Compositions for the delivery of antigens

The present invention relates to compositions and methods for orally delivering antigens. The antigen and an adjuvant are combined with an acylated amino acid or polyamino acid and, a sulfonated amino acids or polyamino acid, or a salt of the foregoing.

FIELD OF THE INVENTION 
The present invention relates to compositions useful for the delivery, and 
preferably the oral delivery, of antigens and adjuvants to animals. 
Methods for the preparation and for the administration of these 
compositions are also disclosed. 
BACKGROUND OF THE INVENTION 
Conventional means for delivering antigens to their intended targets are 
often severely limited by the presence of biological, chemical, and 
physical barriers. Typically, these barriers are imposed by the 
environment through which delivery must take place, the environment of the 
target for delivery, or the target itself. 
Oral delivery of antigens would be the route of choice for administration 
to animals if not for physical barriers such as the mucous layer and the 
epithelial cells of the gastrointestinal (GI) tract. Oral delivery is also 
impeded by chemical barriers such as the pH in the GI tract and the 
presence in the oral cavity and the GI tract of powerful digestive 
enzymes. Furthermore, orally administered soluble and insoluble antigens 
can induce a non-responsive state or tolerance. 
Methods for orally administering antigens have been developed which rely on 
the use of either attenuated microorganisms or polylactide/polyglycocide 
(PLA/PGA) microspheres to increase antigen presentation to and uptake by 
the appropriate antigen presenting cells. Attenuated organisms, unless 
properly delivered, can regain virulence, however. Additionally, broad 
spectrum use of PLA/PGA microspheres is not possible because these 
carriers require organic solvents that may alter or denature antigens. 
Furthermore, PLA/PGA systems are difficult to manufacture. 
More recently, microspheres comprising artificial polymers of mixed amino 
acids (proteinoids) have been described for delivering biologically active 
agents including antigens. Santiago, et al. Pharmaceutical Res. Vol. 10, 
No. 8, (1993). 
Adjuvants have been coadministered with antigens to increase the 
effectiveness of antigens, but adjuvants and antigen/adjuvant compositions 
are susceptible to the common problems of oral delivery described above. 
Consequently, there is still a need in the art for simple, inexpensive, and 
easily prepared systems which can effectively deliver a broad range of 
antigens, particularly via the oral route. 
SUMMARY OF THE INVENTION 
The present invention provides compositions for delivering antigens. These 
compositions are suitable for delivery via the oral route and comprise: 
(a) an antigen; 
(b) an adjuvant; and 
(c) at least one carrier comprising a member selected from the group 
consisting of; 
(i) an acylated amino acid or a salt thereof; 
(ii) a polyamino acid comprising at least one acylated amino acid or a salt 
thereof; 
(iii) a sulfonated amino acid or a salt thereof; 
(iv) a polyamino acid comprising at least one sulfonated amino acid or a 
salt thereof; or 
(v) any combination thereof. 
These compositions can be orally administered to animals to produce or 
prime and/or to boost an immunogenic response and to achieve immunization. 
When these compositions are used to boost immunogenic responses the prime 
can be delivered by the compositions of the present invention or other 
compositions. 
Also contemplated are methods for preparing mixtures of microspheres of an 
antigen, an adjuvant, and a carrier as described above, and optionally, a 
dosing vehicle.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention uses readily available and inexpensive carrier 
starting materials and provides a cost-effective method for preparing and 
isolating immunogenic compositions. The present invention is simple to 
practice and is amenable to industrial scale-up for commercial production. 
The compositions of the subject invention are useful for administering 
antigens to any animals such as birds and mammals, including, but not 
limited to, primates and particularly humans. The compositions elicit an 
immunogenic response and provide immunization. 
Antigens 
Antigens suitable for use in the present invention include, but are not 
limited to, synthetic or naturally derived proteins and peptides, and 
particularly those which by themselves are unable to induce an efficient 
immune response or which induce tolerance; carbohydrates including, but 
not limited to, polysaccharides; lipopolysaccharides; and antigens 
isolated from biological sources such as, for example, microbes, viruses, 
or parasites, and subunits or extracts therefrom; or any combination 
thereof. Special mention is made of the antigens Streptococcus pneumoniae, 
S. typhi VI carbohydrate, Hemophilus influenzae (type B), Acellular B. 
pertussis, Neisseria meningiditis (A,C), H. influenzae (type B, Hib), 
Clostridium tetani (tetanus), Corynebacterium diphtheriae (diphtheria), 
and infectious bursal disease virus (IBDV) (attenuated and virulent). 
Adjuvants 
Adjuvants suitable for use in the present invention include, but are not 
limited to protein carriers such as protein containing appropriate T-cell 
epitopes; hydrophobic antigens such as proteins with a lipid tail or 
antigens in oil with added MDP; polyclonal activators of T-cells such as 
PPD, poly A and poly U; B-cell activators such as antigen-polymerizing 
factors and B-cell mitogens; macrophage (APC) stimulators such as muramyl 
dipeptides (MDP) and derivatives thereof; and lipopolysaccharides (LPS); 
alternate pathway complement activators such as, for example, inulin, 
zymosan, endotoxin, levamisole, C. parvum; or any combinations thereof. 
Other useful adjuvants include lipoidal amines in general; 
polyphophazenes; bacterial toxins such as E-coli heat labile enterotoxin 
(LT-OA), cholera or diphtheria toxin or subunits, thereof, such as, for 
example, cholera toxin .beta.-subunit or E-coli heat labile anterotoxin 
.beta.-subunit; bacterial toxoids; poly or di-saccharides; or any 
combination thereof such as, for example, cholera toxin and cholera toxin 
.beta.-subunit. 
Preferred adjuvants are mucosal adjuvants. 
Carriers 
The carriers of the present invention are modified amino acids; polyamino 
acids; or peptides or salts thereof. Modified amino acids, poly amino 
acids, or peptides are either acylated or sulfonated and include amino 
acid amides and sulfonamides. 
Amino acids are the basic materials used to prepare these carriers. An 
amino acid is any carboxylic acid having at least one free amine group and 
includes naturally occurring and synthetic amino acids. The preferred 
amino acids for use in the present invention are .varies.-amino acids, and 
most preferably are naturally occurring .varies.-amino acids. Many amino 
acids and amino acid esters are readily available from a number of 
commercial sources such as Aldrich Chemical Co. (Milwaukee, Wis, USA); 
Sigma Chemical Co. (St. Louis, Mo., USA); and Fluka Chemical Corp. 
(Ronkonkoma, N.Y., USA). 
Representative, but not limiting, amino acids suitable for use in the 
present invention are generally of the formula 
##STR1## 
wherein: R.sup.1 is hydrogen, C.sub.1 -C.sub.4 alkyl, or C.sub.2 -C.sub.4 
alkenyl; 
R.sup.2 is C.sub.1 -C.sub.24 alkyl, C.sub.2 -C.sub.24 alkenyl, C.sub.3 
-C.sub.10 cycloalkyl, C.sub.3 -C.sub.10 cycloalkenyl, phenyl, naphthyl, 
(C.sub.1 -C.sub.10 alkyl) phenyl, (C.sub.2 -C.sub.10 alkenyl) phenyl, 
(C.sub.1 -C.sub.10 alkyl) naphthyl, (C.sub.2 -C.sub.10 alkenyl) naphthyl, 
phenyl (C.sub.1 -C.sub.10 alkyl), phenyl (C.sub.2 -C.sub.10 alkenyl), 
naphthyl (C.sub.1 -C.sub.10 alkyl), or naphthyl (C.sub.2 -C.sub.10 
alkenyl); 
R.sup.2 being optionally substituted with C.sub.1 -C.sub.4 alkyl, C.sub.2 
-C.sub.4 alkenyl, C.sub.1 -C.sub.4 alkoxy, --OH, --SH, --CO.sub.2 R.sup.3, 
C.sub.3 -C.sub.10 cycloalkyl, C.sub.3 -C.sub.10 cycloalkenyl, heterocyclic 
having 3-10 ring atoms wherein the hetero atom is one or more of N, O, S, 
or any combination thereof , aryl, (C.sub.1 -C.sub.10 alk)aryl, aryl 
(C.sub.1 -C.sub.10 alkyl) or any combination thereof; 
R.sup.2 being optionally interrupted by oxygen, nitrogen, sulfur, or any 
combination thereof; and 
R.sup.3 is hydrogen, C.sub.1 -C.sub.4 alkyl, or C.sub.2 -C.sub.4 alkenyl. 
