U.S. patent application number 12/347332 was filed with the patent office on 2009-05-14 for active immunization using a siderophore receptor protein.
This patent application is currently assigned to Epitopix, LLC. Invention is credited to Daryll A. Emery, Darren E. Straub.
Application Number | 20090123500 12/347332 |
Document ID | / |
Family ID | 22716062 |
Filed Date | 2009-05-14 |
United States Patent
Application |
20090123500 |
Kind Code |
A1 |
Emery; Daryll A. ; et
al. |
May 14, 2009 |
ACTIVE IMMUNIZATION USING A SIDEROPHORE RECEPTOR PROTEIN
Abstract
The invention provides a vaccine for immunizing poultry and
other animals against infection by a gram-negative bacteria, and a
method of immunizing an animal using the vaccine. The vaccine may
contain purified siderophore receptor proteins derived from a
single strain or species of gram-negative bacteria or other
organism, which are cross-reactive with siderophores produced by
two or more strains, species or genera of gram-negative bacteria.
The invention further provides a process for isolating and
purifying the siderophore receptor proteins, and for preparing a
vaccine containing the proteins. Also provided is a method for
diagnosing gram-negative sepsis.
Inventors: |
Emery; Daryll A.; (New
London, MN) ; Straub; Darren E.; (New London,
MN) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581336
MINNEAPOLIS
MN
55458-1336
US
|
Assignee: |
Epitopix, LLC
Willmar
MN
|
Family ID: |
22716062 |
Appl. No.: |
12/347332 |
Filed: |
December 31, 2008 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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12177438 |
Jul 22, 2008 |
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12347332 |
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11147662 |
Jun 8, 2005 |
7413743 |
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12177438 |
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10185498 |
Jun 28, 2002 |
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11147662 |
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09361081 |
Jul 26, 1999 |
6432412 |
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10185498 |
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08903858 |
Jul 30, 1997 |
6027736 |
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09361081 |
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08385273 |
Feb 8, 1995 |
5830479 |
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08903858 |
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08194040 |
Feb 9, 1994 |
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08385273 |
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Current U.S.
Class: |
424/257.1 ;
424/234.1; 424/258.1 |
Current CPC
Class: |
A61K 39/0275 20130101;
A61P 31/04 20180101; Y10S 424/823 20130101; Y02A 50/474 20180101;
A61P 37/04 20180101; Y02A 50/30 20180101; A61K 39/0258 20130101;
Y10S 424/824 20130101; A61K 39/102 20130101; Y02A 50/482 20180101;
Y10S 424/826 20130101; G01N 33/56911 20130101 |
Class at
Publication: |
424/257.1 ;
424/258.1; 424/234.1 |
International
Class: |
A61K 39/108 20060101
A61K039/108; A61K 39/112 20060101 A61K039/112; A61K 39/02 20060101
A61K039/02; A61P 31/04 20060101 A61P031/04; A61P 37/04 20060101
A61P037/04 |
Claims
1-45. (canceled)
46. A method comprising: administering to an animal a composition
comprising gram-negative whole cells, wherein the gram-negative
whole cells express siderophore receptor proteins on the cell
surface, wherein the gram-negative whole cells lack outer
oligosaccharide side chains of the lipopolysaccharide layer of the
outer membrane, and wherein the administration results in an
increased immune response to the siderophore receptor proteins.
47. The method of claim 46 wherein the gram-negative whole cell is
E. coli.
48. The method of claim 46 wherein the gram-negative whole cell is
an avirulent R-mutant.
49. The method of claim 46 wherein the gram-negative whole cell is
E. coli J5.
50. The method of claim 46 wherein the gram-negative whole cell is
Salmonella minnesota.
51. The method of claim 46 wherein the gram-negative whole cell is
chemically altered to eliminate the outer oligosaccharide side
chains.
52. The method of claim 46 wherein the gram-negative whole cell is
a bacterin.
53. A vaccine comprising gram-negative whole cells and an adjuvant,
wherein the gram-negative whole cells express siderophore receptor
proteins on the cell surface, and wherein the gram-negative whole
cells lack outer oligosaccharide side chains of the
lipopolysaccharide layer of the outer membrane.
54. The vaccine of claim 53 wherein the gram-negative whole cell is
E. coli.
55. The vaccine of claim 53 wherein the gram-negative whole cell is
an avirulent R-mutant
56. The vaccine of claim 53 wherein the gram-negative whole cell is
E. coli J5.
57. The vaccine of claim 53 wherein the gram-negative whole cell is
Salmonella minnesota.
58. The vaccine of claim 53 wherein the gram-negative whole cell is
chemically altered to eliminate the outer oligosaccharide side
chains.
59. The vaccine of claim 53 wherein the gram-negative whole cell is
a bacterin.
Description
BACKGROUND OF INVENTION
[0001] The economic impact of infectious diseases in the poultry
industry is well-appreciated. Immunization of birds has helped
reduce the cost of production by decreasing the incidence of
gastrointestinal, respiratory and systemic diseases. While vaccines
provide adequate immunity for those pathogens against which a flock
has been immunized, there are few vaccines which can provide
broad-based cross-protection against unanticipated diseases or
against those diseases for which an animal has not been
specifically vaccinated.
[0002] A number of important diseases of domestic poultry are
caused by bacteria able to invade host tissues, such as Salmonella
spp., Escherichia spp. and Pasteurella spp. While many vaccines are
available for immunization against individual species and
serotypes, none provide cross-protection or stimulate broad-based
immunity against multiple serotypes, species or genera.
[0003] One essential factor required for a bacteria to induce
clinical disease is the ability to proliferate successfully in a
host tissue. Iron is an essential nutrient for the growth of
gram-negative bacteria in vivo, but is virtually unavailable in
mammalian and/or avian tissues because the iron is either
intracellular or extracellular, complexed with high affinity,
iron-binding proteins, for example, transferring in blood and lymph
fluids and lactoferrin in external secretions. In normal tissues,
the concentration of iron is approximately 10.sup.-18M, far below
that required for bacterial growth.
[0004] To circumvent these restrictive conditions, pathogenic
bacteria have evolved high affinity iron transport systems produced
under low iron conditions, which consist of specific ferric iron
chelaters, "siderophores," and iron-regulated outer membrane
proteins (IROMPs) and/or siderophore receptor proteins (SRPS) which
are receptors for siderophores on the outer membrane of the
bacterial cell. Siderophores are synthesized by and secreted from
the cells of gram-negative bacteria under conditions of low iron.
Siderophores are low molecular weight proteins ranging in molecular
mass from about 500 to about 1000 MW, which chelate ferric iron and
then bind to IROMPs in the outer bacterial membrane which, in turn,
transport the iron into the bacterial cell. Although the use of
IROMPs as immunogens has been considered, these proteins have not
been examined for such use, at least in part, due to an inability
to extract these proteins from bacterial membranes in high volume
and with a desired level of purity and immunogenic quality.
[0005] Accordingly, an object of the invention is to provide a
method for obtaining high amounts of immunogenic quality
siderophore receptor proteins from Escherichia coli, Salmonella,
Pasteurella, and other gram-negative bacteria. Another object is to
provide a vaccine for immunizing poultry and other animals against
these bacteria. Yet another object is to provide a vaccine for
cross-protection against multiple serotypes, species and/or genera
of bacteria belonging to the family Enterobacteriaceae and/or
Pasteurellaceae. A further object is to provide a diagnostic assay
to monitor and/or profile sepsis and subclinical disease caused by
gram-negative bacteria under field conditions.
SUMMARY OF THE INVENTION
[0006] These and other objects are achieved by the present
invention which is directed to a vaccine for prevention and
treatment of infection by gram-negative bacteria, and a method of
immunizing poultry and other animals against such infections using
the vaccine. The invention also provides a method for isolating and
purifying outer membrane siderophore receptor proteins from
gram-negative bacteria for producing the vaccine. The invention
further provides an in vitro method of diagnosing infections of
gram-negative bacteria in an animal using antibodies raised to the
isolated receptor proteins.
[0007] The vaccine is useful for immunizing an avian or other
animal against infection by gram-negative bacteria such as
colibacillosis, salmonellosis and pasteurellosis. The vaccine is
composed of a substantially pure siderophore receptor protein
derived from the outer membrane of a gram-negative bacteria, for
example, Salmonella spp., Escherichia spp. and Pasteurella spp. A
siderophore receptor protein, useful according to the invention, is
a protein or antigenic peptide sequence thereof derived from the
outer membrane of a gram negative bacterium, which is capable of
producing an antibody that will react with the siderophore receptor
protein expressed by a gram-negative bacteria of the same or
different strain, species or genus. Preferably, the siderophore
receptor protein is derived from a bacterium belonging to the
family Enterobacteriaceae and/or Pasteurellaceae.
[0008] The vaccine contains siderophore receptor proteins (SRPs)
derived from a gram-negative bacteria, capable of eliciting an
immune response in an animal with the production of anti-SRP
antibodies. These antibodies will react with siderophore receptor
proteins of that bacteria, and may also cross-react with
siderophore receptor proteins of a different strain, species and/or
genera of gram-negative bacteria to provide cross-protection
against infection from such other bacteria. Useful siderophore
receptor proteins having a molecular weight of about 72-96 kDa, as
determined by SDS-PAGE, have been isolated from E. coli, Salmonella
spp., Pasteurella spp., Pseudomonas spp., and Klebsiella spp.
Preferably, the siderophore receptor proteins (SRPs) are derived
from Escherichia coli, Salmonella spp. and/or Pasteurella spp. The
antibodies produced from those SRPs will react with SRPs of those
bacteria and cross-react with SRPs of a different strain, species
and/or genera of bacteria within the family Enterobacteriaceae
and/or Pasteurellaceae.
[0009] The vaccine contains one or more siderophore receptor
proteins extracted from the outer membrane of a single strain or
species, or two or more different strains or species of
gram-negative bacteria. The amount and type of siderophore receptor
protein included in the vaccine is effective to stimulate
production of antibodies reactive with a siderophore receptor
protein of one, preferably two or more strains, species or genera
of gram-negative bacteria. A preferred vaccine is composed of an
amount and profile of siderophore receptor-proteins to effectively
induce antibodies reactive with a majority, preferably all, of the
siderophore receptor proteins of a bacterial population to
effectively enhance opsonization and complement-mediated bacterial
lysis, and/or block the iron binding capacity of the bacteria. The
siderophore receptor protein is combined with a
physiologically-acceptable carrier, preferably a liquid. The
vaccine may further include an adjuvant to enhance the immune
response, and other additives as desired, such as preservatives,
flavoring agents, buffering agents, and the like.
