WO2020041394A1 - Binding proteins, compositions and methods thereof for neutralization of infection of pathogens having a type iii secretion system - Google Patents

Abstract

Provided are binding proteins which comprise heavy-chain-only variable domains (VHH) that specifically bind to the Invasion Plasmid Antigen D (IpaD) and neutralize contact-mediated hemolysis of pathogens having a type III secretion system (T3SS), such as Shigella spp.

Description

BINDING PROTEINS, COMPOSITIONS AND METHODS THEREOF FOR NEUTRALIZATION OF INFECTION OF PATHOGENS HAVING A TYPE III

SECRETION SYSTEM

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and benefit of U.S. Provisional Patent Application No. 62/721,955, filed on August 23, 2018, the contents of which are incorporated by reference herein in their entirety.

BACKGROUND

High affinity binding agents, other than classical antibodies, that neutralize disease causing agents for the treatment of both routine incidents of disease and pandemics are needed worldwide, particularly to combat infection and intoxication of subjects by a variety of pathogenic and toxigenic microorganisms.

Accordingly, there is a need for cost effective alternative therapeutic agents for treating bacterial infections, are increasingly important due to several factors, including the threats of bacterial multi drug resistance and bioterrorism.

SUMMARY OF THE DISCLOSURE

Aspects of the invention provide recombinant binding proteins that bind Invasion Plasmid Antigen D (IpaD) and neutralize or inhibit contact hemolysis of pathogens having a type III secretion system (T3SS). In some embodiments, the invention provides recombinant binding proteins that bind IpaD protein and neutralize or inhibit contact hemolysis of Shigella species. In some embodiments, the binding protein comprises a variable domain of heavy- chain only (VHH) antibody.

In some aspects, the present invention provides a variable domain of a heavy chain- only antibody (VHH) for neutralizing Shigella spp. In one aspect of the invention, the VHH comprises the amino acid sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a variable domain of a heavy chain- only antibody (VHH) for neutralizing Shigella spp , the VHH comprising the amino acid sequence of SEQ ID NO: 2.

In another aspect, the present invention provides a variable domain of a heavy chain- only antibody (VHH) for neutralizing Shigella spp , the VHH comprising the amino acid sequence of SEQ ID NO: 3. In another aspect, the present invention provides a variable domain of a heavy chain- only antibody (VHH) for neutralizing Shigella spp , the VHH comprising the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the VHH binds to structural epitopes clustered within the distal region of the IpaD protein. In some embodiments, the structural epitopes include residues 165-177 and 198-205 of Shigella IpaD. In some embodiments, the VHH binds to amino acids Glu20l and Lys205 of the of Shigella IpaD.

In some embodiments, the VHH inhibits ri%/ge//a-mediated hemolytic activity when tested in a contact-mediated hemolysis assay. In some embodiments, the VHH inhibits from 30% to 50% Shige//a-med\ ated hemolytic activity when tested in a contact-mediated hemolysis assay.

In some aspects, the present invention provides a binding protein comprising a first VHH and a second VHH, wherein the first VHH is a neutralizing VHH comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, wherein the second VHH is a neutralizing VHH comprising the amino acid sequence of SEQ ID NO:

1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and wherein the first and second VHH are different. In some embodiments, the binding protein inhibits Shigella- mediated hemolytic activity when tested in a contact-mediated hemolysis assay by 80% or more. In some embodiments, the binding protein is a heterodimer comprising the amino acid sequence of SEQ ID NO: 1 covalently linked to SEQ ID NO: 2.

In some aspects, the present invention provides a binding protein a first VHH and a second VHH, wherein the first VHH is a neutralizing VHH comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and wherein the second VHH is a non-neutralizing VHH comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the VHHs are linked by a flexible spacer. In some embodiments, the flexible spacer is (GGGGS)₃ (SEQ ID NO: 8).

In some aspects, the present invention provides a binding protein comprising two or more VHHs, wherein the two or more VHHs comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the two or more VHHs are identical VHHs. In other embodiments, the two or more VHHs are different VHHs. In some embodiments, the binding protein comprises two or more VHHs, wherein the two or more VHHs comprise the amino acid sequence of SEQ ID NO: 1. In some embodiments, the VHHs are linked by a flexible spacer. In some embodiments, the flexible spacer is (GGGGS)₃ (SEQ ID NO: 8).

In some aspects, the present invention provides a binding protein comprising the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 1, wherein the binding protein binds IpaD and inhibits Shige//a-med\ ated he olytic activity when tested in a contact-mediated hemolysis assay.

Other aspects of the invention provide a binding protein comprising the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 2, wherein the binding protein binds IpaD and inhibits Shigella-mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

Other aspects of the invention provide a binding protein comprising the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 3, wherein the binding protein binds IpaD and inhibits Shigella-mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

Other aspects of the invention provide a binding protein comprising the first complementary determining regions (CDR1) of SEQ ID NO: 4, wherein the binding protein binds IpaD and inhibits Shigella-mediated hemolytic activity when tested in a contact- mediated hemolysis assay.

According to aspects of the invention, the binding proteins described herein inhibit Shigella- mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

Aspects of the invention provide a pharmaceutical composition comprising the VHHs or the binding proteins described herein and a pharmaceutically acceptable carrier, excipient, or vehicle.

Aspects of the invention provide a method of treating a subject in need thereof comprising administering to the subject an effective amount of the VHHs or the binding proteins described herein.

Aspects of the invention provide a polynucleotide encoding the VHHs or the binding proteins described herein. In some aspects, expression vectors comprising the polynucleotide are provided.

Other features and advantages of the invention will be apparent from the detailed description and from the claims infra. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ah, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By“ameliorate” is meant decrease, reduce, diminish, suppress, attenuate, arrest, or stabilize the development or progression of a disease or pathology.

By“antibody” is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.

By "binding to" a molecule is meant having a physicochemical affinity for that molecule. Binding may be measured by any of the methods practiced in the art, e.g., using an antibody binding assay or an in vitro translation binding assay.

By“disease” is meant any condition, disorder, or pathology that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include, diseases associated with infection by Gram-negative pathogens having a type III secretion system (T3SS), for example, Shigella spp., Salmonella enterica, Pseudomonas aeruginosa,

Burkholderia pseudomallei .

By "effective amount" is meant the amount of a required to ameliorate, or optimally eliminate, the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

As used herein, the terms“polynucleotide,”“DNA molecule” or“nucleic acid molecule” include both sense and anti-sense strands, cDNA, genomic DNA, recombinant DNA, RNA, mRNA, and wholly or partially synthesized nucleic acid molecules. A nucleotide "variant" is a sequence that differs from the recited nucleotide sequence in having one or more nucleotide deletions, substitutions or additions. Such modifications are readily introduced using standard mutagenesis techniques, such as oligonucleotide-directed site- specific mutagenesis as described, for example, in Adelman et al., 1983, DNA 2: 183.

Nucleotide variants are naturally-occurring allelic variants, or non-naturally occurring variants. Variant nucleotide sequences in various embodiments exhibit at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence homology or sequence identity to the recited sequence. Such variant nucleotide sequences hybridize to the recited nucleotide sequence under stringent hybridization conditions. In one embodiment, "stringent conditions" refers to prewashing in a solution of 6 x SSC, 0.2% SDS; hybridizing at 65°C, 6xSSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in lxSSC, 0.1% SDS at 65°C, and two washes of 30 minutes each in 0.2 x SSC, 0.1% SDS at 65°C.

By "isolated polynucleotide" is meant a nucleic acid (e.g., DNA, cDNA, RNA, mRNA) that is free of the genes, which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, e.g., mRNA, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

The terms "protein", "peptide" and "polypeptide" are used herein to describe any chain of amino acid residues, regardless of length or post-translational modification (for example, glycosylation or phosphorylation). Thus, these terms can be used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid. Thus, the term "polypeptide" includes full- length proteins, which may be, but need not be, naturally occurring, as well as recombinantly or synthetically produced polypeptides that correspond to a full-length protein, or to particular domains or portions of a protein, which may be, but need not be, naturally occurring. The term also encompasses mature proteins which have an added amino-terminal methionine to facilitate expression in prokaryotic cells. The binding molecules of the invention are encoded by polynucleotides and can be chemically synthesized or synthesized by recombinant DNA methods.

By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent. By "operably linked" is meant the connection between regulatory elements and one or more polynucleotides (genes) or a coding region. That is, gene expression is typically placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A polynucleotide (gene or genes) or coding region is said to be "operably linked to" or "operatively linked to" or "operably associated with" the regulatory elements, meaning that the polynucleotide (gene or genes) or coding region is controlled or influenced by the regulatory elements. The one or more polynucleotides may be separated by spacers or linkers.

By“pathogen” is meant any harmful microorganism, bacterium, virus, fungus, or protozoan capable of interfering with the normal function of a cell. Pathogens as referred to herein produce toxins, e.g., protein toxins, that intoxicate the cells and tissues of a host or recipient organism and cause disease and pathology, often severe, unless they are neutralized and eliminated from the organism to the extent possible, such as by action of the binding proteins described herein. As described herein, bacterial pathogens include, but are not limited to, Gram-negative pathogens having a type III secretion system (T3SS), for example, Shigella spp., Salmonella enter ica, Pseudomonas aeruginosa, Burkholderia pseudomallei .

By“neutralizing” or“blocking”, is meant the ability of the binding proteins of the invention to specifically bind to structural epitopes of the IpaD protein and to interfere with the biological function of the IpaD protein and blocking the capacity of the bacterium to deliver its effectors of virulence to the target cells.

By“reduces” is meant a negative or lowering alteration of at least 5%, 10%, 25%, 50%, 75%, or 100%.

By "specifically binds" is meant a compound, molecule, or antibody that recognizes and binds a protein, peptide, or polypeptide (e.g., an amino acid sequence of the protein, peptide, or polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which may contain the protein, peptide, or polypeptide that is specifically bound.

"Nucleic acid" (also called polynucleotide herein) refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids (polynucleotides) containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non- naturally occurring, which have similar binding properties as a reference nucleic acid, and which are metabolized in a manner similar to the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (for example, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with suitable mixed base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res, 19:081; Ohtsuka et al., 1985, J Biol. Chem ., 260:2600-2608; Rossolini et al., 1994, Mol. Cell Probes , 8:91-98). The term nucleic acid can be used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

Nucleic acid molecules or polynucleotides useful in the invention include any nucleic acid molecule or polynucleotide that encodes a peptide or polypeptide, e.g., a binding molecule, of the invention or a component or portion thereof. Nucleic acid molecules useful in the methods of the invention include any polynucleotide or nucleic acid molecule that encodes a polypeptide e.g., binding molecule, of the invention or a component or portion thereof that has substantial identity to the binding molecule. Such nucleic acid molecules need not be 100% identical with the nucleic acid sequence of the binding molecule, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to a binding molecule sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By "hybridize" is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger, 1987 , Methods Enzymol. 152:399; Kimmel, A. R., 1987, Methods CnzymoL 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 °C, more preferably of at least about 42°C, and even more preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.

Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York. By "subject" is meant a mammal, including, but not limited to, a human or non human mammal, such as, without limitation, a human, a non-human primate, or a bovine, equine, canine, ovine, or feline mammal. Other mammals include rabbits, goats, llamas, mice, rats, guinea pigs, camels and gerbils. In particular, a“subject” as used herein refers to a human subject, such as a human patient. In some cases, the terms subject and patient are used interchangeably herein.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 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.

A VHH binding molecule as referred to herein is, in general, a single domain immunoglobulin molecule (antibody) isolated from camelid animals. A VHH (or VHH antibody) corresponds to the heavy chain of a camelid antibody having a single variable domain (or single variable region), e.g., a camelid-derived single variable H (V_H) domain antibody. A VHH has a molecular weight (MW) of about 15 kDa. VHH technology is based on fully functional antibodies from camelids that lack light chains. These heavy-chain antibody molecules contain a single variable domain (VHH) and two constant domains (CH₂ and CH₃). A cloned (recombinantly produced) and isolated VHH domain is a stable polypeptide harboring the antigen-binding capacity of the original heavy-chain antibody.

See, e.g., U.S. Patent No. 5,840,526 and U.S. Patent No. 6,015,695, each of which is incorporated by reference herein in its entirety. VHHs, called NANOBODIES™, may be obtained commercially (Ablynx Inc., Ghent, Belgium).

VHHs are efficiently expressed in E. coli , coupled to detection markers, such as a fluorescent marker, or conjugated with enzymes. The small size of VHHs permits their binding to epitopes, e.g.,“hidden epitopes” that are not accessible to whole antibodies of much larger size. As a therapeutic, a VHH is capable of efficient penetration and rapid clearance. Its single domain nature allows a VHH to be expressed in a cell without a requirement for supramolecular assembly, as is needed for whole antibodies which are typically tetrameric (two heavy chains and two light chains, having a MW of about 150 kDa). VHHs are also exhibit stability over time and have a longer half-life versus non- VHH antibody molecules, which comprise disulfide bonds that are susceptible to chemical reduction or enzymatic cleavage.

