WO2014077977A1 - Recombinant polypeptide construct comprising multiple enterotoxigenic escherichia coli fimbrial subunits - Google Patents
Recombinant polypeptide construct comprising multiple enterotoxigenic escherichia coli fimbrial subunits Download PDFInfo
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
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- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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- A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the inventive subject matter relates to a method of inducing an immune response against enterotoxigenic Escherichia coli using bacterial fimbrial components.
- the method contemplates using enterotoxigenic Escherichia coli major and minor fimbrial subunits, incorporated into a stabilizing construct, as immunogens.
- ETEC Enterotoxigenic Escherichia coli
- LT heat-labile
- ST heat-stable enterotoxin
- ETEC typically attach to host cells via filamentous bacterial surface structures known as colonization factors (CFs). More than 20 different CFs have been described, a minority of which have been unequivocally incriminated in pathogenesis (Gaastra and Svennerholm, Trends Microbiol., 4: 444-452 (1996)).
- CFs colonization factors
- CFA/I colonization factor antigen I
- CFA/I assembly takes place through the alternate chaperone pathway, distinct from the classic chaperone-usher pathway of type I fimbrial formation and that of other filamentous structures such as type IV pili (Ramer, et al., J. Bacterid., 184: 3457-65 (2002); Soto and Hultgren., J. Bacteriol., 181 : 1059-1071 (1999).
- CFA/I and related fimbriae have been grouped as class 5 fimbriae.
- CS3 coli surface antigen 3
- CFA/II ETEC colonization factor antigen II
- CS6 coli surface antigen 6
- CSl fimbrial stalk consists of repeating CooA major subunits.
- the CooD minor subunit is allegedly localized to the fimbrial tip, comprises an extremely small proportion of the fimbrial mass, and is required for initiation of fimbrial formation (Sakellaris, et al., J. Bacteriol., 181 : 1694-1697 (1999). Contrary to earlier evidence suggesting that the major subunit mediates binding (Buhler, et al., Infect. Immun.
- the major subunit alleles of CS4, CS14, CS17 and CS19 gene clusters each showed 99-100% nucleotide sequence identity with corresponding gene sequence(s) previously deposited in GenBank, with no more than four nucleotide differences per allele.
- Each locus has four open reading frames that encoded proteins with homology to the CFA/I class chaperones, major subunits, ushers and minor subunits.
- the one exception was for the CS14 gene cluster, which contained two tandem open reading frames downstream of the chaperone gene.
- Type 1 and P fimbriae have been useful models in elucidating the genetic and structural details of fimbriae assembled by the classical chaperone-usher pathway (23, 24, 25).
- An outcome of work with type 1 and P fimbriae (Kuehn, et al., Nature, 356: 252- 255 (1992); Sauer, et al., Science, 285: 1058-1061 (1999); Choudhury, et al., Science, 285: 1061-1066 (1999)) has led to the development of the principle of donor strand complementation, a process in which fimbrial subunits non-covalently interlock with adjoining subunits by iterative intersubunit sharing of a critical, missing ⁇ -strand
- the invention relates to a recombinant polypeptide construct expressing enterotoxigenic Escherichia coli (ETEC) fimbrial subunits.
- ETEC enterotoxigenic Escherichia coli
- the composition is useful as an immunogenic composition against ETEC strains.
- the composition comprises a recombinant polypeptide construct design wherein major or minor subunits, derived from the same ETEC fimbrial type, are connected, via polypeptide linkers, and stabilized by donor strand
- the C-terminal most ETEC major subunit is connected, via a linker, to a donor strand region from an ETEC major subunit, which can be either homologous or heterologous to the C-terminal major subunit.
- the immunogenic composition can comprise a whole or an immunogenic fragment, containing a donor ⁇ strand region, of the ETEC fimbrial major or minor subunits.
- major ETEC fimbrial subunits can contain an N-terminal deletion of 14 to 18 amino acids.
- one or more of the above constructs are connected, via a polypeptide linker, to form a multipartite fusion construct, wherein the subunits derived from multiple fimbrial types are expressed.
- the fimbrial subunits can be derived from any ETEC fimbrial types, including, but not limited to: ETEC class 5 fimbriae type, including class 5a, 5b or 5c; ETEC CS3; and ETEC CS6.
- the embodied multipartite construct can contain a deletion of the N-terminal region of one or more fimbrial subunits to avoid undesirable associations with other monomers or multimers and to remove reduce amino acid sequence length between polypeptides to reduce the protease cleavage.
- DNA encoding the recombinant polypeptide construct can be used to express a polypeptide for inclusion into immunogenic compositions, such as a subunit vaccine or the DNA encoding the recombinant polypeptide construct can be inserted into a suitable expression system such as a DNA plasmid, viral expression or bacterial vector.
- a suitable expression system such as a DNA plasmid, viral expression or bacterial vector.
- an object of the invention also includes a use of the construct for immunizing mammals, including humans, against ETEC strains.
- the embodied use comprises one or more priming administrations of one or more of the immunogenic compositions, either as a subunit vaccine or expressed from a molecular construct inserted into an appropriate expression system, such as a live vaccine.
- the priming dose can be subsequently followed by one or more boosting doses of construct expressed as a subunit vaccine or as a recombinant construct inserted in a DNA plasmid, viral or bacterial expression vector.
- FIG. 1 Illustration of inventive construct design wherein major or minor subunits, derived from the same ETEC fimbrial type are connected, via polypeptide linkers and stabilized by donor strand complementation.
- the construct can contain a deletion of the N-terminal region of the N-terminal subunit. This feature prevents undesirable association with other monomers or multimers. The deletion also reduces amino acid sequence length between polypeptides, that are not involved in domain folding and precludes or reduces the likelihood of inter-subunit protease cleavage.
- the C-terminal subunit is stabilized by a donor ⁇ strand, connected to the subunit via a polypeptide linker, wherein the donor ⁇ strand is either derived from a homolgous subunit, which is defined as a subunit that is the same as the subunit the donor strand is stabilizing or from a heterologous subunit, defined as derived from a subunit that is different still from the same fimbrial type.
- FIG. 2 Illustration of multipartite construct design wherein multiple compositions, illustrated in FIG. 1, are connected via a polypeptide linker.
- the first subunit is a major or minor (e.g., ETEC class 5 adhesin) ETEC fimbrial subunit.
- One or more major ETEC fimbrial subunits are then connected to the first subunit and to each other via a linker, wherein the subunits are stabilized by donor strand complementation.
- the C-terminal most ETEC major subunit is connected, via a linker, to a donor strand region from an ETEC major subunit, which can be either homologous or heterologous to the terminal major subunit.
- major ETEC fimbrial subunits in order to avoid inadvertent association of subunits, especially in CS6 subunits, to each other, major ETEC fimbrial subunits can contain an N-terminal deletion of 14 to 18 amino acids. Deletion of amino acid sequence length, not involved in folding, also reduces the likelihood of proteolytic degradation.
- FIG. 3. Diagram of phylogenetic relationship of strains used.
- FIG. 4 Enhanced immunogenicity of adhesin-pilin fusions compared to the prototype dscCfaE (CfaE) adhesin.
- Panel A shows hemagglutination inhibition (HAI) titers against CFA/I-ETEC (upper), CS14-ETEC (middle), and CS4-ETEC (lower graph).
- Panel B shows serum IgG titers against CFA/I (upper), CS14 (middle) and CS4 (lower) fimbriae. Note that the middle and lower graphs in each panel display a different y-axis scale to that of the corresponding upper graphs.
