to 2 quarts per feeding or at 10% body weight, twice daily
for the entire trial using a commercial calf milk replacer with 30% colostrum
substitute. Special care will be given to avoid the administration of milk
within 2
hours pre or post challenge.
The route of natural infection is oral; therefore, all the challenges will be
administered orally using an esophageal tube.
Group A : non-challenged control calves.
Group B : 1-3x105 C. paywum oocysts (strain Beltsville), diluted in 60 ml of
commercial antibiotics free soy mills.
Group C : Coinoculation of 1-3x105 C. pa~vum oocysts (strain Beltsville),
diluted in 60 ml of commercial antibiotics free soy milk, and of 10 ml bovine
rotavirus inoculum (strain TND BRV G6P5) diluted in 40 ml PBS.
Group D : 10 ml fecal filtrate from bovine rotavirus infected calves (strain
IND BRV G6P5) diluted in 40 ml PBS.
Fecal samples are collected from the collection pan once a day after
thoroughly mixing to ensure a representative sample is obtained.
Oocysts are separated from calves feces by centrifugation on sucrose
cushions and counted using a cell counting chamber (hemocytometer) under a
microscope. For rotavirus shedding, the feces are diluted in buffer and the
rotavirus
antigen is quantified using an ELISA lcit from Le Centre d'Economie Rurale
(CER)
1 rue du Carmel, B6900 Marloie, Belgium.
Calves are observed for clinical signs prior to challenge and then twice daily
for 10 days post-challenge. Observations include rectal temperature, general
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47
condition, anorexia, diarrhea, dehydration and death.
Depression, diarrhea, and dehydration are categorized as follows
General condition
Good The calf is bright, alert and responsive
Apathetic The calf is quiet, alert and responsive
Depression The calf is lying aside, reluctant to
rise, and slow to respond
Prostration The calf is curled up or prostrate and
not responsive
Dehydration
None No dehydration
Moderate Persistent skin fold, dry mouth and depressed
eyeballs
Shock State of shock
Diarrhea
None Normal feces
Loose Pasty or mucous feces
Liquid Liquid feces
anorexia is aezermmea nasea on whether the calt-nurses less than 2 liters of
milk. During the 1 St 48 hours of life, calves may be fed via an esophageal
tube. The
score is derived for each calf ors each day based on the presence of clinical
signs
(rated 1) or absence (rated 0) for each sickness category. Rectal temperature
is
recorded in degrees Fahrenheit.
Two calves died in Group C on days 7 and 8, two in Group B on day 7, none
in Group D and one in Group A on day 3. Results are shown on Figures 7 to 13.
A
synergistic effect on clinical signs and microorganisms excretion in feces is
observed when both microorganisms are administered compare to single
administrations.
Example 13: Production of Bovine Colostrum Containing Antibodies to the E coli
expressed C. ,~aivzcm Subunit Proteins C7~P21) and/or CP15/60
Pregnant dairy cows from 4 different herds were randomly assigned to one of
6 vaccinate groups: GST-P21; 6His-P21; GST-CP15/60; 6His-CP15/60; GST-P21 +
GST-CP15/60, and placebo controls. Upon entering dry-off, each cow received
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48
three 5 ml doses of the assigned vaccine subcutaneously, with each dose given
fourteen days apart. Colostrum from each cow was collected 3 times during the
first
24-36 hours post-calving and labeled; a 10-20 ml sample was withdrawn, and the
balance frozen in individual containers at each collection. Colostrum was
assayed
for total IgG levels by RIDA. ELISA assayed for P21 and CP15/60 subunit
protein
antibodies. Serology analysis by ELISA was conducted for the same subunit
protein
antibodies, both immediately prior to vaccination, and at the time of calving.
Feces
were collected pre-vaccination and were tested with the Prospect test lcit for
the
presence of C. pa~~una; all samples tested were negative for C.
paf°vur~.
