U.S. patent application number 12/988088 was filed with the patent office on 2011-02-10 for vaccine for protection against lawsonia intracellularis, mycoplasma hyopneumoniae and porcine circo virus.
Invention is credited to Antonius Arnoldus Christiaan Jacobs, Carla Christina Schrier, Ruud Philip Antoon Maria Segers, Paul Vermeij.
Application Number | 20110033497 12/988088 |
Document ID | / |
Family ID | 39496087 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110033497 |
Kind Code |
A1 |
Jacobs; Antonius Arnoldus
Christiaan ; et al. |
February 10, 2011 |
VACCINE FOR PROTECTION AGAINST LAWSONIA INTRACELLULARIS, MYCOPLASMA
HYOPNEUMONIAE AND PORCINE CIRCO VIRUS
Abstract
The present invention pertains to a vaccine comprising in
combination non-live antigens of Lawsonia intracellularis, of
Mycoplasma hyopneumniae and Porcine circo virus, and a
pharmaceutically acceptable carrier. The invention also pertains to
a kit comprising a first container having contained therein
non-live antigens of Lawsonia intracellularis, one or more other
containers having contained therein Mycoplasma hyopneumoniae and
Porcine circo virus antigens and instructions for mixing the
antigens of Lawsonia intracellularis, Mycoplasma hyopneumoniae, and
Porcine circo virus to formulate one combination vaccine suitable
for systemic vaccination.
Inventors: |
Jacobs; Antonius Arnoldus
Christiaan; (Boxmeer, NL) ; Vermeij; Paul;
(Boxmeer, NL) ; Segers; Ruud Philip Antoon Maria;
(Boxmeer, NL) ; Schrier; Carla Christina;
(Boxmeer, NL) |
Correspondence
Address: |
Intervet/Schering-Plough Animal Health
Patent Dept. K-6-1, 1990, 2000 Galloping Hill Road
Kenilworth
NJ
07033-0530
US
|
Family ID: |
39496087 |
Appl. No.: |
12/988088 |
Filed: |
April 16, 2009 |
PCT Filed: |
April 16, 2009 |
PCT NO: |
PCT/EP09/54517 |
371 Date: |
October 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61046188 |
Apr 18, 2008 |
|
|
|
Current U.S.
Class: |
424/201.1 |
Current CPC
Class: |
A61K 9/0019 20130101;
C12N 2750/10034 20130101; A61K 39/0241 20130101; A61P 33/02
20180101; A61K 2039/521 20130101; A61P 31/04 20180101; A61K 39/295
20130101; A61K 39/105 20130101; A61K 2039/523 20130101; A61K
2039/55566 20130101; A61P 31/00 20180101; A61P 31/12 20180101; A61K
2039/70 20130101; A61K 39/39 20130101; A61K 2039/552 20130101; A61P
31/20 20180101; A61P 37/04 20180101 |
Class at
Publication: |
424/201.1 |
International
Class: |
A61K 39/295 20060101
A61K039/295; A61P 31/04 20060101 A61P031/04; A61P 31/12 20060101
A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 18, 2008 |
EP |
08154765.5 |
Claims
1. A vaccine comprising in combination non-live antigens of
Lawsonia intracellularis, Mycoplasma hyopneumniae and Porcine circo
virus, and a carrier.
2. The vaccine according to claim 1, characterised in that the
vaccine is in a form suitable for systemic administration.
3. The vaccine according to claim 2, characterised in that the
non-live Lawsonia intracellularis antigens are obtained from a
carbohydrate containing composition, the carbohydrate being also
found in live Lawsonia intracellularis cells in association with
the outer cell membrane of these cells.
4. The vaccine according to claim 3, characterised in that the
carbohydrate containing composition is material resulting from the
killing of Lawsonia intracellularis bacteria.
5. The vaccine according to claim 4, characterised in that the
carbohydrate containing composition contains whole cells of killed
Lawsonia intracellularis bacteria.
6. The vaccine according to claim 5, characterised in that the
vaccine comprises an oil in water adjuvant containing oil droplets
of sub-micrometer size.
7. The vaccine according to claim 6, characterised in that the
adjuvant comprises droplets of biodegradable oil and droplets of
mineral oil, the droplets of biodegradable oil having an average
size that differs from the average size of the droplets of mineral
oil.
8. A kit comprising a first container having contained therein
non-live antigens of Lawsonia intracellularis, one or more other
containers having contained therein Mycoplasma hyopneumoniae and
Porcine circo virus antigens and instructions for mixing the
antigens of Lawsonia intracellularis, Mycoplasma hyopneumoniae, and
Porcine circo virus to formulate one combination vaccine suitable
for systemic vaccination.
9. The kit according to claim 8, wherein the Mycoplasma
hyopneumoniae and Porcine circo virus antigens are contained in one
container, formulated in an oil in water adjuvant.
10. The vaccine according to claim 1, characterised in that the
non-live Lawsonia intracellularis antigens are obtained from a
carbohydrate containing composition, the carbohydrate being also
found in live Lawsonia intracellularis cells in association with
the outer cell membrane of these cells.
11. The vaccine according to claim 10, characterised in that the
carbohydrate containing composition is material resulting from the
killing of Lawsonia intracellularis bacteria.
12. The vaccine according to claim 11, characterised in that the
carbohydrate containing composition contains whole cells of killed
Lawsonia intracellularis bacteria.
