Patent Application
20220233672
The present invention is related to the field of immunology and provides an immunogenic formulation for preventing or treating infectious diseases. In particular, it provides a vaccine against shiga toxin-producing Escherichia coli to be used in animal reservoir and human risk populations.
Escherichia coli (E. coli) is a member of the Enterobacteriaceae family found in the gastrointestinal microbiota of humans and other warm-blooded animals. Most of this family are considered harmless, but some are responsible of serious infectious diseases in humans. Particularly, shiga toxin-producing Escherichia coli (STEC) are etiological agents of acute diarrhea, dysentery and hemolytic uremic syndrome (HUS), a clinical syndrome characterized by progressive renal failure that can be lethal or that might lead to lifelong sequelae.
Although STEC serotype O157:H7 is the most prevalent cause of sporadic outbreaks and cases of severe disease, STEC serogroups such as 026, 0103 and 0113 have been associated with similar outbreaks.
Currently, the treatment of STEC HUS is fundamentally a supportive care and the use of antibiotics is contraindicated because such treatment might favor a release of shiga toxins (Stx1 and/or Stx2), increasing the risk of developing HUS. Alternative therapies based on compounds that bind and block the Stx have been proposed. However, the results of these therapies have been unsuccessful.
Many vaccines have been tested in animal models, such as: recombinant forms of Stx, intimin and SpA, chimeras of subunits A and B of Stx1 and Stx2, and avirulent strains of O157:H7, but not very successfully. The only candidate tested in humans is one based on the fusion of the “O” polysaccharide of E. coli O157:H7 and the exotoxin A of Pseudomonas aeruginosa (O157-rEPA). However, these studies are exclusively directed to STEC O157:H7 and they have not included other serogroups/serotypes (non-O157).
For this reason, there is a need for a broad-spectrum vaccine which confers protection against a wider range of STEC serotypes than just O157.
Currently, vaccine candidates have been unsuccessful because the characterization of antigens has mainly focused on strains O157:H7 as possible targets of STEC vaccines, including Stx, determinants of serological classifications (O antigen and flagellum) and proteins encoded in the locus of enterocyte effacement (LEE). For example, the US 2010/0166788 of Novartis Vaccines & Diagnostic discloses several polypeptides that can be included in immunogenic compositions specific for pathogenic E. coli strains. The polypeptides have cellular locations which render them accessible to the immune system. The genes encoding the polypeptides were initially identified as being present in uropathogenic strain 536 but absent from non-pathogenic strains. This document describes isolated polypeptides but not an immunogenic formulation of chimeric proteins.
CN 101062410 of Army Medical University (former Third Military Medical University, PLA.) discloses a “poly valency fuse type enterorrhagia property Escherichia coli O157:H7 gene engineering vaccine, which comprises the following steps: adopting enterorrhagia property E. coli O157:H7 Vi antigen Shiga's toxin II; combining subunit, compact sticking element and III type secretory protein A; constructing fuse engineering bacteria through gene retooling method; proceeding high density ferment; proceeding a series of purity course; getting fuse protein molecule vaccine with high purity”.
For these reasons, the main objective of this invention is to provide an immunogenic formulation that will substantially prevent or minimize infection caused by several STEC serogroups, where the most frequent serogroups associated with severe clinical symptoms of hemorrhagic diarrhea and HUS are O157, O26, O45, O91, O103, O104, O113, O111, O121, O145 and O174.
The present invention comprises an immunogenic formulation that induces protection from or immunity against shiga toxin-producing Escherichia coli (STEC). In particular, the immunogenic formulation comprises one or more chimeric proteins derived from OmpT and Hes proteins of STEC. The immunogenic formulation of the present invention may also be used to prepare vaccines.
The present invention provides an immunogenic formulation that induces protection or immunity against shiga toxin-producing Escherichia coli (STEC). In particular, the present invention resides in an immunogenic formulation comprising: one or more chimeric proteins derived wholly or in part from OmpT and Hes proteins of STEC; and at least one physiologically or pharmaceutically acceptable excipient, carrier, diluent and/or adjuvant.
OmpT is an aspartyl protease found on the outer membrane of E. coli and is a housekeeping protease that degrades foreign peptide material that the bacteria encounters in its surrounding environment. The Hes protein is a new allelic variant that has been denominated as hemagglutinin from E. coli STEC (Hes). The hes gene is present in STEC LEE-negative strains associated with severe disease and not detected in commensal E. coli strains.
