Variant antiviral polypeptides

Abstract

Provided herein, inter alia, are variant lectin antiviral polypeptides and compositions and methods for expressing and using the same to treat and prevent viral diseases in animals.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 376,393 filed on September 20, 2022 and U.S. Provisional Patent Application No. 63 / 502,297 filed on May 15, 2023, the disclosures of each of which are incorporated herein by reference in their entirety. Technical Field

[0003] Provided herein, inter alia, are variant antiviral polypeptides and compositions and methods for expressing and using the same to treat and prevent viral diseases in animals.

[0004] References to Electronic Sequence Listings

[0005] The contents of the electronic sequence listing (20230831_NB42022-WO-PCT_sequencelisting.xml; size: 65,998 bytes; and creation date: August 31, 2023) are incorporated herein by reference in its entirety. Background Art

[0006] Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically significant diseases of pigs. The disease was first identified in the United States in 1987 (Keffaber 1989) and in Europe in 1990 (Wensvoort et al. 1991). Molecular analysis of the prototype PRRS virus (PRRSV) VR-2332 and Lelystad (US and European isolates, respectively) showed that different evolutionary strains emerged almost simultaneously on both continents, probably due to similar changes in pig management practices (Murtaugh et al. 1995; Nelsen et al. 1999).

[0007] Since its initial appearance, the virus has spread worldwide, and European genotypes of PRRSV have been detected in U.S. swine herds (Ropp et al. 2004). PRRS is characterized by severe and sometimes fatal respiratory disease and reproductive failure, but also renders infected pigs susceptible to bacterial and other viral pathogens (Benfield et al. 1992). This factor is a key component of the economically significant porcine respiratory disease complex (PRDC). The most consistent pathological lesions caused by PRRSV during acute infection are interstitial pneumonia and mild lymphocytic encephalitis. After the acute phase of PRRSV infection, which is typically characterized by viremia and clinical disease, many pigs recover completely but continue to carry low-level viral infection for an extended period of time. These "carrier" pigs remain persistently infected with PRRSV and shed the virus intermittently or continuously and may infect uninfected pigs following direct or indirect contact. Persistent infection with PRRSV has been well documented under experimental conditions (Albina et al. 1994; Allende et al. 2000; Benfield et al. 1998).

[0008] PRRS has caused and continues to cause significant damage to the global swine industry. Therefore, effective and economically sustainable measures are needed to treat or prevent this and other viral diseases in pigs and other economically important livestock.

[0009] The subject matter disclosed herein addresses these needs and provides additional benefits. Summary of the invention

[0010]

[0013] Provided herein, inter alia, are compositions and methods for treating or preventing one or more viral diseases in animals.

[0011] Thus, in one aspect, provided herein is a non-naturally occurring variant lectin polypeptide or a functional fragment thereof, comprising an amino acid sequence at least about 60% identical to SEQ ID NO: 8 or SEQ ID NO: 18 and comprising one or more substitutions at positions 10, 38, 53, 54, and 78. In some embodiments, the polypeptide comprises at least one substitution at position 10. In some embodiments, the substitution at position 10 is selected from the group consisting of: X10D, X10E, X10G, X10H, X10N, X10P, X10Q, and X10T. In some embodiments, the substitution at position 10 is selected from the group consisting of: S10D, S10E, S10G, S10H, S10N, S10P, S10Q, and S10T. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least one substitution at position 38. In some embodiments, the substitution at position 38 is selected from the group consisting of: X38P and X38Q. In some embodiments, the substitution at position A31 is selected from the group consisting of: H38P and H38Q. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least one substitution at position 53. In some embodiments, the substitution at position 53 is selected from the group consisting of: X53A, X53C, X53D, X53E, X53H, X53K, X53L, X53M, X53Q, X53R, X53T, and X53V. In some embodiments, the substitution at position 53 is selected from the group consisting of: G53A, G53C, G53D, G53E, G53H, G53K, G53L, G53M, G53Q, G53R, G53T, and G53V. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least one substitution at position 54. In some embodiments, the substitution at position 54 is selected from the group consisting of: X54P and X54T. In some embodiments, the substitution at position 54 is selected from the group consisting of: S54P and S54T. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least one substitution at position 78. In some embodiments, the substitution at position 78 is selected from the group consisting of: X78W, X78H, X78Q, and X78Y. In some embodiments, the substitution at position 78 is selected from the group consisting of: M / N78W, M / N78H, M / N78Q, and M / N78Y. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least two substitutions at positions 53 and 78. In some embodiments, the substitution at position 53 is X53E, K, or L; and the substitution at position 78 is X78N or Y. In some embodiments, the substitution at position 53 is G53E or K; and the substitution at position 78 is M / N78Y. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least three substitutions at positions 10, 53, and 78.In some embodiments, the substitution at position 10 is X10D or N; the substitution at position 53 is X53K, E, or L; and the substitution at position 78 is X78Q or Y. In some embodiments, the substitution at position 10 is S10D or N; the substitution at position 53 is G53K, E, or L; and the substitution at position 78 is M / N78Q or Y. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least three substitutions at positions 38, 54, and 78. In some embodiments, the substitution at position 38 is X38Q; the substitution at position 54 is X54P; and the substitution at position 78 is X78Q. In some embodiments, the substitution at position 38 is H38Q; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Q. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least three substitutions at positions 10, 38, and 53. In some embodiments, the substitution at position 10 is X10N or D; the substitution at position 38 is X38Q; and the substitution at position 53 is X53E, L, or K. In some embodiments, the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; and the substitution at position 53 is G53E, L, or K. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least three substitutions at positions 10, 53, and 54. In some embodiments, the substitution at position 10 is X10N or D; the substitution at position 53 is X53L or K; and the substitution at position 54 is X54P. In some embodiments, the substitution at position 10 is S10N; the substitution at position 53 is G53L; and the substitution at position 54 is S54P. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least four substitutions at positions 10, 38, 53, and 54. In some embodiments, the substitution at position 10 is X10N or D; the substitution at position 38 is X38Q; the substitution at position 53 is X53E or K; and the substitution at position 54 is X54P. In some embodiments, the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; the substitution at position 53 is G53E or K; and the substitution at position 54 is S54P. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least four substitutions at positions 10, 53, 54, and 78. In some embodiments, the substitution at position 10 is X10N or D; the substitution at position 53 is X53L or K; the substitution at position 54 is X54P; and the substitution at position 78 is X78Y or Q. In some embodiments, the substitution at position 10 is S10N or D; the substitution at position 53 is G53L or K; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Y or Q.In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least four substitutions at positions 10, 38, 53, and 78. In some embodiments, the substitution at position 10 is X10D or N; the substitution at position 38 is X38Q; the substitution at position 53 is X53L, E, or K; and the substitution at position 78 is X78Y or Q. In some embodiments, the substitution at position 10 is S10D or N; the substitution at position 38 is H38Q; the substitution at position 53 is G53L, E, or K; and the substitution at position 78 is M / N78Y or Q. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises at least five substitutions at positions 10, 38, 53, 54, and 78. In some embodiments, the substitution at position 10 is X10N or D; the substitution at position 38 is X38Q; the substitution at position 53 is X53K or L; the substitution at position 54 is X54P; and the substitution at position 78 is X78Y or Q. In some embodiments, the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; the substitution at position 53 is G53K or L; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Y or Q. In some embodiments of any of the embodiments disclosed herein, the polypeptide has antiviral activity. In some embodiments of any of the embodiments disclosed herein, the variant lectin protein or a functional fragment thereof is a fully functional lectin polypeptide that can naturally dimerize (i.e., form a dimer) with other lectin polypeptides (e.g., variant and / or naturally occurring lectin polypeptides).

