Classifications

• • 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
• • 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
  • A61K39/0258—Escherichia
• • 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
  • A61K39/385—Haptens or antigens, bound to carriers
• • 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
  • A61K47/646—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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
  • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
• • 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
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • 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
  • A61K2039/55583—Polysaccharides
• • 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
  • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
• • 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
  • A61K2039/70—Multivalent vaccine
• • 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

• This invention relates to compositions and methods for inducing an immune response against extra-intestinal pathogenic Escherichia coli (ExPEC) to thereby provide immune protection against diseases associated with ExPEC.
• ExPEC extra-intestinal pathogenic Escherichia coli
• embodiments of this invention relate to multivalent vaccines containing conjugates of E. coli polysaccharide antigens 025B, 01 A, 02, and 06A each covalently bound to a detoxified exotoxin A of Pseudomonas aeruginosa carrier protein and uses of the vaccines to provide effective immune protection against ExPEC infection.
• Extra-intestinal pathogenic E. coli are normally harmless inhabitants of human gut.
• ExPEC strains can possess virulence factors for the colonization and infection of sites outside of the gastrointestinal tract to cause diverse and serious invasive diseases, resulting in significant morbidity, mortality, and costs annually (see, e.g., Johnson et al., J Lab Clin Med. 2002;139(3):155-162; Kohler et al., Int JMed Microbiol. 2011 ;301(8):642- 647; Foxman, Am J Med. 2002;113 Suppl 1A:5S-13S; and Russo et al., Microbes Infect.
• ExPEC strains are the most common cause of urinary tract infection (UTI). They are also a contributor to surgical site infections and neonatal meningitis (Johnson et al., 2002; and Russo et al., 2003), associated with abdominal and pelvic infections and nosocomial pneumonia, and are occasionally involved in other extra-intestinal infections such as
• ExPEC sequence type 131 is considered the main driver of increased drug resistance, including multi-drug resistance (Johnson et al., Antimicrob Agents Chemother. 2010; 54(l):546-550; Rogers et al., J Antimicrob Chemother. 2011 ; 66(1): 1-14).
• This clone is found in 12.5% to 30%) of all ExPEC clinical isolates, mostly exhibits serotype 025B:H4, and shows high levels of fluoroquinolone resistance, which is often accompanied by
• trimethoprim/sulfamethoxazole resistance (Rogers et al, 2011, and Banerjee et al., Antimicrob Agents Chemother. 2014; 58(9):4997-5004).
• the O-antigen serotype is based on the chemical structure of the O polysaccharide antigen, the outer membrane portion of the lipopolysaccharide (LPS) in a Gram-negative bacterium. More than 180 E. coli O-antigens have been reported (Stenutz et al., FEMS
• ExPEC infection can be caused by any serotype. Although there is an overrepresentation of certain serotypes in ExPEC infection, surface polysaccharides from ExPEC isolates nonetheless exhibit considerable antigenic diversity, which makes the development of an ExPEC vaccine based on surface polysaccharides extremely challenging (Russo et al., Vaccine. 2007; 25: 3859-3870). Also, certain O-antigens may be poorly immunogenic.
• the vaccine composition such as the choice of serotypes for inclusion in a vaccine and the dosage levels of the included serotypes, can be critical, since use of a vaccine against certain serotypes may potentially increase carriage of and disease from serotypes not included in the vaccine, or even a serotype that is included in the vaccine but only weakly effective in immunizing against the serotype (Lipsitch, Emerging Infectious Diseases; 1999, 5:336-345).
• a vaccine should maximize its beneficial effects in the prevention of disease caused by serotypes included in the vaccine, while minimizing the risk of added disease from increased carriage of non-vaccine serotypes.
• E. coli 025B antigen appears to be somewhat less immunogenic than other E. coli O-antigens (e.g., 01 A, 02, and 06A) when tested as conjugates of the O-antigens each covalently bound to a detoxified exotoxin A of P.
• EPA aeruginosa carrier protein
• vaccination with a composition containing EPA conjugates of E. coli 025B antigen and EPA conjugates of one or more additional E. coli O- antigens at an appropriate dose and ratio provides an improved immune response against the ExPEC 025B serotype and the one or more additional ExPEC O-serotypes.
• EPA aeruginosa
• the invention relates to a composition
• a composition comprising an E. coli 025B antigen at a first concentration of 8 to 48 ⁇ ⁇ , and at least one additional E. coli O-antigen at a second concentration that is 10% to 100% of the first concentration, wherein each of the E. coli 025B antigen and the at least one additional E. coli O-antigen is
• the composition is effective in inducing an immune response against the E. coli 025B antigen and the at least one additional E. coli O-antigen in a subject in need thereof.
• the at least one additional E. coli O-antigen is present at a second concentration of at least 5 ⁇ g/ml, more preferably, at a second concentration of least 8 ⁇ g/ml.
• the invention relates to a composition
• a composition comprising an E. coli 025B antigen at a first concentration of 10 to 36 ⁇ g/ml, and at least one additional E. coli O- antigen selected from the group consisting of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06A antigen, wherein each of the additional E. coli O-antigens has a concentration that is independently 50% to 100% of the first concentration of the E. coli 025B antigen in the composition, and each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA carrier protein.
• the invention relates to a composition
• a composition comprising an E. coli 025B antigen at a first concentration of 10 to 36 ⁇ g/ml, and a second concentration of each of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06 A antigen, wherein each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA carrier protein, and the ratio of the first concentration to the second concentration is 1 : 1 to 2: 1.
• the composition comprises the E.
• the composition comprises 16 or 32 ⁇ g/ml of the 025B antigen.
• the invention in another general aspect, relates to a method of inducing an immune response to extra-intestinal pathogenic E.coli (ExPEC) in a subject in need thereof.
• the method comprises administering to the subject an E. coli 025B antigen at a first effective amount of 4 to 24 ⁇ g per administration, and at least one additional E. coli O-antigen at a second effective amount that is 10% to 100% of the first effective amount, wherein each of the E. coli 025B antigen and the at least one additional E. coli O-antigen is independently covalently bound to an EPA carrier protein, and the administration is effective in inducing an immune response against the E. coli 025B antigen and the at least one additional E.
• the at least one additional E. coli O-antigen is administered at a second effective amount of at least 3 ⁇ g per administration, more preferably, at a second effective amount of least 4 ⁇ g per administration.
• the E. coli 025B antigen and the at least one additional E. coli O-antigen can be administered in one composition or administered in combination from multiple compositions.
• the invention relates to a method of inducing an immune response to ExPEC in a subject in need thereof, comprising administering to the subject an E. coli 025B antigen at a first effective amount of 5 to 18 ⁇ g per administration, more preferably 8 to 16 ⁇ g per administration, and at least one additional E. coli O-antigen selected from the group consisting of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06 A antigen, wherein each of the additional E. coli O-antigens is administered at an effective amount that is independently 50% to 100% of the first effective amount of the E. coli 025B antigen, and each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA carrier protein.
• the invention relates to a method of inducing an immune response to ExPEC in a subject in need thereof, comprising administering to the subject an E. coli 025B antigen at a first effective amount of 5 to 18 ⁇ g per administration, more preferably 8 to 16 ⁇ g per administration, and a second effective amount of each of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06 A antigen, wherein each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA carrier protein, and the ratio of the first effective amount to the second effective amount is 1 : 1 to 2: 1.
• the E. coli 025B antigen at a first effective amount of 5 to 18 ⁇ g per administration, more preferably 8 to 16 ⁇ g per administration, and a second effective amount of each of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06 A antigen, wherein each of the E.
• coli 025B, 01 A, 02 and 06A antigens are administered at a dosage ratio of 1 : 1 : 1 : 1 or 2:1 : 1 : 1 , and the E. coli 025B antigen is administered at 5 ⁇ g, 8 ⁇ g or 16 ⁇ g per administration.
• the E. coli 025B, 01 A, 02 and 06A antigens are administered to the subject in one composition, more preferably, in compositions comprising the E.
• coli 025B, 01 A, 02 and 06A antigens each independently covalently bound to an EPA carrier protein, wherein the concentrations of these O-antigens in the compositions are chosen from respectively 16:8:8:8 ⁇ g/ml (i.e., 16 ⁇ g/ml of 025B antigen, 8 ⁇ g/ml of 01A antigen, 8 ⁇ £/ ⁇ 1 of 02 antigen, and 8 ⁇ g/ml of 06A antigen), 16: 16:16:16 ⁇ , 32: 16:16:16 ⁇ ⁇ 1 and 32:32:32:32 ⁇ g/ml.
• 0.5 ml of such a composition is administered to a subject to achieve a dosage per administration of E.
• coli 025B, 01 A, 02 and 06A antigens respectively at 8:4:4:4 ⁇ g (i.e., 8 ⁇ g of 025B antigen, 4 ⁇ g of 01A antigen, 4 ⁇ g of 02 antigen, and 4 ⁇ g of 06A antigen), 8:8:8:8 xg, 16:8:8:8 ⁇ g or 16: 16:16: 16 ⁇ g.
• the immune response induced by a method of the present invention prevents an invasive ExPEC disease caused by ExPEC serotypes 01 A, 02, 06A and 025B in a human subject in need thereof.
• Diseases associated with ExPEC or ExPEC diseases include, but are not limited to, urinary tract infection, surgical-site infection, bacteremia, abdominal or pelvic infection, pneumonia, nosocomial pneumonia, osteomyelitis, cellulitis, pyelonephritis, wound infection, meningitis, neonatal meningitis, peritonitis, cholangitis, soft- tissue infections, pyomyositis, septic arthritis, and sepsis.
• the human subject is an at- risk human subject, who has or is at risk of having an invasive ExPEC disease, such as an elderly human, a hospitalized patient, a human child, an immunocompromised human, a pregnant woman, people with diabetes or wound injuries, people who recently had or are scheduled to have a surgery, etc.