A VHH-based binding molecule or polypeptide that specifically binds to and neutralizes the activity of a target agent, such as a bacterial toxin, is referred to as a“VHH- based neutralizing agent (VNA)” a“VNA polypeptide molecule” or a“VNA binding molecule” herein.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing, diminishing, abating, alleviating, improving, or ameliorating a disorder and/or symptom associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, a“multimeric binding molecule” refers in general to a multi- component protein or polypeptide containing two or more, same or different, VHH binding molecules, which are coupled or linked, e.g., via spacer sequences, to other components of the molecule. Multimeric binding molecules may be homomultimeric, in that the binding molecule contains more than one, e.g., two, different VNA binding molecule components that bind to the same target agent. The different VNA binding molecule components of a homomultimeric binding molecule may bind to different regions, portions, or epitopes (e.g., non-overlapping epitopes) of the same target agent. In some embodiments, the

homomultimeric binding molecule contains two, three, four or five identical VHH binding molecule components. Alternatively, the multimeric binding molecules may be

heteromultimeric, in that the binding molecule contains more than one, e.g., two, three, four or five, different VHH binding molecule components, each of which specifically binds to a different target agent or to different regions, portions, or epitopes (non-overlapping epitopes) of the same target agent, such that the heteromultimeric binding molecule comprises several different VHH binding molecule components, for example, two different VHH binding molecule components. In some embodiments, the heteromultimeric binding molecule contains two different VHH binding molecule components. A VNA binding molecule can refer to a heteromultimeric binding molecule that comprises two or more different VHH binding molecule components.

As used herein, the terms“prevent,”“preventing,”“prevention,”“prophylactic treatment,”“protection” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but who is at risk of, is susceptible to, or disposed to (e.g., genetically disposed to), developing a disease, disorder, pathology, or condition.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. The term“about” can be understood as within 10%, 9%, 8%, 7%,

6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show multiple Sequence Alignments of the 7 IpaD-specific VHHs 20ipaD (SEQ ID NO: 1), JPSG3 (SEQ ID NO: 2), JMFK11 (SEQ ID NO: 3), JMKE3 (SEQ ID NO: 4), JMK-G3 (SEQ ID NO: 5), JMK-H2 (SEQ ID NO: 6), JMK-H5 (SEQ ID NO: 7) and four X-ray Crystal Structures of VHHs according to embodiments of the invention. FIG. 1A shows multiple amino acid sequence alignments of seven VHHs generated using

ClustalW and rendered with ESPRIPT. The numbers above and below the sequences correspond to JMK-H2 and JPS-G3, respectively. The secondary structure of JMK-H2 is shown above the alignment, and the secondary structure of JPS-G3 is shown below. The bars indicating“CDR1”,“CDR2” and CDR3” below the sequences correspond to the three complementarity determining regions (CDRs). Certain of the amino acid residues show identity in sequence conservation and others show similarity in sequence conservation as judged by the BLOSUM62 matrix. FIG. IB depicts in schematic format the crystal structures of the 4 VHHs according to embodiments of the invention, comprising a-helices, b-sheets and loop regions. All VHHs are oriented in a similar manner and include the CDRs 1-3 as indicated in FIG. 1A.

FIGS. 2A-2C show the identification of unique epitope regions within IpaD. FIG.

2A depicts in schematic ribbon format the crystal structure of IpaD (PDB ID: 2JOO) with individual domains as follows: N-terminal domain (D1-120), central coiled-coil and distal domain. Alpha helices (1-7) are labeled within the IpaD^ image. FIG. 2B shows two immunoblots. IpaD deletion fragment immunoblots were used to identify minimal binding regions of the VHHs. The leftmost immunoblot demonstrates that polyclonal IpaD antisera recognizes each of the 3 protein constructs depicted in FIG. 2A. Immunoblot with 20ipaD is representative of 7/7 VHHs, demonstrating requirement of the distal domain for reactivity. FIG. 2C presents a visual representation of binning data derived from competition ELIS As (FIG. 6) between differentially affinity-tagged VHHs. Clustering of each VHH suggests common or overlapping epitopes are shared between each member. The circled numbers represent bins.

FIG. 3 presents a bar graph showing the inhibition of ri%/ge//a-mediated hemolytic activity. The ability of each VHH to prevent hemoglobin release (% hemolysis) through contact-mediated lysis was evaluated and plotted relative to a wild-type Shigella flexneri control (set at 100%). Data are representative of at least 3 independent tests for each VHH. Relative hemolysis for the evaluated hetero-dimers is plotted on the far right of the graph. Statistical differences between each VHH and WT were assessed using one-way ANOVA (** p <0.01, *** p <0.001, **** p <0.0001), and error bars depict mean ± SD.

FIGS. 4A-C show the crystal structures of IpaD-VHH complexes. FIG. 4A shows the structures of IpaD and their individual domains depicted in schematic ribbon format in complex with VHHs. IpaD is oriented in a similar manner in each complex. FIG. 4B shows surface representations of the equivalent IpaD-VHH complexes in FIG. 4A. In FIG. 4B, IpaD is represented in lighter gray with VHH interacting residues outlined in black and superimposed thereon: 20ipaD, JMK-E3, JPS-G3 and JMK-H2, rotated 90° about the vertical axis). FIG. 4C shows surface representation of IpaD demonstrating overlapping structural epitopes for the 4 VHH complexes. IpaD residues contacting VHHs (see Table 3) are outlined according to the number of represented complexes, denoted as 1, 2, or 3. The two IpaD residues represented in darker gray in FIG. 4C are Glu20l and Lys205.

FIGS. 5A-5D show IpaD-VHH binding interfaces. Residues within hydrogen bonding distance (2.5-3.5 A) between each IpaD-VHH complex (FIG. 5A, 20ipaD; FIG. 5B, JPS-G3; FIG. 5C, JMK-E3 and FIG. 5D, JMK-H2) are depicted as balls-and-sticks (IpaD, and VHH). IpaD secondary structure elements are shown as in Fig. 2A, relevant IpaD a- helices are labeled and each VHH is also shown in the figure. Further information on these distances can be found in Table 4.

FIG. 6 shows VHH Competition ELISA Data. VHHs with a c-myc tag were incubated with IpaD coated in each well of a 96 well plate. VHHs with an E-tag were then probed for the ability to bind to each IpaD/VHH complex and quantified as % binding relative to the no c-myc tagged VHH control (i.e. only IpaD present in the well). The negative VHH was a VHH known to bind to a completely unrelated protein. Negative VHH data was not available for JPS-G3. The competition groups derived from this data were consistent with those from competition data using E-tagged VHH competitors of phage- displayed VHH binding to IpaD (not shown).

FIGS. 7A-7C show Biolayer Interferometry (BLI) Binding Data. The binding of VHHs to recombinant IpaD was assayed by BLI. His-tagged IpaD was coated on Ni-NTA biosensor pins and equilibrated in IX kinetics buffer (IX PBS pH 7.4, 0.01% BSA, 0.002% Tween 20) before being incubated with varying concentrations of each VHH for 180 seconds. The biosensors were then placed back in IX kinetics buffer to allow for dissociation (300 seconds). For each VHH, real time binding profiles using multiple protein concentrations along with fitted curves (1 : 1 Langmuir binding model) are displayed in the left panel, with steady state analysis on the right panel. FIG. 7A presents BLI data for 20ipaD and VHHs JPS-G3 and JMK-F11; FIG. 7B presents BLI data for VHHs JMK-G3, JMK-H2 and JMK- E3; and FIG. 7C presents BLI data for VHH JMK-H5.

FIG. 8 presents high-quality, digital photographs of the eyes of animals for ante mortem ophthalmological evaluation as described in Example 2 herein. Ante-mortem, ophthalmological examinations were conducted at three time-points (pre- infection, 8 hours’ post infection, and 24 hours’ post infection) at which time the digital photographs were taken. In this figure, the eye treated with anti -Shigella VHH is shown on the left, and the un-treated (mock/control) eye is shown on the right, T +24.

FIG. 9 shows a graph of the results of the cumulative ante-mortem severity scoring versus time, based on the ophthalmological examinations as described in FIG. 8 and in Example 2.

FIG. 10 shows a graph of corneal specific ophthalmological ante-mortem evaluation versus lesion severity score at 24 hours post-infection. Corneal lesion severity, which is the hallmark observation in the Serehy assay employed in the study described in Example 2, reveals a statistically significant difference in both corneal edema (p = 0.0010) and corneal ulceration (p = 0.0016) at the 24-hour time point in animals treated with the anti -Shigella VHH versus untreated animals in the study.

FIG. 11 shows a graph of histological, post-mortem corneal lesion ophthalmological evaluation of animals treated with anti -Shigella VHH versus mock treated, control animals. As described in Example 2, the cumulative corneal disease score (left panel) presents the data as the sum of the average foe each pathological change in VHH-treated versus mock-treated, control animals. The corneal % disease (right panel) presents the data as the percentage reduction in disease attributable to treatment of animals with anti -Shigella VHH

(20ipaD_JPS-G3 VHH) and corrects for individual animal variation in response to infection.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Provided herein are binding protein molecules, compositions and methods that are useful for treating or preventing diseases and pathologies associated with infection by Gram negative pathogens having a type III secretion system (T3SS), in particular, Shigella spp ., Salmonella enterica, Pseudomonas aeruginosa, Burkholderia pseudomallei, and Yersinia spp.

Aspects of the invention relates to compositions and methods for blocking entry of Shigella spp. into a cell of a subject, to therefore treat or prevent, or reduce the severity of Shigella infections in the subject.

In some embodiments, the compositions and methods described herein can be used inhibit entry of T3SS-possessing human pathogens by specifically targeting the hydrophobic translocator binding site within the tip protein that is structurally conserved across all T3SS- possessing pathogens.

Aspects of the invention provide recombinant binding proteins that bind Invasion Plasmid Antigen D (IpaD) and neutralize or inhibit contact hemolysis of pathogens having a type III secretion system (T3SS). In some embodiments, the invention provides recombinant binding proteins that bind IpaD protein and neutralize or inhibit contact hemolysis of Shigella species. In some embodiments, the binding protein comprises a variable domain heavy-chain only (VHH) antibody. In some embodiments, the binding protein comprises a camelid recombinant VHH antibody.

VHH domains (also referred herein as VHH) with binding affinity with IpaD and binding protein comprising the VHHs (also referred herein as VHH-based neutralizing agent (VNA)) are described herein. In some embodiments, the VHHs recognize structural epitopes with IpaD that are critical for the type III secretion system T3SS function.

In some embodiments, the VHHs bind to the IpaD distal domain. In some

embodiments, the VHHs bind to structural epitopes clustered within the distal region of the IpaD protein including residues 165-177 (coiled-coil a3) and 198-205 (distal domain a4). In some embodiments, the VHHs form a complex with amino acids Glu20l and Lys205 of the IpaD protein.

In some aspects, the VHH comprises or consists of one or more of the following amino acid sequences:

QVQLVESGGGLVQAGGSLRLSCAVSGLEMQSHAIGWFRQAPGKEREGVSCINDDGS TTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAAKSVWFCSVIRSHEF NSWGQGTQVTVSS (SEQ ID NO: 1);

QVQLAESGGGLVQPGGSLRLSCSASGGVFIIYNMGWYRQAPGKQRELVASIDSYSGS ITNY AD S VKGRFTISRDNVEKRVYLEMNNLKPEDT AVYY CNANLRTNNYW GQGT Q VTVSS (SEQ ID NO: 2);

QVQLVESGGGLVQPGGSLRLSCAVSGLEMQDYAIGWFRQAPGKEREGVSCINNDGS TTRY GD S VKGRF AMSRDNAKNT VYLQMN SLKPEDT AVYFCAAKSVWF C S VIRSDEF GSWGQGIQVTVSS (SEQ ID NO: 3); or

QVQLAETGGGLAQPGGSLRLSCAASGFTFSRAVMNWYRQAPGKERELVARIYDAG GNGSIADP VKGRFTISRDNAKNTVHLQMN SLKPEDT AM YV CNAGIFDGN YRT YW G QGTQ VTVSS (SEQ ID NO: 4). In some embodiments, the binding proteins comprising SEQ ID NO: 1, SEQ ID NO:

2, SEQ ID NO: 3 or SEQ ID NO: 4 decrease hemolytic activity of the pathogen (e.g. Shigella spp.) from 30 to 50%.

In some aspects, the binding protein comprises one or more of the following amino acid sequences:

QVQLVESGGGLVQAGGSLRLSCAVSGLEMQSHAIGWFRQAPGKEREGVSCINDDGS TTRYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAAKSVWFCSVIRSHEF NSWGQGTQVTVSS (SEQ ID NO: 1);

QVQLAESGGGLVQPGGSLRLSCSASGGVFIIYNMGWYRQAPGKQRELVASIDSYSGS ITNY AD S VKGRFTISRDNVEKRVYLEMNNLKPEDT AVYY CNANLRTNNYW GQGT Q VTVSS (SEQ ID NO: 2);

QVQLVESGGGLVQPGGSLRLSCAVSGLEMQDYAIGWFRQAPGKEREGVSCINNDGS TTRY GD S VKGRF AMSRDNAKNT VYLQMN SLKPEDT AVYFCAAKSVWF C S VIRSDEF GSWGQGIQVTVSS (SEQ ID NO: 3), or

QVQLAETGGGLAQPGGSLRLSCAASGFTFSRAVMNWYRQAPGKERELVARIYDAG GNGSIADP VKGRFTISRDNAKNTVHLQMN SLKPEDT AM YV CNAGIFDGN YRTYW G QGTQVTVSS (SEQ ID NO: 4). In some embodiments, the binding proteins comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4 inhibit hemolytic activity of the pathogen (e.g. Shigella spp.) by from 30 to 50%.

