U.S. patent application number 12/584967 was filed with the patent office on 2010-03-25 for oral-intestinal vaccines against diseases caused by enteropathogenic organisms using antigens encapsulated within biodegradable-biocompatible microspheres.
Invention is credited to Edgar C. Boedeker, Frederick J. Cassels, Daniel L. Jarboe, Robert H. Reid.
Application Number | 20100074913 12/584967 |
Document ID | / |
Family ID | 24772650 |
Filed Date | 2010-03-25 |
United States Patent
Application |
20100074913 |
Kind Code |
A1 |
Boedeker; Edgar C. ; et
al. |
March 25, 2010 |
Oral-intestinal vaccines against diseases caused by
enteropathogenic organisms using antigens encapsulated within
biodegradable-biocompatible microspheres
Abstract
This invention is directed to oral-intestinal vaccines and their
use against diseases caused by enteropathogenic organisms using
antigens encapsulated within biodegradable-biocompatible
microspheres.
Inventors: |
Boedeker; Edgar C.; (Chevy
Chase, MD) ; Cassels; Frederick J.; (Gaithersburg,
MD) ; Jarboe; Daniel L.; (Sebastian, FL) ;
Reid; Robert H.; (NicComes, CT) |
Correspondence
Address: |
NASH & TITUS, LLC
21402 UNISON RD
MIDDLEBURG
VA
20117
US
|
Family ID: |
24772650 |
Appl. No.: |
12/584967 |
Filed: |
September 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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08762757 |
Dec 10, 1996 |
7604811 |
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12584967 |
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08396986 |
Mar 1, 1995 |
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08762757 |
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08191374 |
Apr 6, 1994 |
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08396986 |
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07690485 |
Apr 24, 1991 |
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08191374 |
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07521945 |
May 11, 1990 |
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07690485 |
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06590308 |
Mar 16, 1984 |
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07521945 |
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Current U.S.
Class: |
424/185.1 |
Current CPC
Class: |
A61P 37/04 20180101;
C07K 14/245 20130101; A61K 39/39 20130101; A61K 2039/55555
20130101; Y02A 50/30 20180101; Y02A 50/466 20180101; A61K 38/00
20130101; Y02A 50/484 20180101; A61K 9/1647 20130101 |
Class at
Publication: |
424/185.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61P 37/04 20060101 A61P037/04 |
Goverment Interests
I. GOVERNMENT INTEREST
[0002] The invention described herein may be manufactured, licensed
and used by or for govermental purposes without the payment of any
royalties to us thereon.
Claims
1-41. (canceled)
42. A composition for inducing an immune response in primates
comprising of an antigenic synthetic peptide containing CFA-1 pilus
protein epitopes encapsulated within a biodegradable-biocompatible
poly(DL-lactide-co-glycolide) polymeric matrix, wherein said
antigenic synthetic peptide is present in an amount of 0.1% to 1%
by weight of said vaccine and said polymer is present in an amount
of 99% to 99.9% by weight of said vaccine, Wherein the CFA/I pilus
protein epitopes consist of the amino acid sequence: (SEQ ID NO: 2)
8(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu), (SEQ ID NO: 3) 12
(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID NO: 4) 15
(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala), (SEQ ID NO: 5) 20
(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), (SEQ ID NO: 6) 26
(Pro-Ser-Ala-Val-Lys-Leu-Ala-Tyr-Ser-Pro), (SEQ ID NO: 7) 72
(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser), (SEQ ID NO: 8) 78
(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln), (SEQ ID NO: 9) 87
(Gln-Val-Leu-Ser-Thr-Thr-Ala-Lys-Glu-Phe), (SEQ ID NO: 10) 126
(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr,) and mixtures
thereof.
43. The composition according to claim 42 wherein the polymeric
matrix is poly(DL-lactide-co-glycolide).
44. The composition according to claim 42 wherein the relative
ratio of lactide to glycolide component is within the range of
40:60 to 0:100.
45. The composition according to claim 42 wherein the relative
ratio of lactide to glycolide components is within the range of
48:52 to 58:42.
46. A composition for inducing an immune response in primates
comprising of an antigenic synthetic peptide containing CFA-1 pilus
protein B-Cell epitopes encapsulated within a
biodegradable-biocompatible poly(DL-lactide-co-glycolide) polymeric
matrix, wherein said antigenic synthetic peptide is present in an
amount of 0.1% to 1% by weight of said vaccine and said polymer is
present in an amount of 99% to 99.9% by weight of said vaccine,
wherein the CFA/I pilus protein B-cell epitopes consist of the
amino acid sequence: (SEQ ID NO: 12) 3
(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val), (SEQ ID NO: 13) 11
(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID NO: 14) 22
(Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), (SEQ ID NO: 15) 32
(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
(SEQ ID NO: 16) 32 (Ala-Tyr-Ser-Pro-Ala-Ser-Lys-thr-Phe), (SEQ ID
NO: 17) 38 (Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val), (SEQ ID NO: 18) 66
(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser) (SEQ ID NO: 19) 93
(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala), (SEQ ID NO: 20) 124
(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr), (SEQ ID NO: 21) 127
(Gly-thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), (SEQ ID NO: 22) 124
(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and mixtures
thereof.
47. The composition according to claim 46 wherein the polymeric
matrix is poly(DL-lactide-co-glycolide).
48. The composition according to claim 46 wherein the relative
ratio of lactide to glycolide component is within the range of
40:60 to 0:100.
49. The composition according to claim 46 wherein the relative
ratio of lactide to glycolide components is within the range of
48:52 to 58:42.
50. A composition for inducing an immune response in primates
comprising of an antigenic synthetic peptide containing CFA-1 pilus
protein T-Cell and B-Cell epitopes encapsulated within a
biodegradable-biocompatible poly(DL-lactide-co-glycolide) polymeric
matrix, wherein said antigenic synthetic peptide is present in an
amount of 0.1% to 1% by weight of said vaccine and said polymer is
present in an amount of 99% to 99.9% by weight of said vaccine,
wherein the CFA/I pilus protein T_-Cell and B-cell epitopes consist
of the amino acid sequence: (SEQ ID NO: 23) 3
(Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Val-Asp-Pro) (SEQ ID NO: 24) 8
(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID
NO: 13) 11 (Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID
NO: 5) 20 (Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), (SEQ ID NO:
22) 124 (Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
(SEQ ID NO: 25) 126 (Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser),
and mixtures thereof.
51. The composition according to claim 50 wherein the polymeric
matrix is poly(DL-lactide-co-glycolide).
52. The composition according to claim 50 wherein the relative
ratio of lactide to glycolide component is within the range of
40:60 to 0:100.
53. The composition according to claim 50 wherein the relative
ratio of lactide to glycolide components is within the range of
48:52 to 58:42.
54. A method for inducing an immune response in primates comprising
administering a pharmaceutical composition comprising an antigenic
synthetic peptide containing CFA/I pilus protein T-cell epitopes
encapsulated within a biodegradable polymeric matrix comprising
poly(DL-lactide-co-glycolide) having a relative ratio between the
amount of lactide and glycolide components within the range of
48:52 to 58:42, wherein the CFA-I pilus protein T-cell epitopes
consist of the amino acid sequence: (SEQ ID NO: 2)
8(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu), (SEQ ID NO: 3) 12
(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID NO: 4) 15
(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala), (SEQ ID NO: 5) 20
(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), (SEQ ID NO: 6) 26
(Pro-Ser-Ala-Val-Lys-Leu-Ala-Tyr-Ser-Pro), (SEQ ID NO: 7) 72
(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser), (SEQ ID NO: 8) 78
(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln), (SEQ ID NO: 9) 87
(Gln-Val-Leu-Ser-Thr-Thr-Ala-Lys-Glu-Phe), (SEQ ID NO: 10) 126
(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr,) and mixtures
thereof.
55. The method in accordance with claim 54 wherein said antigenic
synthetic peptide is present in an amount of 0.1% to 1% by weight
of said composition and said polymer is present in an amount of 99%
to 99.9% by weight of said composition.
56. A method for inducing an immune response in primates comprising
administering a pharmaceutical composition comprising an antigenic
synthetic peptide containing CFA/I pilus protein B-cell epitopes
encapsulated within a biodegradable polymeric matrix comprising
poly(DL-lactide-co-glycolide) having a relative ratio between the
amount of lactide and glycolide components within the range of
48:52 to 58:42, wherein the CFA-I pilus protein B-cell epitopes
consist of the amino acid sequence: (SEQ ID NO: 12) 3
(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val), (SEQ ID NO: 13) 11
(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID NO: 14) 22
(Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), (SEQ ID NO: 15) 32
(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
(SEQ ID NO: 16) 32 (Ala-Tyr-Ser-Pro-Ala-Ser-Lys-thr-Phe), (SEQ ID
NO: 17) 38 (Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val), (SEQ ID NO: 18) 66
(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser), (SEQ ID NO: 19) 93
(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala), (SEQ ID NO: 20) 124
(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr), (SEQ ID NO: 21) 127
(Gly-thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), (SEQ ID NO: 22) 124
(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and mixtures
thereof.
57. The method in accordance with claim 56 wherein said antigenic
synthetic peptide is present in an amount of 0.1% to 1% by weight
of said composition and said polymer is present in an amount of 99%
to 99.9% by weight of said composition.
58. A method for inducing an immune response in primates comprising
administering a pharmaceutical composition comprising an antigenic
synthetic peptide containing CFA/I pilus protein B-Cell and T-cell
epitopes encapsulated within a biodegradable polymeric matrix
comprising poly(DL-lactide-co-glycolide) having a relative ratio
between the amount of lactide and glycolide components within the
range of 48:52 to 58:42, wherein the CFA-I pilus protein B-cell and
T-cell epitopes consist of the amino acid sequence: (SEQ ID NO: 23)
3 (Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Val-Asp-Pro) (SEQ ID NO: 24) 8
(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID
NO: 13) 11 (Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), (SEQ ID
NO: 5) 20 (Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), (SEQ ID NO:
22) 124 (Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
(SEQ ID NO: 25) 126 (Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser),
and mixtures thereof.
59. The method in accordance with claim 58 wherein said antigenic
synthetic peptide is present in an amount of 0.1% to 1% by weight
of said composition and said polymer is present in an amount of 99%
to 99.9% by weight of said composition.
Description
II. CROSS REFERENCE
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 07/521,945 filed May 11, 1990, which in turn
is a continuation-in-part of U.S. patent application Ser. No.
590,308, filed Mar. 16, 1984.
III. FIELD OF THE INVENTION
[0003] This invention relates to oral-intestinal vaccines against
diseases caused by enteropathogenic organisms using antigens
encapsulated within biodegradable-biocompatible microspheres
(matrix).
IV. BACKGROUND OF THE INVENTION
[0004] Most infectious agents have their first contact with the
host at a mucosal surface; therefore, mucosal protective immune
mechanisms are of primary importance in preventing these agents
from colonizing or penetrating the mucosal surface. Numerous
studies have demonstrated that a protective mucosal immune response
can best be initiated by introduction of the antigen at the mucosal
surface, and parenteral immunization is not an effective method to
induce mucosal immunity. Antigen taken up by the gut-associated
lymphoid tissue (GALT), primarily by the Peyer's patches in mice,
stimulates T helper cell (T.sub.H) to assist in IgA B cell
responses or stimulates T suppressor cells (T.sub.s) to mediate the
unresponsiveness of oral tolerance. Particulate antigen appears to
shift the response towards the (T.sub.H) whereas soluble antigens
favor a response by the (T.sub.S). Although studies have
demonstrated that oral immunization does induce an intestinal
mucosal immune response, large doses of antigen are usually
required to achieve sufficient local concentrations in the Peyer's
patches. Unprotected protein antigens may be degraded or may
complex with secretory IgA in the intestinal lumen.