[0010] The present invention also provides a method for isolating
high quantities of immunogenically effective siderophore receptor
proteins from outer membranes of a single strain or species of
gram-negative bacteria such as E. coli, Salmonella and/or
Pasteurella. The method includes culturing the organism under
conditions of low iron availability, that is, in a culture medium
that lacks iron or includes an iron chelating agent. The
siderophore receptor proteins are then separated from the bacterial
outer membrane and purified by use of the anionic detergent, sodium
dodecyl sulfate, preferably under non-reducing conditions.
[0011] The siderophore receptor proteins may be utilized to raise
polyclonal antibody sera and monoclonal antibodies for use in
passive immunization therapies. Such antibodies may also be used in
an in vitro method of diagnosing a gram-negative bacterial
infection in an animal. The diagnostic method includes contacting a
body material potentially infected with a gram-negative bacteria,
such as a tissue sample or body fluid, with a labelled antibody
raised to a siderophore receptor protein, and detecting the label
in the complex formed between the siderophore receptor protein in
the body material and the labelled antibody. The method may also be
performed by combining the body sample with the antibody to the
siderophore receptor protein, and then contacting the sample with a
labelled anti-species antibody reactive with the protein-specific
antibody, and then detecting the label.
[0012] The siderophore receptor proteins can also be used as
capture antigens in a method of monitoring and profiling gram
negative sepsis. For example, the protein may be used in an ELISA
technique in which the protein is bound to a solid support and
contacted with a sample material to react with and detect
antibodies present in the sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a graphic depiction of the elution profile of
concentrated, solubilized siderophore receptor proteins isolated
from Escherichia coli serotype 078 (ATCC 55652).
[0014] FIG. 2 is a graphic depiction of the quantitative clearance
of Salmonella agona in spleens of turkeys vaccinated with IROMPs
isolated from E. coli and non-vaccinated controls.
[0015] FIG. 3 is a graphic depiction of the serological response to
E. coli siderophore receptor proteins (SRPs) between vaccinated and
non-vaccinated flocks.
[0016] FIG. 4 is a depiction of the total % mortality and culls in
control and E. coli SRP-vaccinated flocks (3-13 weeks of age).
[0017] FIG. 5 is a depiction of the total % mortality and culls in
control and E. coli SRP-vaccinated flocks (3-13 weeks of age).
[0018] FIG. 6 is a graphic depiction of the total mortality in
SRP-vaccinated and non-vaccinated turkeys following challenge with
Pasteurella multocida P-1059.
[0019] FIG. 7 is a graphic depiction of the serological response in
birds vaccinated with purified siderophore receptor proteins from
Salmonella senftenberg, showing cross-reactivity with the SRP of E.
coli.
[0020] FIG. 8 is a graphical depiction of the serological response
in birds vaccinated with purified siderophore receptor proteins
from P. multocida, showing cross-reactivity with the SRP of E.
coli.
[0021] FIG. 9 is a graphic depiction of the total % mortality in
consecutive flocks before and after vaccinating with siderophore
receptor proteins derived from E. coli 078.
[0022] FIG. 10 is a graphic depiction of the serological response
to SRPs from E. coli between SRP-vaccinated and non-SRP-vaccinated
commercial turkey flocks.
[0023] FIG. 11 is a graphical depiction of the serological response
of purified SRP and whole cell of Salmonella heidelberg.
[0024] FIG. 12 is a graphic depiction of the total mortality
between progeny of SRP-vaccinated and non-vaccinated (control)
breeder hens.
[0025] FIG. 13 is a graphical depiction of the serological response
in birds vaccinated with purified siderophore receptor proteins
from Salmonella typhimurium, showing cross-reactivity with the SRP
of E. coli.
[0026] FIG. 14 is a graphical depiction of the serological response
in birds vaccinated with purified siderophore receptor proteins
from Salmonella enteritidis, showing cross-reactivity with the SRP
of E. coli.
[0027] FIG. 15 is a graphical depiction of SRPs of Salmonella
typhimurium as a protective immunogen against a homologous and
heterologous challenge in turkeys.
[0028] FIG. 16 is a graphical depiction of SRPs of Salmonella
enteritidis as protective immunogens against a homologous and
heterologous challenge in turkeys.
DETAILED DESCRIPTION OF THE INVENTION
[0029] As used herein, the term "substantially pure" means that the
siderophore receptor protein has been extracted and isolated from
its natural association with other proteins, lipids, and other like
substances and elements of a bacterial cell or other organism.
[0030] Gram-negative bacteria are frequent pathogens of poultry and
other animals, such as domestic foul, livestock, horses, companion
animals, and humans. In an iron-restricted environment, bacteria
such as Escherichia coli, Salmonella spp. and Pasteurella spp.
produce siderophores that chelate ferric iron and bind to outer
membrane proteins that function as siderophore receptors on the
bacterial membrane.
[0031] The invention provides an improved process for isolating and
separating siderophore receptor proteins from the outer membrane of
gram-negative bacteria. Isolation and purification of
immunogenically intact siderophore receptor proteins from bacterial
membranes in a sufficient quantity and immunogenic quality for
formulating a vaccine against infection by gram-negative bacteria
has been difficult. The structural orientation, or conformation, of
the outer membrane protein necessary to provide antigenicity may be
typically lost when the protein is separated and purified from the
lipopolysaccharide complex. Another problem is that the protein
becomes denatured by the separation process wherein its
immunogenicity is lost. According to the present invention,
however, the isolation and separation of immunogenic quantities of
antigenically effective siderophore receptor proteins from the
outer membrane of gram-negative bacteria has been achieved. This
enables the production of vaccines and hyperimmunized sera for the
treatment of animals infected or susceptible to infection by
gram-negative bacteria, and in vitro diagnostic methods for
detecting such an infection in an animal.
[0032] As a group, gram-negative bacteria possess a common cell
wall structure. Components of the cell wall structure may be used
as immunogens. However, these immunogens may provide only
homologous immune protection. The present vaccine utilizes a
combination of outer membrane siderophore receptor proteins common
to two or more gram-negative bacteria that are capable of
proliferating in the blood or host tissues and causing infection in
an animal. The vaccine may contain two or more siderophore receptor
proteins (SRPs), preferably four or more SRPs derived from the
outer membrane of one or more strains or species of gram-negative
bacteria and/or other organism. Preferably, the SRPs are derived
from a single strain or species of gram-negative bacteria. A
preferred siderophore receptor protein for use in the vaccine has a
common receptor reactive with siderophores produced by two or more
strains, species and/or genera of gram-negative bacteria.
[0033] An example of a useful siderophore receptor protein is the
receptor protein for aerobactin (MW about 72-74 kDa) produced by
members of the family Enterobacteriaceae, for example, Escherichia
coli, Salmonella and Klebsiella. Antibodies produced against an
aerobactin receptor protein of one species, strain or genus of that
family have been found to cross-react with other bacteria within
the family. Species of Pseudomonas of the family Pseudomonadaceae
also express aerobactin siderophore receptor proteins that can be
isolated according to the invention and used in a vaccine to
produce antibodies that cross-react with the aerobactin receptor
proteins of E. coli, Salmonella and Klebsiella, among other members
of the family Enterobacteriaceae.
[0034] Another example of a suitable siderophore receptor protein
for use in the present vaccines is that produced by Pasteurella
multocida for the siderophore multocidin (MW about 500-1000 kDa).
Antibodies to the multocidin receptor protein will react with all
three of the SRPs in Pasteurella multocida. In Western blots, two
of the larger siderophore proteins (96 kDa, 84 kDa) of P. multocida
showed reactivity with hyperimmune E. coli protein antisera.
Antibodies produced to multocidin receptor proteins will
cross-react with the siderophore receptor proteins of Salmonella
spp. and E. coli, as demonstrated by ELISA and Western blot
analysis.
[0035] Other siderophore receptor proteins include those reactive
with the siderophore enterochelin (MW about 81-84 kDa) produced by
E. coli, Salmonella, Pseudomonas and Klebsiella; and the
siderophore citrate (MW about 74-78 kDa) produced by E. coli, among
others. A vaccine containing the enterochelin and/or citrate
receptor proteins will produce antibodies reactive with E. coli,
Salmonella and other bacteria of the family Enterobacteriaceae, and
with Pseudomonas of the family Pseudomonadaceae.
[0036] Another useful SRP is the siderophore receptor protein for
ferrichrome (MW about 78 kDa) produced by E. coli, and Salmonella
spp. In commercial poultry raising facilities, infection by
Aspergillus causes serious respiratory problems in the birds. In
the lungs, Aspergillus will excrete ferrichrome to acquire iron as
a growth nutrient. Under iron restriction or systemic conditions,
E. coli and Salmonella will express ferrichrome receptor protein.
They are also opportunistic bacteria that can scavenge and utilize
ferrichrome produced by Aspergillus as a growth nutrient.
Therefore, it is preferred that the vaccine preparation include a
ferrichrome receptor protein to induce antibodies that will bind
and cross-react with the ferrichrome receptor proteins of
gram-negative bacteria including E. coli and Salmonella, and
fungi/mold. A vaccine containing this SRP will elicit an immune
response to the protein to enhance the bactericidal activity of the
antibody. Also, once the avian or other animal is vaccinated with a
ferrichrome receptor protein, Aspergillus expressing this protein
in vivo in the animal will enhance the antibody response to the
ferrichrome receptor protein which in turn will cross-react with
Salmonella and E. coli and other bacteria that express the
ferrichrome receptor protein.
[0037] Antibody elicited from a ferrichrome receptor protein (MW
about 78 kDa) derived from E. coli can cross-react with the
receptor proteins of fungi, such as Aspergillus flavus, Aspergillus
fumigatus, Penicillium and Fusarium. Western blot analysis against
the outer membrane proteins (OMPs) of A. fumigatus using anti-SRP
antibody revealed three cross-reactive proteins (MW about 45-90
kDa). The inclusion of a ferrichrome receptor protein into a
vaccine preparation will provide inducement of antibodies that will
react with the fungi and/or bacteria to prevent binding and
excretion of the ferrichrome siderophore. Animals such as birds
that are vaccinated with a vaccine preparation containing a
ferrichrome receptor protein will get an elevated antibody titer by
bacteria and/or fungi that challenge the animal and produce a
ferrichrome receptor protein. Also, antibody to the ferrichrome
receptor can be elevated by natural field challenge by bacteria or
fungi which can induce a bactericidal effect that could lessen
system challenge and disease potential.
