Note: Descriptions are shown in the official language in which they were submitted.
WO91/12819 PCT/CA91/00063
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2077~2
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IMPROVED IMMUNOGENIC COMPOSITIONS
Cross-Reference to Related A~lication
This application is a continuation-in-part of
U.S. serial no. 233,674, filed 18 August 1988 and now
pending.
Field of the Invention
The invention relates to improved immunogenic
compositions in which haptens to which an immune response
is desired are conjugated to crystalline or
para-crystalline carriers, especially those represented
by bacterial surface layers, or "S-layers."
Backqround Art
When an immune response is desired to a
particular antigen or hapten, it may be necessary to
supplement the administration of the hapten with a
material which enhances its ability to elicit either or
both a B-cell mediated or T-cell mediated response. One
general class of such supplementing agents has generally
been classified as adjuvants. These are materials
administered along with the hapten which seem to aid in
securing the desired response. The use of killed
bacteria and the products thereof as such adjuvants has a
considerable history, and the use of Freund's Complete
Adjuvant (killed Mycobacteria) or other bacteria and
their components such as peptidoglycan as
immunomodula~ors has been reviewed, for example, by
Warren, H.S., et al., Ann Rev Immunol (1986) 4:369.
Naked or acid-treated bacteria have also been shown to
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act as adjuvants to induce humor~l i~nunity to
carbohydrate moieties on large glycoproteins by
Bellstedt, D.U., et al., J Immunol Meth (1987)
98:249-255; Livingston, PØ, et al., J Immunol (1987)
138:1524-15~9.
European patent application publication no.
180,564 describes t~e~preparation oE a complex from
bacterial substrates designated "Iscom." This complex is
prepared by solubilizing the hydrophobic peptides ~rom
bacteria into detergent, removing the detergent from the
solubilized material and replacing it with glycosides,
such as saponin.
Haptens which are contained in small molecules~
especially carbohydrates, are also rendered more immuno~
genic by conjugation to a carrier such as bovine serum
albumin, keyhole limpet hemocyanin, diphtheria or tetanus
toxoids and the like. The resulting conjugate
vaccination antigens possess increased immunogenic
potential with respect to the low-molecular weight
haptens.
Currently, the only vaccines which are
administered orally are those containing live attenuated
organisms such as the attenu~ted polio virus vaccine.
Vaccines which consist of killed organisms or subunit
vaccines generally are not administered orally because
this route of administration does not induce a strong
systemic immune response. In fact, extensive research
has demonstrated that oral ingestion of an antigen
usually results in an induced state of systemic
hyporesponsiveness referred to as oral tolerance.l It is
believed that active immunosuppression by suppressor T
cells is the mechanism behind the induction of oral
~; tolerance. Another important factor in the induction of
oral tolerance is thought to be the processing of the
antigens by the mucosal reticuloendothelial system.2
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Both cholera toxin (CT) and cationized bovine serum
albumin (cBSA) have heen shown to generate systemic
responses after oral immunization.3'4 Neither CT nor
cBSA are suitable for use as vaccine carriers in humans.
Finally, the immune response to weak
immunogens, such as carbohydrate antigens, is usually an
antibody response, mediated by B-lymphocytes, whereas
effective protection through vaccination would generally
require participation of T-lymphocytes in the immune
response. To modulate the immune response to
carbohydrate haptens in favor of T-lymphocyte-mediated
immunity, is another reason why ~-lymphocyte-dependent
haptens have been attached to protein carriers.
Depending on the nature of chemical linking
process chosen, such binding of haptens to carriers is
often poorly defined and poorly reproducible. Also,
where the carriers are toxoids such as diphtheria or
tetanus toxoids, their formation from the corresponding
toxins is sometimes incomplete so that toxoid
preparations show residual toxic activity.
The invention provides two-dimensional crystal-
line carriers which permit definition of these parameters
so that an ordered structure bearing the
immunostimulatory or immunoregulatory substance can be
obtained, and problems associated with toxicity are
minimized. The invention also demonstrates that S-layers
can also generate a strong systemic immune response a~ter
an oral immunization.
Disclosure of the Invention
In accord with the present invention, the
problems of low or lacking immunogenicity, lack of T-
lymphocyte-mediated response~ defined (poorly
reproducible) coupling of haptens or immunoactive
substances, and residual toxicity of antigen carriers is
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WO91/12819 4 PCT/CA91/00063
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solved by using as the carrier to which the immunoactive
substances are bound two-dimensional crystalline arrays
of proteins or glycoproteins.
These aggregates may also be covalently
cross-linked. By virtue of the crystalline structure of
the two-dimensional arrays, the carrier molecules display
a constant, precisely de~ined spatial orientation with
respect to each other, thus both the nature of the link-
ages and the number and spatial orientation of the bound
haptens or immunoactive substances and the distance
between the binding sites which can carry the haptens or
immunoactive substances are always precisely defined.
Furthermore, by the practice of this invention
it is possible to choose carriers structured so that
they, by virtue of their shape, size, arrangement and
surface properties, are preferentially taken up by
immunologically active accessory cells. Such uptake by
accessory cells, e.g., macrophages, dendritic cells or
Langerhans cells permits a more efficient immune response
to be achieved through enhanced processing and
presentation of the desired hapten.
. Thus, in one aspect, the invention is directed
to pharmaceutical compositions comprising a
two-dimensional crystalline carrier coupled to a moiety
which bears an epitope, e.g., a hapten, to which an
immunological response is desired. The two dimensional
crystalline array may optionally be stabilized by
covalent cross-linking. A particularly preferred
crystailine carrier is obtained from isolation of the
S-layers of microbial cell walls.
In another aspect, the invention is directed to
methods to prepare the pharmaceutical compositions of the
invention which comprises coupling the epitope-bearing
moieties to the above carriers. In still another aspect,
;- the method is directed to methods to elicit a T-cell
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WO91/12819 - PCT/CA91/00063
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mediated or ~-cell mediated immune response to an epitope
of interest, which method compri~es administration of the
compositions of the invention to a vertebrate subject in
an amount effective to elicit the desired response. The
administration of compositions may be done orally to pro-
vide oral immunizations. The present invention further
provides a systemic cell-mediated immune response.
~rief Description of the Drawinqs
Figure 1 is an illustration which shows the
three stages of purification of S-layers from intact
bacteria.
Figure 2 is a graph illustrating one of the
analytical procedures enabling the precise coupling
methods of this invention.
Figure 3 is a graph illustrating the dose
response of the T-disaccharide linked S-layer needed to
prime for a delayed-type-hypersensitivity (DTH) response.
Figure 4 is a graph illustrating the amount of
T-disaccharide needed to elicit a DTH response in primed
mice.
Figure 5 is a graph illustrating the effect
adjuvants have on the T-disaccharide S-layer response.
, Figure 6 shows a comparison of the
delayed-type-hypersensitivity reaction (DTH) as a result
of immunization wi~h hapten linked to a carrier of the
inYention with hapten linked to BSA.
Figure 7 shows data indicating that T-cells
stimulated by i~munization with the invention
compositions can transfer the capacity to exhibit DTH.
Figure 8 shows two bar graphs comparing oral
and intramuscular immunization with S-layer conjugates.
Figure 9 shows two charts showing the tertiary
antibody response to Y hapten coupled to unfixed L lll S-
Layers, antibody response to the Y hapten negative
.
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Wo9l/l28l9 ~3~ -6- PCT/CA91/00063
control and tertiary antibody responses to unfixed Llll
S-Layer coupled with Y hapten, and ant:ibody response to
unfixed Llll, negative control.
Figure lO is a bar graph showing immunoglobulin
isotyping of tertiary antibody respons,e to unfixed Y-
Llll/FA, isotypic antibody response to the Y hapten, and
isotypic antibody response to unfixed Llll.
Figure l~ is a bar graph comparing tertiary
antibody response to T-disaccharide coupled by EDC to
fix~d Llll S-Layer; antibody response to T-disaccharide
at different molar hapten rations, and antibody response
to the Llll S-Layer carrier.
Figure 12 is a graph showing tertiary antibody
response to Streptococcus pneumoniae capsular
polysaccharide ~CPS) type 3-Llll conjugate; antihody
response to CPS type 3; negative control response to CPS
type 3 in mice injected with Llll or PBS.
Figure 13 is a bar graph showing immunoglobulin
isotyping of tertiary antibody response to S. pneumoniae
CPS type 3-Llll S-Layer; isotypic antibody response to
CPS type 3 when mice were immunized with 3-Llll
conjugate; isotypic antibody response to CPS type 3 when
mice were immunized with CPS type 3.
Figure 14 is a graph showing secondary antibody
response to Streptococcus pneumoniae oligosaccharide
type 3.
"
~ Modes of CarrYinq Out the Invention
:
The invention provides pharmaceutical compo-
sitions which are capable of enhancing the immune
response by virtue of employing carriers which are
two-dimensional crystalllne arrays. Various methods of
- attaching the immunoactive substances to these carriers
may also be employed. The resulting compositions may be
;~ used both for stimulation of B-cell and T-cell responses.
.
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WO91/12819 ~7~ 2 0 7 7~3 ~ 2 PCT/CA91/00063
Nature of the Carriers_an~d Compo_ tions
Advantageously, the two-dime.nsional crystalline
arrays may consist of proteins or glyc:oproteins whereby
the structure of the carriers may further approximate the
shape of a bacterium. The appropriate arrays may be
derived from one or several microbial cell wall layers.
