WO2016012951A1 - Immunogenic conjugate - Google Patents

Immunogenic conjugate Download PDF

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Publication number
WO2016012951A1
WO2016012951A1 PCT/IB2015/055537 IB2015055537W WO2016012951A1 WO 2016012951 A1 WO2016012951 A1 WO 2016012951A1 IB 2015055537 W IB2015055537 W IB 2015055537W WO 2016012951 A1 WO2016012951 A1 WO 2016012951A1
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protein
seq
carrier
vaccine
conjugate according
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PCT/IB2015/055537
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French (fr)
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Anna JARZĄB
Andrzej Gamian
Danuta Witkowska
Edmund ZIOMEK
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Wrocławskie Centrum Badań Eit + Sp. Z O.O.
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Publication of WO2016012951A1 publication Critical patent/WO2016012951A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0283—Shigella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the object of the invention is a conjugate of a protein carrier and a peptide antigen, forming an OmpC protein epitope, applicable as a vaccine protecting against infection caused by coliform bacteria of the Enterobacteriaceae family, particularly species of the Shigella genus.
  • the object of the invention is also a pharmaceutical composition, a diagnostic composition and a vaccine comprising said conjugate and a method of obtaining the conjugate and use thereof.
  • Shigella genus bacteria are among the most dangerous pathogenic species belonging to the Enterobacteriaceae family.
  • Shigellosis (dysentery, bacterial dysentery) is a disease characterized by acute gastrointestinitis caused by bacterial penetration into epithelial cells lining the intestinal walls.
  • shigellosis a disease characterized by acute gastrointestinitis caused by bacterial penetration into epithelial cells lining the intestinal walls.
  • Among the most characteristic symptoms of shigellosis are watery, bloody or mucous diarrhea, nausea, vomiting, fever and abdominal pains.
  • Enterobacteriaceae species a group of obligate pathogens deserves particular attention, especially the species of the Shigella, Salmonella and Yersinia genera, responsible for bacterial gastrointestinal infections, especially bacilli being a direct cause of diarrhea, typhoid fever, bacterial dysentery and other intestinal diseases.
  • Coliform bacteria, along with streptococci and staphylococci are referred to as being among the most frequent and most severe etiological factors for diseases with bacterial etiology.
  • WHO World Health Organization
  • gastroenteritis is the cause of death for 1.9 million people per year and rank as the third among diseases with the highest percentage of mortality in the world.
  • WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection.
  • Vaccine, 2011; 29(37): 6371-637 or of killed bacterial cells, inactivated with high temperature, irradiation or chemical factors, showing the ability to induce immunological response associated with the gastrointestinal tract.
  • genes responsible for enterotoxin expression (senA, senB) and the gene responsible for the expression of Lipid A acyltransferase ⁇ msbB2) were removed from S. flexneri 2a bacilli, which, in case of applying to vaccines obtained as a result of these modifications of the strains WRSf2G12 and WRSf2G15, leads to an increase in the safety of the vaccine.
  • Both strains are severely weakened and retain genetic stability, both in in vitro tests done on cell lines and in those in vivo in animal models.
  • the studies conducted on guinea pigs have shown that following the immunization of animals administered ocularly, strong induction of both systemic and mucosal immunological response occurs.
  • sonnei WRSS1 strain mutant was weakened further by inactivating the senA, senB (WRSs2) genes and additionally the msbB2 (WRSs3).
  • WRSs2 and WRSs3 cause stimulation of systemic and mucosal immunological response in animals, on a level similar to the one when the WRSS1 strain is used, while guaranteeing the use of a safer vaccine with a comparable immunogenicity (Barnoy S., Baqar S., Kaminski R.
  • WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection.
  • Attenuated vaccines are comprised of a suitably weakened version of a live microorganism, not capable of causing infection.
  • Using microorganisms of this type in a vaccine is a procedure being the best imitation of causing a natural infection and it leads to formation of a complete immunological response, which in case of this pathogen entering the organism enables the recognition of its antigens.
  • These vaccines elicit both types of immunological response, both cellular and humoral one, and induce a long-term immunity after administering only 1 -2 doses of the vaccine.
  • the vaccines comprising live or weakened strains cannot be administered to persons with acquired immunodeficiency syndrome (e.g. AIDS patients) and to patients having a temporary immunological incapacity, including patients suffering from various types of cancer, receiving chemotherapy.
  • acquired immunodeficiency syndrome e.g. AIDS patients
  • patients having a temporary immunological incapacity including patients suffering from various types of cancer, receiving chemotherapy.
  • inactivated vaccines comprising killed microorganism strains.
  • the vaccine elicits response against LPS, which is specific for the given serotype, also eliciting specific immune response against proteins forming the antigens of the plasmid responsible for bacterial adhesion and invasion of epithelial cells (Ipa - Invasion plasmid antigen).
  • Ipa - Invasion plasmid antigen proteins forming the antigens of the plasmid responsible for bacterial adhesion and invasion of epithelial cells.
  • the advantage is the fact that it induces intestine-associated mucosal response, as well as systemic response, in large part similar to the one induced during natural infection with the pathogen. Preparation thereof is relatively inexpensive, and administration thereof uncomplicated and does not require the use of needles.
  • subunit vaccines form the basis in the development of subunit and conjugate vaccines.
  • subunit vaccines are taken into account, based on surface antigens, such as lipopolysaccharides (LPS) (Lowell G.H., Kaminski R.W., Grate S., Hunt R.E., Charney C, Zimmer S., Colleton C: Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice. Infect.
  • LPS lipopolysaccharides
  • A1 is R
  • A1 is R
  • A3 is D, R, E, N or Q
  • A4 is E, D, N or Q
  • the peptide antigen has a sequence of RYDERYIGC.
  • reaction mixture is subjected to dialysis in 0.1 M sodium bicarbonate with pH of 8.3 and the ester is removed on a column.
  • Another object of the invention is a use of said conjugate for preparation of blood-based, immunoglobulin therapeutic preparations, specific against Enterobacteriaceae, especially bacteria of the Shigella genus.
  • the peptides synthesized on hydrophobic carriers such as polyethylene pins or protein carriers such as tetanus toxoid, diphtheria toxoid or bovine serum albumin, are much better recognized by antibodies than the same peptides linked with polylysine (Fig. 7).
  • An isolated epitope not only without the cell, but also lacking the remaining initial protein, is an active fragment, an element required for the construction of a vaccine without the whole high molecular load.
  • a preparation method moves away from traditional methods of obtaining attenuated vaccines or the ones employing whole bacteria or whole protein antigens.
  • Subunit vaccines wherein the peptide vaccine according to the invention is classified, are safe and do not carry a risk of post-vaccination infection. Moreover, such a vaccine is relatively inexpensive to produce, and also utilizes techniques not carrying a risk of infection with a virulent strain.
  • the vaccine according to the invention is devoid of components inducing an additional inflammation and excessive reactogenicity of the preparation, such as LPS or toxins.
  • An undoubted advantage of using peptide antigens is the handling of a well-defined and standardized component, having the characteristic structure of the native protein antigen.
  • the vaccine according to the invention may be modified very easily and in a controlled manner, by chemical means, which may be crucial for preventing allergy or eliciting auto-immune response directed against own tissues.
  • FIG. 1 A. Electrophoretic image (SDS-PAGE) of the tetanus toxoid monomer (5 g) and the conjugates RYDERYIGC T (5 ⁇ g) and GLNRYDERYIGC T (5 pg); B. Electrophoretic image (SDS-PAGE) of BSA (2 ⁇ Jtg) and the conjugates RYDERYIGC:BSA (2 Mg) and GLNRYDERYIGC: BSA (2 Mg).
  • FIG. 2 A. MALDI-TOF-MS spectrum of the TT monomer and the conjugates RYDERYIGC:TT and GLNRYDERYIGC:TT.