An immunogenic formulation is a composition that induces a cellular and/or humoral immune response. An immune response is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In the present invention, the immune response is specific for an antigen that comprises at least one chimeric protein derived from OmpT and Hes proteins of STEC (an “antigen-specific response”). An immune response may be a T cell response, such as a CD4+ response or a CD8+ response, or a B cell response, and results in the production of specific antibodies.
Expressed in another way, the one or more chimeric proteins of the present invention are immunogenic proteins. An immunogenic protein or polypeptide comprises an allele-specific motif, an epitope, or other sequence such that the protein/polypeptide will bind a MHC molecule and induce an immune response.
In one embodiment, the chimeric protein may further comprise a sequence derived wholly or in part from α-Cah protein of STEC. Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation.
In one example, the chimeric protein may have at least a 90% identity with the amino acid sequence of SEQ ID No. 1. Alternatively, the chimeric protein may have at least a 90% identity with the amino acid sequence of SEQ ID No. 2.
In one embodiment, the formulation may comprise more than one or a second chimeric protein, wherein one chimeric protein has an amino acid sequence having at least 90% identity with SEQ ID NO. 1, and another or a second chimeric protein has an amino acid sequence having at least 90% identity to SEQ ID No. 2. It will be appreciated that sequence identity may be about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.
In such an embodiment, both or the two chimeric proteins may be present in a ratio of between 1:2 to 2:1 in the immunogenic formulation. For example, both or the two chimeric proteins may be present in a ratio of 1:1.
The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods. The percentage sequence identity may be determined over the full length of the relevant sequence (e.g., over the full length of SEQ ID NO:1 or SEQ ID NO:2) or over a fragment thereof, e.g., at least 50, 100, 200, 300, 400 or 500 amino acid residues of SEQ ID NO:1 or SEQ ID NO:2.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, Mol. Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5: 151-3, 1989; Corpet et al., Nuc. Acids Res. 16: 10881-90, 1988; Huang et al., Computer Appls. in the Biosciences 8, 155-65. 1992; and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol. Biol. 215:403-10, 1990, presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX. A description of how to determine sequence identity using this program is available on the NCBI website on the internet. The BLAST and the BLAST 2.0 algorithm are also described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natd. Acad. Sci. USA 89: 10915, 1992).
The formulation of the present invention includes at least one physiologically or pharmaceutically acceptable excipient, carrier, diluent and/or adjuvant. Such carriers, excipients, and other agents that are incorporated into formulations provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations may be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as LIPOFECTINT™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See also Powell et al “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.
By “adjuvant” is meant any substance that is used specifically or non-specifically to potentiate an antigen-specific immune response, perhaps through activation of antigen presenting cells. Examples of adjuvants include an oil emulsion (e.g., complete or incomplete Freund's adjuvant), Montanide incomplete Seppic adjuvant such as ISA, oil in water emulsion adjuvants such as the Ribi adjuvant system, syntax adjuvant formulation containing muramyl dipeptide, aluminum salt adjuvant (ALUM), polycationic polymer, especially polycationic peptide, especially polyarginine or a peptide containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, immunostimulatory oligodeoxynucleotide (ODN) containing non-methylated cytosine-guanine dinucleotides (CpG) in a defined base context (e.g., as described in WO 96/02555) or ODNs based on inosine and cytidine (e.g., as described in WO 01/93903), or deoxynucleic acid containing deoxy-inosine and/or deoxyuridine residues (as described in WO 01/93905 and WO 02/095027), especially Oligo(dIdC)13 (as described in WO 01/93903 and WO 01/93905), neuroactive compound, especially human growth hormone (described in WO 01/24822), or combinations thereof, a chemokine (e.g., defensins 1 or 2, RANTES, MIP1-α, MIP-2, interleukin-8, or a cytokine (e.g., interleukin-1β, -2, -6, -10 or -12; interferon-γ; tumor necrosis factor-α; or granulocyte-monocyte-colony stimulating factor), a muramyl dipeptide variant (e.g., murabutide, threonyl-MDP or muramyl tripeptide), synthetic variants of MDP, a heat shock protein or a variant, a variant of Leishmania major LeIF (Skeiky et al., 1995, J. Exp. Med. 181: 1527-1537), non-toxic variants of bacterial ADP-ribosylating exotoxins (bAREs) including variants at the trypsin cleavage site (Dickenson and Clements, (1995) Infection and Immunity 63 (5): 1617-1623) and/or affecting ADP-ribosylation or chemically detoxified bAREs (toxoids), QS21, Quill A, N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-dipalmitoyl-s-glycero-3-(hydroxyphosphoryloxy)]ethylamide (MTP-PE) and compositions containing a metabolizable oil and an emulsifying agent. An adjuvant may be administered with an antigen or may be administered by itself, either by the same route as that of the antigen or by a different route than that of the antigen. A single adjuvant molecule may have both adjuvant and antigen properties.