[0012] In another aspect, provided herein is a polypeptide dimer comprising two polypeptides or functional fragments thereof, wherein the polypeptides or functional fragments thereof comprise an amino acid sequence at least about 60% identical to SEQ ID NO: 8 or SEQ ID NO: 18. In some embodiments of any of the embodiments disclosed herein, the polypeptide has antiviral activity.

[0013] In another aspect, the present invention provides a variant lectin polypeptide dimer comprising two polypeptides or functional fragments thereof, wherein the polypeptides or functional fragments thereof comprise any of the variant lectin polypeptides disclosed herein. In some embodiments of any of the embodiments disclosed herein, the two polypeptides in the dimer are identical. In some embodiments of any of the embodiments disclosed herein, the two polypeptides in the dimer are separated by a linker amino acid sequence. In some embodiments, the length of the linker is between 2 and 50 amino acids. In some embodiments of any of the embodiments disclosed herein, the linker comprises the amino acid sequence (GTG) n , wherein n=1-7. In some embodiments of any of the embodiments disclosed herein, the linker comprises (i) any one of SEQ ID NOs: 32-57; or (ii) (GGG) n or (GPG)n , wherein n=1-7. In some embodiments of any of the embodiments disclosed herein, each polypeptide of the polypeptide dimer is capable of dimerizing with the Griffithsin protein. In some embodiments of any of the embodiments disclosed herein, the polypeptide has antiviral activity. In some embodiments of any of the embodiments disclosed herein, the dimer comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29, and SEQ ID NO:31.

[0014] In yet another aspect, provided herein is a nucleic acid encoding any of the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein.

[0015] In additional aspects, provided herein is a vector comprising any of the nucleic acids disclosed herein.

[0016] In still other aspects, a recombinant host cell is provided herein, comprising any one of the nucleic acids disclosed herein or any one of the vectors disclosed herein. In some embodiments, the host cell is a fungal cell, an algae cell, a plant cell, a bacterial cell, or a yeast cell. In some embodiments, the host cell is a Bacillus subtilis cell.

[0017] In another aspect, a pharmaceutical composition is provided herein, comprising a therapeutically effective amount of any of the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated for oral, nasal and / or topical administration to an animal.

[0018] In other aspects, the present invention provides a method for treating or preventing animal viral infection, the method comprising administering to the animal any of the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein or any of the pharmaceutical compositions disclosed herein. In some embodiments, the polypeptide or pharmaceutical composition is administered to the animal orally, nasally and / or topically. In some embodiments of any embodiment disclosed herein, the animal is a pig or a horse. In some embodiments, the pig is a sow, a gilt, a boar, a lactating piglet, a weaned piglet and / or a fattening pig. In some embodiments of any embodiment disclosed herein, the viral infection includes a virus (PRRSV) that causes porcine reproductive and respiratory syndrome, porcine epidemic diarrhea virus (PEDV), porcine rotavirus, or equine viral arteritis (EVA) virus.

[0019] In still other aspects, the present invention provides a method for producing a polypeptide, the method comprising culturing any of the host cells disclosed herein in a suitable culture medium under conditions suitable for polypeptide expression. In some embodiments, the method further comprises purifying the polypeptide.

[0020] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of an embodiment or aspect.

[0021] Throughout the specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. The disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Depicted are images of SDS-PAGE gels showing the results of a protease stability assay of the combined Gravesin variants.

[0023] Figure 2 is a graph depicting the PRRSV antiviral effect of Gravesonia variants with low antiviral load at 1 MOI (multiplicity of infection).

[0024] Figure 3 is a graph depicting the PRRSV antiviral effect of Gravesonia variants with high antiviral load at 10 MOI (multiplicity of infection).

[0025] Figure 4 Depicted are SDS-PAGE gel images of intracellular samples of 9 protease deletion strains of Bacillus subtilis that produce Grefsen M78 monomers and dimers with GGG, GPG, GTG linkers. Lane 1 is See Blue Plus2 molecular weight ladder; lane 2 is negative control Bacillus subtilis 9 protease deletion strain; lanes 3, 8, 10 and 12 show Grefsen M78 monomers; lanes 4-5 are Grefsen M78 GGG dimers; lanes 6-7 are Grefsen M78 GPG dimers, and lane 9 is Grefsen M78 GTG dimers.

[0026] Figure 5 is a graph depicting daily body temperatures of pigs from 3 days before challenge to 41 days after challenge. NT / NC = untreated non-challenged pigs; NT / C = untreated challenged pigs; LDT / C = challenged pigs receiving low dose of greverson; HDT / C = challenged pigs receiving high dose of greverson.

[0027] Figure 6is a bar graph depicting viral load in serum samples at different time points after challenge. Treatment groups: NT / C = untreated challenged pigs; LDT / C = challenged pigs receiving low dose of Greifsern; HDT / C = challenged pigs receiving high dose of Greifsern.

[0028] Figure 7 is a bar graph depicting the overall lung score for the presence of lesions in the different lung lobes at day 42 post-challenge. The average score for the 7 lung lobes per pig is shown. Treatment groups: NT / C = untreated challenged pigs; LDT / C = challenged pigs receiving low dose of grefsen; HDT / C = challenged pigs receiving high dose of grefsen.

[0029] Figure 8 is a bar graph depicting microscopic lung scores on day 42 post-challenge. Lung tissue was evaluated microscopically for macrophage infiltration and graded from 0 to 4. 0 = no microscopic lesions, 1 = mild interstitial pneumonia, 2 = moderate multifocal interstitial pneumonia, 3 = moderate diffuse interstitial pneumonia, 4 = severe interstitial pneumonia. Treatment groups: NT / C = untreated challenged pigs; LDT / C = challenged pigs receiving low dose of greverson; HDT / C = challenged pigs receiving high dose of greverson.

