Note: Descriptions are shown in the official language in which they were submitted.
CA 02243~26 1998-07-l~
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- 1
COMPOSI~IONS AND ~ETHODS FOR ADMINI~TERING
Bo~R~r~TA BURGDORFERI ANTIGEN~
~TATEMENT OF GOV~RNM~NT ~u~T
This work was supported by NIH grant RO1
AI37248, and without any admission the U.S. Government
may have certain rights.
RELATED APPLICATIONS
Reference is made to applications Serial Nos.
08/320,416, filed October 3, 1994, 08/137,175, filed
October 26, 1993, 08/262,220, filed June 20, 1994,
PCT/US95/07665, 08/373,455, filed January 17, 1995,
PCT/US92/08697, and WO 90/04411, each of which is hereby
incorporated herein by re~erence. Several documents are
cited in this application, with full citation thereof
where cited, or in the listing headed "References" before
the claims; and, each document cited herein is hereby
incorporated herein by reference.
FIELD OF THE l~v~lON
This invention relates to methods for
administering Borrelia burgdorferi antigens. More
particularly, this invention relates to methods for
administering Borrelia burgdorferi OspA (outer surface
protein A), especially recombinant OspA (rOspA), and/or
OspD (outer surface protein D), especially recombinant
OspD (rOspD), or fragments thereof; and to compositions
employed in such methods. Even more particularly, this
invention relates to methods for mucosally administering
OspA such as rOspA, e.g., for orally administering OspA,
e.g., rOspA, especially to a host mammal susceptible to
Lyme disease infection, e.g., humans, domesticated
animals, and even non-domesticated or wild ~n;m~l s (since
the present invention provides that OspA or rOspA can be
left in the wild with bait so as to allow for
administration without contact with the wild animals,
thereby diminishing the Borrelia burgdorferi population
and ergo the ability for Borrelia burgdorferi and Lyme
CA 02243~26 l998-07-l~
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- 2
disease to be transmitted to domesticated animals and
humans); and, to compositions therefor.
BACRGRO~ND OF THE lNV~r~l lON
Lyme disease is a multisystem illness,
transmitted by ticks of the Ixodes ricinus complex. The
spirochaete Borrelia burgdorferi sensu lato is the
aetiologic agent of Lyme disease, which is now the most
common arthropodborne disease in the United States, and
is endemic in Central Europe (1). Although curable by
antibiotic therapy in its early stages, if Lyme disease
is allowed to progress, cardiac, neurological and joint
abnormalities can arise. Investigations into the
development of a human vaccine for Lyme disease are under
way. The outer surface lipoprotein OspA of Borrelia
burgdorferi is the current major candidate molecule for
development of such a vaccine. Recombinant OspA
lipoprotein (rOspA) is known to elicit a protective
immune response in mice against challenge by infectious
B. ~urgdorferi (2,3). OspA is currently undergoing human
field trials as a subcutaneously administered vaccine in
the United States (4).
Above-cited applications 08/373,455 and
PCT/US92/08697 relate to rOspA vaccines, especially
lipidated rOspA, and methods for expressing DNA encoding
OspA or fragments thereof. Above-cited applications
08/320,416 and WO 90/04411 relate to DNA encoding OspA,
the amino acid se~uence of OspA, synthetic OspA,
compositions containing OspA or synthetic OspA, and
methods of using such compositions. And, the other
above-cited applications relate to DNA encoding other
Borrelia antigens or other Osps, or to DNA encoding
useful fragments of OspA or of other Osps or of other
Borrelia antigens, amino acid sequences thereof,
compositions containing such fragments or other Osps, and
methods for using such compositions; and, such DNA can be
used in the methods of 08/373,455 or PRC/US92/08697 to
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- 3
produce OspA, other Borrelia antigens or osps, or
fragments thereof, for use in this invention.
Alternative vaccination strategies are
desirable as such provide alternative routes to
administration, thereby allowing administration to humans
who may be sensitive to injections, e.g., young children
or infants, or to other hosts with whom there is
difficulty giving injections, e.g., wild animals, and
even domestic animals.
OspA a~m;n;ctered orally in an Escherichia coli
was capable of stimulating a mucosal immune response that
protected mice against challenge with infectious B.
burgdorferi ( 5). More recently, Dunne et al. reported
oral immunization of mice with an attenuated strain of
Salmonella typhimur7~m expressing OspA, which appeared to
protect 80% of the mice from challenge by infectious B.
burgdorferi (6). Mucosal immunity was also demonstrated
following intra-nasal administration of recombinant BCG
expressing OspA (7). However, rOspA in E. coli,
Salmonella expressing OspA, and, BCG expressing OspA, are
not viable products for usefully administering rOspA to
humans or animals - domestic or wild - as E. coli,
Salmonella and BCG are not safe or approved for
administration to humans or animals (and even if
attenuated, there is nonetheless a chance of reversion);
and, one cannot be certain if any immunological response
in these prior publications was not an effect of an
adjuvanting or immunolocial stimulating effect of E.
coli, Salmonella or BCG (note, for instance, how LPS is
known to have an adjuvanting effect).
