U.S. patent application number 12/988083 was filed with the patent office on 2011-02-10 for vaccine for protection against lawsonia intracellulars.
Invention is credited to Antonius Arnoldus Christiaan Jacobs, Carla Christina Schrier, Ruud Philip Antoon Maria Segers, Paul Vermeij.
Application Number | 20110033496 12/988083 |
Document ID | / |
Family ID | 41212183 |
Filed Date | 2011-02-10 |
United States Patent
Application |
20110033496 |
Kind Code |
A1 |
Jacobs; Antonius Arnoldus
Christiaan ; et al. |
February 10, 2011 |
VACCINE FOR PROTECTION AGAINST LAWSONIA INTRACELLULARS
Abstract
The present invention pertains to the use of a non-live
carbohydrate containing composition, the carbohydrate being also
found in live Lawsonia intracellularis cells in association with
the outer cell membrane of these cells, for the manufacture of a
vaccine for protection against an infection with Lawsonia
intracellularis, the vaccine being in a form suitable for systemic
administration.
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: |
41212183 |
Appl. No.: |
12/988083 |
Filed: |
April 16, 2009 |
PCT Filed: |
April 16, 2009 |
PCT NO: |
PCT/EP2009/054516 |
371 Date: |
October 15, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61046161 |
Apr 18, 2008 |
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61111756 |
Nov 6, 2008 |
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Current U.S.
Class: |
424/201.1 ;
424/234.1 |
Current CPC
Class: |
A61K 2039/55566
20130101; A61P 1/04 20180101; A61K 39/105 20130101; A61P 31/12
20180101; A61K 2039/542 20130101; A61P 1/12 20180101; A61K 2039/552
20130101; A61P 31/04 20180101; A61P 37/04 20180101 |
Class at
Publication: |
424/201.1 ;
424/234.1 |
International
Class: |
A61K 39/295 20060101
A61K039/295; A61K 39/02 20060101 A61K039/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 18, 2008 |
EP |
08154764.8 |
Nov 6, 2008 |
EP |
08105738.2 |
Claims
1. A method of manufacturing a vaccine against Lawsonia
intracellularis comprising admixing a non-live carbohydrate
containing composition with a pharmaceutically acceptable carrier,
wherein the non-live carbohydrate containing composition comprises
a carbohydrate that also can be found in association with an outer
cell membrane of a live Lawsonia intracellularis cell; and wherein
the vaccine is in a form suitable for systemic administration.
2. The method according to claim 1, characterised in that the
carbohydrate containing composition is material resulting from the
killing of Lawsonia intracellularis bacteria.
3. The method according to claim 2, characterised in that the
carbohydrate containing composition contains whole cells of killed
Lawsonia intracellularis bacteria.
4. The method according to claim 3, characterised in that the
vaccine comprises an oil in water adjuvant containing oil droplets
of sub-micrometer size.
5. The method according to claim 4, 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.
6. The method according to claim 5, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
7. A non-live carbohydrate containing composition comprising a
carbohydrate that also can be found in association with an outer
cell membrane of a live Lawsonia intracellularis cell; wherein said
composition is for use in the manufacture of a vaccine for
protection against an infection with Lawsonia intracellularis, and
wherein the vaccine is in a form suitable for systemic
administration.
8. The non-live carbohydrate containing composition of claim 7 that
comprises whole cells of killed Lawsonia intracellularis
bacteria.
9. A vaccine comprising the non-live carbohydrate containing
composition of claim 8, and an oil in water adjuvant containing oil
droplets of sub-micrometer size.
10. The vaccine of claim 9 further comprising antigens of
Mycoplasma hyopneumoniae and Porcine circo virus.
11. The method according to claim 2 characterised in that the
vaccine comprises an oil in water adjuvant containing oil droplets
of sub-micrometer size.
12. The method according to claim 1 characterised in that the
vaccine comprises an oil in water adjuvant containing oil droplets
of sub-micrometer size.
