Immunogenic compositions containing ordered carriers

Superior pharmaceutical compositions which comprise carriers coupled to epitope-bearing moieties are described. The carriers are crystalline or paracrystalline glycoproteins, especially those derived from S-layers of microbial cell walls. These conjugates are capable of eliciting the formation of antibodies as well as a T-cell response.

FIELD OF THE INVENTION 
The invention relates to improved immunogenic compositions in which haptens 
to which an immune response is desired are conjugated to a crystalline or 
paracrystalline carriers, especially those represented by bacterial 
surface layers, or "S-layers." 
BACKGROUND 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 immunomodulators 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 act as adjuvants to 
induce humoral immunity to carbohydrate moieties on large glycoproteins by 
Bellstedt, D. U., et al., J Immunol Meth (1987) 98:249-255; Livingston, P. 
O., et al., J Immunol (1987) 138:1524-1529. 
European patent application publication No. 180,564 describes the 
preparation of a complex from bacterial substrates designated "Iscom." 
This complex is prepared by solubilizing the hydrophobic peptides from 
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 immunogenic 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. 
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 B-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 crystalline 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. 
DISCLOSURE OF THE INVENTION 
In accord with the present invention, the problems of low or lacking 
immunogenicity, lack of T-lymphocyte-mediated response, ill-defined 
(poorly reproducible) coupling of haptens or immunoactive substances, and 
residual toxicity of antigen carriers is 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 defined spatial orientation with respect to 
each other; thus both the nature of the linkages 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 
crystalline 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 mediated or B-cell 
mediated immune response to an epitope of interest, which method comprises 
administration of the compositions of the invention to a vertebrate 
subject in an amount effective to elicit the desired response.

MODES OF CARRYING OUT THE INVENTION 
The invention provides pharmaceutical compositions which are capable of 
enhancing the immune response by virtue of employing carriers which are 
two-dimensional crystalline arrays. Various methods of attaching the 
imminoactive substances to these carriers may also be employed. The 
resulting compositions may be used both for stimulation of B-cell and 
T-cell responses. 
NATURE OF THE CARRIERS AND COMPOSITIONS 
Advantageously, the two-dimensional crystalline arrays may consist of 
glycoproteins or proteins 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 composing thse 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. 
FIG. 1 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 S-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. 
In a preferred embodiment of the present invention, the two-dimensional 
crystalline arrays are derived from microbial cell walls as S-layers with 
or without additional peptidoglycan layers. In order to prepare these 
aggregates, bacteria are incubated with detergent at elevated temperatures 
of 30.degree.-60.degree. C., preferably around 50.degree. C., for a 
suitable time period to disintegrate the cytoplasm and plasma membrane of 
the organism. Suitable detergents include, for example Triton X-100, 
various alkylpolyoxyethylene ethers, various acylpolyoxyethylene sorbitol 
esters, such as the Tweens, and the like. Intact bacteria may be treated 
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 is 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 peptidoglycan-degrading enzyme, for 
example lysozyme. The recovered pellet is treated with the enzyme at about 
37.degree. C. in buffer 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 of 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. 
In addition, since the carriers of the invention can be of sufficient 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 
hydrophobic carrier molecules can be mixed with aggregates carrying the 
immunoactive substances causing increased uptake of the pharmaceutical 
structure by accessory cells. 
Carriers comprising different immunoactive substances can be attached to an 
auxiliary matrix which can be cross-linked to the carriers. Thereby, a 
pharmaceutical preparation can be generated that would combine different 
types of carrier molecules such as those differing in their crystalline 
structures. 
Furthermore, carrier aggregates comprising 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 individual 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 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 OF 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 immunospecificity with regard to 
the antibodies obtained. 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 immune response is desired. 
The two-dimensional crystalline 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 of course be employed. 
The choice of the mode of attachment will depend both upon the nature of 
the haptens and/or immunoactive substances and upon the type of 
application of the pharmaceutical composition. Under certain 
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., 
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 enzyme-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, Ill. 
By an advantageous process for the production of the pharmaceutical 
compositions of this invention, such groups on the carrier as would bind 
the immunoactive substances 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 carbohydrate 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 groups of the protein portion of the carrier. 