Colostrum antibodies to P21
A P21-specific antibody response was detected in all groups vaccinated with
the P21 antigen. In contrast, groups vaccinated with CP15/60 and the placebo
group
had no detectable antibody response to P21 (see Figure 14). Interestingly, the
"combo" group (vaccinated with 0.25 mg of GST-P21 in combination with 0.25 mg
of GST-CP15/60) had a very similar P21 response as compared to the monovalent
GST-P21 group (vaccinated with 0.5 mg of GST-P21). The group receiving 0.5 mg
of His-P21 had a P21 response that was slightly, but consistently, lower than
the
groups receiving 0.5 mg of GST-P21, the greatest difference found at the
second
mincing. Further analysis of the individual values shows that the His-P21
group
contained two non-responder cows (G418 and M26) and an outlier value for cow
J54 at the 2°d mincing (1St milking = 0.055; 2"d mincing = 0.296 and
3'~ milking =
0.055). It should be noted that both non-responder cows also had an unusually
low
total IgG level. If the values corresponding to the non-responders and the
outliers
are excluded from the analysis, the group mean of the His-P21 group becomes
very
similar to the GST-P21 groups.
Colostrum antibodies to CP15/60
A C 15/60-specific antibody response was detected in all groups containing
the CP15/60 antigen (Figure 15). By contrast, groups vaccinated with P21 or
the
placebo group had no detectable antibody response to CP15/60. The His-CP15/60
group and the group containing 0.25 mg of GST-P21 in combination with 0.25 mg
of GST-GP15/60 (combo group) had very similar responses. Even though the
monovalent GST-CP15/60 group had twice the amount of GST-CP15/60 antigen as
the combo group (0.5 mg against 0.25 mg), its response was consistently lower
than
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49
the combo group. It is hypothesized that this unexpected outcome is due to
genetic
differences between these two groups, with the monovalent GST-15/60 cows
producing a colostrum of lower quality as compared to the combo group.
Serum antibodies to P21
All groups were seronegative at Day 0. All groups vaccinated with P21
developed similar P21-specific antibody responses with the exception of the
His-P21
group, which had a significantly lower antibody level than the GST-P21 groups
at
day 14 (see Figure 16). By contrast, groups vaccinated with CP15/60 and the
placebo group had no detectable antibody response to P21. As seen with the
colostrum, the combination vaccine containing 0.25 mg of GST-P21 performed
just
as well as the monovalent GST-P21 vaccine containing 0.5 mg of GST-P21. Those
groups receiving either GST-P21 vaccine reached a plateau after the first
injection,
with the day 14 and day 28 values being very similar. The His-P21 group,
however,
did not reach this maximum value before Day 28. As shown with the colostrums,
further analysis of the individual values showed that cows 6418 and M24 were
low
responders. If the corresponding values are excluded from the analysis, the
group
mean of the His-P21 group becomes very similar to the GST-P21 groups. Finally,
a
similar decrease in titer was observed in all P21 groups at the time of
calving. This
is likely due to the active exportations of immunoglobulins in the colostrums
and
peripartum immunosuppression.
Serum antibodies to CP15/60
All groups were seronegative at Day 0. The placebo controls remained
negative throughout the study. Groups vaccinated with P21 were weakly positive
at
Day 14 and Day 28. This is likely to reflect an experimental artifact (non-
specific
background). Groups vaccinated with CplS/60 seroconverted after one
vaccination
(Figure 17). The second injection boosted the antibody response. The His-
CP15/60
group had similar antibody responses. Finally, a similar decrease in titer was
observed in all CP15/60 groups at the time of calving. This is likely due to
the
active exportation of immunoglobulins in the colostrums and peripartum
immunosuppression.
