U.S. patent application number 12/335152 was filed with the patent office on 2009-06-25 for heat treated bacterins, and emulsion vaccines prepared from such heat treated bacterins.
This patent application is currently assigned to PFIZER INC.. Invention is credited to Mark D. Goodyear, Michael J. Huether, Richard Lee Krebs, Nancee L. Oien.
Application Number | 20090162397 12/335152 |
Document ID | / |
Family ID | 40514058 |
Filed Date | 2009-06-25 |
United States Patent
Application |
20090162397 |
Kind Code |
A1 |
Goodyear; Mark D. ; et
al. |
June 25, 2009 |
HEAT TREATED BACTERINS, AND EMULSION VACCINES PREPARED FROM SUCH
HEAT TREATED BACTERINS
Abstract
Heat treated bacterins, a method of producing heat treated
bacterins, and porcine emulsion vaccines prepared from such heat
treated bacterins are disclosed.
Inventors: |
Goodyear; Mark D.; (Portage,
MI) ; Huether; Michael J.; (Lincoln, NE) ;
Krebs; Richard Lee; (Ashland, NE) ; Oien; Nancee
L.; (Kalamazoo, MI) |
Correspondence
Address: |
PHARMACIA & UPJOHN
7000 Portage Road, KZO-300-104
KALAMAZOO
MI
49001
US
|
Assignee: |
PFIZER INC.
New York
NY
|
Family ID: |
40514058 |
Appl. No.: |
12/335152 |
Filed: |
December 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61015718 |
Dec 21, 2007 |
|
|
|
Current U.S.
Class: |
424/201.1 |
Current CPC
Class: |
A61K 39/0225 20130101;
A61K 2039/55566 20130101; A61K 2039/552 20130101; A61K 38/00
20130101; A61P 31/12 20180101; A61P 1/12 20180101; A61P 43/00
20180101; A61K 39/39 20130101; A61K 2039/521 20130101; A61P 31/00
20180101; C07K 14/005 20130101; A61P 37/04 20180101; A61P 31/04
20180101; A61K 2039/70 20130101; A61K 2039/55505 20130101; A61K
39/295 20130101 |
Class at
Publication: |
424/201.1 |
International
Class: |
A61K 39/295 20060101
A61K039/295 |
Claims
1. A vaccine comprising an emulsion, a heat treated bacterin
comprising a suspension of killed bacteria, wherein the killed
bacteria are of the species Leptospira Bratislava and 1 to 13
porcine disease causing viruses selected from the group consisting
of Porcine Adenovirus, Porcine Circovirus, Porcine herpes viruses,
Pseudorabies virus, Classical swine fever virus, Porcine epidemic
diarrhea virus, Porcine hemaglutinating encephalomyelitis virus,
Porcine parvovirus, Porcine Respiratory Corona virus, Porcine
Reproductive and Respiratory Virus, Swine Influenza, Transmissible
gastroenteritis virus, and Vesicular stomatitis virus.
2. A vaccine according to claim 1 further comprising a lecithin
preparation, and alum based adjuvant.
3. A vaccine according to claim 2 further comprising a lecithin in
oil preparation.
4. A vaccine according to claim 1 further comprising heat treated
bacterins comprising a suspension of killed bacteria, wherein the
killed bacteria are one to six bacteria species selected from the
group consisting of Leptospira canicola, Leptospira
icterohaemorrhagiae, Leptospira grippotyphosa, Leptospira hardjo,
Leptospira Pomona, and Erysipelothrix rhusiopathieae.
5. A vaccine according to claim 4 further comprising a lecithin
preparation, and alum based adjuvant.
6. A vaccine according to claim 5 further comprising a lecithin in
oil preparation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority to U.S.
Provisional Application Ser. No. 61/015,718 filed Dec. 21,
2007.
FIELD OF INVENTION
[0002] This invention relates generally to the field of vaccines
and to methods of stabilizing emulsion vaccines. In particular,
this invention relates to heat treated bacterins, a method of
producing heat treated bacterins, and to porcine emulsion vaccines
prepared from such heat treated bacterins.