The preferred naturally occurring amino acids for use in the present 
invention as amino acids or components of a peptide are alanine, arginine, 
asparagine, aspartic acid, citrulline, cysteine, cystine, glutamine, 
glycine, histidine, isoleucine, leucine, lysine, methionine, ornithine, 
phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 
hydroxy proline, .gamma.-carboxyglutamate, phenylglycine, or 
O-phosphoserine. The preferred amino acids are arginine, leucine, lysine, 
phenylalanine, tyrosine, tryptophan, valine, and phenylglycine. 
The preferred non-naturally occurring amino acids for use in the present 
invention are .beta.-alanine, .alpha.-amino butyric acid, .gamma.-amino 
butyric acid, .gamma.-(aminophenyl) butyric acid, .alpha.-amino isobutyric 
acid, 6-aminocaproic acid, 7-amino heptanoic acid, .beta.-aspartic acid, 
aminobenzoic acid, aminophenyl acetic acid, aminophenyl butyric acid, 
.gamma.-glutamic acid, cysteine (ACM), .epsilon.-lysine, .epsilon.-lysine, 
methionine sulfone, norleucine, norvaline, ornithine, d-ornithine, 
p-nitro-phenylalanine, hydroxy proline, 
1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid and thioproline. 
Poly amino acids are either peptides or two or more amino acids linked by a 
bond formed by other groups which can be linked, e.g., an ester, anhydride 
or an anhydride linkage. One or more of the amino acids of a polyamino 
acid or peptide may be modified. Special mention is made of non-naturally 
occurring poly amino acids and particularly non-naturally occurring 
hetero-poly amino acids, i.e., of mixed amino acids. 
Peptides are two or more amino acids joined by a peptide bond. Peptides can 
vary in length from dipeptides with two amino acids to polypeptides with 
several hundred amino acids. See, Walker, Chambers Biological Dictionary, 
Cambridge, England: Chambers Cambridge, 1989, page 215. Special mention is 
made of non-naturally occurring peptides and particularly non-naturally 
occurring peptides of mixed amino acids. Special mention is also made of 
dipeptides, tripeptides, tetrapeptides, and pentapeptides and 
particularly, the preferred peptides are dipeptides and tripeptides. 
Peptides can be homo- or hetero-peptides and can include natural amino 
acids, synthetic amino acids, or any combination thereof. 
Acylated Amino Acid Carriers 
Although the present invention encompasses any of the amino acids discussed 
above which have been acyclated, one group of preferred acylated amino 
acids have the formula 
EQU Ar--Y--(R.sup.4).sub.n --OH II 
wherein Ar is a substituted or unsubstituted phenyl or naphthyl; 
Y is 
##STR2## 
R.sup.4 has the formula 
##STR3## 
wherein: R.sup.5 is C.sub.1 to C.sub.24 alkyl, C.sub.1 to C.sub.24 
alkenyl, phenyl, naphthyl, (C.sub.1 to C.sub.10 alkyl) phenyl, (C.sub.1 to 
C.sub.10 alkenyl) phenyl, (C.sub.1 to C.sub.10 alkyl) naphthyl, (C.sub.1 
to C.sub.10 alkenyl) naphthyl, phenyl (C.sub.1 to C.sub.10 alkyl), phenyl 
(C.sub.1 to C.sub.10 alkenyl), naphthyl (C.sub.1 to C.sub.10 alkyl) and 
naphthyl (C.sub.1 to C.sub.10 alkenyl); 
R.sup.5 is optionally substituted with C.sub.1 to C.sub.4 alkyl, C.sub.1 to 
C.sub.4 alkenyl, C.sub.1 to C.sub.4 alkoxy, --OH, --SH and --CO.sub.2 
R.sup.7, cycloalkyl, cycloalkenyl, heterocyclic alkyl, alkaryl, 
heteroaryl, heteroalkaryl, halogens, or any combination thereof; 
R.sup.7 is hydrogen, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4 
alkenyl; 
R.sup.5 is optionally interrupted by oxygen, nitrogen, sulfur or any 
combination thereof; and 
R.sup.6 is hydrogen, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4 
alkenyl. 
Another group of preferred acylated amino acids have the formula 
##STR4## 
wherein: R.sup.8 is (i) C.sub.3 -C.sub.10 cycloalkyl, optionally 
substituted with C.sub.1 -C.sub.7 alkyl, C.sub.2 -C.sub.7 alkenyl, C.sub.1 
-C.sub.7 alkoxy, hydroxy, phenyl, phenoxy or --CO.sub.2 R.sup.11, wherein 
R.sup.11 is hydrogen, C.sub.1 -C.sub.4 alkyl, or C.sub.2 -C.sub.4 alkenyl; 
or 
(ii) C.sub.1 -C.sub.6 alkyl substituted with C.sub.3 -C.sub.10 cycloalkyl; 
R.sup.9 is hydrogen, C.sub.1 -C.sub.4 alkyl, or C.sub.2 -C.sub.4 alkenyl; 
R.sup.10 is C.sub.1 -C.sub.24 alkyl, C.sub.2 -C.sub.24 alkenyl, C.sub.3 
-C.sub.10 cycloalkyl, C.sub.3 -C.sub.10 cycloalkenyl, phenyl, naphthyl, 
(C.sub.1 -C.sub.10 alkyl) phenyl, (C.sub.2 -C.sub.10 alkenyl) phenyl, 
(C.sub.1 -C.sub.10 alkyl) naphthyl, (C.sub.2 -C.sub.10 alkenyl) naphthyl, 
phenyl (C.sub.1 -C.sub.10 alkyl), phenyl (C.sub.2 -C.sub.10 alkenyl), 
naphthyl (C.sub.1 -C.sub.10 alkyl) or naphthyl (C.sub.2 -C.sub.10 
alkenyl); 
R.sup.10 being optionally substituted with C.sub.1 -C.sub.4 alkyl, C.sub.2 
-C.sub.4 alkenyl, C.sub.1 -C.sub.4 alkoxy, --OH, --SH, --CO.sub.2 
R.sup.12, C.sub.3 -C.sub.10 cycloalkyl, C.sub.3 -C.sub.10 cycloalkenyl, 
heterocyclic having 3-10 ring atoms wherein the hetero atom is one or more 
of N, O, S or any combination thereof, aryl, (C.sub.1 -C.sub.10 alk)aryl, 
aryl(C.sub.1 -C.sub.10 alkyl), halogens, or any combination thereof; 
R.sup.10 being optionally interrupted by oxygen, nitrogen, sulfur, or any 
combination thereof; and 
R.sup.12 is hydrogen, C.sub.1 -C.sub.4 alkyl, or C.sub.2 -C.sub.4 alkenyl. 
Special mention is made of salicyloyl phenylalanine, and the compounds 
having the formulas: 
##STR5## 
Special mention is also made of compounds having the formula: 
##STR6## 
wherein A is Tyr, Leu, Arg, Trp, Phe, Lys, Val, or Cit; and optionally 
wherein if A is Tyr, Arg, Trp, or Cit; A is acylated at 2 or more 
functional groups. Preferably A is Tyr; A is Tyr and is acylated at 2 
functional groups; A is Leu; A is Arg; A is Arg and is acylated at 2 
functional groups; A is Trp; A is Trp and is acylated at 2 functional 
groups; A is Cit; and A is Cit and is acylated at 2 functional groups. 
Special mention is also made of compounds having the formula: 
##STR7## 
wherein A is Arg or Leu; and wherein if A is Arg, A is optionally acylated 
at 2 or more functional groups; 
##STR8## 
where A is Leu or phenylglycine; 
##STR9## 
wherein A is phenylglycine; and 
##STR10## 
wherein A is phenylglycine. 
Acylated amino acids may be prepared by reacting single amino acids, 
mixtures of two or more amino acids, or amino acid esters with an amine 
modifying agent which reacts with free amino moieties present in the amino 
acids to form amides. 
Suitable, but non-limiting, examples of acylating agents useful in 
preparing acylated amino acids include 
acid chloride acylating agents having the formula 
##STR11## 
wherein: 
R.sup.13 is an appropriate group for the modified amino acid being 
prepared, such as, but not limited to, alkyl, alkenyl, cycloalkyl, or 
aromatic, and particularly methyl, ethyl, cyclohexyl, cyclopentyl, phenyl, 
or benzyl, and X is a leaving group. Typical leaving groups include, but 
are not limited to, halogens such as chlorine, bromine and iodine. 