- the shorthand protein names shown in the graph labels and corresponding full names are as follows: CfaE, dsci9CfaE(His) 6 ; CfaEB, dsc 19 CfaE-CfaB[His] 6 ; CfaEB-CsuA2, dsc 15 CfaEB-CsuA2-[His] 6 ; and CfaEB-CsuA2- CsfA, dsc 15 CfaEB-CsuA2-CsfA[His] 6 ; mLT, LTR192G.
- FIG. 5 Enhanced immunogenicity of a Class 5c adhesion-pilin fusion compared to dscCotD (CotD) adhesin.
- Panel (A) shows HAI titer.
- Panel (B) shows anti-CS2 IgG response.
- FIG. 6 Enhanced immunogenicity of Class 5b adhesin-pilin fusion compared to the prototype dscCsbD (CsbD) adhesin.
- Panel (A) shows HAI titer.
- Panel (B) shows anti- CS1 or anti-CS17 IgG responses.
- FIG. 7 Immunity of CS6 constructs, in rabbits. Shown are the serum IgG titers following administration of CS6; dscCssB or dscCssA antigen.
- FIG. 8 Comparison of immunity in mice following administration of CS6 monomers, heterodimers and homodimers.
- FIG. 9 Hemaglutination-inhibition (HAI) titer and serum reactivity of dscCstH compared to non-cvalently linked oligomer (3-14 subunits) of CstH (denoted as CstH(i)).
- CstH(i) was isolated by capture of attached intein ("i"), which was cleaved from the intein-CstH fusion product.
- FIG. 10 Hemaglutining inhibition (HAI) and serum reactivity of dscCstG compared to oligommer (3-14 subunits) of CstG (denoted as CstG(i).
- FIG. 1 Immune and HAI responses of multimeric donor strand complemented CstH.
- H dscCstH
- H2 dscCstH2
- H3 dscCstH3
- H4 dscCstH4
- H5 dscCstHS.
- FIG. 12. Immune response to stabilized CS3-based heterodimers and tetramers. Immune responses, as measured by hemagglutination inhibition (HAI) against CS3-ETEC (Panel A), and serum IgG anti-CS3 titers (Panel (B) are shown for serum drawn two weeks after the last dose (day 42).
- HAI hemagglutination inhibition
- CS3-ETEC Panel A
- serum IgG anti-CS3 titers Panel (B) are shown for serum drawn two weeks after the last dose (day 42).
- FIG. 13 Examples of CS3 multipartite protein construct using construct design of FIG. 1.
- FIG. 14 Examples of CS6 multipartite protein constructs using construct design of FIG.l.
- FIG. 15 Summary of data illustrating that that a multipartite fusion, CsbDA-CooA- CstGH, comprising subunits from a Class 5 ETEC fimbrial type, i.e., CsbDA-CooA and a CS3 fimbrial type, i.e., CstGH, retains the immunogenic effects of the fimbrial types.
- the shorthand protein names shown in the graph labels and corresponding full names are as follows: CsbDA-CooA, dsci 5 CsbDA-CooA [His] 6 ; CstGH, dsci 6 CstGH(His) 6;
- CsbDA-CooA + CstGH an admixture of CsbDA-CooA and CstGH;
- CsbDA-CooA- CstGH the multipartite fusion dsci 4 CsbDA-CooA-ntdi 8 dsci 6 CstGH(His)6.
- FIG. 16 Comparison of immunogenicity of CS3 fusion constructs against admixture of constructs.
- the groups listed on the x-axis are described in Table 9.
- FIG. 17 Comparison of HAI titer of CS3 fusion construct examples against admixture of constructs.
- the groups listed on the x-axis are described in Table 9.
- FIG. 18. Summary of data illustrating that a multipartite fusion, CfaEB-CssBA, comprising subunits from a Class 5 ETEC fimbrial type, i.e., CfaEB and a CS6 fimbrial type, i.e., CssBA, retains the immunogenic effects of the fimbrial types.
- Panel (A) shows the HAI titer for CFA/I and CS14.
- Panel (B) shws the IgG titer for CFA/I, CS6 and CfaE.
- the shorthand protein names shown in the graph labels and corresponding full names are as follows: CfaEB, dsci9CfaEB[His] 6 ; CssBA, dsci6 A CssBA(His) 6; CfaEB + CssBA, an admixture of CfaEB and CssBA; CfaEB-CssBA, the multipartite fusion dsc 1 4 CfaEB-G-dsc i 6 ACSSB A(His) 6 .
- FIG. 19 Anti-CS6 fimbrial subunit IgG immune responses.
- the x-axis represents the antigens administered as in the table in FIG. 19.
- the darkly shaded bars represent response to CssB and the lightly shaded bars indicate response to CssA antigen.
- the bars indicate the IgG response to the component CS6 fimbrial subunits CssB or CssA.
- all mice received doses containing 100 ng of mLT, as in FIG. 19, with the exception of some mice receiving a CfaEB-CssBA (or CssAB) constructs, as indicated in the figure.
- FIG. 20 Anti-CS6 fimbrial subunit IgA immune responses.
- Each bar represents the pooling of murine serum from mice immunized as in FIG. 19. The mice received doses containing 100 ng of mLT with the exception of the bars, indicated in all white.
- polypeptide refers to a polymer formed of two or more amino acid residues, wherein one or more amino acid residues are naturally occurring amino acids.
- amino acid sequence refers to the order of the amino acids within a polypeptide.
- oligomer are polypeptides sequences comprising relatively few amino acids.
- recombinant polypeptide refers to polypeptides or proteins produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or the desired protein.
- recombinant construct refers to the DNA encoding the recombinant polypeptide, recombinant polypeptide construct or recombinant protein.
- donor strand or "donor ⁇ strand” refers to the N-terminal region of an ETEC fimbrial subunit that associates with another ETEC fimbrial subunit in donor strand complementation.
- immunogenic composition refers to a formulation containing proteins or polypeptides and other constitutients that induce a humoral and/or cellular immune response.
- immuno coverage or “spectrum of coverage” refers to the induction of humoral and/or cellular immune response against specific strains of bacteria under the “coverage.”
- immunogenogenic fragment refers to a polypeptide containing one or more B- or T-cell epitopes and is of sufficient length to induce an immune response or to be recognized by T- or B-cells.
- derivative refers to a polypeptide or nucleic acid sequence with at least 80% identity with sequence of the identified gene.
- identity refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when aligned for maximum correspondence. Where some sequences differ in conservative substitutions, i.e., substitution of residues with identical properties, the the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Percent similarity refers to proportion of identical and similar (conserved change) residues.
- Fimbriae are defined as projections or filaments on ETEC bacteria and are composed of major subunits, as in the case of CS3 and CS6 fimbriae or major and minor subunits, as in the case of class 5a, 5b and 5c ETEC. "Fibrillae” are narrow projections from a bacteria. CS3 and CS6 fimbriae can also be termed fibrillae due to their narrow characteristic.
- the term “fimbrial subunit” refers to the proteins that comprise ETEC fimbriae and is used interchangeably with “pilin.” "Pilin”, therefore, can refer to a "major” or “minor” "fimbrial subunit” that comprise ETEC fimbriae.
- a “minor fimbrial subunit” refers to the adhesin protein at the tip of class 5 ETEC fimbriae and is expressed in stoichiometrically low amounts compared to "major” subunits.
- the “minor fimbrial subunits” include, but are not limited to, CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD and CotD.
- Major fimbrial subunits refers to the ETEC fimbrial proteins represented in stoichiometrially larger amounts in ETEC fimbriae, compared to “minor fimbrial subunits.”