  • FIG. 3 A. Reactivity of the TT-absorbed umbilical cord blood serum with the OmpC protein, TT and the conjugates GLNRYDERYIGC:TT and RYDERYIGC:TT.
  • B Reactivity of the umbilical cord blood serum with the OmpC protein, BSA and the conjugates GLNRYDERYIGC:BSA and RYDERYIGC:BSA.
  • Fig. 4 Anti-OmpC IgG class antibody titer in mouse serum after the I, II, III and IV immunization of the animals with the conjugates GLNRYDERYIGC:TT and RYDERYIGC:TT, the TT protein and PBS.
  • Fig. 7 Mice immunization with the conjugate GLNRYDERYIG-poly-Lys and mice immunization with the conjugate GLNRYDERYIGC:TT, respectively in an MPL adjuvant and without using an additional adjuvant.
  • Example 1 Peptide conjugation with a carrier protein.
  • Step 2 Conjugation.
  • Peptides for conjugation containing thiol groups, were suspended in 0.1 M carbonate buffer with 2 mM EDTA with pH of 8.3 in concentration of 10-50 mg/ml.
  • the bromoacetylated protein was added to the peptide solution within 1 minute, pH was adjusted with 0.1 M NaOH to the value of 8.5.
  • the peptide to protein ratio was within 50 to 100 mol/mol. Protein concentration was maintained within 2 mg/ml, while adjusting the concentration of the peptide.
  • Conjugation reaction was conducted overnight, about 16 hours in room temperature on a rotating mixer in a tightly sealed vessel under argon.
  • Step 3 Deactivation of the unreacted bromoacetyl groups of the carrier peptide. Deactivation was performed using 10 ⁇ of 2-mercaptoethanol per 1 ml of the reaction mixture for 1 hour in room temperature.
  • Step 4 Analysis of the conjugate.
  • the conjugation process was monitored in 6% (TT conjugates) or 12.5% (BSA conjugates) polyacrylamide gel in SDS-PAGE (Fig. 1 ).
  • the percentage of the polyacrylamide gel depended on the carrier protein molecular mass.
  • the loading level of the protein molecule with the peptides was measured using MALDI-TOF-MS (Fig. 2).
  • Example 2 Reactivity of the peptide conjugates with human umbilical cord blood antibodies, determined by dot-blot technique.
  • the Immobilon P membrane was wetted by applying 2 ⁇ portions of methanol spotwise. Next, antigen solutions (peptide conjugates, carrier proteins - negative control, OmpC protein - positive control) were applied to the wetted areas in suitable concentrations and left until completely dried. Free spaces on the membrane were blocked in 10 ml of 1 % BSA solution in TBS-T for 1 hour in room temperature. Next, the membrane was incubated in room temperature for 1 hour in 5 ml of antibody/serum solution suitably diluted in TBS-T buffer with 1 % BSA and washed 3 times in TBS-T buffer.
  • antigen solutions peptide conjugates, carrier proteins - negative control, OmpC protein - positive control
  • the membrane was incubated in solution of secondary antibodies conjugated with alkaline phosphatase in 1 :10 000 dilution and washed 3 times in TBS-T buffer.
  • the membrane was placed in substrate solution, containing 5 ml of alkaline phosphatase buffer, 15% BCPI, 30% NBT. The reaction was stopped by immersing the membrane in water.
  • Example 3 Determination of immunogenicity of the peptide conjugates in a mouse model.
  • the level of the induced immune response was determined after 4 immunizations of the animals, measuring IgG class antibodies with ELISA against the OmpC protein (Fig. 5a) and with ELISA on polyethylene pins against the RYDERY epitope (Fig. 5b).
  • the conjugates based on the carrier proteins were prepared and tested.
  • the conjugates comprising the elongated epitope sequence: GLNRYDERYIGC.
  • the peptide with the RYDERY epitope sequence in particular its longer version GLNRYDERYIGC, which has better immunogenic properties, may serve as an antigen in the construction of a conjugate vaccine. This was confirmed inter alia by the experiments concerning determination of the increase in specific anti-RYDERY antibody levels after immunization of mice with the conjugates GLNRYDERYIGC:TT/BSA. Using the TT conjugates resulted in an increase of anti-OmpC antibody titer, after immunization with the conjugate GLNRYDERYIGC:TT.
  • the conjugate comprising the shorter RYDERYIGC:TT peptide was less immunogenic, and the RYDERY peptide without the flanking amino acids, despite having antigenic properties, is a weaker immunogen.
  • Adjuvants were not used for immunizations, indicating that the induced response is highly specific and directed against the epitope molecule in the conjugate.
  • the sera after immunization with the conjugates also comprised antibodies against the carrier (Fig. 5a and Fig. 5b).
  • Example 5 Cyclization of peptides synthesized on polyethylene pins and antigenicity analysis with human umbilical cord blood antibodies. The synthesis was performed rotationally by removing the blocking Fmoc groups from an amino acid and coupling it with other amino acids. The removal of the Fmoc group was done under a fume hood in 20% piperidine solution in DMF (dimethylformamide) for 1 hour in room temperature. Next the pins were washed in DMF, then in methanol and dried under a fume hood.
  • DMF dimethylformamide
  • Aminoacylation was conducted for 4 hours or overnight in room temperature by placing the pins in 100 ⁇ of 60 mM Fmoc amino acid derivative solution with 65 mM HoAt (1 -hydroxy-7- azabenzotriazole) and 60 mM DIC (diisopropylcarbodiimide) in DMF and using 50 ⁇ of bromophenol blue as an indicator of completion for the acylation reaction.
  • all peptides were subjected to N-acetylation using an acetylating solution containing 3% acetic acid anhydride and 0.5% DIEA ( ⁇ , ⁇ -diisopropylethylamine) in DMF for 90 min in room temperature.
  • the pins were washed in methanol and air dried. Removal of the amino acid side-chains blocking groups was done by immersing the pins in the de-blocking solution, containing 5% anisole and 2.5% ethane-1 ,2-dithiol in a concentrated trifluoroacetic acid (TFA). Next, the pins were washed in methanol, then in a 0.5% acetic acid solution in 50% methanol and twice in methanol and dried. Before subjecting the peptides synthesized on polyethylene pins to the cyclization process and biochemical assays, the pins were immersed in denaturing solution, enabling thorough separation of individual peptides and proper exposure thereof.
  • TFA trifluoroacetic acid
  • a sonicator was filled with 0.1 M phosphate buffer (pH 7.2) heated to 55-65°C, containing 1% SDS. Polyethylene blocks with the pins were arranged on the surface of the buffer, so that they were directed downwards, and were sonicated for 10 min (7kW/25kHz). Next the pins were washed in H 2 0 in 60°C and subjected to cyclization. The cyclization was done by oxidizing the -SH groups of Cys side-chains using 0.05% K 3 Fe(CN) 6 solution in 40% acetonitrile in 100 mM NH 4 HC0 3 buffer for 4 hours in room temperature.
  • Peptides synthesized on polyethylene pins were subjected to biological activity assays in interactions with human umbilical cord blood antibodies. To this end, the pins were equilibrated in TBS-T buffer, free places were blocked using 200 ⁇ of 1 % BSA solution in TBS-T for 1 hour in room temperature. Next, the pins were incubated in 100 ⁇ of 9 mixed samples of umbilical serum (1 H-9H) in 500-fold dilution in 1 % BSA solution in TBS-T.
  • the pins were washed 3 times for 5 min in 10 ml of TBS-T buffer and incubated with 100 ⁇ of secondary anti-human IgG antibodies conjugated with alkaline phosphatase (Sigma) in 1 :10000 dilution for 1 hour in room temperature.
  • the pins were washed 3 times for 5 min in 10 ml of TBS-T buffer and placed for 30 min in 200 ⁇ of substrate AP Yellow (pNPP; p-nitrophenylphosphate)/pin in room temperature.
  • mice were immunized 4 times with 10 pg doses of an antigen.