BACKGROUND OF THE INVENTION
[0003] Vaccination is increasingly used to control the infectious
diseases in animals. Adjuvants are frequently used in vaccines
because they are able to increase the humoral and/or cellular
immune response to an antigen. Vaccines are often formulated as
emulsions because the emulsion can act as an adjuvant, and has the
property of retaining the antigen as a depot at the site of
injection. Emulsifiers are commonly used in emulsion vaccines.
Besides using emulsifiers, the stability of the emulsion vaccines
may also be achieved through reducing the droplet size of the
emulsion by mechanical means.
[0004] U.S. Pat. No. 5,084,269 relates to an adjuvant formulation
containing lecithin in combination with mineral oil, which produces
less irritation within the host animal, and simultaneously induces
increased systemic immunity. Compositions according to U.S. Pat.
No. 5,084,269 are in commercial use under the trade name
AMPHIGEN.RTM., a trademark of Pfizer, Inc.
[0005] Generally, bacterial antigens are unstable when heated and
even brief exposure to elevated temperatures can reduce the
activity of the antigens. For example current anthrax vaccines can
lose all biological activity with 48 hours at 37.degree. C. (S.
Sing, N. Ahuja, V. Chauhan, E. Rajasekaran, W. S. Mohsin, R. Bhat,
and R. Bhatnagar, Bioche. Biophys. Res. Commun. 2002 Sep. 6;
295(5): 1058-62).
SUMMARY OF INVENTION
[0006] This invention relates to heat treated bacterins, a method
of producing heat treated bacterins, and porcine emulsion vaccines
prepared from such heat treated bacterins. The method comprises
heating the bacterin to a temperature of about 35 to about
80.degree. C. to form a heat treated bacterin.
DETAILED DESCRIPTION
Definitions
[0007] Acceptable antigenic activity--The term "acceptable
antigenic activity" means the ability to induce a protective immune
response in vaccinated animals after being challenged or by passing
a codified potency test with homologous live organism.
[0008] Bacterin--The term "bacterin" means a suspension of killed
bacteria which may be used as a component of a vaccine.
[0009] Emulsifier--The term "emulsifier" means a substance used to
make an emulsion more stable.
[0010] Emulsion--The term "emulsion" means a composition; of two
immiscible liquids in which small droplets of one liquid are
suspended in a continuous phase of the other liquid.
[0011] Heat treated bacterin--The term "heat treated bacterin"
means a bacterin that has been heat treated and which has a lipase
activity of 50% or less than the lipase activity before the heat
treatment, and has acceptable antigenic activity.
[0012] Invert Emulsion--The term "invert emulsion" means a water in
oil emulsion.
[0013] Lipase--The term "lipase" means enzymes, esterases, lipases,
and phospholipases, which can cause breakdown of an emulsifier in
an emulsion vaccine.
[0014] Normal Emulsion--The term "normal emulsion" means an oil in
water emulsion.
[0015] Oil in Water Emulsion--The term "oil in water emulsion"
means an emulsion in which small droplets of oil are suspended in a
continuous water phase.
[0016] Room Temperature--The term "room temperature" means a
temperature from 18 to 25.degree. C.
[0017] Water in Oil Emulsion--The term "water in oil emulsion"
means an emulsion in which droplets of water are suspended in a
continuous oil phase.
DESCRIPTION
[0018] This invention relates to bacterins with reduced lipase
activity, porcine emulsion vaccines prepared from such bacterins,
and a method of reducing the lipase activity of bacterins. In
addition to antigenic components, some bacterins have lipase
activity. When bacterins with lipase activity are incorporated into
an emulsion, the lipase may break down the emulsifiers used to
create the emulsion. Emulsion vaccines that contain bacterins
having high lipase activity tend to be unstable emulsions, and
those that contain bacterins having low levels of lipase tend to be
stable. Examples of bacteria which may, when killed, produce
bacterins having lipase activity include Erysipeothrix
rhusiopathieae, Listeria monocytogenes, Escherichia coli,
Mycoplasma hyopneumoniae, and Leptospira species, such as the known
pathogens Leptospira canicola, Leptospira grippotyposa, Leptospira
hardjo, Leptospira icterohaemorrhagiae, Leptospira bratislava and
Leptospira pomona. These bacteria can cause diseases in pigs, and
vaccination against these diseases is desirable. The Leptospira
bacterins are more likely to have a high lipase activity while an
Erysipelothrix rhusiopathieae bacterin may have a lower, more
manageable lipase activity.