Examples of the acylating agents include, but are not limited to, acyl 
halides including, but not limited to, acetyl chloride, propionyl 
chloride, cyclohexanoyl chloride, cyclopentanoyl chloride, and 
cycloheptanoyl chloride, benzoyl chloride, hippuryl chloride and the like; 
and anhydrides, such as acetic anhydride, propionic anhydride, 
cyclohexanoic anhydride, benzoic anhydride, hippuric anhydride and the 
like. Preferred acylating agents include benzoyl chloride, hippuryl 
chloride, acetyl chloride, acetylsalicycloyl chloride, cyclohexanoyl 
chloride, cyclopentanoyl chloride, and cycloheptanoyl chloride. 
The amine groups can also be modified by the reaction of a carboxylic acid 
with coupling agents such as the carbodiimide derivatives of amino acids, 
particularly hydrophilic amino acids such as phenylalanine, tryptophan, 
and tyrosine. Further examples include dicyclohexylcarbodiimide and the 
like. 
If the amino acid is multifunctional, i.e., has more than one --OH, 
--NH.sub.2 or --SH group, then it may optionally be acylated at one or 
more functional groups to form, for example, an ester, amide, or thioester 
linkage. 
In the preparation of some acylated amino acids, the amino acids are 
dissolved in an aqueous alkaline solution of a metal hydroxide, e.g., 
sodium or potassium hydroxide and the acylating agent added. The reaction 
time can range from about 1 hour to about 4 hours, preferably about 2 to 
about 2.5 hours. The temperature of the mixture is maintained at a 
temperature generally ranging between about 5.degree. C. and about 
70.degree. C., preferably between about 10.degree. C. and about 50.degree. 
C. The amount of alkali employed per equivalent of NH.sub.2 groups in the 
amino acids generally ranges between about 1.25 moles and about 3 moles, 
and is preferably between about 1.5 moles and about 2.25 moles per 
equivalent of NH.sub.2. The pH of the reaction solution generally ranges 
between about pH 8 and about pH 13, and is preferably between about pH 10 
and about pH 12. The amount of amino modifying agent employed in relation 
to the quantity of amino acids is based on the moles of total free 
NH.sub.2 in the amino acids. In general, the amino modifying agent is 
employed in an amount ranging between about 0.5 and about 2.5 mole 
equivalents, preferably between about 0.75 and about 1.25 equivalents, per 
molar equivalent of total NH.sub.2 groups in the amino acids. 
The modified amino acid formation reaction is quenched by adjusting the pH 
of the mixture with a suitable acid, e.g., concentrated hydrochloric acid, 
until the pH reaches between about 2 and about 3. The mixture separates on 
standing at room temperature to form a transparent upper layer and a white 
or off-white precipitate. The upper layer is discarded, and modified amino 
acids are collected by filtration or decantation. The crude modified amino 
acids are then mixed with water. Insoluble materials are removed by 
filtration and the filtrate is dried in vacuo. The yield of modified amino 
acids generally ranges between about 30 and about 60%, and usually about 
45%. The present invention also contemplates amino acids which have been 
modified by multiple acylation, e.g., diacylation or triacylation. 
If amino acid esters or amides are the starting materials, they are 
dissolved in a suitable organic solvent such as dimethylformamide or 
pyridine and are reacted with the amino modifying agent at a temperature 
ranging between about 5.degree. C. and about 70.degree. C., preferably 
about 25.degree. C., for a period ranging between about 7 and about 24 
hours. The amount of amino modifying agents used relative to the amino 
acid esters are the same as described above for amino acids. 
Thereafter, the reaction solvent is removed under negative pressure and 
optionally the ester or amide functionality can be removed by hydrolyzing 
the modified amino acid ester with a suitable alkaline solution, e.g., 1 N 
sodium hydroxide, at a temperature ranging between about 50.degree. C. and 
about 80.degree. C., preferably about 70.degree. C., for a period of time 
sufficient to hydrolyze off the ester group and form the modified amino 
acid having a free carboxyl group. The hydrolysis mixture is then cooled 
to room temperature and acidified, e.g., with an aqueous 25% hydrochloric 
acid solution, to a pH ranging between about 2 and about 2.5. The modified 
amino acid precipitates out of solution and is recovered by conventional 
means such as filtration or decantation. 
The modified amino acids may be purified by acid precipitation, 
recrystallization or by fractionation on solid column supports. 
Fractionation may be performed on a suitable solid column supports, such 
as silica gel or alumina, using solvent mixtures such as acetic 
acid/butanol/water as the mobile phase; reverse phase column supports 
using trifluoroacetic acid/acetonitrile mixtures as the mobile phase; and 
ion exchange chromatography using water as the mobile phase. The modified 
amino acids may also be purified by extraction with a lower alcohol such 
as methanol, butanol, or isopropanol to remove impurities such as 
inorganic salts. 
The modified amino acids generally are soluble in alkaline aqueous solution 
(pH.gtoreq.9.0); partially soluble in ethanol, n-butanol and 1:1 (v/v) 
toluene/ethanol solution and insoluble in neutral water. The alkali metal 
salts, e.g., the sodium salts of the modified amino acids are generally 
soluble in water at about a pH of 6-8. 
In a poly amino acid or peptide, one or more of the amino acids may be 
modified (acylated). Modified poly amino acids and peptides may include 
one or more acylated amino acid(s). Although linear modified poly amino 
acids and peptides will generally include only one acylated amino acid, 
other poly amino acid and peptide configurations can include more than one 
acylated amino acid. Poly amino acids and peptides can be polymerized with 
the acylated amino acid(s) or can be acylated after polymerization. 
Special mention is made of the compound: 
##STR12## 
wherein A is Arg or Leu and B is Arg or Leu. Sulfonated Amino Acid 
Carriers 
Sulfonated modified amino acids, poly amino acids, and peptides are 
modified by sulfonating at least one free amine group with a sulfonating 
agent which reacts with at least one of the free amine groups present. 
Special mention is made of compounds of the formula 
EQU Ar--Y--(R.sup.14).sub.n --OH LIII 
wherein Ar is a substituted or unsubstituted phenyl or naphthyl; 
Y is --SO.sub.2 --, R.sup.14 has the formula 
##STR13## 
wherein: R.sup.15 is C.sub.1 to C.sub.24 alkyl, C.sub.1 to C.sub.24 
alkenyl, phenyl, naphthyl, (C.sub.1 to C.sub.10 alkyl) phenyl, (C.sub.1 to 
C.sub.10 alkenyl) phenyl, (C.sub.1 to C.sub.10 alkyl) naphthyl, (C.sub.1 
to C.sub.10 alkenyl) naphthyl, phenyl (C.sub.1 to C.sub.10 alkyl), phenyl 
(C.sub.1 to C.sub.10 alkenyl), naphthyl (C.sub.1 to C.sub.10 alkyl) and 
naphthyl (C.sub.1 to C.sub.10 alkenyl); 
R.sup.15 is optionally substituted with C.sub.1 to C.sub.4 alkyl, C.sub.1 
to C.sub.4 alkenyl, C.sub.1 to C.sub.4 alkoxy, --OH, --SH and --CO.sub.2 
R.sup.17 or any combination thereof; 
R.sup.17 is hydrogen, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4 
alkenyl; 
R.sup.15 is optionally interrupted by oxygen, nitrogen, sulfur or any 
combination thereof; and 
R.sup.16 is hydrogen, C.sub.1 to C.sub.4 alkyl or C.sub.1 to C.sub.4 
alkenyl. 
Suitable, but non-limiting, examples of sulfonating agents useful in 
preparing sulfonated amino acids include sulfonating agents having the 
formula R.sup.18 --SO.sub.2 --X wherein R.sup.18 is an appropriate group 
for the modified amino acid being prepared, such as, but not limited to, 
alkyl, alkenyl, cycloalkyl, or aromatics and X is a leaving group as 
described above. One example of a sulfonating agent is benzene sulfonyl 
chloride. 
Modified poly amino acids and peptides may include one or more sulfonated 
amino acid(s). Although linear modified poly amino acids and peptides used 
generally include only one sulfonated amino acid, other poly amino acid 
and peptide configurations can include more than one sulfonated amino 
acid. Poly amino acids and peptides can be polymerized with the sulfonated 
amino acid(s) or can be sulfonated after polymerization. 
Systems 
Delivery of an antigen with an adjuvant and a carrier as described herein 
results in enhanced immune responses. Another advantage of the present 
invention is that smaller amounts of antigen and/or adjuvant may be used 
to achieve an appropriate response. This latter advantage is particularly 
evident when the composition is in microsphere form. 