Thus, heretofore the art has not taught or
suggested mucosal, preferably oral, administration to a
mammalian host - domesticated or wild animal or human -
susceptible to Lyme disease, of Borrelia antigen or
immunological fragment thereof, e.g., OspA, preferably
rOspA, more preferably lipidated OspA or rOspA,
preferably substantially free of other bacterial proteins
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- 4
and substantially free of lipopolysaccharide (LPS), in a
suitable carrier or diluent in an amount sufficient to
induce an immunological response preferably a protective
immunological response, in the host, preferably without
any necessity of using any immunogenicity-enhancing
adjuvant; or compositions therefor; and, the protection
by such administration herein demonstrated has not been
heretofore taught or suggested. Further, heretofore the
advantages of such oral a~m;nictration~ e.g., ease of
administration to domestic animals and young children or
infants by merely droppering into the mouth, ease of
administration to wild animals by dropping bait
containing the OspA or rOspA, has not been taught or
suggested.
08JEC~S AND S~MMARY OF THE lNv~NllON
It is an object of the invention to provide
methods and compositions for mucosally, e.g., orally
administering to a mammalian host susceptible to Lyme
Disease Borrelia burg~orferi isolated and/or purified
Borrelia antigen or a fragment thereof, e.g., OspA,
preferably rOspA, more preferably isolated and/or
purified lipidated OspA or rOspA substantially free of
other bacterial proteins, substantially free of LPS, in a
carrier or diluent, in an amount sufficient to induce an
immune response, preferably a protective immune response,
and preferably without any necessity of adding or using
any immunogenicity-enhancing adjuvant.
More particularly, it has been surprisingly
found that OspA in a carrier or diluent orally
administered induces a protective response against
Borrelia burgdorferi.
Thus, in a broad sense, the invention provides
a method for eliciting an immunological response
comprising mucosally, preferably orally, administering an
isolated and/or purified Borrelia antigen, or
immunologically active fragment thereof in admixture with
a suitable carrier or diluent, to a host susceptible to
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- 5
Lyme disease or Borrelia infection, and to compositions
containing the antigen and carrier or diluent.
Preferably the antigen or fragment is lipidated such that
the composition and method need not contain or use an
adjuvant. The compositions can be in solid or li~uid
form.
The present invention therefore preferably
provides a method for inducing an immunological response,
preferably a protective immunological response, in a host
susceptible to Lyme disease comprising mucosally
administering a composition comprising an immunologically
effective, preferably for protection, amount of Borrelia
burgdorferi OspA and a suitable carrier or diluent. The
OspA can be rOspA, and is preferably lipidated,
substantially free of other bacterial proteins, and
substantially free of LPS. The composition need not, and
preferably does not, contain any adjuvant. The mucosally
administering is preferably by orally administering. The
concepts of substantially free of LPS and substantially
free of other bacterial proteins is as in the
aforementioned and cited "Related Applications". The
carrier or diluent can be a li~uid vehicle such as PBS,
or bait suitable for wild animals. The Borrelia antigen
or fragment, e.g., OspA, can be present in amounts
determined by factors well-known in the medical or
veternary arts, or as disclosed in the aforementioned and
cited related applications, e.g., 0.5-500 ~g; presently
preferably 0.5 to 50 ~g, for instance, 1-10 ~g.
Accordingly, the present invention more
preferably provides a method for inducing an
immunological response, preferably a protective
immunological response, in a host susceptible to Lyme
disease comprising orally administering a composition
consisting essentially of a carrier or diluent and
recombinant lipidated Borrelia burgdorferi OspA which is
isolated and/or purified, substantially free of other
bacterial proteins and substantially free of LPS.
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WO97/26006 PCT~S97/00557
Compositions employed in the methods are
comprehended by the invention.
Fragments of OspA which induce a protective
response can also be used in the practice of the
invention. As to Borrelia antigens and fragments
thereof, the reader is directed to the applications cited
under "Related Applications" and incorporated herein by
reference.
Other objects and embodiments are disclosed or
are obvious from the following Detailed Description.
BRIEF DESCRIPTION OF DRAWING8
In the following Detailed Description reference
is made to the accompanying Figures, incorporated herein
by reference, wherein:
Figure l(A) shows Immunoblots of sera from mice
immunized o.g. with rOspA or rOspD showing antibody to
rOspA in the mice with rOspA (24 ~g rOspA was loaded onto
a preparative PAGE gel which was subsequently
immunoblotted. Sera were diluted 1:100 in PBS containing
0.3~ milk. Positive control (+) was monclonal antibody
(MAb) H5332, specifice for OspA, hybridoma supernatant
diluted 1:50 in P8S/milk. Negative control (-) was MAb
HlC8, specific for OspD, hybridoma supernatant, diluted
1:50 in PBS/milk);
Figure l(B) shows Immunblots of sera from mice
immunized orogastrically (24 ~g rOspD was loaded onto a
preparative PAGE gel for immunoblotting. Sera were
diluted 1:100 in PBS/milk. Positive control (+) was MAb
HlC8); and
Figure ltC) shows T~llnoblots of sera from mice
immunized subcutaneously with 4 ~g rOspD (24 mg rOspD was
loaded onto a preparative PAGE gel for immunoblotting.
