WO2007148229A2 - Particules d'adhésine polyvalentes immunogènes - Google Patents

Particules d'adhésine polyvalentes immunogènes Download PDF

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WO2007148229A2
WO2007148229A2 PCT/IB2007/002430 IB2007002430W WO2007148229A2 WO 2007148229 A2 WO2007148229 A2 WO 2007148229A2 IB 2007002430 W IB2007002430 W IB 2007002430W WO 2007148229 A2 WO2007148229 A2 WO 2007148229A2
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antigen
immunogenic
multivalent complex
adhesin
tag
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PCT/IB2007/002430
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WO2007148229A3 (fr
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Stefan Knight
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Stefan Knight
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • 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
    • A61K2039/6068—Other bacterial proteins, e.g. OMP

Definitions

  • the present invention is directed toward an immunogenic multivalent complex.
  • the complex comprises a receptor-binding domain of a two-domain adhesin antigen coupled with a carrier particle or a single-chain polyadhesin antigen coupled with a carrier particle.
  • the present invention is also directed to immunity-stimulating and adhesion-blocking agents; antibodies; vaccines; immunogenic formulations; immunogenic constructs and compositions comprising an immunogenic multivalent complex.
  • the present invention is further directed to methods for identifying a two- domain adhesin antigen.
  • Infectious diseases constitute a major health and health cost burden in both humans and animals.
  • the World Health Report 2004 estimates that infectious and parasitic diseases caused almost 11 million deaths in 2002.
  • the same report estimates that respiratory and diarrhoeal infections were responsible for 4.0 and 1.8 million deaths respectively in 2002, to be compared to the toll of diseases such as AIDS (2.8 million deaths), TB (1.6 million deaths), and malaria (1.3 million deaths).
  • a significant proportion of respiratory and diarrhoeal infections are caused by Gram negative pathogens such as Salmonella enter ica (typhoid fever, enterocolitis), Haemophilus influenzae (pneumonia), Bordetella pertussis (whooping cough), and Escherichia coli (diarrhea).
  • E. coli a normal inhabitant of the intestinal tract of both humans and animals, can cause a number of diseases both within and outside the intestinal tract.
  • E. coli strains may be subdivided into commensal, non-pathogenic strains, strains that can cause intra-intestinal infections (e.g. ETEC, EHEC, EPEC), and those that can cause extra-intestinal infections such as urinary tract infections (UTIs), abdominal and pelvic infections, pneumonia, surgical site infections, meningitis, and sepsis (ExPEC strains).
  • Extra-intestinal E. coli infections constitute a major but perhaps underestimated health problem.
  • UTIs are estimated to affect at least 50% of women in the western world over life. Each year, 150 million UTIs have been estimated to occur worldwide. The most common cause of UTI in humans is infection by uropathogenic E. coli (UPEC) species, accounting for about 80% of reported cases.
  • UPEC uropathogenic E. coli
  • E. coli infections are common not only in humans but also in animals and occur in almost all newborn farm animals, constituting a major economic loss in live-stock industry.
  • Some examples of extra-intestinal animal diseases caused by E. coli are UTIs in pets (cats and dogs), uterine infections in horses, mastitis in cattle and in pigs, and oedema disease in piglets.
  • Bacterial infections can often be efficiently treated with antibiotics, but the emergence of antibiotic resistant bacterial strains represents a serious and increasing problem. For example, treatment of typhoid fever relies on prompt administration of antibiotics, but strains of S. typhi resistant to one or more of the commonly used antibiotics have emerged worldwide. Treatment of E. coli infections in humans is also affected by the development of antibiotic resistance; e.g. the emergence of resistance to amoxicillin and ampicillin has more or less put an end to the use of these drugs for treatment of cystitis and pyelonephritis. There are currently no E. coli vaccines available for prophylactic or therapeutic use in humans. Current treatment regimes for coliform infections in animals include the use of antibiotics, vaccination, and management of pathogen exposure (e.g. by minimizing the amount of bacteria in bedding).
  • Vaccines targeting some bacterial infectious diseases are available or are being developed. Most of these vaccines have been formulated using either killed whole bacteria or live attenuated bacterial strains. Such whole cell vaccines are associated with a number of problems such as more or less severe side effects (severe local reactions, fever), the need for rigorous safety measures to ensure that live pathogenic bacteria are not transferred with the vaccine or spread from the production plant, or problems of controlling the stability, strength, and nature of immune response owing to variations in the amount of antigen present in the vaccine and in antigen presentation. Additionally, whole cell antigen presentation may shield potentially efficient broad-range antigens from the immune system.
  • bacterial infectious diseases e.g. pertussis, typhoid fever, ETEC diarrhea and bovine mastitis.
  • fibrillar adhesion organelles pli, fimbriae
  • a conserved and intrinsically highly antigenic receptor-binding adhesin is incorporated as a minor component of a large complex protein structure on the bacterial cell surface, leading to efficient production of antibodies directed against the non-conserved bulk components of the organelle but not against the adhesin.
  • the present invention is directed toward an immunogenic multivalent complex.
  • the complex comprises (i) a receptor-binding domain of a two-domain adhesin antigen or a single-chain polyadhesin antigen coupled with (ii) a carrier particle.
  • the present invention is also directed toward an immunogenic construct comprising an N-terminal receptor binding of a two-domain adhesin.
  • the adhesin is prepared in a method in which the receptor-binding site of the antigen is free of any bound ligand.
  • the present invention is further directed to immunity-stimulating and adhesion- blocking agents; vaccines; and compositions comprising an immunogenic multivalent complex or a mixture of antigen and adjuvant.
  • the present invention is further direct to a method for identifying a two-domain adhesin antigen and/or the borders between the pilin and receptor binding domains of the antigen.
  • the method comprises the steps of selecting a sequence of a pilin from a chaperpne/usher system; searching a protein and/or DNA sequence database with the pilin sequence; and identifying a sequence that aligns to the C-terminal portion of the pilin sequence and comprises an unmatched sequence of from about 140 to about 240 amino acid residues preceding the aligned region.