[0005] One possible approach to overcoming these problems is to
homogeneously disperse the antigen of interest within the polymeric
matrix of appropriately sized biodegradable, biocompatible
microspheres that are specifically taken up by GALT. Eldridge et.
al. have used a murine model to show that orally-administered 1-10
micrometer microspheres consisting of polymerized lactide and
glycolide, (the same materials used in resorbable sutures), were
readily taken up into Peyer's patches, and the 1-5 micrometer size
were rapidly phagocytized by macrophages. Microspheres that were
5-10 micrometers (microns) remained in the Peyer's patch for up to
35 days, whereas those less than 5 micrometer disseminated to the
mesenteric lymph node (MLN) and spleen within migrating MAC-1.sup.+
cells. Moreover, the levels of specific serum and secretory
antibody to staphylococcal enterotoxin B toxoid and inactivated
influenza A virus were enhanced and remained elevated longer in
animals which were immunized orally with microencapsulated antigen
as compared to animals which received equal doses of
non-encapsulated antigen. These data indicate that
microencapsulation of an antigen given orally may enhance the
mucosal immune response against enteric pathogens. AF/R1 pili
mediate the species-specific binding of E. coli RDEC-1 with mucosal
glycoproteins in the small intestine of rabbits and are therefore
an important virulence factor. Although AF/R1 pili are not
essential for E. coli RDEC-1 to produce enteropathogenic disease,
expression of AF/R1 promotes a more severe disease. Anti-AF/R1
antibodies have been shown to inhibit the attachment of RDEC-1 to
the intestinal mucosa and prevent RDEC-1 disease in rabbits. The
amino acid sequence of the AF/R1 pilin subunit has recently been
determined, but specific antigenic determinants within AF/R1 have
not been identified.
[0006] Recent advances in the understanding of B cell and T cell
epitopes have improved the ability to select probably linear
epitopes from the amino acid sequence using theoretical criteria. B
cell epitopes are often composed of a string of hydrophilic amino
acids with a high flexibility index and a high probability of turns
within the peptide structure. Prediction of T cell epitopes are
based on the Rothbard method which identifies common sequence
patterns that are common to known T cell epitopes or the method of
Berzofsky and others which uses a correlation between algorithms
predicting amphipathic helices and T cell epitopes.
[0007] In the current study we have used these theortical criteria
to predict probable T or B cell epitopes from the amino acid
sequence of AF/R1. Four different 16 amino acid peptides that
include the predicted epitopes have been synthesized: AF/R1 40-55
as a B cell epitope, 79-94 as a T cell epitope, 108-123 as a T and
B cell epitope, and AF/R1 40-47/79-86 as a hybrid of the first
eight amino acids from the predicted B cell epitope and the T cell
epitope. We have used these peptides as well as the native protein
to stimulate the in vitro proliferation of lymphocytes taken from
the Peyer's patch, MLN, and spleen of rabbits which have received
intraduodenal priming with microencapsulated or non-encapsulatled
AF/R1. Our results demonstrate the microencapsulation of AF/R1
potentiates the cellular immune response at the level of the
Peyer's patch, thus enhancing in vitro lymphocyte proliferation to
both the native protein and its linear peptide antigens. CFA/I
pili, rigid thread-like structures which are composed of repeating
pilin subunits of 147 amino acid found on serogroups 015, 025, 078,
and 0128 of enterotoxigenic E. coli (ETEC) [1-4, 18]. CFA/I
promotes mannose resistant attachment to human brush borders [5];
therefore, a vaccine that established immunity against this protein
may prevent the attachment to host tissues and subsequent disease.
In addition, because the CFA/I subunit shares N-terminal amino acid
sequence homology with CS1, CFA/II (CS2) and CFA/IV (CS4) [4], a
subunit vaccine which contained epitopes from this area of the
molecule may protect against infection with various ETEC.
[0008] Until recently, experiments to identify these epitopes were
time consuming and costly; however, technology is now available
which allows one to simultaneously identify all the T cell and B
cell epitopes in the protein of interest. Multiple Peptide
synthesis (Pepscan) is a technique for the simultaneous synthesis
of hundreds of peptides on polyethylene rods [6]. We have used this
method to synthesize all the 140 possible overlapping actapeptides
of the CFA/I protein. The peptides, still on the rods, can be used
directly in ELISA assays to map B call epitopes [6, 12-14]. We have
also synthesized all the 138 possible overlapping decapeptides of
the CFA/I protein. For analysis of T cell epitopes, these peptides
can be cleaved from the rods and used in proliferation assays [15].
Thus this technology allows efficient mapping and localization of
both B cell and T cell epitopes to a resolution of a single amino
acid [16]. These studies were designed to identify antigenic
epitopes of ETEC which may be employed in the construction of an
effective subunit vaccine.
[0009] CFA/I pili consist of repeating pilin protein subunits found
on several serogroups of enterotoxigenic E. coli (ETEC) which
promote attachment to human intestinal mucosa. We wished to
identify areas within the CFA/I molecule that contain
immunodominant T cell eptiopes that are capable of stimulating the
cell-mediated portion of the immune response in primates as well as
immunodominant B cell epitopes. To do this, we (a) resolved the
discrepancy in the literature on the complete amino acid sequence
of CFA/I, (b) immunized three Rhesus monkeys with multiple i.m.
injections of purified CFA/I subunit in Freund's adjuvant, (c)
synthesized 138 overlapping decapeptides which represented the
entire CFA/I protein using the Pepscan technique (Cambridge
Research Biochemicals), (d) tested each of the peptides for their
ability to stimulate the spleen cells from the immunized monkeys in
a proliferative assay (e) synthesized 140 overlapping octapeptides
which respresented the entire CFA/I protein, and (f) tested serum
from each monkey for its ability to recognize the octapeptides in a
modified ELISA assay. A total of 39 different CFA/I decapeptides
supported a significant proliferative response with the majority of
the responses occurring within distinct regions of the protein
(peptides beginning with residues 8-40, 70-80, and 127-137).
Nineteen of the responsive peptides contained a serine residue at
positions 2, 3, or 4 in the peptide, and a nine contained a serine
specifically at position 3. Most were predicted to be configured as
an alpha holix and have a high amphipathic index. Eight B cell
epitopes were identified at positions 3-11, 11-21, 22-29, 32-40,
38-45, 66-74, 93-101, and 124-136. The epitope at position 11-21
was strongly recognized by all three individual monkeys, while the
epitopes at 93-101, 124-136, 66-74, and 22-29 were recognized by
two of the three monkeys.
V. SUMMARY OF THE INVENTION
[0010] This invention relates to a novel pharmaceutical compositon,
a microcapsule/sphere formulation, which comprises an antigen
encapsulated within a biodegradable polymeric matrix such as poly
(DL-lactide-co-glycolide) (DL-PLG), wherein the relative ratio
between the lactide and glycolide component of the DL-PLG is within
the range of 40:60 to 0:100, and its use, as a vaccine, in the
effective pretreatment of animals (including humans) to prevent
intestinal infections caused by a virus or bacteria. In the
practice of this invention, applicants found that the AF/R1
adherence factor is a plasmid encoded pilus composed of repeating
pilin protein subunits that allows E. coli RDEC-1 to attach to
rabbit intestinal brush borders. To identify an approach that
enhances the immunogenicity of antigens that contact the intestinal
mucosa, applicants investigated the effect of homogeneously
dispersing AF/R1 pili within biodegradable microspheres that
included a size range selected for Peyer's Patch localization. New
Zealand White rabbits were primed twice with 50 micrograms of
either microencapsulated or nonencapsulated AF/R1 by endoscopic
intraduodenal inoculation. Lymphoid tissues were removed and
cellular proliferative responses to AF/R1 and synthetic AF/R1
peptides were measured in vitro. The synthetic peptides represented
possible T and/or B cell epitopes which were selected from the
AF/R1 subunit sequence using theoretical criteria. In rabbits which
had received nonencapsulated AF/R1, Peyer's Patch cells
demonstrated slight but significant proliferation in vitro in
response to AF/R1 pili but not the AF/R1 synthetic peptides. In
rabbits which had received microencapsulated AF/R1, Peyer's Patch
cells demonstrated a markedly enhanced response to AF/R1 and the
synthetic peptides. Cells from the spleen and mesenteric lymph
nodes responded similarly to AF/R1 pili in both groups of animals,
while there was a greater response to the synthetic peptide AF/R1
40-55 in rabbits that had received microencapsulated AF/R1. These
data demonstrate that microencapsulation of AF/R1 potentiates the
mucosal cellular immune response to both the native protein and its
linear peptide antigens.
VI. BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the size destribution of microspheres wherein
the particle size distibution (%) is (a) By number 1-5 (91) and
6-10 (9) and (b) By weight 1-5 (28) and 6-10 (72).
[0012] FIG. 2 shows a scanning electron micrograph of
microspheres.
[0013] FIGS. 3(a) and (b) show the In vitro immunization of spleen
cells and demonstrates that AF/RI pilus protein remains immunogenic
to rabbit spleen cells immunized in vitro after microencapsulation.
AF/R1 pilus protein has been found to be immunogenic for rabbit
spleen mononuclear cells in vitro producing a primary IgM antibody
response specific to AF/RI. Immunization with antigen encapsulated
in biodegradable, biocompatible microspheres consisting of
lactide/glycolide copolymers has been shown to endow substantially
enhanced immunity over immunization with the free antigen. To
determine if microencapsulated AF/RI maintains the immunogenicity
of the free pilus protein, a primary in vitro immunization assay
was conducted. Rabbit spleen mononuclear cells at a concentration
of 3.times.10.sup.5 cells/well. Triplicate wells of cells were
immunized with free AF/RI in a dose range from 15 to 150 ng/ml or
with equivalent doses of AF/RI contained in microspheres.
Supernatants were harvested on days 7, 9, 12, and 14 of culture and
were assayed for free AF/RI pilus protein specific IgM antibody by
the ELISA. Supernatant control values were subtracted from those of
the immunized cells. Cells immunized with free pilus protein showed
a significant positive IgM response on all four days of harvest,
with the antibody response increasing on day 9, decreasing on day
12, and increasing again on day 14. Cells immunized with
microencapsulated pilus protein showed a comparable positive IgM
antibody response as cells immunized with free pilus protein. In
conclusion, AF/RI maintains immunogenicity to rabbit spleen cells
immunized in vitro after microencapsulation.
[0014] FIGS. 4(a) and (b) show in vitro immunization of Peyer's
patch cells. Here the AF/RI pilus protein remains immunogenic to
rabbit Peyer's patch cells immunized in vitro after
microencapsulation. AF/RI pilus protein has been found to be
immunogenic for rabbit Peyer's patch mononuclear cells in vitro
producing a primary IgM antibody response specific to AF/RI.
Immunization with antigen encapsulated in biodegradable,
biocompatible microspheres consisting of lactide/glycolide
copolymers has been shown to endow substantially enhanced immunity
over immunization with the free antigen. To determine if
microencapsulated AF/RI maintains the immunogencity of the free
pilus protein, a primary in vitro immunization assay was conducted.
Rabbit Peyer's patch mononuclear cells at a concentration of
3.times.10.sup.6 cells/ml were cultured in 96-well, round bottom
microculture plates at a final concentration of 6.times.10.sup.5
cells/well. Triplicate wells of cells were immunized with free
AF/RI in a dose range from 15 to 150 ng/ml or with equivalent dose
of AF/RI contained in microspheres. Supernatants were harvested on
days 7, 9, 12, and 14 of culture and were assayed for free AF/RI
pilus protein specific IgM antibody by the ELISA. Supernatant
control values were subtracted from those of the immunized cells.
Cells immunized with free pilus protein showed a significant
positive IgM response on all four days of harvest, with the highest
antibody response on day 12 with the highest antigen dose. Cells
immunized with encapsulated pilus protein showed a positive
response on day 12 with all three antigen doses. In conclusion,
AF/RI pilus protein maintains immunogenicity to rabbit Peyer's
patch cells immunized in vitro after microencapsulation.
