# Patent

## Title
Epitope and its use

## Abstract

An isolated protein containing a common epitope recognized by umbilical blood antibodies specific against enterobacteria, occurring in an extract of bacterial outer membrane proteins, and fragments thereof containing said common epitope, which can be used in medicine and pharmaceutics, particularly in the production of vaccines and diagnostic tests as well as affinity materials.

## Background
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 12, 2015, is named PZ-1722-RW-US_SL.txt and is 18,372 bytes in size.
The subject of the present invention is an epitope recognized by umbilical blood antibodies specific against enterobacteria, which may be used in the production of vaccines against Enterobacteriaceae, particularly opportunistic gastrointestinal bacterial pathogens, particularly bacteria of the genus Shigella. 
Shigellosis, salmonellosis and other diseases caused by members of the family Enterobacteriaceaeare still a considerable medical problem, particularly in developing nations.
The genus Shigella belongs to Gram-negative opportunistic human pathogens and is responsible for colonic infections. Symptoms of infection include lower abdominal pain, fever and bloody diarrhoea which may cause a life threatening dehydration. Bacterial diarrhoea is in third place world wide in terms of causes of juvenile mortality below the age of five years. Infant and child mortality due to bacterial infections are generally caused by low standards of sanitation pervasive in developing nations. Nevertheless, infections by pathogenic intestinal bacteria, including Shigella, also occur in developed nations. Here, a bigger problem consists of strains resistant to antibiotics. For this reason, healthcare authorities both in developing and developed nations are pushing for prevention, including vaccination. Thus far, no one has managed to produce a vaccine against opportunistic bacterial pathogens of the gastrointestinal tract. For approval, such a vaccine must fulfil a series of criteria, such as activity in intestinal mucosa, long-term immune protection and a lack of adverse effects. It is also desirable that the vaccine be easy to administer and relatively inexpensive, because children in nations with poor economies will constitute the major recipient.
Shigella strains against which it has been attempted to produce vaccines include S. flexneri 2a, 3a, S. dysenteriae 1, and S. sonnei. 
Application P.380105 discloses a protein of the external cell wall of Shigella flexneri 3a with a molecular mass of 38 kDa which is immunoreactive with human serum. The application suggests, amongst others, its possible use as a carrier for use in conjugate vaccines.
The goal of the present invention is to obtain novel components which could be used to produce a vaccine against Enterobacteriaceae, particularly against opportunistic pathogens of the gastrointestinal tract, particularly against bacteria of the genus Shigella. 
The subject of the present invention is an epitope with the following amino-acid sequence: A1-A2-A3-A4-A5-A6(SEQ ID NO: 1), where:
A1denotes R,
A2denotes Y,
A3denotes D, R, E, N or Q,
A4denotes E, D, N or Q,
A5denotes R,
A6denotes Y, G or F.
Preferably, an epitope according to the present invention has been selected from a group encompassing peptides with the following amino-acid sequence: RYDERY (SEQ ID NO: 2), RYDDRY (SEQ ID NO: 3), RYEERY (SEQ ID NO: 4), RYQERY (SEQ ID NO: 5) or RYDQRY (SEQ ID NO: 6).
The next subject of the present invention is the use of the epitope defined above or a protein containing this epitope in the production of a vaccine against Enterobacteriaceae, particularly those against bacteria of the genus Shigella. 
The next subject of the present invention is the use of an epitope defined above or a protein containing this epitope in the production of a diagnostic test for detecting antibodies against Enterobacteriaceae, particularly bacteria of the genus Shigella. 
The next subject of the present invention is the use of the epitope defined above or a protein containing such an epitope in the production of blood-derived, immunoglobulin therapeutic preparations specific against Enterobacteriaceae, particularly bacteria of the genus Shigella. 
Unexpectedly, using to the peptides according to the present invention, it is possible to replace a classic vaccine based on thermally attenuated bacteria with a vaccine based on synthetic fragments representing the main surface antigen of bacterial cells isolated from the ompC protein. In contrast to the classic vaccine, the synthetic vaccine is safe, inexpensive and may be produced on a mass scale.
The disclosed peptides can be used to construct a vaccine against Enterobacteriaceae, particularly in cases of humoral immunity insufficiencies. They may also be used to prepare an affinity gel for the isolation of antibodies from donor sera as a therapeutic preparation, and for the diagnosis of specific humoral immunity insufficiencies. In contrast to the native ompC protein, which contains a series of superfluous, neutral or even negative epitopes, an epitope according to the present invention contains only a unique protective property.