[0038] Yet another useful SRP is a coprogen receptor protein (MW
about 74-76 kDa) produced by E. coli. Antibodies produced against
coprogen receptor protein will cross-react with the SRPs of other
E. coli expressing this protein under systemic conditions.
[0039] In one embodiment, the vaccine is formulated with
siderophore receptor proteins (SRPs) of different types and/or
molecular weights, derived from a first gram-negative bacteria, the
SRPs being capable of stimulating production of antibodies that
react with the first gram-negative bacteria as well as a second
gram-negative bacteria of a different strain or species than the
first gram-negative bacteria. The vaccine preferably contains all
SRPs derived from the gram-negative bacteria infectious agent. For
example, P. multocida and Salmonella spp. have been identified as
producing 3 SRPs each, and E. coli produced 2, 3, 4, and 6 SRPs
varying between serotypes. Accordingly, the vaccine is formulated
to contain the SRPs derived from the bacterial causative agent,
i.e., 2-6 or more SRPs. It is preferred that the vaccine also
include siderophore receptor proteins of different types and/or
molecular weights derived from a gram-negative bacteria of a strain
or species different than the first gram-negative bacteria,
preferably 1-15 SRPs, preferably 5-10 SRPs.
[0040] For example, the vaccine may contain a siderophore receptor
protein derived from E. coli, preferably E. coli serotype 01a, 02a
and/or 078, that is capable of stimulating production of an
antibody immunoreactive with that E. coli and a second
gram-negative bacteria such as Salmonella spp., Pseudomonas
aeruginosa, Klebsiella pneumoniae and/or Pasteurella multocida. In
another example, the vaccine may contain a siderophore receptor
protein derived from a species of Pasteurella, such as P.
multocida, that is capable of stimulating production of an antibody
immunoreactive with that species of Pasteurella and a second
gram-negative bacteria such as Salmonella spp. and/or E. coli. In
yet another example, the vaccine may contain a siderophore receptor
protein derived from a species of Salmonella that is capable of
stimulating production of an antibody immunoreactive with that
species of that species of Salmonella, and a second gram-negative
bacteria such as E. coli, Pseudomonas, Klebsiella, and/or
Pasteurella multocida.
[0041] A vaccine formulated with siderophore receptor proteins
derived from E. coli is preferably composed of an aerobactin,
ferrichrome, coprogen, enterochelin and/or citrate SRP, having
molecular weights of about 89 kDa to about 72 kDa, as determined by
SDS-PAGE. The vaccine preferably includes 2-5 receptor proteins,
preferably 3-5 proteins, preferably all five E. coli SRPs. A
preferred vaccine against E. coli infection is prepared with the
SRPs from E. coli 078 (ATCC #55652). E. coli 078 has been
identified as producing up to 6 SRPs ranging in molecular weight
from about 72 to 90 to 92 kDa, as determined by SDS-PAGE. The SRPs
derived from E. coli 078 include aerobactin, ferrichrome, coprogen,
enterochelin and citrate SRPs, having molecular weights of about
91-92 kDa, 89 kDa, 84 kDa, 78 kDa, 74 kDa and 72 kDa, as determined
by SDS-PAGE, 12.5% acrylamide reducing gel. Although the 91-92 kDa
proteins of E. coli 078 are expressed in culture media made with
and without iron, the expression of those proteins is enhanced in
an iron-restricted medium, and as used herein, the 91-92 kDa
proteins are considered to be iron-regulated SRPs. A preferred
vaccine for immunizing an animal against E. coli is formulated with
an aerobactin, ferrichrome, coprogen, enterochelin and citrate SRP
derived from E. coli, preferably E. coli 078, made of at least 5
siderophore receptor proteins, preferably at least 6 receptor
proteins, or more, to induce anti-SRP antibodies to effectively
block a majority, preferably all, iron binding sites of E. coli
serotypes present in an infection, and to induce high antibody
levels to promote bactericidal activity.
[0042] It is further preferred that the vaccine includes one or
more SRPs, preferably about 1-15 SRPs, derived from one or more
additional bacteria, different from the first gram-negative
bacteria. For example, in a vaccine composed of SRPs from E. coli,
it is desirable to include one or more of the SRPs derived from
Salmonella, Pasteurella multocida, Klebsiella and/or
Pseudomonas.
[0043] A preferred vaccine contains each of the SRPs of different
types and/or molecular weights, of a population of gram-negative
bacteria to induce production of antibodies that will effectively
block the iron-binding sites of all of the various SRPs of the
bacterial population so that the bacteria cannot effectively bind
iron as a nutrient for growth. It is also preferred that the
vaccine will induce high SRP antibody levels that will enhance
opsonization and/or complement-mediated bacterial lysis. Due to the
variation in iron-regulated outer membrane proteins (IROMPs)
produced between and within bacterial serotypes, formulating a
vaccine with SRPs isolated and purified from a single isolate
source may provide only a partial profile of the SRPs present in a
bacterial population. Consequently, the effectiveness of the
vaccine to induce anti-SRP antibodies to block bacterial
iron-binding sites and inhibit bacterial infection may be limited
to those serotypes that produce all or less than all of the SRPs
included in the vaccine, while those bacterial serotypes producing
other SRPs may retain an iron-binding capacity. Thus, it is
preferred that a profile, or banding pattern (i.e., SDS-PAGE
protein separations), of a bacterial population is conducted by
examining different field isolates, preferably about 25-100
isolates, to determine the SRPs that are present, and all of the
various SRPs are included in the vaccine.
[0044] Non-iron regulated proteins and polypeptides may also be
included in the vaccine as adjuvants to enhance the effectiveness
of the vaccine and increase opsonization, that is, increase
macrophage activity resulting in increased phagocytosis of
antibody-bound cells, and induce complement-mediated bacterial
lysis. A useful adjuvant protein is a 34-38 kDa group of outer
membrane proteins (porins, i.e., pore-forming proteins) derived
from gram-negative bacteria of the family Enterobacteriaceae and
Pasteurellaceae including E. coli 078, and other gram-negative
bacteria. The transmembrane and porin proteins (MW 34-38 kDa)
identified as OmpA, OmpC, OmpD and OmpF are expressed with and
without iron, are relatively conserved between gram-negative
bacteria, and play a role in iron binding. For example, OmpF and
OmpC will bind lactoferrin (Erdei et al., Infection and Immunity
62:1236-1240 (April 1994)), while OmpA will bind ferrichrome
(Coulton et al., J. Gen. Microbiol. 110:211-220 (1979)). Antibodies
early in infection particularly of the IgM class will cross-react
with outer membrane proteins of E. coli, Salmonella, Pasteurella,
Pseudomonas and Klebsiella, and will bind lactoferrin and/or
ferrichrome, precluding the availability of an iron source for
bacterial growth. Antibodies to these proteins will also bind to
the porin omp on the surface to enhance opsonization and/or
complement-mediated bacterial lysis. Immunogenically intact 34-38
kDa porin outer membrane proteins can be isolated and purified
according to the process of the invention.
[0045] The vaccine may be used to immunize poultry and other
animals such as domestic fowl, livestock, horses, companion
animals, and humans, against infection caused by one or more
gram-negative bacteria. The vaccine is effective for eliciting
antibodies that are immunoreactive with a gram-negative bacteria
that expresses one or more siderophore receptor protein(s).
[0046] Preferably, the vaccine is capable of achieving clinical
efficacy of cross-reactive and cross-protective immunization
against two or more different strains, species and/or genera of
gram-negative bacteria or other organisms capable of expressing
siderophore receptor proteins. For example, a vaccine containing
siderophore receptor proteins for aerobactin, enterochelin,
ferrichrome, coprogen and/or citrate, may be used to stimulate
production of antibodies that cross-react with a number of
different bacteria that express one or more of these receptor
proteins. The effectiveness of the present vaccine is due, at least
in part, to the conservative nature of the outer membrane
siderophore receptor proteins which are cross-reactive with
siderophore receptor proteins produced by two or more different
species, strains and/or genera of Enterobacteriaceae such as E.
coli, Salmonella, and other gram-negative bacteria within other
families such as Pasteurella and/or Pseudomonas.
[0047] Because of the cross-reactivity of the SRPs, the vaccine is
effective in stimulating production of antibodies that react with
the first gram-negative bacteria (from which the SRPs were
derived), as well as a second gram-negative bacteria of a different
strain or species than the first gram-negative bacteria. For
example, a vaccine can be formulated to contain a siderophore
receptor protein derived from E. coli, preferably E. coli serotype
01a, 02a and/or 078, more preferably E. coli 078, that is effective
in stimulating production in vivo of an antibody immunoreactive
with that E. coli serotype (from which the SRP(s) were derived),
and a second gram-negative bacteria such as Salmonella spp.,
Pseudomonas aeruginosa, Klebsiella pneumoniae and/or Pasteurella
multocida. In another example, the vaccine can contain a
siderophore receptor protein derived from a species of Pasteurella,
such as P. multocida, that is effective in stimulating production
of an antibody immunoreactive with that species of Pasteurella and
a second gram-negative bacteria such as Salmonella spp. and/or E.
coli. In yet another example, the vaccine can contain a siderophore
receptor protein derived from a species of Salmonella that is
effective in stimulating production of an antibody immunoreactive
with that species of Salmonella, and a second gram-negative
bacteria such as E. coli, Pseudomonas, Klebsiella, and/or
Pasteurella multocida.
[0048] Advantageously, immunization using the present vaccine
containing an immunogen cross-reactive with multiple species,
strains and genera of gram-negative bacteria, not only minimizes
immunization costs since separate inoculations with a different
immunogen for each type of gram-negative bacteria is not required.
In addition, the present vaccine provides protection against new
strains or unanticipated pathogens of gram-negative bacteria which
produce siderophore receptor proteins that will cross react with
antibodies induced by the siderophore receptor proteins contained
in the vaccine. The vaccine given to an adult animal is highly
efficacious in treating and preventing gram-negative sepsis not
only in the adult animal but also their progeny by the direct
transfer of anti-SRP antibodies.
[0049] Commercial bacterial whole cell vaccines are useful for
treating a particular disease and/or infection but do not provide
effective cross-protection against other infection. For example,
avian pasteurellosis in turkeys caused by Pasteurella multocida is
clinically diagnosed by particular lesions induced by the bacterial
infection. Treating the disease with a commercial whole cell
vaccine stimulates antibodies that are homologous but not
heterologous in their action, and will not cross-protect against
infection by other bacteria.
[0050] Advantageously, the present vaccines provide
cross-protection against a number of infections caused by
gram-negative bacteria. According to the invention, an animal
species suffering from gram-negative bacterial sepsis can be
administered the vaccine containing SRPs derived from the
(causative agent) gram-negative bacteria to induce antibodies
immunoreactive with those SRP(s) to inhibit the disease state. The
antibodies will also cross-react with SRP(s) produced by another
gram-negative bacteria to inhibit a disease state caused by that
other bacteria. Thus, a vaccine containing SRPs of a first gram
negative bacteria will provide protection against an infection
caused by that bacteria and provide cross-protection against
infection caused by a different gram-negative bacteria.