  • Figure 1 is an illustration of an embodiment of an adhesin antigen construct comprising: i) the stable, folded and functional receptor-binding domain of a two-domain adhesin, ii) a linker, and iii) a coupling tag; the construct is of use for formulating an immunogenic multivalent complex;
  • Figure 2 is an illustration of an embodiment of an adhesin antigen construct comprising: i) a stable, folded and functional single-chain polyadhesin, ii) a linker, and iii) a coupling tag; the construct is of use for formulating an immunogenic multivalent complex;
  • FIG 3 is an illustration of an embodiment of an immunogenic multivalent complex comprising an adhesin antigen such as that illustrated in figures 1 and 2 coupled to a carrier particle via a short, rigid linker; and
  • FIG 4 is an illustration of an embodiment of an immunogenic multivalent complex comprising an adhesin antigen such as that illustrated in figures 1 and 2 coupled to a carrier particle via a long, flexible linker.
  • E. coli Most Gram negative bacteria, including E. coli, depend on expression of specific adhesive organelles on the bacterial cell surface to mediate attachment to target tissues.
  • ETEC strains express K88, K99, and F17 fimbriae as well as both fimbrial and nonfimbrial colonization factor antigens (CFAs) to adhere to small bowel tissue.
  • CFAs colonization factor antigens
  • UPEC use Dr adhesins, and type-1, P, S, and FlC pili for attachment to uroepithelium. S and P pili are further associated with persistent coliform mastitis.
  • TDAs two-domain adhesins
  • the fibrillar structures used to display the TDAs consist of linear non-covalent assemblies of one or several different polymerizable protein molecules called 'pilins;' typically a single fibrillar structure contains several hundreds to a thousand or more pilins.
  • TDAs have an N-terminal receptor binding domain joined to a C-terminal pilin domain and can only by added at the tip of a fibrillar structure.
  • a second important class of adhesive organelles assembled via the chaperone/usher pathway comprises structures that do not incorporate a specialized TDA for receptor binding but instead consist of polymerized receptor-binding pilin subunits (single domain adhesins, SDAs), in some cases with a specialized and highly conserved 'invasin' SDA subunit at the tip (Table 2).
  • SDAs single domain adhesins
  • Examples of structures belonging to the class of pblyadhesive SDA organelles include the Dr adhesins mentioned above, Y. pestis pH6 antigen, and S. enteritidis Sefl4 fimbriae.
  • Periplasmic chaperones are sleric chaperones that bind to pilins as they emerge in the periplasm, ensure their correct folding, and deliver the folded subunits to the usher where they are assembled into fibrillar polymeric structures. In the absence of chaperone, subunits are unstable, aggregate, and are digested by periplasmic proteases.
  • the L-shaped periplasmic chaperone molecules consist of two immunoglobulin (Ig)-like domains joined at ⁇ 90° angle.
  • the Fj and Gi ⁇ -strands in the 1 st , N-terminal, domain are connected by a long and flexible loop that protrudes like a handle from the body of the domain.
  • the beginning of the Gi ⁇ -strand harbors a conserved motif of hydrophobic residues that is solvent exposed in the free chaperones and that is critical for subunit binding.
  • Pilins like the chaperone domains, are Ig-like ⁇ -sandwiches. However, the final (G) ⁇ -strand required for a complete Ig-fold is missing, creating a deep hydrophobic cleft on the surface of the subunit.
  • the chaperones bind pilins by inserting their Gi ⁇ -strand into this cleft in a process called donor strand complementation (DSC) (Choudhury et al. (1999). Science, 285, 1061-1066; Sauer et al. (1999). Science, 285, 1058-1061). The two proteins bind via edge strands in the pilin and in the 1 st domain of the chaperone to form a closed super-barrel with a common core.
  • DSC donor strand complementation
  • the hydrophobic side chains in the conserved Gi motif are inserted into the hydrophobic acceptor cleft and become an integral part of the subunit hydrophobic core. While not wishing to be bound by theory, it is believed that the lack of the final, seventh, ⁇ -strand of the Ig fold is why pilins are unstable and tend to aggregate in the absence of chaperone.
  • the N-terminal region of pilus subunits (approximately the first 10-20 residues), which is disordered in chaperone: subunit complexes, harbors a conserved ⁇ -strand motif, similar to the chaperone Gi motif, that constitutes a subunit polymerization sequence.
  • Assembly of subunits proceeds by a donor strand exchange (DSE) mechanism in which the chaperone Gi donor strand bound in the acceptor cleft of one subunit is displaced by the N-terminal region of a second subunit, thereby joining subunits into a fiber (Choudhury et al. (1999). Science, 285, 1061-1066; Sauer et al. (1999). Science, 285, 1058-1061; Zavialov et al. (2003). Cell, 113, 587-596).
  • the N-terminal polymerization sequence becomes ordered and adopts a ⁇ - strand conformation (which is why the N-terminal polymerization sequence is also refered to as Ga for G donor strand).
  • TDAs lack the N-terminal Ga sequence and instead have an entire receptor-binding domain coupled to the N-terminus of the pilin domain, and hence can onoy be located at the tip of a pilin fiber.
  • Binding of a chaperone:TDA complex to an empty usher initiates assembly which then proceeds by sequential addition of pilin subunits to the base of the growing fiber and simultaneous secretion of the fiber through the usher pore. This process results in the surface display of a linear fiber with the TDA capping its tip. Following secretion, many TDA adhesive organelles coil up into more compact helical structures such as in pili or fimbriae.
  • members of the SDA polyadhesin class of adhesins do not display a TDA at their tip but instead some polyadhesins remain uncapped, whilst others are capped by a specialized invasin subunit.
  • Invasin subunits have the same incomplete Ig fold as pilins but lack an N-terminal polymerization sequence and hence can only function as DSC acceptors.