In one embodiment of the present invention, the modified amino acids, poly 
amino acids, peptides, or salts may be used as a carrier by simply mixing 
one or more modified amino acids, poly amino acids, or peptides, or salts 
with the antigen and adjuvant prior to administration. In another 
embodiment, the modified amino acids may be used to form microspheres 
containing the antigen and adjuvant. 
Microspheres containing antigen and adjuvant can generally be of the matrix 
form or the microcapsule form. The matrix form includes both a hollow 
matrix sphere in which the carrier forms a matrix shell around a hollow 
center with the antigen and adjuvant distributed throughout the matrix and 
a solid matrix sphere in which the carrier forms a spherical matrix 
continuum in which the antigen and adjuvant are distributed. 
The microcapsule form is one in which the encapsulated antigen and adjuvant 
independently are either in solution or are solid, with the carrier 
forming a shell around the encapsulated material. The microcapsule form is 
the form most often taken by the self assembly of the carriers of the 
present invention. 
If the delivery composition is to be of the microsphere form, carrier 
microspheres can be prepared by dissolving the carrier in an appropriate 
solute and then stimulating self assembly by contacting the carrier 
solution with a precipitator. Solubility of the carrier can be regulated 
by the selection of the appropriate amino acids. 
Furthermore, the microsphere carriers and, therefore, the compositions of 
the present invention can be pH adapted to be selectively soluble in 
specific acidic, basic, or neutral pH ranges. 
Compositions which are targeted to an acidic environment can be made 
selectively soluble at acidic pH, such as the pH in the stomach. These 
compositions are prepared with an acid-soluble carrier. The acid-soluble 
carrier exists largely in the cation form in at least a portion of the pH 
range from about 1 to about 6.8. However, above about 6.8 or at selected 
ranges above pH 6.8, the carrier is largely unprotonated and insoluble in 
water. Therefore, the carrier could self assemble to microspheres at basic 
or neutral pH, and the antigen in the delivery composition would not be 
released until the carrier solubilizes upon encountering an acidic pH. 
Compositions which are to be targeted to an alkaline environment can be 
made selectively soluble at alkaline pH, such as the pH in the distal 
portion of the intestine. These compositions are prepared with a 
base-soluble carrier. The base-soluble carrier exists largely in an 
anionic form in at least a portion of the pH range of from about 7.2 to 
about 11. However, below and at pH 7.2, the carrier is largely protonated 
and insoluble in water. Therefore, the carrier could self assemble to 
microspheres at acidic or neutral pH, and the antigen in the delivery 
composition would not be released until the carrier solubilizes upon 
encountering a basic pH. 
Compositions which are targeted to a neutral environment can be made 
selectively soluble at neutral pH. These compositions are prepared with a 
neutral-soluble carrier. The neutral-soluble carrier exists largely in a 
neutral form at neutral pH, i,e. from about 6.8 to about 7.2. However, 
above or below this range, the carrier is insoluble in water. Therefore, 
the carrier could self assemble to microspheres at acidic or basic pH, and 
the antigen in the delivery composition would not be released until the 
carrier solubilizes upon encountering a neutral pH. 
In a typical formulation, the final solution can contain from about 10 mg 
to about 2000 mg of carrier per ml of solution, preferably between about 
75 to about 500 mg of carrier per ml of solution, and most preferably from 
about 75 to about 200 mg per ml. Optionally, the mixture is heated to a 
temperature between about 20.degree. C. and about 60.degree. C., 
preferably about 40.degree. C., until the carrier dissolves. Particulates 
remaining in the solution may be filtered out by conventional means such 
as gravity filtration through filter paper. The carrier solution usually 
is maintained at the elevated temperature and is mixed with the antigen 
and/or adjuvant and a precipitator, for example, an acid solution such as, 
for example, aqueous acetic or citric acid at a concentration ranging from 
about 1 N to about 3 N for acid insoluble carriers, a basic solution for 
base insoluble carriers, and a neutralizing solution for neutral insoluble 
carriers. The antigen and/or adjuvant can be mixed with the precipitating 
solution or can be used separately. The resultant mixture is maintained 
for a period of time sufficient for microsphere formation as observed by 
light microscopy. Although it is preferred that the precipitating solution 
is added to the carrier solution, the carrier solution can be added to the 
precipitating solution as well. 
The solutions above may optionally contain additives such as stabilizing 
additives. The presence of such additives promotes the stability and 
dispersability of the active agent in solution. The stabilizing additives 
may be employed at a concentration ranging between about 0.1 and 5% (w/v), 
preferably about 0.5% (w/v). Suitable, but non-limiting examples of 
stabilizing additives include buffer salts, gum acacia, gelatin, methyl 
cellulose, polyethylene glycol, polylysine, carboxylic acids, carboxylic 
acid salts, and cyclodextrins. The preferred stabilizing agents are gum 
acacia, gelatin, and methyl cellulose. 
The amounts of antigen and adjuvant which may be encapsulated by the 
microsphere is dependent upon a number of factors which include the 
concentrations of antigen and adjuvant in the encapsulating solution as 
well as their affinities for the carrier. The concentrations of antigen 
and adjuvant in the final formulation also will vary depending on the 
required dosage for treatment. When necessary, the exact concentrations 
can be determined by, for example, reverse phase HPLC analysis. 
When the present compositions are in microsphere form, the particle size of 
the microsphere can also aid in providing efficient delivery of the 
antigen to the target. Typically, microspheres of the present invention 
will have a diameter of less than 10 .mu.m, preferably in the range of 
from about 0.1 .mu.m to about 10 .mu.m, and most preferably in the range 
of from 0.2 .mu.m to about 10 .mu.m. The size of the microspheres 
containing an antigen can be controlled by manipulating a variety of 
physical or chemical parameters, such as the pH, osmolarity, ionic 
strength of the encapsulating solution, or size of the ions in solution, 
and/or by the choice of the precipitator used in the microsphere forming 
and loading process. 
For example, in the GI tract, it is often desirable to use microspheres 
which are sufficiently small to deliver effectively the antigen to the 
targeted area within the gastrointestinal tract. Small microspheres can 
also be administered parenterally by suspending the spheres in an 
appropriate fluid (e.g. isotonic solution) and injecting the solution 
directly into the circulatory system intramuscularly or subcutaneously. 
The mode of administration of the delivery compositions will vary, of 
course, depending upon the requirement of the antigen administered. It has 
been noted that large amino acid microspheres (greater than 50 .mu.m) tend 
to be less effective as oral delivery systems. 
The compositions of the present invention may also include one or more 
enzyme inhibitors. Such enzyme inhibitors include, but are not limited to, 
compounds such as actinonin or epiactinonin and derivatives thereof. These 
compounds have the formulas below: 
##STR14## 
Derivatives of these compounds are disclosed in U.S. Pat. No. 5,206,384. 
Actinonin derivatives have the formula: 
##STR15## 
wherein R.sup.19 is sulfoxymethyl or carboxyl or a substituted carboxyl 
group selected from carboxamide, hydroxyaminocarbonyl and alkoxycarbonyl 
groups; and R.sup.20 is hydroxyl, alkoxy, hydroxyamino or sulfoxyamino 
group. Other enzyme inhibitors include, but are not limited to, aprotinin 
(Trasylol) and Bowman-Birk inhibitor. 
The compositions of the present invention may be formulated into dosage 
units by the addition of one or more excipient(s), diluent(s), 
disintegrant(s), lubricant(s), plasticizer(s), colorant(s), or dosing 
vehicle(s). Preferred dosage unit forms are oral dosage unit forms. Most 
preferred dosage unit forms include, but are not limited to, tablets, 
capsules, or liquids. The dosage unit forms can include biologically or 
immunogenically effective amounts of the antigen and an biologically or 
immunogenically assisting effective amount of the adjuvant but can include 
less than such an amount if multiple dosage unit forms are to be used to 
administer a total dosage of the antigen and adjuvant. Dosage unit forms 
are prepared by methods conventional in the art. 
The carriers of the present invention do not alter the physiological and 
biological properties of the antigen or the adjuvant. Furthermore, the 
encapsulation process need not alter the structure of the antigen. Any 
antigen can be incorporated within the amino acid microspheres. 