[0051] Gram-negative bacteria suitable for use in obtaining
siderophore receptor proteins according to the invention, are those
capable of producing siderophore receptor proteins when raised
under growth conditions of low iron availability. Examples of
useful gram-negative bacteria include Escherichia coli (serotypes
01a, 02a, and 078), Salmonella agona, Salmonella blockley,
Salmonella enteriditis, Salmonella hadar, Salmonella heidelberg,
Salmonella montevideo, Salmonella senftenberg, Salmonella
cholerasuis, Salmonella typhimurium, Pasteurella multocida
(serotype A:3,4), Klebsiella pneumoniae, Pseudomonas aeruginosa,
and the like. These organisms are commercially available from a
depository such as American Type Culture Collection (ATCC),
Rockville, Md. In addition, such organisms are readily obtainable
by isolation techniques known and used in the art. The
gram-negative bacteria may be derived from an infected animal as a
field isolate, and screened for production of SRPs, and introduced
directly into the preferred iron-depleted media for that bacteria,
or stored for future use, for example, in a frozen repository at
about -20.degree. C. to about -95.degree. C., preferably about
-40.degree. C. to about -50.degree. C., in BHI containing 20%
glycerol, and other like media.
[0052] For producing the siderophore receptor proteins, conditions
of low iron availability are created using culture media that lack
iron or have been supplemented with an iron chelating agent to
decrease iron availability. Suitable culture media for providing
low iron availability and promoting production of the siderophore
receptor proteins in gram-negative bacteria, include media such as
tryptic soy broth (Difco Laboratories, Detroit, Mich.) and/or
brain-heart infusion (BHI) broth which has been combined with an
iron-chelating agent, for example, .alpha.,.alpha.'-dipyridyl,
deferoxamine, and other like agents. In a preferred embodiment,
.alpha.,.alpha.-dipyridyl is added to a BHI culture media in a
concentration of about 1-500 .mu.g/ml, preferably about 50-250
.mu.g/ml, more preferably about 75-150 .mu.g/ml.
[0053] The gram-negative bacteria employed to produce a siderophore
receptor protein are cultured in the preferred media for that
organism using methodologies and apparati known and used in the
art, such as a fermenter, gyrator shaker, or other like apparatus.
For example, a culture may be grown in a gyrator shaker in which
the media is stirred continuously with aeration at about 300-600
rev/minute, for about 15-20 hours, at a temperature and pH
appropriate for growth for that organism, i.e., about 35-45.degree.
C. and about pH 7-7.6, preferably pH 6.5-7.5. The bacterial culture
is then processed to separate and purify the siderophore receptor
proteins from the outer membrane of the bacteria.
[0054] The bacterial culture is concentrated, for example, by
centrifugation, membrane concentration, and the like. For example,
the cell culture may be centrifuged at about 2,450-20,000.times.g,
preferably at about 5,000-16,000.times.g, for about 5-15 minutes at
about 3-6.degree. C. The supernatant is removed by decanting,
suctioning, pipetting and the like, and the concentrated cell
pellet is collected and washed in a compatible buffer solution
maintained at about pH 7-7.6, such as tris-buffered saline (TBS),
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES),
3-N(N-morpholino) propanesulfonic acid (MOPS), and the like. The
washed pellet is resuspended and washed in a compatible buffer
solution, i.e., TBS, HEPES, MOPS and the like. The cell material is
then treated to solubilize the components of the outer membrane by
resuspending the pellet in buffer containing about 0.5-10% sodium
N-lauroyl sarcosinate, preferably about 1-3%, at about 4-10.degree.
C. for about 15 minutes to about 3 hours, preferably about 30
minutes to about 2 hours, preferably with continuous stirring.
[0055] The bacterial cells are then disrupted by sonication, French
pressure, grinding with abrasives, glass bead vortexing, and other
like methods known and used in the art, preferably at a temperature
of about 3-6.degree. C. The cell homogenate is then centrifuged at
about 10,000-20,000.times.g for about 10-45 minutes, to separate
cell debris from the supernatant fraction containing the outer
membrane proteins. The supernatant is collected by decanting,
suctioning, pipetting, or other like method, and then concentrated,
for example, by ethanol precipitation, membrane concentration,
propylene glycol precipitation, and other methods known and used in
the art. In a preferred method, the supernatant is treated by
passing it through a membrane having a molecular weight cut-off of
about 1,000-50,000 MW, preferably about 10,000-25,000 MW, to
concentrate the protein and allow contaminating proteins smaller
than the molecular weight cut-off to pass through the membrane, and
to decrease the amount of detergent. Such membranes are
commercially available, for example, from Amicon, Danvers,
Mass.
[0056] The concentrated supernatant is then reconstituted in a
compatible buffer, i.e., TBS, HEPES, MOPS, and the like, about pH
7-7.6, which contains a detergent for solubilizing the outer
membrane and extracting the siderophore receptor proteins. It was
found that the anionic detergent sodium dodecyl sulfate (SDS), when
used as a solubilizing detergent alone without a reducing agent
such as 2-mercaptoethanol, is particularly effective for extracting
a high quantity of the siderophore receptor proteins without
denaturing or altering their immunogenicity such that the proteins
will function in vivo as effective immunogens to elicit an antibody
response against gram-negative bacteria. The buffer solution
contains about 0.1-4% SDS (0.2%), preferably about 0.1-2% SDS,
preferably about 0.1-2% SDS.
[0057] After about 1-10 minutes, the siderophore receptor proteins
are separated from the buffer solution by affinity, ion exchange,
size exclusion and other like chromatographic methods known and
used in the art. Preferably, the SRP preparation is separated with
a 4% stacking gel on a 12.5t acrylamide reducing gel. The fractions
are then combined, concentrated, for example by centrifuging, and
precipitated, for example with an alcohol (i.e., ethanol, methanol,
acetone), to remove the SDS. The purified proteins may be used
immediately to prepare a vaccine, or may be stored for future use
through lyophilization, cryopreservation, or other like technique
known and used in the art.
[0058] The vaccine of the present invention may be used for
preventing and eliminating infections of gram-negative bacteria in
poultry and other animals, including humans. The vaccine may be
delivered to the animal, for example, by parenteral delivery,
injection (subcutaneous or intramuscular), sustained-released
repository, aerosolization, egg inoculation (i.e., poultry), and
the like, by known techniques in the art. For prophylactic and
anti-infectious therapeutic use in vivo, the vaccine contains an
amount of a siderophore receptor protein to stimulate a level of
active immunity in the animal to inhibit and/or eliminate
gram-negative bacterial pathogenesis and/or sepsis.
[0059] The siderophore receptor proteins are administered in
combination with a pharmaceutical carrier compatible with the
protein and the animal. Suitable pharmacological carriers include,
for example, physiological saline (0.85%), phosphate-buffered
saline (PBS), Tris(hydroxymethyl aminomethane (TRIS), Tris-buffered
saline, and the like. The protein may also be incorporated into a
carrier which is a biocompatible and can incorporate the protein
and provide for its controlled release or delivery, for example, a
sustained release polymer such as a hydrogel, acrylate,
polylactide, polycaprolactone, polyglycolide, or copolymer thereof.
An example of a solid matrix for implantation into the animal and
sustained release of the protein antigen into the body is a
metabolizable matrix, as described, for example, in U.S. Pat. No.
4,452,775 (Kent), the disclosure of which is incorporated by
reference herein.
[0060] Adjuvants may be included in the vaccine to enhance the
immune response in the animal. Such adjuvants include, for example,
aluminum hydroxide, aluminum phosphate, Freund's Incomplete
Adjuvant (FCA), liposomes, ISCOM, and the like. The vaccine may
also include additives such as buffers and preservatives to
maintain isotonicity, physiological pH and stability. Parenteral
and intravenous formulations of the vaccine may include an
emulsifying and/or suspending agent, together with
pharmaceutically-acceptable diluents to control the delivery and
the dose amount of the vaccine.
[0061] Factors bearing on the vaccine dosage include, for example,
the age and weight of the animal. The range of a given dose is
about 25-5000 .mu.g of the purified siderophore receptor protein
per ml, preferably about 100-1000 .mu.g/ml preferably given in
about 0.1-5 ml doses. The vaccine should be administered to the
animal in an amount effective to ensure that the animal will
develop an immunity to protect against a gram-negative bacterial
infection. For example, for poultry, a single dose of a vaccine
made with Freund's Incomplete Adjuvant would contain about 150-300
.mu.g of the purified siderophore receptor protein per ml. For
immunizing a one-day of age bird of about 60 grams weight, the bird
may be subcutaneously or intramuscularly injected with an about
0.25-0.5 ml dose. For an about 3-week old bird of about 1.5 lbs,
the bird may be injected with about 0.25-1 ml dose. A vaccine for
immunizing an about 5-lb piglet against Salmonella cholerasuis
would contain about 100-5000 .mu.g protein per ml, preferably given
in 1-5 ml doses. In each case, the immunizing dose would then be
followed by a booster given at about 21-28 days after the first
injection. Preferably, the vaccine is formulated with an amount of
the siderophore receptor protein effective for immunizing a
susceptible animal against an infection by two or more strains or
species of gram-negative bacteria that express a siderophore
receptor protein.
[0062] For boosting the immunizing dose, the booster may be a
preparation of whole cells as conventionally used, or a chemically
modified cell preparation, among others. For example, a useful
booster is a preparation of a modified E. coli such as avirulent
R-mutants, as for example, E. coli J5 (commercially available from
ATCC as ATCC #43754; described by Overbeck et al., J. Clin.
Microbiol. 25:1009-1013 (1987)), or Salmonella minnesota
(commercially available from ATCC as ATCC #49284; as described by
Sanderson et al., J. Bacteriol. 119:753-759, 760-764 (1974)) that
lack outer oligosaccharide side chains of the lipopolysaccharide
(LPS) layer of the outer membrane. Outer oligosaccharide side
chains tend to mask SRPs on the cell membrane in such a way that
the immune system does not recognize the SRPs and anti-SRP antibody
titers are depressed. To enhance the ability of a booster made with
intact bacterial cells to elicit an anti-SRP immune response, the
cell membrane of the bacteria can be chemically altered to
eliminate the interfering oligosaccharide side chains. Boosting
with chemically-modified bacteria such as an R-mutant,
advantageously provides an anti-SRP antibody titer that is 5-20
times higher than booster made of a non-modified whole cell
bacterial preparation, or a natural field challenge.
[0063] Although not intended as a limitation of the invention, the
mechanism by which immunization with the present vaccine provides
protection against gram-negative bacterial infection is believed to
be as follows. After an animal has been immunized with the vaccine,
upon being challenged with a pathogenic strain of gram-negative
bacteria, the body responds by producing humoral antibodies that
block the siderophore receptor proteins on the outer membrane of
the bacteria. This prevents iron uptake by the cell, which, in
turn, eventually starves the bacteria of required iron nutrients.
Another mechanism is that humoral antibodies produced in response
to the siderophore receptor proteins in the vaccine, bind to the
siderophore receptor protein on the bacterial membrane to cause
activation of compliment (C'). This results in complement-mediated
bacteriolysis, or increased opsonization which leads to increased
phagocytosis by the mononuclear phagocytic system.