  • most SDA polyadhesins remain as flexible thin fibers, sometimes discernible as such in electron microscopic images and sometimes collapsed into a tangle a fibers forming a 'capsule' or 'sheath' surrounding the bacterial surface.
  • Table 1 sets forth examples of TDAs assembled into fibrillar surface organelles via the chaperone/usher pathway.
  • Table 2 sets forth examples of SDA polyadhesins assembled into fibrillar surface organelles via the chaperone/usher pathway.
  • This latter method for identifying TDAs is new and consists of the following steps: 1 ) Select the sequence of a known pilin from a known chaperone/usher system, e.g.
  • PapA, PapE, PapF or PapK from the P-pilus chaperone/usher system, or FimA, FimG or
  • FimF from the type-1 pilus system. Identifying and selecting such a pilin sequence is a trivial task, with many examples in the literature. 2) Use the known pilin sequence as a query sequence to search the protein and or DNA database (e.g. the NCBI non-redundant protein or nucleotide sequence database) for matching protein sequences comprising 260-
  • Matching proteins are those that obtain a statistical significance threshold (E value) greater than a suitable cutoff value, e.g. 0.001. 3) Identify those sequences where the query sequence aligns to the C-terminal part of the hit and where there is an unmatched sequence of 140-240 amino acid residues preceding (N-terminal to) the matched region.
  • This unmatched region provides a preliminary definition of the domain borders for a putative TDA receptor-binding domain that may be used to design primers for cloning.
  • a search of the current database using the method described above identifies in the order of 70 putative TDAs falling into about 25 sequence families, representing a lower bounds estimate on the number of TDAs in the database.
  • the set of TDAs and other adhesins available for expression in a specific bacterial strain provides that strain with a certain range of binding capacities that defines the host and tissue tropism of the strain.
  • the genomic complement of TDAs and other adhesins in a particular bacterial strain determines the range of hosts and tissues that may be infected by that strain. All organelles assembled via the chaperone/usher pathway are believed to be built as linear fibers of subunits linked by DSC but with variations in their gross architecture.
  • the P pilus encoded by the eleven genes of the pap gene cluster ⁇ papA- papK) found in many uropathogenic strains of E. coli, is a composite fiber — a thick rod with a thinner tip fibrillum at its distal end.
  • the rod has a diameter of 7 nm with a hollow core and is composed of PapA subunits wound in a tight right-handed helix; the tip fibrillum consists primarily of PapE subunits wound in an open helical conformation.
  • the PapG TDA which binds to Gal- ⁇ -l,4-Gal sugars found in the human kidney and which is necessary for the development of pyelonephritis in a monkey model, is located at the tip of the fibrillum.
  • the type-1 pilus, encoded by the fim gene cluster (fimA-fimH) has a similar composite structure, but its tip fibrillum is short and stubby.
  • the rod is composed of FimA subunits wound in a tight right-handed helix; the tip fibrillum contains the mannose-binding FimH TDA as well as FimG. FimF is thought to link the tip fibrillum to the rod.
  • Type-1 pili are found in most E. coli strains, including both pathogenic and commensal strains, as well as throughout the Enterobacteriaceae family, and have been shown to play a critical role in the pathogenesis of cystitis.
  • Hif pili encoded by the hif gene cluster (JiifA-hifE) found in pathogenic H. influenzae strains, have rods 6-7 nm in diameter and a short, thinner tip structure.
  • the rods have a cross-over repeat consistent with a double-stranded right- handed helical architecture.
  • the rod is composed primarily of HifA, while the tip contains HifD and HifE.
  • HifE is thought to be the TDA that mediates attachment to human cells.
  • Fl capsular antigen is expressed by the plague pathogen Y. pestis in large quantities at temperatures of 35-37°C to cover the bacterial surface with a massive amount of material forming a gelatinous antiphagocytic capsule.
  • the capsule consists of a tangle of ⁇ 2 nm thin flexible fibers made from the Cafl SDA-type subunit.
  • ExPEC is the major causative agent of urinary tract infections (UTIs) (ExPEC strains isolated from patients with UTI are commonly referred to as uropathogenic E. coli, UPEC).
  • UTIs are a serious health problem affecting as many as every second woman in the western world at least once in their lifetime. The elderly, and diabetics, are particularly susceptible to frequent UTIs. UTIs often recur within the months following the primary infection, despite proper use of antibiotics. Currently no approved UPEC vaccine is available.
  • adhesins are known to be important for UPEC virulence and for establishment of UTIs (Table 3) and are attractive targets for novel therapeutics and vaccines.
  • Type 1 pili recognize mannose-containing receptors present on the luminal surface of the bladder epithelium and are critical for the establishment of cystitis.
  • P pili mediate binding to Gal- ⁇ -l,4-Gal-containing receptors in the upper urinary tract to cause pyelonephritis.
  • the closely related FlC and S pili bind to galactosyl ceramide and globotriaosyl ceramide receptors and to sialic-acid-containing receptors respectively and are implicated in ascending UTTs.
  • Adhesive organelles belonging to the Dr family are frequently found on UPEC strains isolated from patients with cystitis or pyelonephritis.
  • Dr adhesins non- Cystitis, DAF, CEACAMs, DraE (SDA) pilus
  • Pyelonephritis CEA, type IV collagen
  • type-1 pili are by far the most prevalent, being expressed by a wide range of both pathogenic, commensal, and laboratory strains.
  • the FimH TDA of type-1 pili consists of an amino-terminal mannose-binding domain (residues 1-158) joined to a carboxy-terminal pilin domain (residues 159-279) that links the adhesin to the rest of the pilus.
  • UPEC can invade urothelial cells in a type-1 pilus dependent mechanism. Following invasion, they form bacterial communities such as biofilms inside the bladder superficial umbrella cells, creating an environment protected from antibiotics and the immune system of the host. Bacteria within these communities exhibit regional expression of type-1 pili. A subpopulation of UPEC can flux back out of the cells and re- invade to ultimately establish a quiescent reservoir that may serve as a seed for recurrent infections.