Thus, the proteins or glycoproteins composir.g these
arrays are obtained in an especially simple manner. Such
suitable arrays may contain other adhering cell wall
components. In certain cases, microbial cell wall
fragments as such can be used to carry the immunoactive
substances. Particularly suitable as sources of this
type of carrier are those microorganisms which, aside
from the crystalline surface layer proteins, contain
additional rigid layers such as those composed of
peptidoglycan or pseudo-murein.
Figure l shows several stages of purification
of typical bacterial cell wall arrangements. Section a
shows a section of intact bacterial cell wall wherein S
represents the S-layer; PG represents peptidoglycan and
CM represents the cytoplasmic membrane. As shown, the S-
layer is associated with the proteoglycan and cytoplasmic
membrane in a layered structure.
Section b shows the empty peptidoglycan
sacculus after glutaraldehyde fixation. The cytoplasmic
membrane is removed, and the fixed segment contains a
double 5-layer separated by the peptidoglycan.
Section c shows the glutaraldehyde fixed
S-layer composition after lysozyme treatment to remove
the peptidoglycan. The designations iS and oS represent
the inner and outer S-layers. Adapted from Sara
Sleytr, App Microbiol Biotechnol ~l989) 30:l84.
In a preferred embodiment of the present
invention, the two-dimensional crystallin~ arrays are
derived from microbial cell walls as S-layers with or
.
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WO91tl~819 ~r1~t;~ 8- PCT/CA9t/00063
without additional peptidoglycan layers. In order to
prepare these aggregates, bacteria are incubated with
detergent at elevatPd temperatures of -l0-60C, preferably
around 50C, ~or a suitable time period to disintegrate
the cytoplasm and plasma membrane of the organism.
Suitable detergents include, ~or example Triton X-lO0,
various alkylpolyoxyethylene ethers, various
acylpolyoxyethylene sorbitol esters, such as the Tweens,
and the like. Intact bacteria may be t:reated with the
detergent, or the culture may be sonicated or otherwise
disrupted in the presence of buffer when the cellular
shape of the microorganism does not need to be conserved.
The nonsolubilized components are recovered, preferably
through centrifugation, and the pellets are washed. It
i5 desirable to further remove cytoplasmic components and.
nucleic acids by treatment with suitable enzymes
including DNAse, RNAse, and the like. The washed and
pelleted aggregates containing the S-layers are then
utilized as carriers, preferably after treatment with a
cross-linking agent such as glutaraldehyde. The
cross linker is added in an amount which stabilizes the
ordered structure of the S-layer.
Also, optionally, the residual peptidoglycan
can be removed from the recovered S-layers by treatment
with a peptidog~ycan-degrading enzyme, for example
ly50zyme. The recovered pellet is treated with the
enz~me at about 37 C in buf~er for a time suitable to
remove the peptidoglycan.
Thus, the two-dimensional crystalline array
provided as the S-layer from bacterial cells can be
recovered, in general, by extraction o~ nonordered
components using detergent; optionally this structure can
be cross-linked, and optionally the peptidoglycan may be
removed. The preparation can be conducted either on
whole cells or on a disruptate, such as a sonicate.
i
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WO91/12819 2 0 7 7 3 5 2 PCT/CA91/00063
~,
In addition, since the carriers of the
invention can be of suf~icient size to be retained by a
suitable filter, potential pyrogens such as
lipopolysaccharides can be removed by filtration of the
derivatized carrier.
Carriers comprising immunoactive substances may
be combined with other substances and compositions. It
is thus possible to achieve various functions of the
pharmaceutical composition. For example, strongly hydro
phobic carrier molecules can be mixed with aggregates
carrying the immunoactive substances causing increased
uptake of the pharmaceutical structure by accessory
; cells.
i Carriers comprising different immunoactive
substances can be attached to an auxiliary matrix which
can be cross-linked to the carriers. Thereby, a pharma-
ceutical preparation can be generated that would combine
different types of carrier molecules such as those
differing in their crystalline structures.
Furthermore, carrier aggregates ~omprising more
than one hapten can be used so that the profile of
activity of the pharmaceutical composition can be
precisely controlled.
~; In one approach to providing multivalent
vaccines of this type, subunits of the carrier aggregate
can be separated, conjugated separately to the desired
haptens, and then reconstituted to obtain a carrier
aggregate with a multiplicity of haptens. For example,
in the preferred embodiment using prepared S-layers,
separated portions of the S-layer sample can be
conjugated to the individua} haptens or
~ epitope-containing moiety under the conditions described
;~ hereinbelow, and then recombined in the presence of a
chaotropic agent such as guanidine hydrochloride or a
detergent. Upon removal of the detergent or chaotropic
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agent by dialysis, the protomers originating from the
different portions will self-assemble into an S-layer
derivatized with different haptens or immunoactive
substances.
Attachment _f Immunoactive Molecules
The carriers must be attached to the antigenic
determinants to which an immunological response is
desired. As used herein, "epitope-bearing moiety" refers
to a substance which contains a specific determinant to
which the immune response is desired~ The
epitope-bearing moiety may itself be a hapten--i.e., a
simple moiety which, when rendered immunogenic, behaves
as an antigen, or may be a more complex moiety, only
portions of which are responsible for the
immunospeci~icity with regard to the antibodies obtainedO
The need for an appropriate carrier is, of course,
greater when the epitope-bearing moiety is a hapten,
since these simple and low molecular weight substances
are only weakly immunogenic. However, the use of the
carriers of the invention is applicable to any moiety
which contains an epitope to which an immiune response is
desired.
The two-dimensional crystrifflline arrays
frequently contain glycoprotein and thus the hapten or
immunoactive/immunoregulative substances may be linked to
either or both the protein or the carbohydrate portions
of the carrier. For two-dimensional crystalline carrier
arrays which are composed entirely of protein, linkages
to the protein per se must o~ course be employed.
The choice of the mode of attachment will
depfand both upon the nature of the haptens and/or
immunoactive substances and upon the type of application
of the pharmaceutical composition. Under certain
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circumstances, a mixed mode of attachment can be
advantageous.
The immunoactive substances can be attached to
the respective carrier molecules directly or by way of
bridging molecules such as homo- or heterobifunctional
cross-linking agents or peptide chains (e.g., I
polylysine). The introduction of such spacers or
bridging molecules offers the advantage of more precise
control of the release of haptens, etc., and of the
nature of such fragments as would be formed by
enz~me-catalyzed degradation within the endosomes
(lysosomes) of macrophages or other accessory cells.
Using appropriate spacer groups, preferred sites of
cleavage of the immunogenic aggregates may also be
introduced. Commercially available homobifunctional or
heterobifunctional linkers may be obtained, for example,
from Pierce Chemical Co., Rockford, IL.
By an advantageous process for the production
of the pharmaceutical compositions of this invention,
such groups on the carrier as would bind the immunoactive
substanc~s may be activated prior to attaching the
immunoactive substances. Thereby a reliably precise and
reproducibly stable attachment of haptens to the
respective groups is safeguarded.
For attaching immunoactive substances to carbo-
hydrate portions ~glycans) of the S-layer glycoproteins,
binding sites within the glycoproteins may be generated
by oxidation, e.g., using periodate. Binding sites on
the protein portions can also be generated by reacting
with glutaraldehyde, the reagent used for cross linking
and activation. Formation of binding sites can also
occur by the introduction of active groups, whereby a
precise control of the number and kind of binding sites
can be achieved. For an especially stable linkage, the
haptens can be attached by amide linkages to the carboxyl
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groups of the protein portion of the carrier. Attachme~t
of the haptens can also be through aldehy~es in the form
of Schiff bases. The Schiff bases can be reduced to
secondary amines.
Binding sites on the immunoactive substances
can be activated and the immunoactive substances attached
by means of these activated binding sites, to the
carriers. This, too, results in stable linkages. This
avoids the phenomenon of carriertypic suppression of the
immune response.
.-
Utility and Administration
The resulting compositions containing carrier
coupled with one or more epitope-bearing moieties are
then useful in eliciting an immune response.
Administration is by conventional techniques, and the
effect of the carrier is to permit the use of weakly
immunogenic determinants to generate antibodies or to
induce a T-helper response against determinants which,
generally speaking, do not elicit such an immune
response. Suitable epitope-bearing moieties include
those conventionally employed including polypeptides,
carbohydrates, nucleic acids and lipids. The proteins,
glycoproteins and peptides can include cytokines,
hormones, glucagon, insulin-liXe growth factors,
thyroid-stimulating hormone, prolactin, inhibin,
cholecystokin or fragments thereof, calcitonin,
somastatin, thymic hormones, various releasing factors,
as well as antigenic fragments of proteins characteristic
of viruses and other infective agents. Various
carbohydrates and carbohydrate complexes can be used as
well, including bacterial capsules or exopolysaccharides,
for example, from Hemophilus influenza B, blood group
antigens, and the like. Further, lipid materials can be
used such as the steroids or prostaglandins, as well as
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glycolipids. Other molecules of interest include
alkaloids such as vindoline, serpentine, or any other
hapten-containing material to which an immune response is
sought.
