Patent Grant
11077200
The inventive subject matter relates to a recombinant construct against enterotoxigenic Escherichia coli and Campylobacter jejuni comprising a combined anti-ETEC recombinant polypeptide construct and C. jejuni campsule polysaccharide.
Enterotoxigenic Escherichia coli (ETEC), Shigella, spp. and Campylobacter jejuni (CJ) are major causes of bacterial diarrhea worldwide. Both pathogens are a serious health threat to western travelers and young children in resource-limited countries, making them apt target populations for a single or dual pathogen vaccine against ETEC and CJ. No FDA-licensed vaccines are available for either pathogen.
ETEC causes an estimated 210 million cases of diarrhea and 380,000 deaths annually among infants and young children. Moreover, ETEC is the most common cause of travelers' diarrhea. ETEC causes diarrhea ranging in severity from mild illness to severe cholera-like purging. There are two major virulence factors, adhesive fimbriae, dubbed colonization factors (CFs), and enterotoxins. Surface-expressed CFs, consisting of complex protein heteropolymers, mediate adherence to the small intestinal epithelium to initiate colonization within this privileged host niche. ETEC produce one or both of two different enterotoxins, a heat-labile (LT) and a heat-stable enterotoxin (STI). LT and STI intoxicate epithelial cells, resulting in fluid and electrolyte secretion and clinical diarrhea. LT is highly immunogenic and a potent adjuvant, while STI is a small, poorly immunogenic peptide.
Prevalent CFs and a non-toxic form of the LT (or its congener cholera toxin (CT)) have been the focus for several strategies to develop an ETEC vaccine. Such antigens have been used individually or bundled as components of a whole-cell killed vaccine, live vaccines vectored by attenuated ETEC or other enterobacterial species (e.g., Shigella and Vibrio cholerae 01), and purified protein vaccines. None has yet been shown to confer sufficiently high and broad levels of protection. The weight of evidence from clinical trials indicates that anti-LT immunity confers short-term protection against LT-producing ETEC. There is also evidence to show that certain CFs function as protective antigens. There are, however, significant challenges for ETEC vaccine development. For one, about half of all ETEC express only STI, for which anti-LT immunity is not thought to be effective, thus necessitating anti-CF or anti-bacterial immunity. Also, the diversity of ETEC CFs poses issues for achievement of sufficiently broad coverage with inclusion of a realistic number of CFs.
The invention relates to an immunogenic construct comprising a polypeptide construct expressing enterotoxigenic Escherichia coli (ETEC) fimbrial subunits combined with a Campylobacter jejuni capsule polysaccharide or Shigella spp lipopolysaccharide (LPS).
In a preferred embodiment, one or more Camplobacter jejuni capsule polysaccharides are conjugated to one or more Escherichia coli enterotoxigenic recombinant polypeptide constructs. In another embodiment, Shigella LPS is conjugated to the ETEC polypeptide construct.
Campylobacger jejuni is associated with induction of Guillain-Barré Syndrome (GBS), a post-infectious polyneuropathy that can result in paralysis. The association is due to molecular mimicry between the sialic acid containing-outer core of the lipooligosaccharide (LOS) and human gangliosides (5, 6, 89, 91). Thus, antibodies generated against LOS cores result in an autoimmune response to human neural tissue. Use of capsule polysaccharide from C. jejuni can induce an immune response without the possible induction of Guillain-Barré Syndrome.
In a preferred embodiment, the composition comprises an ETEC 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 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 C-terminal major subunit. The immunogenic composition can comprise a whole or an immunogenic fragment, containing a donor β strand region, of the ETEC fimbrial major or minor subunits. In some construct examples, 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.
In another embodiment 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. In this embodiment, the fimbrial subunits can be derived from any ETEC fimbrial type, 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 ETEC recombinant polypeptide construct can be used to express a polypeptide for attachment to C. jejuni or Shigella LPS. As such, an object of the invention also includes a use of a construct for immunizing mammals, including humans, by a composition comprising antigens from multiple bacterial species, including ETEC, C. jejuni and Shigella strains. The embodied use comprises one or more priming administrations of the combination construct. The priming dose can be subsequently followed by one or more boosting doses.
The term enterobacteria, as used herein, refers to enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni or Shigella spp., which include: Shigella dysenteriae, Shigella flexneri, Shigella Boydii or Shigella sonnei. As used herein, an enterobacteria polysaccharide polymer is a polysaccharide polymer derived from enterobacteria. The term “polysaccharide antigen” as used herein refers to a capsule polysaccharide derived from Campylobacter jejuni (C. jejuni or Campylobacter jejuni capsule) or a lipopolysaccharide derived from Shigella spp. As used herein, “polysaccharide” refers to two or more monosaccharide units composing a carbohydrate polymer molecule. A “polysaccharide polymer” refers to two or more polysaccharide molecules connected together.