  • FimH The primary physiological receptor for FimH in the urinary tract is the glycoprotein uroplakin Ia that is abundantly present on differentiated uroepithelial cells.
  • FimH recognizes a wide range of glycoproteins carrying one or more N-linked high-mannose structures. FimH also binds yeast mannans and mediates agglutination of yeast cells. FimH-mediated adhesion can be inhibited by £>-mannose and a variety of natural and synthetic saccharides containing terminal mannose residues. Blocking of the FimH-receptor interaction has been shown to prevent bacterial adhesion to the bladder uroepithelium and thereby infection (see e.g. Langermann and Ballou (2003).
  • FimH and any other TDAs or SDAs known or discovered to be UPEC virulence factors, including but not limited to those listed in Table 3, may be used to provide antigens for formulation of UTI vaccines as disclosed in the current invention.
  • ETEC Enterotoxigenic E. coli
  • ETEC strains colonize the small bowel mucosa and cause disease by expressing at least one of the enterotoxins ST and LT.
  • Adhesion to small intestinal tissue can be mediated by a spectrum of adhesive factors, e.g. K88 fimbriae (pigs), K99 fimbriae (calves, lambs, pigs), Fl 7 (G) fimbriae (ruminants, humans), or CFAs (humans).
  • K99 and Fl 7 are typical chaperone/usher- assembled TDA-carrying fimbriae; many of the CFAs appear to belong to a distantly related class of adhesive surface organelles assembled via the so called alternate chaperone/usher system.
  • ETEC vaccine available. Vaccines against ETEC are being developed but current strategies for antigen selection and presentation are being challenged (Boedeker (2005). Curr Opin Gastroenterol, 21, 15-19).
  • EPEC Enteropathogenic E. coli
  • EPEC Localized adherence of EPEC depends on bundle-forming (type FV) pili, whereas intimate adherence is mediated by intimin.
  • Other adhesive factors e.g. TDA-presenting type-1 and P pili, and Fl 845 SDA polyadhesin, may also contribute to EPEC virulence.
  • EHEC Enterohaemorrhagic E. coli
  • EHEC Enterohaemorrhagic E. coli
  • the only adhesive factor with an established role in EHEC intestinal colonization is intimin.
  • additional adhesive factors most likely are important for establishment of infection since pathogenic EHEC strains lacking intimin have been identified.
  • Genes encoding EHEC adhesins e.g. ToxB, Saa, Sfp fimbriae, Dia, Efal, long polar fimbriae (LPF) have been reported but most of the putative adhesins are not well characterized and their role in pathogenesis is not clear.
  • EAEC Enteroaggregative E. coli
  • AAF aggregative adherence fimbriae
  • EIEC Enteroinvasive E. coli
  • pathogenesis is thought to be essentially identical to that of Shigellosis.
  • Current models for pathogenesis entail endocytotic invasion of epithelial cells, lysis of the endocytic vacuole, intracellular multiplication, directional movement through the cytoplasm, and invasion of adjacent epithelial cells.
  • Genes necessary for invasiveness are carried on the 140-MDa (-220 kb) plnv plasmid. Essentially nothing is known about the role of adhesive factors in EIEC pathogenesis.
  • Diffusely adherent E. coli are a heterogne'ous group characterized by their diffuse adherence pattern to cultured HEp-2 and HELA cells.
  • DAEC strains that express Afa/Dr adhesins are frequently associated with watery diarrhea that can become persisent in young children, and with urinary tract infections, in particular cystitis in children and pyelonephritis in pregnant women.
  • diarrheagenic E. coli commonly express adhesive factors that have been implicated in pathogenesis and that might be targeted for vaccine development. Examination of whole genomes of diarrheagenic E. coli reveals even more potential adhesin targets. Many of these, as well as many of the already identified adhesins, are assembled via the chaperone/usher pathway and may hence be of use for formulation o v f vaccines targeting diarrheagenic E. coli following the procedures provided by the current invention.
  • Salmonella infections are a serious medical and veterinary problem world-wide and cause great concern in the food industry. More than 2500 Salmonella enterica serovars have been identified. They are widely distributed in nature and are commonly found in the intestinal tract of domesticated and wild mammals, as well as in reptiles, birds, and insects. The two most common diseases caused by Salmonella in man are typhoid fever and enterocolitis. Typhoid fever is caused by serovars such as Typhi and Paratyphi A, B, and C, that are strictly adapted to humans and higher primates. Human enterocolitis is an infection of the intestine and mesenteric lymph nodes.
  • Enterocolitis can be caused by any of the Salmonella serovars but about 50% of cases in the USA are caused by Typhimurium and Enteritidis serovars. New multi-drug resistant Salmonella strains are rapidly emerging. Vaccination of chickens is regarded as a measure to limit the spread of Salmonella strains in food and in the environment by increasing the resistance of birds against Salmonella exposure and decreasing the shedding of Salmonella. Live and inactivated Salmonella vaccines are in use for the prevention of Salmonella infections in both animals and humans with variable results.
  • S. typhimurium TDA fimbrial systems have been described in some detail: type-1 fimbriae (fim), and long polar fimbriae (lpf). Adhesion of S. enteritidis isolates to chicken isthmal glandular secretions has been shown to depend on the FimH (S) TDA. The lpf fimbrial operon mediates adhesion of S. typhimurium to murine Peyer's patches. The S.
  • Sefl4 fimbria expressed exclusively by S. enteritidis and closely related serovars, belong to the class of SDA polyadhesins, and appear to be required for a stage of enterocolitis after transit across the intestinal barrier.
  • Salmonella TDAs (Table 1), and SDA polyadhesins (Table 2), as well as other as of yet unidentified Salmonella TDAs or SDAs, may be used as sources to provide antigens for formulation of Salmonella vaccines as disclosed in the current invention.