[0064] In addition, the efficacy of this vaccine is based on the
use of purified siderophore receptor proteins rather than using
whole cells. The immune response in animals vaccinated with a
purified SRP preparation is about 20 times greater than the immune
response to a preparation of whole cell grown under iron-restricted
conditions. During gram-negative sepsis, an animal host mounts an
immune response to an invading bacteria. Since the major
constituent of the cell wall of gram-negative bacteria is made of
lipopolysaccharide (LPS), the immune response of an animal is
directed to this structure inducing an immunodominant role for the
LPS cell wall. Outer membrane proteins such as IROMPs or SRPs that
are not dominant proteins on the surface of the bacterial cell wall
induce limited immune response resulting in low antibody titers.
Thus, the use of a bacterin made of whole bacterial cells grown
under iron restriction to express siderophore receptor proteins
provides a limited immune response to the siderophore receptor
proteins due to competing antigens on the cell surface. By
comparison, immunizing an animal with a vaccine made of purified
SRPs, there is less antigenic competition and the animal's immune
system focuses its response on the receptor proteins. Serological
profiles show a significant increase in antibody titer in the
SRP-vaccinated group compared to the whole cell-vaccinated group
when boosted with whole cell expressing SRP.
[0065] Polyclonal antibodies may be raised to the siderophore
receptor protein by hyperimmunizing an animal with an inoculum
containing the isolated siderophore receptor protein. The blood
serum may be removed and contacted with immobilized siderophore
receptor proteins reactive with the protein-specific antibodies.
The semi-purified serum may be further treated by chromatographic
methods to purify IgG and IgM immunoglobulins to provide a purified
polyclonal antibody sera for commercial use.
[0066] Monoclonal antibodies reactive with the siderophore receptor
protein may be raised by hybridoma techniques known and used in the
art. In brief, a mouse, rat, rabbit or other appropriate species
may be immunized with a siderophore receptor protein. The spleen of
the animal is then removed and processed as a whole cell
preparation. Following the method of Kohler and Milstein (Nature
256:496-97 (1975)), the immune cells from the spleen cell
preparation can be fused with myeloma cells to produce hybridomas.
The hybridomas may then be cultured and the culture fluid tested
for antibodies specific for siderophore receptor proteins using,
for example, an ELISA in which specific siderophore receptor
proteins are attached to a solid surface and act as capture
antigens. The hybridoma may then be introduced into the peritoneum
of the host species to produce a peritoneal growth of the
hybridoma, and ascites fluids containing the monoclonal antibody to
the bacteria may be collected.
[0067] The monoclonal antibodies may be used in diagnostic and
therapeutic compositions and methods, including passive
immunization. For example, immunoglobulins specific towards a
siderophore receptor protein may be used to provide passive
immunity against gram negative sepsis. Animals may be treated by
administering immunoglobulins intramuscularly at about 100/mg/kg
body weight, about every 3-7 days.
[0068] A method for diagnosing an infection by gram-negative
bacteria in a body sample may be carried out with the polyclonal
antibody sera or monoclonal antibodies described hereinabove, in an
enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA),
immunofluorescent assay (IFA), a Northern, Western or Southern blot
assay, and the like. In brief, the antibody or body sample (i.e.,
tissue sample, body fluid) may be immobilized, for example, by
contact with a polymeric material such as polystyrene, a
nitrocellulose paper, or other like means for immobilizing the
antibody or sample. The other antibody or body sample is then
added, incubated, and the non-immobilized material is removed by
washing or other means. A labeled species-specific antibody
reactive with the later is added. The serum antibody or
gram-negative bacteria in the body sample, is then added and the
presence and quantity of label is determined to indicate the
presence and amount of gram-negative bacteria in the body
sample.
[0069] The invention will be further described by reference to the
following detailed examples, wherein the methodologies are as
described below. These examples are not meant to limit the scope of
the invention that has been set forth in the foregoing description.
Variation within the concepts of the invention are apparent to
those skilled in the art. The disclosures of the cited references
throughout the application are incorporated by reference
herein.
EXAMPLE 1
Production and Purification of Siderophore Receptor Proteins
[0070] Escherichia coli serotype 078 (turkey isolate; serotyped by
Pennsylvania State University, deposited with the American Type
Culture Collection (ATCC), Bethesda, Md., U.S.A., as ATCC #55652,
on Jan. 3, 1995) (700 ml at 10.sup.8 colonies/ml) was inoculated
into a Virtis bench-top fermenter (Virtis, Inc., Gardiner, N.Y.),
charged with 20-L of brain-heart infusion (BHI, Difco Laboratories,
Detroit, Mich.) containing 50 .mu.grams/ml of dipyridyl (Sigma
Chemical Co., St. Louis, Mo.) at 41.degree. C. This isolate has
been shown to produce four siderophore receptor proteins for (MW 89
kDa, 84 kDa, 78 kDa, 72 kDa) under iron-restrictive conditions. The
pH was held constant at 7.4 by automatic titration with 5N NaOH.
The fermenter was stirred at 400 rpm. The culture was grown
continuously for 18 hours after which the bacteria were removed by
continuous-flow centrifugation at 20,000.times.g at 4.degree. C.
using a Beckman (Model J2-21M) centrifuge (Beckman Instruments,
Eden Prairie, Minn.). The pelletized bacteria were washed two times
with 1,000 ml physiological saline (0.85%) to remove contaminating
culture media proteins.
[0071] The bacteria were resuspended in tris-buffered saline (TBS)
containing 2.0% sodium N-lauroyl sarcosinate (SARKOSYL.TM., Sigma
Chemical Co., St. Louis, Mo.), optical density 5%, 540 nm. The
suspension was incubated at 4.degree. C. for 45 minutes with
continuous stirring. The cells were then disrupted using a
continuous-flow cell sonicator (Banson 450, Danbury, Conn.) at
4.degree. C., with a maximum flow rate of 5 gph. The disrupted cell
suspension was centrifuged at 16,000.times.g for 20 minutes.
[0072] The effluent from the continuous-flow cell sonicator
containing the outer membrane proteins was collected and
concentrated using ethanol precipitation at -20.degree. C. It is
understood that the supernatant may also be concentrated by
membrane concentration using a 50,000 MW cut off diaflow membrane
(Amicon, Danvers, MASS). The concentrated material (10% T at 540
nm) was solubilized using 0.2 percent sodium dodecyl sulfate (SDS)
in TBS at pH 7.4.
[0073] The elution profile of the concentrated material treated
with 0.2% SDS is shown in FIG. 1. The solubilized material was
applied to a Vantage column (Amicon, Danvers, Mass.) containing
3.2-L of cellufine fast flow GC-700 gel matrix (Amicon, Danvers,
Mass.) equilibrated with TBS containing 0.2% SDS at 25.degree. C.
Purification of the protein was monitored by UV absorption at 280
nm. Flow rate through the column was 3,000 ml/hr and 15-ml
fractions were collected using a UA-5 Detector and Retriever 5
fraction collector (ISCO, Inc., Lincoln, Nebr.). Fractions from
each peak were pooled and concentrated using a Diaflo
ultrafiltration apparatus with a 50,000 MWCO membrane. Concentrated
material from each peak was examined by gel electrophoresis. As
shown in FIG. 1, peak 1 contained approximately 85% pure
siderophore proteins. This solution was ethanol precipitated at
-20.degree. C. for 24 hours to remove the SDS, and then resuspended
in phosphate buffered saline (PBS). The amount of protein was
determined using a Pierce BCA protein assay (Pierce, Rockford,
Ill.).
EXAMPLE 2
Preparation of Vaccine with Siderophore Receptor Proteins
[0074] The precipitate from Example 1, hereinabove, containing
siderophore receptor proteins of E. coli serotype 078, were
resuspended in physiological saline (0.85%) containing 0.1%
formalin as a preservative. The protein concentration was 300
.mu.g/ml. The aqueous protein suspension (1,000 ml) was emulsified
in a water-in-mineral oil adjuvant containing 972 ml Drakeol 6
mineral oil and 28 ml of Anlacel A as an emulsifier. The mixture
was emulsified using an Ultra-Turnax T50 emulsifier (KIKA Works,
Inc., Cincinnati, Ohio) at 4.degree. C. The water-in-oil emulsion
was stored at 4.degree. C.
EXAMPLE 3
Vaccination of Poultry with Siderophore Receptor Protein
Vaccine
[0075] Seventy-two turkey poults were raised in isolation from one
day of age. At three weeks of age, the birds were divided into two
equal groups. Group 1 was vaccinated subcutaneously with the
vaccine from Example 2 above, at a dosage level of 150 .mu.g of
siderophore receptor protein per bird. Group 2 remained as
non-vaccinated controls. Group 1 was given a booster vaccination
with the vaccine at a dosage level of 250 .mu.g siderophore
receptor protein per bird at 18 days after the first
vaccination.
[0076] The vaccinated and non-vaccinated birds were equally divided
among four isolation rooms. Rooms A and B contained the vaccinated
birds, and Rooms C and D contained the non-vaccinated controls. At
seven weeks of age, birds in Groups A and C were challenged
subcutaneously with Salmonella agona at 1.0.times.10.sup.8
cfu/bird. At 24, 48, 72, 96 and 120 hours post-challenge, two
controls and two vaccinated birds were killed. The spleens were
aseptically removed from each bird and individually weighed, and
adjusted to 4 grams/spleen, 10 grams/liver. Each sample was then
homogenized in sterile physiological saline using a Stomacher Lab
Blender, Model 3500 (Seward Medical, London). Serial ten-fold
dilutions of each homogenate was plated in duplicate on brilliant
sulfur green plates (Difco Laboratories, Detroit, Mich.).
[0077] The results show the quantitative clearance of Salmonella
agona in spleens of SRP-vaccinated and non-vaccinated turkeys (FIG.
2). Time 0 represents the number of bacteria given to each bird. At
24-hours post-challenge in the vaccinated birds, the level of
bacteria were reduced to zero and remained at that level throughout
the sampling period. In contrast, the non-vaccinated controls
remained positive for the duration of the experiment.
EXAMPLE 4
Cross-Reactivity of Siderophore IROMPS Produced by Escherichia coli
(Serotype 078)
[0078] Hyperimmunized serum produced against purified siderophore
receptor proteins was examined for its cross-reactivity to bacteria
from different genera and species. Siderophore receptor proteins
were produced in the following bacteria: Escherichia coli
(serotypes 01a, 02a and serotype 078 (ATCC #55652)), Salmonella
agona, Salmonella blockley, Salmonella enteriditis, Salmonella
hadar, Salmonella Heidelberg, Salmonella montevideo, Salmonella
senftenberg, Salmonella cholerasuis, and Pasteurella multocida
(serotype A:3,4; deposited with ATCC as ATCC #______, on Feb.