13. The vaccine according to claim 12, characterised in that the
vaccine comprises an oil in water adjuvant containing oil droplets
of sub-micrometer size.
14. The vaccine according to claim 13, characterised in that the
adjuvant comprises droplets of biodegradable oil and droplets of
mineral oil, the droplets of biodegradable oil having an average
size that differs from the average size of the droplets of mineral
oil.
15. The vaccine according to claim 3, characterised in that the
vaccine comprises an oil in water adjuvant containing oil droplets
of sub-micrometer size.
16. The vaccine according to claim 15, characterised in that the
adjuvant comprises droplets of biodegradable oil and droplets of
mineral oil, the droplets of biodegradable oil having an average
size that differs from the average size of the droplets of mineral
oil.
17. The vaccine according to claim 2, characterised in that the
vaccine comprises an oil in water adjuvant containing oil droplets
of sub-micrometer size.
18. The vaccine according to claim 17, characterised in that the
adjuvant comprises droplets of biodegradable oil and droplets of
mineral oil, the droplets of biodegradable oil having an average
size that differs from the average size of the droplets of mineral
oil.
Description
[0001] The present invention pertains to a vaccine for protection
against Lawsonia intracellularis, Mycoplasma hyopneumoniae and
Porcine circo virus. Protection in this sense means that the
vaccine at least provides a decrease in a negative influence caused
by Lawsonia intracellularis, Mycoplasma hyopneumoniae and Porcine
circo virus, such negative influence being e.g. tissue damage
and/or clinical signs such as decreased weight gain, diarrhea,
coughing, sneezing etc. The present invention also pertains to a
kit comprising a first container having contained therein non-live
antigens of Lawsonia intracellularis, one or more other containers
having contained therein Mycoplasma hyopneumoniae and Porcine circo
virus antigens and instructions for mixing the antigens of Lawsonia
intracellularis, Mycoplasma hyopneumoniae, and Porcine circo virus
to formulate one combination vaccine suitable for systemic
vaccination.
[0002] Proliferative enteropathy (PE or PPE, also called enteritis
or ileitis) in many animals, in particular pigs, presents a
clinical sign and pathological syndrome with mucosal hyperplasia of
immature crypt epithelial cells, primarily in the terminal ileum.
Other sites of the intestines that can be affected include the
jejunum, caecum and colon. Weanling and young adult pigs are
principally affected with typical clinical manifestation of rapid
weight loss and dehydration. Natural clinical disease in pigs
occurs worldwide. The disease is consistently associated with the
presence of intracellular curved bacteria, presently known as
Lawsonia intracellularis.
[0003] Mycoplasmal pneumonia of swine caused by the bacterial
pathogen Mycoplasma hyopneumoniae is a widespread chronic
respiratory disease in pigs. Especially young piglets are
vulnerable to this, non-fatal, disease. The enzootic pneumonia is a
chronic disease that results in poor feed conversion and stunted
growth. The disease is highly contagious and transmission is
usually through direct contact with infected respiratory tract
secretions, e.g. in the form of infected droplets after
coughing/sneezing. The most problematic consequence of this disease
is that it predisposes for all kinds of secondary infections of the
respiratory system. It is estimated that e.g. in the USA, 99% of
all pig farms are infected.
[0004] Porcine circo virus is thought to be linked to the
post-weaning multisystemic wasting syndrome (PMWS) observed in
young pigs. This disease was encountered for the first time in
Canada in 1991. The clinical signs and pathology were published in
1997 and include progressive wasting, dyspnea, tachypnea, and
occasionally icterus and jaundice. Porcine circo virus is a small
(17 nm) icosahedral non-enveloped virus containing a circular
single stranded DNA genome. PDNS (porcine dermatitis and
nephropathy syndrome) is another major problem for pig farmers
which appeared around the same time as PMWS and which is also
related to Porcine circo virus. Characteristic of PDNS are
red/brown circular skin lesions with haemorrhages, usually on the
ears, flanks, legs and hams.
[0005] With respect to PE, oral vaccination against Lawsonia
intracellularis has shown to be an economically efficient measure
to control Ileitis and to allow a better exploitation of the
genetic growth potential of the pig (Porcine Proliferative
Enteropathy Technical manual 3.0, July 2006; available from
Boehringer Ingelheim). Furthermore, oral rather than parenteral
vaccination will reduce the transmission of blood-borne infections
such as PRRS via multi-use needles and the reduction of injection
site reactions and needles retained in carcasses. It will reduce
animal and human stresses, time, labour costs and effort compared
to individual vaccination (McOrist: "Ileitis--One Pathogen, Several
Diseases" at the IPVS Ileitis Symposium in Hamburg, Jun. 28,
2004).
[0006] It is generally understood that the advantage of an
attenuated live vaccine approach is that the efficacy of immunity
is usually relatively good, as the host's immune system is exposed
to all the antigenic properties of the organism in a more "natural"
manner. Specifically for intracellular bacterial agents such as
Lawsonia intracellularis, the live attenuated vaccine approach is
believed to offer the best available protection for vaccinated
animals, due to a full and appropriate T cell based immune
response. This is in contrast with the variable to poor immunity
associated with subunit or killed vaccine types for intracellular
bacteria. This is also specifically true for obligate intracellular
bacteria such as Lawsonia intracellularis or the Chlamydia sp,
which cause pathogenic infections within mucosa. Studies indicate
that whole live attenuated forms of the intracellular bacteria in
question are best delivered to the target mucosa, that they are
required as whole live bacterial forms to produce a fully
protective immune response in the target mucosa but also that they
are immunologically superior compared to use of partial bacterial
components.