[0030] Fig. 9 is a bar graph depicting the weight of pigs recorded at various time points before and after challenge. Treatment groups: NT / NC = untreated non-challenged pigs; NT / C = untreated challenged pigs; LDT / C = challenged pigs receiving low dose of Greifsern; HDT / C = challenged pigs receiving high dose of Greifsern.

[0031] Fig.10 is a bar graph depicting the average daily weight gain of the pigs. Treatment groups: NT / C = untreated challenged pigs; LDT / C = challenged pigs receiving a low dose of Greifsern; HDT / C = challenged pigs receiving a high dose of Greifsern. DETAILED DESCRIPTION

[0032] Porcine reproductive and respiratory syndrome (PRRS) is characterized by severe and sometimes fatal respiratory disease and reproductive failure, but also leaves infected pigs vulnerable to bacterial and other viral pathogens. Therefore, there is a great need in the swine industry for therapeutics that can treat and prevent PRRS infection and spread.

[0033] Lectins are generally defined as carbohydrate-binding proteins that can recognize and bind simple or complex carbohydrates in a reversible and highly specific manner while not displaying catalytic activity (Lagarda-Diaz et al., 2017). Lectin proteins were originally named hemagglutinins due to their ability to agglutinate erythrocytes (and other cells). Recently, lectins such as the red algae (Griffithsia species) Griffithsia rapa (GRFT) protein have been evaluated for antiviral activity (Whitley et al., 2013). However, the economics of recombinant lectin production (e.g., using currently available host expression systems and their downstream recovery processes) significantly limits the acceptance and / or use of lectins as antimicrobial compositions.

[0034] The present invention is based at least in part on the inventors' discovery that introducing one or more substitutions into the amino acid sequence of a lectin (e.g., a Gravesin (GRFT) lectin polypeptide) can improve one or more properties of the lectin (such as, but not limited to, thermal stability, recombinant expression levels, and / or protease stability), thereby improving recombinant production and stability. In addition, as shown in the examples, the engineered lectin has potent antiviral properties.

[0035] I. Definitions

[0036] As used herein, the term "lectin" refers to a carbohydrate binding protein.

[0037] The terms "variant lectin protein" or "variant lectin" used interchangeably herein refer to a polymer having carbohydrate binding activity and containing at least one amino acid modified amino acid residue relative to the wild-type (ie, naturally occurring) lectin amino acid sequence.

[0038] As used herein, "microorganism" or "microbe" refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0039] The terms "protein" and "polypeptide" refer to compounds comprising amino acids connected via peptide bonds and can be used interchangeably. "Protein" or "polypeptide" comprises a polymeric sequence of amino acid residues. Single-letter and 3-letter codes for amino acids defined in accordance with the IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) are used throughout this disclosure. Single letter X refers to any one of the twenty amino acids. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. The amino acid position in a given polypeptide sequence can be named by the single-letter code for amino acids followed by the position number. For example, glycine (G) at position 87 is represented as "G087" or "G87".

[0040] "Variant polypeptides" (as used interchangeably herein as "variant lectin polypeptides" or "variant lectins", e.g., variant Grefsen polypeptides) or "non-naturally occurring polypeptides" are used interchangeably herein and refer to polypeptides that have been engineered to have an amino acid sequence that differs from a reference or parent polypeptide sequence by at least one amino acid. In some embodiments, the reference sequence is the wild-type (i.e., naturally occurring) amino acid sequence of a lectin. In other embodiments, the reference sequence is SEQ ID NO: 8 or SEQ ID NO: 18.

[0041] As used herein, when an "amino acid sequence" is described, it refers to the amino acid sequence of a protein or peptide molecule. An "amino acid sequence" can be derived from a nucleic acid sequence encoding a protein. However, terms such as "polypeptide" or "protein" are not intended to limit the amino acid sequence to the derived amino acid sequence, but may include post-translational modifications of the derived amino acid sequence, such as amino acid deletions, additions, and modifications (such as glycosylation and addition of lipid moieties). In addition, unless otherwise indicated, the use of unnatural amino acids such as D-amino acids to improve stability or pharmacokinetic behavior falls within the scope of the term "amino acid sequence".

[0042] The term "mature" form of a protein, polypeptide or peptide refers to the functional form of the protein, polypeptide or enzyme without the signal peptide sequence and the propeptide sequence.

[0043] With respect to amino acid sequences or nucleic acid sequences, the term "wild-type" indicates that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance (e.g., protein, amino acid or nucleic acid sequence) found in nature. In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant / engineered nucleic acid and protein sequences produced in a laboratory, or modifications of wild-type sequences).

[0044] As used herein, the term "sequence identity" or "sequence similarity" means that two polynucleotide sequences (candidate sequence and reference sequence) are identical (i.e., 100% sequence identity) or similar (i.e., on a nucleotide-by-nucleotide basis) over the length of the candidate sequence. When comparing a candidate sequence to a reference sequence, the candidate sequence may contain additions or deletions (i.e., gaps) as compared to a reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). Optimal sequence alignment for determining sequence identity can be performed using any number of publicly available local alignment algorithms known in the art (e.g., ALIGN or Megalign (DNASTAR)), or by inspection.

[0045] As used herein, the terms "percent (%) sequence identity" or "percent (%) sequence similarity" with respect to a reference sequence are defined as the percentage of nucleotide residues in a candidate sequence that are identical to the residues in a reference polynucleotide sequence, after the sequences have been optimally aligned and gaps introduced, if necessary, to achieve the maximum percent sequence identity.

[0046] As used herein, with respect to amino acid residue positions, "corresponding to" or "corresponds to" or "corresponds to" refers to the amino acid residue at the recited position in a protein or peptide, or an amino acid residue that is similar, homologous, or identical to the recited residue in a protein or peptide.

[0047] As used herein, the term "recombinant" or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, vector, etc. that has at least one engineered genetic change or has been modified by the introduction of a heterologous nucleic acid molecule; or refers to a cell (e.g., a host cell) that has been altered so that the expression of a heterologous nucleic acid molecule or an endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell, or is a descendant of a non-natural cell with one or more such modifications. Genetic changes include, for example, the introduction of modifications of expressible nucleic acid molecules encoding proteins, or the addition, deletion, substitution, or other functional changes of other nucleic acid molecules of the cell's genetic material. For example, a recombinant cell can express genes or other nucleic acid molecules (e.g., polynucleotide constructs) that are not found in the same or homologous form in a natural (wild-type) cell, or can provide an altered endogenous gene expression pattern, such as overexpression, underexpression, minimal expression, or no expression at all.

[0048] "Recombination" or "recombining" or the production of a "recombined" nucleic acid is generally the assembly of two or more nucleic acid fragments, wherein the assembly results in a chimeric DNA sequence that would not otherwise be found in the genome.