______, 1995: These bacteria, except for S. cholerasuis, were field
isolates obtained from clinically diagnosed birds and serotyped by
the State Poultry Testing Laboratory, Willmar, Minn. (Salmonella
spp.) and Pennsylvania State University (E. coli). Salmonella
cholerasuis was obtained from the University of Minnesota
Diagnostic Laboratory. The bacterial isolates were grown in 100 ml
of BHI broth with dipyridyl (175 mM), and without dipyridyl but
containing 200 .mu.m ferric chloride.
[0079] The bacteria were collected from the cell cultures by
centrifugation at 16,000.times.g for 10 minutes at 4.degree. C. The
cell pellets were washed twice in tris-buffered saline (TBS) at pH
7.4 and resuspended in 30 ml TBS. The cells were ultrasonically
disrupted for 2 minutes at 4.degree. C. using a Branson Ultrasonic
Sonicator (Danbury, Conn.). The disrupted cell suspension was
centrifuged at 16,000.times.g for 20 minutes at 4.degree. C. The
supernatant was collected centrifuged at 30,000.times.g for 2 hours
at 4.degree. C. The pellet was resuspended in 10 ml TBS containing
21 sodium n-lauroyl sarcosine and placed on a gyratory shaker for
45 minutes at 4.degree. C. The detergent insoluble outer membrane
protein-enriched fraction was collected by centrifugation at
30,000.times.g for 2 hours at 4.degree. C. The pellet was
resuspended in 1 ml TBS and stored at -90.degree. C. Proteins were
separated by SDS-PAGE with a 4% stacking gel on a 12% resolving
gel. Laemmli, U.K., Nature, 227:680-685 (1970).
[0080] The outer membrane proteins from the different E. coli,
Salmonella and Pasteurella isolates were transferred from the
SDS-PAGE gels to nitrocellulose membranes (Bio-Rad Laboratories,
Hercules, Calif.). The membranes were probed with negative
(control) and positive antisera to the siderophore receptor
proteins.
[0081] The control antisera was collected from the birds in group
2, as described in Example 3 hereinabove. The positive antisera was
collected from birds in group 1 from Example 3 hereinabove, at 5
days after the second vaccination. The sera, 50 ml each, were
absorbed with killed whole cell bacteria (E. coli 078, Salmonella
heidelberg, Pasteurella multocida) grown in iron-replete media (BHI
containing 200 .mu.m ferric chloride) for 1 hour at 4.degree.
C.
[0082] The SDS-PAGE patterns of the outer membrane protein extracts
of the different bacterial isolates, showed expression of
siderophore receptor proteins when grown under conditions of iron
restriction, in contrast to non-iron restricted controls which did
not express siderophore receptor proteins. Pasteurella multocida
produced three siderophore receptor proteins under conditions of
iron restriction which had molecular masses of approximately 96
kDa, 84 kDa and 80 kDa. The E. coli isolates produced slight
variation in their IROMP profiles. Serotype 078 produced four
siderophore receptor proteins with approximate molecular mass of 89
kDa, 84 kDa, 78 kDa and 72 kDa. Serotype 02a produced three bands
with molecular weights of 89 kDa, 78 kDa and 72 kDa. Serotype 01a
produced two bands with molecular weights of 84 kDa and 78 kDa. All
of the Salmonella isolates examined produced three siderophore
receptor proteins with identical banding patterns with approximate
molecular weights of 89 kDa, 81 kDa and 72 kDa.
[0083] Western blot analysis revealed that the positive antisera
prepared against the purified siderophore receptor proteins of E.
coli 078 reacted intensely with the siderophore receptor proteins
of E. coli serotypes 01a, 02a and the receptor proteins of
Salmonella. The 96 kDa and 84 kDa receptor protein of Pasteurella
reacted with the positive E. coli protein antisera. These results
show that the siderophore receptor proteins of E. coli have
complete antigenic homology to Salmonella and partial homology to
Pasteurella multocida. The control sera did not react with any
siderophore receptor proteins of those species.
EXAMPLE 5
Cross-Reactivity of Siderophore Receptor Proteins of Escherichia
coli (Serotype 078)
[0084] Escherichia coli isolates (150 isolates) originating from
colisepticemic birds were screened for reactivity with the positive
antisera of Example 4, hereinabove. The isolates were examined by
direct agglutination using the siderophore receptor antisera and
negative reference sera. Ninety-eight percent (98%) of the E. coli
isolates were agglutinated using the positive antisera in contrast
to the negative sera. The positive antisera also reacted with
Pseudomonas aeruginosa, Klebsiella pneumoniae and five sero groups
of Salmonella (serotype B, C.sub.1, C.sub.2, D.sub.1 and
E.sub.3).
EXAMPLE 6
Serological Response to Siderophore Receptor Proteins (SRP) of E.
coli in Vaccinated and Non-Vaccinated Flocks Under Natural Field
Conditions
[0085] Fifty one thousand, one-day old turkey poults were equally
divided among two barns designated as barns 1 and 2. At six weeks
of age, birds in barn 1 were subcutaneously injected with a
water-in-oil vaccine as described hereinabove in Example 2. Each
bird received 0.5 cc containing 300 .mu.g E. coli serotype 078
siderophore receptor protein (SRP) in the lower neck region. Barn 2
remained as non-vaccinated controls. Blood was drawn from 15 birds
per barn at weekly intervals.
[0086] FIG. 3 represents the serological response to E. coli SRPs
between vaccinated and non-vaccinated flocks. The antibody response
to the SRPs in the vaccinated flock increased steadily with each
sampling period as compared to non-vaccinated controls. At 35 days
following vaccination, the vaccinated group had a 7.1 times greater
antibody response than the control group.
[0087] Table 1, below, shows the average weight of processed birds
between the vaccinated and non-vaccinated flocks. There was a
statistically greater weight advantage between the vaccinated flock
(12.2 lbs/bird) as compared to the non-vaccinated flock (11.8
lbs/bird).
TABLE-US-00001 TABLE I THE AVERAGE BODY WEIGHT BETWEEN
SRP-VACCINATED AND NON- VACCINATED TURKEYS AT TIME OF PROCESSING
Barn 2 (non-vaccinated) Barn 1 (SRP-vaccinated) Ave. Body weight
Ave. Body weight # of Birds/lot (Lbs) # of Birds/lot (Lbs) 2772
11.85 1986 12.00 3108 11.91 3168 12.11 3024 11.92 3072 12.04 3168
11.97 3060 12.25 3256 11.98 3072 12.36 3186 11.75 3072 12.57 3136
11.65 3024 12.31 2112 11.42 3024 12.16 Total 23762 Mean 11.8 Total
23460 Mean 12.2 SD 0.192 SD 0.18 CV 1.63 CV 1.54
[0088] FIGS. 4 and 5 show the total percent mortality and culls in
E. coli SRP-vaccinated sister flocks (i.e., originating from the
same breeder hens or hatchmates), and the non-SRP-vaccinated
controls, from 3-13 weeks of age. These results show the true field
mortality after vaccination, by excluding early poult mortality
which could result in erroneous results. As can be seen, there was
a significant reduction in both mortality and birds culled in the
SRP-vaccinated flocks. These results demonstrate the usefulness of
E. coli-derived siderophore receptor proteins in a vaccine for
controlling systemic infections caused by E. coli under natural
field conditions.
EXAMPLE 7
Cross-Reactivity of SRPs of Salmonella senftenberg and Pasteurella
multocida
[0089] Forty-eight Nicholas turkey poults were raised in isolation
from one day of age. At three weeks of age, the birds were divided
into two equal groups designated as Group 1 and Group 2. Twelve
birds in Group 1 were vaccinated subcutaneously with (0.5 cc) 300
.mu.g purified SRP isolated from Salmonella senftenberg. The
vaccine was prepared as described in Example 2 above. The remaining
twelve birds were used as non-vaccinated controls. Birds in Group 2
were treated the same as in Group 1, except 12 of the birds were
vaccinated with 300 .mu.g purified SRP isolated from Pasteurella
multocida.
[0090] Blood was taken from all of the birds in both groups at 5
day intervals. Fifteen days after the first injection, vaccinated
birds received a second injection of the appropriate SRP. Each
vaccinated bird received 500 .mu.g, (0.5 cc) SRP subcutaneously in
a water-in-mineral adjuvant. All non-vaccinated birds remained as
controls. Birds were bled at 5-day intervals.
[0091] Fifteen days after the second injection, the vaccinated
birds in Group 1 were intravenously challenged with 100 .mu.g S.
heidelberg SRP (FIG. 7). Blood was taken at 2-day intervals post
challenge. There was a high antibody response to challenge at 2-
and 4-days post challenge. This data shows the cross-reactivity of
S. heidelberg to S. senftenberg. These proteins, in turn, both
cross-react with E. coli, as demonstrated by the ELISA using E.
coli SRPs as the capture antigen according to the protocol
described hereinabove in Example 5.
[0092] Likewise, 15 days after the second injection, all birds in
Group 2 were challenged intramuscularly with 1.1.times.10.sup.6 CFU
of P. multocida, ATCC strain P-1059. Mortality was recorded daily
for 2 weeks post-challenge. FIG. 6 and Table 2 below also shows the
mortality between the vaccinated and non-vaccinated birds following
challenge.
TABLE-US-00002 TABLE 2 Mortality of Vaccinated and Non-Vaccinated
Turkeys Following Challenge with Pasteurella multocida P-1059
Numbers of dead/total tested Non-vaccinated Vaccinated 11/12
(91.6%) 1/12 (8.3%)
[0093] Eleven (91.6%) of the non-vaccinated birds died within 14
days after challenge (see, FIG. 6). In contrast, only 1 (8.3w) of
the birds in the vaccinated group died. These results demonstrate
that siderophore receptor proteins can be used as protective
immunogens.
[0094] FIGS. 7 and 8 show the serological response of birds
vaccinated with siderophore receptor proteins isolated from S.
senftenberg and P. multocida, respectively. The siderophore
receptor proteins induced primary and secondary immune responses in
both vaccinated groups at 10 and 20 days post-vaccination as
compared to non-vaccinated control birds. These antibody responses
demonstrate the cross-reactive nature of these protein, which was
confirmed in the ELISA assay using SRPs isolated from E. coli as
capture antigens.
EXAMPLE 8
Cross-Reactivity of Siderophore Receptor Proteins as Evaluated by
ELISA
[0095] The cross-reactivity of E. coli siderophore receptor
proteins from Example 7 above was further examined using an
Enzyme-Linked Immunosorbent Assay (ELISA). The siderophore receptor
proteins (SRPs) were purified from polyacrylamide gels using a
model 422 electro-eluter (Bio-Rad Laboratories, Hercules, Calif.).