13. The method according to claim 11, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
14. The method according to claim 10, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
15. The method according to claim 6, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
16. The method according to claim 5, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
17. The method according to claim 4, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
18. The method according to claim 3, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
19. The method according to claim 2, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
20. The method according to claim 1, characterised in that the
vaccine further comprises antigens of Mycoplasma hyopneumoniae and
Porcine circo virus.
Description
[0001] The present invention pertains to a vaccine for protection
against an infection with Lawsonia intracellularis, a vaccine in
this sense being a composition that at least provides a decrease in
a negative influence of the infection with Lawsonia
intracellularis, such negative influence being e.g. tissue damage
and/or clincal signs such as decreased weight gain, diarrhea,
etc.
[0002] Proliferative enteropathy (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] In general, 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).
[0004] 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 the 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.
[0005] 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.
[0006] It is an object of the present invention to provide an
alternative vaccine to protect against an infection with Lawsonia
intracellularis. To this end it has been devised to use a non-live
carbohydrate containing composition, the carbohydrate being also
found in live Lawsonia intracellularis cells in association with
the outer cell membrane of these cells, for the manufacture of a
vaccine for protection against an infection with Lawsonia
intracellularis, the vaccine being in a form suitable for systemic
administration. Surprisingly, against the persistent general
understanding how to combat Lawsonia intracellularis, it was found
that by using a carbohydrate containing non-live composition, for
example extracted from the outer cell membrane of Lawsonia
intracellularis, as an antigen in a vaccine, one can induce a
protection against Lawsonia intracellularis that is comparable with
or even improved with respect to the protection provided by using
the live vaccine Enterisol .RTM. Ileitis (administered according to
the corresponding instructions), when the antigen 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. Apart from the very good protection obtainable,
an important advantage of the present non-live vaccine is that it
is an inherent safety when compared to a live vaccine.
[0007] In general, the carbohydrate containing composition can be
used to manufacture a vaccine by using art-known methods that
basically comprise admixing the antigenic carbohydrate containing
composition (or a composition derived therefrom, such as a dilution
or concentrate of the original composition or an extract, one or
more purified components etc.) with a pharmaceutically acceptable
carrier, e.g. a liquid carrier such as (optionally buffered) water
or a solid carrier such as commonly used to obtain freeze-dried
vaccines. As such, manufacturing 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. 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.
[0008] 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. Killed bacteria (which inherently contain the
carbohydrate that is also found in live Lawsonia intracellularis
cells in association with the outer cell membrane) are also
suggested as antigens in vaccines against Lawsonia intracellularis
but no vaccines based on killed whole cells have actually been
tested and reported to provide good protection. Apart from that,
systemic administration has not been used in conjunction with these
killed bacteria, because of the general understanding that there is
no reasonable expectation of success for systemic administration of
antigens to locally (i.e. in the intestines) combat Lawsonia
intracellularis.
[0009] In this respect it is noted that in WO 97/20050 (Daratech
PTY Ltd) mentions the use of killed Lawsonia intracellularis
bacteria to immunize pigs. However, systemic administration is not
mentioned. Based on the current knowledge that vaccination is only
effective upon oral administration, it is commonly understood that
the oral route was the administration route chosen for the
experiments described in the Daratech application. Another patent
application that mentions killed bacteria is WO 2005/011731
(Boehringer Ingelheim). However, actually disclosed is only the use
of a live vaccine administered orally. It is not shown that a
killed vaccine may be effective, let alone that the killed vaccine
can be given systemically. EP 843 818 (Boehringer Ingelheim)
describes the intramuscular administration of a killed vaccine
(paragraph [0115] in combination with paragraph [0119]). In par
[0115] it is stated that the bacteria were killed by storing them
at 4.degree. C. at normal atmospheric conditions. As is commonly
known however, under such conditions Lawsonia intracellularis
bacteria survive. Thus, this document does not teach the subject
matter of the present invention. 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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).
[0014] In an embodiment, the vaccine further comprises antigens of
Mycoplasma hyopneumoniae and Porcine circo virus. 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.