[0049] The term "derived" encompasses the terms "originating," "obtained," "obtainable," and "produced," and generally indicates that one specified material or composition finds its origin in, or has characteristics that can be described with reference to, another specified material or composition.

[0050] As used herein, "endogenous gene" refers to a gene that is located in its natural location in the genome of an organism.

[0051] As used herein, a "heterologous" gene, "non-endogenous" gene, or "foreign" gene refers to a gene (or gene coding sequence; CDS / open reading frame; ORF) that is not normally present in the host organism but is introduced into the host organism by gene transfer. The term "foreign" gene includes a native gene (or ORF) inserted into a non-native organism and / or a chimeric gene inserted into a native or non-native organism.

[0052] As used herein, "heterologous control sequences" refer to gene expression control sequences (e.g., promoters, enhancers, terminators, etc.) that do not essentially function to regulate (control) the expression of a gene of interest. Typically, heterologous nucleic acids are not endogenous (natural) to the cell or part of the genome in which they are present, and have been added to the cell by infection, transfection, transduction, transformation, microinjection, electroporation, etc. "Heterologous" nucleic acid constructs may contain control sequence / DNA coding (ORF) sequence combinations that are the same or different from those found in the natural host cell.

[0053] As used herein, the terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can participate in the secretion or directional transport of a mature protein or a precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. There is generally no signal sequence in the mature protein. Typically, the signal sequence is cut from the protein by a signal peptidase during the translocation process.

[0054] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid molecules of the present disclosure. Expression can also refer to the translation of mRNA into polypeptides. Therefore, the term "expression" includes any step involved in the production of polypeptides, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0055] As used herein, "nucleic acid" refers to nucleotide or polynucleotide sequences and fragments or parts thereof, as well as DNA, cDNA and RNA of genomic or synthetic origin, which can be double-stranded or single-stranded, whether representing sense strand or antisense strand. It should be understood that due to the degeneracy of the genetic code, a variety of nucleotide sequences can encode a given protein. It should be understood that the polynucleotides (or nucleic acid molecules) described herein include "genes", "vectors" and "plasmids". Accordingly, the term "gene" refers to a polynucleotide encoding a specific sequence of amino acids, which contains all or part of a protein coding sequence, and may include a regulatory (non-transcribed) DNA sequence, such as a promoter sequence, which determines, for example, the conditions for expressing a gene. The transcription region of a gene may include an untranslated region (UTR) (including introns, 5'-untranslated region (UTR) and 3'-UTR) and a coding sequence.

[0056] As used herein, the term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of a coding sequence are usually determined by an open reading frame (hereinafter, "ORF") that usually begins with the ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0057] As used herein, the term "promoter" refers to a nucleic acid sequence that is capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located (3') downstream of the promoter sequence. The promoter may be derived entirely from a natural gene, or may be composed of different elements derived from different promoters found in nature, or may even comprise a synthetic nucleic acid segment.

[0058] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence (e.g., ORF) when it is able to affect the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense direction. Therefore, when a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked" to another nucleic acid sequence. For example, if the DNA encoding a secretory leader sequence (i.e., a secretory signal sequence) is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA encoding the secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; if a ribosome binding site is positioned to promote translation, the ribosome binding site is operably linked to a coding sequence. Typically, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in the reading phase. However, enhancers need not be continuous. Linking is achieved by ligating at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints are used in accordance with conventional practice.

[0059] As used herein, "a functional promoter sequence that controls the expression of a target gene (or its open reading frame) linked to a target gene protein coding sequence" refers to a promoter sequence that controls the transcription and translation of a coding sequence in a desired host cell. For example, in certain embodiments, the present disclosure relates to a polynucleotide comprising an upstream (5') promoter (or a 5' promoter region, or a tandem 5' promoter, etc.) that is functional in a host cell, wherein the promoter region is operably linked to a nucleic acid sequence (e.g., ORF) encoding a variant lectin or a variant lectin dimer protein.

[0060] As used herein, "suitable regulatory sequence" refers to a nucleotide sequence that is located upstream (5' non-coding sequence), internal or downstream (3' non-coding sequence) of a coding sequence and affects transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences can include promoters, transcription leader sequences, RNA processing sites, effector binding sites and stem-loop structures.

[0061] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include, but are not limited to: (a) introduction, substitution or removal of one or more nucleotides in a gene (or its ORF), or introduction, substitution or removal of one or more nucleotides in a regulatory element required for transcription or translation of a gene or its ORF, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene down-regulation, (f) specific mutagenesis of any one or more genes disclosed herein and / or (g) random mutagenesis of any one or more genes disclosed herein.

[0062] As used herein, "disruption of a gene" or "gene disruption" can be used interchangeably and broadly refers to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., protein). Therefore, as used herein, gene disruption includes, but is not limited to, frameshift mutations, premature termination codons (i.e., so that no functional protein is produced), substitutions that eliminate or reduce protein activity (so that no functional protein is produced), internal deletions, insertions that disrupt coding sequences, mutations that remove the operable connection between the natural promoter required for transcription and the open reading frame, etc.

[0063] As used herein, the term "introduced", as used in phrases such as "introducing into a bacterial cell" or "introducing into a bacterial cell" at least one polynucleotide open reading frame (ORF), or a gene thereof, or a vector thereof, includes methods known in the art for introducing a polynucleotide into a cell, including but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation, and the like.

[0064] As used herein, "transformed" or "transformation" means transforming a cell by using recombinant DNA technology. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., polynucleotides, ORFs, or genes) into a cell. The inserted nucleotide sequence may be a heterologous nucleotide sequence (i.e., a sequence that does not naturally occur in the cell to be transformed). Thus, transformation generally refers to the introduction of exogenous DNA into a host cell such that the DNA remains as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0065] As used herein, "transforming DNA", "transforming sequence" and "DNA construct" refer to DNA for introducing a sequence into a host cell or an organism. Transforming DNA is DNA for introducing a sequence into a host cell or an organism. DNA can be produced in vitro by PCR or any other suitable technique. In certain embodiments, the transforming DNA comprises an input sequence, and in other embodiments, it further comprises an input sequence flanked by homology boxes. In yet other embodiments, the transforming DNA comprises other non-homologous sequences, added to the ends (i.e., stuffing sequences or flanking). The ends can be closed so that the transforming DNA forms a closed loop, such as, for example, inserted into a vector.

[0066] As used herein, "input sequence" refers to a DNA sequence introduced into the host cell chromosome. In certain embodiments, the input sequence is a part of a DNA construct. In other embodiments, the input sequence encodes one or more target proteins. In certain embodiments, the input sequence comprises a sequence that may or may not be present in the genome of the cell to be transformed (that is, it may be a homologous or heterologous sequence). In certain embodiments, the input sequence encodes one or more target proteins, genes and / or mutations or modified genes. In alternative embodiments, the input sequence encodes functional wild-type genes or operons, functional mutant genes or operons or non-functional genes or operons. In certain embodiments, non-functional sequences can be inserted into genes to destroy the function of genes. In another embodiment, the input sequence includes a selective marker. In a further embodiment, the input sequence includes two homology boxes.