To facilitate a better understanding of the nature of the present invention, its description has been illustrated with the following figures:
FIG. 1 represents a visualisation of conformational epitopes from the ompC protein of Shigella flexneri 3a. Epitopes 1-3 (SEQ ID NOS 47-49, respectively, in order of appearance) are loops directed to the interior of the cell, and for this reason are of lesser significance to host-bacteria interactions. Epitopes 4-8 (SEQ ID NOS 50-53 and 11, respectively, in order of appearance) correspond to extracellular loops II-VI respectively (according to the nomenclature of ompC of E. coli).
FIG. 2 represents the observed reactivity of antibodies present in sera 1H-9H mixed with peptides Nos. 1-6 synthesized on HPM-pins.
FIG. 3 represents the reactivity of antibodies present in the individual sera 1H-6H and 13H-14H with peptides Nos. 1-6 synthesized on HPM-pins.
FIG. 4 represents amino-acids that determine the immunological activity of the RYDERY epitope (SEQ ID NO: 2) of ompC (SEQ ID NO: 54).
FIG. 5 represents the activities of peptides derived from the RYDERY peptide (SEQ ID NO: 2), in regard to antibodies present in human umbilical blood sera (SEQ ID NOS 2, 55-60, 2, 61-66, 2, 67-70, 3-4, 71, 2, 5-6 and 72-75, respectively, in order of appearance).
FIG. 6 represents the level of anti-OMP-38 class IgG antibodies in murine sera following immunization with poly-lysine conjugates, OMP-38 protein, as well as non-immunized mice, and determined using an ELISA test (SEQ ID NOS 41 and 44-46, respectively, in order of appearance).