[0007] It has become a general understanding that a vaccine against
Lawsonia intracellularis needs to be administered orally (see i.a.
Technical Manual 3.0 as referred to here-above). This is based on
the fact that the basis of the body's resistance to Ileitis is the
local immunity in the intestine, which is the product of
cell-mediated immunity and local defense via antibodies, especially
IgA. According to current knowledge, serum antibodies (IgG) do not
give any protection simply because they do not reach the gut lumen.
It has been demonstrated in studies that oral vaccination produces
cell-mediated immunity as well as local production of IgA in the
intestine (Murtaugh, in Agrar- und Veterinar-Akademie,
Nutztierpraxis Aktuell, Ausgabe 9, Juni 2004; and Hyland et al. in
Veterinary Immunology and Immunopathology 102 (2004) 329-338). In
contrast, intramuscular administration did not lead to protection.
Moreover, next to the general understanding that a successful
vaccine against intracellular bacteria has to induce cell-mediated
immunity as well as the production of local antibodies, the skilled
practitioner knows that only a very low percentage of orally
ingested antigens are actually absorbed by the enterocytes, and
that the incorporation of Lawsonia intracellularis into the cell is
an active process initiated by the bacterium. Accordingly an
inactivated vaccine would provide the intestine with insufficient
immunogenic antigen (Haesebrouck et al. in Veterinary Microbiology
100 (2004) 255-268). This is why it is believed that only
attenuated live vaccines induce sufficient cell-mediated protection
in the intestinal cells (see Technical Manual 3.0 as referred to
here-above). At present there is only one vaccine on the market to
protect against Lawsonia intracellularis, viz. Enterisol.RTM.
Ileitis marketed by Boehringer Ingelheim. This vaccine is a live
vaccine for oral administration indeed.
[0008] Hitherto combination vaccines of Lawsonia intracellularis
have been suggested in the prior art. However, not many of such
combinations have actually been tested for efficacy. The reason for
this is that it is generally understood that combination of
antigens with antigens of Lawsonia intracellularis can only lead to
successful protection if the Lawsonia antigens are provided as live
(attenuated) cells. In this respect, we refer to WO 2005/011731,
which also suggests all kinds of combination vaccines based on
Lawsonia intracellularis. However, regarding the description and
claim structure the patent application, the assignee (Boehringer
Ingelheim) appears to be convinced that combination vaccines are
only expected to have a reasonable chance of success when the
Lawsonia antigens are present in the form of live cells. The same
is true for WO2006/099561, also assigned to Boehringer Ingelheim.
Indeed, based on the common general knowledge this is an obvious
thought. Combination of live antigens however is not
straightforward given the high chance of interference between the
antigens and the difficulty of manufacturing such a live
combination vaccine.
[0009] It is an object of the present invention to provide a
vaccine to combat Lawsonia intracellularis, and at the same time
combat one or more other swine pathogens. To this end a vaccine has
been devised that comprises in combination non-live antigens of
Lawsonia intracellularis, Mycoplasma hyopneumoniae and Porcine
circo virus, and a pharmaceutically acceptable carrier.
Surprisingly, against the persistent general understanding how to
combat Lawsonia intracellularis and that a combination vaccine
should comprise live Lawsonia intracellularis antigens, it was
found that by using non-live Lawsonia intracellularis antigens, in
combination with antigens from Mycoplasma hyopneumoniae and Porcine
circo virus, a vaccine can be provided that protects against
Lawsonia intracellularis, Mycoplasma hyopneumoniae and Porcine
circo virus.
[0010] In general, a vaccine can be manufactured by using art-known
methods that basically comprise admixing the antigens with a
carrier. Typically the antigen(s) are combined with a medium for
carrying the antigens, often simply referred to as a carrier or
"pharmaceutically acceptable carrier". Such a carrier can be any
solvent, dispersion medium, coating, antibacterial and antifungal
agent, isotonic and absorption delaying agent, and the like that
are physiologically compatible with and acceptable for the target
animal, e.g. by being made i.a. sterile. Some examples of such
carrying media are water, saline, phosphate buffered saline,
bacterium culture fluid, dextrose, glycerol, ethanol and the like,
as well as combinations thereof. They may provide for a liquid,
semi-solid and solid dosage form, depending on the intended mode of
administration. As is commonly known, the presence of a carrying
medium is not essential to the efficacy of a vaccine, but it may
significantly simplify dosage and administration of the antigen. As
such, the manufacturing of the vaccine can take place in an
industrial environment but also, the antigens could be mixed with
the other vaccine constituents in situ (i.e. at a veterinaries', a
farm etc.), e.g. (immediately) preceding the actual administration
to an animal. In the vaccine, the antigens should be present in an
immunologically effective amount, i.e. in an amount capable of
stimulating the immune system of the target animal sufficiently to
at least reduce the negative effects of a post-vaccination
challenge with wild-type micro-organisms. Optionally other
substances such as adjuvants, stabilisers, viscosity modifiers or
other components are added depending on the intended use or
required properties of the vaccine.