Sera were diluted 1:200 in PBS/milk. Positive control
(+) was MAb HlC8); and
Figure l(D) shows Immunoblots of sera ~rom mice
immunized o.g. or s.c. with rOspA and rOspD showing
detection of IgA subclass immunoglobulin with an IgA-
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WO 97/26006 PCT/US97100557
specific conjugate (Mouse sera were diluted 1:400 1:400
PBS/milk. IgG positive control (~-OspA +) control was
MAb H5332 (IgG2a) hybridoma supernatant diluted 1:10 in
PBS/milk, and the strip was incubated with an IgG-
specific conjugate. IgG negative control tIgG -ve) was
H5332 hybridoma supernatant, and the strip was incubated
with the IgA-specific con3ugate. Control for IgA-
specific conjugate background (IgA -ve) was a trip
incubated in PBS/milk and then in IgA-specific conjugate.
IgA positive control was mouse myeloma protein
TEPC15(IgAk)).
DE~TT,~n DESCRIPTION
As discussed above, the invention preferably
provides methods for immunizing or vaccinating a host
susceptible to Lyme disease, e.g., a m~m~lian host,
against Borrelia burgdorferi and accordingly ~yme
Disease, by mucosally, preferably orally, administering
OspA, preferably lipidated Osp~; more pref~-rably
lipidated OspA substantially free of other bacterial
proteins and substantially free of LPS, most preferably
lipidated rOspA substantially free of other bacterial
proteins and substantially free of LPS, in a suitable
carrier or diluent, and preferably without any necessity
of there being any immunogenicity-enhancing adjuvant
present.
Indeed, as shown in Example 1 recombinant outer
surface protein A (rOspA) from Borrelia burgdorferi
strain B31, substantially free of other bacterial
proteins and substantially free of LPS, was administered
orally to mice at doses of 4.0 ~g and 2.0 ~g
respectively, in carrier or diluent (PBS) without any
ad3uvant. Mice were challenged with 104 infectious
Borrelia burgdorferi and organs were cultured to
determine protection. In 2 experiments, 8/8 mice that
received 4.0 ~g of rOspA were protected against infection
by B. burgdorferi, 6/7 mice that received 2.0 ~g rOspA
were protected 0/8 of the mice that received 4.0 ~g rOspD
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- 8
were protected. Enzyme linked immunosorbent assay
(ELISA) and immunoblot analysis revealed that those mice
which received rOspA orally mounted a specific antibody
response against rOspA and that the sera contained
antibodies of the IgA immunoglobulin subclass. In
contrast, no antibody response to rOspD was detectable in
the sera from those mice immunized orogastrically with
OspD. Sera from mice immunized with rOspD
subcutaneously, however, did contain rOspD-specif~c
antibody detectable on immunoblots. Sera from the mice
given rOspA orally also inhibited growth of B.
burgdorferi B31 in vitro.
As shown by present dog and human trials from
the previous work with mice (3), it is clear that mice
are now a suitable animal model with respect to Borrelia
burgdorferi and Lyme disease for extrapolation to
domestic animals, humans, and other animals susceptible
to Lyme disease or Borrelia burgdorferi infection (e.g.,
wild animals such as deer).
In view of the broad nature of the invention,
i.e., that the invention is applicable to Borrelia
antigens and immunologically active fragments thereof,
discussion herein directed to OspA is intended to
encompass the broad nature of the invention, i.e., "OspA"
is exemplary and can be read in this specification to
include "Borrelia antigen or an immunological fragment
thereof".
The mucosal administration in the present
invention is preferably oral a~;n;~tration; but, the
invention broadly comprehends oral, nasal, peroral,
sublingual, perlingual, intragastric, anal, vaginal or
other mucosal routes of administration.
In the present invention OspA (or broadly the
Borrelia antigen or immunologically active fragment
thereof) can be administered in dosages and by techniques
well known to those skilled in the medical or veterinary
arts taking into consideration such factors as the age,
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g
sex, weight, species and condition of the particular
patient, and the route of administration. OspA (or
antigen or fragment thereof) can be administered alone,
or can be co-a~;n;~tered or sequentially a~m;n;ctered
with other antigens; and, the OspA (or antigen or
fragment thereof) can be sequentially a~m;n;ctered, e.g.,
each Spring as the "Lyme disease season" is about to
begin.
