Patent Application
20220088211
The first, general protein glycosylation pathway in bacteria, the N-linked glycosylation system of Campylobacter jejuni, was discovered two decades ago (Szymanski C M, et al. (1999) Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol Microbiol 32(5):1022-1030). Since then, many diverse prokaryotic glycosylation systems have been characterized, including O-linked glycosylation systems that have no homologous counterparts in eukaryotic organisms (Iwashkiw J A, et al. (2013) Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 89(1):14-28). Shortly after these discoveries, glycosylation pathways were recombinantly introduced into E. coli creating the field of bacterial glycoengineering (Wacker M, et al. (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298(5599):1790-1793). Bacterial glycoengineering is an emerging biotechnological tool that harnesses prokaryotic glycosylation systems for the generation of recombinantly glycosylated proteins using E. coli or other Gram-negative organisms as a host. Currently, glycoengineering utilizes two broad approaches to recombinantly glycosylate proteins, both of which can generate N- or O-linkages: oligosaccharyltransferase (OTase)-dependent and OTase-independent.
Protein glycosylation, or the covalent attachment of carbohydrates to proteins, is a ubiquitous posttranslational modification. For the most part, protein glycosylation is characterized as either N-linked with glycans attached to asparagine residues, or as O-linked with glycans attached to serine or threonine residues. While the importance of eukaryotic glycosylation has been and continues to be a source of intensive research, prokaryotic glycosylation has only recently grabbed the attention of the scientific community with the discovery of a general N-linked protein glycosylation system in the ε-proteobacterium Campylobacter jejuni (Szymanski C M, et al. (1999) Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol Microbiol 32(5):1022-1030). Since the initial C. jejuni discovery, prokaryotic glycosylation systems have been described across a plethora of Gram-negative and Gram-positive bacteria and been shown to contribute towards normal bacterial physiology as well as pathogenesis (Iwashkiw J A, et al. (2013) Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 89(1):14-28; Nothaft H & Szymanski C M (2010) Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 8(11):765-778); Schaffer C & Messner P (2017) Emerging facets of prokaryotic glycosylation. FEMS Microbiol Rev 41(1):49-91). Given the straightforward nature of prokaryotic genetics, it was only a matter of time before protein glycosylation systems were engineered and exploited for the production of designer glycoproteins in a process termed “bacterial glycoengineering”.
Much like eukaryotic glycosylation, bacteria have evolved an N-linked OTase pathway, but also employ O-linked OTase systems that are unique to prokaryotic organisms. OTase-independent glycosylation occurs in the cytoplasm and relies on glycosyltransferases to transfer monosaccharides from nucleotide activated precursors for the sequential assembly of glycoproteins. Both OTase-dependent and -independent pathways are exploited for bioconjugating carbohydrates to proteins.
Bacterial surface polysaccharides are some of the first, and most abundant, microbial components encountered by the immune system during infection (Comstock L E & Kasper D L (2006) Bacterial glycans: key mediators of diverse host immune responses. Cell 126(5):847-850). These polysaccharides, usually in the form of capsule or O antigen attached to lipid A, serve a multitude of purposes, including protecting microbial organisms from external threats and immune clearance. Given their abundance on invading organisms as well as their biochemical distinctness from eukaryotic carbohydrates, some microbial surface polysaccharides have been used as antigens for vaccine development. However, when polysaccharides are used alone in vaccine formulations, they usually act as T-cell independent antigens and therefore do not stimulate immunoglobulin class switching and long-term B cell memory. Moreover, polysaccharide vaccines alone do not elicit protection in vulnerable groups like infants and children under two years of age. This poor immune response can be overcome by covalently attaching a polysaccharide to a protein carrier in a process known as conjugation (De Gregorio E & Rappuoli R (2014) From empiricism to rational design: a personal perspective of the evolution of vaccine development. Nat Rev Immunol 14(7):505-514).
Traditionally, glycoconjugate vaccines are synthesized using a semi-synthetic approach where the polysaccharide is extracted from the target bacterium, purified, chemically modified and covalently linked to a carrier protein. This approach has resulted in the commercial licensure of multiple glycoconjugate vaccines to prevent colonization and infection by Haemophilus influenzae type B, and multiple serotypes of Streptococcus pneumoniae and Neisseria meningiditis. For detailed reviews on semi-synthetic or synthetic glycoconjugate vaccine production please refer to the following excellent review article (Berti F & Adamo R (2018) Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification. Chem Soc Rev 47(24):9015-9025). Although conjugate vaccines produced chemically have seen immense commercial success (the glycoconjugate vaccine Prevnar 13 has been Pfizer's best-selling product from 2015-2018 with over 24 billion USD in sales), their manufacturing processes are not without drawbacks; including, batch to batch variation, heterogenous product formation, large scale production of pathogenic organisms, and high manufacturing costs (Frasch C E (2009) Preparation of bacterial polysaccharide-protein conjugates: analytical and manufacturing challenges. Vaccine 27(46):6468-6470).
Over the last two decades, alternative strategies for producing glycoconjugate vaccines have emerged. These techniques are broad in their approach with some yielding vaccines closer to commercial licensure than others. Specifically, the advent of in vivo bacterial conjugations for manufacturing glycoconjugate vaccines have produced some of the most clinically advanced products to date. Commonly referred to as bioconjugation or protein glycan coupling technology (PGCT), the in vivo conjugation of polysaccharides to proteins for glycoconjugate vaccine production relies on OTases (Frasch C E (2009) Preparation of bacterial polysaccharide-protein conjugates: analytical and manufacturing challenges. Vaccine 27(46):6468-6470). It is generally considered that bioconjugation represents a simplification of the production and manufacturing process of glycoconjugate vaccines (Rappuoli R, De Gregorio E, & Costantino P (2019) On the mechanisms of conjugate vaccines. Proc Natl Acad Sci USA 116(1):14-16).
Both N-linking and O-linking OTases have been employed for biologically conjugating polysaccharides to carrier proteins for glycoconjugate vaccine production. Regardless of which OTase is employed, biological conjugations in any Gram-negative bacterium rely on three components: a genetic locus or loci that encode(s) for the polysaccharide biosynthesis proteins, a carrier protein to be glycosylated, and an OTase to transfer the desired carbohydrate to the carrier protein. While these three components are required, they do not necessarily need to be on three separate plasmids.
Recently, a third class of O-linking OTase was employed for bioconjugate vaccine production (Harding C M, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891). Much like the only other known O-linking OTases, PilO and PglL, this third class of OTase, termed PglS, naturally glycosylates a pilin like protein, ComP (Schulz B L, et al. (2013) Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates. PLoS One 8(5):e62768). A follow up study demonstrated that PglS was indeed a pilin specific OTase, likely, only glycosylating ComP as no other glycoproteins were identified using a comprehensive glycoprotein screening approach (Harding C M, et al. (2015) Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins. Mol Microbiol 96(5):1023-1041). Originally characterized as a PglL ortholog from the environmental bacterium Acinetobacter baylyi strain ADP1, PglS is in fact phylogenetically distinct from PglL proteins. Strains of Acinetobacter that encode for a PglS protein also encode for a PglL protein, which has been shown to act as the general OTase glycosylating at least seven membrane-associated proteins in a manner similar to Neisseria species (Iwashkiw J A, et al. (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 8(6):e1002758). In addition, some strains of Acinetobacter also encode for PilO OTases, making Acinetobacter the only known genera of bacteria carrying genes for all three O-OTase families (PilO, PglL, and PglS) (Harding C M, et al. (2015) Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins. Mol Microbiol 96(5):1023-1041; Iwashkiw J A, et al. (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 8(6): e1002758).
Aside from phylogenetic differences, PglS glycosylates its cognate pilin at a unique serine site that is not conserved when compared to the site of glycosylation for PilE (the pilin target of PglL) or PilA (the pilin target for PilO), and is not contained within an LCR (Harding C M, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891). However, the most notable difference lies in the polysaccharide substrates PglS transfers. PglS is the only known OTase, both N- or O-linking, capable of transferring polysaccharides with glucose at the reducing end. Many pathogens, like Streptococcus pneumoniae (Geno K A, et al. (2015) Pneumococcal Capsules and Their Types: Past, Present, and Future. Clin Microbiol Rev 28(3):871-899), Group B Streptococcus (Carboni F, et al. (2017) Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide. Proc Natl Acad Sci USA 114(19):5017-5022), and Klebsiella pneumoniae (Pan Y J, et al. (2015) Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp. Sci Rep 5:15573), produce capsules that contain polysaccharides with glucose at the reducing and are thus potential targets for PglS dependent bioconjugate vaccine development. Indeed, PglS was used to generate a polyvalent pneumococcal bioconjugate vaccine against serotypes 8, 9V, and 14 (all contain glucose at the reducing end) using the natural acceptor, ComP, as a carrier protein. In addition, a fragment of ComP lacking its first 28 amino acids was also able to serve as a glycotag when translationally fused to the C-terminus of exotoxin A of P. aeruginosa paving the way for incorporation of more conventional vaccine carriers in the PglS bioconjugation system (Harding C M, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891).
This disclosure provides for a bioconjugate comprising an oligo- or polysaccharide covalently linked to a fusion protein: wherein the fusion protein comprises a ComP protein (ComP) glycosylation tag; wherein the ComP glycosylation tag comprises both a cysteine residue corresponding to the conserved cysteine residue at position 71 of SEQ ID NO: 2 (ComP110264: ENV58402.1) and a cysteine residue corresponding to the conserved cysteine residue at position 93 of SEQ ID NO: 2 or both a cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) and a cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1; and wherein the fusion protein is glycosylated with the oligo- or polysaccharide on the ComP glycosylation tag at a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 2 or position 84 of SEQ ID NO: 1. In certain embodiments, the ComP glycosylation tag does not comprise a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, the fusion protein of the bioconjugate does not comprise, in relationship to the ComP glycosylation tag, a methionine residue at a position that would correspond to or correspond about to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, the bioconjugate is a conjugate vaccine.