  • adhesin vaccine formulations have been based on intact adhesive organelles (e.g. pili), which are antigenically highly variable and hence induce protection limited to bacteria expressing the same fimbrial variant.
  • adhesive organelles e.g. pili
  • antibodies directed against purified whole type-1 or P pili protect against cystitis and pyelonephritis, respectively, in both murine and primate models for these diseases.
  • protection is limited to either homologous E.
  • vaccines composed predominantly of the major structural proteins of pili (e.g. FimA or PapA) are of limited value because antibodies developed against these highly variable proteins are specific for the strains from which the protein used for immunization was derived.
  • TDA-based vaccines might be useful for the prevention and or treatment of a number of different diseases.
  • a vaccine targeting the FimH TDA from an ExPEC strain might be effective in preventing the wide range of clinical manifestations of ExPEC infection (e.g. UTI, pneumonia, surgical site infection, meningitis, sepsis).
  • TDAs consist of an N-terminal receptor-binding domain joined to a C- terminal pilin domain that serves to link the adhesin to the tip of the fibrillar surface appendage.
  • TDAs Prior to assembly, TDAs exist in the periplasm as complexes with their cognate chaperone. Binding to the periplasmic chaperone is required for stabilization and solubilization of the adhesins, making them difficult to produce in large quantities.
  • TDAs are highly unstable molecules that tend to aggregate and are rapidly degraded by periplasmic proteases, and hence are not suitable for use in subunit vaccine formulations.
  • TDAs could be obtained as stable and soluble complexes with their cognate chaperone inspired new hope for the development of adhesin-based vaccines.
  • a chaperone: adhesin vaccine formulation includes the chaperone, which is not normally presented on the outside of the bacterial cell. Presence of the chaperone in a vaccine formulation might potentially block antigenic determinants on the adhesin and or might diminish the immunogenic effect of the adhesin.
  • the receptor- binding domain of TDAs may be expressed on its own in soluble and functional form, and the structure of three such constructs have been determined using X-ray crystallography (FimH and PapG-II from UPEC, and F17-G (GafD) from ETEC (Bouckaert et al. (2005). MoI Microbiol, 55, 441-455; Buts et al. (2003). MoI Microbiol, 49, 705-715; Dodson et al. (2001). Cell, 105, 733-743; Merckel et al. (2003). J MoI Biol, 331, 897-905).
  • the SDAs of polyadhesins assembled via the chaperone/usher pathway suffer from the same problems of solubility and stability as the full length TDAs. These problems may be circumvented by construction of circularly permuted 'self- complemented' SDAs where the N-terminal donor strand has been moved to the C terminus of the SDA (e.g. Anderson et al. (2004). Molecular Cell, 15, 647-657; Zavialov et al. (2005). Biochem J, 389, 685-694; Chalton et al. (2006). Infect Jmmun, 74, 6624- 6631).
  • Such constructs are able to fold into a functional, soluble, and stable unit even in the absence of a periplasmic chaperone and can be used to formulate antigens for incorporation into multivalent adhesin particles as disclosed in the current invention.
  • receptor binding sites are not necessarily located on individual SDA subunits but may be located e.g. between adjacent subunits, a monomeric construct such as a self-complemented SDA might not be ideal for generation of blocking antibodies.
  • This problem may be overcome by constructing a multidomain single-chain protein comprising between 2-10 self-complemented SDA units, e.g. 2-5 units, e.g. 2-3 units.
  • An invasin subunit may be included to cap the single-chain polyadhesin.
  • Single- chain polyadhesin constructs can easily be made by standard molecular biology techniques by someone reasonably skilled in the art (see e.g., Cota et al (2006) MoI Microbiol, 62, 356-.366) In such a construct, self-complemented SDA units are covalently coupled by a direct link from the C-terminus of the self-complementing G d donor strand of subunit n to the N-terminus of strand A of subunit n-1 ( Figure 2).
  • the single-chain polyadhesin constructs may be used for formulation of adhesin-based vaccines as disclosed in the current invention.
  • Multivalent adliesin presentation Multivalent Adhesin Particles (MAPs)
  • Multivalent antigen presentation generally results in a significantly improved response.
  • Multivalent antigen presentation may be achieved by coupling subunit antigens to a suitable carrier particle.
  • antigens are coupled such that their antigenic capacity is not blocked.
  • antigens are presented on the carrier particle in the same way as they would be on the surface of the pathogen from which they have been derived, so as to present the immune system with a good mimic of the pathogen against which protection is desired.
  • adhesins the receptor binding site should not be destroyed or masked if blocking antibodies are to be raised.
  • tags may be added at either the N-terminus or the C-terminus of the construct.
  • the introduced tags may be useful both for purification of antigen and for coupling to carrier particles.
  • a large number of such tags are known and commercially available and any of which may be employed herein (see e.g. Heam and Acosta (2001). J MoI Recognit, 14, 323-369; Terpe (2003). Appl Microbiol Biotechnol, 60, 523-533; Zhang et al. (2005). J Am Chem Soc, 127, 10136-10137).
  • the tags can easily be introduced either using standard molecular biology techniques and or by chemical modification.
  • a coupling method that is inexpensive and that results in a metabolically stable and nontoxic linkage of the antigen to the carrier particle may be used.
  • Such methods exist and are well known to one skilled in the art, thereby not implying that these known methods are the only ones that could be used.
  • an affinity-tag system utilizing a small peptide tag and that allows one-step affinity purification is used.
  • Tags that are useful both to facilitate purification and for coupling the adhesin antigen to carrier particles for multivalent presentation include, but are not limited to, hexa-histidine, polylysine, polyarginine, FLAG-tag, Strep-tag II, artificial polypeptide scaffolds or a combination thereof.
  • the surface density of adhesin antigens on the carrier particle may influence the immunological properties of the multivalent adhesin-particle complexes.
  • This variable can be modulated.