• an invasive ExPEC disease such as an elderly human, a hospitalized patient, a human child, an immunocompromised human, a pregnant woman, people with diabetes or wound injuries, people who recently had or are scheduled to have a surgery, etc.
• each of the O-antigen is covalently bound to the EPA carrier protein at the Asn residue of a glycosylation sequence comprising Asp (Glu)-X-Asn-Z-Ser (Thr) (SEQ ID NO: 3), wherein X and Z are independently selected from any natural amino acid except Pro.
• the EPA carrier protein comprises the amino acid sequence of SEQ ID NO: 1.
• the O-antigen is covalently bound to the EPA carrier protein at a polysaccharide -to-protein weight ratio of 1 :7 to 1 :2, preferably, 1 : 5 to 1 :2.
• the weight of the O-antigen can be 15% to 50%, or 20% to 40%, of the weight of the EPA.
• Another aspect of the invention relates to a process of making a composition according to an embodiment of the invention, the process comprises combining the E. coli 025B, 01 A, 02 and 06 A antigens, each independently covalently bound to the EPA carrier protein, in one composition.
• Yet another aspect of the invention relates to use of a composition according to an embodiment of the invention for the manufacture of a vaccine or medicament for inducing an immune response to ExPEC or for preventing or treating a disease associated with ExPEC in a subject in need thereof.
• Figure 1 is an exemplary representation of the glycoconjugate vaccine production platform: the cytoplasm (marked in grey) of the host cell contains all the DNA constructs necessary for the recombinant production of the O-antigen/EPA conjugate in the periplasm (marked in white) of the host cell;
• FIG. 2 is a detailed schematic representation of the protein glycosylation process
• Figures 3A-3C depict the opsonization indices (OIs) obtained with sera derived from rats pre-immunization (empty circles) compared to 42 days post-immunization (filled squares) with one priming dose and two booster doses of indicated doses of monovalent vaccine;
• Figure 3A 02-EPA immunization
• Figure 3B 06A-EPA immunization
• Figure 3C 025B-EPA immunization
• Figure 4 shows the ELISA titers obtained with sera from human subjects vaccinated with a placebo or a tetravalent vaccine comprising E. coli antigens 01 A, 02, 06A and 025B at 4 ⁇ g polysaccharide per serotype; a significant increase in the ELISA titers between post- (30 days after injection) and pre-injection (day 1) was observed only in the vaccinated groups (* represents statistical significance, wherein multiple * represent increased degree of significance; ns, no significant difference); and
• Figures 5A-5D depict the OIs obtained with sera derived from human subjects vaccinated with a tetravalent vaccine comprising E. coli antigens 01 A, 02, 06A and 025B at 4 ⁇ g polysaccharide per serotype; immune response as indicated by 01 against placebo and components of the tetravalent vaccine;
• Figure 5A 01 A-EPA;
• Figure 5B 02-EPA;
• Figure 5C 06A-EPA;
• Figure 5D 025B-EPA; pre-injection (Day 1) and post- injection (30 days after injection), wherein a significant increase in the 01 between post- and pre-injection (indicated by *, multiple * represents increased degree of significance) was observed only in the vaccinated groups; ns, no significant difference.
• O polysaccharide As used herein, the terms "O polysaccharide,” “O-antigen”, “O antigen”, “O-antigen polysaccharide,” “O-polysaccharide antigen” and the abbreviation “OPS”, all refer to the O antigen of Gram-negative bacteria, which is a component of the lipopolysaccharide (LPS) and is specific for each serotype or sero(sub)type of the Gram-negative bacteria.
• the O antigen usually contains repeating units (RUs) of two to seven sugar residues. As used herein, the RU is set equal to the biological repeat unit (BRU).
• BRU biological repeat unit
• conjugate and “glycoconjugate” all refer to a conjugation product containing an E. coli O antigen covalently bound to a carrier protein.
• the conjugate can be a bioconjugate, which is a conjugation product prepared in a host cell, wherein the host cell machinery produces the O antigen and the carrier protein and links the O antigen to the carrier protein, e.g., via N-links.
• the conjugate can also be prepared by other means, for example, by chemical linkage of the protein and sugar antigen.
• the term "effective amount" in the context of administering an O antigen to a subject in methods according to embodiments of the invention refers to the amount of the O antigen that is sufficient to induce a desired immune effect or immune response in the subject.
• an "effective amount” refers to the amount of an O antigen which is sufficient to produce immunity in a subject to achieve one or more of the following effects in the subject: (i) prevent the development or onset of an ExPEC infection, preferably an invasive ExPEC disease, or symptom associated therewith; (ii) prevent the recurrence of an ExPEC infection, preferably an invasive ExPEC disease, or symptom associated therewith; (iii) prevent, reduce or ameliorate the severity of an ExPEC infection, preferably an invasive ExPEC disease, or symptom associated therewith; (iv) reduce the duration of an ExPEC infection, preferably an invasive ExPEC disease, or symptom associated therewith; (v) prevent the progression of an ExPEC infection, preferably an invasive ExPEC disease, or symptom associated therewith; (vi) cause regression of an ExPEC infection or symptom associated therewith; (vii) prevent or reduce organ failure associated with an ExPEC infection; (viii) reduce the chance or frequency of hospitalization
• an "effective amount” can vary depending upon a variety of factors, such as the physical condition of the subject, age, weight, health, etc.; route of administration, such as oral or parenteral; the composition administered, such as the target O antigen, the other co-administered O antigens, adjuvant, etc.; and the particular disease for which immunity is desired.
• the effective amount for the O antigen is calculated based on only the O antigen polysaccharide moiety in the conjugate.
• a first composition can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g.
• subject means any animal, preferably a mammal, most preferably a human, to who will be or has been vaccinated by a method or composition according to an embodiment of the invention.
• mammal encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., most preferably a human.
• a subject is a human infant.
• a subject is a human child.
• a subject is a human adult.
• a subject is an at- risk human adult.
• a subject is an elderly human.
• another human infant preferably a mammal, most a human.
• a subject encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., most preferably
• a subject is a human infant, including a premature human infant and a human infant born at term. In another embodiment, a subject is a human toddler.
• the terms “subject” and “patient” may be used herein interchangeably.
• premature human infant refers to a human infant born at less than 37 weeks of gestational age.
• human infant refers to a newborn to 1 year old human.
• human toddler refers to a human that is 1 year to 3 years old.
• human child refers to a human that is 1 year to 18 years old.
• human adult refers to a human that is 18 years or older.
• at-risk human adult refers to a human that is 18 years or older who is more prone to ExPEC infection than the average human adult population.
• At-risk human adult examples include, but not limited to, elderly humans,
• yielderly human refers to a human that is 55, preferably 60, more preferably 65, years or older.
• an "invasive ExPEC disease” is defined as isolation and identification of ExPEC from normally sterile body sites in a subject presenting with an acute illness consistent with bacterial infection.
• an "immunological response” or “immune response” to an antigen or composition refers to the development in a subject of a humoral and/or a cellular immune response to the antigen or an antigen present in the composition.
• E. coli 025B antigen conjugated to an EPA carrier protein appears to be less immunogenic than the other E. coli O- antigens (e.g., OIA, 02, and 06A) conjugated to the EPA carrier protein.
• This discovery leads to further investigation into the dosage of E. coli 025B antigen and the dosage ratios of various E. coli O antigens within a multivalent vaccine, thus the development of multivalent vaccines and immunization methods based on E. coli O antigens for improved immune responses against the 025B serotype and other serotypes of ExPEC.
• OIA, 06A and 025B antigens were determined to be the more frequent subtypes among the analyzed more recent clinical strains or isolates for 01 , 06 and 025 serotypes, respectively. See related disclosure on epidemiology studies in International Patent application No. PCT/EP2015/053739, the disclosure of which is herein incorporated by reference in its entirety.
• Table 1A Distribution of the most common UTI-associated E. coli serotypes from a collection of 1841 urine samples collected in Switzerland in 2012. Shown is the serotype distribution of samples from a relevant subpopulation of 671 subjects, and the distribution from all** samples.
• Table IB Prevalence of most common UTI-associated serotypes from selected literature ranging from 1987- 2011 and from retrospectively analyzed US data from 2000-2011
• Table 1C Distribution of the most common bacteremia-associated E. coli O- serotypes from a collection of 860 blood isolates collected in the US and EU in the period 2011- 2013. Indicated is the relative O-serotype distribution of the samples.
• 025B A novel 025 agglutinating clone has recently emerged in E. coli isolated from hospital settings, and this is named 025B.
• 025B For O-serotype 025, it was found using subtyping analysis by PCR that the vast majority is actually of the 025B subtype (in a study of 24 tested clinical isolates with an 025 agglutination positive phenotype, 20 were assigned to the 025B subtype while the remaining 4 were assigned to the 025A subtype, and in the bacteremia population that was studied then, 56 of 57 studied 025 serotype isolates were typeable as 025B).
• compositions according to embodiments of the invention comprise an E. coli 025B antigen conjugated to an EPA carrier protein and other E. coli O-antigens conjugated to the EPA carrier protein.
• the invention relates to a multivalent vaccine containing 0- antigen serotypes found predominantly among E. coli clinical isolates, which can be used to provide active immunization for the prevention of disease caused by ExPEC having the O- antigen serotypes contained in the vaccine.
• the invention relates to a composition comprising an E. coli 025B antigen at a first concentration of 8 to 48 ⁇ ⁇ , and at least one additional E. coli O-antigen at a second concentration that is 10% to 100% of the first concentration, wherein each of the E. coli 025B antigen and the at least one additional E. coli CD- antigen is independently covalently bound to an EPA carrier protein.
• the at least one additional E. coli O antigen used in compositions according to embodiments of the invention is prevalent among the E. coli clinical isolates.
• additional O antigens include, but are not limited to, E. coli 01 , 02, 04, 06, 07, 08, 015, 016, 018, 021 , 073, 075 and 0153 antigens.