[0019] The lipase, which can break down the emulsifiers used to
create the emulsion, and thus cause emulsion instability and
breakdown, may include one or more emulsion breaking enzymes such
as esterases, lipases, and phospholipases. Collectively these
enzymes, esterases, lipases, and phospholipases are referred to as
lipase. The lipase activity of a bacterin may be measured using a
synthetic substrate called O-pivaloyloxymethyl umbelliferone
(C-POM). The rate of hydrolysis caused by the lipase is the measure
of the lipase activity. The reaction rate of the hydrolysis caused
by the lipase in this reaction is monitored by an increase in the
fluorescence intensity of the product of the lipase activity. The
reaction rate is dependent upon the exact hydrolysis test
conditions chosen, so that comparisons of lipase activity levels,
as measured by hydrolysis rates, should be made using data produced
by the same test conditions. Literature methods are disclosed in
several articles, including Kurioka S. and Matsuda M. (1976) Ana.
Biochem. 75: 281-289, De Silva N. S. and Quinn P. A. (1987) J.
Clin. Microbiol. 25: 729-731 and Grau A. and Ortiz A. (1998) Chem.
Phys. of Lipids. 91: 109-118.
[0020] In an emulsion vaccine, the break down of the emulsion
causes phase separation of the components. This is undesirable
because when there is phase separation the individual doses removed
from the container may not contain the same level of the vaccine
components. In addition, the loss of emulsion can lead to a loss of
the adjuvant activity of the emulsifier and lead to a reduction in
the antigenic effect of the vaccine.
[0021] Attenuated live viruses are frequently included in vaccines
along with bacterins. Such vaccines are useful because a single
vaccine can be used to create immunity to different diseases with
one vaccine. If the lipase activity is present in the bacterin, it
will cause release of the emulsifier from the emulsion. This free
emulsifier can disrupt and inactivate the live vaccine viruses,
thereby leading to a loss of viral infectivity.
[0022] A bacterin useful in vaccines may be formed by culturing the
bacterium of interest, and then killing the bacteria to produce a
bacterin containing a variety of bacterial components, including
cell wall components. The bacteria may be killed by a variety of
methods including exposing them to a compound such as merthiolate,
formalin, formaldehyde, diethylamine, binary ethylenamine (BEI),
beta propiolactone (BPL), and glutaraldehyde. Combinations of these
compounds may be used. In addition, it is possible to kill the
bacteria with sterilizing radiation.
[0023] It has now been found that the lipase activity of a bacterin
having such lipase activity may be reduced by heat treatment.
Specifically, the lipase activity of a bacterin may be reduced by
heating the bacterin to a temperature of about 35 to about
80.degree. C. to form a heat treated bacterin, which has acceptable
antigenic activity. The heat treatment is conducted for a period of
time sufficient so that the lipase activity of the heat treated
bacterin is 50% or less than that found in the bacterin prior to
the heat treatment. For good emulsion vaccine stability it is not
necessary that the lipase activity be reduced to zero. We have
found that vaccines having a good shelf life may be prepared from
heat treated bacterins having lipase activity level that is 50% or
less than of the lipase activity level before the heat
treatment.
[0024] When a rate of hydrolysis of a test substrate has been used
as a measure of the lipase activity of a bacterin, then the rate of
hydrolysis of the test substrate before the heat treatment is
compared to the rate of hydrolysis after the heat treatment. The
heat treatment is conducted so as to reduce the rate of hydrolysis
to 50% or less than the rate of hydrolysis that is observed for the
fresh bacterin.