[0015] The invention will be further explained using the following
examples.
[0016] Example 1 describes a method to obtain a substantially
protein free carbohydrate containing composition and a vaccine that
is made by using this composition.
[0017] Example 2 describes an experiment wherein a second vaccine
according to the present invention is compared with the vaccine
currently on the market and an experimental vaccine comprising
subunit proteins of Lawsonia intracellularis.
[0018] Example 3 describes an experiment wherein two different
vaccines according to the present invention are compared with the
vaccine currently on the market.
[0019] Example 4 describes an experiment wherein a dosage affect of
a vaccine according to the invention is established.
EXAMPLE 1
[0020] 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-oligosaccharde, or even non-lipo
polysaccharides).
[0021] Carbohydrate Fraction for Vaccine Preparation
[0022] 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. 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.
[0023] 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 8M 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=4w)
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=6w 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=9w 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 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=6w.
[0032] At T=0, 4, 6, 7, 8 and 9w 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 was used to evaluate the statistical
significance (tested two-sided and level of significance set at
0.05).
RESULTS OF EXAMPLE 2
[0034] Serology
[0035] 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.RTM. 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
[0036] Real-Time PCR on Faeces Samples
[0037] 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 = Total post- Group 6 weeks T
= 7 weeks T = 8 weeks T = 9 weeks 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
[0038] Histology Scores
[0039] 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
[0040] From the results it can be concluded that systemic
administration of 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
[0041] This experiment was conducted to test a vaccine comprising a
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
[0042] The vaccine based on a substantially protein free
carbohydrate containing composition was obtained as described under
Example 1.
[0043] 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.
[0044] 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.
[0045] The other issues in the experimental design were the same as
described in Example 2, unless indicated otherwise.
RESULTS OF EXAMPLE 3
[0046] Serology
[0047] Before first vaccination all pigs were seronegative for IFT
Lawsonia 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
[0048] 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, comparable to those obtained with the commercially
available vaccine.
[0049] 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 commercially available
vaccines.
[0050] Real-Time PCR on Faeces Samples
[0051] 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 5.
TABLE-US-00006 TABLE 5 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
[0052] Histological Scores
[0053] 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 6). 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-00007 TABLE 6 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
[0054] From the results it can be concluded that systemic
administration of the whole cell Lawsonia bacterin combined with
PCV and Mhyo antigen as well as the vaccine comprising
(substantially protein-free) the carbohydrate, administered at
3-day-old and 25-day-old, both induce partial protection against
experimental Lawsonia intracellularis infection. It is particularly
surprising that the vaccine is effective when the prime
administration takes place before weaning (younger than 21-25
days). It is noted that in the examples 2 and 3 the vaccines as far
as Lawsonia antigens are concerned, per ml contain antigenic
material derived from more than 1 E8 Lawsonia intracellucaris
cells. Given the fact that these vaccines, even though mild
adjuvants are being used (viz. adjuvants containing small droplets
and no or little mineral oil), confer good protection against
ileitis, in particular when compared to the commercially available
vaccine Enterisol.RTM. Ileitis, the dose of antigens could be
lowered. This could be done by administering less vaccine (down to
e.g. 0.2 ml, suitable for e.g. intradermal application), or
decreasing the antigenic content of the vaccine. Based on analogues
in vaccine technology it is believed that with an antigenic dose
(per vaccination) derived from or containing 1 E7 cells, in
particular 2.5E7 cells or higher, still comparable or even better
results can be obtained than with the current commercially
available vaccine. Given the fact that the combination vaccine
provided titres for Mhyo and PCV antibodies to a level comparable
with the levels obtainable with commercially available single
vaccines, it is understood that the combination vaccine also
provides protection against Mycoplasma hyopneumoniae and Porcine
circo virus.
EXAMPLE 4
[0055] This experiment was conducted to establish a dosage affect
of a vaccine according to the invention. Also in this experiment
unvaccinated animals were used as a control.