In certain aspects of this disclosure, the ComP glycosylation tag comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 32 [C1]; SEQ ID NO: 33 [D1]; SEQ ID NO: 34 [E1]; SEQ ID NO: 41 [E2]; SEQ ID NO: 42 [F2]; SEQ ID NO: 43 [G2]; SEQ ID NO: 44 [H2]; SEQ ID NO: 45 [A3]; SEQ ID NO: 46 [B3]; SEQ ID NO: 47 [C3]; SEQ ID NO: 55 [D4]; SEQ ID NO: 56 [E4]; SEQ ID NO: 57 [F4]; SEQ ID NO: 58 [G4]; SEQ ID NO: 59 [A5]; SEQ ID NO: 60 [B5]; SEQ ID NO: 61 [D5]; SEQ ID NO: 62 [E5]; SEQ ID NO: 63 [F5]; SEQ ID NO: 72 [H6]; SEQ ID NO: 73 [B7]; SEQ ID NO: 74 [C7]; SEQ ID NO: 75 [D7]; SEQ ID NO: 76 [E7]; SEQ ID NO: 77 [F7]; SEQ ID NO: 78 [A8]; SEQ ID NO: 79 [B8]; SEQ ID NO: 92 [A10]; SEQ ID NO: 93 [B10]; SEQ ID NO: 94 [C10]; SEQ ID NO: 95 [D10]; SEQ ID NO: 96 [F10]; SEQ ID NO: 97 [G10]; SEQ ID NO: 98 [H10]; SEQ ID NO: 99 [A11]; SEQ ID NO: 100 [B11]; and SEQ ID NO: 101 [C11], or a variant thereof having one, two, three, four, five, six, or seven amino acid substitutions, additions, and/or deletions, wherein the variant maintains both a cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) and a cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1; and wherein the variant maintains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1.
This disclosure provides for a ComP glycosylation tag comprising an isolated fragment of a ComP protein, wherein the fragment comprises a serine residue corresponding to the conserved serine residue at position 84 in SEQ ID NO: 1 (ComPADP1: AAC45886.1) and both a cysteine residue corresponding to the conserved cysteine residue at position 71 of SEQ ID NO: 2 (ComP110264: ENV58402.1) and a cysteine residue corresponding to the conserved cysteine residue at position 93 of SEQ ID NO: 2 or both a cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) and a cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1. In certain embodiments, the ComP glycosylation tag of claim 41, wherein the ComP glycosylation tag does not comprise a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, the ComP glycosylation tag of claim 42, wherein the amino acid sequence of the ComP glycosylation tag does not extend in the C-terminus direction beyond the amino acid residue corresponding to position 103 of SEQ ID NO: 2 (ComP110264: ENV58402.1).
Provided for herein is a fusion protein comprising a ComP glycosylation tag of this disclosure.
Also provided for herein is a method of in vivo conjugation of an oligo- or polysaccharide to an acceptor polypeptide, the method comprising covalently linking the oligo- or polysaccharide to the acceptor polypeptide with a PglS oligosaccharyltransferase (OTase), wherein the acceptor polypeptide comprises the ComP glycosylation tag of this disclosure; optionally, wherein the ComP glycosylation tag is linked to a heterologous carrier protein.
Also provided for herein is a host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a PglS OTase; and (3) an acceptor polypeptide comprising the ComP glycosylation tag of this disclosure.
Also provided for herein is an isolated nucleic acid encoding the ComP glycosylation tag and/or the fusion protein of this disclosure and a host cell comprising said isolated nucleic acid.
Also provided for herein is a composition comprising the conjugate vaccine or the fusion protein of thisi disclosure, and an adjuvant.
A method of inducing a host immune response against a bacterial pathogen comprising administering to a subject in need of the immune response an effective amount of the conjugate vaccine, the fusion protein, or a composition of this disclosure.
Also provided for herein is a method of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof the conjugate vaccine, the fusion protein, or a composition of this disclosure.
Also provided for herein is a method of producing a pneumococcal conjugate vaccine against pneumococcal infection comprising isolating the bioconjugate or glycosylated fusion protein of this disclosure and combining the isolated conjugate vaccine or isolated glycosylated fusion protein with an adjuvant.
To the extent necessary to provide descriptive support, the subject matter and/or text of the appended claims is incorporated herein by reference in their entirety.
It will be understood by all readers of this written description that the exemplary aspects and embodiments described and claimed herein may be suitably practiced in the absence of any recited feature, element or step that is, or is not, specifically disclosed herein.
It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a polysaccharide,” is understood to represent one or more polysaccharides. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
It is understood that wherever aspects are described herein with the language “comprising” or “comprises” otherwise analogous aspects described in terms of “consisting of,” “consists of,” “consisting essentially of,” and/or “consists essentially of,” and the like are also provided.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related.
Numeric ranges are inclusive of the numbers defining the range. Even when not explicitly identified by “and any range in between,” or the like, where a list of values is recited, e.g., 1, 2, 3, or 4, unless otherwise stated, the disclosure specifically includes any range in between the values, e.g., 1 to 3, 1 to 4, 2 to 4, etc.
The headings provided herein are solely for ease of reference and are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole.
As used herein, the term “non-naturally occurring” substance, composition, entity, and/or any combination of substances, compositions, or entities, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the substance, composition, entity, and/or any combination of substances, compositions, or entities that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”
As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
A “protein” as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein.
By an “isolated” polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.
As used herein, the term “non-naturally occurring” polypeptide, or any grammatical variants thereof, is a conditional term that explicitly excludes, but only excludes, those forms of the polypeptide that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”
Disclosed herein are certain binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless specifically referring to full-sized antibodies such as naturally-occurring antibodies, the term “binding molecule” encompasses full-sized antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., naturally-occurring antibody or immunoglobulin molecules or engineered antibody molecules or fragments that bind antigen in a manner similar to antibody molecules.
As used herein, the term “binding molecule” refers in its broadest sense to a molecule that specifically binds an antigenic determinant. As described further herein, a binding molecule can comprise one of more “binding domains.” As used herein, a “binding domain” is a two- or three-dimensional polypeptide structure that cans specifically bind a given antigenic determinant, or epitope. A non-limiting example of a binding molecule is an antibody or fragment thereof that comprises a binding domain that specifically binds an antigenic determinant or epitope. Another example of a binding molecule is a bispecific antibody comprising a first binding domain binding to a first epitope, and a second binding domain binding to a second epitope.
The terms “antibody” and “immunoglobulin” can be used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof as disclosed herein comprises at least the variable domain of a heavy chain and at least the variable domains of a heavy chain and a light chain. Basic immunoglobulin structures in vertebrate systems are relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).
Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, e.g., in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules encompassed by this disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
By “specifically binds,” it is meant that a binding molecule, e.g., an antibody or fragment, variant, or derivative thereof binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, a binding molecule is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” is used herein to qualify the relative affinity by which a certain binding molecule binds to a certain epitope. For example, binding molecule “A” can be deemed to have a higher specificity for a given epitope than binding molecule “B,” or binding molecule “A” can be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”
The term “bispecific antibody” as used herein refers to an antibody that has binding sites for two different antigens within a single antibody molecule. It will be appreciated that other molecules in addition to the canonical antibody structure can be constructed with two binding specificities. It will further be appreciated that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Bispecific antibodies can also be constructed by recombinant means. (Strohlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9 (2010)). A bispecific antibody can also be a diabody.
The term “polynucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide can comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term “nucleic acid” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated” nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, polynucleotide or a nucleic acid can be or can include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.
As used herein, a “non-naturally occurring” polynucleotide, or any grammatical variants thereof, is a conditional definition that explicitly excludes, but only excludes, those forms of the polynucleotide that are well-understood by persons of ordinary skill in the art as being “naturally-occurring,” or that are, or that might be at any time, determined or interpreted by a judge or an administrative or judicial body to be, “naturally-occurring.”
In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, a polynucleotide can be RNA.
A “vector” is nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker gene and other genetic elements known in the art.
A “transformed” cell, or a “host” cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses those techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed cell or a host cell can be a bacterial cell or a eukaryotic cell.
The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into polypeptide(s). If the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide that is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
As used herein the terms “treat,” “treatment,” or “treatment of” (e.g., in the phrase “treating a subject”) refers to reducing the potential for disease pathology, reducing the occurrence of disease symptoms, e.g., to an extent that the subject has a longer survival rate or reduced discomfort. For example, treating can refer to the ability of a therapy when administered to a subject, to reduce disease symptoms, signs, or causes. Treating also refers to mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.
By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals, including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.
The term “pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.
An “effective amount” of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
Conjugate vaccines, consisting of a polysaccharide linked to a protein, are lifesaving prophylactics. Traditionally, conjugate vaccines are manufactured using chemical methodologies. However, in vivo bacterial conjugations have emerged as manufacturing alternatives. In vivo conjugation (bioconjugation) is reliant upon an oligosaccharyltransferase to attach polysaccharides to proteins. Currently, the oligosaccharyltransferases employed for bioconjugations are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end. This limitation has enormous implications as ˜75% of Streptococcus pneumoniae capsules contain glucose as the reducing end sugar. Disclosed herein is the use of an O-linked oligosaccharyltransferase to generate the first ever polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. Pneumococcal bioconjugates were immunogenic, protective, and rapidly produced with recombinant techniques. Certain aspects disclosed herein provide for the engineering, characterization, and immunological responses of a polyvalent pneumococcal bioconjugate vaccine using the natural acceptor protein ComP as a vaccine carrier as well as a monovalent pneumococcal bioconjugate vaccine using a conventional vaccine carrier; e.g., in certain aspects, containing the Pseudomonas aeruginosa exotoxin A protein. This establishes a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.