[0011] In an embodiment, the vaccine is in a form suitable for
systemic administration. To applicants surprise it has been found
that one can induce a protection against Lawsonia intracellularis
that is comparable with or even improved with respect to the
protection provided by using the (single) live vaccine
Enterisol.RTM. Ileitis (administered according to the corresponding
instructions), when the combination vaccine according to the
present invention is administered systemically, i.e. in a way that
it reaches the circulatory system of the body (comprising the
cardiovascular and lymphatic system), thus affecting the body as a
whole rather than a specific locus such as the gastro-intestinal
tract. Systemic administration can be performed e.g. by
administering the antigens into muscle tissue (intramuscular), into
the dermis (intradermal), underneath the skin (subcutaneous),
underneath the mucosa (submucosal), in the veins (intravenous) etc.
For systemic vaccination many forms are suitable, in particular
liquid formulations (with dissolved, emulsified or suspended
antigens) but also solid formulations such as implants or an
intermediate form such as a solid carrier for the antigen suspended
in a liquid. Systemic vaccination, in particular parenteral
vaccination (i.e. not trough the alimentary canal), and suitable
(physical) forms of vaccines for systemic vaccination have been
known for more than 200 years. An advantage of this embodiment is
that the same way of administration can be used that is the current
standard for administering Mycoplasma hyopneumoniae or Porcine
circo virus antigens, viz. parenteral, in particular via
intramuscular or intradermal injection (in the latter case often
needle-less).
[0012] In an embodiment the non-live Lawsonia intracellularis
antigens are obtained from a carbohydrate containing composition,
the carbohydrate being also found in live Lawsonia intracellularis
cells in association with the outer cell membrane of these cells.
Unexpectedly, by using a carbohydrate containing fraction of
Lawsonia intracellularis cells (i.e. a composition containing the
carbohydrates as present in live Lawsonia intracellularis cells) in
the combination vaccine, good protection against PE could be
provided. It is noted that for formulating the vaccine a
carbohydrate containing composition directly obtained from Lawsonia
intracellularis cells could be used but also a composition derived
therefrom, such as a dilution or concentrate of the original
composition or an extract, one or more purified components etc. It
is noted that subunits of Lawsonia intracellularis cells have been
reported as antigens in a vaccine for protection against this
bacterium. However, these are mainly recombinant proteins and
hitherto none of them has proven to be able and provide good
protection. It is also noted that a carbohydrate containing
composition, wherein the carbohydrate is also found in live
Lawsonia intracellularis cells in association with the outer cell
membrane of these cells, is known from Kroll et al. (Clinical and
Diagnostic Laboratory Immunology, June 2005, 693-699). However,
this composition is used for diagnostics. It has not been tested as
a protective antigen for reasons as stated here-above.
[0013] In an embodiment, the carbohydrate containing composition is
material resulting from the killing of Lawsonia intracellularis
bacteria. It has been found that a very convenient way of providing
the carbohydrate for use according to the present invention is to
simply kill Lawsonia intracellularis cells and use the material
resulting from that as a source for the carbohydrate. To extract
the carbohydrate from living cells could in theory also be done
(analogous to the creation of living ghost cells by removing the
cell wall) but requires more sophisticated and thus more expensive
techniques. The material as a whole could be used, e.g. a
suspension of whole cells or a lysate of Lawsonia intracellularis
cells, or one could purify or even isolate the carbohydrate out of
the material. This method can be performed by using relatively
simple art-known techniques.
[0014] In a preferred embodiment the carbohydrate containing
composition contains whole cells of killed Lawsonia intracellularis
bacteria. This has proven to be the most convenient way to provide
the carbohydrate as an antigen in the vaccine. Besides, the
efficacy of the vaccine is even further increased, possibly since
this way of offering the antigen to the immune system of the target
animal better mimics the natural environment of the
carbohydrate.
[0015] In an embodiment the vaccine comprises an oil in water
adjuvant containing oil droplets of sub-micrometer size. In
general, an adjuvant is a non-specific immunostimulating agent. In
principal, each substance that is able to favor or amplify a
particular process in the cascade of immunological events,
ultimately leading to a better immunological response (i.e. the
integrated bodily response to an antigen, in particular one
mediated by lymphocytes and typically involving recognition of
antigens by specific antibodies or previously sensitized
lymphocytes), can be defined as an adjuvant. It has been shown that
using an oil in water adjuvant containing oil droplets of
sub-micrometer size provides a very good protection against
Lawsonia intracellularis. Indeed, the application of oil in water
adjuvants as such is common in connection with non-live antigens.
However, it is generally known that the best immunostimulating
properties are obtained when the oil droplets are large in
diameter. In particular, oil droplets with a diameter beneath 1
micrometer are in particular used when it is believed that safety
is an important issue. In that case, one could use small droplets
since these are known to evoke less tissue damage, clinical signs
etc. However, in the case of obtaining protection for a gut
associated disorder via systemic vaccination (as is the case in the
present invention), one would choose large droplets since one would
expect that the immune response has to be boosted significantly. In
contrast, we found that using small oil droplets in the composition
provided very good results with respect to protection against
Lawsonia intracellularis.
[0016] In an even preferred embodiment, the adjuvant comprises
droplets of biodegradable oil and droplets of mineral oil, the
droplets of biodegradable oil having an average size that differs
from the average size of the droplets of mineral oil. It has been
shown that the use of a mixture of biodegradable oil and mineral
oil provides very good results with regard to efficacy and safety.
In addition to this, stability of the composition is very high,
which is an important economic advantage. The stability has proven
to be very good, in particular when the average (volume weighed)
size of either the biodegradable oil droplets or the mineral
droplets is below 500 nm (preferably around 400 nm).