In some aspects, the binding protein is a multimeric binding protein and comprises two, three, four, or five VHHs. In some embodiments, the combination of two or more VHHs can increase the inhibitory activity of neutralizing VHHs. In some embodiments, the binding protein can comprise two, three, four, five different VHH that bind distinct and non overlapping structural epitopes of IpaD that are critical for the type III secretion system T3SS function. In other embodiments, the binding protein can comprise two, three, four, five identical VHHs. For example, the binding protein can be a homodimer, a homotrimer, a homotetramer, a homopentamer. In some embodiments, the two or more VHHs in the binding protein are covalently linked. In some embodiments, the two or more VHHs in the binding protein can bind to the same or different binding site or epitope sites on IpaD such that the hemolytic activity is inhibited. In some embodiments, the two or more VHHs can bind to the same or different binding site or epitope sites on the distal domain of IpaD (amino acids 198-205) such that the hemolytic activity of the pathogen is inhibited. In some embodiments, the two or more VHHs can be linked by a flexible peptide spacer. For example, the two or more VHHs monomers can be linked a flexible peptide spacer

(GGGGS)₃ (SEQ ID NO. 8)_. In some embodiments, the combination of two or more neutralizing VHHs increases inhibition of the virulence of the pathogen (e.g. hemolytic activity) compared to each monomeric component.

In some embodiments, the binding protein comprises or consist of SEQ ID NO: 1. In some embodiments, the binding protein comprises or consist of the amino acid sequence SEQ ID NO: 1 and binds to amino acids Glu20l and Lys205 of the IpaD protein.

In some embodiments, the binding protein comprises the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 1. In some embodiments, the binding protein comprises the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 2. In some embodiments, the binding protein comprises the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 3. In some embodiments, the binding protein comprises the first complementary determining region (CDR1) of SEQ ID NO: 4.

In some aspects of the invention, the binding protein is a multimer comprising two or more neutralizing identical VHHs. In some embodiments, the binding protein is a multimer comprising two or more neutralizing identical VHHs comprising the amino acids of SEQ ID NO: 1.

In some aspects of the invention, the binding protein is a heterodimer comprising two neutralizing VHHs. In some embodiments, the binding protein is a heterodimer comprising the amino acids of SEQ ID NO: 1. For example, the binding protein can be a heterodimer comprising SEQ ID NO: 1 covalently linked to SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the heterodimers have a synergistic increase of the inhibition of the virulence of the pathogen. For example, the inhibition of the hemolytic activity of heterodimer comprising SEQ ID NO: 1 and SEQ ID NO: 2 can be greater than 80%.

In some embodiments, the heterodimer can comprise a neutralizing VHH (for example a VHH comprising or consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4) and a non-inhibitory VHH. In some

embodiments, the non-inhibitory VHH comprises or consists of one or more of the following amino acid sequences:

QVQLVESGGGLVQPGGSVRLSCAASGFTFSLYLMSWHRQAPGKERELVATISTTDGS TNGVDSVKGRFTISRDNAKNTVDLQMNSLKPEDTAVYYCKAEALLPVLRPREVWG QGTLVTVSS (SEQ ID NO: 5);

QLQLVESGGGLVQPGGSLRLSCAASGFTLDDQPIAWFRQAPGKEREGVSCISIDGNT QSYSDSVKGRFTISRDTANNRVHLQMNNLKPEDTAVYYCAADRYTSVRQMCTMIE GLHRVW GQGTQ VT V S S (SEQ ID NO: 6);

QLQLVETGGGLVQPGGSLRLSCVASGFAFSIYHMKWFRQAPGKDRELVAVIATTGG TATYADSVKGRFTISRDNAKKTVYLQMNSLKPEDTAVYYCNAEGLVSPDVYWGQG TQVTVSS (SEQ ID NO: 7).

In some embodiments, the VHHs having an affinity with IpaD are obtained from immunized alpacas. In some embodiments, the VHHs having an affinity with IpaD are recombinant VHHs. In some embodiments, the VHHs are neutralizing VHHs (e.g. inhibit Shige//a-med\ ated hemolytic activity when tested in a contact-mediated hemolysis assay.

In an aspect, the present invention provides a pharmaceutical composition comprising binding proteins as described herein and a pharmaceutically acceptable carrier, excipient, or vehicle.

In an aspect, the binding proteins as described are used in therapies of infection by Gram -negative pathogens having a type III secretion system (T3SS), in particular, Shigella spp., Salmonella spp., Burkholderia spp. In some embodiments, the binding proteins as described are used in therapies of infection by Shigella spp., Salmonella enterica,

Pseudomonas aeruginosa, Burkholderia pseudomallei .

In some aspect, a method of treating a subject is provided, in which the method comprises administering to a subject in need thereof an effective amount of the VHH or binding protein as described herein.

Described herein are polynucleotides encoding the binding proteins, compositions comprising the binding proteins (or their encoding polynucleotides); methods using the binding proteins and kits comprising the binding proteins. In some embodiments, the polynucleotide is DNA, cDNA, RNA, mRNA or the like.

Type III secretion system (T3SS)

Many Gram-negative bacterial pathogens use a type III secretion system (T3SS) to inject host-modulating proteins during infection. The type III secretion apparatus (T3SA) is comprised of a basal body that spans both bacterial membranes, a cytoplasmic sorting platform and an extracellular needle with an associated tip complex. The tip complex is responsible for assembly of a pore-forming translocon in the host cell membrane through which effector proteins enter the host cell.

At the distal end of the exposed portion of the T3SS, e.g. Shigella T3SS, a tip complex (TC) is assembled that consists of a pentamer of IpaD, but which is ultimately comprised of IpaD and one or both hydrophobic translocator proteins (IpaB and IpaC) depending upon its activation state. To date, the exact stoichiometry and functional topology of these proteins within the TC remains elusive, with many discordant reports suggesting either a pentamer of IpaD or a four-plus-one complex of IpaD and IpaB (6,10).

T3SS tip proteins can be subdivided into three main families, 1) IpaD-SipD-BipD from Shigella , Salmonella and Burkholderia species; 2) LcrV-PcrV-AcrV from Yersinia , Pseudomonas and Aeromonas species; and 3) EspA-Bsp22 from enteropathogenic A. coli and Bordetella species (8). To date, available structural information from tip proteins within each family indicates the presence of a conserved antiparallel coiled-coil with variability in the globular domains at each end of this coiled-coil (14,33). Visualization of TCs from the LcrV-PcrV-AcrV family also indicated that the TC was comprised of a pentamer in a similar orientation described for IpaD (32). Furthermore, protective epitopes derived from both LcrV and PcrV localize to the region equivalent to a4 within the IpaD distal domain (34,35). While structural data is relatively lacking for the EspA-Bsp22 family, both proteins are capable of eliciting protective antibody responses in mice (36,37).

Altogether, these observations in tandem with the data presented herein support a hypothesis that the hydrophobic translocator (e.g. IpaB in Shigella) likely binds to a region within the tip protein that is structurally conserved across all T3SS-possessing pathogens.

Pathogens Using the Type III secretion system (T3SS)

Pathogens that use a T3SS for virulence are particularly attractive due to the structural homology of their apparatus components and the global burden of the diseases they represent. Diarrheal diseases, such as those caused by Shigella spp. and Salmonella enterica have an estimated 1.7 billion cases per year and are the second leading cause of mortality in children under the age of five (28,29). Other pathogens with T3SS are also recognized as significant targets for new therapeutics. For example, use of such therapies could prove important for combating nosocomial infections caused by Pseudomonas aeruginosa or for preventing the bioterror threat of Burkholderia pseudomallei . Shigella

Shigella is a genus of Gram-negative rod-shaped pathogenic bacteria belonging to the family Enterobacteriaceae. Shigella is divided into 4 species, S. sonnei, S.flexneri, S. hoydii and S. dysenteriae and 50 stereotypes. Different serogroups, considered as species, can be differentiated. S. dysenteriae is considered the most virulent, and can produce a potent cytotoxin known as Shigatoxin. Shigella is transmitted by a fecal-oral route, typically through contaminated food or water. S.flexneri is most common in developing countries where proper sanitation and hygiene are lacking while S. sonnei is most common in developed countries.

Shigella spp. are the causative agents of shigellosis, a form of bloody diarrhea often referred to as bacillary dysentery. Shigella uses its T3SS to invade colonic epithelial cells as a first step in causing infection. After initial invasion, the bacteria spread directly from cell to cell and elicit a massive inflammatory response that leads to the symptoms of shigellosis.

The tip complex of Shigella flexneri is comprised of invasion plasmid antigen D (IpaD), which initially regulates T3SS secretion status and upon host cell contact provides a physical platform for IpaB and IpaC to form the translocon pore in the host membrane. The tip complex currently represents a promising point for therapeutic intervention for numerous important pathogens that possess the T3SS as a primary virulence factor.

Multiple models of in situ TC structures have been proposed, however, no definitive structure or universal composition has been identified. A homopentameric array is the most common theme proposed for TC structures with such a structure proposed for the Shigella TC (10,14), the Salmonella enterica TC (31) and the Yersinia TC (32). An alternative four-plus- one (IpaD-plus-IpaB, respectively) model has been proposed for Shigella (6), however, even in those studies, the predominant TC composition is that of a homopentamer (>90% of injectisomes). In some embodiments, the VHHs described herein can provide a definitive test of needle TC composition for nascent apparatuses.

Neutralizing Anti-IpaD VHHs

Aspects of the invention relate to VHHs that can inhibit Shigella virulence activities ( i.e . contact-mediated hemolysis), which are related to translocon pore formation. In some embodiments, the epitopes recognized by these VHHs are localized to the mixed a/b structure distal domain of IpaD. This is consistent with previous studies suggesting that the distal domain is key for steps subsequent to nascent TC formation (5,10). Furthermore, VHHs that fail to impair virulence functions appear to bind to epitopes that localize to the central coiled- coil of IpaD, which is proposed to be buried within the TC (6,10).

In some embodiments, anti-IpaD VHHs that impair Shigella virulence activities can be fused into multimers to further enhance these inhibitory activities. In some embodiments, anti-IpaD VHHs multimers can bind to TCs in a multivalent manner to display an additive effect. Such an effect would be attractive for their use as novel therapeutics. In some embodiments, the additive effect can be due to an increase in the binding affinity, and the neutralization occurs through a form of steric hindrance caused by the added bulk following a single binding event. Such a possibility is supported by the observation that a non-inhibitory VHH fused with an inhibitory VHH also enhances the ability of the latter to impair Shigella contact-mediated hemolysis. In some embodiments, the multimers can be homo- and heterotrimers, tetramers, etc. having an increased neutralizing activity than the individual monomers.

In some embodiments, a panel of single-domain antibodies (VHHs or nanobodies) that recognize distinct epitopes within IpaD have been generated. Through a variety of biochemical, molecular and structural techniques, structural epitopes within IpaD that are critical for proper T3SS function have been identified. The VHHs, according to embodiments of the invention, display a diverse ability to recognize the in situ tip complex and modulate the infectious properties of the T3SS-possessing pathogens, for example Shigella.

The structural elucidation of several IpaD-VHH complexes as shown herein enabled insights into tip complex formation and function, with potential applications across other T3SS-possessing pathogens.

Polynucleotides encoding VHH and VHH containing binding proteins

The compositions and methods described herein in various embodiments include an isolated polynucleotide sequence or an isolated polynucleotide molecule that encodes a binding protein molecule comprising the VHHs described herein. Accordingly, the isolated polynucleotide sequence or isolated polynucleotide molecule comprises or consists of a polynucleotide sequence that encodes a polypeptide molecule having an amino acid sequence of SEQ ID NOs: 1-7, or a functional portion thereof, as described herein. In an embodiment, a composition comprises a combination of the isolated polynucleotide sequences or isolated polynucleotide molecules as described herein. Also encompassed by the present invention are polynucleotide sequences, DNA or RNA, which are substantially complementary to the DNA sequences encoding the polypeptides described herein, and which specifically hybridize with these DNA sequences under conditions of stringency as are known to those of skill in the art. As referred to herein, substantially complementary means that the nucleotide sequence of the polynucleotide need not reflect the exact sequence of the original encoding sequences, but must be sufficiently similar in sequence to permit hybridization with a nucleic acid sequence under high stringency conditions. For example, non-complementary bases can be interspersed in a nucleotide sequence, or the sequences can be longer or shorter than the polynucleotide sequence, provided that the sequence has a sufficient number of bases complementary to the sequence to allow hybridization thereto. Conditions for stringency are described, e.g., in Ausubel, F. M., et ah, Current Protocols in Molecular Biology, (Current Protocol, 1994), and Brown, et ah, Nature, 366:575 (1993); and further defined in conjunction with certain assays.

Vectors, plasmids or viruses containing one or more of the polynucleotide molecules encoding the amino acid sequence of SEQ ID NOS: 1-7 are also provided. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by the skilled practitioner in the art. Additional vectors can also be found, for example, in Ausubel, F. M., et ak, Ibid and in Sambrook et ah, "Molecular Cloning: A Laboratory Manual," 2nd ED. (1989), and other editions.