In addition to the ability to raise antibodies,
the immune com~ositions of the invention can be utilized
to elicit a T-cell response, as can be verified, as
described below, by the ability to elicit a delayed-type
hypersensitive (DTH) response in inoculated subjects.
Furthermore, T-cells obtained from animals immunized with
the compositions of the invention can be transplanted
into other animal subjects, resul~ing in the transfer of
immunity to these subjects.
The pharmaceutical compositions of the present
invention are particularly suitable as immunizing
antigens for achieving high antibody titers and
protective isotypes and for immunological memory. When
antibodies or fragments thereof are used as
epitope-bearing moieties, anti-idiotypic antibodies may
be prepared by this method.
The pharmaceutical compositions can be used to
advantage for primary immunization and boosting when one
and the same immunoactive substance is bound to S-layers
derived from two different bacterial strains. Various
regimens can be used in administering the compositions of
the invention. In typical immunization protocols, a
series of injections is employed wherein subsequent
injections boost the immune response obtained from the
initially injected compositions. The availability of a
multiplicity of two-dimensional crystalline carriers
permits the use of analogous but different carriers in
the series. The problem of inducing carriertypic
tolerance is thereby overcome. In some instances,
especially where low molecular weight haptens are
employed, repeated injections with the hapten conjugated
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WO91/12819 ~ 14- PCT/CA91/00063
to the same carrier results in a modulated immune
response due to this phenomenon. By changing the nature
of the carrier, for example, by using S-layers prepared
from different microorganisms, this problem can be
avoided. Thus, protocols are devised wherein the
epitope-bearing moiety is injected first conjugated to S-
layer prepared from a first microorganism and followed by
injection of the epitope-bearing moiety coupled to a
carrier which is composed of an S-layer derived from a
second microorganism.
The carrier-epitope moiety complexes are
applicable also for use as immunosorbents or as affinity
matrices, e.g., for diagnostic kits or extracorporeal
depletion of undesirable antibodies from human blood.
The invention will be further explained making
reference to the following examples, which are intended
to illustrate, not to limit, the invention. In the
examples, Figures 2-7 are referred to. A more detailed
explanation of these figures is given in the section
entitled "Figure Legends" which is set forth at tne end
of the specification herein.
.
~ Example l
,: :
A. Preparation of the Carrier
; Cells of Clostridium thermoh~drosulfuric~lm
Ll11-69 (2.5 g) are suspended in 50 mM Tris-HCl buffer,
pH 7.l, and sonicated briefly (about l minute).
Followin~ the addition of a 2~ solution of Triton X-lOO
tl2.5 ml), the suspension is incubated at 50 C for 15
. minutes. By this treatment, the cytoplasm and plasma
membrane of the organisms is disintegrated whereas the
; two-dimensional crystalline protein-containing cell wall
layer (henceforth termed "S Layer") and the underlying
peptidoglycan layer are conserved as fragments.
.
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Subsequently, the mixture is centrifuged at 20,000 x g
and the pellets washed three times to remove detergent.
The pellets are then suspended in 5 mM magnesium chloride
solution (25 ml). For removal of cytoplasmic residues
and nucleic acids, DNAse (125 ug) and RNAse t500 ug) are
added and the whole stirred for 15 minutes at 37 CO The
suspension is then centrifugeæ. at 20,000 x g and washed
three times with water. The pellet i-; then suspended in
l.lM cacodylate buffer (pH 7.2; 20 ml) and a 50% aqueous
solution of glutaraldehyde is added at 4 C to a final
concentration of 0.5%. The suspension is well stirred at
4 C for a few minutes, centrifuged, and washed with
water. The pellet is then suspended in water ~25 ml) and
Tris-hydroxymethylaminomethane ("Tris") is added.
Following lO minutes standing at room temperature, the
suspension is again centrifuged (20,000 x g) and washed.
Ultrasonic treatment is omitted when the
cellular shape of the microorganisms is to be conserved
and only the cytoplasmic constituents are to be removed.
When the above procedure is used, the
underlying peptidoglycan layer remains associated with
the protein-containing cell wall layer. Wi~h numerous
organisms, this may result in the formation of an
additional S-Layer. Thus, the fragments or "ghosts",
consisting only of S-Layer and peptidoglycan, now display
S-Layers on the inner face of the peptidoglycan layers
(Figure l); these additional S-Layers can also be coupled
to haptens and/or immunoactive substances.
Should the presence of the peptidoglycan be
undesirable, the latter can be degraded with a
peptidoglycan-degrading enzyme, e.g., lysozyme, and
removed. To this end, the material produced as under
this section A is treated for l h at 36 C with a solution
of lysozyme (O.5 mg lysozyme per ml of a 50 mM solution
of Tris-HCl buffer, pH 7.2). In this case, lO ml of
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WO91/12819 PCT/CA9t/00063
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lyso~me solution is added per 0.5 g wet pellet.
Depending on the microorganism used, the S-Layer
fragments obtained consist of a single or double S-Layer.
Following ultrasonic treatment of cells, open fragments
are formed whereas in the absence of ultrasonic
treatment, the cellular shape, i.e., the crystalline two-
dimensional array is preserved intact.
. Formation of Bindin~ Sites
The pellet prepared according to A is suspended
in water (5 ml) and a O.lM solution of sodium periodate
(5 ml) added. the suspension is allowed to stand for 24
h with exclusion of light to allow oxidation and give
rise to the formation of aldehyde functions as binding
sites. Subsequently, the suspension is centrifuged and
the pellet washed with 10 mM sodium chloride solution, to
remove the iodine-containing salts.
C. Bindina of Proteins to the Modified S-Layers
The pellet of oxidized material obtained
according to B (about 0.2 g) is suspended in water (1 ml)
and the suspension is mixed with a solution (1 ml) of
bovine serum albumin (50 mg) in water (10 ml). 1'his
solution is allowed to stand at room temperature (60
minutes) and is then centrifuged.
To determine the amount of albumin bound to the
carrier, the extinction at 750 nm is measured relative to
; that of a preparation wherein the periodate solution has
been replaced by water (unoxidized control). The result
of this measurement is seen in Figure 2. Clearly, the
attachment to the carrier is significantly higher in the
case involving prior oxidation with periodate.
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WO91/12819 PCT/CA91/00063
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A. Preparation of the Carrier
Cells of Bacillus stearothermophilus PV72 (2.5
g) are suspended in 50 ~M Tris-HCl buffer, pH 7.2, and
sonicated for about 1 minute. Following addition of 2%
Triton X-100 (12.5 ml), the suspension is incubated for
15 min. at 50 C. By means of this treatment, the
cytoplasm of the cells is disintegrated while the S-
Layer and the peptidoglycan layer are preserved. Thus,
fragments are Pormed which correspond in shape more or
less to the original shape of the bacterial cell (so
called "ghosts").
Subsequently, the suspension is centrifuged at
20,000 x g and the pellet washed three times with water
to remove the detergent. The pellet is then suspended in
5 mM magnesium chloride solution (25 ml), DNAse
(deoxyribonuclease, 125 ~g) and RNAse (ribonuclease,
500 ~g) are added, and the mixture is stirred at 37 C for
15 min. Subsequently, the pellet is washed three times
with water, centrifugation in between being at 20,000 x
g. The pellet is then suspended in O.lM cacodylate
buffer (pH 7.2) and the suspension mixed with a 50%
solution of glutaraldehyde in water at 4 C to a final
concentration of 0.5%. The suspension is then vigorously
stirred at 4C for a few minutes, centrifuged, and the
pellet washed with water. Where glutaraldehyde residues
are linked through only one of their two aldehyde
functions, the remaining aldehyde function can serve as a
binding site. This provides aldehyde functions for
binding, similar to those of the oxidation products
described under Example lB.
.
SlJBSTlTUT~ SHEET
,~
. . .
. ..
WO91112819 PCT/CA91/00063
~ 18-
B. Bindina of _rotein(s) to the Modified S-Layers
The modified S-Layers prepared in section 2A
are mixed with a solution of bovine serum albumin as in
Example lC, and the amount of protein bound is determined
as described therF-
Example 3
A. Preparation of the Carrier
Cell walls of Clostrldium thermohydrosulfuricumL111-69 are treated with glutaraldehyde (0.5% in O.lM
sodium cacodylate buffer, pH 7.2) for 20 minutes at 20 C,
so as to stabilize the outermost cell wall layer (S-
layer). The reaction is terminated by the addition of
excess ethanolamine. During cross-linking the cell wall
fragments may be either in suspension or attached to a
porous surface (e.g., an S-Layer ultrafiltration
membrane). The cell wall fragments are then washed with
distilled water to remove the reagent mixture.
B. Creating Binding Sites for Liqands Containina
Thiol lSH) Groups
The pellet of a cross-linked preparation as
under A above, is suspended in distilled water (30 ml)
and to the suspension is added l-ethyl-3,3- ~dimethyl-
aminopropyl)carbodiimide (EDAC; 60 mg) maintaining a pH
of 4.75. This step activates the exposed carboxyl groups
of the S-Layer. Subsequently, an excess of hexamethyl-
enediamine (0.5 g) is added and the pH kept at 8.0 for 60
minutes. Subsequently, the reaction is terminated by
addition of acetic acid. The suspension is centrifuged
at 20,000 x g and the pellet washed three times with
distilled water. The wet pellet (100 mg) is suspended in
50 mM phosphate buffer, pH 7 (9 ml) and a solution of
meta-maleimidobenzoyl-N-hydroxysuccinimide ester (50 mg
.