Attachment of the haptens can also be through aldehydes 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-like 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 
glycolipids. Other molecules of interest include alkyloids 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 compositions 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, resulting 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 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 perpared from different microorganisms, this problem can be 
avoided. Thus, protocols are devised wherein the epitope-bearing moiety is 
injected first conjugated to S-layer perpared 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, FIGS. 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 the end of the specification herein. 
EXAMPLE 1 
A. Preparation of the Carrier 
Cells of Clostridium thermohydrosulfuricum L111-69 (2.5 g) are suspended in 
50 mM Tris-HCl buffer, pH 7.1, and sonicated briefly (about 1 minute). 
Following the addition of a 2% solution of Triton X-100 (12.5 ml), the 
suspension is incubated at 50.degree. 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. Subsequently, the mixture 
is centrifuged at 20,000.times.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 (500 ug) are added and the whole stirred 
for 15 minutes at 37.degree. C. The suspension is then centrifuged at 
20,000.times.g and washed three times with water. The pellet is then 
suspended in 1.1M cacodylate buffer (pH 7.2; 20 ml) and a 50% aqueous 
solution of glutaraldehyde is added at 40.degree. C. to a final 
concentration of 0.5%. The suspension is well stirred at 4.degree. 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 to pH=3. Following 10 minutes standing at room temperature, the 
suspension is again centrifuged (20,000.times.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. With 
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 (FIG. 1); 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 1 h at 36.degree. C. with a solution of lysozyme (0.5 mg 
lysozyme per ml of a 50 mM solution of Tris-HCl buffer, pH 7.2). In this 
case, 10 ml of lysozyme solution is added per 0.5 g wet pellet. Depending 
on the microorganism used, the S-Layer fragments obtained consist of a 
simple 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. 
B. Formation of Binding Sites 
The pellet prepared according to A is suspended in water (5 ml) and a 0.1M 
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. Binding 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). This 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 FIG. 2. Clearly, the attachment to the carrier 
is significantly higher in the case involving prior oxidation with 
periodate. 
EXAMPLE 2 
A. Preparation of the Carrier 
Cells of Bacillus stearothermophilus PV7.2 (2.5/g) are suspended in 50 nM 
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.degree. 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 formed 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.times.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 .mu.g) and RNAse (ribonuclease, 500 .mu.G) are 
added, and the mixture is stirred at 37.degree. C. for 15 min. 
Subsequently, the pellet is washed three times with water, centrifugation 
in between being at 20,000.times.g. The pellet is then suspended in 0.1M 
cacodylate buffer (pH 7.2) and the suspension mixed with a 50% solution of 
glutaraldehyde in water at 4.degree. C. to a final concentration of 0.5%. 
The suspension is then vigorously stirred at 40.degree. C. 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 1B. 
B. Binding of Protein(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 1C, and the amount of protein bound is 
determined as described there. 
EXAMPLE 3 
A. Preparation of the Carrier 
Cell walls of Clostridium thermohydrosulfuricum L111-69 are treated with 
glutaraldehyde (0.5% in 0.1M sodium cacodylate buffer, pH 7.2) for 20 
minutes at 20.degree. 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 Ligands Containing Thiol (SH) 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 
1-ethyl-3,3-(dimethylaminopropyl)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 hexamethylenediamine (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.times.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 
per ml of tetrahydrofuran; 1 ml) is added. The mixture is then incubated 
for 30 minutes at 20.degree. C. 
C. Binding of SH-Containing Proteins to the S-Layers Derivatized as Under B 
Following centrifugation at 20,000.times.g, the pellet is suspended in 50 
mM phosphate buffer (pH 7.0), .beta.-galactosidase (20 mg) is added and 
the mixture is incubated for 2 h at 20.degree. C. After centrifugation at 
20,000.times.g and repeated washing with phosphate buffer, the activity of 
the .beta.-galactosidase covalently linked to the protein matrix is 
determined. 
The reactions of the Example are summarized as follows: 
##STR1## 
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 with 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.degree. C. for 10 minutes, 
centrifuged at 20,000.times.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.times.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.degree. C. 