[0025] The exact method of measuring the lipase activity level is
not critical as long as the same method is used to measure the
activity before the heat treatment and the activity after the heat
treatment. For example, if the rate of hydrolysis of a test
substrate is measured using one substrate, a different substrate
might produce a different rate. However, if the same substrate is
used for the initial activity determination and the activity
determination after treatment, the relative rates will still show
the effect of the heat treatment.
[0026] There are codified tests for antigenic activity for
Leptospira Pomona Bacterin (9 CFR .sctn. 113.101), Leptospira
Icterohaemorrhagiae Bacterin (9 CFR .sctn.113.102), Leptospira
Canicola Bacterin (9 CFR .sctn.113.103), Leptospira Grippotyphosa
Bacterin (9 CFR .sctn.113.104), and Leptospira Hardjo Bacterin (9
CFR .sctn.113.105) (9CFR .sctn.113.101, .sctn.113.102,
.sctn.113.103, .sctn.113.104, and .sctn.113.105). For these species
acceptable antigenic activity may be defined as the ability to
induce a protective immune response in vaccinated hamsters such
that when hamsters are challenged with homologous live bacteria, at
least 75% of the vaccinated hamsters survive in a model where at
least 80% of the non-vaccinated hamsters do not survive. In the
case of the antigen, Leptospira hardjo, acceptable antigenic
activity may be defined as the ability of a vaccine to induce a
serological agglutination geometric mean titer against Leptospira
hardjo of .gtoreq.40 in calves that have been vaccinated with a
vaccine comprising the bacterial antigen, Leptospira hardjo. For
other bacterins acceptable antigenic activity is defined as the
ability to induce a protective immune response in vaccinated
animals after being challenged or by passing a potency test with
homologous live organism.
[0027] The heat treatment may be conducted over a range of
temperatures, and for a variable length of time. Generally, the
heating may be done at a temperature of about 35 to about
80.degree. C. for about 20 minutes to about 24 hours. When the
bacterin is heated to a higher temperature, such as about 75 to
about 80.degree. C., the time of heating is at the short end of the
time range. When the heating is done at a lower temperature, the
heating is done for a longer period of time. Another combination of
temperature and time is heating at a temperature of about 60 to
about 70.degree. C. for about 9 to about 10 hours. Another
combination of temperature and time is heating at a temperature of
about 65 to about 70.degree. C. for about 5 to about 8 hours.
Another combination of temperature and time is heating at a
temperature of about 65 to about 70.degree. C. for about one hour.
Another combination of temperature and time is heating at a
temperature of about 55 to about 65.degree. C. for about 5 to about
8 hours.
[0028] The bacterins, after the heat treatment, have a lower lipase
activity than freshly prepared bacterins but otherwise may be
formulated in the same manner as freshly prepared bacterins.
Accordingly, the heat treated bacterins may be incorporated into
vaccines by ordinary methods of producing vaccines. These methods
are well known in the art.
[0029] Emulsion vaccines may be formed by combining the desired
bacterin with an oils phase and an emulsifier, or emulsifiers. The
combination is then subjected to intense agitation to form an
emulsion. Suitable agitation methods include homogenizing and
subsequently microfluidizing. Preservatives and excipients may also
be included in the combination prior to emulsification.
[0030] Vaccines may include both bacterins and viral antigens. In
preparing a vaccine that includes bacterins and viral antigens, the
bacterins, any viral antigens to be included, the emulsifier, or
emulsifiers and optionally preservatives and excipients are
combined with an oil phase, and emulsified. Following emulsion
formation, the pH of the formulations may be adjusted to an
appropriate pH using either solutions of NaOH or HCl. For vaccine
use, it is generally desirable that the pH be close to neutral to
avoid irritation at the injection site. A pH of about 7.0 to about
7.3 is common.
[0031] Suitable oil phases for emulsion vaccine formation include
non-metabolizable oils and metabolizable oils. The
non-metabolizable oils include mineral oils, such as white mineral
oil, and light mineral oil. The metabolizable oils include
vegetable oils, fish oils and synthetic fatty acid glycerides.