- Major fimbrial subunits include the ETEC class 5 proteins: CfaB, CsfA, CsuA2, CsuAl, CooA, CosA, CsdA, CsbA, CotA; the ETEC CS3 proteins: CstH, CstG; and the ETEC CS6 proteins: CssA, and CssB.
- a "fusion" is defined herein as two molecules covalently connected.
- an "adhesin-pilin" fusion is a major or minor ETEC fimbrial subunit connected, covalently, to a Class 5 adhesin.
- the term "fimbrial type” refers to fimbrial proteins derived from fimbriae of different ETEC types.
- the different "fimbrial types”, as used in this application include, but are not limited to, CS6, which include CssA and CssB; CS3, which include CstH and CstG; ETEC Class 5a; ETEC Class 5b and ETEC Class 5c fimbriae.
- homologous subunit is defined as a subunit that is an identical type to the subunit to which it is connected.
- heterologous subunit refers to a subunit that is a different type from the subunit to, which it is connected but that is derived from the same ETEC fimbrial type.
- the present invention relates to recombinant polypeptide constructs for use in an immunogenic composition and to a method for using the composition to induce an immune response against enterotoxigenic Escherichia coli.
- the inventive composition utilizes a construct design that incorporates ETEC fimbrial subunits from multiple ETEC fimbrial types. Fimbrial types include, but are not limited to Class 5a, 5b, and 5c, as well as CS3, and CS6, in order to obtain broad anti-ETEC immunity.
- the construct also enables association of subunits, such as in CS3 and CS6 and stabilization of the subunits from proteolytic degradation, through donor strand complementation.
- FIG. 1 illustrates the basic recombinant polypeptide construct design.
- the construct design comprises major or minor subunits, derived from the same ETEC fimbrial type, which are connected, via polypeptide linkers and stabilized by donor strand complementation.
- the construct can contain a deletion of the N-terminal region ("ntd") of the N-terminal subunit. This feature prevents undesirable associations with other monomers or multimers.
- the size of the deletion of the N- terminal region is 14 to 18 amino acids.
- the C-terminal subunit is connected to and stabilized by a donor ⁇ strand, connected to the subunit via a polypeptide linker, wherein the donor ⁇ strand is either derived from the adjacent subunit (i.e., homologous) or from a different subunit of the same fimbrial type (i.e., heterologous).
- the size of the N-terminal donor strand depends on the fimbrial type and stabilized subunit. In preferred embodiments, for class 5 fimbrial subunits, the donor ⁇ strand, derived from the N-terminal region of the class 5 subunit stabilized, is 12 to 16 amino acids. In a preferred embodiment, for CS3 and CS6 subunits, the donor ⁇ strand is 14 to 16 amino acids.
- FIG. 2 illustrates the basic multipartite construct, wherein multiple constructs as in FIG. 1, are connected forming a recombinant polypeptide construct comprising two or more fimbrial types. As illustrated in FIG. 1, major or minor subunits from the same fimbrial type are connected via a polypeptide linker sequence. In the multipartite construct, two or more constructs, as in FIG. 1, are connected, via a linker polypeptide.
- the first subunit is a major or minor ETEC fimbrial subunit.
- Each additional subunit is connected to adjacent subunits via a polypeptide linker that enables rotary freedom of the molecular components.
- the subunits are associated with and stabilized via a donor strand complementation from a C-terminally adjacent subunit via a donor ⁇ strand, connected via a linker polypeptide, to the C-terminus of the stabilized subunit.
- subunits can contain a deletion of 14 to 18 amino acids from its N-terminal end.
- constructs can be constructed with or without signal peptides of 18 to 22 amino acids and with or without histidine tags at the C-terminus.
- Fimbrial types include, but are not limited to ETEC class 5a, 5b, 5c, CS3 and CS6.
- a single construct can include subunits derived from any two or more of class 5a, 5b, 5c, CS3 and CS6 fimbrial types.
- linker sequences can be utilized in connecting the individual subunits.
- specific linkers include the tetrapeptide of SEQ ID No. 5.
- Another example is a tri-glycine linker (i.e., G-G-G).
- G-G-G tri-glycine linker
- in cis donor strand complementation is used to stabilize adhesins and adhesin-pilin fusions for representative Class 5a, 5b, and 5c adhesins.
- FIG. 3 A summary of the phylogenetic relationships of Class 5 minor (i.e., adhesins) and major subunits is illustrated in FIG. 3.
- the contemplated composition is designed to enable as wide a range of coverage of ETEC strains as possible.
- the contemplated composition and use is aimed at inducing immunogenic response against class 5a, 5b, 5c ETEC, as well as ETEC strains expressing CS3 or CS6 fimbrial components.
- fimbrial subunits such as CfaE
- CfaE are relatively susceptible to proteolytic degradation outside of the fimbrial structure
- stabilization of the adhesin is also important. Therefore, constructs are designed to express ETEC subunits stabilized from misfolding and degradation by donor strand complementation.
- the donor ⁇ strand is provided by the major fimbrial subunit.
- stabilization is provided by the N-terminal region of CfaB.
- dscCfaE is folded into a native, ⁇ -sandwich conformation, consisting of two half-barrels, comprising the N-terminal adhesin domain (CfaEad) a short a-helical connector, and the C-terminal pilin domain (CfaEpd).
- the molecule is functional in that it directly mediates MRHA of bovine and human erythrocytes, and generates neutralizing antibodies that act to inhibit MRHA and decorate the tips of CFA/I fimbriae on immunoelectron microscopy.
- a fusion protein was engineered by genetic insertion of the coding sequence for mature major structural subunit of ETEC adhesin, such as CfaB, to the 3'-end of the minor subunit, such as CfaE.
- This concept was disclosed in Savarino, U.S. Patent application (1 1/340,003, filed January 10, 2006), which is incorporated, herein.
- This molecule contains all three domains of the CFA/I fimbriae (i.e., ad, pd, and major subunit) in a ratio of 1 : 1 : 1 , rather than that found in native fimbriae (ca. 1 :1 : 1000).
- nonsynonymous nucleotide changes at one site each in only five of these 31 alleles (Chattopadhyay, et al., J. Biol. Chem., 287(9): 6150-6158 (2012)).
- the target protein shows uniformity in natural ETEC bacterial populations.
- CfaE a Class 5a fimbrial adhesin
- CsuD of CS14 fimbriae and CsfD of CS4 fimbriae are 80-81% identical with the other Class 5a minor subunits proteins adhesins CsuD of CS14 fimbriae and CsfD of CS4 fimbriae.
- CsuD and CsfD share 94% identity. This is considerably higher than the average identity with other Class 5b and 5c fimbrial adhesins (mean 50% identity).
- rabbit anti-dscCfaE serum cross-neutralizes CS4- and CS14-ETEC in the hemagglutination assay (HAI).
- HAI hemagglutination assay
- An embodiment includes anti-class 5 ETEC constructs based on the construct design illustrated in FIG. 1 , whereby the N-terminal subunit is an ETEC class 5 minor (i.e., adhesin) subunit, listed in Table 1, including CfaE, CsfD, CsuD, CooD, CsdD, CosD, CsbD and CotD, connected, via a polypeptide linker, to one or more ETEC major subunits, from the same ETEC class 5 type, listed in Table 1.
- the polypeptide linker can be any of a number of polypeptide sizes. In a preferred embodiment, the linker is a tetrapeptide with the polypeptide sequence of SEQ ID No. 5.
- the C-terminal class 5 subunit is connected to a donor ⁇ strand, derived from a homologous subunit and is typically 12-19 amino acids.
- one or more major subunit can include a deletion of 12 to 16 amino acids from the N-terminal region of the subunit.