The terms “polypeptide,” “peptide,” and “protein” as used herein can be interchangeably used, and refer to a polymer formed of two or more amino acid residues, wherein one or more amino acid residues are naturally occurring amino acids. The term “amino acid sequence” refers to the order of the amino acids within a polypeptide. As used, herein, “oligomer” are polypeptides sequences comprising relatively few amino acids.
The term “recombinant polypeptide”, “recombinant polypeptide construct”, or “recombinant protein”, as used herein, 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. The term “recombinant construct” refers to the DNA encoding the recombinant polypeptide, recombinant polypeptide construct or recombinant protein.
The term “donor strand” or “donor 03 strand” refers to the N-terminal region of an ETEC fimbrial subunit that associates with another ETEC fimbrial subunit in donor strand complementation.
The term “immunogenic composition” refers to a formulation containing proteins or polypeptides or polysaccharides or polysaccharide polymers that induce a humoral and/or cellular immune response. The term “immunogenic coverage” or “spectrum of coverage” refers to the induction of humoral and/or cellular immune response against specific strains of bacteria under the “coverage.” The term “immunogenic 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. The term “derivative” refers to a polypeptide or nucleic acid sequence with at least 80% identity with sequence of the identified gene. In this context, “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 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, CsuA1, CooA, CosA, CsdA, CsbA, CotA; the ETEC CS3 proteins: CstH, CstG; and the ETEC CS6 proteins: CssA, and CssB.
The pathogenesis of Campylobacter jejuni remains poorly understood in comparison with ETEC and the organism shares few virulence factors with better-characterized pathogens. C. jejuni is unusual, however, among enteric pathogens in that it expresses a polysaccharide capsule (CPS) that is one of its few confirmed virulence factors.
Because of the importance of ETEC and C. jejuni as pathogenic agents, a combined ETEC-CJ composition was constructed in order to afford protection against both agents. In one embodiment, a recombinant polypeptide construct, comprising fimbrial subunits from Class 5 ETEC strains is fused to a capsule polysaccharided from the C. jejuni strain 18-176.
In a preferred embodiment, one or more recombinant polypeptide ETEC constructs, comprising the ETEC fimbrial adhesion, are conjugated to isolated C. jejuni capsule polysaccharide (CPS). One or more of a number of ETEC recombinant constructs can be conjugated to one or more of a number of C. jejuni capsule polysaccharide structures. In the inventive construct, the ETEC recombinant construct operates both as an immunogen against ETEC and as a protein carrier molecule, presenting the C. jejuni polysaccharide. Examples of ETEC recombinant polypeptides and C. jejuni capsule polysaccharides that can be incorporated into a combined structure are given in the following examples.
In a preferred embodiment, the ETEC polypeptide construct can not only serve as antigen against ETEC but also serve as a protein carrier for polysaccharide antigens, such as C. jejuni capsule polysaccharide.
In a preferred embodiment, ETEC recombinant polypeptides or polypeptide constructs are conjugated to C. jejuni CPS. The CPS can be derived from a number of C. jejuni strains. In the embodiment, any CPS of any C. jejuni strain is envisioned to be conjugated to ETEC recombinant polypeptide constructs. Alternatively, Shigella LPS can be conjugated to ETEC recombinant polypeptide constructs.
The overall method of conjugating includes oxidizing C. jejuni CPS, for example, with NaIO4 in sodium acetate (pH 4.0). Oxidized CPSs were desalted with a 5 kDa cutoff membrane by stirred ultrafiltration, which is subsequently lypholized. ETEC proteins are then added. The stoichiometery protein to CPS can vary, however, a typical ratio is 1:2 protein to CPS by mass. The concentration of components can be by any method. However, for example, polysaccharide concentration was determined by antrhone assay and protein concentration was determined. NaCNBH3 is then added. The conjugates are subsequently desalted by ultrafiltration and lyophilized. CPS (or Shigella LPS), ETEC proteins and conjugates were analyzed, for example by SEC-HPLC. Conjugates were also analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and Gel Code® Blue (Pierce, Biotechnology, Inc, Lombard, Ill.) staining. Conjugates were detected by antibody-based assay using anti-CPS and anti-ETEC protein.
As an example of ETEC recombinant polypeptide and C. jejuni conjugation, the CPS from the C. jejuni strain 81-176 was conjugated to ETEC recombinant polypeptide construct CfaE (class 5 ETEC adhesin) or to the recombinant polypeptide construct CfaE linked, via a polypeptide linker, to the major subunit CfaB.
CPS C. jejuni capsule was purified from Campylobacter jejuni strain 81-176 (PG3208). This mutant, in which the galT gene was insertionally inactivated by chloraphenicol cassette, lacks all ganglioside mimicry in its lipoligosaccharide (LOS) core.