[0032] Examples of emulsifiers that may be used in preparing
emulsion vaccines of this invention are phospholipids, sorbitan
esters, polyethoxylated sorbitan esters, and mannitol derivatives
which are common vaccine emulsifiers. Phospholipid emulsifiers
include lecithin, phosphatidylethanolamine, phosphatidylinisitol,
phosphatidylserine, and lecithin, (e.g. such as AMPHIGEN.RTM.).
Sorbitan ester emulsifiers include sorbitan monolaurate, (e.g.
Span.RTM. 20 and Arlacel.RTM. 20), sorbitan monooleate (e.g.
Span.RTM. 80 and Arlacel.RTM. 80), sorbitan monopalmitate (e.g.
Span.RTM. 40 and Arlacel.RTM. 40), and sorbitan monostearate (e.g.
Span.RTM. 60 and Arlacel.RTM. 60). Polyethoxylated sorbitan esters
include polyethoxy sorbitan monolaurate (e.g. Tween.RTM. 20 and
Tween.RTM. 21), polyethoxy sorbitan monooleate (e.g. Tween.RTM.
80), polyethoxy sorbitan monopalmitate (e.g. Tween.RTM. 40), and
polyethoxy sorbitan monostearate (e.g. Tween.RTM. 60). Mannitol
derivative emulsifiers include mannitol octadecanoic ethers
Span.RTM., Arlacel.RTM., and Tween.RTM. are trademarks of ICI
Americas. AMPHIGEN.RTM. is a trademark of Pfizer, Inc. Generally,
vaccines are formulated as normal oil in water emulsions, although
it is possible to prepare invert water in oil emulsions.
[0033] A variety of adjuvants, such as Quil A, cholesterol,
aluminum phosphate, and aluminum hydroxide, and preservatives such
as merthiolate may be used in vaccines. Quil A is purified mixture
of quillaja saponins extracted from the bark of the South American
tree Quillaja Saponaria Molina. Quil A acts directly on the immune
system to activate a generalized state of sensitivity. In doing so,
it induces both humoral and cell-mediated responses. The lipophilic
chain allows interaction of antigen and adjuvant to be delivered
into the cytosol for processing in an endogenous pathway. Quil A is
often used with cholesterol because cholesterol eliminates the less
desirable side effects when added in the appropriate proportions.
Cholesterol forms insoluble complexes with Quit A that form
helix-like structures as the cholesterol binds with Quil A, thus
exposing the molecule's sugar units that help stimulate the immune
response.
[0034] It is common to add porcine viral antigens to vaccines
containing bacterins. One advantage of this approach is that one
vaccine may be used to create immunity to several diseases instead
of requiring dosages of several different vaccines to achieve the
same result. Both killed viruses and attenuated live viruses may be
used in vaccines. Among the porcine disease causing viruses that
may be used are Porcine Adenovirus, Porcine Circovirus, Porcine
herpes viruses, Pseudorabies virus, Classical swine fever virus,
Porcine epidemic diarrhea virus, Porcine hemaglutinating
encephalomyelitis virus, Porcine parvovirus, Porcine Respiratory
Corona virus, Porcine Reproductive and Respiratory Virus, Swine
Influenza, Transmissible gastroenteritis virus, and Vesicular
stomatitis virus.
[0035] If lipase activity is present in the bacterin, it may cause
release of the emulsifier from the emulsion. This free emulsifier
may disrupt the live virus envelope, and inactivate the live
vaccine viruses, thereby leading to a loss of viral infectivity.
Accordingly, heat treatment of the bacterin serves to stabilize the
emulsion, and preserve its adjuvant effect, as well as preserving
the viral infectivity of the viruses.
[0036] The following examples are provided for the purpose of
further illustration and are not intended to limit the scope of the
claimed invention.
Procedures
Procedure 1 Determination of Turbidity
[0037] Turbidity is determined in Nephelometric Units (NU) by a
light scattering method. The intensity of light scattered by the
sample under defined conditions is compared to the intensity of
light scattered by a standard reference suspension. The higher the
intensity of the scattered light, the higher the turbidity of the
sample. A light source is directed into the sample and the light
scatter is measured at 90.degree. to the direction of the light
source. The instrument is calibrated by measuring the light scatter
from a formazin suspension.