[0015] FIG. 5 shows proliferative responses to AF/RI by rabbit
Peyer's patch cells. Naive rabbits were primed twice with 50
micrograms of either non-encapsulated (rabbits 132 and 133) or
microencapsulated (rabbits 134 and 135) AF/RI pili by endoscopic
intraduodenal inoculation seven days apart. Seven days following
the second priming, Peyer's patch cells were cultured with AF/RI in
96-well plates for four days followed by a terminal six hour pulse
with [.sup.3H]thymidine. Data shown is the SI calculated from the
mean cpm of quadruplicate cultures. Responses were significant for
all rabbits: 132 (p=0.013), 133 (p=0.0006), 134 (p=0.0016), and 135
(p=0.0026). Responses were significantly different between the two
groups. Comparison of the best responder in the nonencapsulated
antigen group (rabbit 133) with the lowest responder in the
microencapsulated antigen group (rabbit 134) demonstrated an
enhanced response when the immunizing antigen was microencapsulated
(p=0.0034).
[0016] Additionally, FIG. 5 relates to the in vitro lymphocyte
proliferation after sensitization of rabbit lymphoid tissues with
encapsulated or non-encapsulated AF/RI pilus adhesion of E. coli
strain RDEC-1. The AF/RI adherence factor is a plasmid encoded
pilus protein that allows RDEC-1 to attach to rabbit intestinal
brush borders. We investigated the immunopotentiating effect of
encapsulating purified AF/RI into biodegradable non-reactive
microspheres composed of polymerized lactide and glycolide,
materials used in resorbable sutures. The microspheres had a size
range of 5-10 microns, a size selected for Peyer's Patch
localizaiton, and contained 0.62% protein by weight. NZW rabbits
were immunized twice with 50 micrograms of either encapsulated or
non-encapsulated AF/RI by intraduodenal later of non-encapsulated
AF/RI by intraduodenal inoculation seven days apart. Lymphocyte
proliferation in response to purified AR/RI was conducted in vitro
at seven days and showed that encapsulating the antigen into
microspheres enhanced the cellular immune response in the Peyer's
Patch; however, no significant increase was observed in spleen or
mesenteric lymph node. These data suggest that encapsulation of
AF/RI may potentiate the mucosal cellular immune response.
[0017] FIGS. 6 a-d show proliferative responses to AF/RI synthetic
peptides by rabbit Peyer's patch cells. Naive rabbits were primed
twice with 50 micrograms of either non-encapsulated (rabbits 132
and 133) or microencapsulated (rabbits 134 and 135) AF/RI pili by
endoscopic intraduodenal inoculation seven days apart. Seven days
following the second priming, Peyer's patch cells from each rabbit
were cultured with AF/R1 40-55 (FIG. 6a), AF/R1 79-94 (FIG. 6b),
AF/R1 108-123 (FIG. 6c), or AF/R1 40-47/79-86 (FIG. 6d) in 96-well
plates for four days followed by a terminal six hour pulse with
[.sup.3H]thymidine. Data shown is the SI calculated from the mean
cpm of quadruplicate cultures. The responses of rabbits 132 and 133
were not significant to any of the peptides tested. Rabbit 134 had
a significant response to (a) AF/R1 40-55 (p=0.0001), (b) AF/R1
79-94 (p=0.0280), and (d) AF/R1 40-57/79-86 (p=0.025), but not to
(c) AF/R1 108-123. Rabbit 135 had a significant response to (a)
AF/R1 40-55 (p=0.034), (b) AF/R1 79-94 (p=0.040), and (c) AF/R1
108-123 (p<0.0001), but not to (d) AF/R1 40-47/79-86. This
demonstrates enhanced proliferative response to peptide antigens
following mucosal priming with microencapsulated pili. AF/RI pili
promotes RDEC-1 attachment to rabbit intestinal brush borders.
Three 16 amino acid peptides were selected by theoretical criteria
from the AF/RI sequence as probable T or B cell epitopes and were
synthesized: AF/R1 40-55 as a B cell epitope, 79-94 as a T cell
epitope, and 108-123 as a T and B cell epitope. We used these
peptides to investigate a possible immunopotentiating effect of
encapsulating purified Af/RI pili into biodegradable, biocompatible
microspheres composed of polymerized lactide and glycolide at a
size range that promotes localization in the Peyer's Patch (5-10
micrometers). NZW rabbits were primed twice with 50 micrograms
AF/RI by endoscopic intraduadenal inoculation and their Peyer's
Patch cells were cultured in vitro with the AF/RI peptides. In two
rabbits which had received encapsulated AF/RI, lymphocyte
proliferation was observed to AF/RI 40-55 and 79-94 in both rabbits
and to 108-123 in one of two rabbits. No responses to any of the
peptides were observed in rabbits which received non-encapsulated
AF/RI. These data suggest that encapsulation of AF/RI may enhance
the cellular response to peptide antigens.
[0018] FIGS. 7a-d show B-cell responses of Peyer's patch cells to
AF/R1 and peptides.
[0019] FIGS. 8a-d show B-cell responses of Peyer's Patch cells to
AF/R1 and peptides.
[0020] FIGS. 9a-d show B-cell responses of spleen cells to AF/R1
and Peptides.
[0021] FIGS. 10a-d show B cell responses of spleen cells to AF/R1
and peptides.
[0022] FIGS. 7 through 10, illustrate enhanced lymphocyte antibody
response by mucosal immunization of rabbits with microencapsulated
AF/R1 pilus protein. The AF/RI pilus protein has been found to be
immunogenic for rabbit spleen and Peyer's patch cells in vitro
producing a primary IgM antibody response. The purpose of this
study was to determine if AR/R1 pilus protein immune response is
enhanced by microencapsulation. The AF/R1 was incorporated into
biodegradable, biocompatible microspheres composed of
lactide-glycolide copolymers, had a size range of 5-10 micrometer
and containing 0.62% pilus protein by weight. Initially, NZW
rabbits were immunized twice with 50 micrograms of either
encapsulated or non-encapsulated AF/RI via intraduodenal route
seven days apart. For in vitro challenge, 6.times.10.sup.5 rabbit
lymphocytes, were set in microculture at final volume of 0.2 ml.
Cells were challenged with AR/RI or three different synthetic 16
amino acid peptides representing, either predicted T, B or T and B
cell epitopes in a dose range of 15 to 150 ng/ml for splenic cells
or 0.05 to 5.0 micrograms/ml for Peyer's patch mononuclear cells
(in triplicate). Supernatants were collected on culture days 3, 5,
7, and 9 assayed by ELISA for anti-AF/R1 antibody response as
compared to cell supernatant control. Significant antibody
responses were seen only from spleen and Peyer's patch cells from
rabbits immunized with microencapsulated AF/R1. The antibody
response tended to peak between days 5 and 9 was mainly an IgM
response. The results for the predicted epitopes were similar to
those obtained with purified AF/RI. In conclusion, intestinal
immunization with AF/RI pilus protein contained within microspheres
greatly enhances both the spleen and Peyer's patch B-cell responses
to predicted T & B-cell epitopes.
[0023] FIG. 11 shows proliferative responses to AF/R1 40-55 by
rabbit MLN cells. Naive rabbits were primed twice with 50
micrograms of either nonencapsulated (rabbits 132 and 133) or
microencapsulated (rabbits 134 and 135) AF/R1 pili by endoscopic
intraduodenal inoculation seven days apart. Seven days following
the second priming, MLN cells were cultured with AF/R1 40-55 for
four days in 24-well plates. Cultures were transferred into 96-well
plates for a terminal [.sup.3H]thymidine pulse. Data shown is the
SI calculated from the mean cpm of quadruplicate cultures.
Responses of rabbits 132 and 133 were not statistically
significant. Responses were significant for rabbits 134
(p=0.0.0051) and 135 (p=0.0055).
[0024] FIG. 12 shows proliferative responses to AF/R1 40-55 by
rabbit spleen cells. Naive rabbits were primed twice with 50
micrograms of either nonencapsulated (rabbits 132 and 133) or
microencapsulated (rabbits 134 and 135) AF/R1 pili by endoscopic
intraduodenal inoculation seven days apart. Seven days following
the second priming, spleen cells were cultured with AF/R1 40-55 for
four days in 24-well plates. Cultures were transferred into 96 well
plates for a terminal [.sup.3H]thymidine pulse. Data shown is the
SI calculated from the mean cpm of quadruplicate cultures.
Responses of rabbits 132 and 133 were not statistically
significant. Responses were significant for rabbits 134
(p=0.0.0005) and 135 (p=0.0066).
[0025] FIG. 16. A. SDS-PAGE of intact CFA/I (lane 1), trypsin
treated CFA/I (lane 2), and S. aureus V8 protease treated CFA/I.
Molecular masses of individual bands were estimated from molecular
weight standards (on left). Multiple lanes of both trypsin and V8
treated CFA/I were transferred to PVDF membranes where bands
corresponding to the approximate molecular masses of 3500 (trypsin
digest, see arrow lane 2) and 6000 (V8 digest, see arrow lane 3)
were excised and subjected to Edman degradation. B. Resulsting
sequence of protein fragments from each lane of A (position of
sequenced portion of fragment in the intact protein. Underlined,
italisized residues are amino acids under dispute in
literature.
[0026] FIG. 17. ELISA assay results testing hyperimmune sera of
monkeys (A) 2Z2 (monkey 3), (B) 184(D) (monkey 1) and (C) 34
(monkey 2) to CFA/I primary structure immobilized on polyethylene
pins. Monkey sera diluted 1:1000. Peptide number refers first amino
acid in sequence of octapeptide on pin from CFA/I primary structure
OD 405 refers to optical density wavelength at which ELISA plates
were reat (405 nm).
[0027] FIG. 18. Complete sequence of CFA/I (147 amno acids) with B
cell recognition site (boxed areas) as defined by each individual
monkey response (2Z2, 184D, and 34). Derived from data in FIG.
17.
[0028] FIGS. 19-21. Lymphocyte proliferation to synthetic
decapeptides of CFA/I. Each monkey was immunized with three i.m.
injections of CFA/I subunits in adjuvant, and its spleen cells were
cultured with synthetic decapeptides which had been constructed
using the Pepscan technique. The decapeptides represented the
entire CFA/I protein. Concentrations of synthetic peptide used
included 6.0, 0.6, and 0.06 micrograms/ml. Values shown represent
the maximum proliferative response produced by any of the three
concentrations of antigen used.+-.the standard deviation. The cpm
of the control peptide for each of the three monkeys was
1,518.+-.50, 931.+-.28, and 1,553.+-.33 respectively. The cpm of
the media control for each of the three monkeys was 1,319.+-.60,
325.+-.13, and 1,951.+-.245 respectively.
[0029] FIGS. 22-24. Lymphocyte proliferation to 6.0, 0.6, and 0.06
micrograms/ml synthetic decapeptides of CFA/I in one monkey. The
monkey (2Z2) as immunized with three i.m. injections of CFA/I
subunits in adjuvant, and its spleen cells were cultured with
synthetic decapeptides which had been constructed using the Pepscan
technique. The decapeptides represented the entire CFA/I protein.
Values shown represent the proliferative response which occurred to
6.0 micrograms/ml (FIG. 22), 0.6 micrograms/ml (FIG. 23), or 0.06
micrograms/ml (FIG. 24) of antigen.+-.the standard deviation. The
cpm of the control peptide was 1,553.+-.33 and the cpm of the media
control was 1,951.+-.245.
[0030] FIGS. 11 and 12 serve to illustrate that inclusion of
Escherichia coli pilus antigen in microspheres enhances cellular
immunogenicity.
[0031] A primary mucosal immune response, characterized by
antipilus IgA, follows infection of rabbits with E. coli RDEC-1.
However, induction of an optimal primary mucosal response by
enteral vaccination with pilus antigen depends on immunogenicity of
pilus protein, as well as such factors as its ability to survive
gastrointestinal tract (GI) transit and to target immunoresponsive
tissue. We tested the effect of incorporating AF/R1 pilus antigen
into resorbable microspheres upon its ability to induce primary
mucosal and systemic antibody responses after direct inoculation
into the GI tract. METHODS: rabbits were inoculated with 50
micrograms of AF/R1 pilus antigen alone or incorporated into
uniformaly sized (5-10 microns) resorbably microspheres (MIC) of
poly(DL-lactide-coglycolide). Inoculation was by intra-duodenal
(ID) intubation via endoscopy or directly into the ileum near a
Peyer's patch via the RITARD procedure (with the cecum ligated to
enhance recovery of gut secretions and a reversible ileal tie to
slow antigen clearance). ID rabbits were sacrificed at 2 weeks for
collection of gut washes and serum. RITARD rabbits were bled and
purged weekly for 3 weeks with Co-lyte to obtain gut secretions.