  • the MPL adjuvant was used due to low immunogenicity.
  • the adjuvant itself is non-specifically increasing antibody titer against the administered antigen, especially the peptide conjugate. Therefore, immunization with conjugates on the tetanus toxoid carrier (TT) was done without using an adjuvant, since the MPL adjuvant itself raised the titer of the studied antibodies.
  • TT tetanus toxoid carrier
  • mice sera after 4 immunizations were tested against the protein antigen (OmpC) in a standard ELISA and against the peptide one (RYDERY) using ELISA on polyethylene pins (Fig. 7).
  • Mice were immunized with the conjugate GLNRYDERYIG-poly-Lys in the MPL adjuvant.
  • Induction of IgG antibodies against the OmpC protein and the RYDERY-pin peptide was shown, wherein the antibody levels against the protein were higher than those for highly specific antibodies against the RYDERY peptide.
  • the antibody levels after immunization with the GLNRYDERYIG-polyLys antigen in the MPL adjuvant were comparable with the levels after administration of MPL alone.
  • the disclosed conjugates are useful for constructing a vaccine for induction of protection against pathogenic Enterobacteriaceae species, in particular the Shigella species.
  • the vaccine actively protecting against development of gastrointestinal infections is intended for humans living in countries having low life and sanitary standards.
  • the vaccine is also useful for use for medical personnel, soldiers, tourists, particularly for persons having a defect of deficiency in humoral immunity, also for persons, especially children with humoral immunity deficiencies.

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Abstract

The object of the invention is a conjugate of a protein carrier and a peptide antigen, forming an OmpC protein epitope, applicable as a vaccine protecting against infection caused by coliform bacteria of the Enterobacteriaceae family, particularly species of the Shigella genus. The object of the invention is also a pharmaceutical composition and a vaccine comprising said conjugate and a method of obtaining the conjugate and use thereof.

Description

Immunogenic conjugate
The object of the invention is a conjugate of a protein carrier and a peptide antigen, forming an OmpC protein epitope, applicable as a vaccine protecting against infection caused by coliform bacteria of the Enterobacteriaceae family, particularly species of the Shigella genus. The object of the invention is also a pharmaceutical composition, a diagnostic composition and a vaccine comprising said conjugate and a method of obtaining the conjugate and use thereof.
The Shigella genus bacteria are among the most dangerous pathogenic species belonging to the Enterobacteriaceae family. Shigellosis (dysentery, bacterial dysentery) is a disease characterized by acute gastrointestinitis caused by bacterial penetration into epithelial cells lining the intestinal walls. Among the most characteristic symptoms of shigellosis are watery, bloody or mucous diarrhea, nausea, vomiting, fever and abdominal pains.
Among the Enterobacteriaceae species, a group of obligate pathogens deserves particular attention, especially the species of the Shigella, Salmonella and Yersinia genera, responsible for bacterial gastrointestinal infections, especially bacilli being a direct cause of diarrhea, typhoid fever, bacterial dysentery and other intestinal diseases. Coliform bacteria, along with streptococci and staphylococci, are referred to as being among the most frequent and most severe etiological factors for diseases with bacterial etiology. According to reports from The World Health Organization (WHO) per 4.5 billion of disease cases, gastroenteritis is the cause of death for 1.9 million people per year and rank as the third among diseases with the highest percentage of mortality in the world. Moreover, it is estimated that about 99% of all deaths occur in developing countries, and this concerns infants and children below five years of age in particular. Gastrointestinal infections caused by coliform bacteria of the Enterobacteriaceae family pose a threat to the population around the world, in particular among refugees, sailors and tourists, who may transmit an infection due to geographical movements of the population. Statistical data indicate that about 50% of all travelers to the third world countries suffer from the so-called 'traveler's diarrhea'.
The spreading of antibiotic resistance mechanisms among the Enterobacteriaceae family bacteria, as well as emergence of strains exhibiting multi-drug resistance, is an important and difficult therapeutic problem. Due to large similarities of microorganisms belonging to this family, differentiating between individual species, particularly identification thereof is impeded. This complicates adopting suitable therapeutic procedures enabling treatment, especially the selection of the suitable, directed and effective antibiotic treatment. It appears therefore that the most effective action should be prevention. Both maintaining proper sanitary conditions as well as development and detailed characterization of additional factors enabling quick diagnostics of infection and preventive measures, aiming at development of vaccines protecting against infections caused by these pathogens, are within the scope of this term.
Known in the prior art are vaccines composed of live, attenuated (weakened) strains (Barnoy S., Baqar S., Kaminski R. W., Collins T, Nemelka K, Hale T.L., Ranallo R.T., Venkatesan M.M.: Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection. Vaccine, 2011; 29(37): 6371-637) or of killed bacterial cells, inactivated with high temperature, irradiation or chemical factors, showing the ability to induce immunological response associated with the gastrointestinal tract. In case of constructing vaccines comprising live but weakened bacterial strains, using mutants obtained as a result of deleting the virG protein gene, responsible for Shigella cell mobility, associated with utilization of actin fibers is considered. Works illustrating the results of conducted clinical studies currently being in the I and II phase were published by Ranallo R.T., Fonseka S., Boren T.L., Bedford L.A., Kaminski R. W., Thakkar S., Venkatesan M.M.: Two live attenuated Shigella flexneri 2a strains WRSf2G12 and WRSf2G15: a new combination of gene deletions for 2nd generation live attenuated vaccine candidates. Vaccine. 2012; 30(34): 5159-5171.
Moreover, genes responsible for enterotoxin expression (senA, senB) and the gene responsible for the expression of Lipid A acyltransferase {msbB2) were removed from S. flexneri 2a bacilli, which, in case of applying to vaccines obtained as a result of these modifications of the strains WRSf2G12 and WRSf2G15, leads to an increase in the safety of the vaccine. Both strains are severely weakened and retain genetic stability, both in in vitro tests done on cell lines and in those in vivo in animal models. The studies conducted on guinea pigs have shown that following the immunization of animals administered ocularly, strong induction of both systemic and mucosal immunological response occurs. It was found that the animals immunized with attenuated WRSf2G12 and WRSf2G15 strains are protected against infection with a homologous wild-type Shigella flexneri strain (Ranallo R. T., Fonseka S., Boren T.L., Bedford L.A., Kaminski R. W., Thakkar S., Venkatesan M.M.: Two live attenuated Shigella flexneri 2a strains WRSf2G12 and WRSf2G15: a new combination of gene deletions for 2nd generation live attenuated vaccine candidates. Vaccine. 2012; 30(34): 5159-5171).
Similar modifications are introduced in the Shigella sonnei genome. Deletions introduced within the VirG protein gene have prevented this pathogen from spreading within the tissues of the host and have also guaranteed the induction of protective immune response against this pathogen. The thus prepared strain formed a basis for the WRSS1 vaccine, which entered the I phase of clinical trials. However, the occurrence of side effects, such as fever and mild diarrhea in some volunteers led to introducing additional mutations aiming at lowering the toxicity caused by the presence of large amounts of lipopolysaccharide in WRSS1. Similarly to the S. flexneri strains, the S. sonnei WRSS1 strain mutant was weakened further by inactivating the senA, senB (WRSs2) genes and additionally the msbB2 (WRSs3). Studies performed on rhesus monkeys have shown that both WRSs2 and WRSs3 cause stimulation of systemic and mucosal immunological response in animals, on a level similar to the one when the WRSS1 strain is used, while guaranteeing the use of a safer vaccine with a comparable immunogenicity (Barnoy S., Baqar S., Kaminski R. W., Collins T., Nemelka K., Hale T.L, Ranallo R.T., Venkatesan M.M.: Shigella sonnei vaccine candidates WRSs2 and WRSs3 are as immunogenic as WRSS1, a clinically tested vaccine candidate, in a primate model of infection. Vaccine, 2011; 29(37): 6371- 6378).