,i
S~ TIT~JT~ S~EET
.. . . . . . .... . . ~ . .. ...
. ~ ;. . . .
WO91/12819 . PCT/CA9lJ00063
1 9--
:~: 2~7~3~j2
per ml of tetrahydrofuran; 1 ml) is added. The mixture
is then incubated for 30 minutes at 29 C.
C. Bindinq of SH-Containinq Proteins to the S-
Layers Derivatized as Under _
Following centrifugation at 20,000 x g, the
pellet is suspended in 50 mM phosphate buffer (pH 7.0),
B-galactosidase (20 mg) is added and the mixture is
incubated for 2 h at 20 C. After cent:rifugation at
20,000 x g and repeated washing with phosphate buffer,
the activity of the B-galactosidase covalently linked to
the protein matrix is determined.
The reactions of the Example are summarized as
f~llo~s:
~_ (
OH ~_ O
CN - / ~
OH ~ 0/ (cyclic Lm:docar~onaee)
. ~
:. ~
: OH
R - N~1 2 '~
O-C~
J 1
, O
.~ /
:
~No!l!~Ub~i 2U~
~a ~ ~D~
.
:'
SUBST~TUTE SIH~ET
:.., . . "
. .
.
WO91/~2819 ~ ?~ PCT/CA91/00063
~ 3 J -20-
Example 4
A. Preparation of Carrier
For the coupling of invertase, the vicinal diol
groupings of the carbohydrate portion (glycan) of S-
Layer glycoprotein are utilized. Cell wall fragments are
treated wlth glutaraldehyde, as described in Section A of
Example 3, to stabilize the outermost cell surface.
B. Generating the Binding Sites
The cell wall fragments from Section A (100 mg)
are suspended in anhydrous tetrahydrofuran (THF),
incubated at 20 C for 10 minutes, centrifuged at 20,000 x
g and suspended again in a 2.5% solution of cyanogen
bromide in anhydrous tetrahydrofuran (10 ml). Following
incubation for 2 h, the cell wall fragments are separated
by centrifugation at 20,000 x g and washed with THF for
removal of residual reagent.
C. Binding of Proteins to the Derivatized S-Layer
The pellet is suspended in 50 mM phosphate
buffer (pH 8.0 ~10 ml), containing invertase (20 mg) and
incubated for 18 h at 4 C. Following centrifugation at
20,000 x g, the pellet is washed twice with phosphate
buffer and the enzyme activity of the invertase bound to
the protein matrix determined.
The reactions of this Example are summarized as
follo~s:
,~
,
~' '
SlJB~TlTUTE S~EET
., ~ . ~ .
. ` . ` ~ . ~ .. -
. ... . . `
.. . .
.. . . . .
`
. .
",` . . ` , ` . ~
: ` ` . : :
W~91/12819 ~ PCT/C~91/00063
-21-
~ 2~73~2
~_COCIl ~ ~DC HO-N~ O-N~J ~ Lig~ d-NH2--
O O
-- ~a~ and
~lkall
'
Example 5
:
A. Cell wall fragments of Clostridium
thermohydrosulfuricum L111-69 are cross-linked with
glutaraldehyde as described in Example 3, section A.
B. Generatinq the_Bindinq Sites
Cell wall fragments (0.1 g) are suspended in
anhydrous dimethylformamide (DMF, 20 ml) and EDC (60 mg)
and N-hydroxysuccinimide (0.5 g) are added to the
suspension. Following incubation for 1 h, the suspension
is centrifuged at 20,000 x g and washed twice with DMF.
C. Binding of_Proteins to the S-Layer Thus
Modified
The pellet obtained as under 5B is suspended in
O.lM sodium hydrogencarbonate (pH 8.8) containing
dissolved dextranase (20 mg) and the reaction mixture is
incubated at 4 C for 18 h. The cell wall fragments
containing the bound dextranase are obtained by
centrifugation at 20,000 x g and washed twice with
distilled water. The dextranase activity contained in
the pellet is then determined.
- SUBSTITUTE SH~ET
.. .. . ~
.
.. .
WO91/12819 ~ , -22- PCT/CA91/00003
The reactions of this Example are summarized as
follows:
~_COON ~ EDC ~O-N~ O~N ~ ~ Ligand-N~ _
0 13
_ ~_C-~H-Lig~nd
~lk~
Example 6
Coupling of a Synthetic Carbohydrate Antiaen
: to Oxidized S-Layers
A. Preparation of Carrier and Generation of the
Bindinq Sites
The preparation of the oxidized glycoprotein S-
Layers was performed as described in Example 1, section A
and B.
.
B. Bindinq of the Carbohydrate Antigen~to the
Carrier
~ he oxidized (polyaldehyde) derivative of the
S-Layer prepared in Section A is incubated with the 3-
(2-aminoethyl)thiopropyl glycoside of a disaccharide
i. whereby Schiff base formation occurs. This step can also
be performed with any other saccharide attached to an
aglycone that contains amino groups.
These reactions are shown as follows:
`:
,~
SUB~TITUT~ S~EET
-
:`
.~.. , - . . ~ -
... .
. . ~ . `
.
.. . . . . . .
.. ` ~ . , :
}
WO 91/12819 23 2 0 7 7 3 ~ 2
. . .
t~-~2~ 91
d~sd~ su~ o~ ~ 3
H
HO--C-CH OH
HO l 2
~
''.
~C2
2~
\--~ O I~St:X CN~NH~ ~J _"
o~jOH ~ CH2~ ~2 2 ~C~12~ 1SlC~2~aNH2
OR
HO-C - H
C~2011
H_e~ O
N- ~C~ ) S ~e~(2~ 2s-N~cN2cNasc1l2cN~
H eg; 2 2 ,~,f
., ~
.:, .
., .
~UBSTIT~TE SHEET
.~
~ ... ~ .... . . . . . .
. .
..
WO91/12819 ~ 24- PCT/CA91~000~3
General recipe for the prepara~ion of 3-(2-aminoethyl-
thio)propyl glycosides from allyl glycosides.
A solution of the allyl glycoside (5 m~) in a
solution of cysteamine hydrochloride (15 milliequivalerlts
of SH-yroups in 10 ml) is allowed to stand for 1.5 h at
room temperature. The duration of this reaction may
vary. rhe reaction mixture is subsequently separated
over a column o~ cation exchange resin (e.g., Rexyn 101,
ammonium form, 200-400 mesh). The column is eluted with
water, 0.5 M ammonia, and 1.0 M ammonia. Unreacted allyl
glycoside appears in the aqueous eluate, and the 3-(2
aminoethylthio) propyl glycoside is eluted in the
fraction corresponding to 1.0 M ammonia. Those fraction~
containing products are subsequently evaporated to
dryness.
The Schiff base derivative of the S-Layer, as
obtained by binding of the 3-(2-aminoethylthio)propyl
glycoside can be used directly for binding of antibodies.
These can be assayed directly if they are labelled with
ferritin, horseradish peroxidase, 125I (iodine-125) or in
any other appropriate manner. The bound antibodies can
also be assayed ~ia a so-called "sandwich" method by
binding of labelled antibodies directed against the
first, hapten-bound antibodies.
The Schiff base derivative of the S-Layers as
obtained by binding of the 3-(2-aminoethylthio)propyl
glycoside may be converted into a secondary amine
derivative o~ the S-Layers by reacting it with
borohydride or another suitable reducing agent.
C~2CH2sC~2c 2 2
N~CN~N3 ~
f~2~3H C~2~ ~aS~ H2C~E)~2_o_
SIJB5TITUTE S~IEET
. .
.. . .. , .
.. .. . .
.. . . .
. .
.
: . .
WO91/12819 PCT/CA91/00063
-25- 2077~
.
The secondary amine derivative of the S-Layer
would be more stable to acid than the Schiff base
derivative.
O~ ,~ O N ~ ~NH2
N02
: \
H~C.~N-NIH ~ N2
02N
C. Determination of Free Aldehyde Groups
-~ The determination of the content of free
aldehyde groups in the polysaccharide portion, following
oxidation with periodate, is performed using
phenylhydrazine or 2,4 dinitrophenylhydrazine, or other
suitable reagents.
Suitable carbohydrate-containing S-Layers are
oxidized with sodium metaperiodate as described in
Example l, sections A and B. Iodine-containing salts are
removed by dialysis against water. Subsequently, a
solution of the corresponding hydrazine reagent in 10%
acetic acid is added and the mixture is allowed to react
for l h. Then the excess reagent is removed by dialysis
and the amount of hydrazone groups determined by
colorimetry. This method can also be applied to
determine residual free aldehyde groups after binding of
a hapten-containing amino groups, or of the immunoactive
substances.
.1 .
.
'' ' .
.~ .
SUB~TITUTE SI~EET
; . ~ .. . . . . .
.. ... . . . .
.
WO91/12819 PCT/CA91/000~3
~ ; -26-
2 ~r~r~ Example 7
A. Preparation and Structure of S-Layer
Glycoprotein
A cross-linked S-Layer preparation from cell
wall fragments of Clostridium thermohydrosul~uricum L111-
69 (hereinafter L111) and Bacillus stearothermo~_ilus
NRS2004/3A (hereinafter 3A) were prepared using the
procedure set forth in Example lA. El~ectron microscopic
studies of these compositions show that both the "L111"
and "3A" preparations exhibit an S-Layer lattice on
either face of a peptidoglycan sacculus (Figure 1).