Anti-pilus IgA and IgG were measured by ELISA.
TABLE-US-00001 TABLE 1 RITARD- RESULTS: *pos/test PILI RITARD-MIC
ID-PILI ID-MIC Anti-pilus IgA (fluid) *7/8 4/8 1/2 0/3 Anti-pilus
IgG (serum) 0/8 3/8 0/2 1/3
[0032] Native pilus antigen led to a mucosal IgA resposne in 7/8
RITARD rabbits. MIC caused a similar response in only 4/8, but the
groups were not statistically different. MIC (but not pili) induced
some systemic IgG responses (highest in animals without mucosal
responses). Results in rabbits inoculated ID were similar for pili,
but no mucosal response to ID-MIC was noted. SUMMARY: Inoculation
with pilus antigen produces a primary mucosal IgA response.
Microencapsulation does not enhance this response, although the
antigen remains immunogenic as shown by measurable mucosal and some
strong serum responses. It must be determined whether priming with
antigen in microspheres can enhance secondary responses.
B Cell Epitope Data
Materials and Methods
[0033] CFA/I PURIFICATION--INTACT CFA/I pili were purified from
H10407 (078:H--) as described by Hall et al, (1989) [20]. Briefly,
bacteria grown on colonization factor antigen agar were subjected
to shearing, with the shearate subjected to differential
centrifugation and isopycnic banding on cesium chloride in the
presence of N-lauryl sarkosine. CFA/I were dissociated to free
subunits in 6M guanididinium HCl, 0.2 M ammonium bicarbonate (2 hr,
25.degree.), passed through an ultrafiltration membrane (Amicon XM
50 stirred cell, Danvers, Mass.), with concentration and buffer
exchange to PBS on a YM 10 stirred cell (Amicon). Examination of
dissociated pili by electron microscopy demonstrated a lack of
pilus structure.
[0034] Protein Sequencing--The primary structure of CFA/I has been
determined by protein sequencing techniques (Klemm, 1982) and
through molecular cloning methods (Karjalainen, et al 1989) [21].
In these two studies there was agreement in all but two of the 147
amino acid residues (at positions 53 and 74). To resolve the
apparent discrepancies, CFA/I was enzymatically digested in order
to obtain internal amino acid sequence. Trypsin or S. aureus V8
protease (sequencing grade, Boehringer Mannheim) was incubated with
CFA/I at a 1:50 w:w ratio (Tris 50 mM, 0.1% SDS, pH 8.5 for 16 h at
37.degree. (trypsin) or 24.degree. C. (V8)). Digested material was
loaded onto precast 16% tricine SDS-PAGE gels (Schagger and von
Jagow, 1987) (Novex, Encinitis, Calif.) and run following
manufacturers instructions. Separated samples were
electrophoretically transferred to PVDF membranes (Westrans,
Schleicher and Schuell, Keene, N.H.) following Matsiduria (1987)
using the Novex miniblot apparatus. Blotted proteins were stained
with Rapid Coomassie stain (Diversified Biotech, Newton Centre,
Mass.). To obtain the desired fragment containing the residue of
interest within a region accessible by automated gas phase
sequencing techniques, molecular weights were estimated from
standards of molecular weights 20,400 to 2,512 (trypsin inhibitior,
myoglobin, and myoglobin cyanogen bromide fragments; Diversified
Biotech) using the corrected molecular weights for the myoglobin
fragments as given in Kratzin et al., (1989) [22]. The estimated
molecular weights for the unknown CFA/I fragments were compared to
calculated molecular weights of fragments as predicted for CFA/I
from the sequence of CFA/I as analysed by the PEPTIDESORT program
of a package developed by the University of Wisconsin Genetics
Computer Group. Selected fragments were cut from the PVDF emebrane
and subjected to gas phase sequencing (Applied Biosystem 470,
Foster City, Calif.).
[0035] Monkey Immunization--Three rhesus monkeys (Macaca mulatta)
were injected intramuscularly with 250 ug of dissociated CFA/I in
complete Freund's adjuvent and subsequently with two injections of
250 ug of antigen in incomplete Freund's adjuvent at weekly
intervals. Blood was drawn three weeks after primary
immunization.
[0036] Peptide Synthesis--Continuous overlapping octapeptides
spanning the entire sequence CFA/I were synthesized onto
polyethylene pins by the method of Geysen et al. [16], also known
as the PEPSCAN procedure. Derivitized pins and software were
purchased from Cambridge Research Biochemicals (Valley Stream,
N.Y.). Fmoc-amino acid pentafluorophenyl esters were purchased from
Peninsular Laboratories (Belmont, Calif.), 1-hydroxybenzotriazole
monohydrate (HYBT) was purchased from Aldrich, and reagent grade
solvents from Fisher. To span the entire sequence of CFA/I with a
single amino acid overlap of from one peptide to the next, 140
total pins were necessary, with a second complete set of 140 pins
synthesized simultaneously.
[0037] ELISA procedure--Sera raised in monkeys to purified
dissociated pili were incubated with the pins in the capture ELISA
assay of Geysen et al., [16] with the preimmune sera of the same
animal tested at the same dilution simultaneously with the
duplicate set of pins. Dilution of sera used on the pins was chosen
by initial titration of sera by standard ELISA assay and immunodot
blot assay against the same antigen.
Results
[0038] It was essential to utilize the correct sequence of CFA/I in
the synthesis of the pins for both T- and B-cell experiments to
carry out the studies as planned. At issue were the amino acids at
position 53 and 74; incorrect residues at those positions would
effect 36 of 138 pins (26%) for T-cell epitope analysis and 30 of
140 pins (21%) for B-cell analysis. To resolve the discrepancy in
the literature, purified CFA/I was proteolytically digested
separately with trypsin and with S. aureus V8 protease (V8). These
enzymes were chosen in order to give fragments with the residues of
interest (53 and 74) relatively near to the N-terminus for
automated Edman degradation (preferably 1-15 residues). These
digests were separated on tricine SDS-PAGE gels (FIG. 16A) and
molecular masses of fragments estimated. A fragment of 3459
calculated molecular mass is expected from the trypsin digest
(corresponding to amino acids 62-94) and a fragment of 5889
calculated molecular mass is expected from the V8 digest (residues
42-95). These fragments were located within each digest (arrows in
FIG. 16), and a companion gel with four lanes of each digest was
run, electrophoreticaly transferred to PVDF, the bands excised and
sequenced. N-terminal sequences of each fragment are given in FIG.
16B. The N-terminal eighteen residues from the trypsin fragment
were determined that corresponded to positions 62-79 in CFA/I.
Position 74, a serine residue was consistent with that determined
by Karjalainen et al., (Karjalainen et al., 1989). Nineteen
residues of the V8 fragment were determined, corresponding to
residues 41-60 of the parent protein. The twelfth residue of the
fragment contained an aspartic acid, also consistent with
Karjalainen et al., (1989). All other residues sequenced were
consistent with those published previously (including residues
1-29, not shown). For the following peptide synthesis were
therefore utilized the complete amino acid sequence of CFA/I
consistent with Karjalainen et al., (1989).
[0039] Sera from monkeys immunized with CFA/I subunits were tested
in a modified ELISA assay, with the preimmunization sera tested
simultaneously with duplicate pins. Assays results are displayed in
FIG. 17. Monkey 2Z2 (FIG. 2A) responded strongly to six regions of
the CFA/I sequence. Peptide 14 (the octapeptide 14-21) gave the
strongest response with four pins adjacent to it (11, 12, 13, and
15) also appearing to bind significant antibody. The other 2Z2
epitopes are centered at peptides 3, 22, 33, 93, and 124. Monkey
184D (FIG. 17B) also responded strongly to peptide 14, although the
maximum response was to peptide 13, with strong involvement of
peptide 12 in the epitope. Additional epitopes recognized by 184d
were centered at peptides 22, 33, 66, and 93. The third monkey
serum tested, 34, responded to this region of the CFA/I primary
structure, both at peptides 1, 12 and weakly at 14. Two other
epitopes were identified by 34, centered at peptides 67 and 128.
FIG. 18 illustrates the amino acids corresponding to the epitopes
of CFA/I as defined by the response of these three monkeys aligned
with the entire primary structure. The entire antigenic
determinants are mapped and areas of overlap with other epitopes
(consensus sites) are displayed. These epitopes are summarized in
Table 1.
T Cell Epitope Data
Materials and Methods
[0040] Animals. Three healthy adult Macaca mulatta (Rehesus)
monkeys (approximately 7 kg each) were used in this study. Their
medical records were examined to assure that they had not been in a
previous protocol which would preclude their use in this study.
Each monkey was sedated with ketamine HCL1 at standard dosage and
blood was drawn to obtain preimmune serium.
[0041] Antigen. CFA/I pili were purified from E. coli strain
H107407 (serotype 078:H11) by ammonium sulfate precipitation using
the method of Isaacson [17]. The final preparation migrated as a
single band on SD-polyacrylamide gel electrophoresis and was shown
to be greater than 95% pure by scanning with laser desitometry when
stained with coomassie blue. The pili were then dissociated into
CFA/I pilin subunits.
[0042] Immunization. Each monkey was given 25 mg of purified CFA/I
pilin subunits, which had been emulsified in Complete Freund's
Adjuvant, by single i.m. injection (0.5 ml). For each animal, the
initial dose of antigen was followed by two similar injections in
Incomplete Freund's Adjuvant at seven day intervals.
[0043] Peptide Antigens. The peptides were synthesized based on the
published sequence of CFA/I [18] using the Geysen pin method
(Pepscan procedure) [16] with equipment and software purchased from
Cambridge Research Biochemicals, Inc. (Wilmington, Del.).
Fmoc-amino acid pentafluorophenyl esters were purchased from
Peninsula Laboratories (Belmont, Calif.) and used without further
treatment or analysis. The activating agent 1-hydroxybenzotriazole
monohydrate (HOBT) was purchased from Aldrich Chemical Company
(Milwaukee, Wis.). Solvents were reagent grade from Fisher
Scientific (Springfield, N.J.).
[0044] Two schemes were used to synthesize the peptides. Peptides
for the B-cell tests were synthesized as octamers and remained
linked to the resin. However, the peptides used to search for
T-cell epitopes were synthesized as decamers with an additional
Asp-Pro spacer between the pins and the peptides of interest. The
Asp-Pro linkage is acid labile allowing cleavage of the decamers
from the pins for T-cell proliferation assays [15]. The peptides
were cleaved in 70% formic acid for 72 hours at 37 degrees C. The
acid solution was removed by evaporation (Speed-Vac, Savant
Instruments, Framingdale, N.Y.) followed by rehydration with
distilled deionized water and lyophilizaiton. The resulting cleaved
peptides were used without further treatment or analysis. The yield
was approximately 10 ug per pin, approximately 10 percent on a
molar basis of the total amount of proline on each pin as
determined by quantitative amino acid analysis.
[0045] Residues 12 and 13 on the CFA-1 protein are Asp and Pro,
respectively, the same sequence used to cleave the peptides from
the pins. Therefore, to prevent truncated peptides from the native
sequence during the cleavage process, two substitutions were made
for Asp-12. One substitution was a glutamic acid residue for the
aspartic acid, a substitution to retain the carboxylic acid
functional group. The second substitution was an asparagine residue
to conserve the approximate size of the side chain while retaining
some hydrophilicity. Each substitution was tested in the T-cell
proliferation assay. Both substitutions as well as the native
sequence were analyzed by ELISA. For both the T cell and B cell
assays, additional sequences not found on the protein were
synthesied and used as control peptides.
