U.S. patent application number 10/731724 was filed with the patent office on 2004-06-24 for use of bacterium for manufacture of a vaccine.
Invention is credited to Goovaerts, Danny, Jacobs, Antonius Arnoldus Christiaan.
Application Number | 20040120970 10/731724 |
Document ID | / |
Family ID | 8239828 |
Filed Date | 2004-06-24 |
United States Patent
Application |
20040120970 |
Kind Code |
A1 |
Jacobs, Antonius Arnoldus
Christiaan ; et al. |
June 24, 2004 |
Use of bacterium for manufacture of a vaccine
Abstract
The present inventon relates to the use of live attenuated
bacteria for the manufacture of a vaccine for submucosal
administration.
Inventors: |
Jacobs, Antonius Arnoldus
Christiaan; (Kessel, NL) ; Goovaerts, Danny;
(Lichtaart, BE) |
Correspondence
Address: |
INTERVET INC
405 STATE STREET
PO BOX 318
MILLSBORO
DE
19966
US
|
Family ID: |
8239828 |
Appl. No.: |
10/731724 |
Filed: |
December 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10731724 |
Dec 8, 2003 |
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09492206 |
Jan 27, 2000 |
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6682745 |
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Current U.S.
Class: |
424/200.1 |
Current CPC
Class: |
A61K 39/05 20130101;
A61P 11/00 20180101; A61K 39/092 20130101; A61K 2039/541 20130101;
A61P 17/02 20180101; A61P 15/06 20180101; A61P 43/00 20180101; A61K
2039/522 20130101; Y10S 424/82 20130101; Y10S 424/829 20130101;
A61P 31/04 20180101 |
Class at
Publication: |
424/200.1 |
International
Class: |
A61K 039/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 26, 1999 |
EP |
99200202.2 |
Claims
1) Use of a live attenuated bacterium for the manufacture of a
vaccine for submucosal administration.
2) Use according to claim 1, characterised in that the vaccine is
for administration in the submucosa of the labiae.
3) Use according to claim 1 or 2, characterised in that the live
attenuated bacterium is an attenuated form of a horse pathogenic
bacterium.
4) Use according to claim 3,characterised in that the live
attenuated bacterium is selected from the group of bacteria
comprising Streptococcus equi, Streptococcus zooepidemicus,
Rhodococcus equi, Corynebacterium pseudotuberculosis, Pseudomonas
mallel, Actinobacillus equili and Pasteurella multocida.
5) Use according to claim 4, characterised in that the live
attenuated bacterium is of the species Streptococcus equi and/or
Streptococcus zooepidemicus.
Description
[0001] The present invention relates to the use of bacteria for the
manufacture of vaccines. Vaccination has been proven through the
years to be a very efficient method for the prevention of diseases
caused by many different bacteria. Vaccines have the advantage,
contrary to e.g. antibiotic or pharmacochemical therapies, that
they are preventing disease rather than curing it. In many fields,
e.g. the field of animal husbandry, vaccination is a standard
routine. Usually, all animals in a group are vaccinated as a
precautionary measure, in order to prevent disease, whereas in
practice often only a few animals would have become infected if no
vaccine had been given. This explains why for most commonly used
vaccines adverse local reactions due to vaccination are not
acceptable: it is not acceptable to cause (severe) physical stress
in many animals to prevent a (mild) disease in few.
[0002] Nevertheless, for most vaccines, especially for the live
vaccines that are in most cases preferable to inactivated vaccines,
there is a delicate balance between a sufficiently strong
triggering of the immune system on the one hand and acceptable
local reactions at the site of administration of the vaccine on the
other hand. As a rule of thumb, the best live vaccine gives the
most severe local reactions, and therefore local reactions are
often unavoidable if efficacious protection is needed.
[0003] It is an object of the present invention to provide ways to
diminish the problem of local reactions of live vaccines without
further attenuating the live vaccines.