The cells were grown in porcine Brain-Heart Infusion (BHI) broth and sonicated to inactivate the cells. The CPS was extracted by hot water/phenol method previously employed for the same organism (Chen, et al., Carbohyd. Res. 243: 1034 (2008)). Cells were immersed in a water/phenol mixture (3:2 ratio by volume), which was heated to 67° C. with stirring for 4 hours. The suspension was cooled and separation of the mixture into two separate layers (the aqueous layer and the phenol layer) and extraction of the aqueous layer was performed. The aqueous layer was removed and the phenol/water extraction was repeated on residue, to maximize the yield. Aqueous layers from two extractions were pooled and boiled fro 1.5 hours with the addition of acetic acid to a pH of 3.5. The aqueous layer was dialyzed against running water for 2 days and concentrated using a Millipore concentrator cell with a 5 kDa cutoff membrane. Trace amounts of residual RNA were removed by digestion with benzonase enzyme at 90 u/ml in 50 mM Tris-CCl/1 mM MgCl2, pH 8 overnight at 37° C. Benzonase was removed from CPS, then desalted and concentrated using stirred ultrafiltration with 30 and 5 kDa MWCO disc membranes, respectively.
The isolated CPS was oxidized with adding 40 mg of CPS to 40 mM NaI04 in sodium acetate pH 4 in the dark at 4° C. for 2 days. Oxidized CPS was desalted with 5 kDa cutoff membrane by stirred ultrafiltration and was subsequently lyophilized.
Prior to conjugation the ETEC proteins, for example dscCfaE and dscCfaEB, were transferred to 0.1M borate buffer at pH 9.0. Oxidized CPS was added to each ETEC protein at a ratio of 1:2, protein to CPS by mass, and then NaCNBH3 was added at 2 times mass equivalent to CPS. The reaction was incubated 1 day at room temperature and 6 days at 37° C. in the dark with continuous stirring. The conjugates were desalted by stirred ultrafiltration with 30 kDa membrane and lyophilized. Conjugates of the CPS to dscCfaE and dscCfaEB was conducted by SEC-HPLC. and polyacrylamide gel (PAGE) (12.5%) electrophoresis.
In PAGE analysis, immunodetected with rabbit polyclonal antibodies to whole cells of 81-176 was used to detect CPS and to CfaE. The results of this study are shown in
The results of
The results of the SEC-HPLC are shown in
Detection of the conjugates by refractive index (RI) on SEC-HPLC revealed that 45% and 50% of the polysaccharide remained unconjugated with the CfaE and CfaEB conjugates respectively. This is summarized in Table 1, which also illustrates that the conjugated molar ratio of CPS to CfaE was 4.8:1 and that of CPS to CfaEB was 4.4:1.
Anti-ETEC constructs that are contemplated to be conjugated to C. jejuni polysaccharide comprise the structures as illustrated in
In the multipartitie construct design, as in the basic design (compare
In the multipartite construct, subunits from the same fimbrial type are directly connected. Groupings of subunits from the same fimbrial type are then connected to other groupings of subunits from other fimbrial types. Fimbrial types include, but are not limited to ETEC class 5a, 5b, 5c, CS3 and CS6. For example a single construct can include subunits derived from any two or more of class 5a, 5b, 5c, CS3 and CS6 fimbrial types.
Multiple linker sequences can be utilized in connecting the individual subunits. Examples of specific linkers include the tetrapeptide of SEQ ID No. 5. Another example is a tri-glycine linker (i.e., G-G-G). In the inventive construct, in cis donor strand complementation is used to stabilize adhesins and adhesin-pilin fusions for representative Class 5a, 5b, and 5c adhesins.
The contemplated composition is designed to enable as wide a range of coverage of ETEC strains as possible. As such, in one embodiment, 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.
In a preferred embodiment, recombinant polypeptide ETEC constructs are conjugated to C. jejuni capsule polysaccharide (CPS). One or more of a number of ETEC recombinant constructs can be conjugated to one or more of a number of C. jejuni capsule polysaccharide structures. Examples of Class 5 ETEC recombinant polypeptides are listed in Table 2. In Table 2, minor subunits are stabilized by connection, via a polypeptide linker, to associated major subunits. Alternatively, a 12-16 amino acid donor strand, derived from the associated major subunit is connected to the minor subunit via a polypeptide linker. These polypeptides can also be linked as per
1“spd” refers to signal peptide. The mature polypeptide sequence, therefore, would be the full length minus the signal peptide.
2DNA sequence encodes mature protein.
1dsc 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. For example, “dsc14CsfA” 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-His6 at the C-terminus.
An important feature of the anti-ETEC construct is the enhanced immune recognition of the fimbrial adhesion. The minor subunits (i.e., ETEC adhesin) of ETEC Class 5 fimbriae are stoichiometrically represented in very low numbers relative to the major subunit. Therefore, an important feature of the recombinant constructs is the vastly improved stoichiometric representation of the minor subunit in order to enhance immune recognition of the minor subunit. Additionally, since fimbrial subunits, such as 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 β strand is provided by the major fimbrial subunit. For example, in the case of CfaE, stabilization is provided by the N-terminal region of CfaB. Engineering of dscCfaE by incorporation of a donor peptide strand from the N-terminus of the CFA/I major subunit CfaB at its C-terminus transformed an insoluble, unwieldy native, recombinant protein into a stable immunogenic composition (Savarino, U.S. Patent application publication no. 20060153878 (13 Jul. 2006)), which is incorporated by reference, herein.