[0046] Lymphocyte proliferation. At day 10-14 following the final
inoculation of antigen, the monkeys were again sedated with
ketamine HCl, and 50 ml of blood was drawn from the femoral artery
for serum preparation. Animals were then euthanized with an
overdose of pentothal and spleen was removed. Single cell
suspensions were prepared and washed in Dulbecco's modified Eagle
medium (Gibco Laboratories, Grand Island, N.Y.) which had been
supplemented with penicillin (100 units/ml), streptomycin (100
ug/ml), L-glutamine (2 mM), and HEPES Buffer (10 mM) all obtained
from Gibco Laboratories, as well as MEM non-essential amino acid
solution (0.1 mM), MEM [50.times.] amino acids (2%), sodium
bicarbonate (0.06%), and 5.times.10.sup.-5 M 2-ME all obtained from
Sigma Chemical Company (St. Louis, Mo.) [cDMEM]. Erythrocytes in
the spleen cell suspension were lysed using standard procedures in
an ammonium chloride lysing buffer. Cell suspensions were adjusted
to 10.sup.7 cells per ml in cDMM, and autologous serum was added to
yield a final concentration of 1.0%. Cells (0.05 ml) were plated in
96-well flat bottom culture plates (Costar, Cambridge, Mass.) along
with 0.05 ml of various dilutions of antigen in cDMEM without serum
(yielding a 0.5% final concentration of autologous serum) and were
incubated at 37 degrees C. in 5% CO.sub.2. Each peptide was tested
at 6.0, 0.6, 0.06 ug/ml. All cultures were pulsed with 1 uci
[.sup.3H]thymidine (25 Ci/mmol, Amersham, Arlington Hights, Ill.)
on day 4 of culture and were harvested for scintillation counting 6
hours later.
Elisa
[0047] Epitope prediction. Software designed to predict B cell
epitopes based on hydrophilicity, flexibility, and other criteria
was developed by the University of Wisconsin Genetics Computer
Group [19]. Software designed to predict T cell epitopes based on
the Rothbard method [7] was written by Stephen Van Albert (The
Walter Reed Army Institute of Research, Washington, D.C.). Software
designed to predict T cell epitopes based on the Berzofsky method
was published as the AMPHI program [9]. It predicts amphipathic
amino acid segments by evaluating 7 or 11 residues as a block and
assigning the score to the middle residue of that block.
[0048] Statistics. All lymphocyte proliferations were conducted in
replicates of four, and standard deviations of the counts per
minute (cpm) are shown. Statistical significance (p value) for the
proliferative assay was determined using the Student's t test to
compare the cpm of quadruplicate wells cultured with the CFA/I
peptides to the cpm of wells cultured with a control peptide.
Results
[0049] Prediction of T cell epitopes within the CFA/I molecule. To
identify possible T cell epitopes within the CFA/I molecule,
amphipathic amino acid segments were predicted by evaluating 7 or
11 residues as a block using the AMPHI program [9]. Possible t cell
epitopes were also identified using criteria published by Rothbard
and Taylor [7]. The sequence numbers of the first amino acid of the
predicted segments are shown in Table 1.
[0050] Lymphocyte proliferation of monkey spleen cells to CFA/I
synthetic peptides. To determine which segments of the CFA/I
protein are able to stimulate proliferation of CFA/I immune primate
lymphocytes in vitro, three Rhesus monkeys were immunized with
CFA/I subunits, and their splenic lymphocytes were cultured with
synthetic overlapping decapeptides which represented the entire
CF/I sequence. Concentrations of peptides used as antigen were 6.0,
0.6, and 0.6 ug/ml. Proliferative responses to the decapeptides
were observed in each of the three monkeys (FIG. 1-3). The majority
of the responses occurred at the 0.6 and 0.06 ug/ml concentrations
of antigen and within distinct regions of the protein (peptides
beginning with residues 8-40, 70-80, and 27-137). A comparison of
the responses at the 6.0, 0.6 and 0.06 ug/ml concentrations
antigenic peptide for one monkey (2& 2) are shown (FIG. 4-6).
Taking into account all concentrations of antigen tested, spleen
cells from monkey 184D demonstrated a statistically significant
response to decapeptides beginning with CFA/I amino acid residues
3, 4, 8, 12, 15, 21, 26, 28, 33, 88, 102, 10, 133, 134, and 136
(FIG. 19). Monkey 34 had a significant response to decapeptides
beginning with residues 24, 31, 40, 48, 71, 72, 77, 78, 80, 87, and
102, 126 and 133 (FIG. 20); monkey 2Z2 responded to decapeptides
which began with residues 4, 9, 11, 12, 13, 14, 15, 16, 17, 20, 27,
35, 73, 79, 18, 127, 129, 132, and 133 (FIG. 19). Peptides
beginning with amino acid residues 3 through 2 were synthesized
with either a glutamic acid or an asparagine substituted for the
aspartic acid residue at position twelve to prevent truncated
peptides. The observed responses to peptides beginning with residue
8 (monkey 184d), and residues 9, 11, 12 (monkey 2Z2) occurred in
response to peptides that had the glutamic acid substitution.
However, the observed responses to peptides beginning with residue
3, 4, and 12 (monkey 184D), a well as residue 4 (monkey 2Z2)
occurred in response to peptides that had the asparagine
substitution. Monkey 34 did not respond to any of the peptides that
had the substitution at position twelve. All other responses shown
were to the natural amino acid sequence of the CFA/I protein.
Statistical significance was determined by comparing the cpm of
quadruplicate wells cultured with the CFA/I peptides to the cpm of
wells cultured with the CFA/I peptides to the cpm of wells cultured
with a control peptide.
[0051] Analysis of decapeptides that supported proliferation of
lymphocytes from CFA/I immune animals. Of the 39 different peptides
that supported proliferative responses, thirty contained a serine
residue, 19 contained a serine at either position 2, 3, or 4, and
nine had a serine specifically at position 3. Some of the most
robust responses were to the peptides that contained a serine
residue at the third position. The amino acid sequence of four such
peptides is shown in Table 3.
VII. DETAILED DESCRIPTION OF THE INVENTION
[0052] Applicants have discovered efficacious pharmaceutical
compositions wherein the relative amounts of antigen to the
polymeric matrix are within the ranges of 0.1 to 1% antigen (core
loading) and 99.9 to 99% polymer, respectively. It is preferred
that the relative ratio between the lactide and glycolide component
of the poly(DL-lactide-co-glycolide) (DL-PLG) is within the range
of 40:60 to 0:100. However, it is understood that effective core
loads for certain antigens will be influenced by its microscopic
form (i.e. bacteria, protozoa, viruses or fungi) and type of
infection being prevented. From a biological perspective, the
DL-PLG or glycolide monomer excipient are well suited for in vitro
drug (antigen) release because they elicit a minimal inflamatory
response, are biologically compatible, and degrades under
physiologic conditions to products that are nontoxic and readily
metabolized.
[0053] Surprisingly, applicants have discovered an extremely
effective method for the protection against bacterial or viral
infections in the tissue of a mammal (human or nonhuman animal)
caused by enteropathogenic organisms comprising administering
orally to said animal an immunogenic amount of a pharmaceutical
composition consisting essentially of an antigen encapsulated
within a biodegradable polymeric matrix. When the polymeric matrix
is DL-PLG, the most preferred relative ratio between the lactide
and glycolide component is within the range of 48:52 to 58:42. The
bacterial infection can be caused by bacteria (including any
derivative thereof) which include Salmonella typhi, Shigella
sonnei, Shigella flexneri, Shigella dysenteriae, Shigella boydii,
Escheria coli, Vibro cholera, yersinia, staphylococcus, clostridium
and campylobacter. Representative viruses contemplated within the
scope of this invention, susceptible to treatment with the
above-described pharmaceutical compositions, are quite extensive.
For purposes of illustration, a partial listing of these viruses
(including any derivative thereof) include hepatitis A,
rotaviruses, polio virus human immunodeficiency viruses (HIV),
Herpes Simplex virus type 1 (cold sores), Herpes Simplex virus type
2 (Herpesvirus genitalis), Varicella-zoster virus (chicken pox,
shingles), Epstein-Barr virus (infectious mononucleosis; glandular
fever; and Burkittis lymphoma), and cytomegalo viruses.
[0054] A further representation description of the instant
invention is as follows: [0055] A. (1) To homogeneously disperse
antigens of enteropathic organisms within the polymeric matrix of
biocompatible and biodegradable microspheres, 1 to 12 microns in
diameter, utilizing equal molar parts of polymerized lactide and
glycolide (50:50 DL-PLG, i.e. 48:52 to 58:42 DL-PLG) such that the
core load is within the range of about 0.1 to 1% by weight. The
microspheres containing the dispered antigen can then be used to
immunize the intestine to produce a humoral immune response
composed of secretory antibody, serum antibody and a cellular
immune response consisting of specific T-cells and B-cells. The
immune response is directed against the dispered antigen and will
give protective immunity against the pathogenic organism from which
the antigen was derived. [0056] (2) AF/R1 pilus protein is an
adherence factor that allows E. coli RDEC-1 to attach to rabbit
intestinal brush borders thus promoting colonization resulting in
diarrhea. AF/R1 pilus protein was homogeneously dispered within a
polymeric matrix of biocompatible and biodegradable microspheres,
1-12 microns in diameter (FIG. 1 and photograph 1) using equal
molar parts of polymerized lactide and glycolide (50:50 DL-PLG)
such that the core load was 0.62% by weight. [0057] (3) The
microspheres were found to contain immunogenic AF/R1 by immunizing
both rabbit spleen (FIG. 2) and Peyer's patch (FIG. 3) B-cells in
vitro. The resultant cell supernatants contained specific IgM
antibody which recognized the AF/R1. The antibody response was
comparable to immunizing with AF/R1 alone. [0058] (4) Microspheres
containing 50 micrograms of AF/R1 were used to intraintestinally
(intraduodenally) immunize rabbits on two separate occasions 1 week
apart. One week later, compared to rabbits receiving AF/R1 alone,
the intestinal lymphoid tissue, Peyer's patches, demonstrated an
enhanced cellular immune response to AF/R1 and to three AF/R1
linear peptide fragments 40-55, 79-94 and 108-123 by both
lymphocyte transformation (T-cells) (FIGS. 4 and 5) and antibody
producing B-cells (FIGS. 6 and 7). Similarly enhanced B-cell
responses were also detected in the spleen (FIGS. 8 and 9). An
enhanced T-cell response was also detected in the mesenteric lymph
node and the spleen to one AF/R1 peptide fragment, 40-55 (FIGS. 10
and 11). The cellular immune response at two weeks was too early
for either a serum or secretory antibody response (See Results in
Table 1); but indicates that a secretory antibody response will
develop such that the rabbits so immunized could be protected upon
challenge with the E. coli RDEC-1.
[0059] B. Microspheres do not have to be made up just prior to use
as with liposomes. Also liposomes have not been effective in
rabbits for intestinal immunization of lipopolysaccharide
antigens.
[0060] C. (1) Only a small amount of antigen is required (ugs) when
dispersed within microspheres compared to larger amounts (mgms)
when antigen is used alone for intestinal immunization. [0061] (2)
Antigen dispersed within microspheres can be used orally for
intestinal immunization whereas antigen alone used orally even with
gastric acid neutralization requires a large amount of antigen and
may not be effective for intestinal immunization. [0062] (3)
Synthetic peptides with and without attached synthetic adjuvants
representing peptide fragments of protein antigens can also be
dispersed within microspheres for oral-intestinal immunization.
Free peptides would be destroyed by digestive processes at the
level of the stomach and intestine. Any surviving peptide would
probably not be taken up by the intestine and therefore be
ineffective for intestinal immunization. [0063] (4) Microspheres
containing antigen maybe placed into gelatin-like capsules for oral
administration and intestinal release for improved intestinal
immunization. [0064] (5) Microspheres promote antigen uptake from
the intestine and the development of cellular immune (T-cell and
B-Cell) responses to antigen components such as linear peptide
fragments of protein antigens. [0065] (6) The development of
intestinal T-cell responses to antigens dispersed within
microspheres indicate that T-cell immunological memory will be
established leading to long-lived intestinal immunity. This
long-lived intestinal immunity (T-cell) is very difficult to
establish by previous means of intestinal immunization. Failure to
establish long-lived intestinal immunity is a fundamental
difficulty for intestinal immunizaiton with non-viable antigens.