  • the surface density may be modulated by adjusting the amount of coupling acceptor groups on the carrier particle (e.g. the amount of Ni when the antigen coupling tag is hexa- histidine).
  • the surface density may be modulated by mixing tagged antigen with specific amounts of empty tag or other blocking substance prior to coupling. An appropriate mixing ratio may be determined separately for each multivalent construct.
  • the mixing ratio is from about 1 :1 to about 1:10000.
  • a further aspect of the invention concerns the flexibility of coupled adhesin antigen(s) on the carrier particle. While not wishing to be bound by theory, it is believed that a multivalent immunogenic complex derives its efficiency in part from its ability to interact multivalently with components of the immune system. As for any multivalent binding " interaction, this depends not only on the one-to-one affinity between the monovalent binding partners taking part in the multivalent interaction, but also on the probability of multiple constructive encounter between binding partners (i.e. collisions in orientations that allow binding). That is to say, the greater the chance is for multiple binding partners to simultaneously obtain relative orientations that allow binding, the greater the probability for multiple simultaneous interactions, and the tighter the interaction (or, phrased differently, the larger the avidity) will be.
  • the flexible linker is a poly amino-acid linker. In another embodiment, the flexible linker is a poly-glycine linker. Such linkers may be introduced by using standard molecular biology protocols. In one embodiment, the length of the linker is less than 30 amino acids. In another embodiment, the length of the linker is from at least 1 to about 20 amino acids. In yet another embodiment, the length of the linker is from about 3 to about 12 amino acids. Methods for evaluating the immunological and protective properties of multivalent adhesin-particle complexes so as to determine the optimal linker exist and are well known (see Examples 5-7).
  • a construct for addition of receptor-binding domains from TDAs to the surface of a carrier particle comprises: i) a stable, folded and functional receptor-binding domain of a TDA, ii) a C- terminal poly amino-acid linker region, and iii) a coupling tag.
  • a construct for addition of single-chain SDA polyadhesins to the surface of a carrier particle comprises: i) a stable, folded and functional single-chain polyadhesin, ii) a C-terminal or N-terminal poly amino-acid linker region, and iii) a coupling tag.
  • an immunogenic multivalent complex comprises an adhesin antigen coupled to a carrier particle via a short, rigid linker.
  • an immunogenic multivalent complex comprises an adhesin antigen coupled to a carrier particle via a long, flexible linker.
  • Carrier particles include, but are not limited to, bacterial ghosts (see e.g. Mayr et al. (2005). Adv Drug Deliv Rev, 57, 1381-1391; Tabrizi et al. (2004).
  • SMBVTM particles e.g. von Hoegen (2001). Adv Drug Deliv Rev, 51, 113-125
  • magnetite nanoparticles e.g. Pan et al. (2005). J Colloid Interface Sd, 284, 1-6, or combinations thereof.
  • Parameters that are important in selecting a carrier particle include size and size distribution, toxicological profile, metabolic stability, adjuvant effects, availability of coupling methods, and production costs.
  • the size of the carrier particle should be in a size range similar to that of viruses and bacteria, typically in the range 10-10000 nm in diameter, e.g. 500-5000 nm, e.g. 1000-2000 nm, or e.g. 10-200 nm, e.g. 20-60 nm.
  • the carrier particle size distribution should be as narrow and as well defined as possible. Obviously, the carrier particle must not be toxic.
  • any carrier particle may be used, provided that a coupling method for functional display of the TDA- or SDA-derived antigen on the surface of the particle can be identified. This might involve chemical modification of the carrier particle surface to provide an efficient receptor for the antigen tag.
  • MAP constructs using ISCOMATRIX ® and ISCOM ® carrier particles have the potential of being highly efficient vaccines against a range of bacterial infections.
  • similar immunological properties can sometimes be achieved by mixing of antigen and ISCOM ® s, without forming a direct physical link between them.
  • antigen in the form of free un-liganded ('naked') TDA receptor-binding domain without a coupling tag, and or in the form of free un-liganded single-chain SDA polyadhesin without a coupling tag may be mixed with ISCOM ® s to produce an efficient immunogenic formulation.
  • Example 1 Expression of recombinant adhesin antigens
  • Standard molecular biology techniques are used to clone and express receptor- binding domains of TDAs or single-chain SDA polyadhesin constructs in e.g. E. coli C43
  • a suitable tag can be introduced at the C-terminus of TDA receptor binding domains, or at either the N-terminus or C- terminus of single-chain SDA polyadhesin constructs.
  • the pilin domain of FimH can be replaced by a six-His tag (Bouckaert et al. (2005). MoI Microbiol, 55, 441- .455; Schembri et al. (2000).
  • Recombinant receptor-binding domains of TDAs or single-chain SDA polyadhesin constructs expressed in E. coli are produced in soluble form in the periplasm since the constructs include an amino terminal signal peptide that targets the protein for export to the periplasm via the Sec machinery.
  • the periplasmic protein content is obtained by standard methods such as e.g. osmotic shock, and the tagged protein constructs are purified using affinity tag chromatography (e.g. Ni chelate chromatography if the construct is His-tagged) followed by e.g. gel filtration or ion exchange depending on the characteristics of the construct. At each stage of the purification, the yield and purity of the protein is monitored by SDS-PAGE and isoelectric focusing.
  • the highly pure protein sample is analyzed e.g. by dynamic light scattering for homogeneity and solubility. Proper folding of the purified antigen can be checked by circular dichroism. Functional receptor binding can be monitored by any of a number of well known standard ligand-binding assay techniques such as, but not limited to, surface plasmon resonance, solution equilibrium techniques with radioactively labeled ligand, ELISA protocols, or dot blot protocols such as that described for mannose-binding proteins in Example 8.
  • standard ligand-binding assay techniques such as, but not limited to, surface plasmon resonance, solution equilibrium techniques with radioactively labeled ligand, ELISA protocols, or dot blot protocols such as that described for mannose-binding proteins in Example 8.