[0004] It was surprisingly found now that when live attenuated
bacteria are used for the preparation of a vaccine for
administration to submucosal tissue, the thus obtained vaccine when
applied submucosally gives good protection and minor local
reactions.
[0005] This invention is widely applicable in the field of
manufacture of systemic vaccines. It is not restricted to any
specific bacterium or a specific disease. Practically all live
attenuated bacteria that are suitable for the manufacture of a live
attenuated vaccine for systemic application are equally suitable
for use in this specific invention. Systemic application comprises
all applications in which the vaccine is not applied to the mucosa
(mucosal application comprises i.a. oral and intranasal
vaccination). Systemic application routes comprise i. a.
intramuscular application (IM), subcutaneous application (SC),
intradermal vaccination (ID), intravenous vaccination (IV) and
intraperitoneal vaccination (IP).
[0006] Of these routes, intramuscular vaccination is in many cases
the preferred application route. This is due to the fact that the
vaccine, possibly mixed with an adjuvant, is only slowly released
from the site of injection. Thus, the immune system is continuously
triggered for a relatively long time with an immunogenic dose of
the vaccine. This way of administration ensures an adequate immune
response. The disadvantage however is, that many bacterial IM
administered vaccines cause large abscesses at the site of
injection. These abscesses may stay there from days to months. In
those cases in which a live attenuated bacterium must behave
relatively virulent in order to trigger an adequate immune
response, the bacterium often replicates at the injection site to
such a level that the abscess even bursts. Large intramuscular or
skin-abscesses are clearly an unacceptable side-effect of
vaccination with bacterial live attenuated strains, but unavoidable
if further attenuation spoils the immunogenic potential of the
bacterium. This causes the dilemma mentioned above, for which the
invention offers a solution.
[0007] It is certainly unexpected that such soft and vulnerable
tissue as submucosal tissue allows the administration of (sometimes
even hardly) attenuated live bacterial vaccines:
[0008] a) without giving the unacceptable abscesses seen with
intradermal or intramuscular application, while
[0009] b) at the same time allowing a sufficient immune response to
be build up.
[0010] This is even more unexpected if the level of damage is
considered, that many relatively virulent attenuated bacteria cause
to their host when given ID or IM. Intradermal or intramuscular
vaccination with such bacteria often causes, next to the formation
of abscesses, severe lesions at the injection site. The tissue
around the injection site often completely disintegrates, leaving
large scars.
[0011] All these disadvantages are hardly or not seen with the uses
according to the invention.
[0012] Therefore this embodiment of the invention relates to the
use of live attenuated bacteria for the manufacture of a vaccine
for submucosal administration. Mucosal tissue is found i.a. in the
mouth, the nose, the lining of the gut, the eye, the vulva and the
lips.
[0013] Submucosal application is understood to be administration
through the upper layer of the mucosa, and into the submucosa. The
submucosa is a well-defined layer, known as such in the art. In
principle, there is no limit to depth at which vaccination takes
place (i.e. the depth of the tip of the needle), with of course the
proviso that vaccination takes place in the submucosa. In practice
however, the vaccine would not likely be applied deeper than about
5 millimetres from the surface of the mucosa. Generally spoken,
smaller distances between the mucosa and the injection site gives
smaller local effects. A very suitable depth would be in the
submucosa between two and four millimetres below the mucosa.
[0014] Another attractive way of application is by using a
so-called needle-less injector. The use of these injectors is known
from intradermal applications, but these injectors are equally
suitable for submucosal applications. Due to the softness of
mucosal tissue the vaccine, when applied through a needle-less
injector, goes straight through the mucosa and will come to a halt
in the submucosal tissue. The depth of the vaccination only depends
on the power applied during administration.
[0015] In principle, all submucosal tissue is suitable for
submucosal application. In practice however, the submucosal tissue
of the lips and, in female animals, the vulva are very practical
sites of administration. Especially in horses, dogs and cattle the
submucosal tissue of the lips would be the preferable site of
administration.
[0016] Therefore, in a preferred form, the live attenuated bacteria
are used for the manufacture of a vaccine for administration in the
submucosa of the labiae.