The compositions are particularly advantageous for oral immunization with 
antigens which otherwise would be destroyed or rendered less effective by 
conditions encountered within the body of the animal to which it is 
administered, before the microsphere reaches its target zone such as 
peptides or proteins, which, by themselves, do not pass or are not taken 
up in the gastro-intestinal mucosa and/or are susceptible to chemical 
cleavage by acids and enzymes in the gastrointestinal tract. Such antigens 
include those used to provide immunization against diseases including but 
not limited to, influenza, diphtheria, tetanus, measles, polio, hepatitis 
and the like. The compositions of the invention are more effective at 
inducing both mucosal and serum antibody responses than antigens which are 
administered without the carriers specified herein and adjuvants. The 
antigens are administered to a mammal for their biological effect, such 
as, for example as immune stimulators. 
Administration of the present compositions or dosage unit forms preferably 
is oral or by intraduodenal injection. 
EXAMPLES 
The invention will now be illustrated in the following non-limiting 
examples which are illustrative of the invention but are not intended to 
limit the scope of the invention. 
Example 1 
Preparation of O,N-Dicyclohexanoyl-(L)-Tyrosine 
(L)-Tyrosine (61.6 g., 0.34 mole) was dissolved in 190 mL of 2 N sodium 
hydroxide. Cyclohexanoyl chloride (49.32 mL, 0.34 mole) was added dropwise 
to the mixture. Additional aqueous 2 N sodium hydroxide was added, and the 
reaction mixture was allowed to stir at room temperature for 2 hours. The 
mixture was then acidified to pH 9.5 with aqueous (4:1) hydrochloric acid. 
A precipitate formed which was separated by vacuum filtration. The solids 
were dissolved in 2 N sodium hydroxide and dried by lyophilization to 
furnish 33.5 g of N,O-dicyclohexanoyl-(L)-tyrosine. The product was 
purified by column chromatography on silica gel using butanol/acetic 
acid/water as the eluent system. The pure product was a white solid. 
Properties are listed below: 
Mass Spectrum: M+23 m/e 314. 
.sup.1 NMR (300 MHz,DMSO-d6): d=6.8 (d, 2H); 6.4 (d, 2H); 4.4 (m, 1H); 2.5 
(ddd, 2H); 2.0 (m, 2H); 1.6 (m, 10H); 1.2(m, 10H). 
IR (KBr) cm-1: 3350, 2900, 2850, 1600, 1520, 1450, 1400, 1300. 
Example 2 
Preparation of N-Cyclohexanoyl-(L)-Tyrosine 
Cyclohexanoyl chloride (7 mL, 47 mmole) was added dropwise to a stirred 
solution of dry pyridine (400 mL) and O-benzyltyrosine benzyl ester (25 g, 
46.8 mmole). The reaction temperature was maintained at 0.degree. C. 
throughout the addition. The reaction mixture was stirred for an 
additional 2 hours after the addition was complete. The reaction mixture 
was concentrated to dryness in vacuo to provide a solid material. The 
solid was washed with aqueous hydrochloric acid (1N, 4.times.400 mL). The 
residue was dissolved in ethyl acetate (300 mL), washed with aqueous 
hydrochloric acid (1N, 2.times.500 ml), aqueous sodium bicarbonate 
(2.times.300 mL), and dried over magnesium sulfate. Filtration, followed 
by concentration in vacuo, provided an oil which was dissolved in 
methanol/tetrahydrofuran (400 mL/70 mL) and was hydrogenated at 
atmospheric pressure and room temperature over 10% palladium on carbon 
(600 mg). The reaction mixture was filtered through Celite and 
concentrated in vacuo to provide a solid which was recrystallized from 
ethyl acetate/hexane. The crystals were collected to provide the 
N-cyclohexanoyl-(L)-tyrosine (8.7 g, 64%) as a white solid. 
NMR results are listed below: 
.sup.1 H NMR (300 MHz, D.sub.2 O) .delta. 6.9 (d, 2H, aromatic), 6.6 (d, 
2H, aromatic), 4.25 (m, 1H, NHCHCOOH), 2.95 (m, 1H, CH.sub.2), 2.7 (m, 1H, 
CH.sub.2) 2.05 (m, 1H, NHC(O)CH), 1.5 (br. m, 5H, cyclohexyl), 1.05 (br. 
m, 5H, cyclohexyl). 
Example 3 
Preparation of N-Cyclohexanoyl-(L)-Leucine 
Cyclohexanoyl chloride (32.7 mL, 232 mmole) was added dropwise to a 
solution of (L)-leucine (37 g, 282 mmole) in aqueous sodium hydroxide (500 
mL, 2 N). During the course of this addition, the reaction temperature was 
maintained below 45.degree. C. using an ice/water bath, as necessary. The 
pH was maintained at about 10 by the addition of aliquots of 14 N NaOH, as 
necessary. After the addition was complete, the reaction mixture was 
stirred for an additional 2 hours at room temperature. The resulting clear 
solution was adjusted to pH 2.5 by the dropwise addition of concentrated 
hydrochloric acid. The precipitate was collected by filtration, 
re-dissolved in a minimum amount of 12 N sodium hydroxide, and 
re-precipitated by dropwise addition of concentrated hydrochloric acid and 
filtered. The crude reaction product was a white solid and contained about 
85% N-cyclohexanoyl leucine sodium salt, about 10% cyclohexane carboxylic 
acid sodium salt, and about 5% N-cyclohexanoylleucylleucine sodium salt, 
by weight. The solid was washed with dilute aqueous hydrochloric acid (750 
mL, 0.1 N) to provide N-cyclohexanoyl-(L)-leucine as a white crystalline 
solid (52.6 g, 77%). 
NMR results are listed below: 
.sup.1 H NMR (300 MHz D.sub.2 O) .delta. 4.2 (t, 1H, NHCHCOOH), 2.0 (m, 1H, 
cyclohexylmethine), 1.6 (m, 7H, ring CH.sub.2, i-Bu CH.sub.2 and CH), 1.3 
(m, 6H, ring CH.sub.2), 0.8 (dd, 6H,CH.sub.3). 
Example 4 
Prearation of N-Cyclohexanol-(L)-Arginine and N.sub..alpha.,N.sub..gamma. 
-Dicyclohexanol-(L)-Arginine 
(L)-Arginine (103.2 g., 0.6 mole) was dissolved in 600 mL of 2 N sodium 
hydroxide. Cyclohexanoyl chloride (87 mL, 0.6 mole) was added dropwise to 
the mixture. The reaction mixture was maintained at 50.degree. C. for 2 
hours. The mixture was then cooled to room temperature and acidified to pH 
2.3 with aqueous (4:1) hydrochloric acid. The precipitate which formed was 
separated by decantation. The solids were dissolved in 2 N sodium 
hydroxide and dried by lyophilization to furnish 64.1 g of crude 
N-cyclohexanoyl-(L)-arginine. The product was purified by column 
chromatography on silica gel/using butanol/acetic acid/water as the eluent 
system. The products isolated were N-cyclohexanoyl-(L)-arginine and 
N.sub..alpha.,N.sub..gamma. -dicyclohexanoyl-(L)-arginine. 
Properties are listed below: 
N-cyclohexanoyl-(L)-arginine: 
Mass Spectrum: M+1 m/e 285. 
.sup.1 H NMR (300 MHz, DMSO-d6): ppm .delta.=8.75 (br, 1H); 7.6 (br, 5H); 
4.0 (m, 1H); 3.05 (m, 2H); 2.15 (m, 1H); 1.1-1.5 (br.m, 14H). 
N.sub..alpha.,N.sub..gamma. -dicyclohexanoyl-(L)-arginine: 
Mass Spectrum: M+1 m/e 395. 
.sup.1 H NMR: (300 MHz, DMSO-d6): d=2.0 (m, 3H); 1.8-1.4 (br. m, 17H); 
1.3-1.0 (br. m, 20H) 
Example 5 
Preparation of N-Cyclohexanol-(L)-Citrulline 
L-Citrulline (35.2 g., 0.2 mole) was dissolved in 200 mL of 2 N sodium 
hydroxide. Cyclohexanoyl chloride (29 mL, 0.2 mole) was added dropwise to 
the mixture. The reaction mixture was maintained at about 25.degree. C. 
for 1 hour. The mixture was then acidified to pH 2.6 with aqueous (4:1) 
hydrochloric acid. The precipitate which formed was separated by 
decantation. The solids were dissolved in 2 N sodium hydroxide to pH 6.5 
and dried by lyophilization to furnish 44.2 g of 
N-cyclohexanoyl-(L)-citrulline. The product was a white solid. 
Properties are listed below: 
Mass Spectrum: M+23 m/e 308. 