Any of a variety of expression vectors (prokaryotic or eukaryotic) known to and used by those of ordinary skill in the art may be employed to express recombinant polypeptides described herein. Expression can be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule that encodes a recombinant polypeptide. Suitable host cells include prokaryotes, yeast and higher eukaryotic cells. By way of example, the host cells employed include, without limitation, E. coli , yeast, insect cells, or a mammalian cell line such as COS or CHO. The DNA sequences expressed in this manner can encode any of the polypeptides described herein, including variants thereof.

Uses of plasmids, vectors or viruses containing polynucleotides encoding the VNA protein molecules as described herein includes generation of mRNA or protein in vitro or in vivo. In related embodiments, host cells transformed with the plasmids, vectors, or viruses are provided, as described above. Nucleic acid molecules can be inserted into a construct (such as a prokaryotic expression plasmid, a eukaryotic expression vector, or a viral vector construct, which can, optionally, replicate and/or integrate into a recombinant host cell by known methods. The host cell can be a eukaryote or prokaryote and can include, for example, yeast (such as Pichia pastoris or Saccharomyces cerevisiae), bacteria (such as E. coli , or Bacillus subtilis), animal cells or tissue (CHO or COS cells), insect Sf9 cells (such as baculoviruses infected SF9 cells), or mammalian cells (somatic or embryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamster ovary (CHO) cells, HeLa cells, human 293 cells and monkey COS-7 cells). Suitable host cells also include a mammalian cell, a bacterial cell, a yeast cell, an insect cell, and a plant cell.

A VHH protein-encoding polynucleotide molecule can be incorporated or inserted into the host cell by known methods. Examples of suitable methods for transfecting or transforming host cells include, without limitation, calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. "Transformation" or "transfection" as appreciated by the skilled practitioner refers to the acquisition of new or altered genetic features by the incorporation of additional nucleic acids, e.g., DNA, into cellular DNA. "Expression" of the genetic information of a host cell is a term of art which refers to the directed transcription of DNA to generate RNA that is, in turn, translated into a polypeptide. Procedures for preparing recombinant host cells and incorporating nucleic acids are described in more detail in Sambrook et ah, "Molecular Cloning: A Laboratory Manual," Second Edition (1989) and Ausubel, et al. "Current Protocols in Molecular Biology," (1992), and later editions, for example.

A transfected or transformed host cell is maintained under suitable conditions for expression and recovery of the polypeptides described herein. In certain embodiments, the cells are maintained in a suitable buffer and/or growth medium or nutrient source for growth of the cells and expression (and secretion) of the gene product(s) into the growth medium.

The type of growth medium is not critical to the invention and is generally known to those skilled in the art, such as, for example, growth medium and nutrient sources that include sources of carbon, nitrogen and sulfur. Examples include Luria-Bertani (LB) broth,

Superbroth, Dulbecco's Modified Eagles Media (DMEM), RPMI-1640, M199 and Grace's insect media. The growth medium can contain a buffering agent, as commonly used in the art. The pH of the buffered growth medium may be selected and is generally a pH that is tolerated by, or optimal for, growth of the host cell, which is maintained under a suitable temperature and atmosphere.

In another aspect, an RNA polynucleotide, in particular, mRNA, encodes the VHH molecules described herein. mRNA encoding the VHH described herein may contain a 5' cap structure, a 5' UTR, an open reading frame, a 3' UTR and poly- A sequence followed by a C30 stretch and a histone stem loop sequence (Thess, A. et al., 2015, Mol Ther , 23(9): 1456- 1464; Thran, M. et al., 2017, EMBO Molecular Medicine, DOI:

l0. l5252/emmm.201707678). Sequences may be codon-optimized for human use. In an embodiment, the mRNA sequences do not include chemically modified bases. mRNAs encoding the VNAs as described herein may be capped enzymatically or further

polyadenylated for in vivo studies/use.

Expression of proteins, which normally have a shortened serum half-life, by encoding mRNA, particularly sequence optimized, unmodified mRNA, advantageously prolongs the bioavailability of these proteins for in vivo activity (see, e.g., K. Kariko et al, 2012, Mol. Ther., 20:948-953; Thess, A. et al., 2015, Mol Ther, 23(9): 1456-1464;). Accordingly, multimeric and heteromultimeric VNAs with an estimated serum half-life of 1-2 days (with albumin-binding) are likely to benefit from being encoded by mRNA. As reported, the half- lives of VNA serum titers at one to three days after treatment were estimated to be, on average, 1.5-fold higher than from day three onward, even without target-specific mRNA optimization. (Mukheijee et al., 2014, PLoS ONE, 9el06422). In general, one to three days after treatment, both mRNA and protein half-lives contribute to the kinetics of serum titers, while after day three forward, the kinetics is almost exclusively determined by the properties of the expressed protein.

In some embodiments, a VHH binding protein monomer, can be modified, for example, by attachment (e.g., directly or indirectly via a linker or spacer) to another VHH binding protein monomer. In some embodiments, a VHH binding protein monomer is attached or genetically (recombinantly) fused to another VHH binding protein monomer. Accordingly, the polynucleotide (DNA) that encodes one VHH binding protein monomer is joined (in reading frame) with the DNA encoding a second VHH binding protein monomer, and so on. In certain embodiments, additional amino acids are encoded within the polynucleotide between the VHH binding protein monomers so as to produce an unstructured region (e.g., a flexible spacer) that separates the VHH binding protein monomers, e.g., to better promote independent folding of each VHH binding protein monomer into its active conformation or shape. Commercially available techniques for fusing proteins (or their encoding

polynucleotides) may be employed to recombinantly join or couple the VHH binding protein monomers into the multimeric binding proteins containing two or more of the same or different VHH binding proteins as described herein.

Polynucleotide sequences encoding the binding proteins comprising the VHH as described herein can be recombinantly expressed and the resulting encoded VHH can be produced at high levels and isolated and/or purified. In an embodiment, the recombinant VHH are produced in soluble form.

Pharmaceutical Compositions

The present invention features methods for treating or preventing pathologies and disease caused by Gram negative pathogens having a type III secretion system. The methods include administering to a subject in need thereof an effective amount of the binding proteins described herein. In an embodiment, the binding proteins are provided or used in a pharmaceutical composition. In an embodiment, the binding proteins specifically binds to the distal portion of IpaD of the tip complex of the T3SS and neutralizes the activity of the T3SS.

Typically, a carrier or excipient is included in a composition as described herein, such as a pharmaceutically acceptable carrier or excipient, which includes, for example, sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous sucrose, dextrose, or mannose solutions, aqueous glycerol solutions, ethanol, calcium carbonate, albumin, starch, cellulose, silica gel, polyethylene glycol (PEG), dried skim milk, rice flour, magnesium stearate, and the like, or combinations thereof. The terms "pharmaceutically acceptable carrier" and a "carrier" refer to any generally acceptable excipient or drug delivery device that is relatively inert and non-toxic.

The preparation of such solutions ensuring sterility, pH, isotonicity, and stability is effected according to protocols established in the art. Generally, a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g ., subcutaneous, intramuscular, intranasal, and the like. Such methods also include administering an adjuvant, such as an oil-in-water emulsion, a saponin, a cholesterol, a phospholipid, a CpG, a polysaccharide, variants thereof, and a combination thereof, with the composition of the invention. Optionally, a formulation for prophylactic administration may also contain one or more adjuvants for enhancing the effect of, or an immune response to, an antigen or immunogen, e.g., binding proteins as described herein. Suitable adjuvants include, without limitation, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum , and the synthetic adjuvants QS-21 and MF59. In an embodiment, the binding protein molecule is provided in a pharmaceutical composition.

The administration of a binding protein as described herein, or a pharmaceutical composition thereof, as a therapeutic for the treatment or prevention of disease or pathology caused by Gram negative pathogens having a type III secretion system infection may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, if desired, is effective in ameliorating, reducing, eliminating, abating, or stabilizing disease, pathology, or the symptoms thereof in a subject. The therapeutic may be administered systemically, for example, formulated in a pharmaceutically-acceptable composition or buffer such as physiological saline.

Routes of administration include, for example and without limitation, subcutaneous, intravenous, intraperitoneal, intramuscular, intrathecal, intraperitoneal, or intradermal injections that provide continuous, sustained levels of the therapeutic in the subject. Other routes include, without limitation, gastrointestinal, esophageal, oral, rectal, intravaginal, etc. The amount of the therapeutic to be administered varies depending upon the manner of administration, the age and body weight of the subject, and with the clinical symptoms of the bacterial infection or associated disease, pathology, or symptoms. Generally, amounts will be in the range of those used for other agents used in the treatment of disease or pathology associated with Gram negative pathogens having a type III secretion system infection, although in certain instances, lower amounts may be suitable because of the increased range of protection and treatment afforded by the binding protein as therapeutic. A composition is administered at a dosage that ameliorates, decreases, diminishes, abates, alleviates, or eliminates the effects of the bacterial (microorganism) infection or disease (e.g., CID or the symptoms thereof) as determined by a method known to one skilled in the art.

In embodiments, a therapeutic or prophylactic treatment agent may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.

Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Pharmaceutical compositions may in some cases be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of a therapeutic agent or drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of a therapeutic agent or drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the gut or gastrointestinal system; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a disease using carriers or chemical derivatives to deliver the therapeutic agent or drug to a particular cell type. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain a therapeutic level in plasma, serum, or blood.

In an embodiment, one or more multimeric binding protein may be formulated with one or more additional components for administration to a subject in need.

Any of a number of strategies can be pursued in order to obtain controlled release of a therapeutic agent in which the rate of release outweighs the rate of metabolism of the therapeutic agent or drug in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic agent or drug may be formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic agent or drug in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

A pharmaceutical composition may be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intradermal,

intraperitoneal, intrathecal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. A pharmaceutical composition may also be provided by oral, buccal, topical (e.g., via powders, ointments, or drops), rectal, mucosal, sublingual, intraci sternal, intravaginal, rectal, ocular, or intranasal administration. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, noted supra.

Compositions for parenteral or oral use may be provided in unit dosage forms (e.g., in single-dose ampules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. The composition may include suitable parenterally acceptable carriers and/or excipients. In some cases, an active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.

In some embodiments, a pharmaceutical composition comprising an active therapeutic is formulated for systemic delivery, intravenous delivery, e.g., intravenous injection, subcutaneous delivery, or local delivery (e.g., diffusion). To prepare such a composition, the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle, excipient, or solvent. Among acceptable vehicles and solvents that may be employed are, for example, water; water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer; l,3-butanediol; Ringer's solution; and isotonic sodium chloride solution and dextrose solution. An aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases in which a therapeutic agent is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.

In some embodiments, compositions comprising the binding proteins are sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds. In some embodiments, the binding proteins are combined, where desired, with other active substances, e.g., enzyme inhibitors, to reduce metabolic degradation.

An effective amount of compositions can vary according to the choice or type of the binding proteins as described herein, the particular composition formulated, the mode of administration and the age, weight and physical health or overall condition of the patient, for example. In an embodiment, an effective amount of the binding proteins is an amount which is capable of reducing one or more symptoms of the disease or pathology caused by the infectious agent/disease target. Dosages for a particular patient are determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol).

In certain embodiments, a composition includes one or more polynucleotide sequences that encode one or more of the binding proteins as described herein. In an embodiment, a polynucleotide sequence encoding a binding protein is in the form of a DNA molecule. In some embodiments, the composition includes a plurality of nucleotide sequences each encoding a binding protein molecule, or any combination of molecules described herein, such that the binding protein molecule is expressed and produced in situ. In such compositions, a polynucleotide sequence is administered using any of a variety of delivery systems known to those of ordinary skill in the art, including eukaryotic, bacterial and viral vector nucleic acid expression systems. Suitable nucleic acid expression systems contain appropriate nucleotide sequences operably linked for expression in a patient (such as suitable promoter and termination signals). Bacterial delivery systems involve administration of a bacterium (such as Bacillus-Calmette-Guerrin) that expresses the polypeptide on its cell surface. In an embodiment, the binding protein molecule-encoding nucleic acid can be introduced using a viral expression system (e.g., vaccinia or other pox virus, retrovirus, or adenovirus), which uses a non-pathogenic (defective), replication competent virus. Techniques for incorporating nucleic acid (DNA) into such expression systems are well known to those of ordinary skill in the art. The nucleic acid (DNA) can also be "naked," as described, for example, in Ulmer et ah, 1993, Science , 259: 1745-1749 and as reviewed by Cohen, 1993, Science 259: 1691-1692. The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into recipient cells.

Therapeutic Methods

Methods of treating disease, conditions, pathology and/or symptoms thereof associated pathogen that use T3SS for virulence are provided. The methods comprise administering a therapeutically effective amount of a binding protein as described herein, or a pharmaceutical composition comprising such one or more binding protein to a subject (e.g., a mammal such as a human). The method includes the step of administering to a mammal a therapeutic amount of the binding protein as described herein sufficient to treat the disease, illness, condition, disorder and/or symptom thereof, under conditions such that the disease or disorder is treated.