[0017] The present invention also pertains to a kit comprising a
first container having contained therein non-live antigens of
Lawsonia intracellularis, one or more other containers having
contained therein Mycoplasma hyopneumoniae and Porcine circo virus
antigens and instructions for mixing the antigens of Lawsonia
intracellularis, Mycoplasma hyopneumoniae, and Porcine circo virus
to formulate one combination vaccine suitable for systemic
vaccination. In this embodiment, a separate container for the
Lawsonia intracellularis antigens is provided in a kit containing
also the other antigens (which are either combined in one container
as known from the prior art or even present in separate containers
that form part of the contents of the kit. An advantage of this
embodiment is that the Lawsonia antigens can be prevented from
having interactions with the other antigens until right before
administration of the vaccine. Also, since the antigens are in a
separate container less production losses will occur. In an
embodiment the Mycoplasma hyopneumoniae and Porcine circo virus
antigens are contained in one container, formulated in an oil in
water adjuvant. In this embodiment, the Lawsonia antigens can be
mixed with the other ones just before use.
[0018] The invention will be further explained based on the
following examples.
[0019] Example 1 describes a method to obtain a substantially
protein free carbohydrate containing composition and a vaccine that
is made by using this composition.
[0020] Example 2 describes an experiment wherein a non-live
Lawsonia intracellularis vaccine is compared with the vaccine
currently on the market and an experimental vaccine comprising
subunit proteins of Lawsonia intracellularis.
[0021] Example 3 describes an experiment wherein two different
vaccines comprising non-live Lawsonia intracellularis antigens are
compared with the live vaccine currently on the market.
EXAMPLE 1
[0022] In this example a method is described to obtain a
substantially protein free carbohydrate composition associated with
the outer cell membrane of Lawsonia intracellularis cells and a
vaccine that can be made using this composition. In general, a
carbohydrate is an organic compound that contains carbon, hydrogen,
and oxygen, usually in the ratio 1:2:1. Examples of carbohydrates
are sugars (saccharides), starches, celluloses, and gums. Usually
they serve as a major energy source in the diet of animals.
Lawsonia intracellularis is a gram negative bacterium, which thus
contains an outer membrane that is not constructed solely of
phospholipid and protein, but also contains carbohydrates, in
particular polysaccharide (usually polysaccharides such as
lipopolysaccharide, lipo-oligosaccharide, or even non-lipo
polysaccharides).
Carbohydrate Fraction for Vaccine Preparation
[0023] Twenty milliliters of buffered water (0.04 M PBS, phosphate
buffered saline) containing Lawsonia intracellularis cells at a
concentration of 3.7E8 (=3.7.times.10.sup.8) cells/ml was taken.
The cells were lysed by keeping them at 100.degree. C. for 10
minutes. Proteinase K (10 mg/ml) in 0.04 M PBS was added to a final
concentration of 1.7 mg/ml. This mixture was incubated at
60.degree. C. for 60 minutes in order to degrade all proteins and
keep the carbohydrates intact. Subsequently, the mixture was
incubated at 100.degree. C. for 10 minutes to inactivate the
Proteinase K. The resulting material, which is a carbohydrate
containing composition, in particular containing the carbohydrates
as present in live Lawsonia intracellularis bacteria in association
with their outer cell membrane (see paragraph below), was stored at
2-8.degree. C. until further use. The composition was formulated in
Diluvac forte adjuvant which also serves as a carrier for the
antigens. This adjuvant (see also EP 0 382 271) comprises 7.5
weight percent vitamine E acetate droplets with an average volume
weighted size of approximately 400 nm, suspended in water and
stabilized with 0.5 weight percent of Tween 80 (polyoxyethylene
sorbitan mono-oleate). Each milliliter vaccine contained material
that had been extracted from 1.2E8 Lawsonia intracellularis
cells.
Immune Precipitation of Lawsonia Carbohydrate Antigens
[0024] Two batches of monoclonal antibodies (MoAb's) raised against
whole cell Lawsonia intracellularis were precipitated with
saturated Na.sub.2SO.sub.4 at room temperature according to
standard procedures. The precipitate was pelleted by centrifugation
(10.000 g for 10 minutes). The pellet was washed with 20%
Na.sub.2SO.sub.4 and resuspended in 0.04 M PBS. Tylosyl activated
Dynal beads (DynaBeads, DK) were pre washed with 0.1 M NaPO.sub.4
(pH 7.4), according the manual of the manufacturer. Of each batch
of MoAb's 140 .mu.g was taken and added to 2E8 pre washed beads and
incubated overnight at 37.degree. C. The beads were pelleted by
centrifugation and non-bound MoAb's were removed by aspiration of
the supernatant. Spectrophotometrical measurements showed that
between 20 and 35% of the added MoAb's had bound to the beads.
[0025] Two batches of 1 ml Lawsonia intracellularis cells
(3.7E8/ml) in 0.04 M PBS were sonicated for 1 minute. The resulting
cell lysates were added to the Tylosyl activated beads--monoclonal
complexes and incubated overnight at 4.degree. C. The Tylosyl
activated beads--monoclonal complexes were washed three times with
0.1 M NaPO.sub.4 (pH 7.4). The bound compounds were eluted by
washing the beads in 0.5 ml 8 M urea in 0.04 M PBS (E1); 0.5 ml 10
mM Glycine pH 2.5 (E2); and 0.5 ml 50 mM HCl (E3), in a sequential
manner. After elution E2 and E3 were neutralized with either 100
.mu.l and 200 .mu.l 1 M Tris/HCl (pH8.0).