Calibration of the Nephelometer Instrument
[0038] Ultra-filtered water is prepared by filtering distilled
water through a membrane filter having a pore size of 0.2 .mu.m. A
first solution is prepared by dissolving 1.00 g hydrazine sulfate,
(NH.sub.2) H.sub.2S0.sub.4, in ultra-filtered water and diluted
with ultra-filtered water to 100 ml, in a volumetric flask. A
second solution is prepared by dissolving 10.00 g. of
hexamethylenetetramine in ultra-filtered water and diluting with
ultra-filtered water to 100 ml, in a volumetric flask. A formazin
suspension is prepared by mixing 5.0 ml of the first solution with
5.0 ml of the second solution. The mixture is allowed to stand for
24 hours at approximately 24.degree. C. The mixture is diluted to
100 ml with ultra-filtered water to form a stock turbidity
suspension having a turbidity of 400 NU. A 40 NU formazin turbidity
suspension is prepared by diluting 10.00 ml of the stock turbidity
suspension to 100 ml with ultra-filtered water. Further calibration
solutions are prepared by diluting the stock solution.
Measurement of Turbidity
[0039] The sample to be measured is diluted with ultra-filtered
water so that the turbidity falls within the calibrated range of
the nephelometer. The turbidity is measured and the original
turbidity is calculated using the following equation:
Original Turbidity in N U = M .times. ( D + O ) O ##EQU00001##
where:
[0040] M is the turbidity of the diluted sample in NU
[0041] D is the volume of dilution water, in mL
[0042] O is the original sample volume, in mL
Procedure 2 Lipase Analysis
[0043] Lipase activity was determined using
O-pivaloxymethylumbelliferone as a fluorogenic substrate Lipase
catalyzed hydrolysis of this non-fluorescent substrate produces a
hydroxymethylether, which is unstable under aqueous conditions. The
decomposition of the unstable hydroxymethylether generates
formaldehyde and the fluorescent product umbelliferone. Monitoring
the fluorescence intensity of umbelliferone produced, as a function
of time, provides a sensitive kinetic measurement of the lipase
enzymatic activity.
[0044] O-pivaloxymethylumbelliferone (Molecular Probes product no.
P35901) solutions were prepared in neat DMSO, at a stock
concentration of 5 mM; unused solution was stored at -20.degree.
C., protected from light. The 5 mM O-pivaloxymethylumbelliferone
solution was diluted to 750 .mu.m using 58 mM to TRIS-HCl buffer
(pH 8.0), and the resulting solution pre-warmed to 37.degree. C.
The bacterin sample or the control buffer/medium was centrifuged
for 10 minutes at room temperature at 6500.times. gravity to form a
pellet and a supernatant. Reactions were performed by combining 15
.mu.L of 100 mM TRIS-HCl buffer (pH 8.0) with 15 .mu.L of the
supernatant at room temperature from the bacterin sample or the
control buffer/medium, in assay wells of low volume 96 well plates
(Corning 3393, black polystyrene non-binding surface, half area);
pre-incubating for 10 minutes at 37.degree. C.; then initiating the
reaction by the addition of 20 .mu.L of 750 .mu.m
O-pivaloxymethylumbelliferone or the control buffer/medium. The
resulting reaction mixtures contained 53 mM TRIS-HCl buffer (pH
8.0) and 0 or 300 .mu.m O-pivaloxymethylumbelliferone. Fluorescence
intensity was measured at 30-45 second intervals over a one-hour
period (Spectramax Gemini XS, 37.degree. C., .lamda..sub.ex=360 nm,
.lamda..sub.em=460 nm, PMT sensitivity setting `medium`, 6 reads
per well). The reaction rate was determined from the slope of the
resulting progress curve.
Procedure 3 Measurement of the Turbity of an Erysipelothrix
rhusiopathieae Preparation
[0045] The turbidity of an Erysipelothrix rhusiopathieae
preparation is measured spectrophotometrically at a wavelength of
600 nm. The result is reported in optical units (OU),
EXAMPLES
Example 1
Reduction of Lipase Activity by Heat Treatment
[0046] A pool of merthiolate killed leptospira containing the
following species Leptospira canicola, Leptospira
icterohaemorrhagiae, Leptospira grippotyphosa, Leptospira hardjo,
and Leptospira pomona was prepared to from individual bacterins.