In the present invention the OspA (or antigen
or fragment thereof) can be in solutions, suspensions,
emulsions, syrups, elixirs, capsules (including gelcaps-
gelatin capsule containing a liquid OspA, antigen or
fragment preparation), tablets, hard-candy-like
preparations, and the like. The OspA (or antigen or
fragment) may be in admixture with a suitable carrier,
diluent, or excipient such as sterile water,
physiological saline, PBS, glucose or the like. The
compositions can also be lyophilized. The compositions
can contain auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, adjuvants,
gelling or viscosity enhancing additives, preservatives,
flavoring agents, colors, and the like, depending upon
the route of administration, antigen and the preparation
desired (e.g., adjuvant is not presently preferred
especially for lipidated antigens or fragments thereof;
but, may be useful for non-lipidated antigens or
fragments thereof).
Standard texts, such as "REMINGTON'S
PHARMACEUTICAL SCIENCE", 17th edition, 198~, incorporated
herein by reference, may be consulted to prepare suitable
preparations, without undue experimentation. Suitable
dosages can also be based upon the examples below, and
upon the documents herein cited. In view of such
standard texts as "REMINGTONS'S", and such commercially
available products as Dristan~ nasal spray, Vancenase~ AQ
nasal spray and the like, no undue experimentation is
required to nasally administer OspA ~or Borrelia antigen
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- 10
or fragment thereof~; or to make preparations therefor.
And, in view of such standard texts as "REMINGTON'S" and
commercially available anal suppository and vaginal
suppository products, no undue experimentation is
required to anally or vaginally administer OspA (or
Borrel ia antigen or fragment thereof); or to make
preparations therefor.
Further, as shown herein, mucosal
administration of OspA (or Borrelia antigen or fragment
thereof~ in accordance with the invention stimulates an
immune or antibody response in humans or animals. This
antibody response means that the inventive method can be
used for merely stimulating an immune response (as
opposed to also being a protective response) because the
resultant antibodies (without protection) are nonetheless
useful. From eliciting antibodies, by techni~ues well-
known in the art, monoclonal antibodies can be prepared;
and, those monoclonal antibodies, can be employed in well
known antibody binding assays, diagnostic kits or tests
to determine the presence or absence of Borrel ia
burgdorf eri or to determine whether an immune response to
the spirochete has simply been stimulated. Those
monoclonal antibodies can also be employed in
immunoadsorption chromatography to recover or isolate
Borrel ia antigens such as OspA. Monoclonal
antibodies are immunoglobulins produced by hybridoma
cells. A monoclonal antibody reacts with a single
antigenic determinant and provides greater specificity
than a conventional, serum-derived antibody.
Furthermore, screening a large number of monoclonal
antibodies makes it possible to select an individual
antibody with desired specificity, avidity and isotype.
Hybridoma cell lines provide a constant, inexpensive
source of chemically identical antibodies and
preparations of such antibodies can be easily
standardized. Methods for producing monoclonal
antibodies are well known to those of ordinary skill in
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11
the art, e.g., Koprowski, H. et al., U.S. Patent No.
4,196,265, issued April 1, 1989, incorporated herein by
reference.
Uses of monoclonal antibodies are known. One
such use is in diagnostic methods, e.g., David, G. and
Greene, H. U.S. Patent No. 4,376,110, issued March 8,
1983; incorporated herein by reference. Monoclonal
antibodies have also been used to recover materials by
immunoadsorption chromatography, e.g., Milstein, C. 1980,
10 Scientific American 243:66, 70, incorporated herein by
reference.
Accordingly, the inventive methods and products
therefrom have several hereinstated utilities. Other
utilities also exist for embodiments of the invention.
A better understanding of the present invention
and of its many advantages will be had from the following
examples, given by way of illustration.
~;!lrl~MpT~E~
EXAMPLE 1 - ORAL ADNINISTRATION
OF O~pA AND OspD
MAT~RT~TS & METHODS
Borrelia burgdorferi: B. burgdorferi strain Sh-
2-82, a strain from the same OspA serogroup as B31, was
used for infectious challenge (8). She-2-82 had been
cloned by limiting dilution and passaged in SCID mice and
was stored frozen at -135~C in BSK II containing 10% of
v/v DMSO (ATCC, Rockville. MD) until needed. Stain B311
is a clonal, high-passage, non-infectious derivative of
B31 (ATCC 35210) that produces OspA and OspB (9). HB19Rl
is a high passage, non-infectious derivative of HB19
selected for by growth in the presence of antibodies to
OspA and OspA (15). HB19R1 does not produce OspA or OspB
but produces OspD.
Recomb~nant lipoproteins: Recombinant,
lipidated outer surface proteins rOspA and rOspD for
Borrelia ~urgdorferi B31 were obtained and purified as
described previously (3) and were provided by Dr. R.
Huebner of Connaught Laboratories, Swiftwater, PA. rOspA
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- 12
was provided in 50 mM Tris, pH 7.5; 10 mM NaC1; 2 mM
EDTA; 0.3% Triton X-100. rOspD buffer was at pH 6.5, but
was otherwise identical to the rOspA buffer.