[0067] As used herein, "homologous box" or "homologous arm" refers to a nucleic acid sequence homologous to a sequence in a host cell chromosome. More particularly, according to the present invention, a homologous box is an upstream or downstream region that has a sequence identity between about 80% and 100%, a sequence identity between about 90% and 100%, or a sequence identity between about 95% and 100% with a gene or a part of a gene to be deleted, destroyed, inactivated, etc. These sequences guide the integration position of the DNA construct in the host cell chromosome, and guide which part of the host cell chromosome is replaced by the input sequence. Although it is not intended to limit the present disclosure, the homologous box may include between about 1 base pair (bp) and 200 kilobases (kb). In some embodiments, the homologous box includes between about 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb; and between 0.25 kb and 2.5 kb. Homology boxes can also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, the 5' and 3' ends of the selectable marker are flanked by homology boxes, wherein the homology boxes contain nucleic acid sequences that closely flank the coding region of the gene.

[0068] As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence that is capable of being expressed in a host cell and wherein expression of the selectable marker confers to the cells containing the expressed gene the ability to grow in the presence of the corresponding selective agent or in the absence of essential nutrients.

[0069] As used herein, the terms "selectable marker" and "selective marker" refer to a nucleic acid (e.g., a gene) that can be expressed in a host cell that allows easy selection of those hosts containing the vector. Examples of such selectable markers include, but are not limited to, antimicrobial agents. Thus, the term "selectable marker" refers to a gene that provides an indication that the host cell has taken up the input DNA of interest or that some other reaction has occurred. Typically, a selectable marker is a gene that confers antimicrobial resistance or a metabolic advantage to a host cell to allow cells containing exogenous DNA to be distinguished from cells that have not received any exogenous sequences during transformation.

[0070] A "residing selectable marker" is a marker located on the chromosome of the microorganism to be transformed. A residing selectable marker encodes a gene that is different from the selectable marker on the transforming DNA construct. Selectable markers are well known to those skilled in the art. As noted above, the marker may be an antimicrobial resistance marker (e.g., ampR, phleoR, specR, kanR, eryR, tetR, cmpR, and neoR). Other markers useful according to the invention include, but are not limited to, auxotrophic markers such as serine, lysine, tryptophan; and detection markers, such as β-galactosidase.

[0071] As defined herein, a host cell "genome" and the like includes chromosomal genes and extrachromosomal genes.

[0072] As used herein, the terms "plasmid", "vector" and "cassette" refer to an extrachromosomal element that usually carries genes that are typically not part of the central metabolism of the cell and is usually in the form of a circular double-stranded DNA molecule. Such an element may be a linear or circular autonomously replicating sequence derived from single-stranded or double-stranded DNA or RNA from any source, a genome integrating sequence, a bacteriophage, or a nucleotide sequence in which multiple nucleotide sequences have been linked or recombined into a single construct that is capable of introducing a promoter fragment and a DNA sequence for a selected gene product, along with appropriate 3' non-translated sequences, into a cell.

[0073] As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct that is used as a cloning vector and forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotic organisms. In some embodiments, the plasmid is incorporated into the genome of the host cell. In some embodiments, the plasmid is present in the parent cell and is lost in the daughter cell.

[0074] As used herein, "transformation cassette" refers to a specific vector that contains a gene (or its ORF) and has elements in addition to the foreign gene that facilitate transformation of a specific host cell.

[0075] As used herein, the term "vector" refers to any nucleic acid that can be replicated (propagated) in a cell and can carry a new gene or DNA segment to a cell. Therefore, the term refers to a nucleic acid construct designed to be transferred between different host cells. Vectors include viruses, phages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes (such as YAC (yeast artificial chromosome), BAC (bacterial artificial chromosome), PLAC (plant artificial chromosome)) etc. that can be integrated into the chromosome of a host organism as "episomes" (i.e., autonomous replication).

[0076] As used herein, the terms "expression cassette" and "expression vector" refer to a nucleic acid construct that is recombinantly or synthetically generated with a series of specific nucleic acid elements (i.e., these are vectors or vector elements, as described above) that allow a specific nucleic acid to be transcribed in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes (in addition to other sequences) a nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the DNA construct also includes a series of specified nucleic acid elements that allow a specific nucleic acid to be transcribed in a target cell. In certain embodiments, the DNA construct of the present disclosure includes a selective marker and an inactivated chromosome, or a gene, or a DNA segment as defined herein.

[0077] As used herein, "targeting vector" is a vector comprising a polynucleotide sequence that is homologous to the region in the chromosome of the host cell to which the targeting vector is transformed and that can drive homologous recombination in the region. For example, the targeting vector can be used to introduce mutations into the chromosome of the host cell by homologous recombination. In certain embodiments, the targeting vector comprises other non-homologous sequences, such as added to the end (i.e., stuffing sequence or flanking sequence). The end can be closed so that the targeting vector forms a closed loop, such as, for example, inserted into a vector. For example, in certain embodiments, the cell is modified (e.g., transformed) by introducing one or more "targeting vectors" in a Bacillus species (host) cell.

[0078] As used herein, "flanking sequence" refers to any sequence upstream or downstream of the sequence in question (for example, for gene ABC, gene B is flanked by A and C gene sequences). In certain embodiments, the flank of the input sequence on each side is a homology box. In another embodiment, the input sequence and the homology box are included in the unit of the flank on each side as a stuffing sequence. In certain embodiments, the flanking sequence is only present on one side (3' or 5'), but in some embodiments, it is on each side of the sequence flanked. The sequence of each homology box is homologous to the sequence in the Bacillus chromosome. These sequences guide in the Bacillus chromosome, and the new construct is integrated, and part of the Bacillus chromosome will be replaced by the input sequence. In other embodiments, the flanks of the 5' and 3' ends of the selective marker are polynucleotide sequences comprising the part of the inactivated chromosome segment. In certain embodiments, the flanking sequence is only present on one side (3' or 5'), and in other embodiments, it is present on each side of the sequence flanked.

[0079] A "host strain" or "host cell" is an organism into which an expression vector, bacteriophage, virus or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., amylase) has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi and yeast) capable of expressing the polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced by the cell. As will be appreciated by those skilled in the art, many host cells are generally recognized as safe (GRAS) according to US FDA guidelines.