Attenuated vaccines are comprised of a suitably weakened version of a live microorganism, not capable of causing infection. Using microorganisms of this type in a vaccine is a procedure being the best imitation of causing a natural infection and it leads to formation of a complete immunological response, which in case of this pathogen entering the organism enables the recognition of its antigens. These vaccines elicit both types of immunological response, both cellular and humoral one, and induce a long-term immunity after administering only 1 -2 doses of the vaccine. Unfortunately, use of the attenuated vaccines always carries a certain risk of a post-vaccination infection, associated with the possibility of the microorganism reverting to a virulent form as a result of mutation or it may also arise from an insufficient inactivation of virulent pathogens, or side effects may occur, inter alia allergies caused by the presence of numerous components included in bacterial cells.
Furthermore, the vaccines comprising live or weakened strains cannot be administered to persons with acquired immunodeficiency syndrome (e.g. AIDS patients) and to patients having a temporary immunological incapacity, including patients suffering from various types of cancer, receiving chemotherapy.
Obtaining weakened viral strains, their genome being relatively small, is a much simpler process than attenuation of bacterial cells, having a substantially larger genome. In case of viruses, an often sufficient mutagenic factor leading to weakening of the microorganism is growing the virus in conditions unfavorable for its development. On the other hand, in case of bacteria having thousands of different genes, the attenuation process is much more complicated and more difficult to control. In order to inactivate key genes for the pathogenic bacteria employing genetic engineering methods is very often required.
An alternative for attenuated vaccines are inactivated vaccines, comprising killed microorganism strains. One promising vaccine, currently being in the I phase of clinical trials, is the SsWC vaccine comprised of Shigella sonnei cells inactivated with formalin. Pre-clinical trials on the SsWC vaccine have shown that it has immunogenic properties and after infecting guinea pigs with a virulent strain of Shigella sonnei has a protective effect against cornea inflammation. Clinical trials conducted on a small group of volunteers were able to ascertain that administering 2.0 x 1010 inactivated bacterial cells, at different time intervals and with different dose numbers for 4 weeks, causes an increase in titer of anti-SsWC, anti-LPS and anti-lpaC antibodies in serum and stool of the volunteers. Furthermore, the vaccine was well tolerated by the body and no side effects associated with occurrence of fever or gastrointestinal infection were reported. After SsWC vaccination the titer of IgG and IgA antibodies in serum of the volunteers showed an upward trend. Among the 7 vaccinated volunteers, the increase in anti-SsWC, anti-LPS and anti-lpaC antibodies was found in 6 (86%), 4 (57%) and 5 (61 %) persons, respectively. The presence of secretory IgA was analyzed in 5 persons, among which 5 (100%) had antibodies directed against SsWC, 3 (42%) against LPS and 3 (42%) against IpaC {McKenzie R., Walker R.I., Nabors G.S., Van De Verg L.L., Carpenter C, Gomes G., Forbes E., Tian J.H., Yang H.H., Pace J.L., Jackson W.J., Bourgeois A.L: Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial. Vaccine. 2006; 24(18): 3735-3745). The vaccine elicits response against LPS, which is specific for the given serotype, also eliciting specific immune response against proteins forming the antigens of the plasmid responsible for bacterial adhesion and invasion of epithelial cells (Ipa - Invasion plasmid antigen). The advantage is the fact that it induces intestine-associated mucosal response, as well as systemic response, in large part similar to the one induced during natural infection with the pathogen. Preparation thereof is relatively inexpensive, and administration thereof uncomplicated and does not require the use of needles. However it should be noted that the oral SsWC vaccine comprising whole, killed bacterial cells is defined and standardized to a small degree, due to comprising a broad panel of bacterial antigens {McKenzie R., Walker R.I. , Nabors G.S., Van De Verg L.L., Carpenter C, Gomes G., Forbes E., Tian J.H., Yang H.H., Pace J.L., Jackson W.J., Bourgeois A.L: Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial. Vaccine. 2006; 24(18): 3735-3745).
In contrast to live and attenuated strains, the killed pathogens are more stable and safer, as well as easier to transport and store. Nonetheless, these vaccines induce a weaker immune response, characterized by a shorter immunological memory. Therefore, an effective immunization most often requires administering several booster doses. Pathogen inactivation may also lead to changes in conformation of antigens present on cell surface, thus lowering their immunogenic properties {Burke C.J., Hsu T.A., Volkin D.B.: Formulation, stability, and delivery of live attenuated vaccines for human use. Crit Rev Ther Drug Carrier Syst. 1999;16(1): 1-83).
Problems associated with using whole bacterial cells in vaccines do not apply to new generation vaccines, comprising only a set of pre-selected antigens eliciting specific immune response, substantially limiting the occurrence of side effects. The progress in modern technologies is the reason why studies concerning development of numerous infectious diseases and the search for vaccine antigens have begun to be considered on a genomic and proteomic level. Selected ingredients of subunit vaccines enable identification of factors directly involved in inducing immune response. Chemical synthesis of antigens being sequences of peptide or polysaccharide epitopes, as well as isolation of specific cellular fractions of the microorganisms and expression of proteins from virulent strains in different systems (e.g. bacterial, viral, yeast, animal, plant) form the basis in the development of subunit and conjugate vaccines. In case of vaccines protecting against the development of bacterial dysentery, subunit vaccines are taken into account, based on surface antigens, such as lipopolysaccharides (LPS) (Lowell G.H., Kaminski R.W., Grate S., Hunt R.E., Charney C, Zimmer S., Colleton C: Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice. Infect. Immun., 1996; 64: 1706-1713), or cell wall outer membrane proteins (Witkowska D., Bartys A., Gamian A.: Biafka osfony komorkowej pateczek jelitowych / ich udziaf w patogennosci oraz odpornosci przeciwbakteryjnej. Postepy Hig. Med. Dosw. 2009; 63: 176- 199), as well as hybrid vaccines, with antigens deriving from many different microorganisms (e.g. Shigella, E. coli, S. typhi), which may protect the organism against several diseases at once (Kotloff K.L., Herrington D.A., Hale T.L., Newland J. W., Van De Verg L, Cogan J.P., Snoy P.J., Sadoff J.C., Formal S.B., Levine MM..: Safety, immunogenicity, and efficacy in monkeys and humans of invasive Escherichia coli K- 12 hybrid vaccine candidates expressing Shigella flexneri 2a somatic antigen. Infect. Immun., 1992; 60: 2218-2224).
Invaplex 50, being a complex of invasins: IpaB, IpaC, IpaD isolated from Shigella flexneri 2a and an LPS, is a subunit vaccine type being promising as inducing protective immune response against infections with bacillary dysentery. Studies in animal models have shown that mice and guinea pigs immunized with this complex were protected against lethal dose of the virulent S. flexneri strain (Turbyfill K.R., Kaminski R. W., Oaks E. V.: Immunogenicity and efficacy of highly purified invasin complex vaccine from Shigella flexneri 2a. Vaccine, 2008; 26: 1353-1364). A method of manufacturing thereof on a large scale will be obtaining recombinant forms of invasion proteins, and the LPS comprised therein will be extracted from native bacterial cells. This however indicates that the costs of developing such a vaccine, associated with the necessity of purification of the antigens comprised therein, will be high (Riddle M.S., Kaminski R. W., Williams C, Porter C, Baqar S., Kordis A., Gilliland T, Lapa J., Coughlin M., Soltis C, Jones E., Saunders J., Keiser P.B., Ranallo R. T., Gormley R., Nelson M., Turbyfill K.R., Tribble D., Oaks E. V.: Safety and immunogenicity of an intranasal Shigella flexneri 2a Invaplex 50 vaccine. Vaccine., 2011; 29(40): 7009-7019).