[0026] Samples were taken from each step and loaded onto SDS-PAGE
gels. Gels were stained using Commassie Brilliant Blue (CBB) and
Silver staining or blotted. The blots were developed using the same
MoAb's as mentioned here-above. Inspection of the gels and blots
showed that the MoAb's recognized bands with an apparent molecular
weight of 21 and 24 kDa that were not seen on the CBB gels but were
visible on de Silver stained gels. Also, it was established that
the fraction of the cells that bound to the MoAb's was Proteinase K
resistant. Thus, based on these results it can be concluded that
this fraction contains carbohydrates (namely: all protein is lysed,
and sonified DNA fractions will not show as a clear band in a
Silver stain), and that the carbohydrates are in association with
(i.e. forming part of or being bound to) the outer cell membrane of
Lawsonia intracellularis (namely: the MoAb's raised against this
fraction also recognized whole Lawsonia intracellularis cells).
Given the fact that Lawsonia intracellularis is a gram-negative
bacterium, the carbohydrate composition is believed to comprise
polysaccharide(s).
EXAMPLE 2
[0027] This experiment was conducted to test a convenient way to
formulate the carbohydrate antigen in a vaccine, viz. via a killed
whole cell (also known as bacterin). As controls the commercially
available vaccine Enterisol.RTM. ileitis and an experimental
subunit vaccine comprising protein subunits were used. Next to this
unvaccinated animals were used as a control.
Experimental Design of Example 2
[0028] An inactivated whole cell vaccine was made as follows. Live
Lawsonia intracellularis cells derived from the intestines of pigs
with PPE were gathered. The cells were inactivated with 0.01% BPL
(beta-propiolactone). The resulting material, which inherently is a
non-live carbohydrate containing composition in the sense of the
present invention (in particular since it contains the
carbohydrates as present in live Lawsonia intracellularis bacteria
in association with their outer cell membrane), was formulated in
Diluvac forte adjuvant (see Example 1) at a concentration of
approximately 2.8.times.10.sup.8 cells per ml vaccine.
[0029] The subunit vaccine contained recombinant P1/2 and P4 as
known from EP 1219711 (the 19/21 and 37 kDa proteins respectively),
and the recombinant proteins expressed by genes 5074, 4320 and 5464
as described in WO2005/070958. The proteins were formulated in
Diluvac forte adjuvant. The vaccine contained approximately 50
.mu.grams of each proteins per milliliter.
[0030] Forty 6-week-old SPF pigs were used. The pigs were allotted
to 4 groups of ten pigs each. Group 1 was vaccinated once orally
(at T=0) with 2 ml live "Enterisol.RTM. ileitis" (Boehringer
Ingelheim) according to the instructions of the manufacturer. Group
2 and 3 were vaccinated twice intramuscularly (at T=0 and T=4 w)
with 2 ml of the inactivated Lawsonia whole cell vaccine and the
recombinant subunit combination vaccine as described here-above,
respectively. Group 4 was left as unvaccinated control. At T=6 w
all pigs were challenged orally with homogenized mucosa infected
with Lawsonia intracellularis. Subsequently all pigs were daily
observed for clinical signs of Porcine Proliferative Enteropathy
(PPE). At regular times before and after challenge serum blood (for
serology) and faeces (for PCR) were sampled from the pigs. At T=9 w
all pigs were euthanized and necropsied. Histological samples of
the ileum were taken and examined microscopically.
[0031] The challenge inoculum was prepared from infected mucosa:
500 grams of infected mucosa (scraped from infected intestines)
were thawed and mixed with 500 ml physiological salt solution. This
mixture was homogenized in an omnimixer for one minute at full
speed on ice. All pigs were challenged orally with 20 ml challenge
inoculum at T=6 w.
[0032] At T=0, 4, 6, 7, 8 and 9 w a faeces sample (gram quantities)
and a serum blood sample of each pig was taken and stored frozen
until testing. The faeces samples were tested in a quantitative PCR
(Q-PCR) test and expressed as the logarithm of the amount found in
picograms (pg). Serum samples were tested in the commonly applied
IFT test (immuno fluorescent antibody test to detect antibodies
against whole Lawsonia intracellularis cells in the serum). For
histological scoring a relevant sample of the ileum was taken,
fixed in 4% buffered formalin, routinely embedded and cut into
slides. These slides were stained with Hematoxylin-Eosin (HE stain)
and with an immunohistochemical stain using anti-Lawsonia
intracellularis monoclonal antobidies (IHC stain). The slides were
examined microscopically. The histology scores are as follows:
TABLE-US-00001 HE stain: no abnormalities detected score = 0
doubtful lesion score = 1/2 mild lesions score = 1 moderate lesions
score = 2 severe lesions score = 3 IHC stain: no L. intracelluaris
bacteria evident score = 0 doubtful presence of bacteria score =
1/2 presence of single/small numbers of bacteria in the slide score
= 1 presence of moderate numbers of bacteria in the slide score = 2
presence of large numbers of bacteria in the slide score = 3
[0033] All data were recorded for each pig individually. The score
per group was calculated as the mean of the positive animals for
the different parameters after challenge. The non-parametric
Mann-Whitney U test were used to evaluate the statistical
significance (tested two-sided and level of significance set at
0.05).