The peptidoglycan of the foregoing preparations
was removed by digestion with lysozyme as described in
Example lA to provide a double layer of S-Layer
glycoprotein fragments held together by the penta-1,5-
diylidene bridges resulting from the glutaraldehyde
cross-linking. Each morphological unit of the S-Layer
lattice consists of two identical subunits in the 3a
preparation. An additional S-Layer preparation from
Bacillus alvei CCM2051 (hereinafter 2051), prepared from
whole cell walls as described in Example 3, part A,
cross-linked with glutaraldehyde and digested with
lysozyme as described in Example lA also showed two
identical subunits.
B. Specific ~ethods for Attachment of Haptens
lo Periodate Oxidation
The glycan chains of the L111 preparation are
known to consist of approximately 60 disaccharide repeats
of 4(-~-D-ManB-(1-3)-~-L-Rhap-(l, which comprises about
10% of the S-Layer glycoprotein. When treated with
periodate at 0.1 M for 2-5 h at pH 5.5, the mannose
residues were oxidized completely as estimated by the
SUBSTlTlJTE SHEET
. : -
.. . . . . .
- .
WO91tl2819 -27-- PCT/CA91/00063
> 2077352
phenol-sulfuric acid assay. Decreasing the pH value to
4.5 shortens the oxidation times and up to 23 molecules
~; of a carbohydrate hapten linked to a spacer terminating
in a primary amino function (for example, A-
; trisaccharide) per S-Layer protomer was achieved.
However, the resulting Schiff bases must be stabilized by
reduction with, for example, sodium borohydride.
The 3A S-Layer protomers have two different
glycan chains and the total carbohydrate content is about
7.5~. These glycans have the structures:
-2)(-~-L-Rhap-(1-2)-~-L-Rhap-(1-3)-B-L-Rhap(1,
` having about 50 repeats; and
; -4)(-~-ManpUA2,3(NAc)2-(1-3)-~-GlcNAc-(1-4)-B- -
ManpUA2,3(NAc)2-(1-6~-~-Glcp-(l, having
approximately 15 units.
oxidation of this S-Layer preparation under the
same conditions as above provided binding sites for up to
17 molecules of hapten per S-Layer subunit. Un~ixed S-
Layers gave lower yields of conjugates or hapten
~;1, contents.
.,~
; 2. E~ichlorohydrin Activation
Glutaraldehyde-fixed S-Layer fragments (2-3 mgj
were suspended in 2.5 mL of either 0.2 M sodium
bicarbonate/sodium carbonate buffer, pH 9.1 - lO.0, or
0.2 M sodium carbonate solution pH 11.4; or 0.4 sodium
hydroxide solution) containing 25 mg sodium borohydride.
Upon addition of epichlorohydrin (0.2 mL), the reaction
mixtures were incubated for 30 min to 16 h at room
temperature or 40, with rotation on a rotary evaporator
(150 rmp). Blanks were prepared similarly, but either
epichlorohydrin or hapten was omitted.
!
SUBSrlTUTE SHEET
.'~'''`''': '
.: , .
.
~,
WO91/12819 PCT/CA91/00063
- -28-
L~r~
After centrifugation and washing with water
(5 X 1.5 mL) to remove all of the activating agent, the
combined supernatants of the first centrifugation and all
washes were analyzed for carbohydrate rnaterial shed into
the supernatant from the S-Layers under the strongly
alkaline reaction conditions. The remaining pellets were
suspended in hapten solution ~1.0-1.4 mL, 1-2 ~moles/mL
0.2 M sodium bicarbonate solution) and incubated for 2-6
h with shaking on a hematology mixer at room temperatureO
Excess of hapten was removed by washing with water t5 X
1.5 mL~ prior to the blocking of unreacted epoxy groups
by treatment with 0.02 M ethanolamine in 50 mM sodium
bicarbonate solution for 4 18 h at room temperature.
Subsequently, the samples were washed with water (5 X
1.5 mL), lyophilized and assayed for carbohydrates, as
described for the S-Layer conjugate prepared via
periodate oxidation.
:
3. Divinylsulfone Activation
Glutaraldehyde-fixed S-Layer fragments (~-3 mg~
were suspended in 2 mL of appropriate buffer (see epoxy
activation, pH 9.1 - 11.4) and then divinyl sulfone (0.2
mL) was added to the suspensions. Treatment of blanks,
estimation of shedded S-Layer glycan, incubation times
with hapten solutions, and blocking of unreacted vinyl
groups was performed exactly as described for the epoxy
activation.
4. Activation with 1-Ethyl-3-(3-
Dimethylaminopropyl)-Carbodimide (EDAC)
Two different reaction conditions were used:
` .
i) About 3 mg of glutaraldehyde fixed S-Layer
fragments were suspended in 3 ml of water.
,
:
., .
.~ .
SUBSTITUTE SHEET
.
.,
,, ` . ~ , .. . , ., . ~
`. ` . . . .
.. . . - ` . .
. .
WO9l/12819 PCT/CA91/00063
f; ~ ~ 7 7 ~ ~ 2
Then the pH was adjusted to ca. 4.6 using
diluted HC1. After addition of 10 mg EDAC the
pH was readjusted with HCl to 4.67 - 4.70. The
suspensions were stirred at room temperature
for 1 h while the pH should not change. Excess
of EDAC was then removed by washing with ice-
cold water (2 X 10 ml) and centrifugat..on
~19,000 rpm, 4C). The pellets were incubated
with hapten solution (1.0 - 1.5 mL, ca. 2
~moles mL 1 0.2 M NaHCO3) at room temperature
for 2 - 18 h. Vpon washing with water (5 x 1.5
mL) the samples were lyophilized and assayed.
ii) About 3 mg S-Layer material was suspended in 2
mL of 0.1 M phosphate buffer (pH 4.0 - 4.7) and
lO mg EDAC was immediately added to the
suspensions. After activation for l h with
rotation on the rotavapor l mL hapten solution
was added and the mixture was incubated
overnight in the presence of EDAC at room
temperature. After washing with water (5 x 1.5
mLj activated free carboxyl groups were blocked
with 10% glycine in 0.2 M NaHCO3 for 1-2 h at
room temperature. The samples were then
washed, lyophilized and assayed.
,
Up to 60 moles of hapten, immobilized to the
carboxyl groups of the protein was found on
strain L111-69 and the difference observed
between both methods was minimal. With strain
NRS 2004/3a the overnight incubation in the
presence of EDAC increased the amount of
immobilized haptens from 14 to 32 moles per
mol.
, i
.;: .
`I SUB~TlTllJTE SI~EET
`~,.:....................... `
.. : .......
.
.
.
W091/12819 PCr/CA91/00063
-30-
Example 8
Preparation of T-Disaccharide A~igen Çon~uqates
T-disaccharide is a tumor marker of the formula
BGalp(1-3)~GalNAc. A composition containing this
disaccharide as a hapten was prepared by coupling the
spacer-linked disaccharide of the formula ~Gal-(1-
3)~GalNAc-0(CH2)8CONHNH2 to S-Layers by either of the
general procedures described under Example 7.B.4.
Example 9
Immobilization of Sy~nthetic Carbohydrate Antiqens
Onto Unfixed Oxidized S-Layers
A. A Formation of Binding Sites
Two identical suspensions are prepared of
purified, unfixed cell walls of Clostridium
thermohydrosulfuricum L111-69 in water (0.25 mL; 0.25
g/ml). Each suspension is mixed with cold (4~) 50 mM
sodium acetate buffer, pH = 5.0 (0.25 ml). To one of the
suspensions is added a cold 0.2 M solution of sodium
periodate in 50 mM sodium acetate buffer, pH = 5.0 (0.5
mL), and the resulting suspension is stirred for 3 h at
4 with exclusion of light (500 rpm). The other
suspension is not oxidized, but mixed with 50 mM sodium
acetate buffer (0.5 mL) and subjected to analogous
workup. Following the oxidation period, the suspensions
are centrifuged (30,000 x g, 10 min) and washed once with
sodium acetate buffer, pH=5, and twice with 0.2 M sodium
borate buffer, pH = 8.5, to remove iodine-containing
materials.
SUBSTlTUlrE SHEET
,,
.
.. . . . ~ . ...
.
WO91/12819 PCT/CA91/00063
31- 2
B. Bindina of Ca~rbohydrate Anti~en to Carrler
The polyaldehyde product of oxidation of the S-
Layers (carrier formed as under A) is suspended in sodium
borate buffer, pH = 8.5 (2 mL) together with sodium
cyanoborohydride (l0 mg). T-disaccharide (hydrazide
form, 5 mg) is added to one of the oxidized samples,
while one oxidi2ed and one unoxidized ~sample are
processed without hapten addition. Thls preparations are
incubated at 37 for 24 h with stirring (500 rpm).
Subse~uently, the S-Layers are washed once with 0.2 M
borate buffer, once with 0.2 M sodium chloride solution,
and twice with distilled water. The centrifugation
pellets of the respective samples may then be ~rozen for
storage, or processed further. During this procedure,
the immobilization of the haptens occurs by way of
reductive amination on oxidized, unfixed S-Layers. The
presence of the peptidoglycan layer provides for a
stabilizing influence similar to the one exerted by
glutaraldehyde fixation of the S-Layer protein.