Even with the introduction and implementation of pneumococcal conjugate vaccines over the last two decades, ˜1.5 million deaths are still attributed to S. pneumoniae each year. This is due in part to the 90+ serotypes of S. pneumoniae and the complex manufacturing methods required to synthesize pneumococcal conjugate vaccines. Together these factors hinder global distribution and development of broader, more protective variations of the vaccines. To expedite development and lower manufacturing costs, disclosed herein is a platform for developing conjugate vaccines, for example pneumococcal conjugate vaccines, using in vivo conjugation. This streamlined process has the potential to complement existing manufacturing pipelines or completely bypass the dependency on chemical conjugation methodologies, enabling the production of a more comprehensive conjugate vaccines.
Traditional, chemical conjugate vaccine synthesis is considered complex, costly, and laborious (Frasch, C. E. Vaccine 27, 6468-6470 (2009)) however, in vivo conjugation has been thoroughly progressing as a viable biosynthetic alternative (Huttner, A. et al. Lancet Infect Dis 17, 528-537 (2017)). These strides are best highlighted by the successes of GlycoVaxyn, (now LimmaTech Biologics AG an independent company with direct ties to GlaxoSmithKline), a clinical stage biopharmaceutical company with multiple bioconjugate vaccines in various phases of clinical trials, one of which (Flexyn2a) has just completed a Phase 2b challenge study. Although GlycoVaxyn has been at the forefront of the in vivo conjugation revolution, the ability to glycosylate carrier/acceptor proteins with polysaccharides containing glucose (Glc) as the reducing end sugar has been elusive and, expectedly, has stymied the development of a pneumococcal bioconjugate vaccine.
The oligosaccharyltransferase PglS—previously referred to as PglL by Schulz et al. (PMID23658772) and PglLComP by Harding et al. 2015 (PMID 26727908)—was only recently characterized as a functional OTase (Schulz, B. L. et al. PLoS One 8, e62768 (2013)). Subsequent mass spectrometry studies on total glycopeptides demonstrated that PglS does not act as a general PglL-like OTase, glycosylating multiple periplasmic and outer membrane proteins (Harding, C. M. et al. Mol Microbiol 96, 1023-1041 (2015)). In fact, the genome of A. baylyi ADP1 encodes for two OTase, a PglL-like ortholog (UniProtKB/Swiss-Prot: Q6FFS6.1), which acts as the general OTase and PglS (UniProtKB/Swiss-Prot: Q6F7F9.1), which glycosylates a single protein, ComP (Harding, C. M. et al. Mol Microbiol 96, 1023-1041 (2015)).
ComP is orthologous to type IV pilin proteins, like PilA from Pseudomonas aeruginosa and PilE from Neisseria meningiditis, both of which are glycosylated by the OTases TfpO (Castric, P. Microbiology 141 (Pt 5), 1247-1254 (1995)) and PglL (Power, P. M. et al. Mol Microbiol 49, 833-847 (2003)), respectively. Although TfpO and PglL also glycosylate their cognate pilins at serine residues, the sites of glycosylation differ between each system. TfpO glycosylates its cognate pilin at a C-terminal serine residue (Comer, J. E., Marshall, M. A., Blanch, V. J., Deal, C. D. & Castric, P. Infect Immun 70, 2837-2845 (2002)), which is not present in ComP. PglL glycosylates PilE at an internal serine located at position 63 (Stimson, E. et al. Mol Microbiol 17, 1201-1214 (1995)). ComP also contains serine residues near position 63 and the surrounding residues show moderate conservation to PilE from N meningiditis. Comprehensive glycopeptide analysis, however, revealed this serine and the surrounding residues were not the site of glycosylation in ComP. PglS glycosylates ComP at a single serine residue located at position corresponding to the conserved serine at position 84 of ComPADP1: AAC4588631 (SEQ ID NO: 1) (also corresponding to the conserved serine at position 82 of ComP110264: ENV58402.1 (SEQ ID NO: 2)), which is a novel glycosylation site not previously found within the type IV pilin superfamily. The ability of PglS to transfer polysaccharides containing glucose as the reducing end sugar coupled with the identification of a novel site of glycosylation within the pilin superfamilies demonstrates that PglS is a functionally distinct OTase from PglL and TfpO.
PglS, but not PglB or PglL, transferred polysaccharides containing glucose at their reducing end to the acceptor protein ComP. Two classes of OTases, PglB and PglL, have previously been employed for in vivo conjugation (Feldman, M. F. et al. Proc Natl Acad Sci USA 102, 3016-3021 (2005); Faridmoayer, A., Fentabil, M. A., Mills, D. C., Klassen, J. S. & Feldman, M. F. J Bacteriol 189, 8088-8098 (2007)). PglB, the first OTase described, preferentially transfers glycans containing an acetamido-group at the C-2 position of the reducing end (i.e. N-acetylglucosamine), as it is believed to play a role in substrate recognition (Wacker, M. et al. Proc Natl Acad Sci USA 103, 7088-7093 (2006)). However, polysaccharides with galactose (Gal) at the reducing end, such as the S. enterica Typhimurium O antigen, can be transferred by an engineered PglB variant (Ihssen, J. et al. Open Biol 5, 140227 (2015)). The second described OTase, PglL from N meningiditis, has more relaxed substrate specificity than PglB, naturally transferring polysaccharides with an acetamido-group at the C-2 position as well as polysaccharides containing galactose (Gal) at the reducing end (Faridmoayer, A., Fentabil, M. A., Mills, D. C., Klassen, J. S. & Feldman, M. F. J Bacteriol 189, 8088-8098 (2007); Pan, C. et al. MBio 7 (2016)). However, there is no evidence available for PglB or PglL mediated transfer of polysaccharides containing glucose (Glc) at the reducing end, which is of particular interest given that the majority of pneumococcal CPSs contain glucose at the reducing end (Geno, K. A. et al. Clin Microbiol Rev 28, 871-899 (2015)). The ability of PglB and PglL to transfer the pneumococcal serotype 14 capsular polysaccharide (CPS14) to their cognate glycosylation targets, AcrA (Wacker, M. et al. Science 298, 1790-1793 (2002)) and DsbA (Vik, A. et al. Proc Natl Acad Sci USA 106, 4447-4452 (2009)), respectively, was tested. As seen in
Acinetobacter species have been describes as containing three O-linked OTases; a general PglL OTase responsible for glycosylating multiple proteins, and two pilin-specific OTases (Harding, C. M. Mol Microbiol 96, 1023-1041 (2015)). The first pilin-specific OTase is an ortholog of TfpO (also known as PilO) and is not employed for in vivo conjugation systems due to its inability to transfer polysaccharides with more than one repeating unit (Faridmoayer, A., Fentabil, M. A., Mills, D. C., Klassen J. S. & Feldman, M. F. J Bacteriol 189, 8088-8098 (2007)). The second pilin specific OTase, PglS glycosylates a single protein, the type IV pilin ComP28. A bioinformatic analysis indicated that PglS is the archetype of a distinct family of OTases. Given that PglS represents a new class of O-OTase, its ability to transfer pneumococcal CPS14 to its cognate acceptor protein, ComP (Harding, C. M. et al. Mol Microbiol 96, 1023-1041 (2015)) was tested. As seen in
There are more than 90 serotypes of S. pneumoniae (Geno, K. A. et al. Clin Microbiol Rev 28, 871-899 (2015)). Many increasingly prevalent serotypes, like serotypes 8, 22F, and 33F are not included in currently licensed vaccines. Therefore, the versatility was tested of PglS to generate a multivalent pneumococcal bioconjugate vaccine against two serotypes included in Prevnar 13 (serotype 9V and 14) and one serotype not included (serotype 8) (Package Insert-Prevnar 13 FDA, on the world wide web at fda.gov/downloads/BiologicsBloodVaccinesNaccines/ApprovedProducts/UCM201669.pdf)). Importantly, all of three of these capsular polysaccharides contain glucose as the reducing end sugar (Geno, K. A. et al. Clin Microbiol Rev 28, 871-899 (2015)). As seen in
Therefore, it was demonstrated that PglS can transfer S. pneumoniae polysaccharides to ComP, wherein PglB and PglL could not. Specifically, PglS is the only OTase in the known universe capable of transferring polysaccharides with glucose at the reducing end. In certain aspects, PglS can be used to transfer any lipid-linked oligosaccharide or polysaccharide (collectively referred to herein as “oligo- or polysaccharide”) containing glucose at the reducing end to ComP or a fusion protein containing a fragment of ComP.