The formulation may comprise one or more chimeric proteins as described herein above in combination with medical injection buffer and/or with adjuvant. Alternatively, or in addition, the formulation may comprise the one or more chimeric proteins in an aqueous buffered solution at a pH of between 6 and 8, e.g., 6.0 to 6.6, 6.4 to 7.1, 6.9 to 7.6 or 7.4 to 8.0.
An immunogenic formulation of the invention will be formulated to be compatible with its intended route of administration. Methods to accomplish the administration are known to those of ordinary skill in the art. It may also be possible to create compositions which may be topically or orally administered, or which may be capable of transmission across mucous membranes. For example, the administration may be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
Solutions or suspensions used for intradermal or subcutaneous application typically include at least one of the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and tonicity agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases. Such preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials.
Solutions or suspensions used for intravenous administration include a carrier such as physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), ethanol, or polyol. In all cases, the formulation must be sterile and fluid for easy syringability. Proper fluidity can often be obtained using lecithin or surfactants. Such a formulation must also be stable under the conditions of manufacture and storage. Prevention of microorganisms may be achieved with antibacterial and antifungal agents, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents (sugar), polyalcohols (mannitol and sorbitol), or sodium chloride may be included in the formulation. Prolonged absorption of the formulation may be accomplished by adding an agent which delays absorption, e.g., aluminum monostearate and gelatin.
Formulations for oral delivery include an inert diluent or edible carrier. The formulation may be enclosed in gelatin or compressed into tablets. For the purpose of oral administration, the immunogenic protein(s) may be incorporated with excipients and placed in tablets, troches, or capsules. Pharmaceutically compatible binding agents or adjuvant materials can be included in the formulation. The tablets, troches, and capsules may contain (1) a binder such as microcrystalline cellulose, gum tragacanth or gelatin; (2) an excipient such as starch or lactose, (3) a disintegrating agent such as alginic acid, Primogel, or corn starch; (4) a lubricant such as magnesium stearate; (5) a glidant such as colloidal silicon dioxide; or (6) a sweetening agent or a flavoring agent.
Formulations may also be administered by a transmucosal or transdermal route. For example, some proteins may be capable of crossing mucous membranes in the intestine, mouth, or lungs. Transmucosal administration may be accomplished through the use of lozenges, nasal sprays, inhalers, or suppositories. Transdermal administration may also be accomplished through the use of formulations including ointments, salves, gels, or creams known in the art. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used. For administration by inhalation, formulations may be delivered in an aerosol spray from a pressured container or dispenser, which contains a propellant (e.g., liquid or gas) or a nebulizer.
The formulation may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions, such as one or more antibiotic or antibacterial agents, particularly those selective for Gram negative bacterial and/or E. coli.
The formulation may be included in a container, pack, or dispenser and/or in kits, together with instructions for administration.
It will be appreciated that the immunogenic formulation of the present invention may be used in the treatment or prophylactic treatment of STEC infection, or for use in the manufacture of a medicament for the treatment or prophylactic treatment of STEC infection. The invention also relates to the immunogenic formulation as described herein for use (in the manufacture of a medicament) for inducing an immune response in a patient.
Expressed in another way, the invention encompasses a method of treating or preventing an STEC infection, or inducing an immune response, the method comprising administering to a subject in need thereof a therapeutically effective amount of the immunogenic formulation described herein. Alternatively, the invention also reside in a method of treating or preventing infection by STEC or a disorder associated with STEC infection, in a subject, comprising, administering to the subject an immunogenic formulation as described herein, in an amount sufficient to inhibit or reduce STEC infection in the subject, thereby treating or preventing the infection or related disorder.