[0080] As used herein, the terms "purified," "isolated," or "enriched" mean that a biomolecule (e.g., a polypeptide or polynucleotide) is altered from its native state by separating it from some or all of the naturally occurring components with which it is associated in nature. Such separation or purification can be accomplished by art-recognized separation techniques, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salt precipitation, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove whole cells, cell debris, impurities, foreign proteins, or enzymes that are not desired in the final composition. Components that provide additional benefits, such as activators, anti-inhibitors, desired ions, compounds that control pH, or other enzymes or chemicals, can then be further added to the purified or isolated biomolecule composition.

[0081] As used herein, a "protein preparation" is any material, typically a solution, usually aqueous, containing one or more proteins.

[0082] As used herein, the terms "broth", "culture broth", "fermentation broth" and / or "whole fermentation broth" are used interchangeably and refer to a preparation produced by cell fermentation that does not undergo processing steps after fermentation is complete. For example, whole fermentation broth is typically produced when a microbial culture is grown to saturation, incubated under carbon-limited conditions to allow protein synthesis (e.g., expression of proteins by host cells; and optionally secretion of proteins into the cell culture medium). Typically, whole fermentation broth is unfractionated and contains spent cell culture medium, metabolites, extracellular polypeptides, and microbial cells.

[0083] As used herein, the phrase "treated broth" refers to broth that has been conditioned by changing the chemical composition and / or physical properties of the broth. Broth "conditioning" can include one or more treatments such as cell lysis, pH modification, heating, cooling, addition of chemicals (e.g., calcium, salts, flocculants, reducing agents, enzyme activators, enzyme inhibitors, and / or surfactants), mixing, and / or timed holding of the broth (e.g., 0.5 to 200 hours) without further processing.

[0084] As used herein, a "cell lysis" process includes any cell lysis technique known in the art, including but not limited to enzymatic treatment (e.g., lysozyme, proteinase K treatment), chemical means (e.g., ionic liquids), physical means (e.g., French press, ultrasound), simply maintaining the culture without feed, etc.

[0085] As used herein, the terms "recovery," "recovered," and "recovering" refer to at least partially separating a protein from one or more components of a microbial broth, and / or at least partially separating a protein from one or more solvents (e.g., water or ethanol) in the broth.

[0086] In certain aspects, the broth in which the host cells have been fermented for the production of variant lectins or variant lectin dimer proteins is clarified, with or without broth treatment. As used herein, "clarified" broth means a broth that has been subjected to at least one clarification process to remove cell debris and / or other insoluble components. As understood in the art, clarification processes include, but are not limited to, centrifugation techniques, cross-flow membrane filtration techniques, solid / liquid filtration techniques, and the like.

[0087] "Cellular debris" refers to cell walls and other insoluble components released or formed after disruption of the cell membrane (eg, after subjecting a cell to a cell lysis process).

[0088] In certain aspects, separation of the solvent includes, but is not limited to, ultrafiltration, evaporation, spray drying, freeze drying, as understood in the art. The solution obtained is referred to as "clarified broth concentrate", "UF concentrate" or "ultrafiltration concentrate".

[0089] As used herein, "performance index" or "PI" refers to the calculated activity per unit of enzyme relative to a parent molecule. In some aspects of any of the embodiments disclosed herein, the parent molecule used in calculating the performance index is a non-engineered Gravesson molecule. In some embodiments, by definition, a parent molecule has a performance index of one. In other embodiments, a performance index greater than one (PI>1.0) indicates improved activity of a variant polypeptide compared to a parent molecule.

[0090] As used herein, an "effective amount" or "therapeutically effective amount" is an amount that provides a nutritional, physiological, or medical benefit to an animal.

[0091] As used herein, the term "animal" includes all non-ruminants (including humans) and ruminants. In specific embodiments, the animal is a non-ruminant, such as a horse and a monogastric animal. Examples of monogastric animals include, but are not limited to, pigs (pig and swine), such as piglets, growing pigs, sows; poultry, such as turkeys, ducks, chickens, broilers, laying hens; fish, such as salmon, trout, tilapia, catfish and carp; and crustaceans, such as shrimp and prawns. In further embodiments, the animal is a ruminant, including but not limited to cattle, calves, goats, sheep, giraffes, bison, moose, elk, yaks, buffalo, deer, camels, alpacas, llamas, antelopes, pronghorns and wildebeests.

[0092] As used herein, the term "pathogen" means any disease-causing agent. Such pathogens may include, but are not limited to, bacteria, viruses, fungal pathogens, etc.

[0093] "Feed" and "food" mean any natural or artificial diet, meal, etc., or a component of such a meal, which is intended or suitable for consumption, ingestion, digestion by non-human animals and humans, respectively. As used herein, the term "food" is used in a broad sense and covers food and food products for humans and food (i.e., feed) for non-human animals. With regard to products fed to animals when raising livestock, the term "feed" is used. The terms "feed" and "animal feed" are used interchangeably. In a preferred embodiment, the food or feed is consumed by non-ruminants and ruminants.

[0094] Certain ranges are presented herein with the term "about" preceding a numerical value. The term "about" is used herein to provide textual support for the exact number that follows it and for numbers that are close or approximate to the number that follows the term. In determining whether a number is close to or approximate to a specifically stated number, the close or approximate unstated number may be a number that provides a substantial equivalent of the specifically stated number in the context in which it is presented. For example, with respect to a numerical value, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context.

[0095] As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0096] It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0097] Still, it should be noted that, as used herein, the term "consisting essentially of" refers to a composition wherein the component following the term, in the presence of other known components, is a total amount of less than 30% by weight of the total composition and does not affect or interfere with the action or activity of the component.

[0098] It is further noted that, as used herein, the term “comprising” is intended to include but is not limited to the components following the term “comprising.” The components following the term “comprising” are required or mandatory, but the composition comprising the components may further include other non-mandatory or optional components.

[0099] Also note that, as used herein, the term “consisting of is meant to include and be limited to the components following the term “consisting of.” Thus, the components following the term “consisting of” are required or mandatory, and no other components are present in the composition.

[0100] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as if such lower numerical limits were expressly written herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as if such higher numerical limits were expressly written herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such wider numerical range, as if such narrower numerical ranges were all expressly written herein.

[0101] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0102] Additional definitions of terms may appear throughout this specification.

[0103] II. Composition

[0104] A. Variant lectin polypeptide

[0105] Lectin is a protein (or glycoprotein) with a non-catalytic carbohydrate binding site. As generally understood in the art, lectins are different from enzymes because their carbohydrate binding properties never change, and they are different from antibodies because they are not induced as immune responses. For example, some of the most well-known lectins are found in leguminous seeds, which are believed to be responsible for innate immunity and defense mechanisms in plants (Peumans and Van Damme, 1998). Recently, the use of lectins in mitigating viral infections (e.g., HIV, MERSCoV, SARS-CoV-2, HCV, Ebola, etc.) has been significantly concerned (PCT Publication Nos. WO 2005 / 118627, WO 2007 / 064844, WO 2010 / 01424, WO 2016 / 130628, WO 2019 / 108656, and US Publication No. US20110263485). However, the economics of recombinant lectin production (eg, using currently available host expression systems and their downstream recovery processes) significantly limits the acceptance and / or use of lectins as antimicrobial compositions.