PglS can transfer capsular polysaccharides of Klebsiella to ComP. Klebsiella pneumonia (K. pneumoniae), a Gram negative opportunistic human pathogen, produces a capsular polysaccharide known to be important for virulence. To date at least 79 antigenically distinct capsular polysaccharides have been described for Klebsiella species (Pan, Y. J. et al. Sci Rep 5, 15573 (2015)). Furthermore, K. pneumoniae is known to produce at least 59 of the 77 capsular polysaccharides, more than half of which contain glucose as the reducing end sugar (Pan, Y. J. et al. Sci Rep 5, 15573 (2015)). To determine if PglS could transfer K. pneumoniae capsular polysaccharides to ComP, the genes encoding for the proteins required for the synthesis of either the K1 or the K2 capsular polysaccharides were cloned into the IPTG inducible pBBR1MCS-2 vector (Kovach, M. E. et al. Gene 166, 175-176 (1995)). The K1 capsule gene locus was cloned from K. pneumoniae NTUH K-2044, a previously characterized K1 capsule producing strain (Wu, K. M. et al. J Bacteriol 191, 4492-4501 (2009)). The K2 capsule gene locus was cloned from K. pneumoniae 52.145, a previously characterized K2 capsule producing strain (Lery, L. M. et al. BMC Biol 12, 41 (2014)). The K1 or the K2 capsular polysaccharide expressing plasmids were then individually introduced into E. coli co-expressing PglS OTase and the acceptor protein ComP from a separate plasmid vector. To enhance expression of K1 and K2 specific polysaccharides, the K. pneumoniae transcriptional activator rmpA from K. pneumoniae NTUH K-2044 was subsequently cloned into pACT3 (Dykxhoorn, D. M., St Pierre, R. & Linn, T. Gene 177, 133-136 (1996)), a low copy, IPTG inducible vector as it has previously been characterized as a regulator of capsule in K. pneumoniae (Arakawa, Y. et al. Infect Immun 59, 2043-2050 (1991)); Yeh, K. M. et al. J Clin Microbiol 45, 466-471 (2007)). Introduction of the rmpA gene into E. coli strains co-expressing PglS and hexa-his tagged ComP variant and either the K1 or K2 capsular polysaccharides from K. pneumoniae, resulted robust expression and detection of higher molecular ComP bioconjugates as indicated by the typical ladder-like pattern of bands compatible with protein glycosylation when analyzed via western blotting (
PglS can transfer K. pneumoniae polysaccharides to ComP. Given that most K. pneumoniae capsular polysaccharides contain glucose as the reducing end sugar, the only other commercially licensed OTases (PglB and PglL) should be unable to generate conjugate vaccines using these polysaccharides. Moreover, co-expression of the transcriptional activator, RmpA, with the capsule gene cluster enhanced capsule expression to detectably levels. In certain aspects, the method for producing Klebsiella conjugates can be used to generate a pan Klebsiella conjugate vaccine encompassing all serotypes—including other species such as K. varricola, K michiganensis, and K. oxytoca.
Mass spectrometry and site directed mutagenesis confirm PglS is an O-linked OTase and reveal that ComP is glycosylated at a serine residue corresponding to position 84 of COMPADP1. N-glycosylation in bacteria generally occurs within the sequon D-X-N-S-T (SEQ ID NO: 21), where X is any amino acid but proline (Kowarik, M. et al. EMBO J 25, 1957-1966 (2006)). On the contrary, O-glycosylation does not seem to follow a defined sequon. Most 0-glycosylation events in bacterial proteins occur in regions of low complexity (LCR), rich in serine, alanine, and proline (Vik, A. et al. Proc Natl Acad Sci USA 106, 4447-4452 (2009)). Alternatively, some pilins are O-glycosylated at a C-terminal serine residue (Comer, J. E., Marshall, M. A., Blanch, V. J., Deal, C. D. & Castric, P. Infect Immun 70, 2837-2845 (2002)). ComP does not appear to have an obvious LCR or a C-terminal serine residue homologous to those found in other pilin like proteins and therefore mass spectrometry was employed to determine the site(s) of glycosylation. Purified CPS14-ComP bioconjugates were subjected to proteolytic digestion, ZIC-HILIC glycopeptide enrichment, and multiple MS analyses. As seen in
It was previously shown that Acinetobacter species predominantly glycosylate proteins at serine residues and thus it was hypothesized that either serine (S) 82 or 84—as numbered in SEQ ID NO: 1—was the site of glycosylation (Scott, N. E. et al. Mol Cell Proteomics 13, 2354-2370 (2014)). To determine which serine residue was the site of glycosylation, these serine residues were individually mutated to alanine (A) and the glycosylation status of both mutant proteins was analyzed. For this experiment, the biosynthetic locus for the C. jejuni heptasaccharide was employed as the donor glycan, as glycosylation is readily detectable with the hR6 anti-glycan antisera as well as by an increase in electrophoretic mobility (Schwarz, F. et al. Nat Chem Biol 6, 264-266 (2010)). As shown in
Bioinformatic features of ComP pilin orthologs. ComP was first described as a factor required for natural transformation in Acinetobacter baylyi ADP1 (Porstendorfer, D., Drotschmann, U. & Averhoff, B. Appl Environ Microbiol 63, 4150-4157 (1997)). In a subsequent study, it was demonstrated that ComP from A. baylyi ADP1 (herein referred to as ComPADP1) was glycosylated by a novel OTase, PglS, located immediately downstream of ComP, and not the general OTase PglL located elsewhere on the chromosome (Harding, C. M. et al. Mol Microbiol 96, 1023-1041 (2015)). The ComPADP1 protein (NCBI identifier AAC45886.1) belongs to a family of proteins called type IV pilins. Specifically, ComP shares homology to type IVa major pilins (Giltner, C. L., Nguyen, Y. & Burrows, L. L. Microbiol Mol Biol Rev 76, 740-772 (2012)). Type IVa pilins share high sequence homology at their N-terminus, which encode for the highly conserved leader sequence and N-terminal alpha helix; however, the C-terminus display remarkable divergences across genera and even within species (Giltner, C. L., Nguyen, Y. & Burrows, L. L. Microbiol Mol Biol Rev 76, 740-772 (2012)). To help differentiate ComP orthologs from other type IVa pilin proteins, such as, PilA from A. baumannii, P. aeruginosa, and Haemophilus influenzae as well as PilE from Neisseria species (Pelicic, V. Mol Microbiol 68, 827-837 (2008)), a BLASTp analysis was performed comparing the primary amino acid sequence of ComPADP1 against all proteins from bacteria in the Acinetobacter genus. Expectedly, many Acinetobacter type IVa pilin orthologs, including COMPADP1, share high homology at their N-termini; however, very few proteins display high sequence conservation across the entire amino acid sequence of ComP. At least six ComP orthologs (
Therefore, features common to ComP proteins are disclosed herein that identify ComP orthologs in different Acinetobacter species. ComP proteins can be differentiated from other pilins by the presence of the conserved glycosylated serine located at position 84 relative to the ADP1 ComP protein and the presence of a disulfide loop flanking the site of glycosylation. In addition, the presence of a pglS homolog immediately downstream of ComP is an indicator of ComP. Further to be classified as a PglS OTase protein rather than a PglL OTase protein, the OTase downstream of ComP must display higher sequence conservation with PglS (ACIAD3337) when compared to PglL (ACIAD0103) in A. baylyi ADP1. It is also evident to one of ordinary skill in the art that in any embodiment disclosed herein, a ComP protein comprises and is capable of being glycosylated on a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1).
ComP from A. soli CIP 110264 is glycosylated by PglS from A. baylyi ADP1. Given the presence of multiple ComP orthologs, whether PglS from A. baylyi ADP1 was able to glycosylate a divergent ComP protein was investigated. The ComP protein from A. soli CIP 110264 (ComP110264) is 71% identical at the amino acid level when compared to ComPADP1. However, consistent with the features above, ComP110264 contains the predicted disulfide bridge between the predicted alpha-beta loop and the second beta strand as well as the conserved serine located at position 84 relative to ComPADP1. Moreover, a PglS ortholog can be found immediately downstream of ComP110264. To determine whether PglS from A. baylyi ADP1 (PglSADP1) could glycosylate ComP110264, PglSADP1 was cloned into pACT3 and ComP110264 into pEXT20 (Dykxhoorn, D. M., St Pierre, R. & Linn, T. Gene 177, 133-136 (1996)) and these plasmids were introduced into E. coli expressing the serotype 8 capsular polysaccharide (CPS8) from S. pneumoniae. Further, the converse experiment was performed by cloning and expressing PglS from A. soli CIP 110264 (PglS110264) with ComPADP1. As seen in
Generation of a soluble, periplasmic fusion protein capable of being glycosylated by PglS. All members of type IVa pilin family are considered membrane proteins as part of their N-terminal alpha helix is embedded within the inner membrane (Giltner, C. L., Nguyen, Y. & Burrows, L. L. Microbiol Mol Biol Rev 76, 740-772 (2012)). Therefore, in order to generate soluble variants of ComP that are able to be glycosylated by PglS, translational fusions were constructed of truncated ComP fragment proteins onto three different carrier proteins. The carrier proteins, DsbA and MalE (also known as maltose binding protein—MBP) from E. coli, were selected as suitable carriers as both have been previously shown to facilitate periplasmic localization and solubility of acceptor proteins fused at their C-termini (Malik, A. Biotech 6, 44 (2016)). Exotoxin A from Pseudomonas aeruginosa (EPA) was also selected as it has been previously shown to act as an immunogenic carrier protein in other conjugate vaccine formulations (Ravenscroft, N. et al. Glycobiology 26, 51-62 (2016)). Fusion proteins consisted of a leader sequence, carrier protein, a short linker peptide, a ComP variant without the first 28 amino acids, and a hexa-histidine tag. The first 28 amino acids of ComPADP1 and ComP110264 were removed as these amino acids contain the leader sequence as well as the hydrophobic region of the N-terminal alpha helix predicted to be embedded into the inner membrane. Fusion constructs were then introduced into E. coli expressing the pneumococcal serotype 8 capsular polysaccharide (CPS8) and either pACT3 alone or pACT3 carrying pglS110264 or pglSADP1. As seen in
Immunization with a glycosylated ComP bioconjugate elicits an immune response. T-cell dependent immune responses to conjugate vaccines are characterized by the secretion of high affinity IgG1 antibody (Avci, F. Y., Li, X., Tsuji, M. & Kasper, D. L. Nat Med 17, 1602-1609 (2011)). The immunogenicity of a CPS14-ComP bioconjugate in a murine vaccination model was evaluated. As seen in
Next, a second vaccination trial was performed comparing the immunogenicity of a trivalent CPS8-, CPS9V-, and CPS14-ComP bioconjugate to the current standard of care, PREVNAR 13®. Serotypes 9V and 14 are included in PREVNAR 13® and elevated IgG titers could be seen in PREVNAR 13® immunized mice against these two serotypes (
Provide herein are bioconjugates comprising an oligo- or polysaccharide linked to a fusion protein. In certain embodiments, the oligo- or polysaccharide is covalently linked to the fusion protein. In certain embodiments, the fusion protein comprises a ComP protein (ComP). In certain other embodiments, the fusion protein comprises a glycosylation tag of a ComP protein (as described in detail elsewhere herein).