- FIG. 1 utilizes the concepts disclosed in Savarino, U.S. Patent application (11/340,003, filed January 10, 2006)), including donor strand
- FIG. 1 further contemplates multiple constructs incorporating the fimbrial subunits of Table 1, or deriviates of these polypeptides or DNA sequences.
- the construct design incorporates the donor strand complementation stabilization features of Savarino (U.S. Patent application (1 1/340,003, filed January 10, 2006)), and furthers it by incorporating multiple major subunits, from a specific ETEC type, into a single adhesin-pilin construct.
- multiple class 5b major subunits can be connected to a class 5b adhesin (i.e, minor subunit).
- Embodiments include adhesin-pilin constructs containing Csb D (ETEC Class 5b fimbrial adhesin) and Cot D (ETEC Class 5c fimbrial adhesin). Examples, for illustration, of embodiments of adhesin-pilin ETEC class 5 adhesin-pilin constructs, representing Class 5a, 5b and 5c are shown in Table 2.
- 'dsc refers to donor strand complementation.
- the number and subunit refers to the N-terminal amino acids of length represented by the number from the subunit indicated that is connected at the C-terminus of the construct and is serving to stabilize the C-terminal construct.
- dscncsfA refers to the N- terminal 14 amino acids of CsfA connect to the C-terminus of the construct.
- 2Linkers polypeptides are GGG rather than DNKQ.
- 3Sequence in example contains a Leu-Glu-His 6 at the C-terminus.
- Panel A shows the hemagglutination inhibition (HAI) titers against CFA/I-ETEC (upper), CS14-ETEC (middle), and CS4-ETEC (lower graph). Elevated HAI titers against CS14-ETEC and CS4-ETEC coincide with the addition of CS14 (CsuA2) and CS4 (CsfA) pilins, respectively.
- Panel B shows serum IgG titers against CFA/I (upper), CS14 (middle) and CS4 (lower) fimbriae.
- FIG. 5 illustrates the enhanced immunogenicity of a Class 5c adhesin- pilin fusion compared to the prototype dscCotD (CotD) adhesin.
- the adhesin-pilin protein shown in FIG. 5 the CS2 pilin (CotA) is fused to the C-terminus of dscCotD, wherein each component protein is stabilized by in cis donor strand complementation.
- mice Groups of ten BALB/c mice were vaccinated intranasally with 25 ⁇ g of each protein, coadministered with the adjuvant LTR192G (1.5 ⁇ g). Mice were vaccinated three times at 0, 14, and 28 days. The displayed titers are from serum collected at day 42.
- Panel A displays serum hemagglutination inhibition (HAI) titers against CS2-ETEC. Addition of the CS2 pilin is associated with an elicitation of higher homologous HAI titers.
- Panel B shows serum IgG titers against CS2 fimbriae. Illustrated in FIG.
- FIG. 6 illustrates the response of the construct example "dsci 4CsbA CsbD-CsbA- ntdisdsc c ooA CooA", shown in Table 2. Similar to that in FIG. 5, FIG. 6 shows enhanced immunogenicity of Class 5b adhesin-pilin fusions compared to the prototype dscCsbD (CsbD) adhesin. In the two Class 5b adhesin-pilin proteins, the following Class 5b pilin components were successively fused to dscCsbD; CS 17 pilin (CsbA), and CS 1 pilin (CooA).
- mice Groups of ten BALB/c mice were vaccinated intranasally with 25 ⁇ g of each protein, co-administered with the adjuvant LTR192G (1.5 ⁇ g). Mice were vaccinated three times at 0, 14, and 28 days. The displayed titers are from serum collected at day 42.
- Panel A displays hemagglutination inhibition (HAI) titers against (from upper to lower panels) CS17-ETEC, CS19-ETEC, and CS1-ETEC, PCF071 -ETEC, and CFA I-ETEC.
- HAI hemagglutination inhibition
- Panel B shows serum IgG titers against CS 17 fimbriae (upper) and CS 1 fimbriae (lower). Augmented serum anti-fimbrial titers to CS17 and CS 1 coincide with the addition of CS17 (CsbA) and CS 1 (CooA) pilins, respectively.
- the fimbrial structures may function as
- ETEC CS6 and CS3 may be mediated by a donor strand complementation mediated process through association of a CS6 or CS3 subunit with the N-terminal donor strand region of an adjacent subunit. Additionally, protection against misfolding and proteolytic degradation may also be afforded through donor strand complementation.
- CS6 fimbriae comprise CssA and CssB. Whereas the two CS3 major subunits show little to no variation in polypeptide sequences, modest variation in CS6 proteins is observed. For example, greater than 90% identity is found in CS6 protein CssA and greater than 95% identity is found in CssB allotypes. Both CS6 structural proteins exhibit a relatively low level of variation (i.e., greather than 90% amino acid
- Monomeric CS6 subunit assembly appears to be mediated by donor strands from adjacent CS6 subunits, as discussed above. It is hypothesized that interaction to form these stable structures is mediated by inter-subunit interaction through donor strand complementation. Donor strand complementation also affords protection against misfolding and proteolytic degradation. Therefore, in a preferred embodiment, multimenc CS6 constructs were developed to take advantage of these attributes of donor strand complementation. Additionally, multimeric expression provides more efficient manufacture over production of monomers.
- CssA and CssB monomers exhibit similar thermal stability, as illustrated in Table 3. However, as also illustrated in Table 3, dimers of CssA or CssB are generally more thermally stable over larger structures. Additionally, multimers comprising both CssA and CssB were generally more thermally stable than homo-multimers (i.e., comprising only CssA or CssB). Furthermore, multimer constructs with CssB subunit that is N- terminal to CssA were generally more thermally stable over construct containing CssA N-terminal to CssB.
- an embodied construct comprises a multimeric CS6 with one or more of the CS6 subunits, CssA and CssB, or allelic variation or derivatives, with the construct design configuration illustrated in FIG. 1.
- the construct comprises a dimer of CssB and CssA with CssB N- terminal to CssA (i.e., CssB-CssA).
- CS6 subunit association is stabilized by in cis donor ⁇ strand complementation.
- Donor strand complementation is afforded by linking a CS6 subunit at its C-terminus, to the donor ⁇ strand region of another CS6 subunit, via a tetrapeptide linker.
- the linker can be any of a number of polypeptide regions. However, in a preferred embodiment, the linker is either as in SEQ ID No. 5 or a triglyicine linker.
- stabilization is provided by donor ⁇ strand, connected at its C-terminus, from a homologous or heterologous CS6 subunit.
- Homologous subunit is defined as two subunits of the same form (e.g., CssA OR CssB). Heteterologous subunits are of different forms (e.g., one is derived from CssA the other from CssB.
- the CS6 donor ⁇ strand is typically the N-terminal 14-16 amino acid region of CS6 subunit.
- the recombinant protein can be constructed with or without
- hexahistidine affinity tags which are typically on the C-terminus.
- the N-terminal 14-16 amino acids of the N-terminal CS6 subunit is deleted.
- "dsc B uCssBA” would contain a heterologous donor strand (i.e., "dsc"), from CS6 CssB, inserted at the C-terminus of the construct.
- the donor strand is 14 amino acids in length, as indicated by the "14.”
- a constructed designated "ntdijdsc A CssBA” would contain a homologous donor strand at the C-terminus of the construct and also comprises a deletion of the N-terminal amino acid region (termed “ntd”).
- constructs comprise one or more CS6 subunits with amino acid sequences sequences selected from the group consisting of SEQ ID No. 2 (CssA) or SEQ ID No. 4 (CssB), or derivatives of these polypeptides.