Studies on a subunit vaccine comprising TTSS of IpaB and IpaD protein were also conducted by a different team, wherein immunogenicity and protective properties of the IpaB and IpaD proteins were confirmed in a mouse model. It was shown that these proteins when administered with an adjuvant in the form of genetically modified heat-labile E. coli toxin have immunogenic and protective properties. Mice immunized with IpaB protein only or IpaB complexed with IpaD and in both cases supported by an adjuvant, were protected against lethal dose of S. flexneri and S. sonnei. Intranasal immunization of animals causes eliciting systemic and mucosal immune response, directed against both these proteins, and against IpaB in particular. The drawback of this vaccine, apart from the high cost of the recombined ingredients mentioned above, are side effects: problems associated with irritation of nose mucous membranes.
Other proteins taken into account as conjugate vaccine antigens are outer membrane proteins (OMPs). They are displayed on bacterial surface and during pathogen invasion they are the first to contact the cells of the infected organism. Therefore, they are characterized by high potential for subunit vaccine construction. Immunochemical analyses of the major outer membrane proteins from S. flexneri, S. sonnei, S. dysenteriae and S. boydii have shown similar protein profiles and cross-reactivity among all four species of the Shigella genus (Mulczyk M., Adamus G., Witkowska D., Romanowska E.: Studies on virulence of Shigella flexneri. Protective effect of outer membrane proteins. Arch. Immunol. Ther. Exp., 1981; 29: 85-90, Witkowska D., Adamus G., Mulczyk M., Romanowska E.: Outer membrane protein composition of Shigella flexneri. FEMS Microbiol. Lett, 1982; 13: 109-1 11). These vaccines hold out a prospect of selective protection against specific pathogens. Unfortunately, vaccines comprising only the selected antigens usually require a larger number of booster doses and adjuvants.
From the P.401502 application an epitope of a cell wall outer membrane protein from Shigella flexneri 3a is known, having a sequence of A1 -A2-A3-A4-A5-A6, wherein:
A1 is R,
A2 is Y,
A3 is D, R, E, N or Q, A4 is E, D, N or Q, A5 is R, A6 is Y, G or F; having immunoreactivity with human serum, particularly umbilical cord blood serum.
Even though studies on a vaccine protecting against bacterial dysentery are conducted utilizing practically all the latest methods, currently no licensed vaccine is available on the pharmaceutical market. This fact not only stresses the need for its development but also shows how this vaccine is not an easy objective. All of the approaches and vaccines above have disadvantages, namely live, devoid of toxins, inactivated, attenuated cellular vaccines comprise whole bacterial cells, posing a risk of a post-vaccination infection, a possibility of the microbe reverting to a virulent form, mutation, inadequate inactivation of virulent pathogens, there may be side effects, allergies due to numerous cellular components, use of genetic constructs, live and weakened strains cannot be administered to persons with decreased immunity. Whereas the disadvantage of the proposed subunit vaccines are high costs and side effects in the form of problems associated with irritation of nose mucous membranes.
The problem in the prior art is the lack of a safe and effective vaccine against the opportunistic bacterial pathogens of the gastrointestinal tract. For the vaccine to be accepted it should fulfill a variety of criteria, such as the activity in the intestinal mucosa, providing a long-term immunological protection, lack of side effects. Moreover, it is desired for it to be simple to administer and relatively inexpensive, since the main recipient will be children in countries with a low economic level.
Simultaneously, the existing approaches to constructing vaccines demonstrate numerous drawbacks, such as lengthy process of purification of native antigens, denaturation thereof during isolation, low standardization of preparations being mixtures of different antigens.
The object of the invention is thus to provide ingredients for a vaccine against Enterobacteriaceae, particularly against opportunistic bacterial pathogens of the gastrointestinal tract, especially against bacteria of the Shigella genus, solving the existing problems and fulfilling the above criteria.
The object of the invention is also to provide a method of constructing a vaccine, excluding the possibility of the occurrence of infections in consequence of weakened strains reverting to a virulent form.
The object of the invention is a conjugate comprised of a carrier of a hydrophobic nature and a peptide antigen forming an epitope of the OmpC protein, having an amino acid sequence of A1 -A2-A3-A4-A5-A6, wherein:
A1 is R,
A2 is Y,
A3 is D, R, E, N or Q, A4 is E, D, N or Q,
A5 is R,
A6 is Y, G or F. Preferably, the peptide antigen is selected from the group comprising peptides having an amino acid sequence of: RYDERY, RYDDRY, RYEERY, RYQERY or RYDQRY.
Preferably, the peptide antigen has a sequence of RYDERYIGC.
Preferably, the peptide antigen has a sequence of GLNRYDERYIGC.
Preferably, the peptide antigen has a loop conformation.
Preferably, the carrier is a protein carrier selected from the group comprising tetanus toxoid, diphtheria toxoid or bovine serum albumin.
Preferably, the protein carrier is tetanus toxoid.
Preferably, the carrier is polyethylene.
The object of the invention is also a pharmaceutical composition comprising said conjugate and a pharmaceutically acceptable carrier.
A further object of the invention is a pharmaceutical composition comprising said conjugate for induction of antibodies for passive immunization in treatment of infections caused by coliform bacteria of the Enterobacteriaceae family.
Preferably, the infections are caused by bacteria of the Shigella genus.
Another object of the invention is a vaccine comprising said conjugate, a pharmaceutically acceptable carrier and optionally an adjuvant.
Preferably, the vaccine induces the production of antibodies directed against coliform bacteria of the Enterobacteriaceae family, especially bacteria of the Shigella genus.
A further object of the invention is a vaccine comprising said conjugate, a pharmaceutically acceptable carrier and optionally an adjuvant for prevention and treatment of diseases caused by coliform bacteria of the Enterobacteriaceae family, especially bacteria of the Shigella genus.
A further object of the invention is a method of obtaining said conjugate, comprising the following steps: a) providing linkers through bromoacetylation of amine group in carrier protein b) conjugating the peptide with the protein carrier; c) deactivating the unreacted bromoacetyl groups of the carrier protein. Preferably, bromoacetylation of the carrier protein is conducted in a carbonate buffer with pH of 8.3, using bromoacetic acid N-hydroxysuccinimide ester, preferably in concentration of 1 mg of ester per 1 mg of protein (maintaining the protein concentration c = 2 mg/ml).
Preferably, after bromoacetylation the reaction mixture is subjected to dialysis in 0.1 M sodium bicarbonate with pH of 8.3 and the ester is removed on a column.
Preferably, peptides containing thiol groups for conjugation are suspended in 0.1 M carbonate buffer with 2 mM EDTA with pH of 8.3, next bromoacetylated protein is added to the peptide solution, followed by adjusting the pH with 0.1 M NaOH to the value of 8.5.
Preferably, deactivation is conducted using 10 μΙ of 2-mercaptoethanol per 1 ml of the reaction mixture during incubation for 1 hour in room temperature.
A further object of the invention is a use of said conjugate for preparation of a vaccine against Enterobacteriaceae, especially bacteria of the Shigella genus.
A further object of the invention is a use of said conjugate for preparation of a diagnostic test for detecting Enterobacteriaceae, especially bacteria of the Shigella genus.
Another object of the invention is a use of said conjugate for preparation of blood-based, immunoglobulin therapeutic preparations, specific against Enterobacteriaceae, especially bacteria of the Shigella genus.
Unexpectedly, it has been found that there is a large difference in antigenicity between conjugates of the OmpC peptide epitope and a carrier, and that it results from the nature of the employed carrier, which has not been observed before. Namely, the peptides synthesized on hydrophobic carriers such as polyethylene pins or protein carriers such as tetanus toxoid, diphtheria toxoid or bovine serum albumin, are much better recognized by antibodies than the same peptides linked with polylysine (Fig. 7).