As disclosed herein, it has been discovered that ComP is glycosylated on a serine (S) residue. This serine residue is conserved in ComP proteins and corresponds to position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1). This serine residue also corresponds to position 82 of SEQ ID NO: 2 (ComP110264: ENV58402.1) (
In certain embodiments of a bioconjugate of this disclosure, the oligo- or polysaccharide comprises a glucose at its reducing end.
One of ordinary skill in the art would recognize that by aligning ComP sequences with SEQ ID NO: 1, (e.g., either full sequences or partial sequences) the conserved serine residue of a non-SEQ ID NO: 1 ComP protein disclosed herein, corresponding to the serine residue at position 84 of SEQ ID NO: 1, can be identified. Further, one of ordinary skill in the art would recognize that by aligning ComP sequences with SEQ ID NO: 1, other residues, regions, and/or features corresponding to residues, regions, and/or features of SEQ ID NO: 1 as referred to herein can be identified in the non-SEQ ID NO: 1 ComP sequence and referenced in relation to SEQ ID NO:1. And, while reference is generally made herein to SEQ ID NO: 1, by analogy, reference can similarly be made to any residue, region, feature and the like of any ComP sequence disclosed herein, for example, in reference to SEQ ID NO: 2.
A ComP protein is a protein that has been identified as ComP protein consistent with the description provided herein. For example, representative examples of ComP proteins include, but are not limited to: AAC45886.1 ComP [Acinetobacter sp. ADP1]; ENV58402.1 hypothetical protein F951_00736 [Acinetobacter soli CIP 110264]; APV36638.1 competence protein [Acinetobacter soli GFJ-2]; PKD82822.1 competence protein [Acinetobacter radioresistens 50v1]; SNX44537.1 type IV pilus assembly protein PilA [Acinetobacter puyangensis ANC 4466]; and OAL75955.1 competence protein [Acinetobacter sp. SFC]. In certain aspects, a ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 (ComPADP1: AAC45886.1) and contains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1). SEQ ID NO: 1 comprises a leader sequence of 28 amino acids. In certain aspects, a ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 (ComPΔ28ADP1), SEQ ID NO: 8 (ComPΔ28110264), SEQ ID NO: 9 (ComPΔ28GFJ-3), SEQ ID NO: 10 (ComPΔ28P50v1), SEQ ID NO: 11 (ComPΔ284466), or SEQ ID NO: 12 (ComPΔ28SFC) that do not include the 28 amino acid leader sequence but do contain a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1). In certain aspects, a ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 (ComPΔ28ADP1) that does not include the 28 amino acid leader sequence but does contain a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1). In certain aspects, the ComP protein comprises SEQ ID NO: 7 (ComPΔ28ADP1), SEQ ID NO: 8 (ComPΔ28110264), SEQ ID NO: 9 (ComPΔ28GFJ-2), SEQ ID NO: 10 (ComPΔ28P50v1), SEQ ID NO: 11 (ComPΔ284466), or SEQ ID NO: 12 (ComPΔ28SFC). In certain aspects, the ComP protein is SEQ ID NO: 1 (ComPADP1: AAC45886.1), SEQ ID NO: 2 (ComP110264: ENV58402.1), SEQ ID NO: 3 (ComPGFJ-2: APV36638.1), SEQ ID NO: 4 (ComP50v1: PKD82822.1), SEQ ID NO: 5 (ComP4466: SNX44537.1), or SEQ ID NO: 6 (ComPSFC: OAL75955.1).
In certain aspects, the bioconjugate is produced in vivo in a host cell such as by any of the methods of production disclosed herein. In certain aspects, the bioconjugate is produced in a bacterial cell, a fungal cell, a yeast cell, an avian cell, an algal cell, an insect cell, or a mammalian cell. In certain aspects, the bioconjugate is produced in a cell free system. Examples of the use of a cell free system utilizing OTases other than PglS can be found in WO2013/067523A1, which in incorporated herein by reference.
It has been discovered that a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1) can have an inhibitory effect on glycosylation when present in a ComP glycosylation tag even though the full length ComP protein comprising this methionine residue is glycosylated. Thus, in certain embodiments, the ComP glycosylation tag of this disclosure does not comprise a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). For example, in certain embodiments, such methionine residue in a ComP amino acid sequence is substituted with another amino acid that does not exhibit an inhibitory effect or is deleted from the ComP glycosylation tag amino acid sequence. In certain embodiments, the amino acid sequence of the ComP glycosylation tag does not extend in the C-terminus direction beyond the amino acid residue corresponding to position 103 of SEQ ID NO: 2 (ComP110264: ENV58402.1). For example, in certain embodiments, the amino acid sequence of the ComP glycosylation tag ends with the residue corresponding to position 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, or 103 of SEQ ID NO: 2 (ComP110264: ENV58402.1). One of ordinary skill in the art would recognize that a fusion protein comprising a ComP glycosylation tag likewise would not comprise a methionine residue at a position corresponding to or corresponding about to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1) in relation to the ComP glycosylation tag, even if the methionine residue is attributed to a sequence of the fusion protein not as belonging to the ComP glycosylation tag sequence. For example, in certain embodiments, the fusion protein of the bioconjugate does not comprise, in relationship to the ComP glycosylation tag, a methionine residue at a position that would correspond to or correspond about to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, the fusion protein of the bioconjugate does not comprise, in relationship to the ComP glycosylation tag, a methionine residue at a position that would correspond to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1).
A ComP glycosylation tag of the current disclosure is generally not a full length ComP protein. In certain embodiments of any ComP glycosylation tag described herein, the ComP glycosylation tag has a length of between 18 and 50 amino acids in length, for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In certain embodiments, the glycosylation tag has length of between 21 and 45 amino acids in length. In certain embodiments, the glycosylation tag has a length of between 23 and 45 amino acids in length.
The ComP glycosylation tag of the current disclosure can be a fragment, a variant, or a variant fragment of a ComP protein as described anywhere herein. In certain embodiments, the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 (ComPΔ28ADP1), SEQ ID NO: 8 (ComPΔ28116264), SEQ ID NO: 9 (ComPΔ28GFJ-2), SEQ ID NO: 10 (ComPΔ28P50v1), SEQ ID NO: 11 (ComPΔ284466), or SEQ ID NO: 12 (ComPΔ28SFC). For example, in certain embodiments, the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 (ComPΔ28ADP1) or SEQ ID NO: 8 (ComPΔ281106264). In certain embodiments, the ComP protein comprises SEQ ID NO: 7 (ComPΔ28ADP1), SEQ ID NO: 8 (ComPΔ28110264), SEQ ID NO: 9 (ComPΔ28GFJ-2), SEQ ID NO: 10 (ComPΔ28P50v1), SEQ ID NO: 11 (ComPΔ284466), or SEQ ID NO: 12 (ComPΔ28SFC). Further, in certain embodiments, the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 (ComPADP1: AAC45886.1), SEQ ID NO: 2 (ComP110264: ENV58402.1), SEQ ID NO: 3 (ComPGFJ-2: APV36638.1), SEQ ID NO: 4 (Comp50v1: PKD82822.1), SEQ ID NO: 5 (ComP4466: SNX44537.1), or SEQ ID NO: 6 (ComPSFC: OAL75955.1). For example, in certain embodiments, the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1 (ComPADP1: AAC45886.1) or SEQ ID NO: 2 (ComP110264: ENV58402.1). Further, in certain embodiments, the ComP protein comprises SEQ ID NO: 1 (ComPADP1: AAC45886.1), SEQ ID NO: 2 (ComP110264: ENV58402.1), SEQ ID NO: 3 (ComPGFJ-2: APV36638.1), SEQ ID NO: 4 (Comp50v1: PKD82822.1), SEQ ID NO: 5 (ComP4466: SNX44537.1), or SEQ ID NO: 6 (ComPSFC: OAL75955.1).
In certain embodiments, a ComP glycosylation tag of the current disclosure can be defined as comprising or consisting of the amino acid consensus sequence of SEQ ID NO: 27:
wherein: X1 is V, A, or no amino acid;
In certain embodiments, a ComP glycosylation tag comprises or consists of a fragment of the amino acid consensus sequence of SEQ ID NO: 27, wherein the fragment retains the cysteine residue at position 13 of SEQ ID NO: 27, the cysteine residue at position 35 of SEQ ID NO: 27, and the serine residue at position 24 of SEQ ID NO: 27. In certain embodiments, a ComP glycosylation tag comprises or consists of a variant of the amino acid consensus sequence of SEQ ID NO: 27 or a fragment thereof, having one, two, three, four, five, six, or seven amino acid substitutions, additions, and/or deletions, however, wherein the variant maintains the cysteine residue at position 13 of SEQ ID NO: 27, the cysteine residue at position 35 of SEQ ID NO: 27, and the serine residue at position 24 of SEQ ID NO: 27. In certain embodiments, the amino acid substitution is a conservative amino acid substitution. As disclosed herein, in certain embodiments, a ComP glycosylation tag comprising SEQ ID NO: 27 does not comprise a methionine residue in a position corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). Further, in certain embodiments, the amino acid sequence of a ComP glycosylation tag comprising SEQ ID NO: 27 does not extend in the C-terminus direction beyond the amino acid residue corresponding to position 44 of SEQ ID NO: 27. In certain embodiments, a ComP glycosylation tag comprising or consisting of the amino acid consensus sequence of SEQ ID NO: 27 or fragment and/or variant thereof is not more than 25, 30, 40, 45, or 50 amino acids in length.