The therapeutic methods of the invention (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the binding protein as described herein, to a subject or patient in need thereof. A subject or patient is meant to include an animal, particularly a mammal, and more particularly, a human. Such one or more multimeric VNA binding protein molecules used as treatment will be suitably administered to subjects or patients suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof caused by or associated with infection by gram negative pathogen that uses T3SS. Determination of patients who are "at risk" can be made by any objective or subjective determination obtained by the use of a diagnostic test or based upon the opinion of a patient or a health care provider (e.g., genetic test, enzyme or protein marker, family history, and the like). The binding proteins as described herein may be also used in the treatment of any other disorders in which the one or more target protein toxins may be implicated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of the binding proteins as described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject himself or herself, or of a health care/medical professional and can be subjective (e.g., opinion) or objective (e.g., measurable or quantifiable by a test or diagnostic method).

Methods of Use

In an aspect, a method of monitoring treatment progress is provided. The method includes determining a level of toxin protein as an indicator of disease or infection in a subject suffering from or susceptible to infection by, or disease or illness associated with infection by T3SS possessing pathogens, in which the subject has been administered a therapeutic amount of one or more of the multimeric VNA binding protein molecules as described herein sufficient to treat the disease or symptoms thereof. According to the method, the level of the toxin protein(s) (which serves as a marker of infection or disease) is detected, measured, or quantified in a biological sample obtained from the subject relative to known levels of the same toxin protein(s) in healthy normal controls and/or in other afflicted patients to establish the subject’s treatment progress, disease progress, or disease status. In embodiments, the levels of toxin protein(s) in the subject’s sample are measured or quantified at one or more later time points (following the previous measurements), relative to the levels previously detected or measured in the subject, and/or relative to the levels in normal/healthy subjects or in other afflicted patient controls to monitor the course of disease or the efficacy of the therapy. In certain embodiments, a pre-treatment level of toxin protein(s) in the subject is determined prior to beginning treatment according to the method; this pre-treatment level of toxin protein(s) can then be compared to the level of the toxin protein(s) in the subject after the treatment commences to monitor or determine the efficacy of the treatment.

Kits

The invention provides kits for the treatment or prevention of an infection or disease caused by or associated with gram negative pathogens that use T3SS for virulence. In some embodiments, the kit includes an effective amount of a binding protein as described herein, in unit dosage form. In other embodiments, the kit includes a therapeutic or prophylactic composition containing an effective amount of a binding protein in unit dosage form. In some embodiments, the kit comprises a device (e.g., an automated or implantable device for subcutaneous delivery; an implantable drug-eluting device, or a nebulizer or metered-dose inhaler) for dispersal of the composition or a sterile container which contains a

pharmaceutical composition. Non-limiting examples of containers include boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired, a pharmaceutical composition of the invention is provided together with instructions for administering the pharmaceutical composition containing a binding protein to a subject having or at risk of contracting or developing an infection or disease or pathology, and/or the symptoms thereof, associated with infection by gram negative pathogens that use T3SS for virulence. The instructions will generally include information about the use of the composition for the treatment or prevention of an infection and intoxication by the gram negative bacteria and the toxin proteins that they produce. In other embodiments, the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of infection, disease or symptoms thereof caused by one or more of gram negative bacteria and/or the toxin proteins that they produce; precautions; warnings; indications; counter-indications; over-dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.

The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989);“Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);“Methods in Enzymology”“Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. EXAMPLES

EXAMPLE 1- Structural Characterization of IpaD in Complex with Single-Chain Antibodies Provide Insight into the Type III Secretion Needle Tip Complex

Many Gram-negative bacterial pathogens use a type III secretion system (T3SS) to inject host-modulating proteins during infection. At the distal end of the exposed portion of the Shigella T3SS, a tip complex (TC) is assembled that consists of a pentamer of IpaD, but which is ultimately comprised of IpaD and one or both hydrophobic translocator proteins (IpaB and IpaC) depending upon its activation state. To date, the exact stoichiometry and functional topology of these proteins within the TC remains elusive, with many discordant reports suggesting either a pentamer of IpaD or a four-plus-one complex of IpaD and IpaB (6,10). To better understand how IpaD is assembled within the Shigella TC, a panel of IpaD- specific VHHs was generated. Through a variety of biochemical, molecular and structural techniques, the VHHs were characterized and the structural epitopes within IpaD that are critical for proper T3SS function were identified.

MA TERIALS AND METHODS

Alpaca Immunizations and VHH Display Library Preparation

One alpaca ( Vicugna pacos) was used in this study. Immunizations and VHH-display library construction were performed as previously described (38,39). Specifically, the alpaca received five successive subcutaneous injections at 3-week intervals, using an immunogen consisting of purified recombinant IpaD (14). Peripheral blood lymphocytes were obtained from blood five days after the final immunization of the alpaca. RNA and then cDNA were produced from the peripheral blood lymphocytes and PCR was used to amplify the VHH coding repertoire from the cDNA. Using high efficiency E. coli transformation methods, more than 10⁶ independent clones were obtained and pooled to generate the VHH-display phage library.

Anti-IpaD VHH Identification. Expression and Purification

Panning, phage recovery, and clone fingerprinting were performed as previously described (18,24,38). Two rounds of panning were performed with purified IpaD coated onto Nunc Immunotubes. A single low stringency panning approach was employed using 10 pg/ml target antigen. After phages were eluted, they were amplified and subjected to a second round of panning at high stringency with 1 pg/ml target antigen; employing lO-fold reduced input phage, shorter binding times and longer washes. Following the second high stringency round of panning, individual E. coli colonies were picked and grown overnight at 37°C in 96-well plates. A replica plate was then prepared, cultured, induced with Isopropyl B- D-l thiogalactopyranoside (IPTG) and the supernatant was assayed for IpaD binding by ELISA.

From each of the two-cycle panning regimens, >50% of VHH clones bound to IpaD as evidenced by ELISA reactivity values that were >2X over negative controls. The strongest positive binding clones for IpaD were characterized by DNA fingerprinting (18). Multiple clone groups with unique fingerprints were identified among the VHHs selected for binding to IpaD. DNA sequences of the VHH coding regions were obtained for representatives from each identified fingerprint group and sequences were compared for homologies. Based on this sequence analysis, IpaD VHHs identified as unlikely to have common B cell clonal origins were selected for protein expression.

Selected VHH coding DNAs were cloned into the pET32b expression vector

(Novagen) for cytosolic expression in E. coli Rosetta-gami 2 (DE3)pLacl (Novagen) as a fusion to thioredoxin. All VHHs were expressed with hexahistidine (His₆) to facilitate subsequent purification using standard Ni-IMAC chromatography methods, and a carboxyl terminal epitope tag for detection, either E-tag (GAPVPYPDPLEPR), (SEQ ID NO: 9) or myc tag (MEQKLISEEDL), (SEQ ID NO: 10).

Enzyme Linked Immunosorbent Assay (ELISA)

Competition ELISAs were performed as follows. Purified VHHs expressed with a c- myc tag were tested for binding in the presence of 40x excess of purified VHHs having an E- tag, or vice versa. Nunc MaxiSorb plates were coated overnight at 4°C with 1.0 pg/ml IpaD antigen. Plates were blocked with 4% non-fat dried milk in PBS-Tween 0.1% for two hours at 37°C. The competitor VHHs, i.e. myc- VHHs, were diluted in blocking solution to 10 pg/ml and added to each well of the plate going down the columns and allowed to incubate for 30 minutes at room temperature on a rocker platform. The plate was then thoroughly washed with PBS-T followed by PBS. E-tag VHHs were added to the washed wells at 0.25 pg/ml in blocking solution going across the rows of the plate and allowed to incubate for 1 hour at room temperature on a rocker platform. The plate was then thoroughly washed with PBS-T followed by PBS. To the clean wells, HRP goat anti-E-tag mAh diluted 1 : 10,000 in blocking solution was added to each well. Following incubation, wells were washed with PBS-T followed by PBS. Bound HRP was detected using the SureBlue Peroxidase Substrate (KPL, Gaitherburg, MD). The reaction was quenched with 1 M phosphoric acid and absorbance was read at 450 nm.

Contact-Mediated Hemolysis

To determine the effect of VHHs on a known Shigella virulence activity a

modification of a standard contact-mediated hemolysis assay was used as described in Picking et al. (40). In the modified assay, the bacteria were incubated with VHHs (0.1 mg/ml) for 30 min prior to the addition of sheep red blood cells.

VHH Affinity Determinations

The binding affinity of purified His-tagged IpaD (residues 39-322^C322S) with VHHs was monitored by biolayer interferometry (BLI) using an Octet RED96 instrument (Pall ForteBio). IpaD was loaded onto Ni-NTA biosensors (ForteBio) at a concentration of 6.25 pg/ml for 5 min, in IX kinetics buffer (IX PBS pH 7.4, 0.01% BSA, 0.002% Tween 20). All reactions were performed at 25 °C. Real-time data were analyzed use Octet Software version 8.2 (ForteBio). Binding kinetics (association and dissociation) as well as steady-state equilibrium concentrations were fitted using a 1 : 1 Langmuir binding model.

Crystallization

IpaD (residues 39-322^C322S) was purified as previously described (25). IpaD-specific VHHs were purified as described above, with one modification. Prior to expression, the thrombin cleavage sequence within pET32b was modified to a TEV cleavage sequence for ease of purification. Prior to crystallization, IpaD and individual VHHs were mixed in a 1 : 1 molar ratio and injected onto a HiLoad 26/60 Superdex 200 column. The primary peak, containing both IpaD and VHH, was collected and concentrated to 10 mg/ml in 10 mM Tris- HC1 (pH 7.5), 50 mM NaCl for crystal screening. Crystals of IpaD-VHH complexes were obtained by vapor diffusion in Compact Jr. (Emerald Biosystems) sitting drop plates at 20 °C by mixing 0.5 mΐ of protein solution with 0.5 mΐ of reservoir solution. Initial crystal hits were obtained for IpaD-20ipaD complex in Hampton Index D4 (0.1 M citric acid (pH 3.5), 25% (w/v) PEG 3350), for IpaD-JMK-E3 complex in Hampton Index F2 (0.2 M trimethylamine N-oxide dehydrate, 0.1 M Tris-HCl (pH 8.5), 20 % (w/v) PEG 2000), for IpaD-JMK-H2 complex in Hampton Index Hl (0.2 M magnesium chloride, 0.1 M Tris-HCl (pH 8.5), 25% PEG 3350) and for IpaD-JPS-G3 complex in Emerald Wizard III D6 (0.2 M lithium sulfate, 0.1 M Tris-HCl (pH 8.5), 30% (w/v) PEG 4000). Crystals were flash-cooled in a cryoprotectant solution consisting of mother liquor supplemented with 20% (w/v) PEG 200. Diffraction Data Collection. Structure Determination. Refinement and Analysis

X-ray diffraction data were collected on all IpaD-VHH crystals at 1.000 A at 100 K using a Dectris Pilatus 6M pixel array detector at IMCA-CAT beamline 17ID at the APS (Table 2). Following data collection, individual reflections from each dataset were integrated with XDS (41) and scaled with Aimless (42).

Experimental phase information was obtained for each IpaD-VHH structure using molecular replacement (PHASER) within the Phenix suite (43,44). Search models for each VHH were generated using PHYRE (45), while the IpaD model was obtained from PDB entry 2JOO (14).

Structure refinement for each IpaD-VHH structures was carried out using Phenix (43,44). One round of individual coordinates and isotropic atomic displacement factor refinement was conducted, and the refined model was used to calculate both 2 F₀-F_c and F₀-F_c difference maps. These maps were used to iteratively improve the model by manual building with Coot (46,47) followed by subsequent refinement cycles. TLS refinement (48) was incorporated in the final stages to model anisotropic atomic displacement parameters.

Hydrogen atoms were included during all rounds of refinement. Ordered solvent molecules were added according to the default criteria of Phenix and inspected manually within Coot prior to model completion. The following residues within each complex were not modeled as a result of poor map quality, IpaD/JMK-H2: IpaD, residues 39-40, JMK-H2, residues 1, 127- 128; IpaD/JMK-E3: IpaD, residues 124-127 (chain A), residues 39-42, 321-322 (chain C), residues 39-41, 124-126 (chain E), residues 39-41, 321-322 (chain G), residues 39-41, 182- 185, 240-241, 322 (chain I), JMK-E3, residue 128 (chain D), residue 128 (chain F) and residues 127-128 (chain J); IpaD/JPS-G3: IpaD, residues 39, 124-127, 182-185, JPS-G3, residue 128; and IpaD/20ipaD: IpaD, residues 39-41, 97-109, 116-118, 122-136, 321-322, 20ipaD, residue 1.

Multiple Sequence Alignments and Figure Modeling

Multiple sequence alignments were carried out using ClustalW (49) and aligned with secondary structure elements using ESPRIPT (50). Three-dimensional structures were superimposed using the Local-Global Alignment method (LGA) (51). Representations of all structures were generated using PyMol (52). Identification of IpaD-specific Mills

A VHH-display phage library representing the hcAb repertoire of one alpaca repeatedly immunized with purified recombinant IpaD from S. flexneri was prepared and subjected to multiple rounds of panning. This allowed for the identification of 12 phagemids encoding unique VHHs with strong IpaD binding activity when displayed by phage. After DNA sequencing and expression as recombinant proteins in E. coli , 7 VHHs (Fig. 1A) were selected based on further characterization (see below).

To characterize the binding epitopes on IpaD for the VHHs, and to identify superior binding candidates for subsequent study, two experimental methods were employed (FIGS. 2A-C and Table 1).