EXPERIMENTAL DESIGN OF EXAMPLE 4
[0056] Inactivated whole cell vaccines were made as indicated in
example 2. The antigenic material was formulated in Diluvac forte
adjuvant at a concentration of approximately 2.0.times.10.sup.8
cells per ml vaccine, respectively 5.0.times.10.sup.7 and
1.25.times.10.sup.7 cells per ml vaccine. Sixty 3-day-old SPF
piglets were used. The pigs were allotted to four groups of 15 pigs
each. The piglets of groups 1, 2 and 3 were vaccinated
intramuscularly (in the neck) at 3-day old and 25-day-old with 2 ml
of the vaccine each time. Group 4 was left as unvaccinated control.
At 46-day-old all pigs were challenged orally with Lawsonia
bacteria as indicated under example 2. At 67-day-old all pigs were
euthanized and examined. Tests were performed as indicated under
example 2. Next to this PCR was peformed on mucosa samples. For
this, an ileum sample was taken from every animal, where applicable
from an area which showed thickening.
RESULTS OF EXAMPLE 4
[0057] Weight Gain
[0058] From 14 days and onwards significant differences in total
weight gain appeared among the groups. Group 1 showed an average
total weight gain of approximately 5350 grams. In group 2 this was
5150 grams. Group 3 showed a weight gain of about 4250 grams,
whereas Group 4 showed a weight gain of 4550 grams.
[0059] Real-Time PCR on Faeces Samples
[0060] Three weeks after challenge positive reactions were found in
all groups. Group 1 and Group 2 had a significantly lower shedding
level compared to the control. A post-challenge overview of the
number of infected animals (as determined by PCR) is given in table
7.
TABLE-US-00008 TABLE 7 Result of PCR on faeces samples after
vaccination and challenge Number of infected Group animals
post-challenge 1 1/15 2 2/15 3 7/15 4 8/15
[0061] Real-Time PCR on Mucosa Samples
[0062] Three weeks after challenge positive reactions were found in
all groups. Group 1 and Group 2 had a significantly lower shedding
level compared to the control. A post-challenge overview of the
number of infected animals (as determined by PCR) is given in table
8.
TABLE-US-00009 TABLE 8 Result of PCR on mucosa samples after
vaccination and challenge Number of infected Group animals
post-challenge 1 0/15 2 2/15 3 5/15 4 6/14 (no sample of pig no
8)
[0063] Histology Scores
[0064] The total histology score and the number of animals which
were confirmed to have PPE are depicted in table 9.
TABLE-US-00010 TABLE 9 Mean histology score for the ileum. Total
Number of animals Group score with PPE 1 0.3 0/15 2 0.8 2/14 3 1.1
4/15 4 1.9 7/15
CONCLUSION OF EXAMPLE 4
[0065] Contrary to what was expected, the results indicate that
there is a very sudden decrease in protective effect around the
lowest dosage used in these experiments. Although a dosage of
antigenic material derived from 2.5.times.10.sup.7 cells still
provided a protective effect comparable with that of the
commercially available vaccine, the fact the decrease between a
dosage that is only 0.6 log higher is so significant (hardly any
effect seen in weight gain, number of infected animals and PCR on
mucosa; however, still a decrease in number of PPE recognized
animals), gave the insight that in general a practical lowest
effective dose of the antigen can be derived at: an amount of
antigens less than derived from or containing 1.times.10.sup.7
cells will in practice, under the current market circumstances, not
lead to economically relevant results. The reason for the existence
of this apparent cut-off value is not 100% clear. Usually one
expects a more gradual decrease in protection when the dosage is
lowered. It might be that combating a local infection in the mucosa
of the intestines via a systemically derived immune response needs
a minimum amount of antigens.
[0066] Next to the above, the surprising effect seen in Example 3,
viz. that a vaccine based on a carbohydrate antigen administered
systemically, is effective when the prime administration takes
place before weaning (younger than 21-25 days), is confirmed in
this experiment with the use of another adjuvant. Therefore it can
reasonably be understood that this feature is generic for a
non-live vaccine comprising a carbohydrate antigen.
* * * * *