[0017] As mentioned above, practically all live attenuated bacteria
that are suitable for the manufacture of a live attenuated vaccine
for systemic application are suitable for use in this specific
invention. There are many important pathogenic bacteria for which
the use according to the invention means a great improvement in
safety, where the severity of local reactions is concerned. Below,
a list of bacteria is presented, all known to cause abscess
formation and thus severe tissue damage and skin lesions, when
administered intramuscularly. And for all these bacteria there is a
reciprocal relation between the decreased immunogenic potential
after attenuation on the one hand, and the acceptability of local
reactions at the site of administration on the other hand. The
invention applies e.g. to the use of live attenuated bacteria that
are attenuated forms of horse pathogenic bacteria.
[0018] The following bacteria are examples of the large family of
well-established horse pathogenic bacteria:
[0019] Streptococcus equi, the cause of "Strangles". This disease
causes abscesses of lymph nodes of head and neck and systemic
infections. The swelling of the lymph nodes causes the horses to be
suffocated. No reliable vaccine without adverse local reactions is
known so far, Streptococcus zooepidemicus, causing respiratory
tract infections and pneumonia, opportunistic infections and
abortion in horses, Rhodococcus equi, causing bronchopneumonia with
abscesses and intestinal abscesses, Corynebacterium
pseudotuberculosis, causing pectoral abscesses and ulcerative
lymphangitis, Pseudomonas mallel, causing: "Glanders", a disease
characterised by pyogranulomatous inflammations, nodular lesions in
lung and ulcerative and nodular lesions in skin and respiratory
mucosa, Actinobacillus equili, a well-known cause of neonatal
death, abortion in mares, stillbirth and foal septicaemia and
finally Pasteurella multocida, causing respiratory tract infections
in horses
[0020] Horses have in many cases both a high emotional and
economical value to their owners, Especially in the field of
thoroughbreds, it would be unacceptable to have horses suffering
from abscesses after vaccination.
[0021] Therefore, in a more preferred form of the invention the use
relates to a use where the live attenuated bacterium is an
attenuated form of a horse pathogenic bacterium.
[0022] In an even more preferred form, the live attenuated
bacterium is selected from the group of bacteria comprising
Streptococcus equi, Streptococcus zooepidemicus, Rhodococcus equi,
Corynebacterium pseudotuberculosis, Pseudomonas mallel,
Actinobacillus equili and Pasteurella multocida.
[0023] In a still even more preferred form, the live attenuated
bacterium is of the species Streptococcus equi and/or Streptococcus
zooepidemicus.
[0024] The invention is equally applicable to a live attenuated
bacterium that is an attenuated form of a bacterium that is
pathogenic for cattle.
[0025] The following list gives a number of examples of frequently
encountered pathogens in cattle:
[0026] Actinomyces pyogenes, Staphylococcus aureus, Streptococcus
agalactiae and Streptococcus uberis, Noccardia asteroides,
Corynebacterium bovis, Mycoplasma bovis, and Mycobacterium bovis,
all well-established causes of bovine mastitis, Escherichia coli,
causing both bovine mastitis and diarrhoea, Pasteurella haemolytica
and P. multocida, both causing pneumonia and septicaemia, Brucella
abortus, causing abortion, Salmonella dublin and S. typhimurium,
causing diarrhoea, pneumonia and systemic infections and finally
Leptospira hardjo as a cause of urinary tract infections.
[0027] The invention also applies to a live attenuated bacterium
that is an attenuated form of a bacterium that is pathogenic for
pigs.