• the composition can include more than one additional E. coli O antigens, such as two, three, four, five, six, seven, eight or nine additional E. coli O antigens, to provide immune protection against multiple E. coli serotypes in addition to E. coli 025B serotype.
• the additional E. coli O-antigen is selected from the group consisting of E. coli 01, 02 and 06 antigens. More preferably, the additional E. coli O-antigen is selected from the group consisting of E. coli 01 A, 02 and 06 A antigens.
• a composition of the invention comprises an E. coli 025B antigen at a first concentration of 10 to 36 ⁇ g/ml, and E. coli 01 A, 02 and 06A antigens each at a concentration that is independently 10% to 100% of the first concentration, wherein each of the E. coli 025B antigen and the additional E. coli O-antigens is independently covalently bound to an EPA carrier protein.
• each of the E. coli 01 A, 02 and 06 A antigens is independently present at a concentration of at least 5 g/ml, more preferably, at a concentration of least 8 ⁇ g/ml.
• a composition according to an embodiment of the invention contains E. coli 025B antigen at a concentration that is same or higher than the concentration of any of the additional O antigens in the composition.
• the composition can have E. coli 025B antigen at a first concentration of, e.g., 10, 16, 24, 32 or 36 ⁇ g/ml, and one or more additional E. coli O- antigens each at a concentration that is, e.g. 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the first concentration.
• all of the additional O antigens can have the same concentration that is 10 to 100% of the E. coli 025B antigen concentration in the composition.
• the additional O antigens can also have different concentrations, each of which is independently 10-100% of the E. coli 025B antigen concentration.
• the composition comprises 32 ⁇ g/ml E. coli 025B antigen and independently 16 to 32 ⁇ g/ml each of the one or more additional O antigens, wherein each of the E. coli 025B antigen and the additional E. coli O-antigens is independently covalently bound to an EPA carrier protein.
• the composition comprises 16 ⁇ g/ml E. coli 025B antigen, and independently 8 ⁇ g/ml to 16 ⁇ g/ml of each of the one or more additional O antigens, wherein each of the E. coli 025B antigen and the additional E. coli O-antigens is independently covalently bound to an EPA carrier protein.
• the invention relates to a composition
• a composition comprising an E. coli 025B antigen at a first concentration of 10 to 36 ⁇ g ml, and a second concentration of each of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06 A antigen, wherein each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA carrier protein, and the ratio of the first concentration to the second concentration is 1 : 1 to 2: 1.
• the composition comprises the E. coli 025B, 01 A, 02 and 06A antigens at a weight ratio of 1 : 1 : 1 : 1 or 2: 1 : 1 : 1 , and the composition comprises 32 ⁇ g/ml of the E. coli 025B antigen, wherein each of the O-antigen is covalently bound to an EPA carrier protein.
• the composition comprises the E. coli 025B, 01 A, 02 and 06A antigens at a weight ratio of 1 : 1 : 1 : 1 or 2 : 1 : 1 : 1 , and the composition comprises 16 ⁇ g/ml of the E.
• each of the O-antigen is covalently bound to an EPA carrier protein.
• each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA carrier protein having the amino acid sequence of SEQ ID NO: 1, and each of the O-antigen and EPA carrier protein conjugate is made in a cell, i.e., being a bioconjugate.
• the E. coli 025B, 01A, 02 and 06A antigens comprise, respectively, the structures of formula 025B', formula 01A', formula 02' and formula 06A' described infra.
• compositions described herein are useful in the treatment and prevention of infection of subjects (e.g., human subjects) with ExPEC.
• the compositions described herein in addition to comprising E. coli O-antigens covalently bound to an EPA carrier protein, the compositions described herein comprise a pharmaceutically acceptable carrier.
• pharmaceutically acceptable means approved by a regulatory agency of a Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W.
• composition comprising an E. coli 025B antigen covalently bound to an EPA carrier protein, and one or more additional E. coli 0 antigens each covalently bound to the EPA carrier protein.
• composition comprising (i) a bioconjugate comprising an E. coli 025B antigen covalently bound to an EPA carrier protein, and (ii) a bioconjugate comprising an E. coli OIA antigen covalently bound to an EPA carrier protein.
• composition comprising (i) a bioconjugate comprising an E. coli 025B antigen covalently bound to an EPA carrier protein, and (ii) a bioconjugate comprising an E. coli 02 antigen covalently bound to an EPA carrier protein.
• composition comprising (i) a bioconjugate comprising an E. coli 025B antigen covalently bound to an EPA earner protein, and (ii) a bioconjugate comprising an E. coli 06A antigen covalently bound to an EPA carrier protein.
• composition comprising an E. coli 025B bioconjugate comprising an E. coli 025B antigen covalently bound to an EPA carrier protein, and two or more bioconjugates selected from the group consisting of: (i) an E. coli OIA bioconjugate comprising an E. coli OIA antigen covalently bound to an EPA carrier protein; (ii) an E. coli 02 bioconjugate comprising an E. coli 02 antigen covalently bound to an EPA carrier protein; and (iii) an E. coli 06A bioconjugate comprising an E. coli 06A antigen covalently bound to an EPA carrier protein.
• a composition provided herein comprises: (i) an E. coli 025B bioconjugate comprising an E. coli 025B antigen covalently bound to an EPA carrier protein; (ii) an E. coli 01 A bioconjugate comprising an E. coli 01 A antigen covalently bound to an EPA carrier protein; (iii) an E. coli 02 bioconjugate comprising an E. coli 02 antigen covalently bound to an EPA carrier protein; and (iv) an E. coli 06A bioconjugate comprising an E. coli 06A antigen covalently bound to an EPA carrier protein, wherein (i), (ii), (iii), and (iv) are formulated in a single formulation.
• a composition provided herein comprises: (i) an E. coli 025B bioconjugate comprising an E. coli 025B antigen covalently bound to an EPA carrier protein; (ii) an E. coli 01 A bioconjugate comprising an E. coli 01 A antigen covalently bound to an EPA carrier protein; (iii) an E. coli 02 bioconjugate comprising an E. coli 02 antigen covalently bound to an EPA carrier protein; and (iv) an E. coli 06A bioconjugate comprising an E. coli 06A antigen covalently bound to an EPA carrier protein, wherein (i), (ii), (iii), and (iv) are formulated in individual compositions that are administered in combination according to a method of an embodiment of the invention.
• compositions optionally comprise an EPA carrier protein covalently linked to an E. coli O-antigen other than E. coli 01 A, 02, 06A, and 025B.
• E. coli O antigens include, but are not limited to, the additional O antigens listed in Tables 1A-1C above.
• compositions provided herein can be used for eliciting an immune response in a host to whom the composition is administered, i.e., are immunogenic.
• the compositions described herein can be used as vaccines against ExPEC infection, and can comprise any additional components suitable for use in a vaccine.
• the compositions described herein are multivalent formulations, e.g., at least tetravalent fomrulations comprising bioconjugates of E. coli O-antigens of the 025B, 01 A, 06A, and 02 serotypes/subserotypes.
• compositions described herein additionally comprise a preservative, such as the mercury derivative thimerosal.
• a preservative such as the mercury derivative thimerosal.
• the pharmaceutical compositions described herein comprise 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.
• the compositions described herein comprise, or are administered in combination with, an adjuvant.
• the adjuvant for administration in combination with a composition described herein may be administered before, concomitantly with, or after administration of said composition.
• the term "adjuvant" refers to a compound that when administered in conjunction with or as part of a composition described herein augments, enhances and/or boosts the immune response to a bioconjugate, but when the adjuvant compound is administered alone does not generate an immune response to the bioconjugate.
• the adjuvant generates an immune response to the poly bioconjugate peptide and does not produce an allergy or other adverse reaction.
• Adjuvants can enhance an immune response by several mechanisms including, e.g., lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.
• the compositions described herein do not comprise an adjuvant besides the bioconjugates, and/or are not administered in combination with an adjuvant besides the bioconjugates (in case the bioconjugates would comprise some intrinsic adjuvant properties, these would be disregarded and no extrinsic adjuvant would be added in these embodiments).
• adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see United Kingdom Patent GB222021 1), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT/US2007/064857, published as International Publication No. WO2007/109812), imidazoquinoxaline compounds (see International Application No.
• alum such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate
• MPL 3 De-O-acylated monophosphoryl lipid A
• MPL 3 De-O-acylated monophosphoryl lipid A
• MF59 Novartis
• AS03 GaxoSmithKline
• AS04 Gax
• the adjuvant is Freund's adjuvant (complete or incomplete).
• Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al., 1997, N. Engl. J. Med. 336, 86-91).
• compositions described herein are formulated to be suitable for the intended route of administration to a subject.
• the compositions described herein may be formulated to be suitable for subcutaneous, parenteral, oral, intradermal, transdermal, colorectal, intraperitoneal, and rectal administration.
• the pharmaceutical composition may be formulated for intravenous, oral, intraperitoneal, intranasal, intratracheal, subcutaneous, intramuscular, topical, intradermal, transdermal or pulmonary administration.
• the compositions described herein are administered by intramuscular injection.
• compositions described herein additionally comprise one or more buffers, e.g. , Tris-buffered saline, phosphate buffer, and sucrose phosphate glutamate buffer.
• buffers e.g. , Tris-buffered saline, phosphate buffer, and sucrose phosphate glutamate buffer.
• compositions described herein additionally comprise one or more salts, e.g. , Tris-hydrochloride, sodium chloride, calcium chloride, potassium chloride, sodium phosphate, monosodium glutamate, and aluminum salts (e.g. , aluminum hydroxide, aluminum phosphate, alum (potassium aluminum sulfate), or a mixture of such aluminum salts).
• a composition of the invention comprises the bioconjugates described herein in a Tris-buffered saline (TBS) pH 7.4 (e.g. containing Tris, NaCl and KC1, e.g. at 25 mM, 137 mM and 2.7 mM, respectively).