Results of Example 2
Serology
[0034] Before first vaccination all pigs were seronegative when
tested for IFT antibody titres. After vaccination with the whole
cell bacterin (group 2) pigs developed high IFT antibody titres
whereas the controls and the pigs vaccinated with the subunit
vaccine remained negative until challenge (Table 1). Two of the
Enterisol vaccinated pigs (group 1) developed moderate IFT titres
whereas all other pigs in this group remained seronegative. After
challenge all pigs developed high IFT antibody titers. Mean results
are depicted in table 1 (with the used dilution, 1.0 was the
detection level on the lower side).
TABLE-US-00002 TABLE 1 Mean IFT antibody titres (2log) of pig serum
after vaccination and challenge Group T = 0 weeks T = 4 weeks T = 6
weeks T = 9 weeks 1 <1.0 1.1 1.7 >11.4 2 <1.0 3.7 >11.8
>12.0 3 <1.0 <1.0 <1.0 >11.6 4 <1.0 <1.0
<1.0 >12.0
Real-Time PCR on Faeces Samples
[0035] Before challenge all faeces samples were negative. After
challenge positive reactions were found in all groups. Group 1
(p=0.02), group 2 (p=0.01) and group 3 (p=0.03) had a significantly
lower shedding level compared to the control. A post-challenge
overview is given in table 2.
TABLE-US-00003 TABLE 2 Mean results of PCR on faeces samples (log
pg) after vaccination and challenge T = 8 T = 9 Total Group T = 6
weeks T = 7 weeks weeks weeks post-challenge 1 0 1.3 3.6 1.8 6.3 2
0 0.8 2.8 1.9 5.5 3 0 0.5 3.8 2.0 5.9 4 0 0.8 4.9 4.9 10.0
Histology Scores
[0036] Group 2 had the lowest histology HE score (p=0.05), IHC
score (p=0.08) and total histology score (p=0.08). The other groups
had higher scores and were not significantly different from the
control group. See table 3.
TABLE-US-00004 TABLE 3 Mean histology score for the ileum. Group HE
score IHC score Total score 1 1.8 1.5 3.3 2 1.3 1.5 2.7 3 1.8 1.6
3.4 4 2.4 2.3 4.7
Conclusions with Regard to Example 2
[0037] From the results it can be concluded that the non-live whole
cell Lawsonia intracellularis vaccine which inherently contains the
carbohydrate as found also in association with the outer membrane
of live Lawsonia intracellularis cells, induced at least partial
protection. All parameters studied and histology scores were
significantly or nearly significantly better compared to the
controls.
EXAMPLE 3
[0038] This experiment was conducted to test a vaccine comprising a
substantially protein free carbohydrate containing composition as
antigen. A second vaccine to be tested contained in addition to
killed whole cells of Lawsonia intracellularis, antigens of
Mycoplasma hyopneumoniae and Porcine circo virus (the "combi"
vaccine). As a control the commercially available Enterisol.RTM.
ileitis vaccine was used. Next to this, unvaccinated animals were
used as a second control.
Experimental Design of Example 3
[0039] The vaccine based on a substantially protein free
carbohydrate containing composition was obtained as described under
Example 1.
[0040] The experimental combi vaccine contained inactivated
Lawsonia intracellularis whole cell antigen (see Example 2 for the
used method of providing the inactivated bacteria) at a level of
1.7.times.10.sup.8 cells/ml. Next to this it contained inactivated
PCV-2 antigen (20 .mu.grams of the ORF 2 encoded protein of PCV 2
per ml; the protein being expressed in a baculo virus expression
system as commonly known in the art, e.g. as described in WO
2007/028823) and inactivated Mycoplasma hyopneumoniae antigen (the
same antigen in the same dose as is known from the commercially
available vaccine Porcilis Mhyo.RTM., obtainable from Intervet,
Boxmeer, The Netherlands). The antigens were formulated in a twin
emulsion adjuvant "X". This adjuvant is a mixture of 5 volume parts
of adjuvant "A" and 1 volume part of adjuvant "B". Adjuvant "A"
consists of mineral oil droplets with an approximate average
(volume weighed) size around 1 .mu.m, stabilised with Tween 80 in
water. Adjuvant "A" comprises 25 weight % of the mineral oil and 1
weight % of the Tween. Rest is water. Adjuvant "B" consists of
droplets of biodegradable vitame E acetate with an approximate
average (volume weighed) size of 400 nm, stabilised also with Tween
80. The adjuvant "B" comprises 15 weight % of vitamine E acetate
and 6 weight % of Tween 80, rest is water.
[0041] Sixty-four 3-day-old SPF piglets were used. The pigs were
allotted to four groups of 14 piglets and one group of 8 piglets
(Group 4). Group 1 was vaccinated intramuscularly at 3 days of age
with 2 ml of the combi vaccine, followed by a second vaccination at
25 days of age. Group 2 was vaccinated intramuscularly once with 2
ml combi vaccine at 25 days of age. Group 3 was vaccinated orally
with 2 ml Enterisol.RTM. ileitis (Boehringer Ingelheim) at 25 days
of age according to prescriptions. Group 4 was vaccinated
intramusculary at 3 and 25 days of age with 2 ml of the non-protein
carbohydrate vaccine. Group 5 was left unvaccinated as a challenge
control group. At 46 days of age all pigs were challenged orally
with homogenized infected mucosa. Subsequently all pigs were daily
observed for clinical signs of Porcine Proliferative Enteropathy
(PPE). At regular times before and after challenge serum blood and
faeces samples were taken from the pigs for serology and PCR
respectively. At 68 days of age all pigs were euthanized and
post-mortem examined. The ileum was examined histologically.