EXAMPLE 1
Production of an Extract of Bacterial Outer Membrane Proteins (OMP)
A fresh bacterial mass from a 7-hour culture on liquid BHI medium at a temperature of 37° C. was obtained by centrifugation and rinsed with 10 mM Tris-HCl buffer pH 7,6 containing 10 mM MgSO4. The bacteria were suspended in the same buffer with an addition of 20 μg RNase and 20 μg DNase per ml and sonified for 10 min. The disrupted bacteria were centrifuged at 7000×g to remove undisrupted cells, and then the resulting supernatant was ultracentrifuged at 150000×g for 1 hour to remove envelope fragments. To dissolve the cell membrane, the ultracentrifuged precipitate was extracted twice at room temperature with 10 mM Tris-HCl, pH 7,6 containing 10 mM MgSO4 and 2% Triton X-100. After centrifugation at 150000×g the resulting precipitate was extracted twice with the same buffer containing 2% Triton X-100 and 5 mM EDTA and centrifuged at 160000×g. The outer membrane proteins dissolved in the supernatant were precipitated with two volumes of 95% ethanol and characterised using PAGE. OMP fractions contained around 20 proteins, and contained no more than 5% lipopolysaccharide, which was determined based on Kdo content.
EXAMPLE 2
Preparation of a 38 kDa Protein Immunoreactive with Human Serum
The preparative electrophoresis of outer membrane proteins was preferably conducted using a Prep Cell 491 apparatus from BioRad using a 37 mm column, which was loaded with 80 ml of 10% or 12.5% separating gel and 20 ml 5% of stacking gel with a buffer containing 25 mM Tris, 0.192 mM glycine and 1% SDS, pH 8.3 was used for electrophoresis and elution. After loading 30-40 mg of protein extract on the stacking gel, electrophoresis was performed at 260 V and 109 mA. Elution was initiated when the dye, bromophenol blue, exited the separating gel. During the elution, we collected fractions of 1.4 ml, and the presence of protein in the fractions was monitored at a wavelength of 280 nm with a UV detector and verified via electrophoresis and immunoblotting. Fractions with appropriate proteins, preferably 38 kDa, were dialysed into water, pooled and concentrated through centrifugation under a vacuum. OMP fractions were characterised in a polyacrylamide gel under reducing conditions using 10% or 12.5% gels and standard methods.
EXAMPLE 3
Sequence Determination of the Protein Temporarily Denoted as omp38 and Establishment of its Homology to a Protein Known as ompC
The ompC sequence was determined at the protein and DNA levels. We isolated and purified omp38 from Shigella flexneri 3a as described above. The purified protein was hydrolysed with trypsin and the resulting fragments were analysed using mass spectrometry (ESI-MS/MS). In this way, we determined 67% of the ompC sequence. This was confirmed and completed following the isolation of the ompC gene and DNA sequence determination. We determined that ompC of Shigella flexneri 3a has an identical sequence to that determined for ompC of Shigella flexneri 2a (GeneBank, AE014073.1), Shigella boydii Sb227 (GeneBank CP000036.1), Shigella flexneri 5 (GeneBank CP000266.1) and Shigella flexneri 2002017 (GeneBank CP001383.1).
EXAMPLE 4
Construction of a 3-dimensional Model of ompC of Shigella flexneri 3a Based on the Structure of Homologous Proteins Available from the PDB Database
In order to build a 3D model of the Shigella flexneri 3a ompC sequence, we threaded the ompC of E. coli into the structure using a method available from the internet at http://swissmodel.expasy.org [Arnold K., Bordoli L., Kopp J., and Schwede T., The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling, Bioinformatics, 2006, 22, 195-201]. The resulting antigen model was subjected to a PEPITO bioinformatic analysis simulating which amino-acids may interact with antibodies [Sweredoski, M. J. and Baldi, P., Bioinformatics Application Note, 2008, 24, 12, 1459-1460]. This method is also available on the Internet (http://pepito.proteomics.ics.uci.edu).
FIG. 1 shows the resulting visualisation of the conformational epitopes.
EXAMPLE 5
Synthesis of Peptides Representing Five (5) Bioinformatically Predicted Potential Epitopic Regions and Determination that Only One of them, Loop V, Reacts with Umbilical Serum
Based on the bioinformatic analysis, we designed the syntheses of five peptides corresponding to extracellular epitopic regions. These were:










1.
GNSAENENNSWT-pin
(62-73)
(SEQ ID NO: 7)


 


2.
EGEGMTNNGREALRQNGDGV-
(157-176)
(SEQ ID NO: 8)



pin




 


3.
GLNRYDERYIGN-pin
(205-216)
(SEQ ID NO: 9)


 


4.
GVINGRNTDDED-pin
(287-299)
(SEQ ID NO: 10)


 


5.
DDNQFTRDAG-pin 
(327-336)
(SEQ ID NO: 11)






Peptides representing the bioinformatically predicted regions were synthesized using an NCP kit with 96 hydroxypropylmethacrylate (HPM) pins (MIMOTOPES, Clayton, Victoria, Australia) according to the manufacturer's specifications (Carter, J.M. (1994) An epitope mapping of a protein using the Geysen (PEPSCAN) procedure. Methods Mol Biol 36: 207-223). As the sixth, control, peptide we synthesised a fragment corresponding to the N-terminal region of ompC (AEVYNKDGNKLD-pin (SEQ ID NO: 12)).
After the completed synthesis, we performed initial reactivity tests for the individual peptides, using a standard ELISA test. The ELISA was performed in a mixture of 9 different umbilical blood sera (1H-9H) according to the following scheme:

    a. Pin equilibration in TBS-T.
    b. Blocking of free sites on the pins using 1% BSA in TBS-T
    c. Incubation with umbilical sera at a dilution of 1:500 in a solution of 1% BSA in TBS-T buffer
    d. Rinsing with TBS-T buffer
    e. Incubation with secondary antibodies, conjugated Anti-Human IgG (Fc)-AP (Promega)
    f. Rinsing with TBS-T
    g. Reaction of AP with the substrate pNPP (p-Nitrophenyl Phosphate, Sigma) and absorbance measurement at 405 nm
    h. Dissociation of proteins/antibodies from peptides synthesized on HPM-pins.


The results obtained are presented in FIG. 2.
Only peptide No. 3, representing a looped region of the ompC protein (loop V) found between amino-acids 205-216 (GLNRYDERYIGN (SEQ ID NO: 9)) turned out to be active immunologically.
We also determined the individual reactivity of umbilical blood sera from various patients (1H-6H, 13H, 14H) against peptides (nr 1-6) synthesized on HPM-pins. The results obtained (FIG. 3) confirmed the earlier result (FIG. 2) which identified peptide No. 3 as the most reactive. They also indicate individual differences at the level of antibodies recognizing peptide No. 3 in different patients.
EXAMPLE 6
Establishment of the Minimal Length of the Immunologically Active Peptide Representing Loop V
The next stage of research meant to demonstrate the sequence of the epitope binding antibodies present in umbilical blood serum was the determination of the full and minimal sequence of the epitope which guarantees antibody binding. For this reason we performed a chemical synthesis of short peptides representing sequences in the region of loop V. We synthesized 29 different twelve amino-acid peptides, which overlapped by one amino-acid. These are shown in Table 1.







TABLE 1







Sequences of peptides representing loop V of ompC responsible 


for the reaction with umbilical blood.









SEQ



ID


N-end-------------------------------------------------C-end-(HPM-pin)
NO:























 1
R
A
E
T
Y
T
G
G
L
K
Y
D
13


 2
D
R
A
E
T
Y
T
G
G
L
K
Y
14


 3
G
D
R
A
E
T
Y
T
G
G
L
K
15


 4
N
G
D
R
A
E
T
Y
T
G
G
L
16


 5
G
N
G
D
R
A
E
T
Y
T
G
G
17


 6
I
G
N
G
D
R
A
E
T
Y
T
G
18


 7
Y
I
G
N
G
D
R
A
E
T
Y
T
19


 8
R
Y
I
G
N
G
D
R
A
E
T
Y
20


 9
E
R
Y
I
G
N
G
D
R
A
E
T
21


10
D
E
R
Y
I
G
N
G
D
R
A
E
22


11
Y
D
E
R
Y
I
G
N
G
D
R
A
23


12
R
Y
D
E
R
Y
I
G
N
G
D
R
24


13
N
R
Y
D
E
R
Y
I
G
N
G
D
25


14
L
N
R
Y
D
E
R
Y
I
G
N
G
26


15
G
L
N
R
Y
D
E
R
Y
I
G
N
9


16
F
G
L
N
R
Y
D
E
R
Y
I
G
27


17
N
F
G
L
N
R
Y
D
E
R
Y
I
28


18
Q
N
F
G
L
N
R
Y
D
E
R
Y
29


19
D
Q
N
F
G
L
N
R
Y
D
E
R
30


20
D
D
Q
N
F
G
L
N
R
Y
D
E
31


21
T
D
D
Q
N
F
G
L
N
R
Y
D
32


22
R
T
D
D
Q
N
F
G
L
N
R
Y
33


23
K
R
T
D
D
Q
N
F
G
L
N
R
34


24
S
K
R
T
D
D
Q
N
F
G
L
N
35


25
S
S
K
R
T
D
D
Q
N
F
G
L
36


26
S
S
S
K
R
T
D
D
Q
N
F
G
37


27
V
S
S
S
K
R
T
D
D
Q
N
F
38


28
A
V
S
S
S
K
R
T
D
D
Q
N
39


29
A
A
V
S
S
S
K
R
T
D
D
Q
40









To exclude the possibility of non-specific interactions, the control consisted of HPM-pins blocked solely with glycine residues. No non-specific antibody binding to HPM-pins were noted.
The ELISA assay conducted on a mixture of umbilical sera 1H-9H demonstrated that peptides denoted with numbers 12-18 (Table 1) are responsible for binding antibodies in these sera. From the immunological sequence analysis of the active peptide it stems that the common element for this series of sequences is the sequence RYDERY (SEQ ID NO: 2). This peptide also turned out to be the shortest active portion of the epitope.
EXAMPLE 7
Determination of Critical and Less Significant Amino-acid Positions for Immunological Activity in the Minimal Peptide RYDERY (SEQ ID NO: 2). Optimization of the Peptide Sequence
Unexpectedly it was shown that the immunological activity of the RYDERY peptide (SEQ ID NO: 2) is not affected by the removal of amino-acids in positions P+1 and P+2, nor in P−1, R−2, P−3 and R−4. The removal of amino-acids in positions P1 or P6, and in the subsequent positions (P2, P3 or P5, P4) caused a complete loss of activity. Likewise, a loss of immunological activity was caused by an exchange of residues at positions P1, P2 and P5. The latter amino-acids must remain unchanged in the final form of the peptide vaccine. Positions P3, P4 and P6 are less restrictive. In this case native amino-acids may be substituted by a series of other residues without any significant loss of immunological activity (FIG. 4). This property of the epitope facilitates the synthesis of a peptide less susceptible to enzymatic hydrolysis, and a higher antigenicity.
Furthermore, it was unexpectedly shown that an alteration of amino-acid D or E for Q yields an increased reactivity with umbilical serum (FIG. 5).
EXAMPLE 8
Setting the Immunologically Active Peptide on Carriers Such as Polymers and Proteins
One of the commonly accepted methods of preparing an artificial antigen is the synthesis of an immunologically active peptide on a resin with previously synthesised and attached poly-lysine. Such a carrier makes it possible to attach eight peptide chains. The resulting synthetic antigen has a molecular mass of about 9 kDa.
We prepared 5 different conjugates differing in terms of peptide sequences synthesized thereon. Four contained the sequence RYDERYIG (SEQ ID NO: 41) (the IG residues occur in the natural protein sequence and were used as linkers facilitating a better exposition of the peptide by separating it from the poly-Lys carrier) and were successively elongated using N, LN, GLN amino-acid residues (from the natural ompC protein sequence and exhibiting antigenicity with regard to class IgG antibodies from umbilical blood sera). One such conjugate, containing the YDERY sequence (SEQ ID NO: 42), which demonstrated no activity in earlier experiments may constitute a control.
The series of the above mentioned artificial antigens was synthesized (Table 2) and used to immunize mice.
It is also possible to use natural proteins as antigen peptides (haptens) carriers. Typical carrier proteins are bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) and ovalbumin (OVA) (see; Bioconjug Chem. 1999 (3):496-501).









TABLE 2






Conjugate No.
Peptide Sequence








1
Ac-YDERYIG-polyLys




(core peptide disclosed as SEQ ID NO: 43)



2
Ac-RYDERYIG-polyLys




(core peptide disclosed as SEQ ID NO: 41)



3
Ac-NRYDERYIG-polyLys




(core peptide disclosed as SEQ ID NO: 44)



4
Ac-LNRYDERYIG-polyLys




(core peptide disclosed as SEQ ID NO: 45)



5
Ac-GLNRYDERYIG-polyLys




(core peptide disclosed as SEQ ID NO: 46)