Example 14: Experimental Challenge of C. ~arvunz in Newborn Calves
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Eight colostrum-deprived beef calves obtained by induced labor were
divided evenly into 2 groups and each group was placed in an isolation room
for the
6-day study period. Each calf occupied a metabolism crate. Each group was
bottle-
fed 2 pints 0960 ml) of colostrum at 3 and 12-15 hours post-partum. At 24
hours
5 post-partum, all calves had blood IgG levels >1000 mg/dL as detected by RIDA
(Radial Immunodiffusion Assay). Each calf in the challenge group was orally
challenged with 108 oocysts of C. pa~vuna. Blood samples were collected daily
and
were tested for serum antibodies to G. parvuyn P21 and CP15/60 antigens,
hematocrit and total protein. Feces (per rectum) were collected 3 times daily,
and
10 dry matter content measured. Oocyst shedding in feces was determined daily
by
ProSpecT ELISA kit. Clinical signs including body temperature, general
condition
(depression, etc.), anorexia, hydration status, fecal consistency (diarrhea,
etc.), and
death were evaluated daily (see Example 12 for clinical signs scoring). All
calves
that died or were euthanized were subjected to necropsy and analysis of gut
and gut
15 content for bovine rotavirus, coronavirus, E. coli, Salmonella spp., and C.
parvuyu.
Oocyst Shedding
Table 1 shows C, panvuna oocyst shedding detection by whole oocyst
ELISA.
+ 205 206 207 208 209 210 211 212
control
Day + - _ _ _ _ _ _
0
Day + _ _ _ _ _ NS _ _
1
Day + - - - + - + -
2
Day + - - - + - + - +
3
Day + - +* - + - + - +
4
Day + - D - D - D - +
5
Day + - D - D - D - +
6
~ = aeaa
20 +* feces collected prior to death on Day 4
NS = no stool sample
Even numbered IDs = unchallenged
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51
Clinical Signs
All unchallenged controls remained healthy during the study period. All
calves in the challenge group developed clinical signs consistent with
cryptosporidiosis by day 4 post-challenge. Three calves in the challenge group
died
prior to the end of the study (1 at Day 4 and 2 at Day 5). The 4t1' calf in
the
challenge group was euthanized at the termination of the study (Day 6) as it
met the
criteria for euthanasia as previously established. Temperature, depression,
diarrhea,
anorexia, and dehydration were monitored and were characterized as described
in
Example 15.
Mean temperature between groups varied less than 1°F at any time
point and
ranged between 101.57 - 103.5°F in the challenge group and between
101.53 -
102.78°F in the unchallenged group, all within clinically normal
limits.
All unchallenged calves remained bright and alert. Challenged calves began
showing depression on day 2 (2/4) and on days 2-5, the remaining 3 calves in
the
challenged group continued to exhibit depression. The one remaining calf on
day 6
was still depressed at the end of the study.
All calves in the challenge group exhibited a diarrhea consistent with
cryptosporidiosis: yellowish, foamy, watery, and in large volumes. Diarrhea in
this
group started on Day 2 and continued through the end of the study. There was a
transient diarrhea in one of the unchallenged calves on Day 3 and again on Day
5,
which was resolved by the end of the study. The diarrhea was likely the result
of
nutritional intake since the calf did not exhibit other signs of
crptosporidiosis.
All calves in the unchallenged group, except one on Day 3, had good
appetites and were aggressive nursers (calf bottle). The calf that was
anorexic on
Day 3 (corresponding with its 1st day of diarrhea) was fed with an esophageal
feeder
on that day only, and then returned to normal calf bottle feedings. All calves
in the
challenged group were anorexic by Day 3 and continued to be anorexic through
the
end of the study.
None of the calves in the unchallenged group exhibited clinical dehydration.
One calf in the challenged group began exhibiting clinical dehydration on day
2 and
all calves in that group were clinically dehydrated by Day 3 and remained so
through the study's end. Results of hematological parameters indicating
dehydration
(hematocrit) are shown in Figure 18.