Based on its atomic structure, dscCfaE is folded into a native, β-sandwich conformation, consisting of two half-barrels, comprising the N-terminal adhesin domain (CfaEad) a short α-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 (Ser. No. 11/340,003, filed Jan. 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).
A number of observations indicate the suitability of dscCfaE (cloned from ETEC strain E7473) as a vaccine antigen. First, sequencing of 31 different wild type alleles of cfaE from ETEC isolates of varying geographic origin and serotypes, show that the gene and predicted polypeptide sequence are nearly invariant, with three different 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)). Hence, the target protein shows uniformity in natural ETEC bacterial populations.
Additionally, CfaE, a Class 5a fimbrial adhesin, is 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).
Moreover, rabbit anti-dscCfaE serum cross-neutralizes CS4- and CS14-ETEC in the hemagglutination assay (HAI). A number of vaccination studies have been performed in small (rabbit and mice) and large (monkeys and cows) animals with various routes of administration and adjuvant combinations showing that dscCfaE is a potent immunogen that can elicit systemic and mucosal antibodies which recognize dscCfaE and CFA/I and are neutralizing (as measured by HAI assay).
An embodiment includes anti-class 5 ETEC constructs based on the construct design illustrated in
The design in
The construct design, illustrated in
CS6 and CS3
Rabbit model (RITARD) studies suggest the colonization factor CS6 and CS3 has immune-protective potential (Svennerholm, et al., Infect. Immun. 56: 523-528 (1988); Svennerholm, et al., Infect. Immun. 58: 341-346 (1990)). As such, an important technical goal is to reproduce a stabilized CS6 expressing recombinant structure expressing CS6 antigens that maximally elicits antibody responses inhibitory to CS6-directed adhesion.
Unlike class 5 ETEC fimbriae, the fimbrial structures may function as polyadhesins rather than monadhesins (Zavialov, et al., FEMS Microbiol. Rev. 31: 478-514 (2007)). Extrapolation from related fimbriae, assembly of 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.
Association of monomers of CS3 and CS6 was evaluated by visualization of the subunit proteins under denaturing and non-denaturing conditions in polyacrylamide gel electrophoresis (PAGE). For both CS3 and CS6 monomers, under denaturing conditions the proteins migrating at the expected sizes. Under non-denaturing conditions multiple size (i.e., ladders) are seen formed by multimeric association of the subunits.
CS6 Fimbriae
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., greater than 90% amino acid conservation), with greater variation in CssA and the mutations randomly distributed along the CssA polypeptide.
In order to design an effective immunogenic composition that would be suitable for inclusion in a vaccine formulation a number of criteria were devised for determination of suitable constructs. These included the ability to maintain a structure without unwanted self-association or assembly; thermostability; and ability to generate anti-CS6 IgG and IgA antibody levels similar to those elicited by immunization with CS6.
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, multimeric 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.
In one embodiment, a construct conjugated to C. jejuni 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
As illustrated in
Additionally, to prevent recombinant polypeptide constructs forming molecular associations resulting in un-desirable non-covalent oligomer formation, in a preferred embodiment, the N-terminal 14-16 amino acids of the N-terminal CS6 subunit is deleted. As an illustration, “dscB14CssBA” would contain a heterologous donor strand (i.e., “dsc”), from CS6 CssB, inserted at the C-terminus of the construct. In this case, the donor strand is 14 amino acids in length, as indicated by the “14.” Similarly, a constructed designated “ntd15dscACssBA” 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”).
Examples of constructs comprise one or more CS6 subunits with amino acid 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. In a preferred embodiment, the linker is a tetrapeptide with the amino acid sequence of SEQ ID No. 5.
CS3 Fimbriae
Savarino (U.S. patent application Ser. No. 11/340,003 (2006)) discloses donor strand complementation stabilized ETEC constructs. Embodiments of this application incorporate the donor strand stabilization of CstH and adds the second CS3 subunit, CstG. Embodiments herein add additional features found to be important for stabilization of the CS3 subunits and immunogenicity against CS3. 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. Similarly, although some variation CstG is observed, it is also relatively invariant, with 99-100% amino acid conservation.