Immunization: Female C3H/HeN mice were obtained
from Harlan laboratories (Indianapolis, IN). At age 10
weeks, mice were vaccinated with rOspA or rOspD from
Borrelia b~rgdorferi B31. Administration of vaccine was
via the oral route, using 0.5 ml rOspA diluted in sterile
PBS (pH 7.4). Vaccine was delivered through 20 guage,
1.5 in stainless gavage needles (Popper ~ Son Inc., NY).
Challsnge ~ith infectious: B. burgdorferi Mice
were injected intra-dermally at the base of the tail with
104 (100 times the ID50) B. burgdorferi Sh-2-82 (3). 10
days after challenge with infectious ~. burgdorferi Sh-2-
82, mice were sacrificed. Mice were anaesthetized withMetofane (Pitman-Moore Inc., Mundelein, IL),
exsanguinated by cardiac puncture and were euthanized by
cervical dislocation. Heart, urinary bladder and cross-
cuttings of the tibiotarsal joints were aseptically
removed. ~hese organs, and 0.5 ml plasma, were cultured
in BSK II containing 10% rabbit serum, and 35~C.
Cultures were examined for the presence of spirochetes by
phase contrast microscopy on day 5 after sacrifice and
were ~;ned up to day 16. Cultures were considered
negative if no spirochetes were seen in 20 high power
fields.
Enzyme linked i lunosorbent assay (ELI8A):
Mouse sera were sub3ected to a whole wet cell ELISA,
described previously (9). Plates were coated at 4~C for
48h with 107 B. burgdorferi strain B311 cells (9) per
well, in bicarbonate coating buffer (15 mM Na2C03), 35 mM
NaHC03, 3 mM NaN3, pH 9.6) or with rOspA as described by
Erdile et al. (3). Serial dilutions of mouse sera were
made in PBS (pH 7.4) containing 1% w/v non-fat dried
milk. Secondary antibody was either goat anti-mouse
IgG+IgA+IgM (H+L) or goat anti-mouse IgA (H+L),
conjugated to alkaline phosphatase (Zymed Laboratories,
-
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13
South San Francisco, CA) used at a dilution of 1: 1000 in
PBS/1% milk.
Po}yacrylamide gel electrophoresis (PAGE) and
immunoblotting: PAGE and immunoblotting were carried out
as described (10,11). 24~g of recombinant proteins rOspA
and rOspD were run on preparatives SDA-PAGE gels and were
then transferred onto nitrocellulose membranes.
Immunoblots were dried and stored at 4~C until needed.
IgA positive control was purified mouse myeloma protein
TEPC15 (IgAk). Monoclonal antibody hybridoma supernatants
from H5332 and HlC8 were used at a dilution of 1: 10 in
1% PBS/milk, and served as positive controls for rOspA
and rOspD, respectively.
Growth Inhi~ition Assay: In vitro growth
inhibitory activity of mouse sera was assessed as
described by Sadziene et al (lZ). 2 hemolytic units (HU)
of unheated guinea pig complement (Calbiochem-Novabiochem
Corporation, San Diego, CA) was added to each well to
give a final concentration of 10 HU/ml of medium after
addition of antibody. Microtiter wells were monitored
visually for changes in the color of the phenol red
indicator and by phase-contrast microscopy of wet mounts.
The growth inhibitory (&I) titer was defined as the
lowest dilution of antiserum that resulted in pink
instead of yellow wells and represented at least 20-fold
fewer cells than in control (no immune serum) wells.
Trypsin digestion: Lipidated recombinant
proteins rOspA and rOspD were both diluted to a
concentration of
250 ~g/ml in Osp dilution buffer. One hundred microliter
volumes were dispensed into wells of a 96-well microtiter
plate. L-1 Tosylamide-2-phenylethyl chloromethyl ketone
(TPCK) - treated trypsin (Sigma Chemical co., St. Louis,
MO) stock solution was diluted in trypsin digestion
buffer,
pH 8.0, (13) (10 mM sodium phosphate pH 6.0, 50 mM NaCl,
20 mM Tris-HCL). To each well was added 100 ~l 3 X SDS-
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14
PAGE solubilization bu~fer (O.19M Tris, pH 6.~, 30% v/v
glycerol, 3% w/v S~S, 0.0015% w/v bromophenol blue)
containing 15 ~1 1 M dithiothreitol (DTT) was added to
each well, and the samples were ; mme~; ately frozen.
Samples were then boiled for 3 minutes immediately prior
to loading onto a 15% polyacrylamide gel for
electrophoresis.
RE8ULT~
T ni zation:
Experiment 1: Mice were vaccinated with either
4 ~g rOspA, 2 ~g rOspA or 4 ~g rOspA on day 1, and were
given an identical dose of vaccine on days 2 and 4. 12
days after the initial vaccination, the mice were bled
from the tail. Mice were boosted with identical vaccine
on day 21 and were bled again from the tail on day 31.