Following centrifugation at 20,000.times.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 follows: 
##STR2## 
EXAMPLE 5 
A. 
Cell wall fragments of Clostridium thermohydrosulfuricum L111-69 are 
cross-linked with glutaraldehyde as described in Example 3, section A. 
B. Generating the Binding 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.times.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 0.1M sodium 
hydrogencarbonate (pH 8.8) containing dissolved dextranase (20 mg) and the 
reaction mixture is incubated at 4.degree. C. for 18 h. The cell wall 
fragments containing the bound dextranase are obtained by centrifugation 
at 20,000.times.g and washed twice with distilled water. The dextranase 
activity contained in the pellet is then determined. 
The reactions of this Example are summarized as follows: 
##STR3## 
EXAMPLE 6 
Coupling of a Synthetic Carbohydrate Antigen to Oxidized S-Layers 
A. Preparation of Carrier and Generation of the Binding Sites 
The preparation of the oxidized glycoprotein S-Layers was performed as 
described in Example 1, section A and B. 
B. Binding of the Carbohydrate Antigen to the Carrier 
The oxidized (polyaldehyde) derivative of the S-Layer prepared in Section A 
is incubated with the 3-(2-aminoethyl)thiopropyl glycoside of a 
disaccharide 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: 
##STR4## 
General recipe for the preparation of 3-(2-aminoethylthio)propyl 
glycosides from allyl glycosides. 
A solution of the allyl glycoside (5 mM) in a solution of cysteamine 
hydrochloride (15 milliequivalents of SH-groups in 10 ml) is allowed to 
stand for 1.5 h at room temperature. The duration of this reaction may 
vary. The reaction mixture is subsequently separated over a column of 
cation exchange resin (e.g., Rexyn 101, ammonium form, 200-400 mesh). The 
column is eluted with water, 0.5M ammonia, and 1.0M ammonia. Unreacted 
allyl glycoside appears in the aqueous eluate, and the 
3-(2-aminoethylthio) propyl glycoside is eluted in the fraction 
corresponding to 1.0M ammonia. Those fractions 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, .sup.125 I (iodine-125) or in any other 
appropriate manner. The bound antibodies can also be assayed via 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 of the S-Layers by reacting it with borohydride or 
another suitable reducing agent. 
##STR5## 
The secondary amine derivative of the S-Layer would be more stable to acid 
than the Schiff base derivative. 
##STR6## 
The determination of the content of free aldehyde groups in the 
polysaccharide portion, following oxidation with periodate, is perforemd 
using phenylhydrazine or 2,4-dinitrophenylhydrazine, or other suitable 
reagents. 
Suitable carbohydrate-containing S-Layers are oxidized with sodium 
metaperiodate as described in Example 1, 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 1 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. 
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. 
After oxidation with sodium metaperiodate as above described, 
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 1 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. 
EXAMPLE 7 
A. Preparation and Structure of S-Layer Glycoprotein 
A cross-linked S-Layer preparation from cell wall fragments of Clostridium 
thermohydrosulfuricum L111-69 (hereinafter L111) and Bacillus 
stearothermophilus NRS2004/3A (hereinafter 3A) were prepared using the 
procedure set forth in Example 1A. Electron 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 (FIG. 1). 
The peptidoglycan of the foregoing preparations was removed by digestion 
with lysozyme as described in Example 1A 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 six 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 1A also showed two 
identical subunits. 
B. Specific Methods for Attachment of Haptens 
1. Periodate Oxidation 
The glycan chains of the L111 preparation are known to consist of 
approximately 60 disaccharide repeats of 
4(-.alpha.-D-Man.beta.-(1-3)-.alpha.-L-Rhap-(1, which comprises about 10% 
of the S-Layer glycoprotein. When treated with periodate at 0.1M for 2-5 h 
at pH 5.5, the mannose residues were oxidized completely as estimated by 
the 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)(-.alpha.-L-Rhap-(1-2)-.alpha.-L-Rhap-(1-3)-.beta.-L-Rhap(1, having 
about 50 repeats; and 
-4)(-.beta.-ManpUA2,3(NAc)2-(1-3)-.alpha.-GlcNAc-(1-4)-.beta.-ManpUA2,3(NAc 
)2-(1-6)-.alpha.-Glcp-(1, 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. Unfixed S-Layers gave lower yields of conjugates or hapten 
contents. 