The proteins were then used as capture molecules in an indirect
ELISA test.
[0096] The optimum working concentrations of SRP and conjugate was
determined by several chequerboard titrations using positive and
negative control serums from Example 6 above. A prediction curve
was then established to calculate SRP ELISA titers at a 1:200
dilution. All subsequent tests were performed at a single serum
dilution (1:200) and SRP titers were calculated from the average of
duplicate test absorbance values.
[0097] The ELISA was performed by adding 100 .mu.l of diluted SRP
of E. coli in 0.05 M (0.1 ug) carbonate buffer (pH 9.6) to each
well of a 96-well flat-bottom, easy wash microtiter plate (Corning,
Corning, N.Y.). After overnight incubation at 4.degree. C., excess
SRP was removed and the plate was washed. All subsequent washing
steps were done three times in phosphate-buffered saline (pH 7.4)
with 0.05% Tween 20. The plates were blocked for one hour at
37.degree. C. with 4% Fish Gelatin (Sigma) in PBS and then
washed.
[0098] Duplicate serum samples from Example 7 were tested in
parallel at single-point dilutions using 100 .mu.l/well and
incubated for 40 minutes at 37.degree. C. Each plate contained
positive and negative control sera obtained from birds from Example
4 above. After washing, 100 .mu.l peroxidase-labeled conjugate was
added to each well. After incubation for 40 minutes at 37.degree.
C., the plates were washed and 100 .mu.l of ABTS peroxidase
substrate in buffered H.sub.2O.sub.2 solution (Kirkegaard &
Perry Laboratories Inc., Gaithersburg, Md.) was added to each well.
The substrate was allowed to react for 15 minutes at room
temperature. The reaction was terminated with 50 .mu.l of 1% SDS
and the absorbance read directly using a MR650 microtiter plate
reader (Dynatech Laboratories, Alexandria, Va.).
EXAMPLE 9
Fermentation Protocol for Production of Siderophore Receptor
Proteins
[0099] The following protocol was used to culture E. coli 078 (ATCC
#55652) resulting in expression of six (6) siderophore receptor
proteins.
[0100] An E. coli master seed stock was prepared by growing the
organism in 2000 ml of sterile BHI broth containing 1-500 .mu.g
2,2'-dipyridyl for 8 hours at 37.degree. C. The bacteria were
harvested by centrifugation at 10,000.times.g for 30 minutes. The
culture is washed twice by centrifugation and resuspending the
pellet in sterile PBS. The final pellet was resuspended into 500 ml
sterile BHI containing 20' sterile glycerol. One milliliter of
culture was transferred to a 2-ml cryovial and stored at
-85.degree. C.
[0101] A cryovial (1 ml) of the E. coli master seed stock was used
to inoculate a 100-ml culture flask containing tryptone (10 g/l),
yeast extract (5 g/l), dextrose (2 g/l), NaCl (10 g/l), and
2,2'-dipyridyl (15.0 .mu.g/ml). The culture was incubated at
37.degree. C. for 7 hours, at which time it was inoculated into 2
liters of the above media and allowed to grow for an additional 4
hours at 37.degree. C. The 2-liter culture was used to inoculate a
20-liter Virtis bench-top fermenter (Model 233353, Virtis,
Gardiner, N.Y.) charged with 13 liters of the above-described
media. The pH was held constant between 6.9 and 7.2 by automatic
titration with 30% NaOH and 10% HCl. The stirring speed was 250
rev/minute, and the culture was aerated with 11 liters/minute at
34.degree. C. Foaming was controlled automatically by the addition
of 0.4% silicone defoamer (Antifoam-B, J. T Baker, N.J.). The
culture was allowed to grow continuously at these conditions for 12
hours (O.D. 600 nm=7.10) at which time it was pumped into a
150-liter fermenter (W. B. Moore, Easton PN) charged with 110
liters of the above-described media containing 26.7 .mu.g/ml
dipyridyl and 0.2% defoamer. The conditions in the fermenter were
as follows: 450 rpm, 50 slpm air, 10 psi backpressure, 34.degree.
C., and pH held at 6.9 with NaOH.
[0102] After 12 hours of fermentation, the bacteria were
inactivated by the addition of 0.15% formalin. The bacteria were
harvested by continuous flow centrifugation (20,000.times.g at
4.degree. C.) using two Beckman (Model J2-21M) centrifuges equipped
with JCF-Z continuous flow rotors.
[0103] The pelletized bacteria were then washed to remove
contaminating culture media proteins and further processed as
described above in Example 1. The concentrated material was treated
with 0.2% SDS and eluted as described above in Example 1. The peak
from the elution profile containing approximately 85% pure
siderophore receptor proteins was ethanol precipitated to remove
SDS, and resuspended in PBS.
[0104] The material was separated by SDS-PAGE as described above in
Example 4 with a 4% stacking gel on 12.5% acrylamide gel. The
SDS-PAGE pattern of the outer membrane protein extract showed
expression of SRPs having molecular weights of 91-92 kDa, 89 kDa,
84 kDa, 78 kDa, 74 kDa and 72 kDa.
EXAMPLE 10
Efficacy of Vaccine of SRPs from Escherichia coli Under Natural
Field Conditions
[0105] The efficacy of vaccinating turkeys with E. coli siderophore
receptor proteins (SRPs) under natural field conditions was shown
as follows. A farm complex with a history of disease was chosen for
experimental trials. The facility was a three state operation,
having two brooding barns and eight finishing farms.
[0106] Data was collected for one year prior to vaccination to
establish an accurate profile on mortalities and bird performance
(flocks 1-16 before vaccination). Vaccinating with SRPs was
evaluated for a period of 6 months (flocks 17-24 after
vaccination). A total of 24 flocks comprising 1,160,864 birds was
examined. Vaccination trials began in January and ran through July,
considered to be a critical time period for E. coli infections and
other natural field challenges.
[0107] Brooder barns 1 and 2 were divided in half and designated as
A and B (barn-1) and C and D (barn-2). Approximately 50,000
randomized hens were placed in each barn so that each flock
contained 25,000 birds. All flocks were vaccinated by subcutaneous
injection at 3 weeks of age with a vaccine preparation containing
SRPs (MW 91-92 kDa, 89 kDa, 84 kDa, 78 kDa, 74 kDa and 72 kDa,
SDS-PAGE on 12.5% acrylamide gel) isolated and purified from E.
coli 078 as described above in Example 1. Flocks A and C were
vaccinated with a dosage level of 300 .mu.g SRP and 10.sup.9
TCID.sub.50 Newcastle Disease Virus (NDV) in a water-in-oil
emulsion. Flocks B and D were the controls, and given a dosage
level of 10.sup.9 TCID.sub.50 NDV only.
[0108] At 4 weeks of age, the birds were moved into four
second-stage barns while maintaining identity. At nine weeks of
age, the birds were moved to four finishing barns, keeping identity
on each 25,000 bird flock. Birds were marketed at 12- and 14-weeks
of age and identity was maintained throughout processing.
[0109] Table 3 shows the cumulative farm history before and after
SRP-vaccination. Twenty-four flocks were evaluated, the 16 before
vaccination (1-16) and the 8 vaccinated flocks (17-24) including
controls. Flocks 1-16 were not SRP-vaccinated and included as a
farm history to show the performance advantage to SRP-vaccinated
flocks 17-24.
[0110] Table 3 below, shows the age at which each flock was
marketed, the head count, total percent mortality, condem (i.e.,
condemnation at processing), and average bird weight/lot
processed.
TABLE-US-00003 TABLE 3 Age Head Mortality Flocks (days) Count (%)
Condem (%) Ave. wt. Flock History Before SRP-vaccination 1 97 47818
8.37 1.13 13.88 2 94 45638 12.53 1.17 13.80 3 95 51443 12.87 3.44
13.58 4 96 49999 4.20 1.23 13.86 5 92 49733 4.68 0.96 13.25 6 96
48303 7.36 1.25 13.49 7 101 48722 16.50 2.12 15.10 8 103 51456
12.26 1.41 15.60 9 98 50423 7.84 1.63 14.73 10 96 50880 7.04 1.59
13.81 11 95 46710 14.85 1.16 14.04 12 98 48994 11.32 1.09 13.89 13
92 43433 21.28 1.74 13.23 14 94 49806 9.59 1.08 13.64 15 93 39216
28.08 2.35 12.92 16 96 46119 15.95 1.45 13.76 Flock History After
SRP-vaccination 17 99 48323 8.08 1.45 15.37 18 96 48091 8.15 1.16
14.93 19 96 48748 6.89 1.07 16.16 20 90 48462 7.36 1.06 14.11 21 92
49175 6.11 1.00 15.08 22 90 48261 7.86 0.83 14.38 23 94 51813 5.95
0.92 15.52 24 98 49296 9.44 1.08 16.10 Mean 96/94 48043/49021
12.2/7.5 1.6/1.07 13.9/15.3 SD 2.9/3.5 6.2/1.2 0.63/0.18 0.70/0.77
CV 3.1/3.7 51.4/15.5 40.9/17.2 5.0/5.0
[0111] As shown above in Table 3, the average percent mortality
before vaccination was 12.2.+-.6.2 with a coefficient of variation
(cv) of 51.4% as compared to the average mortality after
vaccination of 7.5.+-.1.2 with a cv of 15.5%. This is a 4.7%
decrease in mortality, which equates to 4700 birds for every
100,000. The decrease in the coefficient of variation (51.4% as
compared to 15.5%) on total mortality illustrates a positive effect
on bird livability and uniformity. FIG. 9 is a graphical
representation of mortalities in consecutive flocks before and
after vaccination.
[0112] Condemnation was also positively effected showing
1.6.+-.0.63 percent before vaccination as compared to 1.07.+-.0.18
percent after vaccination (Table 3 above). The difference, 0.53% is
significant considering the number of birds processed.
[0113] A dramatic effect that was observed by the SRP vaccination
was the increased weight advantage, as seen above in Table 3.
Before vaccination the average bird weight was 13.9.+-.0.70 pounds,
with an average growing time of 96 days. The average weight per
bird after vaccination was 15.3.+-.0.77 pounds, with an average
growing time of 94 days. These results demonstrate the advantage in
performance that can be obtained through SRP-vaccination.
[0114] FIG. 10 shows the serological response to SRPs of E. coli
between the SRP-vaccinated and non-SRP-vaccinated flocks as
determined by ELISA, using purified E. coli SRPs as the capture
molecule. The assay was conducted as described above in Example 8.
The profile was consistent between the vaccinated and
non-vaccinated flocks under natural field conditions. As the
profile illustrates, once the bird's immune system becomes focused
to recognize these proteins, continuous field challenge by bacteria
expressing SRPs causes a steady rise in antibody titer to a level
which provides protection and/or to the point where systemic
challenge does not effect performance.