~ xperiment 2: Mice were immunized as in
Experiment 1, except the first tail bleed was done on 8
days after the initial immunization, there was an
additional booster injection on day 22, and the second
bleed was done on day 25.
Challenge with infe~tious B. burgdor~eri: To
determine whether oral immunization with the different
recombinant osps resulted in protection of mice against
infectious challenge, 32 days after the initial
vaccination, mice were challenged intradermally with 104
B. burgdorferi Sh2. Following sacrifice, cultures from
mice were first ~;ned by phase contrast microscopy
after 5 days, and were ~;ned again on days 7, 8, lO,
12 and 16, for the presence of spirochetes. By day 5,
spirochetes were evident in the heart, bladder and joint
cultures ~rom all 5 of the mice that were given rOspD,
and all other cultures were negative. The culture data
on day 16 is presented in Table 1.
~T.T~ Sera from the immunized mice were
subjected to ELISA to investigate the humoral response to
oral immunization with rOspA or rOspD. Titers were
compared to those from sera of mice immunized
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WO g7/26006 PCT/US97/00557
subcutaneously. Sera were diluted by two-fold serial
dilution beginning at 1:20, through 1:40,960. Titers
obtained from the second bleed in both Experiments 1 and
2 are shown in Table 1. Sera from Experiment 2 were also
subjected to ELISA with non-lipidated rOspA, using a
second antibody response specific for murine IgA.
Immunoblots: Specificity of the antibody
response of the mice to oral immunization with rOspA or
rOspD was investigated by immunoblot. When sera from
orally immunized mice was required, only sera from
experiment 2 were used. Sera from the mice immunized via
the oral route with 4 ~g rOspA had a titer of 1:1600 by
immunoblot. Sera from 2 of the mice immunized with 2 ~g
rOspA had an immunoblot titer of 1:1600, and the titer of
the sera from the other 3 mice was 1:400. Fig. lA shows
binding of antibodies in the sera from orally immunized
mice to rOspA. Sera from the mice that were immunized
orally with rOspD did not have antibodies that bound to
rOspD in immunoblots (Fig. lB). However, sera from mice
that had been immunized subcutaneously with 4.0 ~g rOspD
alone did contain antibodies that bound rOspD in
immunoblots ~Fig. lC). Sera from the mice immunized by
either the oral or subcutaneous route (diluted 1 in 400)
were also subjected to immunoblot using a conjugate
specific for murine IgA to compare the relative amounts
of this immunoglobulin subclass in sera from animals
immunized via different routes (Fig. lD).
Growth Inhibition Ass~ys: Sera from mice
immunized orally with OspA or rOspD and those immunized
subcutaneously with rOspD were assessed by GIA to
determine whether the antibodies in the sera could
inhibit growth of B. burgdorferi in vitro. Sera from the
mice immunized orally with 4 ~g rOspA inhibited growth of
the OspA-producing stain B . burgdorferi B311 in vitro at
a dilution of 1 in 128. The same sera, however, did not
inhibit the growth of strain HBl9-R1 in vitro. This
strain does not produce OspA, but produces an increased
CA 02243~26 1998-07-1~
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16
amount of OspD compared to the parent HB19 strain (ref).
Sera from the mice that received 2 ~g rOspA orally had a
reciprocal GI titer 4 fold less than that of the mice
that received 4 ~g rOspA by this route, and did not have
any effect on the growth of strain HB19-R1. Sera from
the mice that received rOspD via the oral route did not
inhibit the growth of either B311 or HB19-R1 in vitro,
whereas sera from mice immunized subcutaneously with
rOspD inhibited growth of HB19-Rl up to a dilution of 1
in 32. The GI titers obtained are summarized in Table 2.
Sensitive~ of rOspA and rO pD to tryp~in: The
immunoblot, ELISA and GI data suggested that rOspA
administered subcutaneously resulted in the production of
OspD-specific antibodies, but rOspD administered orally
did not. It was decided to investigate the relative
trypsin sensitivities of rOspA and rOspD in an attempt to
explain why rOspA stimulated antibody production when
given orally but rOspD did not. rOspA at a concentration
of 250 ~g.ml~l in Osp buffer appeared to be susceptible to
digestion by 0.03125 ~g.ml~l trypsin under the same
conditions. Bovine serum albumin (BSA) at concentration
of 250 ~g.ml~l in Osp buffer was incubated with 25 ~g.ml~
trypsin to determine whether the trypsin would be active
in the Osp buffer. BSA was digested by trypsin under
these conditions.