.sup.1 H NMR (300 MHz,DMSO-d6): d=4.1 (dd, 1H); 2.9 (t, 2H); 2.1 (m, 2H); 
1.6-1.2 (br.m, 14H). 
IR (KBr) cm-1: 3400, 3300, 2950, 2850, 1700, 1650, 1600, 1450, 1400 cm-1. 
Example 6 
Preparation of N-Cyclopentanoyl-(L)-Arginine 
(L)-Arginine (32.8 g., 0.19 moles) was dissolved in 188 mL of 2 N sodium 
hydroxide. Cyclopentanoyl chloride (22.9 mL, 0.19 moles) was added 
dropwise to the mixture. The reaction mixture was maintained at about 
25.degree. C. for 2 hours. The mixture was then acidified to pH 1.5 with 
aqueous (4:1) hydrochloric acid. The precipitate which formed was 
separated by decantation. The solids were dissolved in 2 N sodium 
hydroxide to pH 7.5 and dried by lyophilization to furnish 67.4 g of 
N-cyclopentanoyl-(L)-arginine. The product was a white solid. 
Properties are listed below: 
Mass Spectrum: M+1 m/e 271. 
Example 7 
Preparation of 4-(4-Phenylsulfonamido)Phenylbutyric Acid 
4-(4-Aminophenyl)butyric acid, (20 g 0.11 moles) was dissolved in 110 mL of 
aqueous 2 N sodium hydroxide solution. After stirring for about 5 minutes 
at room temperature, benzene sulfonyl chloride (14.2 mL, 0.11 moles) was 
added dropwise into the amino acid solution over a 15 minute period. After 
stirring for about 3 hours at room temperature the mixture was acidified 
to pH 2 by addition of hydrochloric acid. This furnished a light brown 
precipitate which was isolated by filtration. The precipitate was washed 
with warm water and dried. The yield of 
4-(phenylsulfonamido)4-phenylbutyric acid was 24.3 g (69%). The melting 
point was 123-25.degree. C. 
Example 8 
Preparation of 4-Phenylsulfonamidobenzoic Acid 
Following the procedure of Example 7, 4-aminobenzoic acid was converted to 
4-(phenylsulfonamido)benzoic acid. 
Example 9 
Preparations of 4-(4-Phenylsulfonamido)Phenylacetic Acid, 
4-(4-Phenylsulfonamido) Hippuric Acid, and 
4-(4-Phenylsulfonamidomethyl)Benzoic Acid 
Following the procedure of Example 7, 4-aminophenylacetic acid, 
4-aminohippuric acid, and 4-aminomethylbenzoic acid were converted to 
4-(4-phenylsulfonamido)phenylacetic acid, 4-(4-phenylsulfonamido)hippuric 
acid, and 4-(4-phenylsulfon-amidomethyl)benzoic acid respectively. 
If necessary, the sulfonated amino acids can be purified by 
recrystallization and/or chromatography. 
Example 10 
Reaction of Mixed Amino Acids with Benzene Sulfonyl Chloride 
A mixture of sixteen amino acids were prepared prior to chemical 
modification. The constituents of the mixture are summarized in Table 1. 
65 grams of the amino acid mixture (total concentration of --NH.sub.2 ! 
groups=0.61 moles) was dissolved in 760 mL of 1 N sodium hydroxide 
solution (0.7625 equivalents) at room temperature. After stirring for 20 
minutes, benzene sulfonyl chloride (78 ml, 1 eq.) was added over a 20 
minute period. The reaction mixture was then stirred for 2.5 hours, 
without heating. As some precipitation had occurred, additional NaOH 
solution (2 N) was added to the solution until it reached pH 9.3. The 
reaction mixture stirred overnight at room temperature. Thereafter, the 
mixture was acidified using dilute hydrochloric acid (38%, 1:4) and a 
cream colored material precipitated out. The resulting precipitate was 
isolated by decantation and dissolved in sodium hydroxide (2 N). This 
solution was then reduced in vacuo to give a yellow solid, which was dried 
on the lyophilizer. 
TABLE 1 
______________________________________ 
Amino Acid Composition 
No. of moles 
Weight % of Total 
of each Amino 
No. of Moles 
Amino Acid 
(g) Weight Acid (.times. 10.sup.-2) 
of - --NH.sub.2 ! 
______________________________________ 
Thr 2.47 3.8 2.07 2.07 
Ser 2.25 3.46 2.1 2.1 
Ala 4.61 7.1 5.17 5.17 
Val 4.39 6.76 3.75 3.75 
Met 0.53 0.82 0.35 0.35 
Ile 2.47 3.8 0.36 0.36 
Leu 3.86 5.94 2.95 2.95 
Tyr 1.03 1.58 0.56 0.56 
Phe 4.39 6.76 0.27 0.27 
His 2.47 3.8 1.6 3.2 
Lys 4.94 7.6 3.4 6.8 
Arg 5.13 7.9 2.95 5.90 
Glutamine 
9.87 15.18 6.76 13.42 
Glutamic 
9.87 15.18 6.70 6.70 
Acid 
Asparagine 
3.32 5.11 2.51 5.02 
Aspartic 
3.32 5.11 2.50 2.50 
Acid 
______________________________________ 
Example 11 
Reaction of Five Mixed Amino Acids with Benzene Sulfonyl Chloride 
An 86.1 g (0.85 moles of NH.sub.2) mixture of amino acids (see Table 2) was 
dissolved in 643 mL (1.5 eq.) of aqueous 2 N sodium hydroxide solution. 
After stirring for 30 minutes at room temperature, benzene sulfonyl 
chloride (108 mL, 0.86 moles) was added portionwise into the amino acid 
solution over a 15 minute period. After stirring for 2.5 hours at room 
temperature, the pH of the reaction mixture (pH 5) was adjusted to pH 9 
with additional 2 N sodium hydroxide solution. The reaction mixture 
stirred overnight at room temperature. Thereafter, the pH of the reaction 
mixture was adjusted to pH 2.5 by addition of dilute aqueous hydrochloric 
acid solution (4:1, H.sub.2 O: HCl) and a precipitate of modified amino 
acids formed. The upper layer was discarded and the resulting yellow 
precipitate was isolated by decantation, washed with water and dissolved 
in 2 N sodium hydroxide (2 N). The solution was reduced in vacuo to give a 
yellow solid which was lyophilized overnight. The yield of crude modified 
amino acid was 137.9 g. 
Example 12 
Reaction of Five Mixed Amino Acids with Benzoyl Chloride 
An 86 g (0.85 moles of NH.sub.2) mixture of amino acids (see Table 2 in 
Example 11) was dissolved in 637 mL (1.5 eq.) of aqueous 2 N sodium 
hydroxide solution. After stirring for 10 minutes at room temperature, 
benzoyl chloride (99 mL, 0.85 moles) was added portionwise into the amino 
acid solution over a 10 minute period. After stirring for 2.5 hours at 
room temperature, the pH of the reaction mixture (pH 12) was adjusted to 
pH 2.5 using dilute hydrochloric acid (4:1, H.sub.2 O:HCl) and a 
precipitate of modified amino acids formed. After settling for 1 hour, the 
resulting precipitate was isolated by decantation, washed with water and 
dissolved in sodium hydroxide (2 N). This solution was then reduced in 
vacuo to give crude modified amino acids as a white solid (220.5 g). 
TABLE 2 
______________________________________ 
Amino Acid Composition 
Moles of Amino 
Moles of 
Amino Acid Acid (.times. 10.sup.-2) 
--NH.sub.2 ! .times. 10.sup.-2 
______________________________________ 
Valine 7.5 7.5 
Leucine 10.7 10.5 
Phenylalanine 
13.4 13.4 
Lysine 21.0 42.0 
Arginine 6.0 12.0 
______________________________________ 
Example 13 
Preparation of N-Phenylsulfonylvaline 
L-Valine (50 g, 0.43 mole) was dissolved in 376 mL (0.75 eq.) of aqueous 2 
N sodium hydroxide by stirring at room temperature for 10 minutes. Benzene 
sulfonyl chloride (68.7 mL, 0.38 mole, 1.25 eq.) was then added to the 
amino acid solution over a 20 minute period at room temperature. After 
stirring for 2 hours at room temperature, a precipitate appeared. The 
precipitate was dissolved by adding 200 mL of additional 2 N sodium 
hydroxide solution. After stirring for an additional 30 minutes, dilute 
aqueous hydrochloric acid solution (4:1, H.sub.2 O:HCl) was added until 
the pH of the reaction mixture reached 2.6. A precipitate of modified 
amino acid formed was recovered by decantation. This material was 
dissolved in 2 N sodium hydroxide and dried in vacuo to give a white 
solid. The yield of crude modified amino acid wad 84.6 g, 77%. 