• compositions described herein can be included in a container, pack, or dispenser together with instructions for administration.
• compositions described herein can be stored before use, e.g. , the compositions can be stored frozen (e.g. , at about -20°C or at about -70°C); stored in refrigerated conditions (e.g. , at about 4°C); or stored at room temperature.
• the invention in another general aspect, relates to a method of inducing an immune response to ExPEC in a subject in need thereof.
• the immune response is effective to prevent or treat a disease associated with ExPEC in the subject in need thereof.
• the method comprises administering to the subject an E. coli 025B antigen at a first effective amount of 4 to 24 ⁇ g per administration, and at least one additional E. coli O antigen at a second effective amount that is 10% to 100% of the first effective amount, wherein each of the E. coli 025B antigen and the at least one additional E. coli O-antigen is independently covalently bound to an EPA carrier protein, and the composition is effective in inducing an immune response against the E. coli 025B antigen and the at least one additional E. coli O-antigen in the subject in need thereof.
• the at least one additional E. coli O antigen used in the methods and uses of the invention is prevalent among the E. coli clinical isolates.
• additional 0 antigens include, but are not limited to, E. coli 01 , 02, 04, 06, 07, 08, 015, 016, 018, 021, 073, 075 and 0153 antigens.
• more than one additional E. coli 0 antigens such as two, three, four, five, six, seven, eight or nine additional E. coli O antigens, can be administered to provide immune protection against multiple E. coli serotypes in addition to E. coli 025B serotype.
• a method of the invention induces an immune response in a subject in need thereof against ExPEC serotype 025, and one or more additional E. coli 0- antigens selected from the group consisting of E. coli 01, 02 and 06 antigens.
• a method of the invention induces an immune response in a subject in need thereof against ExPEC serotypes 025B, and one or more additional E. coli O-antigens selected from the group consisting of E. coli 01 A, 02 and 06A antigens.
• the method comprises administering to a subject in need thereof an E. coli 025B antigen at a first effective amount of 5 to 18 ⁇ g per administration, and E.
• each of the E. coli 01 A, 02 and 06 A antigens is independently administered at an effective amount of at least 3 ⁇ g per administration, more preferably, at an effective amount of at least 4 ⁇ g per administration.
• the administered effective amount of E. coli 025B antigen is same or higher than the administered effective amount of any of the additional O antigens.
• the E. coli 025B antigen can be administered at a first effective amount of 4 to 24 ⁇ g per administration, and the at least one additional E. coli O- antigen can be administered at a second effective amount that is, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the first effective amount.
• all of the additional O antigens can be administered at the same effective amount that is 10-100% of the first effective amount for the E. coli 025B antigen.
• the additional O antigens can also be administered at different effective amounts each of which is independently 10-100% of the first effective amount for the E. coli 025B antigen.
• the E. coli 025B antigen is administered at the first effective amount of 5 ⁇ g to 18 ⁇ g, and the at least one additional O antigen is administered at a second effective amount that is independently 50% to 100% of the first effective amount.
• a method of inducing an immune response to ExPEC in a subject in need thereof comprises administering to the subject an E. coli 025B antigen at a first effective amount of, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ⁇ g per administration, and E. coli 01 A, 02 and 06 A antigens each at an effective amount that is independently, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the first effective amount, wherein each of the E. coli 025B antigen and the additional E. coli O-antigens is independently covalently bound to an EPA carrier protein.
• the invention relates to a method of inducing an immune response to ExPEC in a subject in need thereof, comprising administering to the subject an E. coli 025B antigen at a first effective amount of 5 to 18 ⁇ g per administration, and a second effective amount of each of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06 A antigen, wherein each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA carrier protein, and the ratio of the first effective amount to the second effective amount is 1 : 1 to 2: 1.
• the E. coli 025B antigen at a first effective amount of 5 to 18 ⁇ g per administration
• a second effective amount of each of an E. coli 01 A antigen, an E. coli 02 antigen and an E. coli 06 A antigen wherein each of the E. coli 025B, 01 A, 02 and 06A antigens are independently covalently bound to an EPA
• coli 025B, 01 A, 02 and 06A antigens are administered to the subject at a dosage ratio of 1 : 1 : 1 : 1 or 2: 1 : 1 : 1 , and the E. coli 025B antigen is administered at 5 ⁇ g, 8 ⁇ g or 16 ⁇ g per administration. Also preferably, the E. coli 025B, 01 A, 02 and 06A antigens are administered to the subject in one composition.
• compositions of the invention for inducing an immune response to ExPEC in a subject in need thereof, comprising administering to the subject an E. coli 025 B antigen covalently bound to an EPA carrier protein, and at least one additional E. coli O antigen covalently bound to the EPA carrier protein.
• the compositions described herein are used to vaccinate a human subject to induce a protective immunity against ExPEC infection of the human subject.
• said subject has an ExPEC infection at the time of
• said subject does not have an ExPEC infection at the time of administration.
• infections caused by ExPEC include, but are not limited to, urinary tract infection, surgical-site infection, bacteremia, abdominal or pelvic infection, pneumonia, nosocomial pneumonia, osteomyelitis, cellulitis, wound infection, meningitis, neonatal meningitis, peritonitis, cholangitis, soft-tissue infections, pyomyositis, septic arthritis, and sepsis.
• the infection caused by ExPEC is an invasive ExPEC disease caused by ExPEC serotypes of which antigens are included in the compositions or methods according to embodiments of the invention.
• the methods of inducing an immune response in a subject described herein result in vaccination of the subject against infection by the ExPEC strains whose O-antigens are present in the composition(s).
• a method of the invention can also induce immune response to another O-antigen subtype having similar antigenicity.
• the immune response induced by a method or composition described herein is effective to prevent and/or treat an infection caused by E. coli of the 025 serotype.
• said 025 serotype is 025B.
• said 025 serotype is 025A.
• the immune response induced by a method or composition described herein is effective to prevent and/or treat an infection caused by E. coli of the 025 serotype, e.g. 025B serotype, and 01 serotype, e.g. 01 A serotype.
• the immune response induced by a method or composition described herein is effective to prevent and/or treat an infection caused by E. coli of the 025 serotype, e.g. 025B serotype, and 02 serotype.
• the immune response induced by a method or composition described herein is effective to prevent and/or treat an infection caused by E. coli of the 025 serotype, e.g. 025B serotype, and 06 serotype, e.g. 06A serotype.
• the immune response induced by a method or composition described herein is effective to prevent and/or treat an infection caused by E. coli of the 025 serotype (e.g. 025B and/or 025 A), and two or more of the following E. coli serotypes: 01 (e.g., 01A, 01B, and/or 01C), 02, and/or 06 (e.g., 06A and/or 06GlcNAc).
• 01 e.g., 01A, 01B, and/or 01C
• 02, and/or 06 e.g., 06A and/or 06GlcNAc
• the immune response induced by a method or composition described herein is effective to prevent and/or treat an infection caused by each of the following E. coli serotypes: 025 (e.g., 025B and/or 025A), 01 (e.g., 01A, 01B, and/or 01C), 02, and 06 (e.g., 06A and/or 06GlcNAc).
• 025 e.g., 025B and/or 025A
• 01 e.g., 01A, 01B, and/or 01C
• 02, and 06 e.g., 06A and/or 06GlcNAc
• the immune response induced by a method or composition described herein is effective to prevent and/or treat an infection caused by E. coli of the 025 serotype, e.g. 025B serotype, and an E. coli serotype other than 01, 02, 06, or 025, including, but not limited to, the additional O serotypes listed in Tables 1A-1C.
• the subject can be administered a single composition described herein, wherein said composition comprises E. coli 025B antigen, and one, two, three, four, or more additional E. coli O antigens described herein, each covalently bound to an EPA carrier protein.
• the subject in order to treat a subject having an ExPEC infection or immunize a subject against an ExPEC infection, the subject can be administered multiple compositions described herein in combination.
• a subject can be administered a composition comprising E. coli 025B antigen conjugated to an EPA carrier protein, in combination with the administration of two, three, four, or more compositions comprising additional O antigen conjugates according to embodiments of the invention.
• the immune response induced in a subject following administration of a composition described herein is effective to prevent or reduce a symptom resulting from an ExPEC infection, preferably in at least 30%, more preferably at least 40%, such as at least 50%, of the subjects administered with the composition.
• Symptoms of ExPEC infection may vary depending on the nature of the infection and may include, but are not limited to: dysuria, increased urinary frequency or urgency, pyuria, hematuria, back pain, pelvic pain, pain while urinating, fever, chills, and/or nausea (e.g., in subjects having a urinary tract infection caused by ExPEC); high fever, headache, stiff neck, nausea, vomiting, seizures, sleepiness, and/or light sensitivity (e.g., in subjects having meningitis caused by ExPEC); fever, increased heart rate, increased respiratory rate, decreased urine output, decreased platelet count, abdominal pain, difficulty breathing, and/or abnormal heart function (e.g., in subjects having sepsis caused by ExPEC).
• the immune response induced in a subject following administration of a composition described herein is effective to reduce the likelihood of hospitalization of a subject suffering from an ExPEC infection. In some embodiments, the immune response induced in a subject following administration of a composition described herein is effective to reduce the duration of hospitalization of a subject suffering from an ExPEC infection.
• Embodiments of the invention relate to compositions and methods relating to E. coli 025B antigen and one or more additional E. coli O antigens.
• the additional O antigen is prevalent among the clinical isolates of E. coli.
• E. coli antigens that can be used in the invention include, but are not limited to, the E. coli 025B, 01 A, 02, and 06A antigens.
• an "E. coli 025B antigen” refers to an O antigen specific to the E. coli 025B serotype.
• an E. coli 025B antigen comprises the structure of Formula 025B:
• the E. coli 025B antigen comprises the structure of Formula 025B':
• n in Formula 025B or Formula 025B' is an integer of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
• the n in Formula 025B or Formula 025B' is an integer of 10-20.