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- 17
Table 1. ELISA titers and protection data from study in which
mice were ;~11~; zed orogastrically with rOspA or rOspD of
Borrelia burgdorferi B31
Immunogen ELISA Positive Cultures
titera plasma heart bladder joint
Expt. 1:
rOspD, 4 ~g 20 0/3 3/3 3/3 3/3
rOspA, 4 ~g 640 0/3 O/3 0/3 0/3
rOspA, 4 ~g 320 O/2 1/2 1/2 1/2
Expt. 2:
rOspD, 4 ~g 20 3/5 5/5 5/5 5/5
rOspA, 4 ~g 1470 0/5 O/5 0/5 0/5
rOspA, 2 ~g 485 0/5 0/5 0/5 O/5
a ELISA titers presented are the geometric mean titers from the second
bleed (3 day~ post-boost).
* PositLve cultures were obtained ~rom the same mouse
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~ 18
Table 2. Growth inhibitory titers o~ sera from mice immunized
orally or subcutaneously with recombinant Borrelia burgdorferi
lipoproteins
Growth inhibitory titers
Immunogen Route B311a HB19Rlb
4.0 ~g rOspD oral S8 S8
4.0 ~g rOspA oral 128 S8
2.0 ~g rOspA oral 32 S8
4.0 ~g rOspD subcutaneousS8 32
a B311 expresses OspA but not OspD
b HB19Rl expresses OspD but not OspA
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19
DISCUSBION
Investigations into the development of a
vaccine for Lyme disease currently focus on OspA as a
candidate vaccine. In human field trials, rOspA was
a~m;ni~tered subcutaneously (3). The present invention
provides alternative routes of a~m;nistration of a
vaccine or immunological composition. Stimulation of the
mucosal immune system by intra-nasal a~;n;stration of a
recombinant BCG vector expressing OspA has been
demonstrated (7) and Dunne and co-workers recently
reported oral immunization of mice with an attenuated
strain of Salmonella ty~himl~rium expressing OspA (6).
However, in addition to safety and other issues whereby
the work of Dunne and Langermann provide nothing of any
practical utility and only mere laboratory curiosities,
without any true proof of results being from OspA (and
not from immunity enhancement from other materials
present in their preparations a~min;~tered), in the work
of Dunne, Langermann and others, not all the experimental
animals were protected against infectious challenge by
the alleged vaccine in their studies, whereas the herein
Applicants report 100% protection of mice against
infection by B. burgdorferi by oral administration of
rOspA alone (without ad~uvant or additional ingredients
which could enhance immunogenicity).
ELISA and immunoblot studies showed a strong
antibody response to OspA in the immunized animals, of
both IgG and IgA immunoglobulin subclasses following
booster administrations of vaccine or immunological
composition of the invention. The lipoprotein control
group, that received rOspD via the same route, did not
demonstrate a detectable antibody response to rOspD
either by ELISA or immunoblot. In contrast, mice that
were immunized subcutaneously with 4 ~g rOspD produced an
3~ antibody response detectable by immunoblot at a serum
dilution of 1:100. Of course, if lipidated or if
CA 02243~26 1998-07-1~
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~ 20
adjuvanted, the OspD should elicit an immunological
response.
Sensitivities of rOspA and rOspD to trypsin and
low pH were investigated in an attempt to explain the
difference in immunogenicity of these lipoproteins when
administered orally. It was thought that the gastric
acidity or trypsin in the small intestine may affect
these proteins differently and thereby influence the
antibody response to them. It was found that, under the
conditions ~Am; ned here, that is, rOspA and rOspD
resuspended in the Osp buffer, there was a four-fold
difference in their sensitivities to trypsin. Although
rOspA was slightly more resistant to trypsin digestion
than rOspD, it did not appear that rOspD was dramatically
more sensitive. rOspA appeared to be more sensitive to
trypsin than had been reported by Dunn and co-workers,
but in that case, the rOspA was not resuspended in Osp
buffer. ~his buffer contains Triton X-100, which could
increase the sensitivity of rOspaA to proteolytic
cleavage.
The use of a single protein mucosal, preferably
oral, vaccine or immunological composition has not been
described or suggested for B. burgdorferi infection.
Studies in which oral immunization against B. ~urgdorferi
has been investigated have made use of bacterial carrier
systems, namely, Escherichia coli and attenuated
Salmonella ty~ i 7n77 rium ( 5, 6). Because of safety,
certainty of the result being from OspA, and ease and
cost of preparation, it should be considered that the
present invention is surprisingly more safe, certain,
efficient and effective than the prior art. It is also
possible that the host immune system would mount a
response against antigens of the prior art carrier
systems, and therefore reduce efficiency of vaccine
antigen delivery upon repeated administrations of prior
art compositions (or repeated performance of the prior
art~.
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21
The molecular structure of rOspA lipoprotein
may facilitate its absorption in the gastrointestinal
tract, and stimulation of the mucosal immune system. It
appears that gastric acidity does not affect the ability
of OspA to be taken up and transported by cells in the
gastrointestinal tract. There is evidence that proteins
that are relatively resistant to gastric acid are good
candidates for oral vaccines; for example, recombinant
urease of Nelicobacter pylori (14). It is not known
whether rOspD is stable in acidic conditions. Such
differences in biochemical properties of these proteins
may explain the apparent failure of rOspD to elicit a
detectable antibody response when administered via the
oral route. The protection data from this study show
that the rOspA administered mucosally, preferably orally,
is 100% effective in eliciting a protective immune
response in mice against challenge by infectious B.
burgdorferi.