- the DNA sequence for CssA is SEQ ID No. 1 and for CssB, SEQ ID No. 3.
- the subunits are connected by a polypeptide linker sequences.
- the linker is a tetrapeptide with the amino acid sequence of SEQ ID No. 5.
- rabbits were immunized every 28 days over an 84 day period, with two rabbits per group.
- the animals were primed with 400 ⁇ g and boost three times with 200 ⁇ g of either CS6, dscCssA or dscCssB, in
- CS6 elicits high homologous antibody titer, as well as high anti-CS6 (i.e., CssA or B) subunit titer.
- Immunization of rabbits with donor strand stabilized CssB (dscCssB) yielded high anti-CS6 and anti-CssB titers.
- immunization with dscCssA elicited somewhat lower anti-CS6 titers and intermediate levels of anti-CssA titers of IgG.
- FIG. 8A shows anti-CssA IgG titer and FIG. 8B shows anti-CssB igG titer, in response to different CS6 antigens administered (FIG. 8C).
- FIG. 8A shows anti-CssA IgG titer
- FIG. 8B shows anti-CssB igG titer, in response to different CS6 antigens administered (FIG. 8C).
- ntdisdsceCssBA This construct contains a 16 amino acid donor strand region of CssB at the C-terminus of the construct, which is heterologous to the C-terminal subunit (i.e., CssA).
- CssA C-terminal subunit
- ntdi5dsci6 B CssBA, and ntdisdsc A CssBA yielded similar titer.
- ntdi 5 dsci 6A CssBA which contains the homologous donor strand on CssA, is likely less stable.
- Het refers to the donor strand origination for the terminal (i.e., C- terminal) subunit.
- CssA-CssB "Het" construct contains a donor strand of 16 amino acids connected at the C-terminus derived from CssA.
- CssB-CssA "Het” construct contains a donor strand of 16 amino acids connected at the C-terminus derived from CssB.
- 3 ntd refers to N-terminal deletion.
- CssA-CssB CssA contains a deletion of 15 amino acids from its N-terminus.
- CssB-CssA CssB contains a deletion of 14 amino acids its N-terminus.
- CS3 comprises CstH and CstG.
- the CS3 structural protein CstH is invariant.
- CstG is also highly conserved, showing 99- 100% identity in polypeptide sequence for 39 wildtype CS3 genes sequenced. Similiarly, although some variation CstG is observed, it is also relatively invariant, with 99-100% amino acid conservation.
- H refers to dsc ⁇ CstH (His whereby the construct is in cis donor strand complemented at its C-terminus with a tetrapeptide linker, connected to a 16 amino acide polypeptide sequence of the N-terminal beta strand of CstH.
- the construct also contains a string of six (6) histidine (His) 6 at its C-terminus.
- H2 refers to dsci 6 CstH2, whereby the construct comprises two CstH fimbrial subunits linked by a tetrapeptide linker and where the C-terminal CstH is connected, via a tetrapeptide linker, and in cis donor complemented at its C-terminus, to a 16 amino acide polypeptide sequence of the N-terminal beta strand of CstH.
- Similar constructs are referred to for H3 (but with three tandemly linked CstH fimbrial subunits), H4 (four tandemly linked CstH subunits) and H5 (five tandemly linked CstH subunits).
- FIG. 12 A summary of the results of the analysis of these constructs is illustrated in FIG. 12.
- Immune responses as measured by hemagglutination inhibition (HAI) against CS3-ETEC (Panel (A)), and serum IgG anti- CS3 titers (Panel (B)) are shown for serum drawn two weeks after the last dose (day 42).
- HAI hemagglutination inhibition
- A CS3-ETEC
- B serum IgG anti- CS3 titers
- a preferred embodiment for a polypeptide construct that can be used to elicit an immune response against CS3 comprises constructs designed according to FIG. 1.
- CS3 constructs comprise one or more CS3 fimbrial subunits connected via a polypeptide linker.
- the C-terminal fimbrial subunit is connected, via a polypeptide linker, to a donor ⁇ strand region of a CS3 fimbrial subunit.
- the C-terminal donor ⁇ strand can be derived from the same CS3 subunit to which it is connect (i.e., homologous) or derived from a different subunit (i.e., heterologous).
- the polypeptide linker can be any number of polypeptide regions, however, in a preferred embodiment, the linker is a tetrapeptide of the sequence of SEQ ID No. 5, or a triglycine (i.e., G-G-G).
- the donor ⁇ strand region is the N-terminal 14 - 16 amino acids of the mature CstH or CstG protein.
- the first 14 - 18 amino acids of the N-terminal region of the N-terminal most subunit is deleted to avoid undesirable associations.
- the CS3 construct is a dimer.
- the recombinant polypeptide construct can be configured as "dsci6 C st H CstG-(linker)-CstH".
- the mature CstG polypeptide (SEQ ID No. 101) or full length polypeptide sequence (SEQ ID No. 87) is connected at its C-terminus to CstH
- polypeptide (SEQ ID No. 99), via a polypeptide linker.
- the CstH polypeptide is connected, at its C-terminus, to a donor ⁇ strand region of 16 amino acids derived from CstH via a polypeptide linker.
- C-terminal donor ⁇ strand can be either homologous (derived from the same subunit) or heterologous (derived from a different subunit) to the C-terminal most CS3 fimbrial subunit.
- the fimbrial type "CS3" comprises CstH and CstG.
- the fimbrial type “CS6” comprises CssA and CssB.
- the fimbrial types of Class 5 ETEC include the fimbrial types Class 5a, Class 5b and Class 5c.
- major and/or minor subunits, derived from the same ETEC fimbrial type are connected, via polypeptide linkers, and stabilized by donor ⁇ strand complementation, as illustrated in FIG. 1 and Examples 1-4.
- a multipartite fusion comprises one or more fimbrial subunits of the same fimbrial type, as in FIG. 1, connected to one or more fimbrial subunits derived from a different fimbrial type as illustrated in FIG. 2.
- the multipartite fusion construct can include a deletion of the N-terminal region of one or more fimbrial subunits, but is preferably on the N-terminal most fimbrial subunit for a given ETEC fimbrial type, as illustrated in FIG. 2. This feature prevents undesirable associations with other monomers or multimers.
- the size of the deletion of the N-terminal region is 14 to 18 amino acids.
- multipartite fusion constructs comprising Class 5 adhesins do not contain a deletion of the N-terminal region.
- the C-terminal subunit for an ETEC fimbrial type, is connected to and stabilized by a donor ⁇ strand, connected to the subunit via a polypeptide linker, wherein the donor ⁇ strand is either that derived from the adjacent subunit (i.e., homologous) or from a different subunit of the same fimbrial type (i.e., heterologous).
- the size of the N-terminal donor strand depends on the fimbrial type and subunit stabilized. In preferred embodiments, for class 5 fimbrial subunits, the donor ⁇ strand, derived from the N-terminal region of the class 5 subunit stabilized, is 12 to 16 amino acids.
- the donor ⁇ strand is 14 to 16 amino acids.
- the construct can contain a deletion of the N-terminal region of the N- terminal subunit. This feature prevents undesirable associations with other monomers or multimers.
- the size of the deletion of the N-terminal region is 14 to 18 amino acids.
- FIG. 2 multiple constructs as in FIG. 1 are connected forming a recombinant polypeptide construct comprising two or more ETEC fimbrial types.
- one or more major or minor subunits derived from the same ETEC fimbrial type, are connected via polypeptide linkers and stabilized by donor strand complementation.
- one or more glycine residues separates different ETEC fimbrial types, acting as a "swivel" means between the ETEC types.