• a population of E. coli 025B antigens having the structure of Formula 025B, more preferably Formula 025B' is used in compositions and methods according to embodiments of the invention, wherein the n of at least 80% of the E. coli 025B antigens in the population is an integer of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
• the n of at least 80% of the E. coli 025B antigens in the population is an integer of 10- 20.
• an "E. coli 01 antigen” refers to an O antigen specific to the E. coli
• an E. coli 01 antigen is an E. coli OIA antigen.
• an "E. coli OIA antigen” refers to an O antigen specific to the E. coli
• an E. coli OIA antigen comprises the structure of Formula
• the E. coli OIA antigen comprises the structure of Formula OIA' :
• n in Formula OIA or Formula OIA' is an integer of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
• the n in Formula 01 A or Formula OIA' is an integer of 7-15.
• a population of E. coli OIA antigens having the structure of Formula OIA, more preferably Formula OIA' is used in compositions and methods according to embodiments of the invention, wherein the n of at least 80% of the E. coli OIA antigens in the population is of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
• the n of at least 80% of the E. coli 01 A antigens in the population is an integer of 5-15.
• an "E. coli 02 antigen” refers to an O antigen specific to the E. coli 02 serotype.
• an E. coli 02 antigen comprises the structure of Formula 02:
• the E. coli 02 antigen comprises the structure of Formula 02' : ⁇ a a ⁇
• n in Formula 02 or Formula 02' is an integer of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
• the n in Formula 02 or Formula 02' is an integer of 8-16.
• a population of E. coli 02 antigens having the structure of Formula 02, more preferably Formula 02' is used in compositions and methods according to embodiments of the invention, wherein the n of at least 80% of the E. coli 02 antigens in the population is of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
• the n of at least 80% of the E. coli 02 antigens in the population is an integer of 5-20.
• an "E. coli 06 antigen” refers to an O antigen specific to the E. coli 06 serotype.
• an E. coli 06 antigen is an E. coli 06A.
• an E. coli 06A antigen comprises the structure of Formula 06A:
• the E. coli 06A antigen comprises the structure of Formula 06A' :
• the n in Formula 06 A or Formula 06A' is an integer of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20. In one embodiment, the n in Formula 06A or Formula 06A' is an integer of 8-18.
• a population of E. coli 06 A antigens having the structure of Formula 06A, more preferably Formula 06A', is used in compositions and methods according to embodiments of the invention, wherein the n of at least 80% of the E. coli 06A antigens in the population is of 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 10 to 30, 15 to 30, 20 to 30, 25 to 30, 5 to 25, 10 to 25, 15 to 25, 20 to 25, 10 to 20, or 15 to 20.
• the n of at least 80% of the E. coli 06A antigens in the population is an integer of 5-20.
• a composition of the invention comprises E. coli 025B antigens having the structure of formula 025B', wherein the n of at least 80% of the E. coli 025B antigens in the population is an integer of 10-20; E. coli 01 A antigens having the structure of formula 01 A', wherein the n of at least 80% of the E. coli 01 A antigens in the population is an integer of 5-15; E. coli 02 antigens having the structure of formula 02', wherein the n of at least 80% of the E. coli 02 antigens in the population is an integer of 5-20; and E.
• coli 06A antigens having the structure of formula 06A', wherein the n of at least 80% of the E. coli 06 A antigens in the population is an integer of 5-20, wherein each of the O-antigens is covalently bound to an EPA carrier protein having the amino acid sequence of SEQ ID NO: 1.
• E. coli O antigen useful in the invention can be produced by methods known in the art in view of the present disclosure.
• they can be produced from a cell, preferably a recombinant cell that is optimized for the biosynthesis of the O antigen.
• a cell preferably a recombinant cell that is optimized for the biosynthesis of the O antigen.
• E. coli O antigen biosynthesis in WO 2006/1 19987, WO 2009/104074, International Patent Application No. PCT/EP2015/053739, Ihssen et al, 2010, Microbial Cell Factories 9, 61, the disclosures of which are herein incorporated by reference in their entirety.
• each E. coli O antigen is covalently bound to an EPA carrier protein (see, e.g., Ihssen et al., 2010, Microbial Cell Factories 9, 61).
• EPA carrier protein see, e.g., Ihssen et al., 2010, Microbial Cell Factories 9, 61.
• EPA carrier proteins see, e.g., Ihssen et al., 2010, Microbial Cell Factories 9, 61.
• EPA carrier protein see, e.g., Ihssen et al., 2010, Microbial Cell Factories 9, 61.
• the EPA carrier proteins used in the conjugates described herein are EPAs modified in such a way that the protein is less toxic and/or more susceptible to glycosylation.
• detoxification can be achieved by mutating and deleting the catalytically essential residues, such as L552V and ⁇ 553, according to Lukac et al., Infect Immun, 56: 3095-3098, 1988 and Ho et al., Hum Vaccin, 2:89-98, 2006.
• the carrier proteins used in the generation of the conjugates described herein are EPAs modified such that the number of glycosylation sites in the carrier proteins is optimized in a manner that allows for lower concentrations of the protein to be administered, e.g., in an immunogenic composition, in its bioconjugate form.
• the EPA carrier proteins are EPAs modified to include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more glycosylation sites than would normally be associated with the carrier protein (e.g., relative to the number of glycosylation sites associated with the carrier protein in its native/natural, e.g., "wild-type" state).
• glycosylation sites introduction of glycosylation sites is accomplished by insertion of glycosylation consensus sequences (e.g., Asn- X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 2); or preferably Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO:3) (see WO 2006/1 19987)) anywhere in the primary structure of the EPA protein.
• the EPA carrier protein comprises 4 consensus glycosylation sequences Asp/Glu-X-Asn-Z-Ser/Thr (SEQ ID NO: 3), and has an amino acid sequence as provided in SEQ ID NO: 1.
• the EPA carrier protein can be produced together with a signal sequence (such as a signal peptide for E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), etc.) that targets the carrier protein to the periplasmic space of the host cell that expresses the carrier protein.
• a signal sequence such as a signal peptide for E. coli DsbA, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), etc.
• the EPA carrier protein can also be modified to a "tag," i.e., a sequence of amino acids that allows for the isolation and/or identification of the carrier protein.
• An EPA carrier protein useful in the invention can be produced by methods known in the art in view of the present disclosure. See, e.g., relevant disclosure in e.g., Ihssen et al., 2010, Microbial Cell Factories 9, 61, and in WO 2006/1 19987, WO 2009/104074, and International Patent application No. PCT/ EP2015/053739, the disclosure of which are herein incorporated by reference in their entirety.
• a host cell can produce an E. coli O antigen and an EPA carrier protein, and covalently bind the O antigen to the EPA carrier protein to form a bioconjugate useful in the invention.
• a bioconjugate useful in the invention See, e.g., relevant disclosure in e.g., Ihssen et al., 2010, Microbial Cell Factories 9, 61 , and in WO 2006/119987, WO 2009/104074, and International Patent application No. PCT/ EP2015/053739, the disclosures of which are herein incorporated by reference in their entirety.
• the glycoconjugates can be prepared by chemical synthesis, i.e., prepared outside of host cells (in vitro).
• the E. coli O-antigens described herein, e.g., 025B antigen can be conjugated to carrier proteins using methods known to those of skill in the art, including by means of using activation reactive groups in the polysaccharide/ oligosaccharide as well as the protein carrier. See, e.g., Pawlowski et al, 2000, Vaccine
• Such approaches comprise extraction of antigenic polysaccharides/ oligosaccharides from host cells, purifying the
• polysaccharides/oligosaccharides chemically activating the polysaccharides/oligosaccharides, and conjugating the polysaccharides/ oligosaccharides to a carrier protein.
• Bioconjugates have advantageous properties over glycoconjugates made in vitro, e.g., bioconjugates require less chemicals in manufacture and are more consistent and homogenous in terms of the final product generated. Thus, bioconjugates are preferred over chemically produced glycoconjugates.
• the EPA carrier protein is N-linked to an E. coli O-antigen useful in the invention.
• the E. coli O antigen is linked to the Asn residue in a glycosylation sequence of a carrier protein, such as Asn-X-Ser(Thr), wherein X can be any amino acid except Pro (SEQ ID NO: 2), preferably Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO: 3).
• the conjugates described herein can be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, anionic exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. See, e.g., Saraswat et al., 2013, Biomed. Res. Int. ID#312709 (p. 1-18); see also the methods described in WO 2009/104074.
• the actual conditions used to purify a particular conjugate will depend, in part, on the synthesis strategy (e.g., synthetic production vs. recombinant production) and on factors such as net charge, hydrophobicity, and/or hydrophilicity of the bioconjugate, and will be apparent to those having skill in the art.
• a composition described herein is administered to a subject in combination with one or more other therapies (e.g. , antibacterial or immunomodulatory therapies).
• the one or more other therapies can be beneficial in the treatment or prevention of an ExPEC infection or can ameliorate a symptom or condition associated with an ExPEC infection.
• the one or more other therapies are pain relievers or anti-fever medications.
• the therapies are administered less than 5 minutes apart to less than 1 week apart. Any anti-bacterial agents known to one of skill in the art (e.g. antibiotics) may be used in combination with a composition described herein.
• a composition (or method) described herein is administered (or applied) to a naive subject, i.e., a subject that does not have an ExPEC infection or has not previously had an ExPEC infection.
• a composition (or method) described herein is administered (or applied) to a na ' ive subject that is at risk of acquiring an ExPEC infection.
• composition (or method) described herein is administered (or applied) to a subject who has been or was previously diagnosed with an ExPEC infection.
• a composition (or method) described herein is administered (or applied) to a subject infected with ExPEC before symptoms manifest or symptoms become severe (e.g., before the patient requires hospitalization).
• a composition (or method) described herein is administered (or applied) to a subject who has been diagnosed with an uropatho genie E.coli (UPEC) infection.