The term “treatment” refers to a therapeutic or preventative (prophylactic) measure, or passive immunization. The treatment may be administered to a subject who is already infected, or who ultimately may acquire or be at risk of acquiring such an infection, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the infection or a disorder or recurring disorder associated with the infection, or to prolong the survival of a subject beyond that expected in the absence of such treatment.
A “therapeutically effective amount” is the amount of agent, such as the immunogenic formulation disclosed herein that is sufficient to prevent, treat (including prophylaxis), reduce and/or ameliorate the symptoms and/or underlying causes of a disorder or disease, for example to prevent, inhibit, and/or treat an infection. In some embodiments, a therapeutically effective amount is sufficient to reduce or eliminate a symptom of a disease, such as an infection. For instance, this can be the amount necessary to inhibit or prevent bacterial replication or to alter measurably outward symptoms of the bacterial infection. In general, this amount will be sufficient to inhibit measurably bacterial replication or infectivity.
It is understood that to obtain a protective immune response against a pathogen may require multiple administrations of the immunogenic formulation. Thus, a therapeutically effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining a protective immune response. For example, a therapeutically effective amount of an agent may be administered in a single dose, or in several doses, for example daily, during a course of treatment (such as a prime-boost vaccination treatment). However, the therapeutically effective amount may depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent may be packaged in a therapeutic amount, or in multiples of the therapeutic amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.
Formulations of the invention may be administered in therapeutically effective amounts as described. Therapeutically effective amounts may vary with the subject's age, condition, sex, and severity of medical condition. Appropriate dosage may be determined by a physician based on clinical indications. The formulations may be given as a bolus dose to maximize the circulating levels of immunogen for the greatest length of time. Continuous infusion may also be used after the bolus dose.
As used herein, the term “subject” is intended to include human and non-human animals. Subjects may include a human patient having a disorder characterized by STEC infection. The term “non-human animals” of the invention includes all vertebrates, such as non-human rodents, camelids, primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.
Examples of dosage ranges that may be administered to a subject may be chosen from: 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 10 μg/kg to 1 mg/kg, 10 μg/kg to 100 μg/kg, 100 μg to 1 mg/kg, 500 μg/kg to 1 mg/kg.
It may be advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited for the patient or subject. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the aforesaid antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, the aforesaid antibody may be contained in about 5 to about 100 mg and in about 10 to about 250 mg for the other dosage forms. Thus, each dosage unit contains a predetermined quantity of immunogenic agent calculated to produce a therapeutic effect in association with the carrier. The dosage unit depends on the characteristics of the immunogenic agent and the particular therapeutic effect to be achieved.
Toxicity and therapeutic efficacy of the formulation may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
Various delivery systems are known and may be used to administer the formulation of the invention. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The formulation may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration may be systemic or local.
The formulation of the present invention may be administered with one or more additional active agents. Such additional agents may be administered together, separately or sequentially with the immunogenic formulation of the present invention. Separate administration refers to two compositions or active ingredients being administered at different times, e.g., at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One may also administer active ingredients at 24 hours apart, or even longer apart. Alternatively, two or more active ingredients may be administered simultaneously, e.g., less than 10 or less than 5 minutes apart. Compositions administered simultaneously may, in some aspects, be administered as a mixture, with or without similar or different time release mechanism for each of the components.
In methods of treatment described herein, one or more doses may be administered. In some cases, a single dose may be effective to achieve a long-term benefit. Thus, the method may comprise administering a single dose of the formulation. Alternatively, multiple doses may be administered, usually sequentially and separated by a period of days, weeks or months.
In another embodiment, the immunogenic formulation of the present invention may also be used to generate antibodies. Thus, the present invention relates to a method for inducing immunity in an animal, such as a mammal, against shiga toxin-producing E. coli (STEC) or pathology caused by STEC comprising administering the immunogenic formulation as described herein to the animal. Expressed in another way, there is provided the immunogenic formulation as described herein for use in inducing immunity against shiga toxin-producing E. coli (STEC) or pathology caused by STEC in an animal.
Encompassed within this is also a method for protecting an animal, such as a mammal, against infection of shiga toxin-producing E. coli (STEC) or pathology caused by STEC comprising administering an immunogenic formulation to the animal. Alternatively, there is provided the immunogenic formulation as described herein for use in protecting an animal, such as a mammal, against shiga toxin-producing E. coli (STEC) or pathology caused by STEC in the animal.