CS3 contains both CstG and CstH, in near equal amounts. Therefore, dimeric constructs were devised incorporating CstG and CstH, according to the template construct design of
In one embodiment a polypeptide construct conjugated to C. jejuni capsule polysaccharide comprises a CS3 s construct designed according to
In a preferred embodiment, the CS3 construct is a dimer. Although other examples are contemplated using the design of
Other examples can include constructs, according to
Construction of Multipartite Fusion Constructs
Immunity to multiple strains of ETEC is important to obtain the greatest extent of anti-ETEC immunity. Toward this goal, recombinant polypeptide constructs were developed comprising two or more subunits derived from different ETEC fimbrial types according to the design illustrated in
In a preferred embodiment, major and/or minor subunits, derived from the same ETEC fimbrial type are connected, via polypeptide linkers, and stabilized by donor β strand complementation, as illustrated in
In one embodiment, 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
As illustrated in
As illustrated in
In other embodiments, 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
The inventive compositions can utilize different linker sequences. In a preferred embodiment, the linker contains the amino acid sequence of SEQ ID No. 5. In another embodiment, the linker is a tri-glycine linker. In other embodiments, the C-terminal end of the construct contains a histidine tag for purification of the construct.
In the inventive construct, in cis donor strand complementation is used to stabilize adhesins and adhesin-pilin fusions for representative Class 5a, 5b, and 5c adhesins. For each adhesin target group, in a preferred embodiment, the compositions are constructed with the intent of eliciting anti-adhesive immune responses. Further towards this goal, 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 β-strand of the last major subunit.
Other embodiments include 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 PCFO71 pilin subunit CosA; and Class 5c adhesin CotD tandemly linked at its C-terminus to CotA (CS2 major subunit).
Embodiments of ETEC multipartite fusion constructs are illustrated in Table 4 and 5. In this embodiment, constructs comprise any major or minor ETEC fimbrial subunit from Table 2 in multiple combinations, connected by linker polypeptides and stabilized from proteolytic degradation by donor strand complementation utilizing the design illustrated in
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 2.
The strategy for selecting and developing specific genetic fusion constructs is guided, in part, by the phylogenetic and antigenic relatedness of subunits. For example, 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 such, adhesin (i.e., minor fimbrial subunit) from a specific fimbrial type (e.g., Class 5a) are linked to Class 5a major subunits. Further selection of subunits is guided and based on epidemiological study analysis in order to achieve optimum immunogenic coverage of ETEC strains. What C. jejuni capsule polysaccharide to conjugate is predicated primarily on epidemiological data suggesting pathogenicity of the strain providing the capsule polysaccharide. Although many C. jejuni strains exist, most are not pathogenic.
In the multipartite constructs listed in Table 4 and 5 the linker polypeptide, depending on the example construct, can comprise a four (4) amino acid sequence (tetrapeptide) or a tri-glycine. Also, as illustrated in
In Table 4 and 5 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 4 or 5 (or Table 3), other combinations of major and minor subunits are contemplated utilizing the construct design illustrated in
1All combinations can include a histidine (i.e., His6) at the C-terminal end.
2Subunits can be linked via either DNKQ or tri-glycine linker.
3(G) refers to glycine residue introduced to provide a “swivel.”
4“ntd” refers to N-terminal deletion (excised from mature protein) with extent of deletion (i.e., amino acids) indicated.
5“dsc” refers to span of N-terminal residues from donor β-strand, its amino acid length and its source.
1All combinations can include a histidine (i.e., His6) at the C-terminal end.
2Subunits can be linked via either DNKQ or tri-glycine (GGG) linker. In preferred embodiments, DNKQ is used, except where indicated with (GGG).
3(G) refers to glycine residue introduced to provide a “swivel.”
4“spd” refers signal peptide. Number indicates number of amino acids.
5“ntd” refers to N-terminal deletion (excised from mature protein) with extent of deletion (i.e., amino acids) indicated.
6“dsc” refers to span of N-terminal residues from donor β-strand, its amino acid length and its source.
In another embodiment, recombinant polypeptide constructs can contain a C-terminal toxin A subunit, such as cholera toxin A2 (CTA) to form a chimeric molecule. In this embodiment, a full-length or truncated CTA2 is connected to CS6 or CS3 multimeric recombinant polypeptide construct, such as a CS6 or CS3 dimer.
Examples of these toxin constructs are illustrated in Table 4 and 5. In these constructs, the LTB gene and the CS3 or CS6-toxin chimera are separately expressed. LTB, once expressed, would self assemble to form a pentameric structure. The ensuing LTB multimeric composition (i.e., LTBs) and CS3 or CS6-toxin chimera then non-covalently associate to form a holotoxin-like heterohexamer.
Although other examples are contemplated, the sequences of examples of illustrative chimeric constructs, containing a C-terminal toxin component, are illustrated in Table 4 (for CS3) and Table 5 (for CS6).
For CS3-chimeric molecules, one or more CS3 fimbrial subunits are connected, as in
CS6 toxin chimera examples are also illustrated in Table 5. For 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 β 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). In a preferred embodiment, like for CS3, 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.