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52
Hematocrit levels in unchallenged calves remained constant after Day 1,
while hematocrit in challenged calves increased on Day 2 and remained higher
than
the control calves on Days 4, 5, and 6. On the day of birth (Day -1), mean
hematocrit of unchallenged calves was 41.0%. It decreased on Day 0 and
maintained at levels of between 32.0 - 37.5% for the remainder of the study
period.
Challenged calves had a mean hematocrit of 37.8% on the day of birth (Day -1)
which then dropped on days 0-3 to between 29.5 - 35.0%. On Day 4 post-
challenge,
mean hematocrit had a clinically significant increase to 41.7%. Figure 18
shows the
daily differences in hematocrit by group. The values for Days 5 and 6
represent the
results for one calf.
Total plasma protein (TP) in unchallenged calves remained constant through
the study, ranging from a mean of 6.45 on Day -1 (pre-challenge) to a low of
5.85
on Day 0 (time of challenge-24 hours of age). Challenged calves started at 6.4
on
Day -1 and reached their highest level at Day 2 (7.0) and remained higher than
the
control calves throughout the study period.
Fecal dry matter content, as a % of volume, remained fairly constant in the
control calves, while the challenged calves began a downward trend (lower %
dry
matter equaling diarrhea) on Day 2, which continued through the end of the
study.
Challenged calves had consistently lower dry matter content, by 6 - 39%, than
control calves. Mean fecal diy matter content in unchallenged calves ranged
from
39.9% at the 24-hour post-partum time point to 51.7% at the Day 2 morning
sample
collection. Mean fecal dry matter content in challenged calves ranged from
28.4%
at the 24 hour post-partum time point to 41.0% at the Day 2 morning sample
collection, steadily decreasing thereafter to a mean low of 9.6% at the Day 4
evening
sample collection. Figure 19 illustrates the daily differences in % fecal dry
matter
by group.
Control calves remained negative to C. pa~~vum infection throughout the
study period. Challenged calves shed C. pa~~um oocysts and calves challenged
with
C. pa~vum developed clinical signs of cryptosporidiosis. Unchallenged controls
remained healthy.
Example 15: Demonstration of efficacy of various G parvurra subunit protein
vaccines via calf challenge
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53
Based upon the significantly less sever clinical signs observed in calves fed
colostrums from vaccinated cows versus calves fed colostrums from control
cows,
six groups of calves were selected: GST-P21 (group 1); His-P21 (group 2); GST-
15/60 (group 3); His-15/60 (group 4); GST-C& + GST-C15/60 (group 5); Placebo
vaccine (group 6). Approximately eight animals were in each treatment group.
Prior to the first colostrum intalce, newborn calves were bled for serology,
and
observed for body weight, body temperature, fecal matter, and other clinical
observations (i.e. anorexia, depression, diarrhea). The first colostrum was
fed at
approximately 3 hours of age by calf nurser or esophageal tube. The second
colostrum was administered approximately 12 hours later. The C. pa~vun~
challenge
(10' oocysts) was provided at approximately 24 hours of age. Observation of
the
calves occurred four times daily, during which time blood samples were
obtained,
body temperature and clinical observations were monitored and feces collection
occurred.
All calves were challenged by oral administration of 108 oocysts of C.
paywurn 24 hours after time of birth. Sixty to 100 ml of calf mill: replacer
was
administered to the calf via clean calf nurser or clean esophageal tube
immediately
prior to challenge. This was followed by the challenge material, which was
then
followed by a rinse of 40-100 ml of water or calf milk replacer.
Calves were observed for clinical signs immediately prior to challenge and
then four times daily at approximately the same time every day for 6 days post-
challenge. Clinical observations included rectal temperature, general
condition,
anorexia, diarrhea, dehydration, and death.
Serology
The P21 antibody-detection ELISA used to generate the data for the chart
shown in Figure 20 is an indirect competitive ELISA, meaning that higher OD's
correspond with lower antibody levels, and lower OD's correspond with higher
antibody levels. The calves in this study were naive at day -l, but showed
seroconversion after receiving test colostrums containing P21 antibodies (GST-
P21,
His-P21, and the combo). The calves that received colostrum containing GST-
15/60, His-15/60, and placebo antibodies all remained negative for P21
antibodies
throughout the 6-day observation period.