Unexpectedly, replacing the peptide carrier from hydrophilic polylysine with a more hydrophobic carrier, e.g. polyethylene or tetanus toxoid (TT) protein, substantially increased the antigenicity of the conjugate with umbilical cord serum and immunogenicity in mice. Therefore, it was not obvious that the biologically active conformation of the peptide is dependent on the carrier type used in the conjugate. As a result, for peptides with an epitope structure only such carriers that provide a highly immunogenic conjugate were used. This indicated the peptide adopting the correct epitope conformation in hydrophobic conditions. Such carriers with pronounced hydrophobicity are tetanus toxoid, diphtheria toxoid, commonly used in human vaccines, and also bovine serum albumin as a model protein. The not obvious solution was found to be synthesizing the peptide in a loop conformation, according to the model present in the OmpC protein molecule, as an epitope for conjugation with a carrier. It was not obvious that the RYDERY peptide requires adopting the correct conformation, in the form of a loop, in order to form an active epitope. Such an epitope, recognized by umbilical antibodies is formed in hydrophobic conditions, as provided by polyethylene or the TT, BSA carriers but not the polylysine carrier.
Selecting the suitable carrier was found to be extremely important, since it allowed eliciting specific response directed against said epitope, without the use of an adjuvant. Previous experiments have shown that a peptide synthesized on a polylysine carrier does not show as good immunogenic properties, as a peptide conjugated with hydrophobic carriers TT and BSA, enabling the synthesis of the conjugate with the peptide in a loop conformation.
The prepared conjugates according to the invention have thus shown their immunogenicity, manifested by induction of high titer of highly specific immunoglobulins, recognizing the peptide antigen.
The proposed solution provides an efficient and safe vaccine based on a peptide epitope bound to a carrier, a vaccine devoid of genetic material or toxins, being stable and much less expensive in production and storage.
An isolated epitope, not only without the cell, but also lacking the remaining initial protein, is an active fragment, an element required for the construction of a vaccine without the whole high molecular load. A preparation method moves away from traditional methods of obtaining attenuated vaccines or the ones employing whole bacteria or whole protein antigens.
A synthetic peptide vaccine comprising said epitope displays protein antigen sequences showing a particular antigenic and immunogenic activity, while conjugating the peptide epitope with larger carriers enhances their presentation to the cells of the immune system.
Subunit vaccines, wherein the peptide vaccine according to the invention is classified, are safe and do not carry a risk of post-vaccination infection. Moreover, such a vaccine is relatively inexpensive to produce, and also utilizes techniques not carrying a risk of infection with a virulent strain. The vaccine according to the invention is devoid of components inducing an additional inflammation and excessive reactogenicity of the preparation, such as LPS or toxins.
An undoubted advantage of using peptide antigens is the handling of a well-defined and standardized component, having the characteristic structure of the native protein antigen. The vaccine according to the invention may be modified very easily and in a controlled manner, by chemical means, which may be crucial for preventing allergy or eliciting auto-immune response directed against own tissues.
Using the conjugate for immunization allows replacing the classic vaccine based on employing thermally inactivated 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, effective, inexpensive and can be produced on a large scale. The vaccine will replace all the current vaccines against infections with Shigella and other pathogenic enterobacteria.
Conjugates comprised of carriers and peptides immobilized thereon according to the invention may then be used for preparing an affinity gel for isolation of protective antibodies from donor blood/serum. The thus obtained antibodies may be used for preparing blood-derived, immunoglobulin therapeutic preparations, specific against enterobacteria.
The carriers comprising peptides immobilized thereon according to the invention may also be used for diagnostics of specific deficiencies in humoral immunity. Due to their specificity, diagnostic tests comprising peptides according to the invention are particularly useful for determination of the levels of specific antibodies against important pathogens of the gastrointestinal tract, especially significant in pediatrics.
Description of figures
Fig. 1. A. Electrophoretic image (SDS-PAGE) of the tetanus toxoid monomer (5 g) and the conjugates RYDERYIGC T (5 \ g) and GLNRYDERYIGC T (5 pg); B. Electrophoretic image (SDS-PAGE) of BSA (2 \Jtg) and the conjugates RYDERYIGC:BSA (2 Mg) and GLNRYDERYIGC: BSA (2 Mg).
Fig. 2. A. MALDI-TOF-MS spectrum of the TT monomer and the conjugates RYDERYIGC:TT and GLNRYDERYIGC:TT. B. MALDI-TOF-MS spectrum of BSA and the conjugates RYDERYIGC:BSA and GLNRYDERYIGC:BSA.
Fig. 3. A. Reactivity of the TT-absorbed umbilical cord blood serum with the OmpC protein, TT and the conjugates GLNRYDERYIGC:TT and RYDERYIGC:TT. B. Reactivity of the umbilical cord blood serum with the OmpC protein, BSA and the conjugates GLNRYDERYIGC:BSA and RYDERYIGC:BSA.
Fig. 4. Anti-OmpC IgG class antibody titer in mouse serum after the I, II, III and IV immunization of the animals with the conjugates GLNRYDERYIGC:TT and RYDERYIGC:TT, the TT protein and PBS.
Fig. 5. A. IgG class antibody levels in mouse serum* after 4 immunizations with respective antigens in the OmpC test. B. IgG class antibody levels in mouse serum after immunization with the conjugates of the GLNRYDERYIGC peptide with TT/BSA in the test with the RYDERY peptide synthesized on 15 identical polyethylene pins.
Respective group designations introduced in Fig. 5 are: (Gr. 1 , G1 in fig.) - after 4 immunizations with the conjugate GLNRYDERYIGC:TT
(Gr. 2, G2 in fig.) - after 4 immunizations with the conjugate GLNRYDERYIGC:BSA
(Gr. 3, G3 in fig.) - after 2 immunizations with the conjugate GLNRYDERYIGC:TT and after 2 immunizations with the conjugate GLNRYDERYIGC:BSA
(Gr. 4, G4 in fig.) - after 4 immunizations with the TT carrier
(Gr. 5, G5 in fig.) - after 4 immunizations with the BSA carrier
(Gr. 6, G6 in fig.) - after 4 immunizations with PBS
Fig. 6. ELISA results showing the binding strength of individual peptides after cyclization with antibodies of human umbilical cord blood.
Fig. 7. Mice immunization with the conjugate GLNRYDERYIG-poly-Lys and mice immunization with the conjugate GLNRYDERYIGC:TT, respectively in an MPL adjuvant and without using an additional adjuvant.
The present invention is illustrated with embodiments which do not limit the scope of protection thereof.
Example 1. Peptide conjugation with a carrier protein.
Step 1 . Protein bromoacetylation. In order to protect Cys-containing peptides against dimerization before conjugating with a protein carrier, the peptide was dissolved in methylphosphine (approx. 10-fold excess of methylphosphine in relation to the peptide amount) and dried under argon, tightly sealing the vial. Free amine groups in the carrier protein before conjugation were bromoacetylated in 0.1 M carbonate buffer with pH of 8.3 using bromoacetic acid N-hydroxysuccinimide ester, in concentration of 1 mg of ester per 1 mg of protein (maintaining the protein concentration c = 2 mg/ml) for 3 hours in room temperature on a rotating mixer. During bromoacetylation pH was kept constant within 8.3 using 0.1 M NaOH. Following the reaction, the mixture was dialyzed in 0.1 M sodium bicarbonate with pH of 8.3 and the ester was removed on the PD-10 column. The bromoacetylated protein was concentrated on a membrane until the concentration of 10-20 mg/ml. The level of bromoacetylation was determined by measuring free amine groups before and after bromoacetylation using TNBS (2,4,6-Trinitrobenzenesulfonic acid).
Step 2. Conjugation. Peptides for conjugation, containing thiol groups, were suspended in 0.1 M carbonate buffer with 2 mM EDTA with pH of 8.3 in concentration of 10-50 mg/ml. The bromoacetylated protein was added to the peptide solution within 1 minute, pH was adjusted with 0.1 M NaOH to the value of 8.5. The peptide to protein ratio was within 50 to 100 mol/mol. Protein concentration was maintained within 2 mg/ml, while adjusting the concentration of the peptide. Conjugation reaction was conducted overnight, about 16 hours in room temperature on a rotating mixer in a tightly sealed vessel under argon.