Although many additional combinations are possible, in the examples shown in Table 5, 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 β strand. The donor β strand can be homologous or heterologous to the C-terminal most fimbrial subunit. However, in the examples in Table 5 the donor strands are heterologous to the C-terminal fimbrial subunit. The donor strand is then connected to a cholera toxin A2 (CTA2) subunit. The polypeptide sequences of one of the examples is as in SEQ ID No. 43, which is encoded by the nucleotide sequence of SEQ ID Nos. 42. In this example, the N-terminal subunit is CssA, with the N-terminal 15 amino acids of the mature CssA sequence deleted. In this example, a pelB leader sequence (22 amino acids) was also added, which is illustrated in
Recent development of a molecular CPS typing system re-enforced the strong correlation between CPS and Penner types (Poly, et al., J. Clin. Microbiol. 49: 1750 (2011)). Both Penner serotyping and molecular CPS typing have revealed the predominance of a handful of CPS types worldwide. Also, despite over 60 Penner serotypes having been identified, most Campylobacter diarrheal disease is caused by C. jejuni expressing only a limited number of serotypes. Therefore, only selected strains of C. jejuni, predicated on epidemiological studies, provides suitable candidate strains for development of vaccine compositions. However, despite the importance of this organism to human disease, there are no licensed vaccines against C. jejuni.
C. jejuni capsule polysaccharide (CPS) was extracted from C. jejuni strains selected based on their association with diarrheal disease. CPS from bacteria was extracted by hot water-phenol extraction for 2 h at 70° C. The aqueous layer was dialyzed (1000 Da) against water followed by ultracentrifugation to separate the CPS from the LOS. The supernatant material containing the CPS was subjected to size-exclusion chromatography (Sephadex G50) for further purification to yield the intact CPSs. Monosaccharide composition was performed using a procedure amenable to the alditol acetate method (Chen, et al., Carbohydr. Res. 343: 1034 (2008)) with the alditol acetates being analyzed in a ThermoFinnigan POLARIS™-Q (Thermo Fisher Scientific, Inc, Waltham, Mass.) gas chromatograph/mass spectrometer (GC/MS) using a DB-17 capillary column. The sugar linkage types were characterized by characterization of the permethylated alditol acetates by GC/MS as previously described (Chen, et al., Carbohydr. Res. 343: 1034 (2008)). The NMR experiments were performed on a Bruker 400 MHz spectrometer (Bruker Corporation, Billeria, Mass.) equipped with a Bruker cryo platform at 295 K with deuterated trimethylsilyl propanoic acid and orthophosphoric acid as external standards. The structures of important pathogenic C. jejuni capsule polysaccharides are shown in Table 6.
Additionally, the capsule polysaccharide from the HS5 strain of C. jejuni can be attached. HS 5 contains a complex of variations of polysaccharides. These include the following structures:
Induction of an immune response by the conjugates was evaluated. In these studies, BALB/c mice were immunized with escalating amounts of vaccines administered with Alhydrogel® (Sergent Adjuvants, Clifton, N.J.). Mice received a total of two immunization at a 4-week interval.
The results of these studies is illustrated in
Furthermore, as illustrated in
The data shown in
To determine the levels of functional anti-adhesive antibody generated by HS36 conjugate vaccines, serum samples were tested by hemagglutination inhibition assay (HAI) assay in order to measure functional anti-adhesive antibodies present in the immune mouse serum.
The HAI assays were conducted by evaluating samples from each animal. The samples were initially diluted 1:8, then diluted two-fold over a wide range of dilutions. Each serum dilution was incubated with an equal volume of CFA/I+ ETEC bacteria (strain H10407), which further diluted the serum 1:2. The final lowest dilution tested was 1:16, which was the limit of detection (LOD). The pre-incubated mixture was subsequently mixed with bovine erythrocytes in the presence of 0.5% mannose in U-bottom 96-well plates. In the absence of anti-adhesive antibodies, the erythrocytes formed visible agglutinated “buttons” of cells. In the presence of anti-adhesive antibodies, agglutination was inhibited. The HAI titer was the highest serial dilution that completely inhibited agglutination. If there was not detectable inhibition at the lowest serum dilution of 1:16, the samples were assigned a value of one-half of the detection limit (i.e., 8) for computational purposes.