  • Example 2 The need for active ('naked') adhesin antigens in MAP vaccine formulations; expression and purification of such antigens
  • Recombinantly expressed carbohydrate binding proteins such as e.g. bacterial adhesins can co-purify with ligands, derived from e.g. the medium used to grow bacteria expressing the recombinant adhesin, bound in their receptor binding pocket.
  • ligands derived from e.g. the medium used to grow bacteria expressing the recombinant adhesin
  • One example of this is the co-purification of a tight-binding butyl mannoside in the mannose-binding pocket of the E. coli type-1 pilus TDA (FimH) (Bouckaert et al. 2005, MoI Microbiol, 55, 441-455).
  • an adhesin sample purified without consideration to this will consist of 'dressed' adhesin or a mixture of 'naked' and 'dressed' adhesin. The exact amounts of each species in such a mixture will not be defined and will vary between batches depending on the exact composition of e.g. the bacterial growth medium.
  • antibodies generated using such a mixture will contain an undefined mixture of blocking and non-blocking antibodies directed against the 'naked' and 'dressed' adhesin antigen, respectively.
  • the external ligand is capable of very tight binding to the adhesin, all or nearly all adhesin molecules will be 'dressed' and hence blocking antibodies will not be efficiently generated by this antigen sample.
  • FimCrFimH chaperone:adhesin vaccine development of which was dropped during phase II clinical trials because of limited protection, might be one example of this.
  • the FimCrFimH complex used to formulate the vaccine was obtained from recombinant protein produced in bacteria grown in LB broth, and will thus most likely have consisted of an undefined mixture of FimC:FimH complexes with and without butyl mannoside bound in the receptor binding pocket. That failure to use defined antigen with an empty receptor binding site contributed to the lack of protection is supported by the fact that better protection was afforded when a mutant antigen incapable of receptor binding was used for immunization.
  • the present invention provides a method for producing 'naked' FimH antigen, defined as either intact FimH alone or complexed to a periplasmic chaperone, or to a complementary peptide that may be free or covalently attached to the C-terminus of full- length FimH to produce 'self-complemented' FimH, or a mannose binding fragment of FimH such as the FimH N-terminal mannose-binding domain.
  • the externally derived butyl mannoside ligand present in FimH obtained from cultures grown in LB medium is derived from the LB medium. No such ligand is present in e.g. M9 minimal medium.
  • butyl mannoside in LB medium is detected using mass spectroscopy and chemical methods.
  • the presence of specific external ligands in any culture medium or in solutions used to dissolve the protein sample during purification can be determined using similar techniques.
  • the presence of butyl mannoside in the binding pocket of FimH is detected using crystallographic, chemical, and mass spectroscopic methods (Bouckaert et al. 2005, MoI Microbiol, 55, 441-455).
  • external ligands bound to any adhesin can be detected using e.g. mass spectroscopic methods.
  • An alternative method for producing ligand-free adhesin antigens is to construct site directed mutants where one or more critical receptor-binding residues in the receptor- binding pocket have been changed such that the mutant is no longer able to bind receptor.
  • the desired point mutations may be introduced using standard molecular biology techniques.
  • Critical receptor-binding residues can be identified e.g. by crystallographic techniques and or site directed mutagenesis.
  • the ligand binding pocket is a relatively small and deep pocket, as in e.g. FimH, such changes may be introduced without significantly changing the protein surface accessible to antibodies and hence without affecting the ability to generate blocking antibodies that recognize the wild type antigen.
  • the procedures described herein can be used to obtain any adhesin antigen in the desired form.
  • ISCOM ® -linked adhesin antigens are prepared from ISCOMATRIX ® formulations and purified adhesin antigens following the procedures prescribed by the
  • ISCOMATRIX ® manufacturers For example, a purified His-tagged adhesin construct is coupled to Ni-containing ISCOMATRIX ® formulations.
  • the carrier ISCOMATRIX ® is produced by mixing the selected fraction of quillaja saponin with detergent (MEGA-10) solubilized cholesterol, phosphatidyl choline, and chelating lipid. The detergent is removed using dialysis or ultrafiltration and ISCOMATRIX ® particles are formed. ISCOMATRIX ® particles are activated with Ni 2+ and mixed with adhesin antigen(s) under conditions that allow binding. After binding, the construct is transferred to a physiological buffer such as PBS. His-tagged FimH receptor-binding domain coupled to ISCOM ® s retain receptor binding functionality, showing that this approach for production of MAPs is feasible.
  • VLPs are particles made from viral coat proteins. Preparation of VLPs relies on the ability of viral coat proteins to spontaneously self- assemble following recombinant expression. VLPs resemble live viruses but do not contain genetic material. Methods for preparation of VLPs are well known to those skilled in the art. VLPs are commonly used for incorporation or surface display of small molecules and peptides. Surface display of peptides depends on the ability of capsid proteins to accept insertion of foreign peptides into surface exposed loops while retaining their ability to self assemble. DNA that codes for the foreign sequence is then inserted into the gene for the capsid protein using standard molecular biology tools.
  • Loop insertion is however not amenable for display of TDA-derived or SDA-derived antigens partly because of their larger size but chiefly because the N- and C- terminii of these constructs are at opposite ends of the protein making loop insertion impracticable.
  • an alternative approach as disclosed here must be used for preparation of VLP MAPs.
  • This method consists of a two step process.
  • capsid proteins are engineered to display on their surface (a) suitable tag receptor(s).
  • Tag receptors that may serve this purpose include but are not limited to natural as well as synthetic, engineered amino acid side chains incorporating suitable functional groups (see e.g. Wang et al. 2006, Annu Rev Biophys Biomol Struct, 35, 225-249; Hendrickson et al.
  • a modified capsid protein containing a tag receptor for surface display of proteins is an engineered version of the Tobacco Mosaic Virus (TMV) coat protein (Smith et al. 2006, Virology, 348, 475- 488), where introduction of a reactive lysine at the surface exposed amino terminus allows capsid biotinylation.