Example 14 
Preparation of N-Hippurylphenylalanine 
L-Phenylalanine methyl ester hydrochloride (15 g, 0.084 mole) was dissolved 
in dimethylformamide (DMF) (100 mL) and to this was added pyridine (30 
mL). A solution of hippuryl chloride (16.6 g, 0084 moles in 100 mL DMF) 
was immediately added to the amino acid ester solution in two portions. 
The reaction mixture was stirred at room temperature overnight. The 
reaction mixture was then reduced in vacuo and dissolved in 1 N aqueous 
sodium hydroxide. The solution was heated at 70.degree. C. for 3 hours in 
order to hydrolyze the methyl ester to a free carboxyl group. Thereafter, 
the solution was acidified to pH 2.25 using dilute aqueous hydrochloric 
acid solution (1:3 HCl/H.sub.2 O). A gum-like precipitate formed and this 
was recovered and dissolved in 1 N sodium hydroxide. The solution was 
reduced in vacuo to afford 18.6 g of crude modified amino acid product 
(Yield 18.6 g). After recrystallization from acetonitrile, pure modified 
phenylalanine (12 g) was recovered as a white powder. m.p. 223-225.degree. 
C. 
Example 15 
Preparation of Antigen/Delivery System 
A carrier solution of 300 mg of the mixture of modified amino acids, 
prepared in Example 11, was added to 1.5 ml of water and mixed. 
Cholera toxin (CT) adjuvant solution was prepared by reconstituting it in 
water at a concentration of 1 mg/ml. 
Ovalbumin (3 mg) (OVA) antigen was dissolved in 1.2 ml of a solution of 1.7 
N citric acid/1% gum acacia, and 0.3 ml of the cholera toxin solution was 
added. 
The carrier solution and the OVA/CT solution were warmed to 40.degree. C. 
and mixed together. The sample had a carrier concentration of 100 mg/mL 
and an OVA concentration of 1 mg/mL. 
Example 16 
Antigen in Vivo Experiments in Mice 
Following the procedure in Example 15, a preparation of antigen (1 mg/ml of 
OVA), adjuvant (100 .mu.g/ml of CT) with carrier (100 mg/ml of modified 
amino acid carrier) was prepared. Fasted mice were anesthetized with 
Ketamine, and administered, by oral gavage, a dose containing 100 .mu.g 
OVA, 10 .mu.g CT, and 10 mg of carrier. 
Intestinal secretions were collected on days 18, 32, 46, and 67 after 
dosing with the antigen/adjuvant/carrier preparation. The mice were dosed 
with a hypertonic solution prior to collection of the secretion samples. 
The secretions were then placed in a solution containing protease 
inhibitors. The resultant solution was cleared by centrifugation and 
assayed for total and OVA-specific IgA. IgA titer was determined by 
analyzing the secretions for the total IgA in the secretions and the 
OVA-specific IgA using the ELISA procedure below. The OVA-specific IgA 
could then be calculated from the results. IgA was expressed as "units" of 
specific anti-OVA IgA. 
Elisa for Total IgA in Intestinal Secretions 
1. Coat plate with 100 .mu.l per well of affinity purified goat anti-mouse 
IgA (1 .mu.g/ml) in carbonate buffer (pH 9.6). 
Incubate overnight at 4.degree. C. 
2. Wash with an imidazole buffer having 0.05% Tween 20). 
3. Add 1/10 diluted BSA blocking solution, 300 ml per well. Incubate, with 
shaking, 30 minutes at room temperature. 
4. Wash with an imidazole buffer having 0.05% Tween 20. 
5. Add 100 .mu.l per well of serially diluted samples starting at 1/1000. 
Standard reference Mouse IgA is run at 6 dilutions: 1/150,000 (10 .mu.l of 
Mouse IgA to 10 ml of buffer (1/1000). Add 100 .mu.l of this solution to 
14.9 ml of buffer (final dilution 1/150,000)) (16.32 ng/ml) (standard 
(1)); 200,000 3 ml of standard (1)+1 ml of buffer (1/200,000) (12.25 
ng/ml) (standard (2)); 300,000 2 ml of standard (1)+2 ml of buffer 
(1/300,000) (8.16 ng/ml) (standard (3)); 400,000 2 ml of standard (2)+2 ml 
of buffer (1/400,000) (6.125 ng/ml) (standard (4)); 600,000 1 ml of 
standard (3)+1 ml of buffer (1/600,000) (4.08 ng/ml) (standard (5)); and 
800,000 1 ml of standard (4)+1 ml of buffer (1/800,000) (3.06 ng/ml) 
(standard (6)). Incubate for one hour at room at room temperature, shaking 
at high speed. 
6. Wash 8 times with an imidazole buffer having 0.05% Tween 20. 
7. Add 100 .mu.l per well of a 1/10,000 dilution of Rabbit anti-Mouse IgA 
in 1/15 diluent containing 4% PEG 6000. Mix briefly on shaker. Incubate 
overnight at 4.degree. C. 
8. Wash 8 times with an imidazole buffer having 0.05% Tween 20. 
9. Add 100 .mu.l per well of a 1/10,000 dilution of Alkaline-Phosphatase 
conjugated-Goat anti-Rabbit IgG in 1/15 diluent containing 4% PEG 6000. 
Incubate one hour at room temperature, with rapid shaking. 
10. Wash 8 times with an imidazole buffer having 0.05% Tween 20. 
11. Add 100 .mu.l per well of PNPP/DEA, pH 9.8. Incubate 30 minutes at room 
temperature (so that maximum OD=1.8-2.0). Read OD 405, subtracting OD of 
appropriate background well. 
12. Calculate total IgA in samples from standard curve (OD vs. logIgA!, 
taking the average of the values calculated for all dilutions whose OD's 
fall within the standard curve (i.e., find OD for sample which falls 
within the linear range of the curve and interpolate to find its 
concentration on the curve. Multiply this value by the appropriate 
dilution factor for that value). 
Elisa for Specific Anti-OVA IgA in Intestinal Secretions 
1. Coat plate with OVA. Add 100 .mu.l of a 4 .mu.g/ml solution of OVA in 
carbonate buffer (pH 9.6) to each well. 
2. Incubate overnight at 4.degree. C. or two hours at room temperature with 
rapid shaking. 
3. Empty wells and wash 4 times with an imidazole buffer having 0.05% Tween 
20. 
4. Add 300 .mu.l of BSA solution to each well and incubate 30 minutes at 
room temperature with shaking. 
5. Wash 4 times with a imidazole buffer having 0.05% Tween 20. 
6. Place intestinal secretion samples in 37.degree. C. water bath until 
almost thawed, and centrifuge at 4.degree. C. at 4000 rpm for 10 minutes 
to remove any precipitate. 
7. Add 100 .mu.l per well of appropriately diluted samples (three-fold 
serial dilutions from 1/2 up to 1/486). 
Leave at least two "background" wells (all reagents except sample). 
Negative control: pooled secretions collected from naive mice (diluted as 
with samples.) 
Reference: Rabbit anti-OVA IgG diluted 1/200,000 2 wells) 
8. Incubate one hour at room temperature with rapid shaking. 
9. Wash 8 times with a imidazole buffer having 0.05% Tween 20. 
10. (a) To secretions: Add 100 .mu.l of dilute (1/1000) 
Alkaline-Phosphatase conjugated anti-mouse IqA in 1/15 diluent containing 
4% PEG 6000. 
(b) To reference and one background well: 100 .mu.l of 1/10,000 diluted A-P 
conjugated Goat anti-rabbit IgG in 1/15 diluent+4% PEG 6000. 
11. Incubate one hour with rapid shaking. 
12. Wash 8 times with an imidazole buffer having 0.05% Tween 20. 
13. Add 100 .mu.l of freshly prepared p-NPP substrate in diethanolamine 
buffer to each well. 
14. Incubate 30 minutes at room temperature in the dark. 
15. Read OD.sub.405 subtracting the average OD of the appropriate 
background wells. 
16. Define the number of "antibody units" in the sample as 1/dilution of 
the sample whose OD.sub.405 =average OD.sub.405 of the IgG reference 
wells.times.100. 
Express IgA content of samples as: 
##EQU1## 
Results are illustrated in FIG. 1. 