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54
The CP15/60 antibody-detection ELISA is a direct ELISA, so high OD's
correspond with high antibody levels, and low OD's correspond with low
antibody
levels (Figure 21). All calves were naive at Day-1. The calves that received
colostrum containing 15160 antibodies (GST-15/60, His-15/60 and the combo) all
showed rapid seroconversion. The slightly increased values for the cattle
receiving
colostrums containing P21 and placebo antibodies is common, and hypothesized
to
be background due to cross-reactivity, a limitation of the ELISA. Regardless,
the
calves that received the colostrum containing P21 and placebo antibodies
remained
negative throughout the 6-day observation period.
Overall Sickness Score Ghart
The overall sickness score is an accumulation of all the clinical signs
(diarrhea, anorexia, and depression) observed in this study over a 6-day
period (four
observations per day). This chant, in conjunction with other data, indicated
that the
GST-P21 and His-P21 vaccines had no protective effect. However, the GST-15160
vaccine shows a modest but significant reduction in clinical signs. This
protection
can be more clearly seen in Figure 22. With the other vaccine data removed, it
is
apparent that the calves that received colostrums containing GST-15/60
antibodies
were consistently less sick (i.e., showed fewer clinical signs) throughout
most of the
6-day observation period (Figure 23).
Diarrhea
Four times a day the calves were given a score correlating with the type of
diarrhea observed. At observation 10, there were four calves in the placebo
group
with diarrhea scores of one, so the total score for that observation is 4. All
calves
became symptomatic for diarrhea, regardless of which group they were in, but
for
the majority of the 6-day study, the GST-15/60 group scored lower than the
placebo
group. The difference between the groups was especially apparent in
observations
6-11.
Figure 24 is a cloud diagram that shows the relative distribution of diarrhea
for all the calves in the study. The cloud diagram shows the relative
distribution of
all the calves and was generated by averaging the 24 sickness scores for each
calf
(each filled black circle represents one calf in that treatment group), and
then
averaging those values to obtain an average for the treatment group
(represented by
a filled purple square). If more than one data point occupies the same space,
the
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number of overlapping data points is indicated by the superscript. The average
for
GST-15/60 is lower than that of the placebo and the GST-15/60 values are more
closely grouped (four of the data points overlap with the average for the
group). The
His-15/60 group also did well, having an average much lower than the placebo
or
5 other groups, although the overall grouping of the values is not as close as
GST-
15/60.
Anorexia
After the second day of study, any calf nursing less than 2 liters of milk and
requiring an esophageal tube was scored as anorexic (anorexia observations
during
10 the first two days of life were recorded, but not analyzed). The calves in
the GST-
15/60 group had no anorexia throughout most of the study, in contrast with the
placebo group, which often contained two or three anorexic calves. Figure 25
shows
a cloud diagram depicting the relative distribution of all the calves' total
anorexia
scores, for all vaccines. The GST-15/60 has the closest grouping as well as
the
15 lowest average of all the groups in the study.
Depression
Four times a day, calves were observed and given a score correlating to their
condition. The number of healthy calves in the GST-15/60 group was greater
than
that of the placebo group. It should be noted that none of the calves in
either group
20 scored higher than a 1 (apathetic) condition score at any observation.
Thus, the
score of 3 on observation 21 for the placebo group indicates three calves with
scores
of l, not one calf with a score of 3. Figure 26 shows the distribution of the
total
general condition scores for each calf. The GST-15/60 group shows a much
closer
grouping than the other vaccine groups, as well as having a very low average
25 occurrence as compared to the placebo. Interestingly, the combo vaccine
group
(which consisted of GST-P21 and GST-15/60) also did well, although the results
for
the GST-P21 vaccine alone look similar to those of the placebo. The results
suggest
that the GST-15/60 vaccine improves the general condition.