In certain embodiments, a ComP glycosylation tag of the current disclosure can be defined as comprising or consisting of the amino acid consensus sequence of SEQ ID NO: 28:
wherein:
In certain embodiments, a ComP glycosylation tag comprises or consists of a variant of the amino acid consensus sequence of SEQ ID NO: 28 having one, two, three, four, five, six, or seven amino acid substitutions, additions, and/or deletions, however, wherein the variant maintains the cysteine residue at position 1 of SEQ ID NO: 28, the cysteine residue at position 23 of SEQ ID NO: 28, and the serine residue at position 12 of SEQ ID NO: 28. In certain embodiments, the amino acid substitution is a conservative amino acid substitution.
In certain embodiments, a ComP glycosylation tag comprising SEQ ID NO: 28 does not comprise a methionine residue in a position corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). Further, in certain embodiments, the amino acid sequence of a ComP glycosylation tag comprising SEQ ID NO: 28 does not extend in the C-terminus direction beyond the amino acid residue corresponding to position 103 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, a ComP glycosylation tag comprising the amino acid consensus sequence of SEQ ID NO: 28 or variant thereof is not more than 25, 30, 40, 45, or 50 amino acids in length.
In certain embodiments, the ComP glycosylation tag comprises or consists of a variant thereof having one, two, three, four, five, six, or seven amino acid substitutions, additions, and/or deletions of an amino acid sequence selected from the group consisting of: SEQ ID NO: 32 [C1]; SEQ ID NO: 33 [D1]; SEQ ID NO: 34 [E1]; SEQ ID NO: 41 [E2]; SEQ ID NO: 42 [F2]; SEQ ID NO: 43 [G2]; SEQ ID NO: 44 [H2]; SEQ ID NO: 45 [A3]; SEQ ID NO: 46 [B3]; SEQ ID NO: 47 [C3]; SEQ ID NO: 55 [D4]; SEQ ID NO: 56 [E4]; SEQ ID NO: 57 [F4]; SEQ ID NO: 58 [G4]; SEQ ID NO: 59 [A5]; SEQ ID NO: 60 [B5]; SEQ ID NO: 61 [D5]; SEQ ID NO: 62 [E5]; SEQ ID NO: 63 [F5]; SEQ ID NO: 72 [H6]; SEQ ID NO: 73 [B7]; SEQ ID NO: 74 [C7]; SEQ ID NO: 75 [D7]; SEQ ID NO: 76 [E7]; SEQ ID NO: 77 [F7]; SEQ ID NO: 78 [A8]; SEQ ID NO: 79 [B8]; SEQ ID NO: 92 [A10]; SEQ ID NO: 93 [B10]; SEQ ID NO: 94 [C10]; SEQ ID NO: 95 [D10]; SEQ ID NO: 96 [F10]; SEQ ID NO: 97 [G10]; SEQ ID NO: 98 [H10]; SEQ ID NO: 99 [A11]; SEQ ID NO: 100 [B11]; and SEQ ID NO: 101 [C11], wherein the variant maintains both a cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) and a cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1 and the variant maintains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1. In certain embodiments, the amino acid substitution is a conservative amino acid substitution. Further, in certain embodiments, the ComP glycosylation tag comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 32 [C1]; SEQ ID NO: 33 [D1]; SEQ ID NO: 34 [E1]; SEQ ID NO: 41 [E2]; SEQ ID NO: 42 [F2]; SEQ ID NO: 43 [G2]; SEQ ID NO: 44 [H2]; SEQ ID NO: 45 [A3]; SEQ ID NO: 46 [B3]; SEQ ID NO: 47 [C3]; SEQ ID NO: 55 [D4]; SEQ ID NO: 56 [E4]; SEQ ID NO: 57 [F4]; SEQ ID NO: 58 [G4]; SEQ ID NO: 59 [A5]; SEQ ID NO: 60 [B5]; SEQ ID NO: 61 [D5]; SEQ ID NO: 62 [E5]; SEQ ID NO: 63 [F5]; SEQ ID NO: 72 [H6]; SEQ ID NO: 73 [B7]; SEQ ID NO: 74 [C7]; SEQ ID NO: 75 [D7]; SEQ ID NO: 76 [E7]; SEQ ID NO: 77 [F7]; SEQ ID NO: 78 [A8]; SEQ ID NO: 79 [B8]; SEQ ID NO: 92 [A10]; SEQ ID NO: 93 [B10]; SEQ ID NO: 94 [C10]; SEQ ID NO: 95 [D10]; SEQ ID NO: 96 [F10]; SEQ ID NO: 97 [G10]; SEQ ID NO: 98 [H10]; SEQ ID NO: 99 [A11]; SEQ ID NO: 100 [B11]; and SEQ ID NO: 101 [C11]. In certain embodiments, such a ComP glycosylation tag comprising one of the above sequences or variants thereof does not comprise a methionine residue in a position corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). Further, in certain embodiments, the amino acid sequence of such a ComP glycosylation tag comprising one of the above sequences or variants thereof does not extend in the C-terminus direction beyond the amino acid residue corresponding to position 103 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, a ComP glycosylation tag comprising an amino acid sequence and/or variant thereof listed above is not more than 25, 30, 40, 45, or 50 amino acids in length. In certain embodiments, a ComP glycosylation tag consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 32 [C1]; SEQ ID NO: 33 [D1]; SEQ ID NO: 34 [E1]; SEQ ID NO: 41 [E2]; SEQ ID NO: 42 [F2]; SEQ ID NO: 43 [G2]; SEQ ID NO: 44 [H2]; SEQ ID NO: 45 [A3]; SEQ ID NO: 46 [B3]; SEQ ID NO: 47 [C3]; SEQ ID NO: 55 [D4]; SEQ ID NO: 56 [E4]; SEQ ID NO: 57 [F4]; SEQ ID NO: 58 [G4]; SEQ ID NO: 59 [A5]; SEQ ID NO: 60 [B5]; SEQ ID NO: 61 [D5]; SEQ ID NO: 62 [E5]; SEQ ID NO: 63 [F5]; SEQ ID NO: 72 [H6]; SEQ ID NO: 73 [B7]; SEQ ID NO: 74 [C7]; SEQ ID NO: 75 [D7]; SEQ ID NO: 76 [E7]; SEQ ID NO: 77 [F7]; SEQ ID NO: 78 [A8]; SEQ ID NO: 79 [B8]; SEQ ID NO: 92 [A10]; SEQ ID NO: 93 [B10]; SEQ ID NO: 94 [C10]; SEQ ID NO: 95 [D10]; SEQ ID NO: 96 [F10]; SEQ ID NO: 97 [G10]; SEQ ID NO: 98 [H10]; SEQ ID NO: 99 [A11]; SEQ ID NO: 100 [B11]; and SEQ ID NO: 101 [C11].
In certain embodiments, the oligo- or polysaccharide for conjugation to the glycosylation tag, fusion protein, and/or bioconjugate is produced by a bacteria from the genus Streptococcus. For example, in certain embodiments, the polysaccharide is a S. pneumoniae, S. agalactiae, or S. suis capsular polysaccharide. Further, in certain embodiments, the capsular polysaccharide is CPS14, CPS8, CPS9V, or CPS15b. In certain other embodiments, the oligo- or polysaccharide is produced by a bacteria from the genus Klebsiella. For example, in certain embodiments, the polysaccharide is a Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca capsular polysaccharide. In certain embodiments, the polysaccharide is a Klebsiella pneumoniae capsular polysaccharide. Further, in certain embodiments, the polysaccharide is a serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae.
In certain embodiments, the bioconjugate is produced in vivo. For example, in certain embodiments, the bioconjugate is produced in a bacterial cell.
As the bioconjugate comprises an oligo- or polysaccharide covalently linked to a fusion protein, in certain applications, it may be advantageous to form a fusion protein with a carrier protein or fragment thereof. In certain embodiments, the carrier protein is one recognized in the art as useful in producing conjugate vaccines. In certain embodiments, when a ComP glycosylation tag fragment is fused to a carrier protein or fragment thereof, the glycosylation tag fragment and thus the fusion protein, can be glycosylated at the conserved serine residue described elsewhere herein. In certain embodiments, the fusion protein comprises a carrier protein selected from the group consisting of diphtheria toxoid CRM197, tetanus toxoid, Pseudomonas aeruginosa Exotoxin A (EPA), tetanus toxin C fragment, cholera toxin B subunit, Haemophilus influenza protein D, or a fragment thereof. In certain embodiments, the carrier protein or fragment thereof is linked to the ComP glycosylation tag via an amino acid linker, for example (GGGS). (SEQ ID NO: 23), wherein n is at least one or AAA (SEQ ID NO: 24). In order to increase the potential immunogenicity of a ComP fusion protein, it may be advantageous to include more than one glycosylation tag. Thus, in certain embodiments, the fusion protein comprise two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more ComP glycosylation tags. In certain embodiments, the fusion protein comprises any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 to any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 ComP glycosylation tags. In certain embodiments, multiple glycosylation tags are arranged in tandem to one another in the fusion protein. In certain embodiments, multiple glycosylation tags are arranged apart from one another in the fusion protein, for example separated by sequences of carrier protein. In certain embodiments, the glycosylation tag(s) can be, for example, located at the N-terminal end of the carrier protein and/or fusion protein. In certain embodiments, the glycosylation tag(s) can be, for example, located at the C-terminal end of the carrier protein and/or fusion protein. In certain embodiments, the glycosylation tag(s) can be located internally within the carrier protein and/or fusions protein, for example, wherein a glycosylation tag is located between multiple carrier proteins in a fusion protein. In certain embodiments, the multiple carrier proteins can be identical in type or different in type. In certain embodiments, the glycosylation tags can be identical in type or different in type. In certain embodiments, these ComP glycosylation tags are identical. In certain embodiments, at least two of the ComP glycosylation tags differ from each other. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation tags all differ from each other. Further, in certain embodiments, none of the ComP glycosylation tags are the same.