C. Degradation of Peptidoqlycan ln a ~Sterlle~
Ultrafiltration Cell
The (frozen) S-Layer pellets (produced
according to B) are suspended in 50 mM Tris-hydrochloride
buffer p~ = 7.2 (5 mL) in a l0 mL ultra~iltration cell
(AMICON) equipped with a magnetic stirrer and an
ultrafiltration membrane (e.g. BIOFILTM produced from S-
Layers o~ Bacillus stearothermophilus PV72). The
suspension is mixed with lysozyme (5 mg) and incubated at
room temperature for 90 min with stirring (500 rpm).
Subsequently, pressure is applied to the cell (0.2 MPa)
whereby the high molecular weight S-Layer protein
(containing the bound hapten) becomes enriched in the
supernatant. The turbid suspension is washed with water
(ca. 60 mL) to remove degraded peptidoglycan and lysozyme
SUB~TIITUTE SH~ET
,~ ~
WO91/12819 -32- PCT/CA91/00063
~ 3~ h ~ ~
(until no extinction is detectable at ~80 nm). To test
for possible loss of (haptenated) S-Layer material, all
washes were combined, dialyzed exhaustively against
water, lyophilized, and analyzed by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE). This analysis has
confirmed the absence of S-Layer materials from the wash
fluids.
D. Purification of S-Laver-Hapten Coniuqates
The enriched suspension in the ultraflltration
cell (ca. 1 mL) is mixed with 5 M guanïdine hydrochloride
(5 mL) and incubated for 1 h at room temperature with
stirring. By this treatment, the S-Layers are
dissociated into protomers (MW ca. 100 kDa) and protein
impurities are dissolved. The process is accompanied by
a clearing of the suspension. The protein-containing
solution is enriched by the application o~ pressure (0.2
MPa) with stirring (500 rpm) and the guanidine
hydrochloride washed out with water until extinction at
280 nm is no longer detectable. ~he clear washes were
combined, dialyzed and analyzed for S-Layer fragments by
S~S-PAGE. During washing of the clear solution in the
filtration cell, the S-Layer protein reaggregates. The
suspension is removed from the cell, lyophilized and
examined by SDS-PAG~. The aggregates consist only of S- !
Layer protein containing bound hapten. The quantity of
bound hapten is estimated by the difference in
phenolsulfuric acid reactivity between the activated and
non-activated samples.
Using this process for im~obiliza~ion of hapten
on an unfixed S-Layer, followed by the removal of the
peptidoglycan and filtration over an ultrafiltration
membrane of defined pore size, S-Layer-hapten-conjugates
are produced aseptically in a sterilizable reaction
vessel. By virtue of special precautions, the star~ing
, ~ .
SU~TITUTE SHEET
.
: . - : . .. - .
.. . . ~. . . . ..
. . . . . . .
.. . .
WO91/12819 PST/CA91/00063
~33~ 2 7 73 ~2
. , .
material (S-Layer-containing cell walls) is practically
free of lipopolysaccharide (LPS; endotoxin)
contaminations; moreover, LPS fragments would conceivably
be removed during filtration after dissociation of the S-
Layer protein. Therefore, the S-Layer hapten conjugates
thus obtained can be considered pyrogen-free.
Example lO
EfflcacY of the T-Disaccharide S-Layer Compositions
Groups of 5-lO mice were prelreated with
cyclophosphamide (CP) and two days later were immunized
with the T-disaccharide coupled, 3a-S-Layer preparations
described in Example 8. Injection was made
intramuscularly into the hind leg muscle. Seven days
later, the mice were footpad-challenged with the T-
disaccharide-coupled Llll S-Layer preparation, also
prepared according to the procedure outlined in Example
8. Changes in footpad thickness were determined 24 h
later using a Mitutoyo Engineering micrometer. By
varying the amounts of T-disaccharide-3a-S-Layer
administered, it was found that the minimum concentration
of T-disaccharide-3a S-Layer required to prime the mouse
for a subsequent DTH response to challenge with T-
disaccharide-Llll was 5 ~g/mouse, and a maximal response
was obtained when immunization was performed at lO ~g of
disaccharide T-disaccharide-3a-S-Layer per mouse (Figure
3). At this level, a footpad swelling of approximately
5.5 mm 1 was obtained. An increase in the dose to 20 ~g
resulted in a lowering of swelling to 4 mm l.
In further experiments, CP-pretreated mice were
immunized with lO ~g of T-disaccharide-3a-S-Layer, and
seven days later were footpad-challenged with
varying amounts of T-disaccharide Llll S-Layer. Maximum
response (4.5 mm l) was obtained using 20 ~g of T-
disaccharide Llll S-Layer (Figure 4).
. ~
.
~,
S~B~ITUTE SHE~T
. ~ `
..
`
WO91/12819 34 PCT/CA91iO0063
t~
The foregoing results show that the T-
disaccharide S-Layer preparations induced a hapten-
specific cellular response to the carbohydrate hapten,
and give results comparable to the response seen with
strong immunogens such as complex viral antigens.
Example ll
SPecificitv of the T-Cell Respon~ses Generated
by S-Layer Coniuqates
A further control confirmed that the DTH
response was specific to the T-disaccharide hapten. In
these controls, CY-pretreated mice were immunized with T-
disaccharide-3a-S-Layer in the presence of lO0 ~g
dimethyl- dioctadecyl ammonium bromide (DDA). They were
then challenged with PBS and with Llll S-Layer lacking
the T-disaccharide, as well as T-disaccharide Llll S-
Layer. The results are shown in Table l. Only the T-
disaccharide-Llll resulted in significant swelling.
Thus, the results show an absence of cross-reactions
between different S-Layer preparations and make it
possible to utilize different S-Layers for multiple
immunization with the same hapten.
' This can be useful in immunotherapy or in
immunopotentiation of the immune response to weak
antigens, avoiding the phenomenon of carrier-typic
` suppression.
.. ..
SUB~TITIJTE ~I~EET
.
. . .. .. . ~
. . . . . . . . .
. . .
WO91~12819 35 PCT/CA91/00063
7 ~ 3 ~ ~
Table 1
S~ecificity of the Haptenated S-Layer
Immunization Footpad
Antiqena Challenqe Anti~en Swellinq
(mm x lO-l)
T-3a lO ~g/mouse PBS 0.8 + 0.8
T-3a lO ~g/mouse EDAC Llll lO ~g/mouseb 0.37 + 9.~
T-3a lO ~g/mouse T-Llll lO ~g/mouse 3.8 i 0.8C
PBS & DDA T-Llll lO ~g/mouse l.89 i 0.65
NT T-Llll lO ~g/mouse l.33 * 0.4
a CP-pretreated mice were immunized with antigen
mixed with lO0 ~g DDA.
, b Llll was sham-treated with EDAC to control for
the posslble modification of Llll by the EDAC
reagent.
c Significantly different from all other groups
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Example 12
Comparison to Other Carriers and Ad~uvant Systems
The immunization protocols of Example l0 were
followed where mice were CP treated or not, then
immunized with T-disaccharide-3a-S-Layer. DDA or
Freund's Incomplete Adjuvant (FIA) were used as
adjuvants. Seven days after immunizat:ion, the mice were
footpad-challenged with T-disaccharide-Llll (analogous S-
Layer preparations according to Example l0), and the
footpad swelling was measured 24 h later. These results
indicate that although T-disaccharide-3a is effective in
priming for DTH, the strongest response is observed when
mice were treated with CP (Figure 5). To compare the
immunopotentiating response of S-Layers with other
carriers, mice were immunized as outlined in example l0
using T-disaccharide-3a-S-Layers, T-disaccharide bovine
serum albumin (T-disaccharide BSA) or 8-
methyloxycarbonyloctanol (the linker arm attached to the
T-disaccharide) linked to BSA. Seven days after
immunization, mice were footpad challenged with T-
disaccharide-Llll according to example lO. The results
in Figure 6 demonstrates the immunopotentiating
properties of the S-Layer carrier, since administration
of the T-disaccharide coupled to bovine serum albumin
(BSA) produced no DTH response. Although other workers
have reported DTH responses to carbohydrate antigens, the
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protocols were complex, and a DT~ response could not be
obtained by immunization with hapten conjugates alone.
The foregoing results provide evidence for DTH against
the T-disaccharide by a single immunization with the T-
disaccharide conjugated to S-Layer.
ExamPle 13
Induction of In-Vitro Lympho~roliferative Response
The mice immuni~ed with both T-disaccharide-3a
and T-disaccharide-Llll, as described above ~using DDA
after CP treatment), were sacri~iced, and the immune
lymphocytes were isolated and cultured. The isolated
lymphocytes were then cultured with varying amounts of
2051 S-Layer, T-disaccharide-2051, T-disaccharide-BSA, or
T-disaccharide-KLH. As shown in Table 2, only T-2051 was
capable of inducing a response, as measured by tritiated
thymidine uptake. The response was dose dependent and
peaked at lO ~g/ml. The failure of T-disaccharide-KLH or
T-disaccharide-BSA to elicit a blastogenic response may
be explained by the notion that the S-Layer conjugate may
be taken up preferentially by the macrophages because S-
Layers take on the shape of oacteria.