Example 17 
Antigen in Vivo Experiment 
Following the procedure in Example 15, a composition containing antigen (1 
mg/ml of OVA), adjuvant (100 .mu.g/ml of CT) and carrier (100 mg/ml of 
cyclohexanoyl-Arg) was prepared. Mice were administered, by oral gavage, a 
dose containing 100 .mu.g OVA, 10 .mu.g CT and 10 mg of carrier. Blood 
samples were taken at six weeks post dose. Serum was assayed using an 
ELISA to measure anti-OVA serum IgG. The procedure was as described below: 
Serum IgG Titer Determination 
1. Add 100 .mu.l OVA solution (4 .mu.g/ml in carbonate buffer, pH 9.6) to 
each well. 
2. Incubate at 4.degree. C. overnight, or 2 hours at room temperature with 
shaking. 
3. Empty and wash plate 4 times with imidazole buffer having 0.05% Tween 20 
and one 5 minute soak. 
4. Add 300 .mu.l of BSA solution and incubate 30 minutes at room 
temperature. 
5. Wash as above. 
6. Add 100 ml of 1/15 diluted BSA solution to each well except first row of 
samples, first standard curve well, and wells for positive and negative 
controls. 
7. Add samples and controls. 
Samples: Place 150 .mu.l of a 1/200 dilution of each sample in first well 
of sample rows. 
Serially dilute 50 .mu.l for 3-fold dilutions. 
Positive Controls: Place 200 .mu.l of hyper immune serum at 1/2000 dilution 
in first well. Serially dilute 100 .mu.l two-fold to 1/64000 (6 wells). 
Negative control: pooled serum from naive mice (1/200 dilution): 100 .mu.l. 
"Background": all reagents except serum in at least two wells. 
8. Incubate two hours at room temperature with shaking. 
9. Wash 8 times with imidazole buffer having 0.05% Tween 20 and one 5 
minute soak. 
10. Add 100 .mu.l of Goat anti-Mouse IgG Alkaline Phosphatase Conjugate 
(diluted 1/1000 in 1/15 PBS/BSA solution containing 4% PEG 6000) 
11. Incubate overnight at 4.degree. C. after shaking for a few minutes. 
12. Wash 8 times with imidazole buffer having 0.05% Tween 20. 
13. Add 100 .mu.l of freshly prepared pNPP solution to each well and 
develop at room temperature in the dark. 
14. Read OD.sub.405. (Subtract blank i.e., empty well, not background). 
15. Record when OD.sub.405 of 1/2000 standard=1.2 (about 0.5-1 hour). 
16. Calculate antibody titers in samples by interpolation of OD's of 
dilutions. (max dilution at which OD.sub.405 =3.times.background). 
Results are illustrated in FIG. 2. 
Comparative Example 17A 
Antigen in Vivo Experiment 
A composition of antigen (OVA) and adjuvant (CT) was prepared. Mice were 
administered, by oral gavage, a dose containing antigen (100 .mu.g OVA) 
and adjuvant (10 .mu.g CT). Blood samples were collected and analyzed as 
described in Example 17. 
Results are illustrated in FIG. 2. 
Example 18 
Antigen in Vivo Experiment 
Following the procedure of Example 15, a composition of antigen (1 mg/ml of 
OVA), adjuvant (100 .mu.g/ml of CT) and carrier (100 mg/ml of 
cyclohexanoyl-Arg) was prepared. Mice were administered, by oral gavage, a 
dose containing 100 .mu.g of OVA, 10 .mu.g of CT and 10 mg of carrier. 
Secretion samples were collected and analyzed at 46 days post dose as 
described in Example 16. 
Results are illustrated in FIG. 3. 
Comparative Example 18A 
Antigen in Vivo Experiment 
Following the procedure of comparative Example 17A mice were administered a 
composition containing antigen (100 .mu.g OVA) and adjuvant (10 .mu.g CT). 
Secretion samples were collected and analyzed at 46 days post dose as 
described in Example 16. 
Results are illustrated in FIG. 3. 
Example 19 
Antigen in Vivo Experiment 
Mice were administered, by intraperitoneal injection, an antigen 
preparation containing antigen (10 .mu.g OVA) and adjuvant (10 .mu.g CT). 
This was followed by a booster administered by oral gavage, containing 
antigen (100 .mu.g OVA), adjuvant (10 .mu.g CT) and 10 mg of 
cyclohexanoyl-Arg. Secretion samples were collected and analyzed at 46 
days post dose as described in Example 16. 
Results are illustrated in FIG. 3. 
Example 20 
Preparation of Antigen/Carrier Composition 
A carrier solution is prepared by adding 90 mg of 
N-cyclohexanoyl-(L)-tyrosine and 135 mg of N-cyclohexanoyl-leucine to 1.5 
ml of water. 
Cholera toxin (CT) adjuvant solution is prepared by reconstituting CT in 
water at a concentration of 1 mg/ml. 
Ovalbumin (3 mg) (OVA) antigen is dissolved in 1.2 ml of a solution of 1.7 
N citric acid/1% gum acacia, and 0.3 ml of the cholera toxin solution is 
added. 
The carrier solution and the OVA antigen/CT adjuvant solution are warmed to 
40.degree. C. and mixed together. The sample has a carrier concentration 
of 75 mg/mL, an OVA antigen concentration of 1 mg/mL, and an CT adjuvant 
concentration of 100 .mu.g/mL. 
Example 21 
Antigen in Vivo Experiments in Mice 
Fasted mice are anesthetized with Ketamine, and administered, by oral 
gavage, a dose of a composition prepared according to the method of 
Example 20, containing 100 .mu.g OVA, 10 .mu.g CT, and 7.5 mg of carrier. 
Intestinal secretions were collected on days 18, 32, 46, and 67 after 
dosing with the antigen/adjuvant/carrier composition. The mice were dosed 
with a hypertonic solution prior to collection of the secretion samples. 
The secretions are then placed in a solution containing protease 
inhibitors. The resultant solution is cleared by centrifugation and is 
assayed for total and OVA-specific IgA's using the ELISA procedure 
described in Example 15. The OVA-specific IgA titer is calculated from the 
results. 
Example 22 
Immunization of Chickens 
A solution containing formalin-inactivated Infectious Bursal Disease Virus 
(IBDV) (Maine Biological Laboratories, Winslowe, Me.) was prepared by 
diluting a buffered solution of IBDV (2.5.times.10.sup.9 TCID.sub.50 /mL) 
to 1/10 of the original concentration (2.5.times.10.sup.8 TCID.sub.50 
/mL). 
A mixture of five sulfonated amino acids (2.0 g) prepared according to the 
method of Example 11 and sodium 2-cyclohexylbutyrate (8.0 g) were 
dissolved in 50 mL of the IBDV solution prepared above. 1 mL of a 0.5 
mg/mL solution of cholera toxin .beta.-subunit (CTB) and 0.25 mL of a 0.1 
mg/mL solution of cholera holotoxin (CT) was added, and the resultant 
solution (solution 1) was warmed to 40.degree. C. and incubated for 10 
minutes. 
50 mL of 1.7 N citric acid/1% gum acacia/2% .beta.-cyclodextrin solution 
(solution 2) was warmed to 400 .degree. C. 
Solutions 1 and 2 were mixed to provide a suspension of IBDV/CTB/CT 
containing microspheres. 
Nineteen chickens were each dosed, by oral gavage, with 2 ml per bird of 
the microspheres suspension (prepared daily) on three consecutive days. 
Each daily dose contained 2.5.times.10.sup.8 TCID.sub.50 /mL of IBDV, 10 
.mu.g of CTB, and 0.5 .mu.g of CT. 
After four weeks, each bird immunized as above and twenty-four unimmunized 
birds were challenged via intraocular administration of live IBDV. Four 
days after challenge the immunized birds, the unimmunized birds, and birds 
that were unchallenged and unimmunized were sacrificed, and their bursae 
were removed. Examination for gross bursal lesions and comparison among 
the three groups of birds revealed that all of the birds that were 
administered the microsphere suspension (the immunized/challenged birds, 
19 of 19) were uninfected by IBDV upon challenge, while only 25% of the 
unimmunized/challenged birds (6 of 24) were uninfected after challenge. 
All patents, applications, publications, and test methods cited herein are 
hereby incorporated by reference. 
Many variations of the present invention will suggest themselves to those 
skilled in the art in light of the above detailed disclosure. All such 
modifications are within the full intended scope of the appended claims.