A bioconjugate of this invention may have one of numerous uses including, but not limited to, use as a conjugate vaccine. For example, in certain embodiments, the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8, Streptococcus pneumoniae serotype 1, Streptococcus pneumoniae serotype 2, Streptococcus pneumoniae serotype 4, Streptococcus pneumoniae serotype 5, Streptococcus pneumoniae serotype 6A, Streptococcus pneumoniae serotype 6B, Streptococcus pneumoniae serotype 7F, Streptococcus pneumoniae serotype 9N, Streptococcus pneumoniae serotype 9V, Streptococcus pneumoniae serotype 10A, Streptococcus pneumoniae serotype 11A, Streptococcus pneumoniae serotype 12F, Streptococcus pneumoniae serotype 14, Streptococcus pneumoniae serotype 15B, Streptococcus pneumoniae serotype 17F, Streptococcus pneumoniae serotype 18C, Streptococcus pneumoniae serotype 19F, Streptococcus pneumoniae serotype 19A, Streptococcus pneumoniae serotype 20, Streptococcus pneumoniae serotype 22F, Streptococcus pneumoniae serotype 23F, Streptococcus pneumoniae serotype 33F, Klebsiella pneumoniae serotype K1, Klebsiella pneumoniae serotype K2, Klebsiella pneumoniae serotype K5, Klebsiella pneumoniae serotype K16, Klebsiella pneumoniae serotype K20, Klebsiella pneumoniae serotype K54, Klebsiella pneumoniae serotype K57, Streptococcus agalactiae serotype Ia, Streptococcus agalactiae serotype Ib, Streptococcus agalactiae serotype II, Streptococcus agalactiae serotype III, Streptococcus agalactiae serotype IV, Streptococcus agalactiae serotype V, Streptococcus agalactiae serotype VI, Streptococcus agalactiae serotype VII, Streptococcus agalactiae serotype VIII, Streptococcus agalactiae serotype IX, Streptococcus pyogenes Group A Carbohydrate, Enterococcus faecalis serotype A, Enterococcus faecalis serotype B, Enterococcus faecalis serotype C, Enterococcus faecalis serotype D, Enterococcus faecium capsular polysaccharide and lipotechoic acid, Moraxella catarrhalis lipooligosaccharide A, Moraxella catarrhalis lipooligosaccharide B, Moraxella catarrhalis lipooligosaccharide C, and Staphylococcus aureus lipotechoic acid. In certain embodiments, the conjugate vaccine is useful because it induces an immune response when administered to a subject. In certain embodiments, the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. For example, in certain embodiments the antibody response can be an IgG response, and in certain embodiments, an IgG1 response. In certain embodiments, the conjugate vaccine generates immunological memory in a subject administered the vaccine.
Provided for herein is a fusion protein as disclosed in further detail elsewhere herein and comprising a ComP glycosylation tag as disclosed in detail elsewhere herein. In certain embodiments, the fusion protein is glycosylated at a serine residue on the glycosylation tag corresponding to the serine residue at position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1). In certain embodiments, the fusion protein is glycosylated with an oligo- or polysaccharide. In certain embodiments, the oligo- or polysaccharide is produced by a bacteria from the genus Streptococcus such as, for example, a S. pneumoniae, S. agalactiae, or S. suis capsular polysaccharide. In certain embodiments, the capsular polysaccharide is CPS14, CPS8, CPS9V, or CPS15b. In certain embodiments, the oligo- or polysaccharide is produced by a bacteria from the genus Klebsiella, for example, a Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca capsular polysaccharide. In certain embodiments, the polysaccharide is a Klebsiella pneumoniae capsular polysaccharide. In certain embodiments, the polysaccharide is a serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae. In certain of any embodiments disclosed herein, the oligo- or polysaccharide comprises a glucose at its reducing end. Certain embodiments are drawn a fusion protein wherein the fusion protein is produced in vivo. For example, in certain embodiments, the fusion protein is produced in a mammalian cell, fungal cell, yeast cell, insect cell, avian cell, algal cell, or bacterial cell. In certain embodiments, the fusion protein is produced in a bacterial cell, for example, E. coli.
Disclosed herein are methods for the in vivo conjugation of an oligo- or polysaccharide to a polypeptide (in vivo glycosylation). In certain embodiments, the method comprises covalently linking the oligo- or polysaccharide to the polypeptide with a PglS oligosaccharyltransferase (OTase) (described elsewhere herein). In certain embodiments, the polypeptide comprises a ComP protein or a glycosylation tag thereof. In certain embodiments, the polypeptide comprises a ComP protein or a glycosylation tag thereof linked to a heterologous polypeptide such as a carrier protein. Representative examples of PglS OTases include, but are not limited to PglS110264, PglSADp1, PglSGFJ-2, PglS50v10, PglS4466, and PglSSFC. ComP proteins are described in detail elsewhere and representative examples include, but are not limited to ComP110264, ComPADP1, ComPGFJ-2, ComP50v10, ComP4466, and ComPSFC. It will be recognized that while a PglS OTase from an organism would naturally glycosylate the ComP protein from that organism (e.g., PglS110264 glycosylates ComP110264) in certain embodiments, a PglS from one organism glycosylates a ComP from a different organism (e.g., PglSADP1 glycosylates ComP110264). For example, in certain aspects, the PglS OTase is PglSADP1. In certain embodiments, where the PglS OTase is PglSADP1, the ComP protein glycosylated is not ComPADP1. For example, in certain embodiments where the PglS OTase is PglSADP1, the ComP protein is ComP110264. Of course, it will be recognized that a PglS OTase does not naturally glycosylate a ComP protein or a glycosylation tag fragment thereof, even from the same organism as the PglS Otase, when the ComP protein or glycosylation tag fragment thereof is linked to a heterologous carrier protein.
In certain embodiments for any combination of PglS and ComP, the ComP protein or glycosylation tag fragment thereof is glycosylated at a serine residue corresponding to the serine residue at position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1).
In certain embodiments disclosed herein, the in vivo glycosylation occurs in a host cell. In certain embodiments, for example, the host cell can be a mammalian cell, fungal cell, yeast cell, insect cell, avian cell, algal cell, or bacterial cell. In certain embodiments, the host cell is a bacterial cell, for example, E. coli.
In certain embodiments, the method comprises culturing a host cell comprising the components necessary for the conjugation of the oligo- or polysaccharide to the polypeptide. In general, these components are the oligosaccharyltransferase, the acceptor polypeptide to be glycosylated, and the oligo- or polysaccharide. In certain embodiments, the method comprises culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a PglS OTase; and (3) the acceptor polypeptide. Further, it has been discovered that production of the oligo- or polysaccharide can be enhanced by a transcriptional activator. In certain embodiments, the production of the oligo- or polysaccharide is enhanced by the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044) or a homolog of the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044). In certain embodiments, the method further comprises expressing and/or providing such a transcriptional activator in the host cell along with the other components.
In certain embodiments, the carrier protein linked to the ComP glycosylation tag is, for example, diphtheria toxoid CRM197, tetanus toxoid, Pseudomonas aeruginosa Exotoxin A (EPA), tetanus toxin C fragment, cholera toxin B subunit, Haemophilus influenza protein D, or a fragment thereof.
Certain embodiments, are directed to a method a conjugate vaccine comprising a bioconjugate of this disclosure or a method of producing such conjugate vaccine.
Certain embodiments also provide for a host cell comprising the components for in vivo glycosylation of an acceptor ComP protein or glycosylation tag fragment thereof. In certain embodiments, a host cell comprises: (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a PglS OTase; and (3) an acceptor polypeptide comprising a ComP protein or a glycosylation tag fragment thereof. In certain embodiments, the acceptor polypeptide is a fusion protein. In certain embodiments, the host cell further comprises a transcriptional activator such as described above along with the other components.
In certain embodiments, a host cell comprises an isolated nucleic acid encoding a PglS OTase. In certain embodiments a host cell comprises an isolated nucleic acid encoding the ComP acceptor polypeptide. In certain embodiments, a host cell comprises a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide. In certain embodiments, a host cell comprises at least two of an isolated nucleic acid encoding a PglS OTase, an isolated nucleic acid encoding the ComP acceptor polypeptide, and genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide. In embodiments aspects, a host cell comprises a nucleic acid encoding a PglS OTase of one organism and a nucleic acid encoding the ComP acceptor polypeptide from a different organism.
Certain embodiments also provide for an isolated nucleic acid encoding the ComP protein, ComP glycosylation tag fragment, and/or ComP fusion protein described anywhere herein. In certain embodiments, an isolated nucleic acid referred to herein is a vector or is contained within a vector. In certain embodiments, an isolated nucleic acid referred to herein is inserted and/or has been incorporated into a heterologous genome or a heterologous region of a genome.
Disclosed herein is a pneumococcal bioconjugate vaccine containing a conventional vaccine carrier. Certain embodiments comprise the use of a ComP fragment as a glycosylation tag (aka “glycotag”). In certain embodiments, the glycosylation tag can be added to the C-terminus and/or N-terminus of a carrier protein. For example, in certain embodiments, the glycosylation tag is added to the C-terminus of the conventional carrier protein Pseudomonas aeruginosa Exotoxin A (EPA). It has been demonstrated that in certain embodiments, the glycosylation tag/carrier fusion protein can be paired with the CPS8 polysaccharide and use of PglS, generating a carrier protein-CPS8 bioconjugate, a first of its kind pneumococcal bioconjugate vaccine. For example, in certain embodiments, an EPA fusion can be paired with the CPS8 polysaccharide and use of PglS, generating an EPA-CPS8 bioconjugate. It was demonstrated that the EPA-CPS8 bioconjugate vaccine elicited high IgG titers specific to serotype 8 specific that were protective as determined via bactericidal killing. Importantly, vaccination with as little as 100 ng of polysaccharide in the EPA-CPS8 bioconjugate was able to provide protection. Thus, certain embodiments provide for a CPS8 pneumococcal bioconjugate vaccine.