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2Q~7~ ~ Table 2
Stimulation of LymphocYte from Mice
Immunized with Both T-Disaccharide-3a and T~Disaccharlde-L111
Antigen in Culture CPM 3H-Thymidine Statistical
20 ~q/ml Uptake_ _ Probabilitiesa
PBS 2303 + 692 tND
2051b 2549 + 1258 NSC
T-BSA 1840 + 866 NS
T-KHL 2091 + 498 NS
T-2051 4654 ~ 1294 P = o.oolb
a All statistics were compared to the PBS values.
b 2051 is the unbound S-Layer isolated from B.
alvei and is the control for the T-
disaccharide-bound 2051.
c NS not significant
d P<0.01 when compared to 2051 stimulation alone.
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Example 14
Transf2r of Activated Lympho~ytes
To clearly show that the DTH response generated
by the T-disaccharide conjugate was actually due to
helper T-cells, an adoptive transfer experiment was
carried out. In these series of experiments CP-
pretreated mice were immunized with 10 ~g of T-3a in DDA,
sacrificed seven days later, and their draining lymph
nodes and spleens removed. Primed lymphocytes were then
isolated and cultured for three days with T-Llll and
control antigens. The stimulated cells were then
depleted of the specific populations of T-cells by
treatment with monoclonal antibodies and complement.
The depleted, bulk-stimulated cells were then washed,
mixed with T-S-Layer 2051 or PBS, and injected into the
hind footpad of naive mice. The DTH response was then
measured 24 h later. The results of this experiment are
shown in Figure 7. It can be seen that a strong DTH
response was observed in the mice adoptively transferred
with primed cells stimulated with T-disaccharide-L111 and
mixed with T-disaccharide-2051. However, the similarly
primed cells stimulated with L111 alone and then mixed
with T-disaccharide-2051 did not induce a strong DTH
response. Even though the primed lymphocytes at one
point were exposed to all three different S-Layers, no
cross-reactive DTH response directed towards the S-Layer
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was observed. only when the three S-Layers were
haptenated with the T-disaccharide was a significant DTH
response observed. Also, when the pri;med lymphocytes
were depleted of specific T-cell populations, it became
evident that the primed cells involved in the DTH
response were helper T-cells.
The pharmaceutical structures constituting the
embodiment of the present invention are particularly
suitable as immunizing antlgens for achieving high
antibody titres and protective isotypes. When antibodies
are used as immunoactive substances, anti-idiotypic
antibodies may be prepared by this method. Furthermore,
the pharmaceutical structures can be used to advantage
for primary immunization and boosting when one and the
same immunoactive substance is bound to S-Layer proteins
or glycoproteins derived from two different strains. The
structures are applicable also as immune sorbents or
affinity matrices, e.gO, for diagnostic kits or
extracorporeal depletion of undesirable antibodies from
human blood.
., .
Example 15
Induction of a Cell-Mediated Immune Response
After Oral Immunization
i Briefly, groups of mice were sham-immunized or
immunized with l0 ~g of a preparation of T-disaccharide
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bound to 2051, or 10 ~g of the S. pneumoniae type 8 CPS
antigen bound to Llll, either intramuscularly or orally.
The oral immunization was carried out as described by
Smith et al., Infect. Immun. 31:129 (1980). Seven days
later each group of mice was footpad-challenged with 10
~g of a preparation of T-disaccharide bound to Llll, or 8
CPS coupled to PV72. Footpad swelling was measured 24 h
later with a Mitutoyo Engineering micrometer.
To demonstrate that T-S-layer con~ugates were
capable of generating a DTH response after oral i~nuni-
zation, groups of mice were immunized with T~S-Layer 2051
alone, either intramuscularly or orally. Seven days after
immunization the groups of mice were subsequently chal-
lenged with T-S-Layer L111. Twenty-four hours later the
DTH swelling response was measured. The results are
shown in Figure 8a. Both routes of immunization were
able to prime for the development of an antigen-specific
DTH response. Interestingly, the oral immunization
appears to elicit a slightly stronger DTH response than
does the IM immunization. This result demonstrates the
efficacy of S-Layer conjugates for induction of a
systemic immune response after oral administration.
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Example 16
Induction of a Cell Mediated Immune ResPonse After Oral
~ . , .
Immunization~.w`ith S. pneumoniae t~e 8 Polysaccharide
S-Layer coniuqates
Groups of mice were immunized with type 8
polysaccharide linked to the S-Layer Llll, either
intramuscularly or orally. Seven days after immunization
the groups of mice were challenged with type 8
polysaccharide linked to the S-Layer PV72. Twenty four
hours later the DTH swelling response was measured. The
results indicate that both routes of immunization are
able to prime for the development of an antigen-specific
DTH response (Figure 8b). Again, the oral immunization
appears to generate a slightly stronger DTH response than
does the IM immunization.
Desirable characteristics for a conjugate
vaccine would include enhanced immunogenicity
~predominantly IgG class response) and an anamnestic
response to a booster vaccination. Included here are
some examples of T and Y carbohydrate S-Layer conjugates
and a $treptococcus pneumoniae S-Layer conjugate which
meet these criteria.
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Example l7
Efficacy of Carbohydrate Hapten - Unfixed S-Layer
Con~ugates to Induce Speclfic Antibody_Responses
S-Layer preparations of Y-hapten Llll/SA (SA =
self assembly or unfixed) conjugates described under
Example 9) in 50% Freund's complete acljuvant were
intraperitoneally injected in mice on day 0 ~primary
immunization). Freund's complete adjuvant alone was
injected in control mice. Secondary and tertiary booster
injections were administered as in previous examples.
Fig. 9(a),(b) shows the responses to the hapten Y and to
the carrier Llll/SA respectively. Isotype responses are
illustrated in Figure lO. Immunoprotective IgG isotypes
to the Y hapten and Llll/SA carrier were elicited by this
procedure.
Example 18
The Effect of Varyina the Molar Hapten Ratio on
Antibody Responses
Different hapten molar ratios were produced by
varying the tlme S-Layer pellets were incubated with the
hapten solution. Fixed Llll S-Layers with 3.6, 12.7,
23.8 or 37.6 moles of coupled T hapten were injected
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intraperitoneally at 20 ~g/mouse with Adjuvax (Alfa-Beta
Technology). The antibody response to the T hapten and
to the Llll carrier, assayed by ELISA, is shown in Figure
ll. Antibody responses to the T disaccharide were
directly proportional to the amount of haptenation.
These results demonstrate that one advantage of S-layers
over conventional carriers is their ability to precisely
and reproducibly bind known amounts of hapten.
Example 19
Ability of S-Layer Carrier to Induce Speci~ic
Antibody to Streptococcus Pneumoniae Type 3
Capsular Polysaccharide (CPS)
Type 3 S. pneumoniae CPS (ATCC #169-X) was ~ -
activated by EDC and coupled to glutaraldehyde fixed Llll
as in Example 7B. (Specific methods for attachment of
haptens). Mice were injected with Type 3 -Llll
conjugates in PBS (0.6 ~g 3/20 ~g Llll), Type 3 (20 ~g)
in PBS, Llll (20 ~g) in PBS in P8S or PBS alone as per
previous examples. Results from a capture ELISA (Figure
12) show the specific anti-type 3 tertiary response to
the 3-Llll conjugate. Sera from control mice immunized
with Llll or inje~ted only with PBS reacted negatively.
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Isotyping ELISA results of these tertiary responses
(Figure 13) demonstrate the presence of immunoprotective
', ' - -
IgG subclasses. Antiserum to CPS type 3 alone was found
to be only of the IgA and IgM isotypes.
Example 20
AbilitY_of Unfixed S-Layer Carriers to Elicit
Antibodies to Oligosaccharides from Streptococcu~
Pneumoniae Type ~8 CPS
: - Oliqosaccharides were prepared from Type 8 S.
pneUmOnlae cps Dslng methods modified from Albersheim et
al., Carbohydrate Research 5:340, 1967. Oligosaccharide
preparations were coupled to unfixed P~72, or 20~,1 S-
~: Layers and.tetanus toxoid (TT) as. described in the
.evious-example. ~At ~ D a d:day.~1~4 :of the expe ime~t, ..
mIcé were i-mmuniz;ed ;in~rap;eritonéally with oligo 8-FV72~
-
(8 ~g 8j20 ~g PV72) in ~BS, oligo 8-2051 (10 ~g 8/20 ~g
2051) in PBS, oligo ~-TT t4 ~g 8/20 ~g TT), oligo 8 (9
: ~g) in PBS or PBS aione. Mice were bled on day 21 and an
-ELISA was performed to compare the different anti-oligo.
.. . .
responses. As can be seen from Figure 14, the antibody
. response to the 8 oligo as assayed by ELISA, was greater
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when 8-PV72 or 8-2051 were the immunogens than when
uncoupled 8 or 8-TT were injected.
ELI8~ Protocol:
ELISA plates (Maxisorb, NUNC #4-39450) were
coated with l ~g/well of antigen diluted in 0.05 M
carbonate-bicarbonate coating buffer (pH 9.6). Plates
were incubated overnight at 4C and then blocked with 1%
BSA (Sigma A-7030) in PBS (pH 7.2). After 1 hour
incubation at room temperature, plates were washed three
times with PBS and 0.05% Tween 20.