The results of the HAI analysis are illustrated in
Synthesis of Polysaccharide Construct
A polysaccharide constructed was synthesized as shown in
As shown in
The strategy for the introduction of MeOPN group was inspired by a similar reaction initially proposed by C. Mara et al, Bioorg. Med. Chem. Lett. 6180-6183 (2011). Compound 6 (
Allyl and phthlimido protecting groups were removed with palladium (II) chloride and hydrazine respectively, generating product 9 (
Induction of Immunity Against MeOPN-6-Gal
In one embodiment, galactose modified at the 6 carbon with O-methyl phosphoramidate (MeOPN-6-Gal) is used to induce immunity against multiple C. jejuni strains, even those strains not expressing MeOPN-6-Gal. As illustrated in
The strong cross-reactivity with MeOPON-6-Gal exhibited against HS23/36 and HS4 antibody may be explained by the fact that MeOPN-6-Gal share epitopic structures with HS23/36 and HS4 capsule polysaccharides. One explanation may be that the MeOPN group in both HS23/36 and HS4 is to a primary hydroxyl. The cross reaction of MeOPN-6-Gal (HS23/36) with HS4, which contains MeOPN-7-6d-β-D-ido-Heptose, was unexpected, but may be due to the linkage of MeOPN to primary hydroxyl positions on both sugars. This feature is illustrated in
A synthetic conjugate vaccine strategy can be developed to protect against multiple enteric pathogens. Most efforts at development of vaccines against bacterial enteric pathogens are limited to a specific pathogen. The ability to combine vaccines against multiple, antigenically variable pathogens in a single, multi-valent, injectable vaccine would greatly simplify approaches to prevent acquisition and transmission of these pathogens worldwide. Globally, ETEC and C. jejuni are among the leading causes of bacterial diarrheal disease. In addition CJ has been causally linked to several serious sequelae including Guillain Barré Syndrome, irritable bowel syndrome, and reactive arthritis. Moreover, recent studies have indicated an association between CJ infections and malnutrition and growth stunting in young children in resource-limited settings.
Using conventional methods, we have developed conjugate vaccines containing CJ polysaccharide capsules that have proven to be immunogenic in multiple animal species and to confer protection against C. jejuni diarrhea in NHP. The newer synthetic approach is based on recent data that the immunodominant epitope on CJ polysaccharide capsule conjugate vaccines is the MeOPN modification found on different sugars in different capsule types.
Therefore, an immunogenic platform against both C. jejuni and ETEC can be created by linking synthetic MeOPN-sugars to different ETEC protein antigens. The approach could also be extended to include Shigella lipopolysaccharides (synthetic or detoxified) conjugated to ETEC proteins. Thus, this platform could form the basis of a multivalent vaccine against three major bacterial diarrheal pathogens. Conjugation can also serve as a protein carrier to enhance immunogenicity of the Campylobacter construct.
It is envisioned to conjugate the construct of Examples 3-5 to an ETEC construct. The overall method of conjugating includes oxidizing C. jejuni CPS, for example, with NaIO4 in sodium acetate (pH 4.0). Oxidized CPSs were desalted with a 5 kDa cutoff membrane by stirred ultrafiltration, which is subsequently lypholized. ETEC proteins are then added. The stoichiometery protein to CPS can vary, however, a typical ratio is 1:2 protein to CPS by mass. The concentration of components can be by any method. However, for example, polysaccharide concentration was determined by antrhone assay and protein concentration was determined by Pierce 660 protein assay or the BCA assay. NaCNBH3 is then added. The conjugates can then be subsequently desalted by ultrafiltration and lyophilized. CPS, ETEC proteins and conjugates were analyzed, for example by SEC-HPLC or by SDS polyacrylamide gel electrophoresis (PAGE), or other methods.
The immunogenicity of CfaE-HS23/36 and CfaEB-HS23/36 conjugates was observed in mice, as well as induction of hemagglutination inhibition (HAI) titers against CfaI in mice. The amino acid sequence of the dscCfaE construct used is SEQ ID No. 138 (nucleotide sequence is SEQ ID No. 139). The dsc19CfaE amino acid sequence is SEQ ID No. 143 (nucleotide sequence is SEQ ID No. 142). The amino acid sequence for dsc19CfaEB is SEQ ID No. 141 (nucleotide sequence is SEQ ID No. 140).
The CfaEB-HS23/36 conjugate was down-selected in order to proceed to studies in Aotus nancymaae. This non-human primate (NHP) model was selected because it has been used as a diarrheal disease model for both ETEC and C. jejuni. We synthesized a lot of the CfaEB-HS23/36 vaccine that was sufficient in size for three NHP studies by reductive amination. The first such study, which is the only one that has been completed, was a dose finding study followed by a C. jejuni challenge.
The design of this NHP study is shown in Table 7. Animals (6 per group) were immunized three times at days 0, 42, and 84. The CfaEB-HS23/36 vaccine was given subcutaneously at either 0.5 ug or 3.5 ug polysaccharide (PS) adjuvanted with aluminum hydroxide. The ratio of PS to protein in the vaccine was roughly 1:1 so this was equivalent to 0.5 or 3.5 ug of CfaEB per dose. The 3.5 ug dose was also given intradermally (ID) with poly-IC as adjuvant. This was done to bridge to previous work done using ID immunizations with CfaEB alone. Similarly, another group was given HS23/36-CRM197 subcutaneously to bridge to previous work with the same capsule conjugated to another protein. Finally, the control group was immunized with PBS. On day 148 the animals were all challenged with 4×1011 CFU of CG8421, an HS23/36 strain.