The simplicity and efficacy of oral
administration in accordance with the present invention,
as herein demonstrated, shows that oral immunization or
vaccination of humans and animals - domestic or wild - is
provided by the present invention.
Indeed, the present invention can diminish
infection of wild animals. Infection of wild animals
leads to infection of humans and domestic animals.
Borrelia burgdorferi is well known for being transmitted
by Borrelia burgdorferi infected deer bearing ticks. The
ticks carry the bacteria to domestic animal or human
hosts by the ticks being left by the deer in areas
contacted by domestic animals or humans, and the ticks
then biting and thus infecting the domestic animals or
humans. Accordingly, suitable bait (e.g., food of wild
animals susceptible to Borrelia burgdorferi infection
such as food of deer) containing a Borrelia antigen or
immunological fragment thereof, e.g., OspA could lead to
vaccination of wild hosts, with the resultant diminishing
CA 02243~26 l998-07-l~
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22
of infection in such wild hosts, and the diminishing of
spread of infection from such wild hosts to humans and
domestic animals.
Additionally, since the herein protective
results are from isolated, purified recombinant lipidated
OspA without any adjuvant, it is believed, without
wishing to necessarily be bound by any one particular
theory, that the lipidation of the OspA may be providing
the protective results.~0 EXAMPL~ 2 - INTRA-~T AND INTRA-GASTRIC ~ORAL)
ADMINI~TRATION OF OSDA
Recombinant lipidated OspA (3) prepared as
above was administered as follows: Groups of 5 C3H/HeN
mice were immunized twice on days 0 and 28 with 25 ~g of
the lipidated OspA, either intra-nasally or intra-
gastically, in carrier (PBS) without adjuvant. At day 44
both groups had extremely strong serum IgG and good serum
IgA responses by ELISA. There was also an excellent
secretory IgA response in saliva, particularly in the
intra-nasally immunized group. The mucosal response was
confirmed on day 64 by an ELISPOT analysis. This is
particularly useful because it directly measures the
number of antibody secreting cells, thus allowing a
~uantitative comparison of the response to different
antigens. For mice immunized intra-nasally, the salivary
glands had 1,470-5,700 (average=3,3903 anti-OspA IgA
cells/106 total lymphocytes. Mice immunized intra-
gastrically had 350-1,310 (average=660) anti-OspA IgA
cells/106 total lymphocytes in the salivary glands. As a
comparison, 20 ~g of jack-bean urease also given at days
0 & 28 produced no response when administered
unformulated and only ca. ~00 spots by intra-nasal and
10-50 spots by intra-gastric when given in liposomes.
Even the classic mucosal immunogen cholera toxin B
subunit (10 ~g dose~ gives only 500 spots/106 lymphocytes
by intra-nasal administration and a very few spots by
intra-gastric administration. Thus, lipidated Borrelia
CA 02243~26 1998-07-1~
W097126006 PCT~S97/00557
23
antigens or immunologically active fragments thereof,
especially lipidated OspA, are extremely strong mucosal
immunogens; and, can generate or elicit useful
antibodies, including protective antibodies.
Having thus described in detail preferred
embodiments of the present invention, it is to be
understood that the invention defined by the appended
claims is not to be limited by particular details set
forth in the above description as many apparent
variations thereof are possible without departing from
the spirit or scope thereof.
CA 02243526 l998-07-l5
W097/26006 PCT~S97/00557
24
:RI~:NCES
1. Barbour, A.G. and Fish, D. The biological and social
phenomenon of Lyme disease. Science. 1993, 260, 1610-
1616
2. Fikrig, E., Barthold, S.W., Kantor, F.S. and Flavell,
R.A. Protection of mice agains~ the Lyme disease agent
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3. Erdile, L.f., Brandt, M., Warakomski, D.J., Westrack,
G.J., Sadziene, A., Barbour, A.G. and Mays, J.P. Role
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also USSN 08/373,455.
4. Keller, D., Kister, F.T., Marks, D.H., Hosback, P.,
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6. Dunne, M., Al-Ramadi, B.K., Barthold, S.W., Flavell,
R.A. and Fikrig, e. Oral vaccination with an attenuated
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SU~IllUTESHEET (RULE 26)
CA 02243526 1998-07-15
W097/26006 PCT~S97/OOSS7
11. Barbour, A.G., Tessier, S.L. and Stoenner, H. Variable
major proteins of Borrelia hermsii. ~.Exp.Med. 1982,
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12. Sadziene, A., Thompson, P.A. and Barbour, A.G. In vitro
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Sl,~ JTE SHEET (RULE 26)