2. Epichlorohydrin Activation 
Glutaraldehyde-fixed S-Layer fragments (2-3 mg) were suspended in 2.5 mL of 
either 0.2M sodium bicarbonate/sodium carbonate buffer, pH 9.1-10.0, or 
0.2M 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.degree., with rotation on a rotary evaporation (150 
rpm). Blanks were prepared similarly, but either epichlorohydrin or hapten 
was omitted. 
After centrifugation and washing with water (5.times.1.5 mL) to remove all 
of the activating agent, the combined supernatants of the first 
centrifugation and all washes were analyzed for carbohydrate material 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 .mu.moles/mL 0.2M sodium bicarbonate solution) 
and incubated for 2-6 h with shaking on a hematology mixer at room 
temperature. Excess of hapten was removed by washing with water 
(5.times.1.5 mL) prior to the blocking of unreacted epoxy groups by 
treatment with 0.02M ethanolamine in 50 mM sodium bicarbonate solution for 
4-18 h at room temperature. Subsequently, the samples were washed with 
water (5.times.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 (2-3 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. Then the pH was adjusted to ca. 4.6 using diluted HCl. 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.times.10 ml) and centrifugation (19,000 rpm, 4.degree. C.). The pellets 
were incubated with hapten solution (1.0-1.5 mL, ca. 2 .mu.moles mL.sup.-1 
/0.2M NaHCO.sub.c) at room temperature for 2-18 h. Upon washing with water 
(5.times.1.5 mL) the samples were lyophilized and assayed. 
ii) About 3 mg S-Layer material was suspended in 2 mL of 0.1M phosphate 
buffer (pH 4.0-4.7) and 10 mg EDAC was immediately added to the 
suspensions. After activation for 1 h with rotation on the rotavapor 1 mL 
hapten solution was added and the mixture was incubated overnight in the 
presence of EDAC at room temperature. After washing with water 
(5.times.1.5 mL) activated free carboxyl groups were blocked with 10% 
glycine in 0.2M NaHCO.sub.3 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. 
EXAMPLE 8 
Preparation of T-Disaccharide Antigen Conjugates 
T-disaccharide is a tumor marker of the formula 
.beta.Galp(1-3).alpha.GalNAcp. A composition containing this disaccharide 
as a hapten was prepared by coupling the spacer-linked disaccharide of the 
formula .beta.Gal-(1-3).alpha.GalNAc-O(CH.sub.2).sub.8 CNANA.sub.2 to 
S-Layers by either of the general procedures described under Example 
7.B.4. 
EXAMPLE 9 
Immobilization of Synthetic Carbohydrate Antigens 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.degree.) 50 mM sodium acetate buffer, 
pH=5.0 (0.25 ml). To one of the suspensions is added a cold 0.2M 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.degree. 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.times.g, 10 min) and washed once with sodium acetate buffer, pH=5, 
and twice with 0.2M sodium borate buffer, pH=8.5, to remove 
iodine-containing materials. 
B. Binding of Carbohydrate Antigen to Carrier 
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 (10 mg). T-disaccharide (hydrazide form, 5 mg) is 
added to one of the oxidized samples, while one oxidised and one 
unoxidized sample are processed without hapten addition. The preparations 
are incubated at 37.degree. for 24 h with stirring (500 rpm). 