Animals were observed for diarrheal disease daily for 10 days following challenge. Diarrhea was defined as two or more days of consecutive of stools that were ≥grade 3. The results are summarized in Table 8. Only 3/5 animals in the PBS group developed diarrhea for an attack rate of 60%. Note that one animal was eliminated because it developed diarrhea prior to challenge. The mean time to onset of disease in this negative control group was 2.3 days and the mean duration of illness was 5.3 days. The attack rate in the animals immunized with the HS23/36-CRM197 vaccine was 33% (2/6), with a mean onset of disease of 2 days and a mean duration of illness of 4 days (45% efficacy). Animals that were immunized with CfaEB-HS23/36 intradermally with poly IC also showed an attack rate of 33% with a mean onset of 1.5 days and a duration of two days (45% efficacy). The animals immunized subcutaneously with CfaEB-CPS showed between 67-100% efficacy against diarrheal disease. The attack rate in the group immunized with 0.5 ug of the vaccine was 0 (0/5, with one animal that vomited after challenge being eliminated) and the attack rate in the group immunized with 3.5 ug of the vaccine was 20% (1/5, with one animal being eliminated due to diarrhea prior to challenge). The single animal in this group that did develop diarrhea had a later onset of disease (day 9). There were no significant differences among the control group and any of the immunized animals due to the small numbers of animals per group.
Serology results are shown in
CssBA-HS3 Vaccine.
CssBA is a recombinant form of the two subunits of CS6 that are fused together. This protein was conjugated to capsule from an HS3 strain by TEMPO oxidation. The conjugates were analyzed by SDS-PAGE and immunoblotting. Purified CssBA has a predicted Mr of 31.8 kDa. The conjugate of CssBA-HS3 CPS runs as two bands, one slightly smaller than CssBA and one that runs at approximately 60 kDa. The bands in the conjugate react with both anti-CssBA antiserum and antibodies to whole cells of HS3, indicating that polysaccharide has been conjugated to the protein.
Mice were immunized subcutaneously with three doses of the vaccine given at 4 week intervals. Doses were 5 ug by weight or 25 ug by weight. Animals were bled at day 0 and two weeks after each immunization and the response to CssBA and to CPS were determined by ELISA. The results, shown in
LTB-HS4 Vaccine.
LTB is the binding component of the heat labile enterotoxin of ETEC. Recombinant LTB, which is not toxic, was conjugated to the polysaccharide capsule of an HS4 strain by reductive amination. The conjugate was analyzed by immunoblotting as shown in
Mice were immunized with three doses of either 5 or 25 ug (by weight) of the LTB-HS4 conjugate subcutaneously at 4 week intervals and the serum immune response was determined. The results, shown in
There are four species of Shigella, a human pathogen cause diseases such as diarrhea and bacilliary dysentaery: Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei are important enteropathogens Strains of Shigella spp. Express long-chain lipopolysaccharides. The chemical structures for many strains has been determined (see Liu, et al., FEMS Microbiol. Rev. 32: 627-653 (2008)).
An object of this invention, is a Shigella LPS-ETEC construct. The construct comprises an ETEC construct, as the above examples, conjugated, to a Shigella LPS, as an alternative or in addition to C. jejuni capsule polysaccharide. It is envisioned that any of the Shigella spp. can be conjugated to the ETEC construct. As an example, the Shigella flexneri 2a LPS is illustrated, as a potential LPS structure that can be conjugated to an ETEC construct, as follows:
This application is a Continuation application of application Ser. No. 14/721,656, filed May 26, 2005, which is a Continuation-in-Part application of U.S. Nonprovisional application Ser. No. 11/340,003, filed Jan. 10, 2006, which claims priority to U.S. Provisional application 60/642,771 filed Jan. 11, 2005, and a Continuation-in-Part to U.S. Nonprovisional application Ser. No. 11/524,057 filed Sep. 20, 2006, which claims priority to U.S. Provisional application 60/722,086, filed Sep. 21, 2005, and a Continuation-in-Part to U.S. Nonprovisional application Ser. No. 14/048,264, filed Oct. 8, 2013, which claims priority to U.S. Provisional application 61/727,943, filed Nov. 19, 2012, the contents of which are herein incorporated by reference. This application also claims priority to U.S. Provisional application 62/054,454, filed 24 Sep. 2014, U.S. Provisional application 62/127,927, filed Mar. 4, 2015, U.S. Provisional application 62/165,301, filed May 22, 2015, U.S. Provisional application 62/127,935, filed Mar. 4, 2015, and U.S. Provisional application 62/075,399, filed Nov. 5, 2014, the contents of which are herein incorporated by reference.
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| 10105448 | Guerry | Oct 2018 | B2 |
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| WO 2007117339 | Oct 2007 | WO |
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| 20190008979 A1 | Jan 2019 | US |
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| 60722086 | Sep 2005 | US | |
| 60642771 | Jan 2005 | US |
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