[0042] The other issues in the experimental design were the same as
described in Example 2, unless indicated otherwise.
Results of Example 3
Serology
1-Lawsonia
[0043] Before first vaccination all pigs were seronegative for IFT
antibody titres. After vaccination with the combi vaccine (groups 1
and 2) and the non-protein carbohydrate vaccine (group 4), many
pigs developed IFT antibody titres whereas the controls and the
pigs vaccinated with Enterisol remained seronegative until
challenge. After challenge all pigs (except two in the Enterisol
group) developed IFT antibody titres. For an overview of the mean
values obtained, see table 4 (due to the higher dilution when
compared to example 2, the detection level was 4.0).
TABLE-US-00005 TABLE 4 Mean IFT Lawsonia antibody titres (2log) of
pig serum after vaccination and challenge Group T = 3 days T = 25
days T = 46 days T = 67 days 1 <4.0 <4.0 7.9 10.3 2 <4.0
<4.0 4.8 9.8 3 <4.0 <4.0 <4.0 8.5 4 <4.0 <4.0 6.9
10.6 5 <4.0 <4.0 <4.0 9.0
2-Mycoplasma Hyopneumoniae
[0044] With respect to Mhyo, at the start of the experiment as well
as day of booster (25-day-old) all pigs were seronegative for Mhyo.
After booster vaccination group 1 developed high Mhyo antibody
titres, at the same level of those obtained with the commercially
available prime-boost vaccine Porcilis Mhyo.RTM.. The results are
given in Table 5 below. Apparently, under these circumstances
(given intramuscularly) only when a booster vaccination is given
the antibody titres at 46 days are above detection level (6.0 for
the method used). It is known however that a single shot
vaccination with Mhyo antigens may provide sufficient protection,
in particular when administered intradermally (see e.g. WO
2007/103042).
TABLE-US-00006 TABLE 5 Mean IFT Mhyo antibody titres (2log) of pig
serum after vaccination Group T = 3 days T = 25 days T = 46 days 1
Below detection level Below detection level 8.3 2 Below detection
level Below detection level Below detection level 5 Below detection
level Below detection level Below detection level
3-Porcine Circo Virus
[0045] With respect to PCV, at 3-day-old the piglets had high
maternally derived PCV antibody titres. At day of booster
(25-day-old) the vaccinates (group 1) had a similar titre compared
to group 2 and the control group. The PCV titre at 25-day-old was
slightly lower compared to the titre at 3-day-old. After the
vaccination at 25-day-old the titres of group 1 (2 vaccinations: at
day 3 and 25) and group 2 (one vaccination at day 25) remained at a
high level whereas control piglets showed a normal decrease in
maternally derived antibodies. The PCV titres obtained are
comparable to the titres obtainable with a single vaccine
containing the same antigen (e.g. Intervet's Circumvent PCV, which
vaccine provides very good protection against PCV). For an overview
of the mean values, see the table given below.
TABLE-US-00007 TABLE 6 Mean IFT PCV antibody titres (2log) of pig
serum after vaccination Group T = 3 days T = 25 days T = 46 days 1
11.5 9.6 10.1 2 12.1 9.5 10.8 5 10.9 9.1 7.0
Real-Time PCR on Faeces Samples
[0046] Three weeks after challenge, pigs of group 1, 2 and 4 had
less Lawsonia (DNA) in their feces compared to groups 3 and 5. Only
the differences between group 1 and 3 (Enterisol) and group 4 and 3
were statistically significant (p<0.05, Mann-Whitney U test).
For the mean results, see table 7.
TABLE-US-00008 TABLE 7 Mean results of PCR on faeces samples (log
pg) after vaccination and challenge Group Mean value 1 1.0 2 1.2 3
2.0 4 0.6 5 1.8
Histological Scores
[0047] Histology scores of group 1 and 4 were significantly lower
compared to those of groups 3 and 5 (p<0.05, two-sided
Mann-Whitney U test (see table 8). The number of pigs with
confirmed PPE were 2/13 in group 1, 6/12 in group 2, 12/14 in group
3, 2/7 in group 4 and 12/14 in the control group 5. Groups 1 and 4
had a significantly lower incidence of PPE compared to groups 3 and
5 (p<0.05, two-sided Fischers' exact test).
TABLE-US-00009 TABLE 8 Mean histology score for the ileum. Group HE
Score IHC Score Total Score 1 0.4 0.6 1.0 2 0.7 0.7 1.4 3 1.6 1.4
3.0 4 0.4 0.4 0.8 5 1.9 1.5 3.4
Conclusion of Example 3
[0048] From the results it can be concluded that the carbohydrate
outer cell membrane antigen offers a relatively good protection
against ileitis. It is also found that the whole cell Lawsonia
bacterin is a good means of offering the carbohydrate antigen in a
vaccine to combat ileitis. Moreover, given the fact that the
combination vaccine provided titres for Mhyo and PCV antibodies to
a level comparable with the levels obtainable with available single
vaccines that are adequate to combat these micro-organims, it has
been demonstrated that a combination vaccine comprising non-live
Lawsonia intracellularis antigens in combination with Mhyo and PCV
antigens is suitable to combat Lawsonia intracellularis as well as
Mycoplasma hyopneumoniae and Porcine circo virus.
* * * * *