  • TMV Tobacco Mosaic Virus
  • adhesin antigen with a matching N-terminal or C-terminal tag is coupled to the engineered VLP.
  • biotinylated TMV VLPs are used as carrier particle
  • C-terminally streptavidin-coupled TDA antigen is added to the biotinylated VLP to allow surface display of the TDA antigen.
  • MAPs are quality controlled both in terms of antigen incorporation (biochemical) and in terms of immunological properties.
  • Analytical ultracentrifugation is used to study the resulting particle size (for TSCOM ® MAPs typically 40 run structures or aggregations thereof) and the relative proportion of incorporated adhesin.
  • the antigenicity of MAPs and the correct exposure of adhesin antigens is verified and quantified by ELISA techniques using polyclonal antibodies, monoclonal antibodies, or carbohydrate ligands (immobilized or in solution).
  • mice are immunized and bled and the sera is tested as described below.
  • Groups of mice typically 8-10 Balb/c and or NMRI mice
  • mice are immunized subcutaneously at the base of the tail (100 microliters) or intrabasally by inhalation of 20-30 microliters of experimental vaccine.
  • a typical regimen is immunizations at weeks 0 and 4 with bleeding at weeks 3 and 6.
  • urine, mucosal washings or mucosal extracts are tested as described for sera.
  • the immunological capacity in terms of innate response in terms of innate response (TNF ⁇ , IL-I, IL-6 and
  • GM-CSF and or other cytokines and chemokines is determined as required using available ELISA techniques.
  • the evaluation of acquired immune response includes the determination of ThI cells (IFN- ⁇ , IL-2) and Th2 cells (IL-4, IL-5 and IL-10).
  • Antibody determinations include specific IgG and subclasses (IgGl, IgG2a and IgG3). If required, other Ig classes such as IgM and IgA may be determined.
  • Adhesion blocking is investigated using ligands immobilized on e.g. polystyrene, or various types of cultivated cells, or relevant tissue samples, as receptor substrates. Briefly, receptor substrates are coated on microtiter wells and after washing with phosphate buffered saline (PBS), and quenching with bovine serum albumin (BSA), target bacteria are added and allowed to adhere. Non-adherent bacteria are removed by washing with PBS. In these assays, several clinical isolates of bacteria are used since antigenic variation is anticipated within a species. Bacterial binding can be detected by e.g.
  • Example 7 Evaluation of opsonization properties of antibodies generated by MAP vaccines
  • Example 8 A simple 'dot blot' assay to determine functional mannose binding
  • a simple 'dot blot' assay may be employed (Berglund 2004, Doctoral thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden).
  • the assay is based on the fact that horseradish peroxidase is a mannosylated protein. Horseradish peroxidase catalyses the reduction of H 2 O 2 to O 2 , and this conversion can be coupled to the oxidation of 4-chloro-l-napthol into an insoluble blue- colored product.
  • H 2 O 2 in the presence of 4-chloro-l-napthol can thus be used to detect peroxidase by the appearance of a blue color.
  • Different concentrations of FimH are blotted onto nitrocellulose filter. Blocking buffer containing 2% Tween is applied to block further binding to the filter. After washing, peroxidase at a concentration of 50 ⁇ g/ml is added, and allowed to bind to FimH. Excess peroxidase is washed away. Finally, 4-chloro-l-napthol is added in the presence of 0.01% H 2 O 2 to detect bound peroxidase.
  • the assay is performed with concanavalin A (a mannose-specific lectin) as a positive control, and lysozyme as a negative control, both in the absence and presence of mannose.
  • concanavalin A a mannose-specific lectin
  • lysozyme a negative control

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Abstract

L'invention concerne des complexes polyvalents immunogènes comportant un domaine de liaison au récepteur d'un antigène de l'adhésine à deux domaines couplé à une particule support, des agents stimulant l'immunité et bloquant l'adhésion, des vaccins et des compositions comprenant un complexe polyvalent immunogène. Des procédés pour identifier un antigène de l'adhésine à deux domaines incluent la sélection d'une séquence d'une piline dans un système chaperon/huissier (chaperone/usher); la recherche d'une base de données de séquences protéiques et/ou d'ADN avec la séquence de piline; et l'identification d'une séquence qui s'aligne à la partie C-terminale de la séquence de piline et comporte une séquence non appariée d'environ 140 à environ 240 résidus d'acide aminé précédant la région alignée.
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US9328150B2 (en) 2005-01-11 2016-05-03 The United States Of America As Represented By The Secretary Of The Navy Recombinant polypeptide construct comprising multiple enterotoxigenic Escherichia coli fimbrial subunits
CN102716475A (zh) * 2012-06-29 2012-10-10 黑龙江省科学院微生物研究所 一种奶牛乳房炎疫苗的制备方法
WO2014077977A1 (fr) * 2012-11-19 2014-05-22 The United States Of America As Represented By The Secretary Of The Navy Construction de polypeptide recombiné comprenant de multiples sous-unités fimbriales d'escherichia coli entérotoxigène
US9925254B2 (en) 2014-11-05 2018-03-27 The United States Of America As Represented By The Secretary Of The Navy Synthetic antigen constructs against Campylobacter jejuni
US10500261B2 (en) 2014-11-05 2019-12-10 The United States Of America As Represented By The Secretary Of The Navy Synthetic antigen constructs against campylobacter jejuni
WO2018174957A1 (fr) * 2017-03-22 2018-09-27 Timothy Andrew Erickson Mimétiques microbiens à taille ajustable pour immunothérapie du cancer
WO2021211698A1 (fr) * 2020-04-14 2021-10-21 Scaled Microbiomics, Llc Traitement ou prévention de la diarrhée des voyageurs
WO2024097817A1 (fr) * 2022-11-02 2024-05-10 Design-Zyme LLC Complexes multi-protéiques à auto-adjuvant pour la production modulaire de vaccins

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