Step 3. Deactivation of the unreacted bromoacetyl groups of the carrier peptide. Deactivation was performed using 10 μΙ of 2-mercaptoethanol per 1 ml of the reaction mixture for 1 hour in room temperature.
Step 4. Analysis of the conjugate. The conjugation process was monitored in 6% (TT conjugates) or 12.5% (BSA conjugates) polyacrylamide gel in SDS-PAGE (Fig. 1 ). The percentage of the polyacrylamide gel depended on the carrier protein molecular mass. The loading level of the protein molecule with the peptides was measured using MALDI-TOF-MS (Fig. 2).
Example 2. Reactivity of the peptide conjugates with human umbilical cord blood antibodies, determined by dot-blot technique.
The Immobilon P membrane was wetted by applying 2 μΙ portions of methanol spotwise. Next, antigen solutions (peptide conjugates, carrier proteins - negative control, OmpC protein - positive control) were applied to the wetted areas in suitable concentrations and left until completely dried. Free spaces on the membrane were blocked in 10 ml of 1 % BSA solution in TBS-T for 1 hour in room temperature. Next, the membrane was incubated in room temperature for 1 hour in 5 ml of antibody/serum solution suitably diluted in TBS-T buffer with 1 % BSA and washed 3 times in TBS-T buffer. In the next step of the procedure, the membrane was incubated in solution of secondary antibodies conjugated with alkaline phosphatase in 1 :10 000 dilution and washed 3 times in TBS-T buffer. Next, the membrane was placed in substrate solution, containing 5 ml of alkaline phosphatase buffer, 15% BCPI, 30% NBT. The reaction was stopped by immersing the membrane in water.
Example 3. Determination of immunogenicity of the peptide conjugates in a mouse model.
Six-weeks old BALB/c mice in groups, each of 6 animals, were immunized intraperitoneally with the antigens, the antigens (10 μg) being administered 4 times in 200 μΙ PBS buffer with 7-day intervals, according to the scheme:
Gr. 1 - 4 immunizations with the conjugate GLNRYDERYIGC:TT Gr. 2 - 4 immunizations with the conjugate GLNRYDERYIGC:BSA
Gr. 3 - 2 immunizations with the conjugate GLNRYDERYIGC:TT and 2 immunizations with the conjugate GLNRYDERYIGC:BSA Gr. 4 - 4 immunizations with the TT carrier (control group)
Gr. 5 - 4 immunizations with the BSA carrier (control group) Gr. 6 -4 administrations of PBS (control group)
The level of the induced immune response was determined after 4 immunizations of the animals, measuring IgG class antibodies with ELISA against the OmpC protein (Fig. 5a) and with ELISA on polyethylene pins against the RYDERY epitope (Fig. 5b). The conjugates based on the carrier proteins were prepared and tested. To this end, GLNRYDERYIGC and RYDERYIGC peptides were conjugated with the tetanus toxoid (M = 158.5 kDa) and BSA (M = 66.97 kDa) proteins, as well as with OVA and DT. All conjugates have shown antigenic properties, confirmed by their interaction with umbilical antibodies. Tested in a mouse model they were found to be immunogenic, in particular the conjugates comprising the elongated epitope sequence: GLNRYDERYIGC. The peptide with the RYDERY epitope sequence, in particular its longer version GLNRYDERYIGC, which has better immunogenic properties, may serve as an antigen in the construction of a conjugate vaccine. This was confirmed inter alia by the experiments concerning determination of the increase in specific anti-RYDERY antibody levels after immunization of mice with the conjugates GLNRYDERYIGC:TT/BSA. Using the TT conjugates resulted in an increase of anti-OmpC antibody titer, after immunization with the conjugate GLNRYDERYIGC:TT. The conjugate comprising the shorter RYDERYIGC:TT peptide was less immunogenic, and the RYDERY peptide without the flanking amino acids, despite having antigenic properties, is a weaker immunogen. Adjuvants were not used for immunizations, indicating that the induced response is highly specific and directed against the epitope molecule in the conjugate. The sera after immunization with the conjugates also comprised antibodies against the carrier (Fig. 5a and Fig. 5b).
Example 4. Studying the spatial structure of the cyclic peptides with in silico methods.
Peptide conformation was analyzed utilizing their structural model using in silico methods. To this end, using the pep-fold service (lif o::;b;oserv.rpbs.un;v-paris-diderot?r;PbP-hOLD a variety of peptide models were generated comprising the RYDERY motif. When selecting the best model, the sOPEP (Optimized Potential for Efficient Structure Prediction) value was employed as the key value indicative of the model being the most native or the closest to native, constituting the peptide spatial structure. Therefore, in in silico studies, epitope conformations in aqueous solutions were determined only on the basis of calculations obtained for peptide models with the lowest sOPEP energy. Conformation analysis of the obtained models was done with the Chimera software.
Example 5. Cyclization of peptides synthesized on polyethylene pins and antigenicity analysis with human umbilical cord blood antibodies. The synthesis was performed rotationally by removing the blocking Fmoc groups from an amino acid and coupling it with other amino acids. The removal of the Fmoc group was done under a fume hood in 20% piperidine solution in DMF (dimethylformamide) for 1 hour in room temperature. Next the pins were washed in DMF, then in methanol and dried under a fume hood. Aminoacylation was conducted for 4 hours or overnight in room temperature by placing the pins in 100 μΙ of 60 mM Fmoc amino acid derivative solution with 65 mM HoAt (1 -hydroxy-7- azabenzotriazole) and 60 mM DIC (diisopropylcarbodiimide) in DMF and using 50 μΜ of bromophenol blue as an indicator of completion for the acylation reaction. After the synthesis has finished, all peptides were subjected to N-acetylation using an acetylating solution containing 3% acetic acid anhydride and 0.5% DIEA (Ν,Ν-diisopropylethylamine) in DMF for 90 min in room temperature. Next, the pins were washed in methanol and air dried. Removal of the amino acid side-chains blocking groups was done by immersing the pins in the de-blocking solution, containing 5% anisole and 2.5% ethane-1 ,2-dithiol in a concentrated trifluoroacetic acid (TFA). Next, the pins were washed in methanol, then in a 0.5% acetic acid solution in 50% methanol and twice in methanol and dried. Before subjecting the peptides synthesized on polyethylene pins to the cyclization process and biochemical assays, the pins were immersed in denaturing solution, enabling thorough separation of individual peptides and proper exposure thereof. To this end, a sonicator was filled with 0.1 M phosphate buffer (pH 7.2) heated to 55-65°C, containing 1% SDS. Polyethylene blocks with the pins were arranged on the surface of the buffer, so that they were directed downwards, and were sonicated for 10 min (7kW/25kHz). Next the pins were washed in H20 in 60°C and subjected to cyclization. The cyclization was done by oxidizing the -SH groups of Cys side-chains using 0.05% K3Fe(CN)6 solution in 40% acetonitrile in 100 mM NH4HC03 buffer for 4 hours in room temperature.
Peptides synthesized on polyethylene pins were subjected to biological activity assays in interactions with human umbilical cord blood antibodies. To this end, the pins were equilibrated in TBS-T buffer, free places were blocked using 200 μΙ of 1 % BSA solution in TBS-T for 1 hour in room temperature. Next, the pins were incubated in 100 μΙ of 9 mixed samples of umbilical serum (1 H-9H) in 500-fold dilution in 1 % BSA solution in TBS-T. The pins were washed 3 times for 5 min in 10 ml of TBS-T buffer and incubated with 100 μΙ of secondary anti-human IgG antibodies conjugated with alkaline phosphatase (Sigma) in 1 :10000 dilution for 1 hour in room temperature. The pins were washed 3 times for 5 min in 10 ml of TBS-T buffer and placed for 30 min in 200 μΙ of substrate AP Yellow (pNPP; p-nitrophenylphosphate)/pin in room temperature. The color reaction was stopped by removing the polyethylene pins from the substrate solution. Absorbance was read for λ = 405 nm. After completing the assay the proteins/antibodies were dissociated from the peptides synthesized on pins in denaturation buffer in 55-65°C (0.1 M phosphate buffer with pH of 7.2, containing 1 % SDS) for 10 min using ultrasounds (7kW/25kHz). Next, the pins were washed in water in 60°C, dried and stored over a desiccant resin. ELISA results are shown in Fig. 6. Example 6. Comparison in a mouse immunogenicity model and the diagnostic potential of the peptide conjugates with poly-lysine and tetanus toxoid.