Table 1. Summary of IpaD-VHH Binding Data and Interface Information

(a) IpaD-specific VHHs were tested for the ability to inhibited contact-mediated hemolysis by S. flexneri. The number of plus signs indicates the degree of inhibition relative to wild-type bacteria. - = no reduction, + = 10-20% reduction, ++ = 20-40% reduction, +++ = 40-60% reduction and ++++ = >60% reduction.

(b) Values indicate effective VHH concentration for half-maximal binding to IpaD as measured by Bio-Layer Interferometry (BLI).

(c) Similar epitope clusters based upon competition ELISA (see Fig. 2C and Fig. 6).

(d) Number of hydrogen bonding interactions involving each complementarity- determining region (CDR).

Initial identification of binding regions within IpaD recognized by the different VHHs was completed using IpaD deletion fragments in an immunoblot analysis (Fig. 2B). The crystal structure of the IpaD monomer has been previously determined (14) and was found to have three predominant structural features (Fig. 2A). These include a core central coiled-coil (residues 131-177 and 273-332) flanked by an N-terminal helix-turn-helix domain previously suggested to have a self-chaperoning role (residues 1-130) and globular distal domain comprised of mixed a/b components (residues 178-272).

Purified full-length (FL), D1-120 and Adi stal -do ain IpaD proteins were used to probe for binding within each VHH cluster (Fig. 2B). As expected, all selected VHHs were capable of binding to IpaD^KL, however, they were also capable of binding to IpaD^{A1 120}, indicating there were no paratopes binding within the N-terminal domain for any identified VHH. Intriguingly, none of the VHHs were capable of binding to IpaD^{Adlstal doma} (Fig. 2B) indicating that this region is required for an interaction to occur.

Competition ELISAs were then performed with recombinant VHHs differentially encoding either c-Myc or E-tag epitopes (Fig. 6). From these experiments, it appears that there are 3 unique competition groups present within the 7 selected VHHs: bin 1) JMK-E3, JMK-F11, JPS-G3 and 20ipaD; bin 2) JMK-H2 and bin 3) JMK-H5 and JMK-G3 (Fig. 2C). Partial competition with JMK-H2 was observed with VHHs from bin 1, especially JMK-F11 and 20ipaD. Therefore, this VHH is categorized as belonging to bins 1 and 2 (Table 1). VHH-Specific Inhibition of Shigella Virulence Traits

S. flexneri formation of translocon pores in target cell membranes requires a functional TC protein (IpaD) and functional translocator proteins (IpaB and IpaC), and this activity is routinely determined using a contact-mediated hemolysis assay. The ability of each IpaD-specific VHH to inhibit contact-mediated hemolysis was measured to determine whether any were capable of neutralizing a known virulence trait (Fig. 3 and Table 1). Four VHHs (20ipaD (SEQ ID NO: 1), JPS-G3 (SEQ ID NO: 2), JMK-F11 (SEQ ID NO: 3) and JMK-E3 (SEQ ID NO: 4)) were capable of significantly decreasing hemolytic activity between 30-50% when compared to wild-type S. flexneri in multiple studies. For

comparison, rabbit anti-IpaD IgG has been shown to neutralize S. flexneri contact-hemolysis activity by over 75% (9). In contrast to the VHHs described herein, the anti-IpaD sera appears to exclusively recognize epitopes within the N-terminal domain (residues 1-120) (9). Therefore, it appears that both the N-terminal and distal domains contribute to productive assembly of the T3SS translocon pore. Surprisingly, despite also requiring the presence of the IpaD distal domain, the other 3 IpaD-specific VHHs were essentially incapable of altering hemolysis. Without wishing to be bound by theory, it is unclear whether this results from structural epitopes that either are buried in the IpaD pentamer within the TC or are not important to proper T3SS function.

It was previously demonstrated that heterodimers created by covalent linkage of two inhibitory VHH monomers to create bi-specific antibodies typically leads to agents with increased affinity and neutralization properties (18-24). The Shigella T3SS TC is generally believed to be comprised of five molecules of IpaD for the vast majority of the injectisomes on a given bacterium (6, 10). This suggests that there are potentially multiple VHH binding sites available, which means that physically linking VHHs together could increase binding affinity and neutralization potency. Therefore, three VHH heterodimers were designed, each having a flexible peptide spacer (GGGGS)₃ and consisting of two monomer VHHs with varying efficacies. Two of the VHH heterodimers included a potent inhibitor VHH, 20ipaD or JPS-G3, linked to a non-neutralizing VHH, JMK-G3, that binds a non-overlapping epitope, while the third heterodimer consisted of both neutralizing VHHs, 20ipaD and JPS-G3. The combination of two neutralizing VHHs was predicted to provide increased inhibition potency compared to each monomeric component.

Inhibition of Shigella contact-mediated hemolysis by the linked VHHs was then evaluated for each of the VHH heterodimers in a similar manner as the individual VHHs. Strikingly, the 20ipaD/JPS-G3 heterodimer consistently reduced hemolytic activity by >80% (Fig. 3 and Table 1), indicating that physical linkage of two inhibitory VHHs resulted in increased potency over either monomeric component. Heterodimers involving JMK-G3, a non-inhibitory VHH, were capable of inhibiting hemolysis by 60% (20ipaD/JMK-G3) and 50% (JPS-G3/JMK-G3), -10% more than the monomeric inhibitory VHH alone (p-values < 0.06 for both heterodimers). Taken together, these data suggest that binding to IpaD at multiple sites, whether within a single polypeptide or in the context of the TC pentamer leads to increased inhibition of Shigella T3SS activity. Alternatively, because a dimer consisting of a neutralizing and non-neutralizing VHH showed enhanced inhibition of contact- hemolysis, the increased mass caused by the binding of the dimers at a single site might also contribute to increased inhibitory activity.

Characterization of VHH Binding to Recombinant IpaD

Binding kinetics for the IpaD-specific VHHs were analyzed using biolayer

interferometry (BLI) analysis to determine the relationship between functional attributes and dissociation constants ( K_D ). Intriguingly, despite clear differences in the ability to inhibit Shigella pathogenesis, the binding affinities of each VHH were quite similar (Table 1 and Figs. 7A-7C), with all VHHs exhibiting sub-20 nM dissociation constants. Nearly all of the VHHs had similar k_on rates (between 1-5 xlO⁵ M ¹ s ¹). Further insight into the relationship between binding affinity and inhibitory virulence is apparent when comparing k₀ff rates across VHHs with similar K_D values. For example, the VHHs with the highest inhibitory capability (20ipaD, JPS-G3 and JMK-E3) all have K_D values between 1-10 nM and relatively similar k_on rates (~3 x 10⁵ M ¹ s ¹), however, the Aerates between these three VHHs vary by nearly 15- fold, with 20ipaD displaying the slowest off rate and highest inhibition of contact-hemolysis activity. It is also clear that the K_D and Aerates are not the only mediators of inhibitory activity, as the VHHs with no detectable ability to prevent ShigeHa-med\ ated hemolysis have binding kinetics in the same range as the neutralizing VHHs. Thus, these data indicate that differences in the ability to interact with S. flexneri are most likely reflective of unique structural epitopes that are surface exposed within the TC rather than the affinity of the interaction.

Structural Investigation of V I 111 Binding to IpaD

To identify the regions of IpaD that are targets for antibody neutralization of virulence and must therefore be exposed within the TC in situ , the crystal structures of IpaD in complex with four VHHs was determined (Table 2).

Table 2. Diffraction Data and Refinement Statistics _

IpaD 39-322 IpaD 39-322 IpaD 39-322 IpaD 39-322

Data Collection

Unit-cell parameters (A) o=56.84, a=79.01, a=55.84, 0=58.64, b=l08.22, b=l63.35, b=8l .66, b=83.51, c=l72. l6 c=2l6.02 c=93.00 c=l8l .6l a=b=g=90 a=b=g=90 a=b=g=90 a=b=g=90

Space group 7222 R2i2i2i R2i2i2 C222i Resolution (A)¹ 45.96-2.90 48.62-2.50 47.87-2.80 47.99 (2.05)

(3.08-2.90) (2.54-2.50) (2.95-2.80)

Wavelength (A) 1.000 1.000 1.000 1.000

Temperature (K) 100 100 100 100

Observed reflections 81,449 651,001 71,850 182,091

(12,861) (33,653) (10, 187) (14,325)

Unique reflections 12,317 (1,940) 96, 196 (4,787) 11,000 28,469

(1,573) (2, 191)

<//s(7)>¹ 10.7 (1.7) 10.8 (2.0) 7.3 (1.9) 13.0 (1.9) IpaD 39-322 IpaD 39-322 IpaD 39-322 IpaD 39-322

JMK-H2 JMK-E3 JPS-G3 20ipaD

)

Refinement

Resolution (A) 38.1-2.55 45.0-2.50 47.8-2.80 34.4-2.05

(2.63-2.55) (2.53-2.50) (2.95-2.80) (2.12-2.05)

Reflections 33,611/1,650 183,858/9228 20,098/980 28,416/1,356

(working/test) (2,641/149) (6,019/264) (2,728/143) (2,673/151)

-//factor / //free (%)³ 20.21/27.29 19.72/26.10 20.17/27.31 22.61/25.18

(28.11/36.10) (30.66/37.78) (28.72/32.03) (31.09/35.99)

No. of atoms (Protein 3,117/17 15,133/186 2,985/7 2,683/24 /Solvent)

Model Quality

R.m.s deviations

Bond lengths (A) 0.002 0.002 0.002 0.002

Bond angles (°) 0.536 0.513 0.419 0.457

Average 5-factor (A²)

All Atoms 65.6 50.2 46.3 54.3

Protein 65.8 50.3 46.4 54.4

IpaD 67.5 51.8 49.3 58.9

VHH 61.7 47.0 39.5 45.7

Solvent 45.1 39.1 21.9 48.0

Coordinate error, 0.37 0.37 0.45 0.28 maximum likelihood (A)

Ramachandran Plot

Most favored (%) 94.5 95.8 94.0 98.3

Additionally allowed (%) 4.22 3.70 6.00 1.40

Outliers (%) 1.24 0.52 0.00 0.28

Values in parenthesis are for the highest resolution shell.

²//mcigc = å_///,:/å; \h(hkl) - <I(hkl)>\ / åM/å; h(hkl), where I, (hkl) is the intensity measured for the zth reflection and <I(hkl)> is the average intensity of all reflections with indices hkl.

3 //factor = åw/ 1 |/’₀bs (hkl) | - (Neale (hkl) || / å/_//,:/ |5obs (hkl) |; //n_Cc is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement.

⁴5_meas = redundancy -independent (multiplicity-weighted) //_magc(42,53). R_pim = precision- indicating (multiplicity-weighted) //_magc(54,55).

⁵CC_I/2 is the correlation coefficient of the mean intensities between two random half-sets of data (56,57).

Three of the VHHs (JMK-E3 (SEQ ID NO: 4), JPS-G3 (SEQ ID NO: 2) and 20ipaD (SEQ ID NO: 1)) displayed varying levels of neutralization, ranging from 30-50% inhibition, while the other VHH (JMK-H2 (SEQ ID NO: 6)) had essentially no effect on virulence. All four VHHs were capable of binding to recombinant IpaD with low nM affinity (Table 1). These four VHHs adopted the classical immunoglobulin fold with CDRs 1-3 located on one end of the b-sheet (Fig. IB). Variability within the length of CDR3 ranged from nine (JPS- G3) to 21 (JMK-H2) amino acids. The VHHs with the longest CDR3 region (JMK-H2 and 20ipaD) encoded an additional disulfide bond between CDR2 and CDR3. The structure of IpaD within each of the four complexes (Fig. 4A) was essentially unchanged from previously determined apo-forms [PDB IDs: 2JOO (14), 3R9V (25)] with overall RMSD values < 1.2 A for each structure, indicating minimal structural perturbations had occurred upon VHH binding. Analysis of the four IpaD-VHH crystal structures indicated that structural epitopes were clustered near the region connecting the IpaD distal domain and central coiled-coil (Fig. 4B and 4C), in support of the immunoblots with IpaD domain deletion fragments (described in Fig. 2B).

Three of the four IpaD-VHH crystal structures interact within a very tightly clustered region of the IpaD distal domain. All three of these VHHs were capable of inhibiting contact-mediated hemolysis, indicating that this region is important for Shigella T3SS activity. The most potent inhibitory VHH, 20ipaD (-50% inhibition), also made contact with a3 of the coiled-coil and a4 of the distal domain (Fig. 5), involving 12 intermolecular interactions that bury 610 A² of available surface area. Three residues within CDR2 and four residues within CDR3 predominantly drive the 20ipaD-IpaD interaction. Of potential importance, three IpaD residues within the distal domain (Glu200, Glu20l and Lys205) are involved in 8/12 interactions in the complex, reinforcing that this region is important for T3SS activity.