- the glycine residue due to its small, unbranched molecular characteristics, enables rotary freedom of the molecular components.
- Subunits derived from the same fimbrial type (as in FIG.
- each ETEC fimbrial type is connected by a polypeptide linker, with the subunits stabilized by donor strand complementation.
- the C-terminal subunit of each ETEC fimbrial type is stabilized by a donor ⁇ strand that is homologous or heterologous to the C-terminal subunit of that fimbrial type.
- the construct can contain an N-terminal deletion at the N- terminus of the entire construct as well as an additional deletion, of 14 to 18 amino acids, at the N-terminus of the first "internal" subunit that is of a different fimbrial type. This is illustrated in FIG. 2.
- the deletion serves to shorten the length between subunits, thus reducing the likelihood of misfolding and proteolytic cleavage.
- a donor ⁇ strand derived from a homologous or heterologous subunit, is inserted at the C-terminus of the C-terminal CS6 or CS3 subunit.
- the donor ⁇ strand derived from the N-terminal region of the class 5 subunit that is stabilized, is 12 to 16 amino acids.
- CfaB is stabilized by a 14 amino acid donor ⁇ strand; CsfA by a 14 amino acid donor ⁇ strand; CsbA by a 15 amino acid donor ⁇ strand, Coo A by a 14 amino acid donor ⁇ strand and CotA by a 14 amino acid donor ⁇ strand.
- the donor ⁇ strand is 14 to 16 amino acids, with preferred embodiments of CS3 fimbrial subunits (i.e., CstH or CstG) stabilized by a 16 amino acid donor ⁇ strand derived from CstH or CstG; and CS6 fimbrial subunits (i.e., CssA or CssB) stabilized with a 16 amino acid donor ⁇ strand derived from CssA or CssB.
- CssA or CssB CS6 fimbrial subunits
- other donor ⁇ strand lengths are envisioned.
- the inventive compositions can utilize different linker sequences.
- the linker contains the amino acid sequence of SEQ ID No. 5.
- the linker is a tri-glycine linker.
- the C-terminal end of the construct contains a histidine tag for purification of the construct.
- compositions are constructed with the intent of eliciting anti-adhesive immune responses.
- Class 5 multipartite fusions comprising Class 5 adhesin minor subunits are typically construct such that the adhesin (i.e., minor fimbrial subunit) is located at the N-terminus of the constructed with the minor fimbrial subunit linked at its C-terminus to one or more major subunits, followed at the terminal end of the construct with the donor ⁇ -strand of the last major subunit.
- adhesin i.e., minor fimbrial subunit
- constructs comprising Class 5a adhesin CfaE tandemly linked at its C-terminus to one or more of CfaB (CFA/I major subunit), CsuA2 (CS14 major subunit) and CsfA (CS4 major subunit); Class 5b adhesin CsbD tandemly linked at its C-terminus to one or more of CsbA (CS17 major subunit), which shares high identity to the CS19 pilin subunit CsdA, and CooA (CS1 major subunit), which shares high identity to the PCF071 pilin subunit CosA; and Class 5c adhesin CotD tandemly linked at its C-terminus to CotA (CS2 major subunit).
- CfaB CFA/I major subunit
- CsuA2 CS14 major subunit
- CsfA CS4 major subunit
- Class 5b adhesin CsbD tandemly linked at its C-terminus to one or more of CsbA CS17
- ETEC multipartite fusion constructs are illustrated in Table 7 and 8.
- constructs comprise any major or minor ETEC fimbrial subunit from Table 6 in multiple combinations, connected by linker polypeptides and stabilized from proteolytic degradation by donor strand complementation utilizing the design illustrated in FIG. 2.
- Table 6 lists the ETEC fimbrial subunits (major and minor subunits) than can be used and incorporated into the multipartite fusion construct design of FIG. 2. Any subunit, therefore, is combined with one or more other ETEC major subunits from any ETEC fimbrial phenotypic type, including Class 5a, 5b, 5c, CS3 and CS6.
- the recombinant polypeptide construct motif comprises a whole or immunogenic fragment of a minor or major ETEC fimbrial subunit connected at its C-terminal end to a linker.
- the linker is connected at its C-terminus to a whole major ETEC fimbrial subunit or a polypeptide donor strand of an ETEC major structural subunit, derived from the same fimbrial type.
- the whole ETEC major subunit or donor strand polypeptide is then connected, via a linker at its C-terminal end, to one or more additional major structural fimbrial subunits, derived from the same fimbrial type, from Table 6.
- sequence refers to signal peptide.
- the mature polypeptide sequence therefore, would be the full length minus the signal peptide.
- 2DNA sequence encodes mature protein.
- constructs containing Class 5a, 5b and 5c pilin subunits are selected based on the relatedness of minor and major subunits within a particular ETEC fimbrial class (i.e., class 5a, 5b or 5c), as illustrated in FIG. 3.
- adhesin i.e., minor fimbrial subunit
- a specific fimbrial type e.g., Class 5a
- Further selection of subunits is guided and based on epidemiological study analysis in order to achieve optimum immunogenic coverage of ETEC strains.
- the linker polypeptide can comprise a four (4) amino acid sequence (tetrapeptide) or a tri-glycine.
- the subunits are interconnected and stabilized by donor strand complementation, which is denoted, as in Table 7 and 8, by "dsc". In this nomenclature, the fimbrial subunit derivation is also indicated.
- the N-terminal CS3 subunit "CstG” is connected, via a linker, to the CS3 subunit "CstH", which is connected, via a linker, to a donor strand of 16 amino acids derived from "CstH.”
- the N-terminal CS6 subunit "CssB” is connected, via a linker, as illustrated in FIG.
- donor strand complementation of the "CssB" subunit is via a heterologous donor strand (i.e., derived from "CssA).
- the examples contain a "G” (i.e., glycine) to provide a "swivel.” Also, in some examples, the N-terminal region of N-terminal CS6 subunit is deleted (delineated by "ntd") to avoid undesirable association with other CS6 subunits, as described above. It should be noted that, in addition to the examples illustrated in Table 7 or 8, other combinations of major and minor subunits are contemplated utilizing the construct design illustrated in FIG. 2 and the fimbrial subunits of Table 6. In some sequences listed, a six (6) histidine (i.e., His 6 ) tag is inserted.
- G i.e., glycine
- constructs can be designed to include the histidine (i.e., His6) tag or designed without this tag region. Additionally, some sequences contain the signal peptide (designated "spd” in Table 2 and 3) region. Constructs can be constructed with or without this region, as well, which may be added to improve manufacturing efficiency of the multipartite fusion construct. Table 7
- All combinations can include a histidine (i.e., His 6 ) at the C-terminal end.
- Subunits can be linked via either DNKQ or tri-glycine linker.
- ntd refers to N-terminal deletion (excised from mature protein) with extent of deletion (i.e., amino acids) indicated.
- dsc refers to span of N-terminal residues from donor ⁇ -strand, its amino acid length and its source.
- All combinations can include a histidine (i.e., Hise) at the C-terminal end.
- Hise histidine
- Subunits can be linked via either DNKQ or tri-glycine (GGG) linker.
- DNKQ is used, except where indicated with (GGG).
- spd refers signal peptide. Number indicates number of amino acids.
- ntd refers to N-terminal deletion (excised from mature protein) with extent of deletion (i.e., amino acids) indicated.
- dsc refers to span of N-terminal residues from donor ⁇ -strand, its amino acid length and its source.
- Example 5 ETEC fimbrial subunit - toxin chimeric constructs
- recombinant polypeptide constructs can contain a C- terminal toxin A subunit, such as cholera toxin A2 (CTA) to form a chimeric molecule.