Mice were immunized 4 times with 10 pg doses of an antigen. During immunization with peptide antigens synthesized on the poly-Lys carrier, the MPL adjuvant was used due to low immunogenicity. At that time, it was found that the adjuvant itself is non-specifically increasing antibody titer against the administered antigen, especially the peptide conjugate. Therefore, immunization with conjugates on the tetanus toxoid carrier (TT) was done without using an adjuvant, since the MPL adjuvant itself raised the titer of the studied antibodies. Activity of mice sera after 4 immunizations were tested against the protein antigen (OmpC) in a standard ELISA and against the peptide one (RYDERY) using ELISA on polyethylene pins (Fig. 7). Mice were immunized with the conjugate GLNRYDERYIG-poly-Lys in the MPL adjuvant. Induction of IgG antibodies against the OmpC protein and the RYDERY-pin peptide was shown, wherein the antibody levels against the protein were higher than those for highly specific antibodies against the RYDERY peptide. The antibody levels after immunization with the GLNRYDERYIG-polyLys antigen in the MPL adjuvant were comparable with the levels after administration of MPL alone. In case of immunization with the conjugate GLNRYDERYIGC:TT without an adjuvant, a better induction of antibodies with high specificity against the RYDERY-pin peptide was achieved than in case of reacting with the whole protein antigen. The levels of specific antibodies directed against the RYDERY-pin peptide was higher after immunization with the conjugate GLNRYDERYIGC:TT than after immunization with the TT protein alone. The titer of antibodies against the OmpC protein was also increased after immunization with the carrier protein alone, which may suggest induction of additional antibodies cross-reacting with the OmpC protein, not necessarily being advantageous. The best advantageous response was achieved after immunization with the peptide conjugated with TT without an adjuvant.
The carriers tested thus far demonstrate that the peptide having an epitope sequence exhibits properties of inducing immune response manifested by secretion of specific antibodies.
It is significant, that immunizations in animal models were conducted without the use of adjuvants in order to exclude non-specific increases of the levels of these antibodies by the adjuvant itself, which was observed in these studies.
The disclosed conjugates are useful for constructing a vaccine for induction of protection against pathogenic Enterobacteriaceae species, in particular the Shigella species. The vaccine, actively protecting against development of gastrointestinal infections is intended for humans living in countries having low life and sanitary standards. The vaccine is also useful for use for medical personnel, soldiers, tourists, particularly for persons having a defect of deficiency in humoral immunity, also for persons, especially children with humoral immunity deficiencies.

Claims

Claims
1 . A conjugate comprised of a carrier of a hydrophobic nature and a peptide antigen forming an epitope of the OmpC protein, having an amino acid sequence of A1 -A2-A3-A4-A5-A6, wherein:
A1 is R,
A2 is Y,
A3 is D, R, E, N or Q, A4 is E, D, N or Q, A5 is R, A6 is Y, G or F.
2. The conjugate according to claim 1 , characterized in that the peptide antigen is selected from the group comprising peptides having an amino acid sequence of: RYDERY (SEQ ID NO: 1 ), RYDDRY (SEQ ID NO: 2), RYEERY (SEQ ID NO: 3), RYQERY (SEQ ID NO: 4) or RYDQRY (SEQ ID NO: 5) and GLNRYDERYIGC (SEQ ID NO: 6) and cyclic ones CGGGRYDERYGGGCGG(SEQ ID NO: 7), CGGRYDERYGGCGG (SEQ ID NO: 8), CGRYDERYGCGG (SEQ ID NO: 9).
3. The conjugate according to claim 1 , characterized in that the peptide antigen has a sequence of RYDERYIGC (SEQ ID NO: 10).
4. The conjugate according to claim 1 , characterized in that the peptide antigen has a sequence of GLNRYDERYIGC (SEQ ID NO: 6), CGGGRYDERYGGGCGG (SEQ ID NO: 7), CGGRYDERYGGCGG(SEQ ID NO: 8), or CGRYDERYGCGG (SEQ ID NO: 9).
5. The conjugate according to claim 1 , characterized in that the peptide antigen has a loop conformation.
6. The conjugate according to claim 1 , characterized in that the carrier is a protein carrier selected from the group comprising tetanus toxoid, diphtheria toxoid or bovine serum albumin.
7. The conjugate according to claim 1 , characterized in that the protein carrier is tetanus toxoid.
8. The conjugate according to claim 1 , characterized in that the carrier is polyethylene.
9. A pharmaceutical composition comprising the conjugate according to any one of claims 1 -8 and a pharmaceutically acceptable carrier.
10. A pharmaceutical composition comprising the conjugate according to any one of claims 1 -8 for isolation of antibodies for passive immunization in treatment of infections caused by coliform bacteria of the Enterobacteriaceae family.
1 1. Pharmaceutical composition according to claim 10, characterized in that the infections are caused by bacteria of the Shigella genus.
12. A vaccine characterized in that it comprises the conjugate according to any one of claims 1 -8, a pharmaceutically acceptable carrier and optionally an adjuvant.
13. The vaccine according to claim 12, characterized in that it induces the production of antibodies directed against coliform bacteria of the Enterobacteriaceae family, especially bacteria of the Shigella genus.
14. A vaccine comprising the conjugate according to any one of claims 1 -8, a pharmaceutically acceptable carrier and optionally an adjuvant for prevention and treatment of diseases caused by coliform bacteria of the Enterobacteriaceae family, especially bacteria of the Shigella genus.
15. A method of obtaining the conjugate according to claims 1 -8, comprising the following steps: a) providing linkers through bromoacetylation of amine group in carrier protein b) conjugating the peptide with the protein carrier; c) deactivating the unreacted bromoacetyl groups of the carrier protein.
16. The method according to claim 15, characterized in that bromoacetylation of the carrier protein is conducted in a carbonate buffer with pH of 8.3, using bromoacetic acid N- hydroxysuccinimide ester, preferably in concentration of 1 mg of ester per 1 mg of protein (maintaining the protein concentration c = 2 mg/ml).
17. The method according to claim 15, characterized in that after bromoacetylation the reaction mixture is subjected to dialysis in 0.1 M sodium bicarbonate with pH of 8.3 and the ester is removed on a column.
18. The method according to claim 15, characterized in that the peptides containing thiol groups for conjugation are suspended in 0.1 M carbonate buffer with 2 mM EDTA with pH of 8.3, next bromoacetylated protein is added to the peptide solution, followed by adjusting the pH with 0.1 M NaOH to the value of 8.5.
19. The method according to claim 15, characterized in that deactivation is conducted using 10 μΙ of 2-mercaptoethanol per 1 ml of the reaction mixture during incubation for 1 hour in room temperature.
20. Use of the conjugate according to claims 1 -8 for preparation of a vaccine against Enterobacteriaceae, especially bacteria of the Shigella genus.
21. The conjugate according to claims 1 -8 for use in diagnostic test for detecting Enterobacteriaceae, especially bacteria of the Shigella genus.
22. Use of the conjugate according to claims 1 -8 for preparation of blood-based, immunoglobulin therapeutic preparations specific against Enterobacteriaceae, especially bacteria of the Shigella genus.
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