Two moderately neutralizing VHHs were JPS-G3 and JMK-E3, which were capable of inhibiting Shigella he olytic activity by 40% and 30%, respectively. Similar to the interaction of 20ipaD with IpaD, both JPS-G3 and JMK-E3 bind to IpaD at a3 of the coiled- coil and a4 of the distal domain (Fig. 5). The JPS-G3 interaction contributes 12

intermolecular interactions that bury 624 A² of available surface area. CDR2 is

predominantly responsible for this interaction, contributing four residues within CDR3 including a single interacting residue (Argl02) that forms an extensive electrostatic interaction with Glu20l of IpaD. There are 12 intermolecular interactions formed between the JMK-E3 and IpaD complex, burying 736 A² of available surface area, the most of the 3 neutralizing VHHs (Fig. 5). Additionally, JMK-E3 is the only inhibitory VHH-IpaD complex with binding interactions that involve either CDR1 or the N-terminal IpaD domain (Lys72).

Intriguingly, the VHH with minimal biological effect on Shigella (Fig. 3), JMK-H2, interacts almost exclusively with residues from the central coiled-coil (a3 and a7) of IpaD (Fig. 5), burying 708 A² of available surface area. There are 11 intermolecular interactions within this complex, with only a single hydrogen bond contributed outside of CDR3. The elongated CDR3 of JMK-H2 makes extensive contacts with IpaD residues 273-287 of a7. Despite favorable binding properties in vitro (KD 2 nM), the JMK-H2/IpaD complex suggests that this region of the coiled-coil is not important for inhibiting T3SS function, perhaps because of inaccessibility to the IpaD pentamer within the native TC.

The degree of inhibition by a VHH during contact-mediated hemolysis did not correlate well with binding affinity in vitro or the association network in the described crystallized structures (e.g. number of hydrogen bonds or buried surface area). The key to inhibitory activity appeared to require binding that included interactions with the IpaD distal domain. Analysis of the VHH-IpaD crystal structures revealed that all four complexes involved structural epitopes clustered within the distal region of the protein (Fig. 4C), predominantly including residues 165-177 (coiled-coil a3) and 198-205 (distal domain a4). Furthermore, two specific IpaD residues (Glu20l and Lys205) were involved in all three complexes with inhibitory VHHs, suggesting that this region of the protein is critical to proper function during maturation of the TC and could reflect a potential binding site for the translocator IpaB (5).

Multiple models of in situ TC structures have been proposed, however, no definitive structure or universal composition has been identified. A homopentameric array is the most common theme proposed for TC structures with such a structure proposed for the Shigella TC (10,14), the Salmonella enterica TC (31) and the Yersinia TC (32). An alternative four-plus- one (IpaD-plus-IpaB, respectively) model has been proposed for Shigella (6), however, even in those studies, the predominant TC composition is that of a homopentamer (>90% of injectisomes). It is possible that the VHHs prepared here will allow future studies to provide a definitive test of needle TC composition for nascent apparatuses. Regardless, the VHHs prepared here clearly can inhibit Shigella virulence activities (i.e. contact hemolysis), which are related to translocon pore formation. The epitopes recognized by these VHHs are largely localized to the mixed a/b structure distal domain of IpaD. This is consistent with previous studies suggesting that the distal domain is key for steps subsequent to nascent TC formation (5,10). Furthermore, VHHs that fail to impair virulence functions appear to bind to epitopes that localize to the central coiled-coil of IpaD, which is proposed to be buried within the TC (6,10).

The fact that anti-IpaD VHHs that impair Shigella virulence activities can be fused into multimers to further enhance these inhibitory activities suggest that they may be able to bind to TCs in a multivalent manner to display an additive effect. Such an effect would be attractive for their use as novel therapeutics. It cannot be ruled out, however, that this additive effect is not due to multi -valency but rather to an increase in the binding affinity, and the neutralization occurs through a form of steric hindrance caused by the added bulk following a single binding event. Such a possibility is supported by the observation that a non-inhibitory VHH fused with an inhibitory one also enhances the ability of the latter to impair Shigella contact-mediated hemolysis.

Table 3. Overlapping Epitopes within IpaD-VHH Complexes

Table 4. IpaD-VHH Interaction Information

EXAMPLE 2- In vivo assay for assessing the activity of anti-Shigella VHH molecules as therapeutic agents

In general, humans and higher primates comprise the host range of Shigella and

Shigella-related disease and infection. Thus, a suitable, small-animal enteric model is not readily available for screening the VHH therapeutic agents as newly-described herein.

However, it has been observed that the corneal epithelium of guinea pigs provides an acceptable environment for the study of virulence in an in-vivo system; such a model is known as the Serehy keratoconjunctivitis model. The Serehy test and model, first published by B. Serehy (1957, Acta Microbiol. Acad. Sci. Hung. 4:367-376), remains the most reliable in-vivo indicator of virulence of Shigella strains and of immunogenicity, therapeutic and protective efficacy of Shigella vaccine and immunogen candidates. The model is effective in evaluating the ability of Shigella strains to invade the corneal epithelia of guinea pigs and spread to contiguous cells causing ulcerative keratoconjunctivitis.

For the experiment described in this Example, the standard procedure was modified slightly for testing and assessing a therapeutic anti -Shigella toxin VHH agent for efficacy in treating an infection by Shigella in vivo. To determine whether the anti -Shigella VHHs could reduce the severity of keratoconjunctivitis in this guinea pig model, Shigella flexneri 2a strain 2457T bacteria were pre-incubated with a representative anti -Shigella VHH heterodimer, i.e., 20ipaD_JPS-G, prior to inoculation of the guinea pig’s eye. Additional 20ipaD_JPS-G3 anti- Shigella VHH was administered topically every four hours for the duration of the study (24- hours). In each of the animals, one eye was inoculated with Shigella that had been pre- incubated with 20ipaD_JPS-G3, and the contralateral eye was inoculated with the same number of Shigella bacteria that had not been pre-incubated with anti -Shigella VHH.

Treatment was administered to the animals every four hours. The eye that had been inoculated with pre-incubated Shigella bacteria was treated with additional topically administered 20ipaD_JPS-G3 anti -Shigella VHH. The contralateral eye, which had been inoculated with bacteria that had not been pre-incubated with 20ipaD_JPS-G3, received a mock treatment with sterile PBS, which was also the diluent admixed with the anti -Shigella VHH.

Efficacy within this model is generally evaluated by assigning a severity score based on gross observation of the eyes. A typical scoring system is based on a five-point scale ranging from 0 to 4, each with a pre-assigned severity definition. For such evaluations, a typical example would be a score of 0 = normal eye; 1 = lacrimation or eyelid edema; 2 = 1 plus mild conjunctival hyperemia; 3 = 2 plus slight exudate; and 4 = full purulent

keratoconjunctivitis. In this Example, ante-mortem scoring was performed by a blinded, board-certified veterinary ophthalmologist and was based on an ophthalmological examination performed under magnification with a slit-lamp. In addition, post-mortem histological severity scoring was conducted by a blinded, board-certified veterinary pathologist. FIG. 8 shows the results of ante-mortem, ophthalmological examinations that were conducted on the eyes of the animals at three time-points, namely, pre-infection, 8 hours’ post infection, and 24 hours’ post infection, at which time high quality digital photographs were taken to be evaluated at a later time. A comprehensive severity score system was subsequently created assigning scores of 0 to 3 (i.e. 0 = normal, 1= mild, 2= moderate, and 3 = severe) to each of the following findings: exudate, conjunctival chemosis, conjunctival hemorrhage, conjunctival infiltrates, corneal edema, corneal ulceration, and corneal roughness. The results of the cumulative ante-mortem severity scoring findings are presented in FIG. 9, which shows: 1) the progression of disease severity over a period of 24-hours and 2) the treatment regimen with anti -Shigella VHH 20ipaD_JPS-G3 resulted in a statistically significant reduction in disease severity at the 24-hour time point (p = 0.0001 ***). In particular, corneal lesion severity, which is the hallmark observation in the Serehy assay, revealed a statistically significant difference in both corneal edema (p = 0.0010) and corneal ulceration (p = 0.0016) at the 24-hour time point (FIG. 10).

Upon euthanasia of the guinea pigs at the 24-hour post infection time point, the eyes were collected en-bloc and formalin fixed. Following fixation, a mid-sagittal incision was made and submitted for histological preparation with subsequent staining using the dye combination Haemotoxylin and Eosin (H&E staining) as commonly used in the art. The prepared and stained slides were presented to the blinded, board-certified veterinary pathologist for evaluation. Both the conjunctiva and cornea were scored at 3 random sites. 10-20X magnification was used for three pathological changes: edema, neutrophil infiltration, and ulceration based on a severity scale of none, mild, moderate, and severe. The severity scale evaluations were assigned corresponding point values of 0, 5, 10, or 15, respectively. Based on the evaluation, no significant differences were found in the conjunctiva; however, significant reductions in pathological changes and disease were found in corneal lesions in the eyes of animals that had been treated with the anti -Shigella VHH 20ipaD_JPS-G3 VHH. The cumulative corneal disease score (FIG. 11, left panel) presents the data as the sum of the average for each pathological change. Alternatively, the corneal percent (%) disease (FIG. 11, right panel) presents the data as the percentage reduction in disease attributable to treatment with anti -Shigella VHH 20ipaD_JPS-G3. The latter corrects for individual animal variation in response to infection. Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

References

1. Hueck, C. J. (1998) Type III protein secretion systems in bacterial pathogens of

animals and plants. Microbiol Mol Biol Rev 62, 379-433

2. Diepold, A., and Wagner, S. (2014) Assembly of the bacterial type III secretion

machinery. FEMS Microbiol Rev 38, 802-822

3. Menard, R., Sansonetti, P. J., and Parsot, C. (1993) Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 175, 5899-5906

4. Watarai, M., Tobe, T., Yoshikawa, M., and Sasakawa, C. (1995) Contact of Shigella with host cells triggers release of Ipa invasins and is an essential function of invasiveness. EMBO J 14, 2461-2470

5. Dickenson, N. E., Arizmendi, O., Patil, M. K., Toth, R. T. t., Middaugh, C. R.,

Picking, W. D., and Picking, W. L. (2013) N-terminus of IpaB provides a potential anchor to the Shigella type III secretion system tip complex protein IpaD.

Biochemistry 52, 8790-8799

6. Cheung, M., Shen, D. K., Makino, F., Kato, T., Roehrich, A. D., Martinez-Argudo, T, Walker, M. L., Murillo, T, Liu, X., Pain, M., Brown, J., Frazer, G., Mantell, J., Mina, P., Todd, T., Sessions, R. B., Namba, K., and Blocker, A. J. (2015) Three-dimensional electron microscopy reconstruction and cysteine-mediated crosslinking provide a model of the type III secretion system needle tip complex. Mol Microbiol 95, 31-50

7. Kaur, K., Chatteijee, S., and De Guzman, R. N. (2016) Characterization of the

Shigella and Salmonella Type III Secretion System Tip-Translocon Protein-Protein Interaction by Paramagnetic Relaxation Enhancement. Chembiochem 17, 745-752

8. Cornells, G. R. (2006) The type III secretion injectisome. Nat Rev Microbiol 4, 811- 825

9. Espina, M., Olive, A. J., Kenjale, R., Moore, D. S., Ausar, S. F., Kaminski, R. W., Oaks, E. V., Middaugh, C. R., Picking, W. D., and Picking, W. L. (2006) IpaD localizes to the tip of the type III secretion system needle of Shigella flexneri. Infect Immun 74, 4391-4400 Epler, C. R., Dickenson, N. E., Bullitt, E., and Picking, W. L. (2012) ETltrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri. J Mol Biol 420, 29-39

Dickenson, N. E., Zhang, L., Epler, C. R., Adam, P. R., Picking, W. L., and Picking, W. D. (2011) Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion. Biochemistry 50, 172-180

Epler, C. R., Dickenson, N. E., Olive, A. J., Picking, W. L., and Picking, W. D.

(2009) Liposomes recruit IpaC to the Shigella flexneri type III secretion apparatus needle as a final step in secretion induction. Infect Immun 77, 2754-2761

Gazi, A. D., Charova, S. N., Panopoulos, N. J., and Kokkinidis, M. (2009) Coiled- coils in type III secretion systems: structural flexibility, disorder and biological implications. Cell Microbiol 11, 719-729

Johnson, S., Roversi, P., Espina, M., Olive, A., Deane, J. E., Birket, S., Field, T., Picking, W. D., Blocker, A. J., Galyov, E. E., Picking, W. L., and Lea, S. M. (2007) Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD. J Biol Chem 282, 4035-4044

Yip, C. K., Finlay, B. B., and Strynadka, N. C. (2005) Structural characterization of a type III secretion system filament protein in complex with its chaperone. Nat Struct Mol Biol 12, 75-81

Lunelli, M., Hurwitz, R., Lambers, J., and Kolbe, M. (2011) Crystal structure of Prgl- SipD: insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands. PLoS Pathog 7, el 002163

Muyldermans, S. (2013) Nanobodies: natural single-domain antibodies. Annu Rev Biochem 82, 775-797

Tremblay, J. M., Mukherjee, J., Leysath, C. E., Debatis, M., Ofori, K., Baldwin, K., Boucher, C., Peters, R., Beamer, G., Sheoran, A., Bedenice, D., Tzipori, S., and Shoemaker, C. B. (2013) A single VHH-based toxin-neutralizing agent and an effector antibody protect mice against challenge with Shiga toxins 1 and 2. Infect Immun 81, 4592-4603

Moayeri, M., Leysath, C. E., Tremblay, J. M., Vrentas, C., Crown, D., Leppla, S. EL, and Shoemaker, C. B. (2015) A heterodimer of a VHH (variable domains of camelid heavy chain-only) antibody that inhibits anthrax toxin cell binding linked to a VHH antibody that blocks oligomer formation is highly protective in an anthrax spore challenge model. J Biol Chem 290, 6584-6595

Hultberg, A., Temperton, N. J., Rosseels, V., Koenders, M., Gonzalez-Pajuelo, M., Schepens, B., Ibanez, L. T, Vanlandschoot, P., Schillemans, J., Saunders, M., Weiss, R. A., Saelens, X., Melero, J. A., Verrips, C. T., Van Gucht, S., and de Haard, H. J. (2011) Llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules. PLoS One 6, el7665

Vrentas, C. E., Moayeri, M., Keefer, A. B., Greaney, A. J., Tremblay, J., O'Mard, D., Leppla, S. H., and Shoemaker, C. B. (2016) A Diverse Set of Single-domain

Antibodies (VHHs) against the Anthrax Toxin Lethal and Edema Factors Provides a Basis for Construction of a Bispecific Agent That Protects against Anthrax Infection.