If Streptococcus pneumoniae CPS (type
specific)(Merck, Sharp ~ Dohme) antigen plates were
required, ELISA plates were first coated with l/250
rabbit anti-CPS (type specific; Statens Seruminstitut),
diluted in coating buffer and incubated at room
temperature for 2 hours. Plates were then blocked with
1% BSA for l h at room temperature and washed three times
with PBS Tween. S. pneumoniae CPS (type specific) was
then added to the plates at l~g/well in PBS-Tween and
incubated overnight at 4C. Plates were then washed
three times with PBS-Tween.
Mouse sera were diluted in PBS-Tween, placed
into duplicate columns of the antigen plates, and
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incubated at room temperature for 2 h. After three
washes with PBS-Tween, goat anti-mouse IgG and IgM-
alkaline phosphatase (Tago #4653) diluted in PBS-Tween
was added to the wells and incubated for 2 h at room
temperature. Plates were again washed three times with
PBS-Tween. Disodium p-nitrophenyl phosphate (Sigma #104-
105) (lmg~ml) diluted in 10% diethanolamine substrate
buffer (pH 9.8) was added to the wells. Absorbance at
405 nm (A 405) was read by the BioTek Microplate
AutoReader (Model EL309) after plates were incubated in
t~e dark at room temperature for 30 min.
When ELISAs were done to isotype the antibody
response, the BioRad sub-isotyping panel (#172-2055) was
;i'
; used.
While the invention has been described with
reference to the above embodiments, it will be unders~ood
that its scope is defined by the following claims.
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FIGURE LEGENDS
Figure 1. Preparation scheme of glutaraldehyde-fixed S-
Layer fragments; (a) intact bacterial cell; (b) empty
peptidoglycan sacculus after glutaraldehyde fixation. A
second S-Layer has been formed on the inner surface; (c)
glutaraldehyde-fixed S-Layer fragments after lysozyme
treatment consisting of two S-layers. PG, Peptidoglycan;
CM, cytoplasmic membrane; S, S-layer - iS and oS, inner
and outer S-Layer (adapted from Sara and Sleytr). J.
Bacterial. (1987) 169: 4092-4098.
Figure 3. Effect of the dose of T-disaccharide-linked to
B. stearothermophilus S-Layer ~T-3a) on the immunological
priming for DTH to the T-hapten. Mice were treated with
cyclophosphamide (200 mg/kg) and immunized two days later
with varying concentrations of the T-3a. Seven days
later all groups of mice were footpad-challenged with
lO~g of a preparation of T-disaccharide bound to C.
thermohydrosulfuricum (T-Llll) S-layer. Footpad swelling
was measured 24 h later. The increase in footpad
thickness is expressed in mm x 10 1.
,
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Figure 4. Effect of the concentration of T-disaccharide-
linked to C. thermohvdrosulfuricum (T--Llll) on the
induction of the DTH response to the T-hapten. Mice were
treated with cyclophosphamide (200 mgJkg~ and immunized
two days later with lOI~g of a preparation containing T-
disaccharide bound to B. stearothermophilus (T-3a) S-
layers. seven days later groups of mice were footpad-
challenged with varying amounts of T-disaccharide bound-
to Llll S-Layers. Footpad swelling was measured 24 h
later. The increase in footpad thickness is expressed in
mm x lO l.
Figure 5. Groups of mice were treated with
cyclophosphamide or not and subsequently sham-immunized
or immunized with lO~g of a preparation of T-disaccharide
bound to B. stearothermophilus (T-3a) S-Layers, mixed
with PBS, DDA or FIA. Seven days later each group of
mice were footpad challenged with lO~g of a preparation
of T-disaccharide bound to C. thermohydrosulfuricum (T-
Llll) S-Layers. Footpad swelling was measured 24 h
later. The increase in footpad thickness is expressed in
mm x lO-l.
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Figure 6. Groups of lO mice were treated, or not, with
cyclophosphamide and subsequently immunized with lO~g of
a preparation of T-disaccharide bound to B.
stearothermophilus (T-3a) S-Layers, lO~g T-disaccharide
bound to bovine serum albumin (BSA) or lO~g of the 8-
methylony carbonyal octanol space arm (that attaches the
T-disaccharide to the S-Layer) bound to BSA (G-BSA) with
or without DDA. Seven days later each group of mice were
footpad challenged with lO~g of a preparation of T-
disaccharide bound to C. thermohydrosulfuricum (T-Llll)
S-Layers or lO~g of the 8-methylony carbonyal actonal
space arm bound to BSA. Footpad swelling was measured 24
h later. The increase in footpad thickness is expressed
in mm x lO l plus l Standard deviation.
`
Figure 7. Transfer of the lymphocytes mediating DTH
response against the T-disaccharide. A group of lO mice
were pretreated with cyclophosphamide and subsequently
immunized with lO ~g of a preparation of T-disaccharide-
3a-S-Layer (T-3a). Seven days later mice were sacrificed
and draining lymph nodes and spleens were isolated.
Purified lymphocytes wer;è~then isolated and cultured in
RPMI 1640 supplemented with 2% Ultroser Hy, and 1%
penicillin and streptomycin. The T-disaccharide-3a
,
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WO91/12819 PCT/CA91/00063
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2077~2
primed lymphocytes were bulk stimulated with either P~S,
Llll S-Layers, or T-disaccharide-Llll-S-Layer conjugates
(T-Llll). Four days later the lymphocytes were collected
and washed in PBS. Primed lymphocytes stimulated with T- ;
disaccharide-L111 were then divided into 4 groups and
sham treated or treated with monoclonal antibodies L3T4,
(specific for helper T-cells), Ly 2.2 (specific for
suppressor T-cells) and Thy 1.2 (specific for all T-
cells). The 4 groups cells were then treated with
complement and then washed and counted. The primed cells
(5 x 105) were then mixed with either PBS or T-
disaccharide 2051 S-Layer (T-2051) and then injected into
naive mice. Footpad swelling was measured 24 h later.
The increase in footpad thickness is expressed in mm x
10 1.
Figure 8. Comparison of Oral and Intramuscular
; immunization with S-layer conjugates.
a) Groups of mice were immunized with 10 ~q of
i a preparation of T-disaccharide bound to L111 S-layers
(T-L111), orally or intramuscularly. Seven days later,
each group of mice were footpad challenged with 10 ~g of
a preparation of T-disaccharide bound to 2-51 S-Layers
.
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(T-2051). Footpad swelling was measured 24 h later. The
increase in footpad thickness is expressed in mm l.
; b) Groups of mice immunized with lO ~g of a
preparation of S. pneumoniae type 8 capsular
polysaccharide bound to Llll S-Layers t8CPS-Llll), orally
or intramuscularly. Seven days later each group of mice
were footpad challenged with lO ~g of a preparation of S.
puemoniae type 8 capsular polysaccharide bound PV72 S-
Layers (8 CPS-PV72). Footpad swelllng was measured 24 h
later. The increase in footpad thickness is expressed in
--1
, ~m .
Figure 9
a) Tertiary antibody response to Y hapten
coupled to unfixed L-lll S-Layers (-~-)antibody response
, to the Y hapten, (-o-) negative control.
,;` b) Tertiary antibody responses to unfixed Llll
S-Layer coupled with Y hapten. (-~-)antibody response to
unfixed Llll, (-o-) negative control.
. .
Figure lO
Immunoglobulin isotyping of tertiary antibody
response to unfixed Y-Llll/FA, t~) isotypic antibody
response to the Y hapten, (~) and isotypic antibody
response to unfixed Llll.
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2~773 ~7 2
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Figure 11
Tertiary antibody response to T-disaccharide
coupled by EDC to fixed L111 S-Layer (~) antibody
response to T-disaccharide at different molar hapten
ratios, (~) antibody response to the Llll S-Layer
carrier.
Figure 12
Tertiary antibody response to S. pneumoniae CPS
type 3-Llll conjugate (-~-)antibody response to CPS type
3, negative control response to CPS type 3 in mice
injected with Llll (-~-)or PBS (---).
Figure 13
Immunoglobulin isotyping of tertiary antibody
response to S. pneumoniae CPS type 3-Llll S-Layer (~)
isotypic antibody response to CPS type 3 when mice were
immunized with 3-Llll conjugate (~) isotypic antibody
response to CPS type 3 when mice were immunized with CPS
type 3.
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WO91/12819 PCT/CA91/00063
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Figure 14
Secondary antibody response to S. pneu~oniae
oligosaccharide type 3. Response to CPS type 8 when 8-
PV72 (-~-),8-2051 (-~-),8-TT (-~-), 8-oligo t-o-) or
PBS (-O-)was injected.
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~- REF~RENCE5:
1. Mowat, A. M. (1987) Immunol. Today 8, 93.
2. Bruce, M. G. Ferguson, A. (1986) Immunology 57,
627.
3. Elsun, C.O. Ealding, W. (1984) J. Immuno. 132
6, 2736.
4. Muckerheide, A., Apple R.J., Pesce, A.J.,
Michael, J.G. (1987) J. Immuno. 138 3, 833.
5. Smith, R.H., Ziola, B., (1984) Cell Immuno.
89 20.
6. Smith, R.H., Babiuk, L.A., Stockdale, P.H.G.
i (1980) Infect. Immun. 31 129.
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