Without intestinal long-lived immunity only a short lived secretory
antibody response is established lasting a few weeks after which no
significant immunological protection may remain.
[0066] D. (1) Oral intestinal immunization of rabbits against E.
coli RDEC-1 infection using either whole killed organisms, pilus
protein preparations or lipopolysaccharide preparations. [0067] (2)
Microspheres containing adherence pilus protein AF/R1 or its
antigen peptides for oral intestinal immunization of rabbits
against RDEC-1 infection. [0068] (3) Oral-intestinal immunization
of humans against enterotoxigenic E. coli infection using either
whole killed organisms, pilus protein preparations or
lipopolysaccharide preparations. [0069] (4) Microspheres containing
adherence pilus proteins CFA/I, II, III and IV or their antigen
peptides for oral intestinal immunization of humans against human
enterotoxigenic E. coli infections. [0070] (5) Oral-intestinal
immunization of humans against other enteric pathogens as
salmonella, shigella, camphlobacter, hepatitis-A virus, rota virus
and polio virus. [0071] (6) Oral-intestinal immunization of animals
and humans for mucosal immunological protection at distal mucosal
sites as the bronchial tree in lungs, genito-urinary tract and
breast tissue.
[0072] E. (1) The biocompatible, biodegradable co-polymer has a
long history of being safe for use in humans since it is the same
one used in resorbable suture material. [0073] (2) By using the
microspheres, we are now able to immunize the intestine of animals
and man with antigens not normally immunogenic for the intestinal
mucosa because they are either destroyed in the intestine, unable
to be taken up by the intestinal mucosa or only weakly immunogenic
if taken up. [0074] (3) Establishing long-lived immunological
memory in the intestine is now possible because T-cells are
immunized using microspheres. [0075] (4) Antigens that can be
dispersed into microspheres for intestinal immunization include the
following: proteins, glycoporteins, synthetic peptides,
carbohydrates, synthetic polysaccharides, lipids, glycolipids,
lipopolysaccharides (LPS), synthetic lipopolysaccharides and with
and without attached adjuvants such as synthetic muramyl dipeptide
derivatives. [0076] (5) The subsequent immune response can be
directed to either systemic (spleen and serum antibody) or local
(intestine, Peyer's patch) by the size of the microspheres used for
the intestinal immunization. Microspheres 5-10 microns in diameter
remain within macrophage cells at the level of the Peyer's patch in
the intestine and lead to a local intestinal immune response.
Microspheres 1-5 microns in diameter leave the Peyer's patch
contained within macrophages and migrate to the mesenteric lymph
node and to the spleen resulting in a systemic (serum antibody)
immune response. [0077] (6) Local or systemic antibody mediated
adverse reactions because of preexisting antibody especially
cytophyllic or IgE antibody may be minimized or eliminated by using
microspheres because of their being phagocytized by macrophages and
the antigen is only available as being attached to the cell surface
and not free. Only the free antigen could become attached to
specific IgE antibody bound to the surface of mast cells resulting
in mast cell release of bioactive amines necessary for either local
or systemic anaphylaxis. [0078] (7) Immunization with microspheres
containing antigen leads to primarily IgA and IgG antibody
responses rather than an IgE antibody response, thus preventing
subsequent adverse IgE antibody reactions upon reexposure to the
antigen.
[0079] In addition to the above, the encapsulation of the following
synthetic peptides are contemplated and considered to be well
within the scope of this invention: [0080] (1) AF/R1 40-55; [0081]
(2) AF/R1 79-94; [0082] (3) AF/R1 108-123; [0083] (4) AF/R1 1-13;
[0084] (5) AF/R1 pepscan 16AA; [0085] (6) CFA/I 1-13; and [0086]
(7) CFA/I pepscan 16AA. [0087] (8) Synthetic Pepetides Containing
CFA/I Pilus Protein T-cell Epitopes (Starting Sequence # given)
[0088] 4(Asn-Ile-Thr-Val-Thr-Ala-Ser-Val-Asp-Pro), [0089]
8(Thr-Ala-Ser-Val-Asp-Pro-Val-Ile-Asp-Leu), [0090]
12(Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), [0091]
15(Ile-Asp-Leu-Leu-Gln-Ala-Asp-Gly-Asn-Ala), [0092]
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), [0093]
26(Pro-Ser-Ala-Val-Lys-Leu-Ala-Tyr-Ser-Pro), [0094]
72(Leu-Asn-Ser-Thr-Val-Gln-Met-Pro-Ile-Ser), [0095]
78(Met-Pro-Ile-Ser-Val-Ser-Trp-Gly-Gly-Gln), [0096]
87(Gln-Val-Leu-Ser-Thr-Thr-Ala-Lys-Glu-Phe), [0097]
126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr), and [0098]
133(Gly-Asn-Tyr-Ser-Gly-Val-Val-Ser-Leu-Val), and mixtures thereof.
[0099] (9) Synthetic Peptides Containing CFA/I Pilus Protein B-cell
(antibody) Eptiopes (Starting Sequence # given) [0100]
3(Lys-Ana-Ile-Thr-Val-Thr-Ala-Ser-Val), [0101]
11(Val-Asp-Pro-Val-Ile-Asp-Leu-Leu-Gln-Ala-Asp), [0102]
22(Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), [0103]
32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe-Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val),
[0104] 32(Ala-Tyr-Ser-Pro-Ala-Ser-Lys-Thr-Phe), [0105]
38(Lys-Thr-Phe-Glu-Ser-Tyr-Arg-Val), [0106]
66(Pro-Gln-Leu-Thr-Asp-Val-Leu-Asn-Ser), [0107]
93(Ala-Lys-Glu-Phe-Glu-Ala-Ala-Ala), [0108]
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr), [0109]
127(Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and [0110]
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
mixtures thereof. [0111] (10) Synthetic Peptides Containing CFA/I
pilus Protein T-cell and B-cell (antibody) Epitopes (Starting
Sequence # given) [0112]
3(Lys-Asn-Ile-Thr-Val-Thr-Ala-Ser-Bal-Asp-Pro), [0113]
8(Thr-Ala-Ser-Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp), [0114]
11(Bal-Asp-Pro-Bal-Ile-Asp-Leu-Leu-Gln-Ala-Asp), [0115]
20(Ala-Asp-Gly-Asn-Ala-Leu-Pro-Ser-Ala-Val), [0116]
124(Lys-Thr-Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
[0117] 126(Ala-Gly-Thr-Ala-Pro-Thr-Ala-Gly-Asn-Tyr-Ser), and
mixtures thereof.
[0118] We contemplate that the peptides can be used in vaccine
constructed for systemic administration.
VIII. EXAMPLES
[0119] The peptides in (8), (9), and (10) above can be made by
classical solution phase synthesis, solid phase synthesis or
recombinant DNA technology. These peptides can be incorporated in
an oral vaccine to prevent infection by CFA/I bearing
enteropathogenic E. coli.
[0120] The herein offered examples provide methods for
illustrating, without any implied limitation, the practice of this
invention in the prevention of diseases caused by enteropathogenic
organisms.
[0121] The profile of the representative experiments have been
chosen to illustrate the effectiveness of the immunogenic polymeric
matrix-antigen composites.
[0122] All temperatures not otherwise indicated are in degrees
Celcius (.degree. C.) and parts or percentages are given by
weight.
IX. MATERIALS AND METHODS
[0123] Animals. New Zealand White male rabbits were purchased from
Hazelton Research Products (Denver, Pa.), and were shown to be free
of current RDEC-1 infection by culture of rectal swabs. Animals
were 1-2 kg of body weight and lacked agglutinating anti-AF/R1
serum antibody at the time of the study.
[0124] Antigens. AF/R1 pili from E. coli RDEC-1 (015:H:K
non-typable) were purified by an ammonium sulfate precipitation
method. The final preparation migrated as a single band on
SDS-polyacrylamide gel electrophoresis and was shown to be greater
than 95% pure by scanning with laser densitometry when stained with
coomassie blue. Briefly, equal molar parts of DL-lactide and
glycolide were polymerized and then dissolved to incorporate AF/R1
into spherical particles. The microspheres contained 0.62% protein
by weight and ranged in size from 1 to 12 micrometers. Both the
microencapsulated and non-encapsulated AF/R1 were sterilized by
gamma irradiation (0.3 megarads) before use.
[0125] Synthetic peptides (16 amino acids each) were selected by
theoretical criteria from the amino acid sequence of AF/R1 as
deduced from the nucleotide sequence. Three sets of software were
used for the selections. Software designed to predict B cell
epitopes based on hydrophilicity, flexibility, and other criteria
was developed by the University of Wisconsin Genetics Computer
Group. Software designed to predict T cell epitopes was based on
the Rothbard method was written by Stephen Van Albert (The Walter
Reed Army Institute of Research, Washington, D.C.). Software
designed to predict T cell epitopes based on the Berzofsky method
is published as the AMPHI program. The selected peptides were
synthesized by using conventional Merrifield solid phase
technology. AF/R1 40-55
(Thr-Asn-Ala-Cly-Thr-Asp-Ile-Gly-Ala-Asn-Lys-Ser-Phe-Thr-Leu-Lys)
was chosen as a probable B cell epitope for two reasons: (a) due to
its high hydrophilic and flexibility indices, and (b) because it
was not predicted to be a T cell epitope by either the Rothbard or
Berzofsky method. AF/R1 79-94
(Val-Asn-Gly-Ile-Gly-Asn-Leu-Ser-Gly-Lys-Ala-Ile-Asp-Ala-His-Val)
was selected as a probable T cell eptiope because it contained
areas predicted as a T cell epitope by both methods and because of
its relatively low hydrophilic and flexibility indices. AF/R1
108-123
(Asp-Thr-Asn-Ala-Asp-Lys-Glu-Ile-Lys-Ala-Gly-Gin-Asn-Thr-Val-Asp)
was selected as both a T and B cell epitope. AF/R1 40/47/79-86 was
produced in continuous synthesis
(Thr-Asn-Ala-Cly-Thr-Asp-Ile-Gly-Val-Asn-GlyIle-Gly-Asn-Leu-Ser)
and represents a hybrid of the first eight amino acids from the
predicted B cell epitope and the T cell epitope. The purity of each
peptide was confirmed by C-8 reverse phase HPLC, and all peptides
were desalted over a Sephadex G-10 Column before use. Using a
standard ELISA method, all peptides were assayed for their ability
to specifically bind anti-AF/R1 IgG antibody in hyperimmune serum
from a rabbit which had received intramuscular injections of AF/R1
pili in Freund adjuvant. Only the peptide chosen as a probable B
cell epitope (AF/R1 40-55) was recognized by the hyperimmune
serum.
Example 1
[0126] Immunization. Rabbits were primed twice with 50 micrograms
of either microencapsulated or non-encapsulated AF/R1 by endoscopic
intraduodenal inoculation seven days apart by the following
technique. All animals were fasted overnight and sedated with an
intramuscular injection of xylazine (10 mg) and Ketamine HCl (50
mg). An Olympus BF type P10 endoscope was advanced under direct
visualization through the esophagus, stomach, and pylorus, and a 2
mm ERCP catheter was inserted through the biopsy channel and
threaded 2-3 cm into the small intestine. Inoculums of pili or pili
embedded in microspheres were injected through the catheter into
the duodenum and the endoscope was withdrawn. Animals were
monitored daily for signs of clilnical illness, weight gain, or
colonization by RDEC-1.
Example 2
[0127] Lymphocyte Proliferation. Seven days following the second
priming, the rabbits were again sedated with a mixture of xylazine
and katamine HCl, and blood was drawn for serum preparation by
cardiac puncture. Animals were then euthanized with an overdose of
pentothal and tissues including Peyer's patches from the small
bowel, MLN, and spleen were removed. Single cell suspension were
prepared and washed in Dulbeco's modified Eagle medium (Gibco
Laboratories, Grand Island, N.Y.) which had been supplemented with
penicillin (100 units/ml), streptomycin (100 micrograms/ml),
L-glutamine (2 mM), and HEPES Buffer (10 mM) all obtained from
Gibco Laboratories, as well as MEM non-essential amino acid
solution (0.1 mM), MEM [50.times.] amino acids (2%), sodium
bicarbonate (0.06%), and 5.times.10.sup.-5 micrograms 2-ME all
obtained from Sigma Chemical Company (St. Louis, Mo.) [cDMEM].