Subsequently, the S-Layers are washed once with 0.2M borate buffer, once 
with 0.2M sodium chloride solution, and twice with distilled water. The 
centrifugation pellets of the respective samples may then be frozen 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 Peptidoglycan in a (Sterile) Ultrafiltration Cell 
The (frozen) S-Layer pellets (produced according to B) are suspended in 50 
mM Tris-hydrochloride buffer pH=7.2 (5 mL) in a 10 mL ultrafiltration cell 
(AMICON) equipped with a magnetic stirrer and an ultrafiltration membrane 
(e.g. BIOFIL.TM. produced from S-Layers of 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 (until no extinction is detectable at 280 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-Layer-Hapten Conjugates 
The enriched suspension in the ultrafiltration cell (ca. 1 mL) is mixed 
with 5M guanidine hydrochloride (5 mL) and incubated for 1 h at room 
temperature with stirring. By this treatment, the S-Layers are dissociated 
into protomers (MWA 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 of 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. The 
clear washes were combined, dialyzed and analyzed for S-Layer fragments by 
SDS-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-PAGE. 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 immobilization 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 starting 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 10 
Efficacy of the T-Disaccharide S-Layer Compositions 
Groups of 5-10 mice were pretreated 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 L111 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-L111 was 5 
.mu.g/mouse, and a maximal response was obtained when immunization was 
performed at 10 .mu.g of disaccharide T-disaccharide-3a-S-Layer per mouse 
(FIG. 3). At this level, a footpad swelling of approximately 5.5 mm.sup.-1 
was obtained. An increase in the dose to 20 .mu.g resulted in a lowering 
of swelling to 4 mm.sup.-1. 
In further experiments, CP-pretreated mice were immunized with 10 .mu.g of 
T-disaccharide-3a-S-Layer, and seven days later were footpad-challenged 
with varying amounts of T-disaccharide L111 S-Layer. Maximum response (4.5 
mm.sup.-1) was obtained using 20 .mu.g of T-disaccharide L111 S-Layer 
(FIG. 4). 
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 11 
Specificity of the T-Cell Responses Generated by S-Layer Conjugates 
A further control confirmed that the DTH reponse 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 100 .mu.g 
dimethyldioctadecyl ammonium bromide (DDA). They were then challenged with 
PBS and with L111 S-Layer lacking the T-disaccharide, as well as 
T-disaccharide L111 S-Layer. The results are shown in Table 1. Only the 
T-disaccharide-L111 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. 
TABLE 1 
______________________________________ 
Specificity of the Haptenated S-Layer 
Footpad 
Immunization Swelling 
Antigen.sup.a 
Challenge Antigen (mm .times. 10.sup.-1) 
______________________________________ 
T-3a 10 .mu.g/mouse 
PBS 0.8 .+-. 0.8 
T-3a 10 .mu.g/mouse 
EDAC L111 10 .mu.g/mouse.sup.b 
0.37 .+-. 9.4 
T-3a 10 .mu.g/mouse 
T-L111 10 .mu.g/mouse 
.sup. 3.8 .+-. 0.8.sup.c 
PBS & DDA T-L111 10 .mu.g/mouse 
1.89 .+-. 0.65 
NT T-L111 10 .mu.g/mouse 
1.33 .+-. 0.4 
______________________________________ 
.sup.a CPpretreated mice were immunized with antigen mixed with 100 .mu.g 
DDA. 
.sup.b L111 was shamtreated with EDAC to control for the possible 
modification of L111 by the EDAC reagent. 
.sup.c Significantly different from all other groups P &gt; 0.001. 
EXAMPLE 12 
Comparison to Other Carriers and Adjuvant Systems 
The immunization protocols of Example 10 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 immunization, the mice were footpad-challenged with 
T-disaccharide-L111 (analogous S-Layer preparations according to Example 
10), 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 (FIG. 
5). The results also show that T-disaccharide-3a is best administered in 
conjunction with DDA, in contrast to no adjuvants or FIA. To compare the 
immunopotentiating response of S-Layers with other carriers, mice were 
immunized as outlined in example 10 using T-disaccharide-3a-S-Layers, 
T-disaccharide bovine serum albumin (T-disaccharide BSA) or 8-methyloxy 
carbonyal octanol (the linker arm attached to the T-disaccharide) linked 
to BSA. Seven days after immunization, mice were footpad challenged with 
T-disaccharide-L111 according to example 10. The results in FIG. 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 protocols were complex, and a DTH 
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 Lymphoproliferative Response 
The mice immunized with both T-disaccharide-3a and T-disaccharide-L111, as 
described above (using DDA after CP treatment), were sacrificed, 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 
10 .mu.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 bacteria. 