[0028] The following list gives a few examples of pig-pathogenic
bacteria
[0029] Streptococcus suis causing polyserositis, Staphylococcus
aureus causing exudative epidermitis, Actinobacillus
pleurnopneumoniae causing pleuropneumonia, Pasteurella multocida
causing atrophic rhinitis and pneumonia, Bordetella bronchiseptica
also causing atrophic rhinitis and pneumonia, Escherichia coli
causing diarrhoea and edema disease, Clostridium perfringens as a
cause of diarrhoea and septicaemia, Salmonella cholerasuis also a
known cause of diarrhoea, Haemophilus parasuis also known as the
cause of "Glassers disease", Erysipelothrix rhusiopathiae causing a
disease known as "Erysipelas", Mycoplasma hyopneumoniae causing
pneumonia, Serpulina hyodysentriae as a cause of diarrhoea and
Leptospira pomona that gives abortion.
[0030] Also, the invention applies to a live attenuated bacterium
that is an attenuated form of a bacterium that is pathogenic for
dogs.
[0031] Examples of such bacteria are inter alia the following
bacterial dog pathogens:
[0032] Staphylococcus aureus, pyoderma, Streptococcus pneumoniae,
septicaemia Bordetella bronchiseptica, tracheobronchitis,
Eschenchia coli, diarrhoea Leptospira canicola and
icterohaemorrhagiae, general and urinary tract infections.
[0033] The manufactured vaccines comprise at least an
immunogenically effective amount of a live attenuated bacterium.
Immunogenically effective means that the amount of live attenuated
bacterium administered at vaccination is sufficient to induce in
the host an effective immune response to virulent forms of the
bacterium.
[0034] The useful dosage to be administered will vary depending of
age, weight and mammal to be vaccinated and the type of pathogen
against which vaccination is sought. The vaccine may comprise any
dose of bacteria sufficient to evoke an immune response. Doses
ranging between 10.sup.3 and 10.sup.10 bacteria are e.g. very
suitable doses.
[0035] In addition to an immunogenically effective amount of the
live attenuated bacterium described above, the manufactured vaccine
also contains a pharmaceutically acceptable carrier. Such a carrier
may be as simple as water, but it may e.g. also comprise culture
fluid in which the bacteria were cultured. Another suitable carrier
is e.g. a solution of physiological salt concentration. Other
examples of pharmaceutically acceptable carriers or diluents useful
in the present invention include stabilisers such as SPGA,
carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose,
dextran), proteins such as albumin or casein, protein containing
agents such as bovine serum or skimmed milk and buffers (e.g.
phosphate buffer).
[0036] Optionally, one or more compounds having adjuvant activity
may be added to the vaccine. Adjuvants are non-specific stimulators
of the immune system. They enhance the immune response of the host
to the invading pathogen. Examples of adjuvants known in the art
are Freunds Complete and Incomplete adjuvants, vitamin E, non-ionic
block polymers, muramyldipeptides, ISCOMs (immune stimulating
complexes, cf. for instance European Patent EP 109942), Saponins,
mineral oil, vegetable oil, and Carbopol (a homopolymer). Other
suitable adjuvants are for example aluminium hydroxide, phosphate
or oxide, oil-emulsions (e.g. of Bayol F.sup.(R) or Marcol
52.sup.(R), saponins or vitamin-E solubilisate.
EXAMPLES
EXAMPLE 1
[0037] Comparison of Safety of Intramuscular and Submucosal
Administration of Two Different Attenuated Streptococcus equi
Strains.
[0038] In this experiment the safety and efficacy of live S. equi
strain TW 928 deletion mutant vaccine and of strain TW 928/sls
double mutant vaccine in Diluvac Forte.RTM. (obtainable through
Intervet Int. B. V., P.O.Box 31, 5830 AA Boxmeer, The Netherlands),
both administered submucosally in the lip, were tested. A
comparison with the safety of a similar intramuscular vaccination
has been made.
[0039] After a 2 weeks acclimatisation period, 5 horses were
vaccinated submucosally in the lip with strain TW 928 deletion
mutant. Vaccination was done at 2 spots in the upper lip and 2
spots lower lip. A needle was used that was provided with a disc of
about 1 centimetre diameter, attached at right angles to the
needle, and located at about 2.5 millimetres from the tip of the
needle. This prevented the tip of the needle to enter the submucosa
for more than about 2 millimetre.