In this embodiment, the animal is one suitable for the production and harvesting of suitable antibodies generated by the animal in response to the immunogenic protein. Suitable animals include rodent, camelids and some sharks. It will be appreciated that the immune response of the animal may “primed” by way of pre-treatment with an adjuvant to increase the desired immune response to a later administered immunogenic agent. Alternatively or in addition, immune response of the animal may be “enhanced” through co-administration of an adjuvant and an immunogenic agent, wherein the adjuvant increases the desired immune response to the immunogenic agent compared to administration of the immunogenic agent to the subject in the absence of the adjuvant. Methods of generating antibodies in animals is well known in the art.
In a yet further embodiment, there is provided a chimeric protein derived from OmpT and Hes proteins of STEC comprising immunogenic epitopes, the chimeric protein having an amino acid sequence as set out in SEQ ID No. 1.
In another embodiment, the chimeric protein further comprises a sequence derived wholly or in part from α-Cah protein of STEC and having the amino acid sequence as set out in SEQ ID No. 2.
The invention will now be illustrated by the following non-limiting examples which are not restrictive of the invention as claimed. The accompanying figures constitute a part of this specification and, together with the description, serve only to illustrate embodiments and not limit the invention.
The immunogenic formulation of the present invention comprises one or more chimeric proteins that includes at least one epitope of OmpT, α-Cah and Hes. Chimeric protein Q1 comprises a fusion of OmpT and Hes proteins, while the Q2 chimeric protein comprises carrier protein α-Cah which has been modified and includes in its structure epitopes of OmpT and Hes.
Twenty sera from convalescent pediatric patients who presented a prodrome of diarrhea within 20 days prior to the diagnosis of HUS were used; and two sera from patients without a clinical history of HUS were used as control sera.
The chimeric proteins Q1 and Q2 were prepared with a ?85% of purity percentage. Then, the genes coding for these proteins were optimized for their expression in E. coli and cloned in the plasmid pET30a, allowing the addition of a Tag of 6 histidine residues at the N-terminal end of the protein a Tag of 6 histidine and a processing site for TEV protease. The strain E. coli BL21 (DE3) strain was transformed with the resulting plasmids and recombinant colonies were selected in agar LB containing kanamycin.
The induction of protein synthesis was carried out by growing the recombinant bacteria in TB medium (Terrific Broth) containing kanamycin at 37° C. and, when the culture reached an OD600 of 1.2, the culture was complemented with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 hours. Bacteria were collected by centrifugation and resuspended in lysis buffer followed by sonication. After centrifugation, the sediment was dissolved using urea and was centrifugated again. The supernatant was used for purification of the protein. The denatured proteins were purified by Immobilized Metal Chelate Affinity Chromatography (IMAC-Ni affinity columns). The white proteins were renatured, sterilized in 0.22 μm filters and stored in aliquots. The protein concentration obtained was determined by Bradford assay using bovine serum albumin (BSA) as standard. The purity of the proteins was analyzed by densitometry on SDS-PAGE (
ELISA microplates (96-well) (Nunc Immobilizer Amino Plates, ThermoFisher) were incubated with 1.2 μg of each protein diluted in 100 μl of phosphate buffered saline solution (PBS, pH 7.2) overnight at 4° C. Then, to make the standard curve, purified human IgG (Cat. 02-7102, Invitrogen) or IgA (Cat. 3860-1AD-6, Mabtech) was diluted in PBS from 5 μg/ml to 0.0048 μg/ml or from 0.1 μg/ml to 0.00019 μg/ml, respectively, overnight at 4° C. The plates were washed 3 times with PBS (400 μl/well) containing 0.05% Tween 20 (T-PBS). They were then incubated with 300 μl/well of blocking solution (T-PBS+0.5% bovine serum albumin) for 15 min at room temperature. Patient sera were diluted 1:1000 or 1:500 in blocking solution (100 μl/well) and incubated for 60 min at 37° C. After 5 washes with T-PBS (400 μl/well), Human Anti-IgG (H+L) (conjugated with peroxidase, developed in goat) (Cat. 04-10-06, KPL Inc) or iHuman Anti-IgA alpha chain (conjugated with alkaline phosphatase, developed in goat) (Cat. Ab97212, Abeam), diluted 1:1000 in blocking solution (100 μl/well) were incubated for 60 min at 37° C. After 5 washes with Tris saline solution (TBS) (400 μl/well, pH 7.5) containing 0.05% Tween 20, plates were incubated with the substrate 2,2′-azino-di (3-ethylbenzthiazoline-6-sulfonate) (100 μl/well; ABTS Peroxidase Substrate, Cat. 50-66-18, KPL Inc) or p-nitrophenyl phosphate (100 μl/well, pNPP Substrate, Cat. N2600-10, USBiological) for 10 or 20 min at room temperature, respectively. The reaction was stopped with 5% sodium dodecyl sulfate or 3M sodium hydroxide dissolved in distilled water (100 μl/well). The absorbance of the solution in each well was determined at 405 nm using a Synergy HT microplate reader (Biotek Instruments, USA). Each sample was determined in duplicate and with at least two independent experiments. The relationship between the absorbance values and the IgG or IgA concentration of each sample and control was calculated from each corresponding standard curve using a 4-parameter logistic regression equation determined in the GraphPad Prism 7 software.