• a composition (or method) described herein is administered (or applied) to a subject suffering from reoccurring urinary tract infections.
• a composition (or method) described herein is administered (or applied) to a subject suffering from reoccurring urinary tract infections, but is healthy at the moment of treatment.
• a composition (or method) described herein is administered (or applied) to a subject having or at risk of acquiring bacteremia or sepsis.
• a subject to be administered (or applied) a composition (or method) described herein is an animal.
• the animal is a canine.
• the animal is a feline.
• the animal is a horse.
• the animal is a cow.
• the animal is a mammal, e.g., a horse, swine, rabbit, mouse, or primate.
• the subject is a human.
• a subject to be administered (or applied) a composition (or method) described herein is a human subject, preferably, a human subject at risk of having an invasive ExPEC disease.
• a subject to be administered (or applied) a composition (or method) described herein is a human adult more than 50 years old.
• a subject to be administered (or applied) a composition (or method) described herein is a human adult more than 55, more than 60 or more than 65 years old.
• a subject to be administered (or applied) a composition (or method) described herein is a human child. In certain embodiments, a subject to be administered (or applied) a composition (or method) described herein is a human child. In certain embodiments,
• a subject to be administered (or applied) a composition (or method) described herein is a human infant, including a premature human infant. In some embodiments, a subject to be administered (or applied) a composition (or method) described herein is a human toddler. In certain embodiments, a subject to be administered (or applied) a composition (or method) described herein is not an infant of less than 6 months old.
• a subject to be administered (or applied) a composition (or method) described herein is an individual who is pregnant. In certain embodiments, a subject to be administered (or applied) a composition (or method) described herein is a woman who has given birth 1, 2, 3, 4, 5, 6, 7, or 8 weeks earlier.
• a subject to be administered (or applied) a composition (or method) described herein is an individual at increased risk of ExPEC, e.g., an
• a subject to be administered (or applied) a composition (or method) described herein is an individual in close contact with an individual having or at increased risk of ExPEC infection.
• a subject to be administered (or applied) a composition (or method) described herein is a health care worker.
• a subject to be administered (or applied) a composition (or method) described herein is immunocompromised (e.g., suffers from HIV infection) or immunosuppressed.
• a subject to be administered (or applied) a composition (or method) described herein has diabetes. In certain embodiments, a subject to be administered (or applied) a composition (or method) described herein has multiple sclerosis.
• a subject to be administered (or applied) a composition (or method) described herein has a condition that requires them to use a catheter, such as a urinary catheter.
• a subject to be administered (or applied) a composition (or method) described herein has a spinal cord injury.
• the subject is a male who will undergo or has recently undergone a prostate biopsy.
• the subject to be administered (or applied) a composition (or method) described herein is an at-risk human adult in need of immunization for the prevention of invasive ExPEC disease caused by ExPEC serotypes 01 A, 02, 06A and 025B.
• at-risk human include, but are not limited to, those described herein supra.
• At-risk human examples include, e.g., individuals having transrectal ultrasonography with prostate needle biopsy (TRUS-PNB) or recurrent urosepsis, residents of a long term care facility (LTCF), long term care (LTAC)-assisted living, pre-surgery patients (including but not limited to, patients scheduled for genito-urinary/abdominal surgery); pre-dialysis patients, pre-dialysis, etc.
• TRUS-PNB transrectal ultrasonography with prostate needle biopsy
• LTCF long term care facility
• LTAC long term care
• pre-surgery patients including but not limited to, patients scheduled for genito-urinary/abdominal surgery
• pre-dialysis patients pre-dialysis, etc.
• Administration of the conjugates of O-antigens and an EPA carrier protein and/or composition thereof can be done via various routes known to the clinician, for instance subcutaneous, parenteral, intravenous, intramuscular, topical, oral, intradermal, transdermal, intranasal, etc. In one embodiment, administration is via intramuscular injection.
• the dosage level of E. coli 025B antigen covalently should be no less, preferably more, than the dosage levels of the other E. coli 0 antigens used in a composition or method of the invention, wherein each of the E. coli O antigens is covalently bound to an EPA carrier protein.
• the precise dosage to be employed in the formulation and method will depend on the route of administration, and the seriousness of the infection, and should be decided according to the judgment of the practitioner and each subject's circumstances.
• exemplary dosages for E. coli 025B antigen range from 4 to 24 ⁇ g of 025B antigen per administration, and the exemplary dosages for each of the additional E. coli O antigens to be used in combination with the E. coli 025B antigen range from 10% to 100% of the dosage of E. coli 025B antigen, wherein each of the E. coli O antigens is covalently bound to an EPA carrier protein.
• coli 025B glycoconjugate is, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 ⁇ g of 025B antigen per administration, and an exemplary dosage for another E. coli O glycoconjugate to be used in combination with the E. coli 025B glycoconjugate is, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the dosage for the E. coli 025B glycoconjugate, wherein the dosage is calculated by the amount of the O antigen in the O glycoconjugates per administration.
• an exemplary dosage for per administration to a human subject corresponds to 0.5 ml of a composition containing a first concentration of about 8-48 ⁇ g/mL, e.g., about 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 or 48 ⁇ g/mL, of £ coli 025B antigen covalently bound to an EPA carrier protein, and a concentration of 10% to 100% of the first concentration of one or more additional E. coli O antigens covalently bound to the EPA carrier protein.
• an exemplary dosage for per administration to a human subject corresponds to 0.5 ml of a composition containing a concentration of about 16 ⁇ g mL of E. coli 025B antigen covalently bound to an EPA carrier protein, about 8 ⁇ g/mL of E. coli 01 A antigen covalently bound to an EPA carrier protein, about 8 ⁇ g/mL of E. coli 02 antigen covalently bound to an EPA carrier protein, and about 8 ⁇ g mL of E. coli 06A antigen covalently bound to an EPA carrier protein.
• an exemplary dosage for per administration to a human subject corresponds to 0.5 ml of a composition containing a concentration of about 16 ⁇ g/mL of E. coli 025B antigen covalently bound to an EPA carrier protein, about 16 ⁇ g/mL of E. coli 01 A antigen covalently bound to an EPA carrier protein, about 16 ⁇ g/mL of E. coli 02 antigen covalently bound to an EPA carrier protein, and about 16 ⁇ g/mL of E. coli 06 A antigen covalently bound to an EPA carrier protein.
• an exemplary dosage for per administration to a human subject corresponds to 0.5 ml of a composition containing a concentration of about 32 ⁇ g/mL of E. coli 025B antigen covalently bound to an EPA carrier protein, about 16 ⁇ g/mL of E. coli 01 A antigen covalently bound to an EPA carrier protein, about 16 ⁇ g/mL of E. coli 02 antigen covalently bound to an EPA carrier protein, and about 16 ⁇ g/mL of E. coli 06 A antigen covalently bound to an EPA carrier protein.
• an exemplary dosage for per administration to a human subject corresponds to 0.5 ml of a composition containing a concentration of about 32 ⁇ g/mL of E. coli 025B antigen covalently bound to an EPA carrier protein, about 32 ⁇ g/mL of E. coli OIA antigen covalently bound to an EPA carrier protein, about 32 ⁇ g/mL of E. coli 02 antigen covalently bound to an EPA carrier protein, and about 32 ⁇ g/mL of E. coli 06 A antigen covalently bound to an EPA carrier protein.
• E. coli 025B antigen covalently bound to an EPA carrier protein about 32 ⁇ g/mL of E. coli OIA antigen covalently bound to an EPA carrier protein
• E. coli 02 antigen covalently bound to an EPA carrier protein corresponds to 0.5 ml of a composition containing a concentration of about 32 ⁇ g/mL of E. coli 025B antigen covalently bound to an
• E. coli O-antigen conjugates preferably bioconjugates, described herein or a composition described herein is administered to a subject once as a single dose.
• E. coli O-antigen conjugates, preferably bioconjugates, described herein or a composition described herein is administered to a subject as a single dose followed by a second dose 3 to 6 weeks later.
• booster inoculations can be administered to the subject at 6 to 24 month intervals following the second inoculation.
• the booster inoculations can utilize a different E. coli O-antigen, bioconjugate, or composition.
• an E. coli O-antigen conjugate described herein or a composition described herein is administered to a subject as a single dose once per year.
• an E. coli O-antigen conjugate described herein or a composition described herein is administered to a subject as a single dose once per n years, n being for instance about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 years.
• an E. coli O-antigen conjugate described herein or a composition described herein is administered to a subject as 2, 3, 4, 5 or more doses 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks apart. In some embodiments, 2, 3, 4, 5 or more doses of an E. coli O-antigen conjugate described herein or a composition described herein are administered to a subject 2, 3, 4, 5 or 6 weeks apart. In certain embodiments, the E. coli O-antigen conjugate, or composition administered is the same each time. In certain embodiments, the E. coli O- antigen conjugate, or composition administered is different each time.
• the ability of a bioconjugate to generate an immune response in a subject can be assessed by immunizing a subject (e.g. , a mouse) or set of subjects with the bioconjugate and immunizing an additional subject (e.g., a mouse) or set of subjects with a control (e.g., a placebo).
• a subject e.g. , a mouse
• an additional subject e.g., a mouse
• a control e.g., a placebo
• the subjects or set of subjects can subsequently be challenged with ExPEC and the ability of the ExPEC to cause disease (e.g., UTI) in the subjects or set of subjects can be determined.
• UTI disease
• bioconjugate(s) or composition thereof described herein suffer less from or do not suffer from disease
• the bioconjugate is able to generate an immune response in a subject.
• the ability of a bioconjugate(s) or composition thereof described herein to induce antiserum that cross-reacts with an O-antigen from ExPEC can be tested by, e.g., an immunoassay, such as an ELISA.
• opsonophagocytotic killing assay which represents an established and accepted method that has been used to obtain approval of glycoconjugate-based vaccines.