[0040] A volume of 200 .mu.I of the vaccine, comprising 10.sup.8.8
bacteria was given at each spot. Three other horses were vaccinated
subcutaneously in the same way, but with a double mutant: strain TW
928/sls comprising 10.sup.8.2 bacteria in Diluvac Forte.
[0041] Three horses were vaccinated IM in the neck with comparable
doses of the TW 928 deletion mutant strain.
[0042] Two horses were left as controls.
[0043] At 4 weeks after priming vaccination the vaccinates were
boosted as described above with the same amount of bacteria at
similar vaccination sites. At 2 weeks after booster vaccination,
all horses were challenged intranasally with 7.7.times.10.sup.8 CFU
of the challenge strain S. equi strain Arnica in a 2 ml volume.
After vaccination the horses were observed for any systemic or
local reactions and after challenge, the horses were examined for
clinical signs of strangles or any other abnormality.
[0044] Results:
[0045] Horses subjected to intramuscular vaccination in the neck
developed large abscesses that reached diameters ranging between 10
and 30 centimetres within weeks after vaccination. These abscesses
were persistent and kept growing until they burst. Horses subjected
to submucosal vaccination appeared in a good condition and had a
normal appetite and no significant further systemic reactions were
observed.
[0046] After submucosal priming and boosting with the 928 deletion
mutant only small and translent local reactions were found. Most
reactions had disappeared at 3 weeks after priming vaccination and
at 2 weeks after booster vaccination. The same minor local
reactions, but to an even lesser extend, were observed after both
vaccinations with the 928/sls double mutant.
[0047] After challenge, the five horses vaccinated submucosally
with the TW 928 deletion mutant appeared completely protected.
Complete protection was also obtained in the horses vaccinated
intramuscularly with the TW 928 deletion mutant.
[0048] Therefore it can be Concluded That
[0049] full protection can be obtained with suitable vaccine
strains regardless the site of administration; intramuscularly or
submucosally.
[0050] hardly any adverse local reactions are found at the site of
submucosal administration, whereas intramuscular administration
causes large persistent abscesses at the site of
administration.
Example 2
[0051] Comparison of Submucosal and Intramuscular Administration of
A Strain of the Horse Pathogenic Bacterium Streptococcus
zooepidemicus
[0052] In this experiment the safety of submucosal administration
of Strep. z. was compared to that of intramuscular administration,
especially with respect to adverse local reactions.
[0053] Two horses were vaccinated submucosally in the lip with
7.times.10.sup.7 CFU Strep. z. in a total volume of 0.2 ml. Two
other horses were vaccinated intramuscularly in the neck with the
same dose, but in a total volume of 1 ml.
[0054] Results:
[0055] The intramuscularly vaccinated horses developed large
abscesses from the fourth day after vaccination, that grew to an
average size, at ten days after vaccination, of about 20
centimetres diameter. These abscesses were persistent.
[0056] The submucosally vaccinated horses only developed minor
abscesses with an average size of 2.5 centimetres, beginning at day
5 after vaccination. The abscesses completely disappeared after six
days, leaving no traces behind.
Example 3
[0057] Comparison of Submucosal and Intramuscular Administration of
a Virulent Strain of the Bovine Pathogenic Bacterium Actinomyces
pyogenes.
[0058] In this experiment the safety of submucosal administration
of A. pyogenes was compared to that of intramuscular
administration, especially with respect to adverse local
reactions.
[0059] Two cows were vaccinated submucosally in the vulva with
1.2.times.10.sup.10 CFU in a total volume of 0.2 ml. Two other cows
were vaccinated intramuscularly in the neck with the same dose, but
in a total volume of 1 ml.
[0060] Results:
[0061] In the submucosally vaccinated animals, small abscesses
developed after three days, reaching an average size of about 3.5
centimetres diameter. These abscesses decreased in size after a few
days.
[0062] In the two cows vaccinated intramuscularly in the neck with
the same dose, large and more persistent abscesses developed after
three days, reaching a diameter of between 9 and 14
centimetres.
* * * * *