J Biol Chem 291, 21596-21606

Yang, Z., Schmidt, D., Liu, W., Li, S., Shi, L., Sheng, J., Chen, K., Yu, H., Tremblay, J. M., Chen, X., Piepenbrink, K. H., Sundberg, E. J., Kelly, C. P., Bai, G., Shoemaker, C. B., and Feng, H. (2014) A novel multivalent, single-domain antibody targeting TcdA and TcdB prevents fulminant Clostridium difficile infection in mice. J Infect Dis 210, 964-972

23. Vance, D. J., Tremblay, J. M., Mantis, N. J., and Shoemaker, C. B. (2013) Stepwise engineering of heterodimeric single domain camelid VHH antibodies that passively protect mice from ricin toxin. J Biol Chem 288, 36538-36547

24. Mukheijee, J., Tremblay, J. M., Leysath, C. E., Ofori, K., Baldwin, K., Feng, X., Bedenice, D., Webb, R. P., Wright, P. M., Smith, L. A., Tzipori, S., and Shoemaker,

C. B. (2012) A novel strategy for development of recombinant antitoxin therapeutics tested in a mouse botulism model. PLoS One 7, e2994l

25. Barta, M. L., Guragain, M., Adam, P., Dickenson, N. E., Patil, M., Geisbrecht, B. V., Picking, W. L., and Picking, W. D. (2012) Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure. Proteins 80, 935-945

26. Rossmann, F. S., Laverde, D., Kropec, A., Romero-Saavedra, F., Meyer-Buehn, M., and Huebner, J. (2015) Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria. PLoS One 10, eOl 18405

27. Kuhn, P., Fuhner, V., Unkauf, T., Moreira, G. M., Frenzel, A., Miethe, S., and Hust, M. (2016) Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display. Proteomics ClinAppl 10, 922-948

28. Liu, L., Johnson, H. L., Cousens, S., Perin, J., Scott, S., Lawn, J. E., Rudan, T,

Campbell, H., Cibulskis, R., Li, M., Mathers, C., Black, R. E., Child Health

Epidemiology Reference Group of, W. H. O., and Unicef. (2012) Global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since 2000. Lancet 379, 2151-2161

29. Black, R. E., Morris, S. S., and Bryce, J. (2003) Where and why are 10 million

children dying every year? Lancet 361, 2226-2234

30. de Marco, A. (2015) Recombinant antibody production evolves into multiple options aimed at yielding reagents suitable for application-specific needs. Microb Cell Fact 14

31. Rathinavelan, T., Lara-Tejero, M., Lefebre, M., Chatteijee, S., McShan, A. C., Guo,

D. C., Tang, C., Galan, J. E., and De Guzman, R. N. (2014) NMR model of Prgl-SipD interaction and its implications in the needle-tip assembly of the Salmonella type III secretion system. JMol Biol 426, 2958-2969

32. Broz, P., Mueller, C. A., Muller, S. A., Philippsen, A., Sorg, T, Engel, A., and

Cornelis, G. R. (2007) Function and molecular architecture of the Yersinia injectisome tip complex. Mol Microbiol 65, 1311-1320

33. Espina, M., Ausar, S. F., Middaugh, C. R., Baxter, M. A., Picking, W. D., and

Picking, W. L. (2007) Conformational stability and differential structural analysis of LcrV, PcrV, BipD, and SipD from type III secretion systems. Protein Sci 16, 704-714

34. Sawa, T., Katoh, H., and Yasumoto, H. (2014) V-antigen homologs in pathogenic gram-negative bacteria. Microbiol Immunol 58, 267-285

35. De Tavernier, E., Detalle, L., Morizzo, E., Roobrouck, A., De Taeye, S., Rieger, M., Verhaeghe, T., Correia, A., Van Hegelsom, R., Figueirido, R., Noens, J., Steffensen, S., Stohr, T., Van de Velde, W., Depla, E., and Dombrecht, B. (2016) High

Throughput Combinatorial Formatting of PcrV Nanobodies for Efficient Potency Improvement. J Biol Chem 291, 15243-15255

36. Gu, J., Liu, Y., Yu, S., Wang, H., Wang, Q., Yi, Y., Zhu, F., Yu, X. J., Zou, Q., and Mao, X. (2009) Enterohemorrhagic Escherichia coli trivalent recombinant vaccine containing EspA, intimin and Stx2 induces strong humoral immune response and confers protection in mice. Microbes Infect 11, 835-841

37. Medhekar, B., Shrivastava, R., Mattoo, S., Gingery, M., and Miller, J. F. (2009)

Bordetella Bsp22 forms a filamentous type III secretion system tip complex and is immunoprotective in vitro and in vivo. Mol Microbiol 71, 492-504

38. Moayeri, M., Leysath, C. E., Tremblay, J. M., Vrentas, C., Crown, D., Leppla, S. EL, and Shoemaker, C. B. (2015) A Heterodimer of a VHH Antibody that Inhibits Anthrax Toxin Cell Binding Linked to a VHH that Blocks Oligomer Formation is Highly Protective in an Anthrax Spore Challenge Model. The Journal of biological chemistry

39. Maass, D. R., Sepulveda, J., Pernthaner, A., and Shoemaker, C. B. (2007) Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). J Immunol Methods 324, 13-25

40. Picking, W. L., Nishioka, H., Hearn, P. D., Baxter, M. A., Harrington, A. T., Blocker, A., and Picking, W. D. (2005) IpaD of Shigella flexneri is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes. Infect Immun 73, 1432-1440

41. Kabsch, W. (2010) Xds. Acta Crystallogr D Biol Crystallogr 66, 125-132

42. Evans, P. R. (2011) An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr D Biol Crystallogr 67, 282-292

43. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography 66, 213-221

44. Adams, P. D., Grosse-Kunstleve, R. W., Hung, L. W., Ioerger, T. R., McCoy, A. J., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K., and Terwilliger, T. C. (2002) PHENIX: building new software for automated crystallographic structure determination. Acta crystallographica. Section D, Biological crystallography 58, 1948-1954

45. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and Sternberg, M. J. (2015)

The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10, 845-858

46. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools for molecular

graphics. Acta Crystallogr D Biol Crystallogr 60, 2126-2132

47. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and

development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486-501

48. Painter, J., and Merritt, E. A. (2006) Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr D Biol

Crystallogr 62, 439-450

49. Thompson, J., Higgins, D., and Gibson, T. (1994) CLETSTAL W: improving the

sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673- 4680

50. Gouet, P., Courcelle, E., Stuart, D., and Metoz, F. (1999) ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305-308 51. Zemla, A. (2003) LGA: A method for finding 3D similarities in protein structures. Nucleic Acids Res 31, 3370-3374

52. DeLano, W. L. (2002) The PyMOL Molecular Graphics System. 2009,

http://www.pymol.org

53. Evans, P. (2006) Scaling and assessment of data quality. Acta Crystallogr D Biol

Crystallogr 62, 72-82

54. Diederichs, K., and Karplus, P. A. (1997) Improved R-factors for diffraction data analysis in macromolecular crystallography. Nat Struct Biol 4, 269-275

55. Weiss, M. S. (2001) Global indicators of X-ray data quality. Journal of Applied Crystallography 34, 130-135

56. Karplus, P. A., and Diederichs, K. (2012) Linking crystallographic model and data quality. Science 336, 1030-1033

57. Evans, P. (2012) Biochemistry. Resolving some old problems in protein

crystallography. Science 336, 986-987

Claims

What is claimed is:

1. A variable domain of a heavy chain-only antibody (VHH) for neutralizing Shigella spp , the VHH comprising the amino acid sequence of SEQ ID NO: 1.

2. A variable domain of a heavy chain-only antibody (VHH) for neutralizing Shigella spp , the VHH comprising the amino acid sequence of SEQ ID NO: 2.

3. A variable domain of a heavy chain-only antibody (VHH) for neutralizing Shigella spp , the VHH comprising the amino acid sequence of SEQ ID NO: 3.

4. A variable domain of a heavy chain-only antibody (VHH) for neutralizing Shigella spp , the VHH comprising the amino acid sequence of SEQ ID NO: 4.

5. The variable domain of the heavy chain-only antibody of any one of claims 1 -4, wherein the VHH binds to structural epitopes clustered within the distal region of the IpaD protein.

6. The variable domain of the heavy chain-only antibody of claim 5, wherein the structural epitopes include residues 165-177 and 198-205 of Shigella IpaD.

7. The variable domain of the heavy chain-only antibody of claim 5, wherein the VHH binds to amino acids Glu20l and Lys205 of the of Shigella IpaD.

8. The variable domain of the heavy chain-only antibody of any one of claims 1-4, wherein the VHH inhibits ri%/ge//a-mediated hemolytic activity when tested in a contact- mediated hemolysis assay.

9. The variable domain of the heavy chain-only antibody of any one of claims 1 -4, wherein the VHH inhibits from 30 to 50% Shige//a-med\ ated hemolytic activity when tested in a contact-mediated hemolysis assay.

10. A binding protein comprising a first VHH and a second VHH, wherein the first VHH is a neutralizing VHH comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, wherein the second VHH is a neutralizing VHH comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID

NO: 3 or SEQ ID NO: 4, and wherein the first and second VHH are different.

11. The binding protein of claim 10, wherein the binding protein is a heterodimer comprising the amino acid sequence of SEQ ID NO: 1 covalently linked to SEQ ID NO: 2.

12. The binding protein of claim 11, wherein the binding protein inhibits ri7z/ge//a-mediated hemolytic activity when tested in a contact-mediated hemolysis assay by 80% or more.

13. A binding protein comprising a first VfflT and a second VHH, wherein the first VHH is a neutralizing VHH comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and wherein the second VHH is a non neutralizing VHH comprising the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

14. A binding protein comprising two or more VHHs, wherein the two or more VHHs comprise the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

15. The binding protein of claim 14, wherein the two or more VHHs are identical VHHs.

16. The binding protein of claim 14, wherein the two or more VHHs are different VHHs.

17. The binding protein of claim 14, comprising two or more VHHs, wherein the two or more VHHs comprise the amino acid sequence of SEQ ID NO: 1.

18. The binding protein of any one of 10-17, wherein the VHHs are linked by a flexible spacer.

19. The binding protein of claim 18, wherein the flexible spacer is (GGGGS)₃.

20. The binding protein of any one of claims 13-18, wherein the binding protein inhibits ri%/ge//a-mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

21. A pharmaceutical composition comprising the VHH of any one of claims 1-4 and a pharmaceutically acceptable carrier, excipient, or vehicle.

22. A pharmaceutical composition comprising the binding proteins of any one of claims 10-17 and a pharmaceutically acceptable carrier, excipient, or vehicle.

23. A method of treating a subject in need thereof comprising administering to the subject an effective amount of the VHH of any one of claims 1-4 or the pharmaceutical composition of claim 21.

24. A method of treating a subject in need thereof comprising administering to the subject an effective amount of the binding protein of any one of claims 10-17 or the pharmaceutical composition of claim 22.

25. A polynucleotide encoding the VHH of any one of claims 1-4.

26. A polynucleotide encoding the binding protein of any one of claims 10-17.

27. An expression vector comprising the polynucleotide of claim 25.

28. An expression vector comprising the polynucleotide of claim 26.

29. A binding protein comprising the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 1, wherein the binding protein binds IpaD and inhibits ri%/ge//a-mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

30. A binding protein comprising the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 2, wherein the binding protein binds IpaD and inhibits ri%/ge//a-mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

31. A binding protein comprising the second and the third complementary determining regions (CDR2 and CDR3) of SEQ ID NO: 3, wherein the binding protein binds IpaD and inhibits ri%/ge//a-mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

32. A binding protein comprising the first complementary determining regions (CDR1) of SEQ ID NO: 4, wherein the binding protein binds IpaD and inhibits Shigella- mediated hemolytic activity when tested in a contact-mediated hemolysis assay.

33. A pharmaceutical composition comprising the VHH of any one of claims 29-32 and a pharmaceutically acceptable carrier, excipient, or vehicle.

34. A polynucleotide encoding the binding protein of any one of claims 29-32.

35. An expression vector comprising the polynucleotide of claim 34.

36. A method of treating a subject in need thereof comprising administering to the subject an effective amount of the VHH of any one of claims 29-32 or the pharmaceutical composition of claim 33.