Fecal Dry Matter
30 The total fecal matter was collected (four times daily), pooled, and dried
for
that day. The amount of fecal dry matter was slightly higher in the GST-15/60
group than in the placebo group for most of the study, indicating a reduced
occurrence of diarrhea in the GST-15/60 group, although all animals became
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56
symptomatic. Figure 27 is a cloud diagram showing the average fecal dry matter
score for each calf, for all vaccines. The 15/60-containing vaccine groups all
show
close grouping and a higher average amount than the placebo group.
Oocyst Shedding
S Figure 28 shows the oocyst shedding as determined by the Prospect ELISA
kit (not direct microscopic oocyst counts). As seen before in other clinical
signs
(such as diarrhea), all animals in the study became symptomatic. However,
oocyst
shedding in the His-15/60 group appears to be delayed as compared to the
placebo
group.
Example 16: Immuno~enicity and Safety of Vaccines Containing Rotavirus
Coronavirus, E. coli K99 and F41 and Containin tg he C paf~uuua GST-CP15/60
antigen
The objective of this study was to assess, in susceptible calves, the safety
and
the antibody response induced by two combination vaccines. A specific
objective of
the study was to determine if addition of a C. pamum subunit antigen
interferes with
the immune response to other antigens, such as bovine rotavirus, bovine
coronavirus, and E. coli antigens K99 and F41. To answer these questions, two
vaccines were tested: both were aluminum/saponin adjuvanted and contained the
following inactivated antigens: bovine rotavirus, bovine coronavirus, E. coli
K99
and E. coli F41. Additionally, one of the vaccines contained a crude GST-
CP15/60
subunit antigen of C. parwum, produced in E. coli. Two groups of calves were
vaccinated twice with 5 ml of their respective treatment, at a 28-day
interval.
Another 2 calves served as environmental controls.
Rectal Temperatures
Figure 29 shows the evolution of average rectal temperature in vaccinates
and controls following the first and second vaccinations. A transient phase of
hyperthermia was observed in the two vaccinated groups, with a peak within 24
hours after the ftrst and second vaccinations. The average maximal increase of
rectal temperature after 1St vaccination (Amax 1 = T° at peakl -
T° at DO) were 1.4
and 1.3°C for the combination + crypto and for the combo alone,
respectively. None
of the calves had a Amax >_ 2.0°C. The average maximal increase of
rectal
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57
temperature after second vaccination (Omax 2 = T° at peak 2 - T°
at D28) were 1.4
and 1.1 °C. None of the calves had a 0 max >_ 2.0°C.
Interestingly, the control calves also had an increase of temperature
following vaccinations. The increase was limited (0.4 to 0.5°C on
average) and was
likely due to the handling of animals. This suggests that maximal hyperthermia
specifically attributable to the vaccines is approximately 1.0°C.
Local Reactions (in vivo2
Figure 30 shows the evolution of the average size of local reactions
following first vaccination. Figure 31 shows the evolution of average size of
local
reactions following the second vaccination. With the exception of the first
vaccination in a calf receiving the combo + crypto, a strong local reaction
appeared
shortly after both injections in all vaccinates. Local reactions were maximal
approximately 24-48 hours post vaccination and remained strong for 1 week.
Then,
a rapid reduction of the reaction size was observed. In all cases, local
reactions had
disappeared, or were very limited, 3 weeks after vaccination. Local reactions
were
sometimes accompanied with a transient and slight enlargement of the draining
lymph node. Vaccinated groups were compared by ANOVA for local reaction at
different time points (1St injection Dl, D21; 2"d injection D29, D49). None of
the
differences were significant.
Serolo~y
Mean C. parwuryz antibody titers are depicted in Figure 32. As expected, all
of the non-crypto vaccinated calves remained negative for antibodies to C.
pa~vum.