[0106] PCT publications WO 2005 / 118627 and WO 2007 / 064844 describe methods for isolating natural Griffithsia (GRFT) lectin from red algae (Griffithsia species), cloning its wild-type (grft) gene, generating its recombinant polynucleotide, fermenting and producing it in E. coli host cells, followed by isolating recombinant His-tagged GRFT protein from the E. coli host and characterizing its antiviral activity. However, as described in WO 2005 / 118627 and WO 2007 / 064844, the recombinant GRFT protein encoded by the nucleic acid of Example 2 (and its C-terminal His-tagged GRFT protein) was not efficiently translocated to the periplasmic fraction of E. coli after GRFT protein expression, wherein most of the produced GRFT protein accumulated in the inclusion bodies of E. coli without cleavage of the pelB signal sequence located at the N-terminus of the Griffithsia protein. Therefore, steps were taken to express GRFT in the cytosolic fraction of E. coli using N-terminally (His) tagged GRFT or a His-tagged variant of GRFT.

[0107] Similarly, PCT Publication No. WO 2010 / 01424 generally describes methods for inhibiting hepatitis C virus infection in a host, which methods include administering to the host an effective amount of a glycosylation-resistant GRFT protein (or its polypeptide conjugate) in combination with another antiviral protein. For example, as described in this disclosure, the inventors noted that the antiviral protein combination of scytovirin (SVN) and GRFT (GRFT) has (nanomolar) activity against hepatitis C virus (HCV). U.S. Patent Publication No. US20110263485 further describes methods for inhibiting human immunodeficiency virus (HIV) viral infection in a host, which methods include administering to the host an effective amount of gp120 GRFT and a peptide selected from gp41 binding protein, CCR5 binding protein, gp120 binding protein or another GRFT, which combinations are potent inhibitors of HIV infection.

[0108] PCT Publication No. WO 2016 / 130628 discloses variant GRFT proteins having mutations that alter the isoelectric point of the GRFT protein, which reportedly alter their solubility in various pH ranges, thereby allowing for improved product release.

[0109] PCT Publication No. WO 2019 / 108656 generally describes a microbicidal composition comprising an endosperm extract and an anti-HIV lectin, an anti-HIV antibody or an antigen-binding antibody fragment thereof. More particularly, the inventors utilized transgenic plants expressing two or more cyanobacterial antiviral proteins-N (CVN) proteins, GRFT proteins, scytovirin (SVN) proteins, and other anti-HIV lectin proteins. However, as described in WO 2019 / 108656, the production of such microbicidal components is expensive because an expression platform based on a fermenter is required, and downstream processing facilities must comply with good manufacturing practices (GMP) to ensure the removal of viruses or endotoxins, wherein when each microbicide requires two or three separate products with a separate manufacturing process, the capacity, scalability, and cost issues affecting the fermenter are exacerbated.

[0110] Recombinant production of GRFT in tobacco plants (Nicotiana benthamiana) has been described by O'Keefe et al. (2009), where GRFT accumulated to levels of about 1 gram of recombinant GRFT per kilogram of Nicotiana benthamiana leaf material when expressed via an infectious tobacco mosaic virus (TMV)-based vector. For example, as contemplated in the O'Keefe et al. disclosure, while it is hoped that biologics such as GRFT will act as HIV prophylactics, their practical use as topical microbicides is hampered by high production costs, with any manufacturing system that relies on growth under sterile conditions unlikely to be price competitive with male condoms, which are necessary if the product is to be used by persons at risk for sexual transmission of HIV.

[0111] Therefore, as contemplated and described herein, certain embodiments of the present disclosure relate in particular to nucleic acids encoding variant lectin proteins (e.g., Gravesin proteins), recombinant cells expressing / producing one or more variant lectin proteins, recovery of variant lectin proteins, purification of variant lectin proteins, lectin (protein) preparations, etc. More particularly, in certain embodiments, variant lectin proteins and / or DNA (nucleic acid) sequences encoding them can be derived from / obtained from known lectin proteins. In certain aspects, variant lectin proteins are derived from host organisms that naturally produce lectin proteins. Therefore, in certain embodiments, variant lectin proteins of the present disclosure are derived from eukaryotic cells or cyanobacterial cells. In certain aspects, eukaryotic cells are photosynthetic plant cells or insect cells.

[0112] In certain embodiments, the variant lectin protein is derived from one or more antiviral lectins described in U.S. Patent Publication Nos. US 20040204365, US20020127675, US20110189105, and US20110263485 and / or PCT Publication Nos. WO 2005 / 118627, WO2008 / 022303, WO 2010 / 014248, WO 2014 / 197650, WO 2016 / 130628, and WO 2019 / 108656 (each of which is incorporated herein by reference in its entirety). Thus, in certain aspects, the variant lectin protein is derived from scytovirin (SVN), GRFT, cyanobacterial antiviral protein-N (CVN), and / or functional fragments thereof.

[0113] In other embodiments, the variant lectin protein is derived from one or more antiviral lectins described in PCT Publication No. WO 2019 / 108656, such as Artocarpus heterophyllus (jackfruit lectin) lectin, Musa acuminata (banana) lectin, Boodlea coacta lectin, Microcystis viridis lectin, etc., and / or functional fragments thereof that retain the ability to bind to carbohydrates on the viral envelopes described therein.

[0114] In other embodiments, the variant lectin protein is derived from a eukaryotic lectin source as described in Singh and Sarathi (2012), including but not limited to Aaptos papilleta (sponge) lectin, Abrus precatorius (love bean) lectin, Aegapodium podagraria (Ground elder) lectin, Agaricus bisporus (common mushroom) lectin, Albizzia julibrissin (mimosa seed) lectin, Allomyrina dichotoma (Japanese beetle) lectin, Aloearborescens (aloe plant) lectin, Amphicarpaea bracteata (isomorphic bean) lectin, Anguilla (eel) lectin, Aplysia dichotoma (Japanese beetle) lectin, depilans) (mollusk; Mediterranean) lectin, Arachis aconitifolia (peanut) lectin, Jackfruit (jackfruit lectin) lectin, Bauhinia purpurea (camel hoof tree) lectin, Bryonia diocia (white bryonia) lectin, Caragana arborescens (Siberian pea tree) lectin, Carcinoscorpius rotundacauda (horseshoe crab) lectin, and / or functional fragments thereof.