- CTA cholera toxin A2
- a full-length or truncated CTA2 is connected to CS6 or CS3 multimeric recombinant polypeptide construct, such as a CS6 or CS3 dimer.
- FIG. 13 and FIG. 14 Examples of these toxin constructs are illustrated in FIG. 13 and FIG. 14 (and in Table 7 and 8).
- the LTB gene and the CS3 or CS6 - toxin chimera are separately expressed.
- LTB once expessed, would self assemble to form a pentameric structure.
- the ensuing LTB multimeric composition (i.e., LTB 5 ) and CS3 or CS6 -toxin chimera then non-covalently associate to form a holotoxin-like heterohexamer.
- CS3-chimeric molecules For CS3-chimeric molecules, one or more CS3 fimbrial subunits are connected, as in FIG. 1, via a polypeptide linker, preferably a tetrapeptide or triglycine.
- the C-terminal most CS3 fimbrial subunit is then connected to a donor ⁇ strand, via a polypeptide linker.
- the donor strand can be homologous or heterologous to the C-terminal fimbrial subunit.
- the donor strand is then connected to a toxin fragment, such as CTA2.
- the CS3-chimera example shown in Table 7, comprise the polypeptide sequence of SEQ ID No. 37, which is encoded by the DNA sequence of SEQ ID No. 36.
- the N-terminal fimbrial subunit is CstG with a pelB leader (22 amino acids) connected at its N-terminal end (see FIG. 13).
- the CstH is connected, via a polypeptide linker, to a 16 amino acid donor strand derived from the N-terminal 16 amino acids of CstH, which is connected to an A2 toxin fragment (i.e., CTA2).
- LTB is also expressed.
- LTB comprises the amino acid sequence of SEQ ID No. 39 and is encoded by the nucleotide sequence of SEQ ID No. 38. Once expressed, the LTB sequence would self assemble into a pentamer and associate, non-covalently, with the CS3-chimera to form a hetero-hexameric holotoxin-like structure.
- CS6 toxin chimera examples are also illustrated in Table 8 and FIG. 14.
- CS6 chimeras as in CS3, one or more CS6 fimbrial subunits are connected via a polypeptide linker, preferably a tetrapeptide or triglycine.
- the C-terminal most CS6 fimbrial subunit is then connected to a donor ⁇ strand, via a polypeptide linker.
- the donor strand can be homologous or heterologous to the C-terminal fimbrial subunit.
- the donor strand is then connected to a toxin component (e.g., CTA2).
- CTA2 toxin component
- the chimera is co-expressed, with LTB, which self assembles into a pentamer to form a non-covalent association with the chimeric adhesion-toxoid fusion molecule.
- the constructs are dimers of CS6 subunits, connected via a tetrapeptide linker, with the C-terminal fimbrial subunit connected, via a tetrapeptide linker to a donor ⁇ strand.
- the donor ⁇ strand can be homologous or heterologous to the C-terminal most fimbrial subunit.
- the donor strands are heterologous to the C-terminal fimbrial subunit.
- the donor strand is then connected to a cholera toxin A2 (CTA2) subunit.
- CTA2 cholera toxin A2
- N-terminal subunit 43 which is encoded by the nucleotide sequence of SEQ ID Nos. 42.
- the N-terminal subunit is CssA, with the N-terminal 15 amino acids of the mature CssA sequence deleted.
- a pelB leader sequence 22 amino acids was also added, which is illustrated in FIG. 14.
- ETEC fimbrial types were constructed, as described in Example 5. As mentioned above, broad immunogenicity in a single construct is highly advantageous due to ease of manufacture and standardization of administration, compared to compositions comprising multiple individual components.
- the multipartite examples listed in Table 7 and 8 maintained immunity to each of its fimbrial components.
- the recombinant, multipartite fusion CsbDA-CooA-CstGH retains immunogenicity against the fimbrial subunits of the multiple fimbrial types, i.e., Class 5b and CS3.
- FIG. 15 illustrates the the immunogenicity of the multiple fimbrial components of the construct CsbDA-CooA- CstGH, comprising fimbrial subunits derived from ETEC Class 5b, and CS3. [0097] In FIG.
- CsbDA-CooA-CstGH comprises a tandem fusion (from N- to C- terminus) of CsbD, CsbA (adhesin and pilin subunits of CS17 fimbriae, respectively), CooA (pilin subunit of CS1 fimbriae), CstG and CstH (two major subunits of CS3), wherein each subunit is stabilized by in cis donor strand complementation.
- mice were vaccinated intradermally with the multipartite fusion or an admixture of CsbDA-CooA and CstGH ('CsbDA- CooA+CstGH'), the individual component proteins at molar equivalent doses (all matched to a 25 ⁇ g dose of CsbDA-CooA-CstGH), and co-administered with the adjuvant LTR192G (100 ng).
- Mice were vaccinated two times at 0 and 21 days. The displayed titers are from serum collected at day 32.
- Panel A shows hemagglutination inhibition (HAI) titers against CS3-ETEC (upper), CS17-ETEC (middle), and CSl-ETEC (lower graph).
- HAI hemagglutination inhibition
- Panel B shows serum IgG and IgA titers against CS3 (upper two graphs), and IgG and IgA anti-CsbD titers (lower two graphs).
- high serum anti-CS3 titers were elicited by all preparations containing CstGH, including the multipartite fusion.
- Both the multipartite fusion and admixture preparations elicited high anti-CsbD IgG titers, while only the fusion elicited detectable IgA anti-CsbD titers.
- Data is displayed as the geometric mean titer plus standard error of the mean. The horizontal dotted line in each graph shows the limit of assay detection.
- FIG. 16 The immune response of the CS3 component in several examples, described in FIG. 13, is summarized in FIG. 16.
- the immune induction following the administration of CS3 or CS3 donor strand complentation stabilized constructs was evaluated.
- mice were immunized intradermally on day 0 and day 21 and bled on day 28.
- ELISA enzyme-linked immunosorbant assay
- FIG. 16 similar levels of IgG and IgA anti-CS3 immune responses were elicited by each of the multipartite fusion constructs.
- FIG. 16 Also shown in FIG. 16 is the induction of an anti-CS3 response by a dscCsGH-CTA2/LTB adhesion-toxoid chimera (see Example 5).
- FIG. 17 summarizes the results of studies evaluating the ability of different ETEC multipartite fusion constructs to inhibit mannose resistant
- HAI hemagglutination inhibition assays
- the bacterial strain (CS3 + ETEC strain WS201 OA) was used at a concentration corresponding to two times the minimal hemagglutination titer (2xMHT).
- the MHT was determined at the start of the HAI assay by making serial two-fold dilutions of the bacterial suspension. A total of 25 ⁇ - of each dilution ws added to equal volumes of 3% erythrocyte suspension and PBS with a 0.5% D-mannose and rocked on ice.
- the MHT was defined as the reciprocal of the lowest concentration of bacterial showing at least 1 + MRHA.
- HAI titer of each antiserum preparation, a two-fold dilution series of antibody was made. A 25 ⁇ , volume of each dilution was add to an equal volume of 2xMHT bacterial suspension and pre-incubated at room temperature with rocking for 20 minutes. An equal volume of erythrocyte suspension (3%) was then added to each well and rocked on ice for 20 minutes, after which the MRHA was scored. The HAI titer is expressed as the reciprocal of the highest dilution of antiserum that completely inhibited MRHA.
- dscCstG-CstH (dscCstGH) and CstGH multipartite fusion constructs exhibited significant HAI.