Erythrocytes in the spleen cell suspension were lysed using
standard procedures in an ammonium chloride lysing buffer. Cell
suspension were adjusted to 5.times.10.sup.6 cells per ml in cDMEM,
and autologous serum was added to yield a final concentration of
0.5%. Cells (0.1 ml) were placed in 96-well flat bottom culture
plates (Costar, Cambridge, Mass.) along with 0.1 ml of various
dilutions of antigen and were incubated at 37.degree. C. in 5%
CO.sub.2. In other experiments, cultures were conducted in a
24-well plates. In these experiments, 5.times.10.sup.6 cells were
cultured with or without antigen in a 2 ml volume. After 4 days,
100 microliters aliquots of cells were transferred to 96-well
plates for pulsing and harvesting. Previous experiments have
demonstrated that optimal concentrations of antigen range from 150
ng/ml to 15 micrograms/ml in the 96-well plate assay and 1.5 ng/ml
to 150 ng/ml in the 24-well plate assay. These were the
concentrations employed in the current study. All cultures were
pulsed with 1 Ci [.sup.3H]thymidine (25 Ci/mmol, Amersham,
Arlington Heights, Ill.) on day 4 of culture and were harvested for
scintillation counting 6 hours later.
[0128] Statistics. All cultures were conducted in replicates of
four, and standard deviations of the counts per minute (cpm)
generally range from 5-15% of the average cpm. In experiments where
comparison of individual animals and groups of animals is
desirable, data is shown as a stimulation index (SI) to facilitate
the comparison. SI were calculated by dividing the mean of cultures
with antigen by the mean of cultures without antigen (media
control). Statistical significance (p value) was determined by
comparing the maximum response for each antigen to the media
control using the Student's t test.
IX. RESULTS
[0129] Lymphocyte proliferation in response to protein and peptide
antigens of AF/R1. To determine if lymphoid tissues from AF/R1
immune animals respond in vitro to the antigens of AF/R1, the
immunity in a rabbit with preexisting high levels of anti-AF/R1
serum IgG was boosted twice by injection of 50 micrograms of
purified AF/R1 pili i.p. seven days apart. A week after the final
boost, in vitro lymphocyte proliferation of spleen and MLN cells
demonstrated a remarkable response to AF/R1 pili (FIG. 13). In
response to the synthetic peptides, there was a small, but
significant proliferation of the spleen cells to all the AF/R1
peptides tested as compared to cell cultures without antigen (FIG.
14). Cells from the spleen and Peyer's patches of non-immune
animals failed to respond to either AF/R1 or the synthetic
peptides.
[0130] Microencapsulation of AF/R1 potentiates the mucosal cellular
immune response. To evaluate the effect that microencapsulation of
AF/R1 may have on the cellular mucosal immune response to that
antigen, naive rabbits were primed twice with 50 micrograms of
either microencapsulated or non-encapsulated AF/R1 by endoscopic
intraduodenal inoculation seven days apart. All rabbits were
monitored daily and showed no evidence of clinical illness or
colonization by RDEC-1. One week following the last priming, the
rabbits were sacrificed and lymphoid tissues were cultured in the
presence of AF/R1 pili or peptide antigens. In rabbits which had
received non-encapsulated AF/R1, Peyer's Patch cells demonstrated a
low level but significant proliferation in vitro in response to
AF/R1 pili (FIG. 5), but not to any of the AF/R1 synthetic peptides
(FIG. 6a-6d). However, in rabbits which had received
microencapsulated AF/R1, Peyer's Patch cells demonstrated a
markedly enhanced response not only to AF/R1 (FIG. 5), but now
responded to the AF/R1 synthetic peptides 40-55 and 79-94 (FIGS. 6a
and 6b). In addition, one of two rabbits primed with
microencapsulated AF/R1 (rabbit 135) responded to AF/R1 108-123,
but not AF/R1 40-47/79-86 (FIGS. 6c and 6d). In contrast, the other
rabbit in the group (rabbit 134) responded to AF/R1 40-47/79-86,
but not to AF/R1 108-123 (FIGS. 6d and 6c).
[0131] Response of MLN cells to the antigens of AF/R1. Studies have
shown that cells undergoing blastogenesis in the MLN also tend to
home into mucosal areas, but experiments requiring in vitro
lymphocyte proliferation of rabbit MLN cells are difficult to
conduct and to interpret due to non-specific high background cpm in
the media controls. Our studies have shown that this problem can be
avoided by conducting the proliferative studies in 24-well plates,
and then moving aliquots of cells into 96-well plates for pulsing
with [.sup.3H]thymidine as described in materials and methods. This
method of culture was employed for the remainder of the studies.
The MLN cells of all rabbits demonstrated a significant
proliferation in vitro in response to AF/R1 pili regardless of
whether they had been immunized with microencapsulated or
non-encapsulated AF/R1 (FIG. 15). However, only the rabbits which
had received microencapsulated AF/R1 were able to respond to the
AF/R1 synthetic peptide 40-55 (FIG. 11). The MLN cells of rabbit
134 also responded to AF/R1 79-94 (p<0.0001), AF/R1 108-123
(p<0.0001), and AF/R1 40-47/79-86 (p=0.0004); however, none of
the other rabbits demonstrated a MLN response to those three
peptides (data not shown).
[0132] Response of spleen cells to the antigens of AF/R1.
Proliferative responses of spleen cells to AF/R1 were very weak in
all animals tested (data not shown). However, in results which
paralleled the responses in MLN cells, there was a significant
response to AF/R1 40-55 in rabbits which had been primed with
microencapsulated AF/R1 (FIG. 12). There was no response to the
other AF/R1 synthetic peptides by spleen cells in either group of
animals. The weak response of spleen cells to AF/R1 provides
further evidence that these animals were naive to AF/R1 before the
study began, and indicates that the observed responses were not due
to non-specific stimulative factors such as lipopolysaccharide.
XI. SUMMARY
[0133] We have shown that there is an enhanced in vitro
proliferative response to both protein and its peptide antigens by
rabbit Peyer's patch cells following intraduodenal inoculation of
antigen which had been homogeneously dispersed into the polymeric
matrix of biodegradable, biocompatible microspheres. The
immunopotentiating effect of encapsulating purified AF/R1 pili as a
mucosal delivery system may be explained by one or more of the
following mechanisms: (a) Microencapsulation may help to protect
the antigen from degradation by digestive enzymes in the intestinal
lumen. (b) Microencapsulation has been found to effectively enhance
the delivery of a high concentration of antigen specifically into
the Peyer's patch. (c) Once inside the Peyer's patch,
microencapsulation appears to facilitate the rapid phagocytosis of
the antigen by macrophages, and the microspheres which are 5-10
micrometers become localized within the Peyer's patch. (d)
Microencapsulation of the antigen may improve the efficiency of
antigen presentation by decreasing the amount of enzymatic
degradation that takes place inside the macrophage before the
epitopes are protected by combining with Class II major
histocompatibility complex (MHC) molecules. (e) The slow,
controlled-release of antigen may produce a depot effect that
mimics the retention of antigen by the follicular dendritic cell.
(f) If the antigen of interest is soluble, microencapsulation
changes the antigen into a particulate form which appears to assist
in producing an IgA B cell response by shifting the cellular immune
response towards the T.sub.H and thereby not encouraging a response
by the T.sub.s. There is evidence that the GALT may be able to
discriminate between microbial and non-microbial (food) antigens in
part by the form of the antigen when it is first encountered, and
thus bacterial antigens do not necessarily have special antigenic
characteristics that make them different from food antigens, but
they are antigenic because of the bacterial context in which they
are presented. The particulate nature of microspheres may serve to
mimic that context. It may be important to note that we also
observed a significant response to AF/R1 in animals inoculated with
non-encapsulated pili; thus, some of this antigen which was still
in its native form was able to enter the Peyer's patch. This may be
explained by the fact that AF/R1 is known to mediate the attachment
of RDEC-1 to the Peyer's patch M-cell. If the antigen employed in
this type of study was not able to attach to micrometer M-cells,
one would expect to see an even greater difference in the responses
of animals which had received microencapsulated versus
non-encapsulated antigen.
[0134] The microspheres used in these experiments included a size
range from 1 to 12 micrometers. The 1 to 5 micrometer particles
have been shown to disseminate to the MLN and spleen within
migrating macrophages; thus, the observed proliferative responses
by cells from the MLN and spleen may reflect priming of MLN or
splenic lymphocytes by antigen-presenting/accessory cells which
have phagocytosed 1 to 5 micrometer antigen-laden microspheres in
the Peyer's patch and then disseminated onto the MLN.
Alternatively, these responses may be a result of the normal
migration of antigen stimulated lymphocytes that occurs from the
Peyer's patch to the MLN and on into the general circulation before
homing to mucosal sites. Proliferative responses by MLN cells are
of interest because it has been shown that cells undergoing
blastogenesis in the MLN tend to migrate onto mucosal areas.
However, studies involving in vitro lymphocyte proliferation of
rabbit MLN cells can be very difficult to conduct and to interpret
due to non-specific high background cpm in the media controls. By
simultaneously conducting experiments using different protocols, we
have found that this problem can be prevented by avoiding the use
of fetal calf serum in the culture and by initially plating the
cells in 24-well plates. Using this method, the blasting
lymphocytes are easily transferred to a 96-well plate where they
receive the [.sup.3H]thymidine, while fibroblasts and other
adherent cells remain behind and thus do not inflate the background
cpm.
[0135] The proliferative response to the peptide antigens was of
particular interest in these studies. The rabbits that received
non-encapsulated AF/R1 failed to respond to any of the peptides
tested either at the level of the Peyer's patch, the MLN, or the
spleen. In contrast, Peyer's patch cells from the animals that
received microencapsulated AF/R1 responded to all the peptides
tested with two exceptions: Rabbit 134 did not respond to AF/R1
108-123, and rabbit 135 did not respond to AF/R1 40-47/79-86. The
reason for these non-responses is not clear, but it probably is not
due to MHC restrictions as evidenced by the fact that rabbit 134
was able to respond to AF/R1 108-123 at the level of the MLN. The
non-responses may be due to varing kinetics of sensitized T cell
migration in different rabbits, or they may reflect differences in
the efficiency of antigen presentation by cells from different
lymphoid tissues of these animals. Of all the synthetic peptides
tested, only AF/R1 40-55, (the one selected as a probable B cell
epitope), was recognized by serum from an AF/R1 hyperimmune rabbit.
In addition, this peptide was the only one that was uniformly
recognized by Peyer's patch, MLN, and spleen cells from both
rabbit. In addition, this peptide was the only one that was
uniformly recognized by Peyer's patch, MLN, and spleen cells from
both rabbits that were immunized with microencapsulated AF/R1. The
recognition by anti-AF/R1 serum antibodies indicates that the amino
acid sequence of this peptide includes an immunodominant B cell
epitope. Thus AF/R1 40-55 may readily bind to antigen-specific B
cells thereby leading to an efficient B cell presentation of this
antigen to sensitized T cells. Even though AF/R1 40-55 was not
selected as a probable T cell epitope by either the Rothbard or
Berzofsky methods, the current study clearly indicates that this
peptide can also stimulate a proliferative immune response.
Although further studies are required to definitively show that the
proliferating cells are indeed T cells, the responses observed in
this study are most likely due to the blast transformation of cells
from the lineage. Therefore, AF/R1 40-55 appears to contain a T
cell epitope in addition to the immunodominant B cell epitope, and
this area of the AF/R1 protein may thereby play an important role
in the overall immune response and subsequent protection against
RDEC-1.
[0136] The proliferative responses of spleen cells was low in all
animals tested; however, we feel that this may be simply a matter
of the kinetics of cellular migration. The rabbits in this study
were sacrificed only two weeks after their first exposure to
antigen. This relatively short time period may not have provided
sufficient time for cells that were produced by Peyer's patch and
MLN blasts to have migrated as far as the spleen in sufficient
numbers.