The reactivity of the two chimeric proteins, hereinafter referred to as chimeric protein 1 (Q1) and chimeric protein 2 (Q2), was determined against IgG and IgA present in 20 HUS sera and 2 control sera by ELISA (
As seen in
Particularly, in the case of IgG, Q1 was more sero-reactive (average=253 μg/ml, p<0.005) compared to Q2 (average=174 μg/ml). Similarly, in the case of IgA again Q1 had a higher level of sero-reactivity (average=4.871 ng/ml, p<0.005) compared to Q2 (average=1.390 ng/ml), see
These results indicate that Q1 and Q2 chimeras were recognized specifically by antibodies class IgG and IgA present in HUS sera, with Q1 being the one with the highest sero-reactivity chimeric protein.
BALB/c females whose age ranged between 5-6 weeks were randomly distributed in 7 experimental groups with food and water ad libitum.
Of the 7 groups, 6 were immunized with 20 μg of each vaccinal antigen (Q1, Q2 and an equimolar mixture of both chimeras—hereafter named Q3), using as adjuvants Inject Alum (IA, Thermo Fisher Scientific) for intramuscular immunizations (IM) (50 μl) and Sigma Adjuvant System® oil (Sigma-Aldrich, SA) for intranasal immunizations (IN)(20 μl). The remaining group was immunized only with PBS, negative control. All experimental groups were immunized 3 times with 15-day intervals as shown in the following table:
CFU count in mice immunized with Imject Alum Adjuvant challenged with E. coli O157:H7: To determine the effectiveness of the different vaccine antigens; mice were infected with 109 CFU/ml of E. coli O157:H7 and feces were collected daily for 12 days.
CFU count in mice immunized with an adjuvant (Sigma Adjuvant System) challenged with E. coli O157:H7: To determine the effectiveness of the different vaccine antigens, fresh feces were collected from the challenged mice for 12 days on a daily basis.
In
In summary, seven immunogenic proteins present in STEC strains of different serogroups and/or serotypes were identified in O26:H11, O103, O113: H21 and O157:H7. Notably, these proteins are absent in the commensal E. coli HS strain and are reactive to sera from patients who developed HUS (HUS sera). The antigens OmpT and α-Cah proteins showed seroreactivity to immunoglobulins IgG and IgA class in HUS sera, but not in control sera, obtained from patients not infected with STEC. In addition, the prevalence of genes encoding these two proteins is statistically higher in STEC strains associated with severe disease than in commensal E. coli strains. Additionally, the protein initially identified as Hek, later characterized as a new allele of the family of heat-resistant agglutinin proteins (Hra) and shows reactivity against IgG and IgA in HUS sera (unpublished results). This new allelic variant was denominated as hemagglutinin from E. coli STEC (Hes). The hes gene is present in STEC LEE-negative strains associated with severe disease. Conversely, in commensal E. coli strains the gene was not detected. Consequently, considering the seroreactivity and distribution of the coding genes in a variety of STEC serotypes associated with severe disease, the OmpT, α-Cah and Hes antigens may form the basis for an immunogenic formulation developed as a vaccine against this pathotype.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2019/054554 | 5/31/2019 | WO | 00 |