Six samples of the combined bacterins were stored overnight
(approximately 12 hours) at 4.degree. C., 37.degree. C., 45.degree.
C., 56.degree. C., 65.degree. C., and 80.degree. C. The sample
stored at 4.degree. C. served as the non-treated control. The
samples stored for 12 hours at 37.degree. C., 45.degree. C.,
56.degree. C., 65.degree. C., and 80.degree. C. were heat treated
samples. After storage, the rate at which a test substrate
hydrolysed in the presence of each bacterin was measured according
to the method of Procedure 2. The rate of hydrolysis for a sample
divided by the rate of hydrolysis of the sample stored at 4.degree.
C. multiplied by 100 is the percentage of the original lipase
activity of each bacterin that remains after storage. The following
chart shows the temperature of storage and the percentage of the
original lipase activity that remains after storage.
TABLE-US-00001 Storage Temperature (12 hours) 4.degree. C.
37.degree. C. 45.degree. C. 56.degree. C. 65.degree. C. 80.degree.
C. Percent of 100% 55.4% 32.5% 15.7% 10.8% 8.4% Original Lipase
Activity
[0047] Two Leptospira Bratislava serials were prepared. The serials
were inactivated with merthiolate and then subjected to heat
treatment at 65.degree. C. for 8 hours. Samples were pulled
pre-treatment and every two hours during treatment. Lipase activity
was determined at each time point according to the method of
Procedure 2. When a sample was taken, the rate at which a test
substrate hydrolysed in the presence of each sample was measured.
The rate of hydrolysis for a sample divided by the rate of initial
hydrolysis rate multiplied by 100 is the percentage of the original
lipase activity of each bacterin that remains after heat treatment
The following chart shows the sample time and the average
percentage of the original lipase activity that remains at that
time.
TABLE-US-00002 Sample time (hours) Initial 2 4 6 8 Percent of 100%
41% 34% 28% 24% Original Lipase Activity
Example 2
Preparation of Experimental Vaccine Formulations
[0048] Cultures of Leptospira canicola, Leptospira
icterogorrhagiae, Leptospira grippotyphosa, Leptospira hardjo,
Leptospira pomona, Leptospira Bratislava, Erysipelothrix
rhusiopathieae, and Porcine parvovirus were grown.
[0049] The turbidity of each Leptospira culture was measured in
nephelometric units (NU). The turbidity of the Erysipelothrix
rhusiopathieae culture was measured in optical units (OU). The
bacteria were killed with merthiolate to form bacterins. Each
Leptospira bacterin was heat treated at 65.degree. C. for 8 hours
to reduce the lipase activity. The Erysipelothrix rhusiopathieae
bacterin was not heat treated. The Leptospira bacterins were
combined with killed Porcine parvovirus and killed Erysipelothrix
rhusiopathieae then mixed with AMPHIGEN.RTM., adjuvants,
preservatives, and diluting buffer so that each 2 ml dose of the
vaccine contained the components set forth in the chart below.
Concentrations of Antigens
TABLE-US-00003 [0050] Concentration of Component Component/Dose L.
canicola 1200 NU/2 ml dose L. icterohaemorrhagiae 1200 NU/2 ml dose
L. grippotyphosa 1200 NU/2 ml dose L. hardjo 2400 NU/2 ml dose L.
pomona 1200 NU/2 ml dose L. Bratislava 1200 NU/2 ml dose
Erysipelothrix rhusiopathieae 14 OU/2 ml dose Porcine parvovirus
17,920 HA/.05 ml
Example 3
Potency Testing in Hamsters and Pigs
[0051] The vaccine of Example 2 was administered to hamsters and
rabbits to test for potency using standard lab animal models. The
test hamsters were then challenged with a dose of Leptospira
canicola, Leptospira icterohaemorrhagiae, Leptospira grippotyphosa,
Leptospira Bratislava, or Leptospira pomona to test potency of the
vaccines. The numbers of survivors were measured as a demonstration
of efficacy. Rabbit microscopic agglutination titers were measured
against Leptospira hardjo to demonstrate the potency of that
fraction of the vaccine. The table below shows that vaccines
prepared from heat treated Leptospira bacterins are capable of
producing an antigenic response that passes efficacy criteria.