[0115] Using purified IROMPs in a vaccine optimizes the animal's
immune system to focus on those proteins. The birds vaccinated with
300 .mu.g purified SRP at three weeks of age showed an increase in
titer at 11 weeks of age which was 10,000 times greater than the
titer in the non-SRP-vaccinated controls. This increase in titer is
the result of focusing the immune system to recognize these
proteins. Once vaccinated, the bird establishes a population of
memory cells that are activated upon each field challenge. Under
natural field conditions, the bird is continuously challenged by
gram-negative bacteria such as E. coli, which express SRPs that
cross-react and cause a continuous rise in antibody titer (as was
seen in the SRP-vaccinated birds). By comparison, the control birds
under the same conditions, show low antibody titers even though
exposed to the same field challenges.
EXAMPLE 11
Vaccination with SRP-Vaccine and Vaccine Made with Bacterial Whole
Cells
[0116] A comparison was made between turkeys injected with a
vaccine made of purified SRPs derived from Salmonella Heidelberg
prepared as described above in Example 1, and a vaccine made of
bacterial whole cells of the same organism grown under
iron-restrictions so as to express SRP on the cell surface. The
whole cell bacteria was prepared as described in Example 1, except
for the following modification: after the fermentation process 0.3%
formalin were added to the vessel to kill the organism. The killed
bacteria were collected as described in Example 1, washed and
resuspended in physiological saline, and adjusted to an optical
density of 35% T at 540 mm to give approximately 10.sup.7
bacteria/ml. The vaccine was prepared as described above in Example
2.
[0117] Forty-five thousand one-day old hybrid turkey poults (hens)
were raised to 4 weeks of age, on a brooding facility. At four
weeks of age, the birds were moved to a growing facility and
equally divided among two barns designated as barns 1 and 2. At 6
weeks of age, birds in barn 1 were vaccinated subcutaneously in the
lower neck with 0.5 cc of the SRP vaccine while the birds in barn 2
were vaccinated with the whole cell preparation. Blood was taken
from 12 birds/barn at weekly intervals to monitor the serological
response to SRP between the two groups.
[0118] FIG. 11 shows the titer to SRP between whole cell and
SRP-vaccinated birds. The immunological response to SRP was
significantly greater in purified SRP-vaccinated group as compared
to the whole cell vaccinated group. These results clearly
demonstrate the efficacy of using a substantially pure preparation
of SRP for inducing an immune response in an animal in contrast to
using whole cell expressing the same SRP.
EXAMPLE 12
Transfer of Anti-SRP Antibodies to Breeder Hen Progeny
[0119] The 10-day mortality in progeny from SRP-vaccinated and
non-vaccinated breeder hens was evaluated to assess the transfer of
anti-SRP antibodies from adult to progeny.
[0120] Twenty thousand randomized Nicholas turkey poults (hens)
were equally divided among two brooder barns designated as barns 1
and 2. At four weeks of age, all birds in barn 1 were vaccinated
with 300 .mu.g of E. coli SRP and Newcastle Disease Virus (NDV) in
a water-in-oil vaccine. Birds in barn 2 were given NDV only and
acted as controls. At 24 weeks of age, the birds from barn 1 were
given a second injection of SRP at 300 .mu.g/bird. Birds from barn
2 remained as non-vaccinated controls. At thirty weeks of age, the
birds were placed in barns 1 and 2 of a laying farm. At mid-lay,
eggs were collected from the SRP-vaccinated and non-vaccinated
hens. Eggs were set in separate incubators and hatchers. At hatch
time, all poults were treated the same and identity was maintained
throughout sexing and servicing.
[0121] Five thousand poults (hens) from each group were placed in a
commercial brooding barn and kept in brooding rings at 7
rings/group containing 714 poults/ring. Poult mortality was
monitored for each ring/group for a period of 10 days.
[0122] The total 10-day mortality in poults originating from the
SRP-vaccinated hens was 105 (2.1%) as compared to 160 (3.2%) in the
non-vaccinated progeny (FIG. 12). This is a 1.1% advantage in poult
livability, which equates to 1100 poults for every 100,000. This is
significant considering that there are 200 million turkeys in the
United States and 7 billion broilers worldwide.
[0123] These results show the beneficial effect of vaccinating
breeding stock to induce maternal antibody to SRP in progeny to
reduce gram-negative infections that are responsible for much of
the early poult mortality.
EXAMPLE 13
Cross-Reactive and Cross-Protective Nature of Siderophore Receptor
Proteins (SRP) Between Different Serogroups of Salmonella
[0124] The SRP of Salmonella enteritidis (Se), serogroup D.sub.1
and Salmonella typhimurium (St), serogroup B were examined for
their ability to cross-react and cross-protect. Briefly, 160
randomized hybrid turkey poults (hens) were raised in isolation. At
three weeks of age, the birds were equally divided among 4
isolation rooms, 40 birds/room, designated as A, B, C and D. Birds
in group C were subcutaneously injected with a water-in-oil
vaccine, as described hereinabove in Example 2, containing 300
.mu.g SRP of S. typhimurium. Birds in room D were subcutaneously
injected with 300 .mu.g SRP of S. enteritidis. Birds in rooms A and
B remained as non-vaccinated controls. Blood was taken from 10
birds/group at weekly intervals to monitor the serological response
to SRP.
[0125] Twenty one days after the first injection, birds in groups C
and D were given a second injection containing 300 .mu.g of the
appropriate SRP. Blood was taken at 5 and 10 days after the second
injection. The serological response to SRP was examined by ELISA
using E. coli SRP as the capture molecule as described above in
Example 8.
[0126] FIGS. 13 and 14 show the serological response of birds
vaccinated with SRP isolated from S. typhimurium and S.
enteritidis. The immunological response to SRP increased steadily
in both groups with each sampling period as compared to the
non-vaccinated controls, showing the immunogenicity of these
proteins. Importantly, these results show the cross-reactive nature
of these proteins since the ELISA is using E. coli SRP as the
capture molecule.
[0127] Fifteen days after the second injection, all birds were
intravenously challenged with a nalidixic acid resistant strain of
S. enteritidis or S. typhimurium at 5.0.times.10.sup.7 colony
forming units (CFU)/bird. These bacteria were made resistant to
nalidixic acid to enhance their isolation by incorporating
nalidixic acid in the recovery media which eliminated any
contamination. Bacteria resistant to nalidixic acid were prepared
as follows: One ml of a 12-hour Tryptic soy broth (TSB) culture of
S. enteritidis and/or S. typhimurium containing approximately
10.sup.8 viable organisms, was spread over the surface of a
brilliant sulfur green (BSG) agar (Difco) plate containing 500
.mu.g/ml nalidixic acid (Sigma). The plates were incubated at
37.degree. C. for 24 hours and the colonies that grew were cloned
by plating on BSG containing 250 .mu.g/ml nalidixic acid. The
nalidixic acid-resistant strains of salmonella were incubated in
100 ml of TSB at 37.degree. C. for 12 hours. At the end of
incubation, the culture was centrifuged (10,000.times.g) and washed
twice in PBS (pH 7.4), and the optical density was adjusted to 35%
transmission at 540 nm to obtain 5.0.times.10.sup.7 CFU/ml. These
isolates were then used for challenge.
[0128] To evaluate homologous and heterologous protection, twenty
birds in room C (vaccinated with St-SRP) were wing banded and moved
into room D, and 20 birds in room D (vaccinated with Se-SRP) were
wing banded and moved to room C. All birds in room C (20
St-vaccinated and 20 Se-vaccinated) were challenged with S.
typhimurium, while birds in room D (20 Se-vaccinated and
St-vaccinated) were challenged with S. enteritidis.
[0129] At 24, 48, 72 and 96 hours post-challenge, two birds from
each group were killed. The spleens were aseptically removed from
each bird and individually weighed, and adjusted to 4 grams/spleen.
A fecal sample from the cecal junction from each bird was also
taken. Each sample was weighed and adjusted to 0.5 grams. Four
milliliters of sterile saline was added to each spleen and 0.5 ml
to each fecal sample. Each sample was homogenized using a Stomacher
Lab Blender (Sewert Medical, London) for 1 minute. Serial ten-fold
dilutions of each homogenate were plated in duplicate on BSG plates
containing 250 .mu.g/ml nalidixic acid.
[0130] The results show the quantitative clearance of S.
typhimurium (St) (FIG. 15) and S. enteritidis (Se) (FIG. 16) in
spleens of SRP-vaccinated and non-vaccinated turkeys. As shown in
FIGS. 15 and 16, there was a steady decline in the number of
bacteria/spleen. At 96 hours after challenge (chlg), the difference
between the vaccinated and non-vaccinated groups was approximately
2.5 logs. An important aspect of these results is the
cross-protective nature induced by these proteins. FIG. 15 shows
the cross-protective nature of the birds vaccinated with the SRP of
Se but challenged with St. FIG. 16 shows this same cross-protective
effect of birds vaccinated with SRP of Se and then challenged with
St. All vaccinated groups showed a significant reduction in the
number of bacteria in spleens in contrast to the non-vaccinated
birds.
[0131] At 72 and 96 hours after challenge, intestinal shedding of
Salmonella was detected in the non-vaccinated birds at greater then
log 4. In contrast, all of the vaccinated birds were negative for
Salmonella within this same sampling period. These results indicate
that these proteins may have some beneficial effect in preventing
the intestinal colonization of Salmonella.
EXAMPLE 16
Preparation and Use of the 37-38 kDa Transmembrane and Porin
Proteins in a Vaccine
[0132] The transmembranes and porin proteins (MW 34-38 kDa),
identified as OmpA, OmpC, OmpD and OmpF are expressed with and
without iron. These proteins can be purified as described above in
Example 1, by collecting fractions 1650-2250 as shown in FIG. 1.
These proteins can be combined with peak 1 (FIG. 1) to obtain a
combination of SRP and porin proteins that are conserved among
Salmonella, E. coli, and Pasteurella.
[0133] A vaccine containing E. coli SRPs (MW 89 kDa, 84 kDa, 78 kDa
and 72 kDa) was combined with porins (MW 34 kDa-38 kDa) to give a
total protein content of 600 .mu.g/ml, and prepared as described
above in Example 2. The vaccine was used to induce hyperimmunized
sera. Briefly, six (6) three-week old turkeys were given a single
subcutaneous injection in the lower neck region followed by a
second injection 15 days after. Serum was collected 10 days after
the second injection.
[0134] Western blot analysis, as described above in Example 4,
using sarcosine cell wall extracts of E. coli, Salmonella and
Pasteurella and probed with the above sera revealed cross-negative
proteins in the 34 kDa and 38 kDa region as well as the SRPs from
each isolate examined.
[0135] These results indicate the potential of using conserved
protein (SRP and porins) as an effective method for vaccinating
against gram-negative infections.
* * * * *