It is contemplated that a conjugate vaccine (such as the EPA vaccine construct) can comprise additional/multiple sites of glycosylation to increase the glycan to protein ratio as well as expand upon the number of serotypes in order to develop a comprehensive pneumococcal bioconjugate vaccine.
In certain embodiments, a bioconjugate or glycosylated fusion protein disclosed herein is a conjugate vaccine that can be administered to a subject for the prevention and/or treatment of an infection and/or disease. In certain embodiments, the conjugate vaccine is a prophylaxis that can be used, e.g., to immunize a subject against an infection and/or disease. In certain embodiments, the bioconjugate is associated with (such as in a therapeutic composition) and/or administered with an adjuvant. Certain embodiments provide for a composition (such as a therapeutic composition) comprising a conjugate vaccine described herein and an adjuvant. In certain embodiments, when the conjugate vaccine is administered to a subject, it induces an immune response. In certain embodiments, the immune response elicits long term memory (memory B and T cells). In certain embodiments, the immune is an antibody response. In certain embodiments, the antibody response is a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments where the antibody response is an IgG response, the IgG response is an IgG1 response. Further, in certain embodiments, the conjugate vaccine generates immunological memory in a subject administered the vaccine.
Certain embodiments also provide for producing a vaccine against an infection and/or disease. In certain embodiments a method comprises isolating a bioconjugate or fusion protein disclosed herein (conjugate vaccine) and combining the conjugate vaccine with an adjuvant. In certain embodiments, the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature. In certain embodiments, the vaccine is a conjugate vaccine against pneumococcal infection. In certain embodiments, the disease is pneumonia. In certain embodiments, the infection is a systemic infection and/or an infection of the blood. In certain embodiments, the subject is a mammal. For example, in certain embodiments, a pig or a human.
Importantly, the aspects disclosed herein are not limited to pneumococcal polysaccharides, but in fact, have vast applicability for generating bioconjugate vaccines for many important human and animal pathogens that are incompatible with PglB and PglL. Notable examples include the human pathogens Klebsiella pneumoniae and Group B Streptococcus as well as the swine pathogen S. suis, all immensely relevant pathogens with no licensed vaccines available.
Provided herein are methods of inducing a host immune response against a pathogen. In certain embodiments, the pathogen is a bacterial pathogen. In certain embodiments, the host is immunized against the pathogen. In certain embodiments, the method comprises administering to a subject in need of the immune response an effective amount of a ComP conjugate vaccine, glycosylated fusion protein, or any other therapeutic/immunogenic composition disclosed herein. Certain embodiments provide a conjugate vaccine, glycosylated fusion protein, or other therapeutic/immunogenic composition disclosed herein for use in inducing a host immune response against a bacterial pathogen and immunization against the bacterial pathogen. Examples of immune responses include but are not limited to an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses. In certain embodiments, the immune response is an antibody response. In certain embodiments, the immune response is an innate response, a humoral response, an antibody response, a T cell response, or a combination of two or more of said immune responses.
Also provided herein are methods of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof a conjugate vaccine, a fusion protein, or a composition disclosed herein. In certain embodiments, the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature. In certain embodiments, the disease is pneumonia. In certain embodiments, the infection is a systemic infection and/or an infection of the blood. In certain embodiments disclosed herein, the subject is a vertebrate. In certain embodiments the subject is a mammal such as a dog, cat, cow, horse, pig, mouse, rat, rabbit, sheep, goat, guinea pig, monkey, ape, etc. And, for example, in certain embodiments the mammal is a human.
In any of the embodiments of administration disclose herein, the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.
Previously, it was demonstrated that the ComP protein from Acinetobacter baylyi ADP1 (ComPADP1) and A. soli strain 110264 (ComP110264) are glycosylated at a homologous serine residue located at position 84 or 82, respectively, by the O-linking OTase PglS (Harding C M, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891). Specifically, it was shown that the S84A point mutant of ComPADP1 and the S82A point mutant of ComP110264 were not able to be glycosylated with the serotype 8 pneumococcal capsular polysaccharide by PglS. To further analyze the role of other amino acids important for PglS-dependent glycosylation, a series of point mutants were generated to alter the conserved cysteine residues flanking the site of glycosylation located at positions 75 and 95 of ComPADP1 (
A series of point mutants was first generated replacing cysteine 75 with either alanine or glycine as these mutants would block the formation of a disulfide bond that may be formed between cysteines 75 and 95. The point mutants were then introduced into E. coli SDB1 co-expressing the C. jejuni heptasaccharide biosynthetic gene cluster and PglSADP1. As seen in
It was demonstrated that a translational fusion containing ComP110264 lacking the first 28 amino acids (herein referred to as ComPΔ28110264) fused at the C-terminus of a genetically inactivated variant of the exotoxin A protein from Pseudomonas aeruginosa (EPA) was efficiently glycosylated by PglS with multiple pneumococcal and K. pneumoniae capsular polysaccharides (Harding C M, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891; Feldman M F, et al. (2019) A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae. Proc Natl Acad Sci USA). In order to shorten and define the minimal recognition site required for PglS dependent glycosylation, a reductive cloning strategy was pursued whereby fragments of ComP110264 were translationally fused to the C-terminus of the EPA protein in between a glycine-glycine-glycine-serine (GGGS) linker and a hexahistidine tag (
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67SSGNCTGVTQIASGASAATTNVASA91
68SGNCTGVTQIASGASAATTNVASAQ92
72TGVTQIASGASAATTNVASAQCSDS96
73GVTQIASGASAATTNVASAQCSDSD97
74VTQIASGASAATTNVASAQCSDSDG98
75TQIASGASAATTNVASAQCSDSDGV99
62GTSMPSSGNCTGVTQIASGASAATTNVASA91
63TSMPSSGNCTGVTQIASGASAATTNVASAQ92
72TGVTQIASGASAATTNVASAQCSDSDGVIT101
73GVTQIASGASAATTNVASAQCSDSDGVITV102
74VTQIASGASAATTNVASAQCSDSDGVITVT103
75TQIASGASAATTNVASAQCSDSDGVITVTM104
76QIASGASAATTNVASAQCSDSDGVITVTMT105
57IMNAGGTSMPSSGNCTGVTQIASGASAATTNVASA91
58MNAGGTSMPSSGNCTGVTQIASGASAATTNVASAQ92
70NCTGVTQIASGASAATTNVASAQCSDSDGVITVTM104
71CTGVTQIASGASAATTNVASAQCSDSDGVITVTMT105
72TGVTQIASGASAATTNVASAQCSDSDGVITVTMTD106
73GVTQIASGASAATTNVASAQCSDSDGVITVTMTDK107
74VTQIASGASAATTNVASAQCSDSDGVITVTMTDKA108
75TQIASGASAATTNVASAQCSDSDGVITVTMTDKAK109
52TVSENIMNAGGTSMPSSGNCTGVTQIASGASAATTNVASA91
53VSENIMNAGGTSMPSSGNCTGVTQIASGASAATTNVASAQ92
65MPSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTM104
66PSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMT105
67SSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTD106
68SGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDK107
69GNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKA108
70NCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAK109
71CTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKG110
72TGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGV111
73GVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVS112
74VTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVSI113
75TQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVSIK114
48AMKATVSENIMNAGGTSMPSSGNCTGVTQIASGASAATTNVASAQ92
60AGGTSMPSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTM104
61GGTSMPSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMT105
62GTSMPSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTD106
64SMPSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKA108
65MPSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAK109
66PSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKG110
67SSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGV111
68SGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVS112
69GNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVSI113
70NCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVSIK114
71CTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVSIKL115
72TGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVSIKLT116
O-linking glycosylation recognition motifs can be glycosylated by PglSAADP1 when translationally fused N-terminally, in tandem at the N- or C-terminus, or simultaneously at the N- and C-terminus. Based on the data presented above, the D5 (SEQ ID NO: 61) fragment of ComP110264 was selected for follow up experiments whereby the D5 (SEQ ID NO: 61) fragment or a derivative thereof (D5′) was translationally fused to N-terminus and C-terminus in different combinations as outlined in
EPA fusion constructs were built containing a tandem, C-terminally fused double ComPΔ28110264 glycosylation tag. The ComPΔ28110264 glycosylation tags were separated by either a glycine-glycine-glycine-glycine-serine (GGGS) linker (SEQ ID NO: 23) or by a proline-alanine-proline-alanine-proline (PAPAP) linker (SEQ ID NO: 25). Both constructs contained a hexahistidine tag to aid downstream purification. As a positive control, the EPA fusion containing the ComPΔ28110264 fragment was included as this protein has previously been established to be glycosylated by PglSADP1 with the CPS8. The double tag EPA fusion constructs were then introduced into E. coli SDB1 co-expressing the pneumococcal CPS8 in the presence of absence of PglSADP1. As can be seen in
The present disclosure is not to be limited in scope by the specific aspects described or preceding Examples which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
This PCT application claims the benefit of U.S. Provisional Appl. No. 62/783,971, filed on Dec. 21, 2018. This application is related to U.S. application Ser. No. 15/553,733, filed Aug. 25, 2017, which is a U.S. national stage application of PCT/CA2016/050208, filed Feb. 26, 2016, which claims the benefit of U.S. Provisional Appl. No. 62/121,439, filed on Feb. 26, 2015. This application is also related to PCT/US2019/037251, filed Jun. 14, 2019, which claims the benefit of U.S. Provisional Appl. No. 62/685,970, filed on Jun. 16, 2018 and U.S. Provisional Appl. No. 62/783,971, filed on Dec. 21, 2018.
This invention was made with government support under the R41 AI142928-01 grant awarded by the National Institute for Allergy and Infectious Disease (NIAID). The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/059893 | 11/5/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62783971 | Dec 2018 | US |