TABLE-US-00004 Leptospira Rabbit- Thermal SEROL- Condition- HAMSTER
SURVIVORS OGY ing Canicola Bratislava Ictero Grippo Pomona Hardjo
65.degree. C. 10/10 10/10 10/10 10/10 10/10 Pass (8 hours)
Untreated 10/10 10/10 10/10 10/10 10/10 Pass
[0052] Erysipelothrix rhusiopathieae was tested in rabbits by
comparing the vaccine serological titer to the titer of a reference
vaccine. The vaccine had an RP (relative potency) of 3.0. PPV was
tested in the hemagglutination assay and had an HA titer of 1024
HA/0.05 ml. A titer of 320 HA/0.05 ml is an acceptable value for a
vaccine.
Example 4
Physiochemical Testing of Vaccines
[0053] A vaccine was prepared with heat treated Leptospira
bacterins and other components according to the formulation listed
in Example 2. A similar vaccine was prepared from non-heat treated
Leptospira bacterins according to the method of Example 2. Both
vaccine formulations were stored at 4.degree. C. for 0, 6, 12, 15
and 18 months of age. Particle size analysis was done for each
vaccine at each time point using a laser diffractometer.
[0054] The charts shown below show particle size distributions for
each vaccine over several months of monitoring (0, 6, 12, 15 and 18
months).
Particle Size Analysis of Freshly Prepared Vaccine Containing Heat
Treated Leptospira Bacterins Day 0
[0055] The vaccine prepared from non-heat treated Leptospira
bacterins (upper graph) shows an increase in particle size
indicating emulsion breakdown The vaccine prepared from heat
treated Leptospira bacterins (lower graph) shows particle size
retention through 18 months of age indicating emulsion
stability.
Example 5
PPV Hemagglutination Assay (HA)
[0056] The vaccine formulations listed in Example 2 (the vaccines
in the list in Example 2 contained all of the antigens listed--same
vaccines for all work) prepared from non-heat treated Leptospira
bacterins and heat treated Leptospira bacterins were initially
tested for HA titer and hemolysis titer. The stability of HA titers
at various time points. The HA assay was performed by adjusting a
sample to pH 11-11.2 to extract the PPV (Porcine parvovirus) virus
from the aluminum hydroxide gel. The sample was then centrifuged
and the supernatant collected for use in the assay. Guinea pig red
blood cells were added to a 96 well plate to serve as the
agglutination indicator. The sample supernatant was diluted 2 fold
across duplicate rows with a starting dilution of 1:5. The plate is
incubated at 5.+-.3 C for 16-24 hours. The degree of
hemagglutination is scored from 0-4 for each well. The titer is
recorded as the last dilution containing a score of 2 or above.
During the test of vaccines, which were not heat treated, it was
observed that the vaccine was causing hemolysis. The hemolysis
titer was the highest dilution at which hemolysis was observed.
Heat treated vaccines did not produce hemolysis.
[0057] The chart below shows the average HA titers and hemolysis
titers over time. CTC is a vaccine with heat treated Leptospira
bacterins. OOP is a vaccine without heat treated Leptospira
bacterins
TABLE-US-00005 CHART 1 PPV HA Titers Over Time OOP and CTC PPV HA
Tracking Over Time INITIAL 1 2 3 1 mo. 2 mos. 3 mos. 4 mos. 5 mos.
7 mos. 10 mos. OOP 160 1280 1280 1280 1280 1280 640 1280 1280 1280
CTC 1280 1280 1280 1280 1280 640 640 640 640 1280 Hemolysis Titer
OOP 80 80 80 160 160 160 160 160 80 160 CTC 0 0 0 0 0 0 0 0 0 0
* * * * *