Seroconversion was observed in 3 of the 6 crypto-vaccinated animals after
first
vaccination. Fourteen days after the second vaccination, a strong
seroconversion
was observed in all vaccinated calves. At D42, average CP15/60 antibody titers
were 2.66; one calf being a poor responder with a titer of 1.4.
ELISA results for antibody responses to bovine coronavirus (BCV) are
shown in Figure 33. Seroconversions were observed in all vaccinated calves 14
days after the first vaccination. At D42 (14 days after booster vaccination),
all
vaccinated calves had very high ELISA antibody titers. In seroneutralization
assays,
serum from almost all calves neutralized the virus at all tested dilutions
(titer > 3.84
(log CCID 50/m1) (Figure 34). Mean ELISA antibody titers to BCV were
approximately 10% lower with the Combo + Crypto vaccine as compared to the
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58
combo vaccine alone at each time point. This difference was significant
(p=0.01) at
D49.
ELISA results for antibody responses to bovine rotavirus (BRV) are shown
in Figure 35. At D28, seroconversions were detectable in 4/6 and 5/5 of the
vaccinates from the combo + crypto and the combo group, respectively. At D49,
all
vaccinated calves had high ELISA antibody titers. ELISA titers at D49 were
more
homogeneous in the Combo group (StD = 0.12) than in the Combo + Crypto group
(StD = 0.46), and mean ELISA titers were approximately 15% higher. Differences
between the groups (ANOVA - repeated measures) were significant (p=0.05).
Evolution of the neutralization titers for BRV is shown in Figure 36. All
vaccinated
calves had abnormally high antibody titers at D14. Those titers reduced to
more
normal values at D28, with all calves having seroconverted at that time. At
D49, the
average titer (log CCID 50/m1) was 1.9 for the combo + crypto and 2.2 for the
combo group alone. This difference of approximately 18% was significant
(p=0.01).
Evolution of ELISA titers for E. coli FS-K99 is presented in Figure 37. At
D28, seroconversions were detectable in 4/6 and 3/5 of vaccinates from the
combo +
crypto and combo group, respectively. However, ELISA titers of the calves that
had
seroconverted were much higher in the combo group. At D49, average titer (log
OD
50%) were 2.56 for the combo + crypto and 3.91 for the combo group alone. This
difference of approximately 40% was highly significant (p--'0.002).
Evolution of ELISA titers for E. coli F41 is presented in Figure 38. At D28,
seroconversions were detectable in all vaccinates from both groups. At D49,
titers
were much more homogeneous and were higher in the combo group alone. Average
titers at D49 (log OD 50%) were 2.57 for the combo + crypto and 3.67 for the
combo group alone. This difference of approximately 40% was highly significant
(p=0.005).
On average, both vaccines induced a transient and moderate hyperthermia
after each of the injections. No other systemic reaction was observed. Both
vaccines induced strong local reactions that reduced to very acceptable sizes
within
2 weeks. Reactions were more pronounced at the second vaccination, regardless
of
the nature of the vaccine.
Both vaccines induced production of antibodies against all their respective
antigen components. With the exception of antibodies to C. pafwurn, antibody
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59
responses were always higher with the combo alone vaccine than with the combo
+
crypto vaccine. This difference was particularly clear when looking at
antibodies to
E. coli K99 and to E. coli F41, for which addition of the Crypto antigen in
the
vaccine was associated with a reduction of 40% (in log) of the antibody
response.
These results clearly suggest that interference of the crypto antigen
(especially on E. coli K99 and F41, and possibly BRV antibody responses) is
significant, and might impact on protection. Consequently, this addition may
require redefinition of the antigen dose for E. coli K99, F41, and BRV
fractions.
Although the foregoing invention has been described in some detail by way
of illustration and example for purposes of clarity and understanding, it will
be
apparent to those skilled in the art that certain changes and modifications
can be
practiced. Therefore, the description and examples should not be construed as
limiting the scope of the invention, which is delineated by the appended
claims.
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