TABLE 2 
______________________________________ 
Stimulation of Lymphocytes from Mice 
Immunized with Both T-Disaccharide-3a and T-Disaccharide-L111 
Antigen in Culture 
CPM .sup.3 II-Thymidine 
Statistical 
20 .mu.g/ml Uptake Probabilities.sup.a 
______________________________________ 
PBS 2303 .+-. 692 tND 
2051.sup.b 2549 .+-. 1258 
.sup. NS.sup.c 
T-BSA 1840 .+-. 866 NS 
T-KHL 2091 .+-. 498 NS 
T-2051 4654 .+-. 1294 
P = 0.001.sup.b 
______________________________________ 
.sup.a All statistics were compared to the PBS values. 
.sup.b 2051 is the unbound SLayer isolated from B. alvei and is the 
control for the Tdisaccharide-bound 2051. 
.sup.c NS not significant 
.sup.d P &lt; 0.01 when compared to 2051 stimulation alone. 
EXAMPLE 14 
Transfer of Activated Lymphocytes 
To clearly show that the DTH reponse generated by the T-disaccharide 
conjugate was actually due to helper T-cells, an adopted transfer 
experiment was carried out. In these series of experiments CP-pretreated 
mice were immunized with 10 .mu.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-L111 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 reponse was then measured 24 h later. The results of this 
experiment are shown in FIG. 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 was observed. Only when the three S-Layers were haptenated with 
the T-disaccharide was a significant DTH response observed. Also, when the 
primed lymphocytes were depleted of specific T-cell populations, it became 
evident that the primed cells involved in the DTH repsonse were helper 
T-cells. 
The pharmaceutical structures constituting the embodiment of the present 
invention are particularly suitable as immunizing antigens 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.g., for diagnostic kits or 
extracorporeal depletion of undesirable antibodies from human blood. 
While the invention has been described with reference to the above 
embodiments, it will be understood that its scope is defined by the 
following claims. 
FIGURE LEGENDS 
FIG. 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. 
FIG. 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 10 .mu.g of a 
preparation of T-disaccharide bound to C. thermohydrosulfuricum (T-L111) 
S-layer. Footpad swelling was measured 24 h later. The increase in footpad 
thickness is expressed in mm.times.10.sup.-1. 
FIG. 4. Effect of the concentration of T-disaccharide-linked to C. 
thermohydrosulfuricum (T-L111) on the induction of the DTH response to the 
T-hapten. Mice were treated with cyclophosphamide (200 mg/kg) and 
immunized two days later with 10 .mu.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 L111 S-Layers. Footpad swelling was measured 24 h 
later. The increase in footpad thickness is expressed in 
mm.times.10.sup.-1. 
FIG. 5. Groups of mice were treated with cyclophosphamide or not and 
subsequently sham-immunized or immunized with 10 .mu.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 10 .mu.g of a preparation of T-disaccharide bound to C. 
thermohydrosulfuricum (T-L111) S-Layers. Footpad swelling was measured 24 
h later. The increase in footpad thickness is expressed in 
mm.times.10.sup.-1. 
FIG. 6. Groups of 10 mice were treated, or not, with cyclophosphamide and 
subsequently immunized with 10 .mu.g of a preparation of T-disaccharide 
bound to B. stearothermophilus (T-3a) S-Layers, 10 .mu.g T-disaccharide 
bound to bovine serum albumin (BSA) or 10 .mu.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 10 .mu.g of a preparation of 
T-disaccharide bound to C. thermohydrosulfuricum (T-L111) S-Layers or 10 
.mu.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.times.10.sup.-1 plus 1 Standard deviation. 
FIG. 7. Transfer of the lymphocytes mediating DTH response against the 
T-disaccharide. A group of 10 mice were pretreated with cyclophosphamide 
and subsequently immunized with 10 .mu.g of a preparation of 
T-disaccharide-3a-S-Layer (T-3a). Seven days later mice were sacrificed 
and draining lymphnodes and spleens were isolated. Purified lymphocytes 
were then isolated and cultured in RPMI 1640 supplemented with 2% Ultroser 
Hy, and 1% penicillin and streptomycin. The T-disaccharide-3a primed 
lymphocytes were bulk stimulated with either PBS, L111 S-Layers, or 
T-disaccharide-L111-S-Layer conjugates (T-L111). 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.times.10.sup.5) 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.times.10.sup.-1.