[0137] An ideal mucosal vaccine preparation would not only assist
in the uptake and presentation of the immunogen of interst, but it
would also be effective without requiring carrier molecules or
adjuvants which may complicate vaccine production or delay
regulatory approval. The incorporation of antigen into microspheres
appears to provide an ideal mucosal delivery system for oral
vaccine immunogens because the observed immunopotentiating effect
is achieved without the need for carriers of adjuvants. This
ability may prove to be of great value, particularly to enhance the
delivery of oral synthetic peptide vaccines to the GALT.
TABLE-US-00002 TABLE 1 Linear B-Cell Epitopes of CFA/I in Monkeys
Sequence Individuals Position Responding Consensus Site 1. 11-21 3
VDPVIDLLQ 2. 93-101 2 AKEFEAAA 3. 124-136 2 GPAPT 4. 66-74 2
PQLTDVLN 5. 22-29 2 GNALPSAV 6. 32-40 1 KTF* 7. 38-45 1 8. 3-11 1
*Overlap between epitope 6 and 7
TABLE-US-00003 TABLE 2 Prediction of T cell epitopes within the
CFA/I molecule.sup.a Predicted Amphipathic Segments 7 aa blocks 11
aa blocks Rothbard Criteria 22-25 8-11 16 34-38 32-44 30 40-46
51-71 38 50-53 86-92 44 56-62 102-108 57 64-71 130-131 61 104-108
135-137 70 131-137 116 124 127 137 .sup.aThe sequence numbers of
the first amino acid of the predicted T cell epitopes are shown.
Software designed to predict T cell epitopes based on the Berzofsky
method was published as the AMPHI program. It predicts amphipathic
amino acid segments by evaluating 7 or 11 residues as a block and
assigning a score to the middle residue of that block. Software
designed to predict T cell epitopes based on the Rothbard method
was written by Stephen Van Albert (The Walter Reed Army Instituteof
Research, Washington, D.C.).
TABLE-US-00004 TABLE 3 Amino acid sequence of immunodominant T cell
epitopes.sup.a Residue Numbers Amino Acids 8-17 Thr Ala Ser Val Asp
Pro Val Ile Asp Leu 40-49 Phe Glu Ser Tyr Arg Val Met Thr Gln Val
72-81 Leu Asn Ser Thr Val Gln Met Pro Ile Ser 134-143 Asn Tyr Ser
Gly Val Val Ser Leu Val Met .sup.aOf the 19 decepeptides that
supported a significant proliferative response and contained a
serine at either position 2, 3, or 4, nine has a serine
specifically at position 3. Some of the most robust responses were
to the peptides that contained a serine residue at the third
position. The amino acid sequence of four such decapeptides which
are believed to be immunodominant T cell epitopes is shown.
LITERATURE CITED
[0138] 1. Mooi, F. R., and F. K. de Graaf. 1985. Molecular biology
of fimbriae of enterotoxigenic Escherichia coli. Curr. Top.
Microbial. Immunol. 118:119-138. [0139] 2. Evans, D. G., D. J. Jr.
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characterization of the CFA/I antigen of enterotoxigenic
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and characterization of colonization factor enterotoxigenic
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coli-surface-associated antigens (Cs)1, Cs2, Cs4, and Cs17, FEMS
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Boedeker. 1983. Adherence of an enterotoxigenic Escherichia coli
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sequence pattern common to T cell epitopes. EMBO. J. 7:93-100.
[0145] 8. DeLisi, C., and J. A. Berzofsky. 1985. T-cell antigenic
sites tend to be amphipathic structures. Proc. Natl. Acad. Sci, USA
82:7048-7052. [0146] 9. Margalit, H., J. L. Spounge, J. L.
Cornette, K. B. Cease, C. DeLisi, and J. A. Berzofsky. 1987.
Prediction of Immunodominant helper T cell antigenic sites from the
primary sequence. J. Immunol. 138:2213-2229. [0147] 10. Berzofsky,
J. A. 1988. Structural basis of antigen recognition by T
lymphocytes. J. Clin. Invest. 82:1811-1817. [0148] 11. Stille, C.
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Bracci, A. Santucci, P. Soldani, A. Spreafico, and P. Neri. 1990.
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Troalen, F., A. Razafindratsita, A. Puisieux, T. Voeltzel, C.
Bohuon, D. Bellet, and J. M. Bidart. 1990. Structural probing of
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Ratnam, S. M. Huang, P. L. Smith, and J. H. Freisheim. 1990.
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Meloen, A. Noordzij, and J. Van Embden. 1989. Efficient mapping and
characterization of a T cell epitope by the simultaneous synthesis
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H. M., R. H. Meloen, and S. J. Barteling. 1984. Use of peptide
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Sequence CWU 1
1
39110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Asn Ile Thr Val Thr Ala Ser Val Asp Pro 1 5
10210PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Thr Ala Ser Val Asp Pro Val Ile Asp Leu 1 5
10310PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Asp Pro Val Ile Asp Leu Leu Gln Ala Asp 1 5
10410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Ile Asp Leu Leu Gln Ala Asp Gly Asn Ala 1 5
10510PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Ala Asp Gly Asn Ala Leu Pro Ser Ala Val 1 5
10610PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Pro Ser Ala Val Lys Leu Ala Tyr Ser Pro 1 5
10710PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Leu Asn Ser Thr Val Gln Met Pro Ile Ser 1 5
10810PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Met Pro Ile Ser Val Ser Trp Gly Gly Gln 1 5
10910PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Gln Val Leu Ser Thr Thr Ala Lys Glu Phe 1 5
101010PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Ala Gly Thr Ala Pro Thr Ala Gly Asn Tyr 1 5
101110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Gly Asn Tyr Ser Gly Val Val Ser Leu Val 1 5
10129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Lys Ala Ile Thr Val Thr Ala Ser Val 1
51311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Val Asp Pro Val Ile Asp Leu Leu Gln Ala Asp 1 5
10148PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Gly Asn Ala Leu Pro Ser Ala Val 1
51517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Ala Tyr Ser Pro Ala Ser Lys Thr Phe Lys Thr Phe
Glu Ser Tyr Arg 1 5 10 15Val169PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16Ala Tyr Ser Pro Ala Ser Lys
Thr Phe 1 5178PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 17Lys Thr Phe Glu Ser Tyr Arg Val 1
5189PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Pro Gln Leu Thr Asp Val Leu Asn Ser 1
5198PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Ala Lys Glu Phe Glu Ala Ala Ala 1
5208PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Lys Thr Ala Gly Thr Ala Pro Thr 1
52110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gly Thr Ala Pro Thr Ala Gly Asn Tyr Ser 1 5
102213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 22Lys Thr Ala Gly Thr Ala Pro Thr Ala Gly Asn Tyr
Ser 1 5 102311PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 23Lys Asn Ile Thr Val Thr Ala Ser Val
Asp Pro 1 5 102414PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 24Thr Ala Ser Val Asp Pro Val Ile Asp
Leu Leu Gln Ala Asp 1 5 102514PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 25Ala Gly Thr Ala Pro Thr Ala
Pro Thr Ala Gly Asn Tyr Ser 1 5 102616PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Asp
Thr Asn Ala Asp Lys Glu Ile Lys Ala Gly Gln Asn Thr Val Asp 1 5 10
152716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 27Thr Asn Ala Gly Thr Asp Ile Gly Val Asn Gly Ile
Gly Asn Leu Ser 1 5 10 152816PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 28Thr Asn Ala Gly Thr Asp Ile
Gly Ala Asn Lys Ser Phe Thr Leu Lys 1 5 10 152916PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Val
Asn Gly Ile Gly Asn Leu Ser Gly Lys Ala Ile Asp Ala His Val 1 5 10
15309PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 30Val Asp Pro Val Ile Asp Leu Leu Gln 1
5315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 31Gly Pro Ala Pro Thr 1 5328PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Pro
Gln Leu Thr Asp Val Leu Asn 1 53310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Phe
Glu Ser Tyr Arg Val Met Thr Gln Val 1 5 103410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 34Asn
Tyr Ser Gly Val Val Ser Leu Val Met 1 5 103518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 35Leu
Ala Asp Thr Pro Gln Leu Thr Asp Val Leu Asn Ser Thr Val Gln 1 5 10
15Met Pro3619PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 36Ser Tyr Arg Val Met Thr Gln Val His
Thr Asn Asp Ala Thr Lys Lys 1 5 10 15Val Ile Val37147PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Val
Glu Lys Asn Ile Thr Val Thr Ala Ser Val Asp Pro Val Ile Asp 1 5 10
15Leu Leu Gln Ala Asp Gly Asn Ala Leu Pro Ser Ala Val Lys Leu Ala
20 25 30Tyr Ser Pro Ala Ser Lys Thr Phe Glu Ser Tyr Arg Val Met Thr
Gln 35 40 45Val His Thr Asn Asp Ala Thr Lys Lys Val Ile Val Lys Leu
Ala Asp 50 55 60Thr Pro Gln Leu Thr Asp Val Leu Asn Ser Thr Val Gln
Met Pro Ile 65 70 75 80Ser Val Ser Trp Gly Gly Gln Val Leu Ser Thr
Thr Ala Lys Glu Phe 85 90 95Glu Ala Ala Ala Leu Gly Tyr Ser Ala Ser
Gly Val Asn Gly Val Ser 100 105 110Ser Ser Gln Glu Leu Val Ile Ser
Ala Ala Pro Lys Thr Ala Gly Thr 115 120 125Ala Pro Thr Ala Gly Asn
Tyr Ser Gly Val Val Ser Leu Val Met Thr 130 135 140Leu Gly
Ser14538147PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 38Val Glu Lys Asn Ile Thr Val Thr Ala Ser Val Asp
Pro Val Ile Asp 1 5 10 15Leu Leu Gln Ala Asp Gly Asn Ala Leu Pro
Ser Ala Val Lys Leu Ala 20 25 30Tyr Ser Pro Ala Ser Lys Thr Phe Glu
Ser Tyr Arg Val Met Thr Gln 35 40 45Val His Thr Asn Asp Ala Thr Lys
Lys Val Ile Val Lys Leu Ala Asp 50 55 60Thr Pro Gln Leu Thr Asp Val
Leu Asn Ser Thr Val Gln Met Pro Ile 65 70 75 80Ser Val Ser Trp Gly
Gly Gln Val Leu Ser Thr Thr Ala Lys Glu Phe 85 90 95Glu Ala Ala Ala
Leu Gly Tyr Ser Ala Ser Gly Val Asn Gly Val Ser 100 105 110Ser Ser
Gln Glu Leu Val Ile Ser Ala Ala Pro Lys Thr Ala Gly Thr 115 120
125Ala Pro Thr Ala Gly Asn Tyr Ser Gly Val Val Ser Leu Val Met Thr
130 135 140Leu Gly Ser14539147PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Val Glu Lys Asn Ile Thr Val
Thr Ala Ser Val Asp Pro Val Ile Asp 1 5 10 15Leu Leu Gln Ala Asp
Gly Asn Ala Leu Pro Ser Ala Val Lys Leu Ala 20 25 30Tyr Ser Pro Ala
Ser Lys Thr Phe Glu Ser Tyr Arg Val Met Thr Gln 35 40 45Val His Thr
Asn Asp Ala Thr Lys Lys Val Ile Val Lys Leu Ala Asp 50 55 60Thr Pro
Gln Leu Thr Asp Val Leu Asn Ser Thr Val Gln Met Pro Ile 65 70 75
80Ser Val Ser Trp Gly Gly Gln Val Leu Ser Thr Thr Ala Lys Glu Phe
85 90 95Glu Ala Ala Ala Leu Gly Tyr Ser Ala Ser Gly Val Asn Gly Val
Ser 100 105 110Ser Ser Gln Glu Leu Val Ile Ser Ala Ala Pro Lys Thr
Ala Gly Thr 115 120 125Ala Pro Thr Ala Gly Asn Tyr Ser Gly Val Val
Ser Leu Val Met Thr 130 135 140Leu Gly Ser145
* * * * *