- similar HAI was exhibited whether CstGH was a component of a fusion construct or was part of an admixture of the two components. This is graphically illustrated in Panel B in FIG. 17.
- Multipartite constructs of comprising class 5 and CS6 subunits were also prepared and evaluated for reactivity for anti-CS6 immunogencity.
- mice were immunized against different constructs, containing ETEC Class 5, and CS6 subunits, with or without the adjuvant mLT (genetically modified heat-labile
- CfaEB-CssBA comprises a tandem fusion (from N- to C-terminus) of CfaE, CfaB (minor and major subunits of CFA/I fimbriae, respectively), CssB and CssA (two major subunits of CS6), wherein each subunit is stabilized by in cis donor strand complementation.
- mice were vaccinated intradermally with the multipartite fusion or an admixture of CfaEB and CssBA ('CfaEB + CssB A'), the individual component proteins at molar equivalent doses (all matched to a 25 ⁇ g dose of CfaEB-CssBA), and co-administered with the adjuvant LTR192G (100 ng).
- Mice were vaccinated three times at 0, 14, and 28 days. The displayed titers are from serum collected at day 42.
- Panel A displays hemagglutination inhibition (HAI) titers against CFA/I- ETEC (upper), and CS14-ETEC (lower graph). Similarly elevated homologous (CFA I- ETEC) and within subclass heterologous (CS14-ETEC) HAI titers were observed after vaccination with the multipartite fusion, the admixture, and CfaEB alone (historical control).
- HAI hemagglutination inhibition
- Panel B shows serum IgG titers against CS6 (upper), CFA/I fimbriae (middle) and dscCfaE (lower) adhesin.
- high serum anti-CS6 titers were elicited by all preparations containing CssBA, including the multipartite fusion.
- all preparations containing CfaEB, including the multipartite fusion elicited high anti-CFA/I and anti-CfaE (i.e., anti-adhesin) IgG titers, while CssBA predictably did not elicit anti- CfaE IgG titers.
- Data is displayed as the geometric mean titer + standard error of the mean. The horizontal dotted line in each graph shows the limit of assay detection.
- the CS6 subunits CssA and CssB as heterodimers, elicit a potent IgG response against the individual CS6 subunits.
- Panel A illustrates the antigens administered.
- Panel B shows the anti-CS6 IgG response, for either CssA or CssB, elicited against the administered antigenic construct shown along the x-axis.
- the CS6 subunits fused to Class 5 subunits elicit a similar or even higher antibody response to the CS6 subunits in comparision to admixture of CfaEB and CssAB or CssBA (i.e., groups 6 and 7).
- the IgA response is shown in FIG. 20, for the same panel of antigens as in FIG. 19.
- the adhesins are an important component for the induction of diarrheagenic E. coli bacterial immunity.
- An aspect of this invention is the construction of stable polypeptide constructs for use as immunogens against enterotoxigenic Escherichia coli mediated diarrhea.
- An additional aspect is the ability to induce immunity in mammals, such as in humans, against as many ETEC types as possible.
- mammals such as in humans
- ETEC types as possible.
- Recombinant polypeptide constructs produced using the design of FIG. 1 and FIG. 2, as well as the examples listed in FIG. 13 and 14, can be used in formulations for the induction of immunity to multiple ETEC types.
- constructs as immunogen constructed based on FIG. 1 and/or FIG. 2 or the examples given in FIG. 13 and 14, comprise the following steps: a. priming by administration of an immunogenic composition containing the polypeptide construct described in Examples 1 through 6, above.
- the immunogenic composition can be administered orally, nasally, subcutaneously, intradermally, transdermally, sublingually, transcutaneously intramuscularly, or rectally.
- the range of a unit dose of immunogen is 1 ⁇ g to 1 mg of the polypeptide construct.
- the immunogenic composition can be administered in any number of solutions with or without carrier protein or adjuvant or adsorbed onto particles such as microspheres; b.
- 2 to 4 boosting doses are also administered with unit dose range of 1 ⁇ g to 1 mg of polypeptide construct in a buffered aqueous solution or other suitable solution.
- An alternative vaccine approach is the administration of a recombinant DNA construct capable of expressing the recombinant polypeptide.
- the recombinant DNA encoding the immunogen is inserted into a suitable expression system and expressed in host bacterial cells. The recombinant host cells can then be
- Representative host cells include, but are not limited to Escherichia coli, members of the genus Shigella, members of the genus Campylobacter, members of the genus Salmonella, and members of the genus Vibrio including Vibrio cholerae.
- a method for the induction of whole cell immunity contains the following steps: a. administration of a priming dose of comprising an adequate number of whole cell bacteria, containing DNA encoding and capable of expressing the polypeptide construct described in Examples 1 through 6, above, whereby the bacteria are selected from the group consisting of Escherchia coli, Shigella spp, Salmonella spp, Campylobacter spp, Vibrio spp and Vibrio cholera. b.
- boosting doses of whole cell bacteria, selected from the group consisting of Escherchia coli, Shigella spp, Campylobacter spp, Vibrio spp and Vibrio cholerae, containing and capable of expressing DNA encoding the recombinant polypeptide described in Examples 1 through 6, above.
- the boosting doses can be protein comprising the recombinant polypeptide described in Examples 1 through 6, above, at unit dose range of 1 ⁇ g to 1 mg of immunogen in a buffered aqueous solution.
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CA2890380A CA2890380C (en) | 2012-11-19 | 2013-10-08 | Recombinant polypeptide construct comprising multiple enterotoxigenic escherichia coli fimbrial subunits |
AU2013345316A AU2013345316B2 (en) | 2012-11-19 | 2013-10-08 | Recombinant polypeptide construct comprising multiple enterotoxigenic Escherichia coli fimbrial subunits |
JP2015543046A JP6403680B2 (en) | 2012-11-19 | 2013-10-08 | Recombinant polypeptide constructs containing multiple toxigenic E. coli pilus subunits |
EP13855228.6A EP2920203B1 (en) | 2012-11-19 | 2013-10-08 | Recombinant polypeptide construct comprising multiple enterotoxigenic escherichia coli fimbrial subunits |
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US201261727943P | 2012-11-19 | 2012-11-19 | |
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US14/048,264 | 2013-10-08 | ||
US14/048,264 US9328150B2 (en) | 2005-01-11 | 2013-10-08 | Recombinant polypeptide construct comprising multiple enterotoxigenic Escherichia coli fimbrial subunits |
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WO2016048810A1 (en) | 2014-09-24 | 2016-03-31 | The United States Of America As Represented By Th Secretary Of The Navy | Combined enterotoxigenic escherichia coli and campylobacter jejuni recombinant construct |
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 |
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CN107693786B (en) * | 2017-11-07 | 2021-06-29 | 聊城大学 | Compound vaccine for specifically preventing and treating escherichia coli diarrhea of horse colt |
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US20160052975A9 (en) | 2016-02-25 |
US10689422B2 (en) | 2020-06-23 |
CA2890380A1 (en) | 2014-05-22 |
EP2920203A4 (en) | 2016-07-13 |
JP2015536348A (en) | 2015-12-21 |
AU2013345316B2 (en) | 2016-04-14 |
US9328150B2 (en) | 2016-05-03 |
CA2890380C (en) | 2021-11-30 |
EP2920203A1 (en) | 2015-09-23 |
AU2013345316A1 (en) | 2015-05-21 |
JP6403680B2 (en) | 2018-10-10 |
US20150098961A1 (en) | 2015-04-09 |
EP2920203B1 (en) | 2019-06-05 |
US20160194362A1 (en) | 2016-07-07 |
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