• Such assays are well- known in the art and, briefly, comprise the steps of generating and isolating antibodies against a target of interest (e.g., an O-antigen, e.g., 025B, of E. coli) by administering to a subject (e.g., a mouse) a compound that elicits such antibodies.
• a subject e.g., a mouse
• the bactericidal capacity of the antibodies can be assessed by, e.g., culturing the bacteria in question (e.g., E. coli of the relevant serotype) in the presence of said antibodies and complement and - depending on the assay - neutrophilic cells and assaying the ability of the antibodies to kill and/or neutralize the bacteria, e.g., using standard microbiological approaches.
• a pack or kit comprising one or more containers filled with one or more of the ingredients of the compositions described herein, such as one or more E. coli O antigens and/or conjugates of the E. coli O antigens covalently bound to an EPA carrier protein according to embodiments of the invention.
• Optionally associated with such container(s) can be a notice or instructions in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• the kits encompassed herein can be used in the above methods of treatment and immunization of subjects.
• Embodiment 1 is a composition comprising a first concentration of an E. coli 025B antigen polysaccharide, and a second concentration of each of an E. coli 01 A antigen polysaccharide, an E. coli 02 antigen polysaccharide and an E. coli 06A antigen polysaccharide, wherein the ratio of the first concentration to the second concentration is 1 : 1 to 2:1, each of the E. coli 025B, 01 A, 02 and 06A antigen polysaccharides are independently covalently bound to a detoxified exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein, and the first concentration is 10 to 36 ⁇ g/ml.
• EPA Pseudomonas aeruginosa
• Embodiment 2 is the composition of embodiment 1, comprising the E. coli 025B, 01 A, 02 and 06A antigen polysaccharides at a weight ratio of 1 : 1 : 1 : 1.
• Embodiment 3 is the composition of embodiment 1, comprising the 025B, 01A, 02 and 06 A antigen polysaccharides at a weight ratio of 2: 1 : 1 : 1.
• Embodiment 4 is the composition of any one of embodiments 1 to 3, comprising 16iglm ⁇ of the 025B antigen polysaccharide.
• Embodiment 5 is the composition of any one of embodiments 1 to 3, comprising 32 ⁇ g/ml of the 025B antigen polysaccharide.
• Embodiment 6 is a multivalent immune composition
• EPA Pseudomonas aeruginosa
• Embodiment 7 is a multivalent immune composition comprising an E. coli 025B antigen polysaccharide having the structure of Formula 025B' :
• n is independently an integer of 5 to 25, and
• each of the E. coli 025B, 01 A, 02 and 06A antigen polysaccharides are independently covalently bound to a carrier protein having the amino acid sequence of SEQ ID NO: 1 ; and the concentrations of the E. coli 025B, 01 A, 02, 06A antigen polysaccharides in the compositions are respectively 16:8:8 ⁇ g/ml, 16: 16:16: 16 ⁇ g/ml, 32:16: 16: 16 ⁇ g/ml or 32:32:32:32 ⁇ .
• Embodiment 8 is a method of inducing an immune response to extra-intestinal pathogenic E.coli (ExPEC) in a subject in need thereof, comprising administering to the subject a composition of any one of embodiments 1 to 7.
• Embodiment 9 is a method of inducing an immune response to extra-intestinal pathogenic E.coli (ExPEC) in a subject in need thereof, comprising administering to the subject a first effective amount of an E. coli 025B antigen polysaccharide, and a second effective amount of each of an E. coli 01 A antigen polysaccharide, an E. coli 02 antigen polysaccharide and an E. coli 06A antigen polysaccharide, wherein the ratio of the first effective amount to the second effective amount is 1 : 1 to 2: 1 , each of the E. coli 025B, 01A, 02 and 06A antigen polysaccharide
• polysaccharides are independently covalently bound to a detoxified exotoxin A of Pseudomonas aeruginosa (EPA) carrier protein, and the first effective amount is 5 to 18 ⁇ g per administration.
• EPA Pseudomonas aeruginosa
• Embodiment 10 is the method of embodiment 9, wherein the E. coli 025B, 01 A, 02 and 06A antigen polysaccharides are administered at a dosage ratio of 1 : 1 : 1 : 1.
• Embodiment 1 1 is the method of embodiment 9, wherein the E. coli 025B, 01 A, 02 and 06A antigen polysaccharides are administered at a dosage ratio of 2: 1 : 1 : 1.
• Embodiment 12 is the method of any one of embodiments 8 to 1 1 , wherein 8 ⁇ g of the 025B antigen polysaccharide is administered per administration.
• Embodiment 13 is the method of any one of embodiments 8 to 1 1 , wherein 16 ⁇ g of the 025B antigen polysaccharide is administered per administration.
• Embodiment 14 is the method of any one of embodiments 8 to 13, wherein the E. coli 025B, 01 A, 02 and 06A antigen polysaccharides are administered together in one composition.
• Embodiment 15 is a method of inducing an immune response to extra-intestinal pathogenic E.coli (ExPEC) in a subject in need thereof, comprising administering to the subject an E. coli 025B antigen polysaccharide having the structure of Formula 025B' :
• n is independently an integer of 5 to 25,
• each of the E. coli 025B, 01 A, 02 and 06A antigen polysaccharides are independently covalently bound to a carrier protein having the amino acid sequence of SEQ ID N0:1, and the E. coli 025B, OIA, 02 and 06A antigen polysaccharides are administered at 8:4:4:4 ⁇ g, 8:8:8:8 ⁇ g, 16:8:8:8 ⁇ g or 16: 16: 16: 16: 16 g per administration.
• Embodiment 16 is the method of any one of embodiments 8 to 15, wherein the immune response limits the severity of or prevents an invasive ExPEC disease caused by ExPEC serotypes OIA, 02 and 06A and 025B in an at-risk human subject.
• Embodiment 17 is the method of embodiment 16, wherein the adult human subject has or is at risk of having an invasive ExPEC disease selected from the group consisting of urinary tract infection, a surgical-site infection, an abdominal or pelvic infection, pneumonia, nosocomial pneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, a wound infection, pyelonephritis, meningitis, neonatal meningitis, peritonitis, cholangitis, soft-tissue infections, pyomyositis and septic arthritis.
• an invasive ExPEC disease selected from the group consisting of urinary tract infection, a surgical-site infection, an abdominal or pelvic infection, pneumonia, nosocomial pneumonia, osteomyelitis, cellulitis, sepsis, bacteremia, a wound infection, pyelonephritis, meningitis, neonatal meningitis, peritonitis, cholangitis, soft-tissue infections
• Embodiment 18 is a process of making a composition of any one of embodiments 1 to 7, comprising combining the E. coli 025B antigen polysaccharide, the E. coli OIA antigen polysaccharide, the E. coli 02 antigen polysaccharide and the E. coli 06A antigen polysaccharide to thereby obtain the composition.
• 01 A-EPA, 02-EPA, 06A-EPA and 025B-EPA bioconjugates containing, respectively, E. coli 01 A, 02, 06A and 025B covalently linked to the glycosylation sites of an EPA protein carrier can be produced, purified, and characterized as described in, e.g., Ihssen et al., 2010, Microbial Cell Factories 9, 61, and in WO 2006/119987, WO 2009/104074, and International Patent application No. PCT/ EP2015/053739, the disclosure of which are herein incorporated by reference in their entirety.
• the bioconjugates are synthesized using recombinant E.
• glycoconjugate vaccine can be expressed in the periplasm of E. coli, extracted and purified through a biochemical process illustrated in Figure 1 and Figure 2.
• Table 2 indicates host strains used for the production of conjugates according to an embodiment of the invention.
• Table 2 Host strains for production of preclinical, toxicology study and clinical batches
• a strain with a genomically integrated 025B cluster was constructed: W31 10 AwaaL AgtrABS Ar/3 ⁇ 4016::r 3 ⁇ 4(upecl38), which was transformed with plasmids pGVXN1076 (which expresses the EPA having the amino acid sequence of SEQ ID NO: 1) and pGVXN970 (which expressed the oligosaccharyl transferase PelB) (WO/2009/104074).
• This strain was constructed starting from strain W31 10 by the methods of Datsenko and Wanner (2000, Proc Natl Acad Sci USA 97: 6640-6645) and a homologous recombination technique for site directed integration of large inserts into bacterial chromosomes (see WO 2014/057109).
• the rfb cluster related to the 025B antigen was cloned from E. coli strain upecl 38, which is positive for 025B.
• the recombinant host cells produced 025B/EPA bioconjugates in the periplasm. The resulting 025B bioconjugates were
• Bioconjugates were purified using two consecutive anionic exchange and size exclusion chromatography steps, yielding 98.1 % pure 025B bioconjugate preparations.
• the bioconjugates and an un-glycosylated EPA reference standard were analyzed by size-exclusion chromatography with multi-angle light scattering (SEC-MALS), in order to quantify the degree of mono- and di-glycosylation of the individual bioconjugates, and to determine the molecular mass (MW) of the protein carrier and of the O-PS attached to it.
• SEC-MALS size-exclusion chromatography with multi-angle light scattering
• Table 3-1 Vaccine Compositions Ingredient Amount ⁇ g/mL
• Active substance Composition 1 Composition 2 O-antigen polysaccharide
• the active substances in the vaccine composition are glycosylated proteins
• the dose of the EPA carrier protein depends on the polysaccharide-to-protein ratio.
• the estimated polysaccharide-to-protein ratio was between 15% and 50% depending on the O- antigen serotypes, i.e., the weight of polysaccharide in a conjugate is about 15% to 50% of the weight of the EPA protein carrier in the conjugate.
• the polysaccharide-to-protein ratio was quantified, e.g., the amount of polysaccharide (O-antigen) was measured by the anthrone assay (see Laurentin and Edwards, 2003, Anal Biochem 315, 143- 145) and the bicinchoninic acid (BCA) assay was used to measure the protein concentration.
• the value of EPA provided in Tables 3-1 and 3-2 is the expected value based on the analytical results from ExPEC4V.