Carriers bearing immobilized peptides according to the present invention may be then used to prepare affinity gels for isolating the protective antibodies from the blood/sera of donors. Antibodies produced in this way may be used in the production of blood-derived, immunoglobulin therapeutic preparations specific against enterobacteria.
Carriers bearing immobilized peptides according to the present invention may also be used in the diagnosis of specific humoral immunity insufficiencies. Due to their specificity, the diagnostic tests containing peptides according to the present invention are particularly useful for determining the level of antibodies specific against important pathogens of the gastrointestinal tract, which have a great significance particularly in paediatrics.
EXAMPLE 9
Immunogenicity of the Synthesized Peptide Conjugates in a Mouse Model
BALB/c mice were divided into 7 experimental groups, of which groups 1-4 received poly-lysine conjugates (gr. 1: RYDERYIG-polyLys (core peptide disclosed as SEQ ID NO: 41), gr. 2: NRYDERYIG-polyLys (core peptide disclosed as SEQ ID NO: 44), gr.3: LNRYDERYIG-polyLys (core peptide disclosed as SEQ ID NO: 45) and gr.4: GLNRYDERYIG-polyLys (core peptide disclosed as SEQ ID NO: 46)), group 5received the OMP-38 protein, and the control groups received only the MPL adjuvant-gr. 6 or were not immunized at all—gr. 7. The mice were immunized intraperitoneally with a single dose of 200 μl of vaccine containing the appropriate antigen suspended in PBS with MPL adjuvant (5:1, vol/vol). The mice were immunized over 1.5 months at weekly intervals. After 7 days from each immunization, one mouse was bled from each group, and then the serum specific antibody level was ascertained which was evidence of an induced immune response using a standard ELISA test which measured the titre of antibodies directed against whole OMP-38 protein, wherein we determined the level of highly specific antibodies directed against the RYDERY epitope (SEQ ID NO: 2) present on the surface of OMP-38. The results are summarised in FIG. 6.
EXAMPLE 10
ELISA Test with Human Serum Facilitating the Rapid Detection of Differences in Anti-RYDERY (SEQ ID NO: 2) Antibody Concentrations in Patients' Sera
Peptides with the same sequence (RYDERY (SEQ ID NO: 2)) were equilibrated in TBS-T buffer. Solid phase free space blocking was performed using a 1% BSA solution in TBS-T (1h, room temperature, 200 μl/well). We consecutively tested 100-fold of serum dilutions in TBS-T with 1% BSA (1h, room temperature, 100 μl/well). Next, the reactivity of antibodies present in the sera from patients were detected using anti-Human IgG conjugated with alkaline phosphatase (Sigma) at a dilution of 1:10000 (1h, room temperature, 100 μl/well), using pNPP (p-Nitrophenyl Phosphate, AP Yellow-Sigma) as a substrate.

## Claims

1. A conjugate comprising poly-Lys conjugated to a peptide of no more than twelve amino acids that comprises an amino-acid sequence selected from RYDERY (SEQ ID NO: 2), RYDDRY (SEQ ID NO: 3), RYEERY (SEQ ID NO: 4), RYQERY (SEQ ID NO: 5), and RYDQRY (SEQ ID NO: 6).

2. The conjugate of claim 1, wherein the peptide is selected from the group consisting of the peptides RYDERY (SEQ ID NO: 2), RYDDRY (SEQ ID NO: 3), RYEERY (SEQ ID NO: 4), RYQERY (SEQ ID NO: 5), and RYDQRY (SEQ ID NO: 6).

3. A method of inducing production of antibodies against the OMP-38 protein of Shigella flexneri 3a, comprising immunizing a host with the conjugate according to claim 1.

4. A method of inducing production of antibodies against the OMP-38 protein of Shigella flexneri 3a, comprising immunizing a host with the conjugate according to claim 2.

5. A method of making the conjugate according to claim 1, comprising conjugating the poly-Lys to the peptide.

6. A method of making the conjugate according to claim 2, comprising conjugating the poly-Lys to the peptide.