• EPA ExoProtein PJPseudomonas aeruginosa exotoxin A, detoxified form used as protein carrier;
• a dose of 4 ⁇ g per O-antigen polysaccharide (PS) of ExPEC4V was tested in this study. This dose is equivalent to the maximum dose that was evaluated in the Phase 1 clinical study described below. Hence the full human dose as used in Phase 1 was administered in the rat GLP toxicology study. The study was performed with a nonclinical batch which was representative for the batch used in the Phase 1 clinical study.
• Immunogenicity of the vaccine has been confirmed, inducing higher serum immunoglobulin G (IgG) titers towards the 4 O-antigens in the vaccinated group compared with vehicles that received only formulation buffer.
• IgG immunoglobulin G
• a GLP repeated dose toxicity study with a 3 -week recovery period was conducted in NZW rabbits (TOX11 163, draft report) to assess the toxicity and local tolerance of ExPEC4V following 3 i.m. injections (Days 0, 14, and 28) given 2 weeks apart. Reversibility, persistence, and delayed occurrence of any changes were assessed on Day 49, after a 3 -week treatment- free period following the 3rd injection on Day 28.
• the design of the Phase 2-enabling repeated dose toxicity study in the rabbit can be found in Table 5.
• ADM1 left - lower part m. biceps femoris; ADM2: right - lower part m. biceps femoris;
• ADM3 left - upper part m. biceps femoris
• ADM4 right - upper part m. biceps femoris
• ADM5 left - m. quadriceps femoris
• ADM6 right - m. quadriceps femoris
• a maximum dose of 16 ⁇ g per O-antigen PS of ExPEC4V (total PS dose of 64 ⁇ g, together with 218 ⁇ g EPA carrier protein) was tested in this study. This dose is 4 times higher than the maximum dose that was tested previously in the Phase 1 -enabling GLP toxicity study in the rat and is equivalent to the maximum PS (and EPA) dose that is evaluated in the Phase 2 clinical study described below. Hence the full (maximum) human dose as to be used in Phase 2 was administered in this rabbit GLP toxicology study.
• lymph nodes Within the draining medial iliac lymph nodes, production (in germinal centers) and sequestration of lymphoblastic cells was seen within the paracortex and/or medullary cords of ExPEC4V -dosed rabbits at the end of the treatment period, resulting in increased overall cellularity. Furthermore lymph nodes were larger in both sexes which correlated with an increased weight in females. These findings were not seen at the end of the recovery period. An increased number of germinal centers was noted in the spleen of treated males and females at the end of the treatment and recovery period, and was accompanied by an increase in spleen weight in both sexes at the end of the treatment period.
• the OPK assay measures the ability of serum to facilitate opsonophagocytosis and killing of different E. coli serotypes.
• E. coli serotypes defined dilutions of the sample sera were incubated, in each well, with bacteria from one of the four vaccine-specific E. coli serotypes, a defined amount of HL60 cells, and baby rabbit complement. After incubation, a proportion of the mixture was spotted onto tryptic soy agar (TSA) and the number of bacterial colonies was counted.
• TSA tryptic soy agar
• the ability of the antibodies to bind the bacterial cells and activate deposition of the complement and mediate uptake and killing of the bacteria by HL60 cells was expressed as opsonic titer.
• the opsonic titer or opsonization index (OI) corresponds to the dilution of the sera killing 50% of the bacterial cells.
• Opsonic indices for pre- and post-immune sera are provided. At least a 4-fold increase of OI from pre- to post-immune is considered significant.
• E. coli was pre-opsonized with dilutions of serum from vaccinated rats, incubated with complement and phagocytes (differentiated HL60 cells), and the colony forming units (CFUs) were determined.
• E. coli selected for OPK testing were OC 24453 (serotype 02), OC 24781 (serotype 06 A) and OC 24176 (serotype 025B).
• Table 6 shows the total OI titers for the O-antigens 02, 06A and 025B from rats immunized with the tetravalent vaccine with either 0.4 or 4 ⁇ g per O-antigen.
• the titers were determined in two separate experiments.
• the 0.4 ⁇ g dose induced significant OIs in all animals for the 02 and 06A serotypes.
• 025B 3/8 animals showed a significant increase in 01 following immunization with the 0.4 ⁇ g dose.
• the 4 ⁇ g dose induced lower 01 increases for 02 in all animals. 3/8 animals showed 01 increases when the sera from the 4 ⁇ g dose group were tested on 025B E. coli.
• Table 6 OIs against E. coli 02, 06A and 025B. OIs for individual pre-vaccination and post 3 vaccination sera from two separate experiments are shown for all animals.
• ExPEC4V has been tested in a first-in-human Phase 1 study, which enrolled a total of 194 subjects.
• This Phase 1 study is a randomized, placebo-controlled, multicenter study. The study was conducted in a single-blind manner as the investigator and study staff knew the randomization group of the subjects while the subjects were required to be blinded to their randomization group at all times.
• the primary objective of the study was the comparison of solicited and unsolicited adverse events (AEs) and serious adverse events (SAEs) between subjects who received ExPEC4V and subjects who received placebo.
• the main secondary objectives included immunogenicity parameters, the number of symptomatic UTI episodes caused by E. coli vaccine-serotypes, and the rate of occurrence and clinical symptoms of vaccine-serotype-specific E. coli UTI.
• Eligible subjects were required to have at least 3 independent UTI episodes in the last 12 months or at least 2 independent UTI episodes in the previous 6 months; at least 1 of the independent UTI episodes had to be due to a culture-confirmed E. coli infection.
• a total of 194 subjects were enrolled and randomized to a single i.m. dose of 0.5 mL of ExPEC4V or placebo (see Table 7). The enrollment was done in a staggered approach to assess Day 14 safety data before proceeding to the next phase:
• the first 8 subjects were randomized (3 : 1 ) to a dose of 1 ⁇ g of each PS, or placebo
• the remaining 178 subjects were randomized (1 : 1) to a dose of 4 ⁇ g of each PS, or placebo.
• Table 7 the demographic and baseline characteristics of the subjects enrolled in the Phase 1 study.
• each subject received one intramuscular injection of 0.5 ml of solution (ExPEC4V or placebo) in the deltoid muscle.
• the reduced dose of the candidate vaccine contained 1 ⁇ g of each polysaccharide (total 4 ⁇ g polysaccharide).
• the target dose of the candidate vaccine contained 4 ⁇ g of each polysaccharide (total 16 ⁇ g polysaccharide).
• Safety was evaluated based on solicited local (pain, erythema, and swelling at the injection site) and systemic (fever, i.e., body temperature >38°C) AEs collected in a diary from Day 1 postvaccination until Day 7 and on AEs and SAEs collected until Day 270 (end of study visit). Immunogenicity was evaluated by qualified enzyme-linked immunosorbent assays (ELISA) and opsonophagocytic killing (OPK) assays using serum from blood samples taken prevaccination on Day 1 and postvaccination on Days 30 and 270.
• ELISA enzyme-linked immunosorbent assays
• OPK opsonophagocytic killing
• Descriptive statistics (n, mean, standard deviation, median and ranges for continuous variables, frequencies and percentages for categorical variables) are provided by treatment group and/or visit, where applicable. All data are listed by subject, treatment group and, where applicable, visit. All subjects from Group B receiving placebo are combined to form the placebo treatment group.
• Table 8 GMT and 95% Confidence Intervals in ELISA-Determined Total Antibody Titers from Day 1 (Prevaccination) to Day 30 - Phase 1 Study ExPEC4V
• N 95 serotype 3 serotype'
• the proportion of subjects with a >4-fold increase in antibody titers ranged from 57%> (serotypes OIA and 06A) to 80% (serotype 02), which is lower than the proportion with a 2-fold increase, but notably higher than observed with the dose containing 1 ⁇ g PS per serotype.
• Serotypes OIA and 06A serotypes
• 80% serotype 02
• a robust immune response to each of OIA, 02, 06A, and 025B was observed, and that a significant increase in the ELISA titers between post (30 days after injection) and pre-injection (day 1) was observed only in the vaccinated groups (VJDay 30 v.s. V_Day 1), but not in the placebo groups (P__Day 30 v.s.
• OPK assays were used to assess the functional antibody response of women participating in the clinical study. Sera were collected from study participants. E. coli was pre- opsonized with dilutions of serum from the vaccinated women, incubated with complement and phagocytes (differentiated HL60 cells), and the remaining colony forming units (CFUs) was determined. Subsequently, the maximum percent killing and Opsonization Indices (OI: serum dilution killing of 50% of E. coli) were calculated. E. coli selected for OPK testing were OC 24452 (serotype 01 A), OC 24453 (serotype 02), OC 24454 (serotype 06A), and OC 24176 (serotype 025B).
• OPK titers were determined for the 194 subjects, including 95 subjects receiving placebo, 6 subjects receiving the ExPEC4V 1 ⁇ g PS (per serotype) vaccine and 93 subjects receiving the ExPEC4V 4 ⁇ g PS (per serotype) vaccine.
• N 95 serotype 6 serotype
• the GMT value in OPK-determined functional antibody titer for 025B antigen is lower than those for the other antigens (OIA, 02 and 06A).
• the OPK assay has been accepted as a better surrogate assay for immune protection induced by the PS conjugate vaccine against Streptococcus pneumoniae (Prevenar ® ), since the ELISA may not differentiate nonprotective low-avidity antibodies from protective high-avidity antibodies (Kim et al., Clin Diagn Lab Immunol. 2003 10(4) :616-21)
• composition 1 contains 32, 32, 32, 32 ⁇ g/ml per O-antigen polysaccharide (PS) of the E. coli serotypes OIA, 02, 06A and 025B, respectively, without adjuvant.
• Composition 2 contains 16, 16, 16, 32 ⁇ per O-antigen PS of the E. coli serotypes OIA, 02, 06A and 025B, respectively, without adjuvant.

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