[0115] In certain aspects, the variant lectin proteins of the present disclosure can be divided into multiple groups, including but not limited to "galactose (Gal)" specific lectin, "glucose (Glu)" specific lectin, "fucose (Fuc)" specific lectin, "mannose (Man)" specific lectin, "N-acetylgalactosamine (GalNAc)" specific lectin, "N-acetylglucosamine (GluNAc)" specific lectin, "sialic acid" specific lectin, etc.

[0116] Thus, in certain aspects, variant lectins suitable for use in accordance with the present disclosure can be derived / isolated from a eukaryotic lectin source organism. For example, in certain embodiments, the lectin (protein) can be isolated from a eukaryotic (source) organism using affinity chromatography techniques known to those skilled in the art (i.e., one of the above carbohydrate moieties (Gal, Man, GalNAc, etc.) is attached to an inert (chromatographic) matrix such that the lectin protein having binding specificity for the carbohydrate moiety will be retained).

[0117] In certain other embodiments, the variant lectin protein is a microvirin (MVN) lectin derived from the cyanobacterium Microcystis aeruginosa (PCC7806), which has a mannose-specific affinity. In other embodiments, the variant lectin protein is a scytovirin (SVN) derived from the cyanobacterium Scytonema varium, which binds to the mannose residues on the envelope glycoprotein of the virus with high affinity and inhibits viral replication (e.g., see Breitenbach Barroso Coelho et al., 2018). In yet other aspects, the lectin protein is an ESA-2 lectin derived from the red alga Eucheuma serra (e.g., see Breitenbach Barroso Coelho et al., 2018). In another embodiment, the lectin protein is the BanLec (Jalapenopsis agglutinin-related) lectin derived from the fruit of the banana (Musa acuminate), which recognizes high mannose glycans found on viral envelopes (see, e.g., Breitenbach Barroso Coelho et al., 2018).

[0118] In certain other embodiments, the variant lectin protein is a mannose-binding lectin derived from the rhizome of Aspidistra elatior (AEL), which has been shown to have significant in vitro inhibitory activity against vesicular stomatitis virus, coxsackievirus B4, and respiratory syncytial virus (see, e.g., Breitenbach Barroso Coelho et al., 2018).

[0119] In certain other embodiments, the lectin protein is the CVL lectin (β-galactose specific) derived from the marine worm Chaetopterus variopedatus (see, e.g., Breitenbach Barroso Coelho et al., 2018).

[0120] In certain other embodiments, the variant lectin proteins of the present disclosure can be derived from seeds of Vicia faba (fava bean), Lens culinaris (lentil), and Pisum sativum (pea), as generally described in El-Araby et al., 2020 (incorporated herein by reference). As generally described in the El-Araby et al. (2020) reference, crude extracts of the three legumes were purified by affinity chromatography using mannose agarose, wherein the purified faba, lentil, and pea lectins had molecular weights of 18 kDa, 14 kDa, and 17 kDa, respectively, as determined by amino acid sequence analysis. For example, the minimum inhibitory concentration (MIC) values ​​of these purified lectins ranged from 1.95 μg / ml to 250 μg / ml when tested against bacteria (Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumonia) and fungi (Candida albicans).

[0121] As contemplated herein, one or more variant lectins can be evaluated for function or activity by means including, but not limited to, hemagglutination activity assays, carbohydrate / glycan binding affinity assays, antimicrobial inhibition assays, combinations thereof, and the like, as described and illustrated in El-Araby et al. (2020). Thus, in certain aspects, the variant lectins of the present disclosure have antimicrobial activity (e.g., antiviral activity, antifungal activity, antibacterial activity).

[0122] In other embodiments, the present invention provides an isolated variant lectin protein or a functional fragment thereof, which comprises a sequence similar to SEQ ID NO: 8 or SEQ ID NO:18 is at least about 60% identical (such as about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) amino acid sequence, wherein the variant lectin comprises one or more (such as 1, 2, 3, 4 or 5) substitutions at positions 10, 38, 53, 54 and 78. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermostability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78. In one embodiment, the variant lectin exhibits an improvement in thermal stability (e.g., an improvement in thermal stability of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% or more, including any values ​​in between these percentages) compared to a parent lectin protein that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78. In another embodiment, the variant lectin exhibits improved expression (e.g., recombinant expression; such as an improvement in expression of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% or more, including any values ​​in between these percentages) compared to a parent lectin protein that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78.In yet another embodiment, the variant lectin exhibits improved protease stability (e.g., an improvement in protease stability of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% or more, including any values ​​between these percentages) compared to a parent lectin protein that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78. In some embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence of one of SEQ ID NOs: 10, 12, or 14. Thermal stability, expression, and protease stability can be assessed by any means known in the art, including those set forth in Examples 2 and 3 herein.

[0123] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, wherein ... comprises a substitution at position 10. The substitution may be one of X10D, X10E, X10G, X10H, X10N, X10P, X10Q or X10T. In some embodiments, the substitution is selected from the group consisting of S10D, S10E, S10G, S10H, S10N, S10P, S10Q and S10T. In some embodiments, the variant lectin polypeptide exhibits an improvement in one or more properties, including but not limited to improved thermal stability, improved expression and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise a substitution at position 10.

[0124] In further embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, wherein the variant lectin comprises a substitution at position 38. The substitution can be one of X38P or X38Q. In some embodiments, the substitution is selected from the group consisting of: H38P and H38Q. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise a substitution at position 38.

[0125] In still other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, wherein the variant lectin comprises a substitution at position 53. The substitution may be one of X53A, X53C, X53D, X53E, X53H, X53K, X53L, X53M, X53Q, X53R, X53T, or X53V. In some embodiments, the substitution is selected from the group consisting of G53A, G53C, G53D, G53E, G53H, G53K, G53L, G53M, G53Q, G53R, G53T, and G53V. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise a substitution at position 53.

[0126] In further embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (e.g., about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, wherein the variant lectin comprises a substitution at position 54. The substitution can be one of X54P or X54T. In some embodiments, the substitution is selected from the group consisting of: S54P and S54T. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise a substitution at position 54.

[0127] In another embodiment, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical to SEQ ID NO: 8 or SEQ ID NO: 18 (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical), wherein the variant lectin comprises a substitution at position 78. The substitution can be one of X78W, X78H, X78Q or X78Y. In some embodiments, the substitution is selected from the group consisting of: M / N78W, M / N78H, M / N78Q, and M / N78Y. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise a substitution at position 78.

[0128] In still further embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:8 or SEQ ID NO:18, and comprises at least two substitutions at positions 53 and 78. The substitution at position 53 may be X53E, K or L; and the substitution at position 78 may be X78N or Y. In other embodiments, the substitution at position 53 is G53E or K; and the substitution at position 78 is M / N78Y. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 53 and 78.

[0129] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:8 or SEQ ID NO:18, and comprises at least three substitutions at positions 10, 53, and 78. The substitution at position 10 may be X10D or N; the substitution at position 53 may be X53K, E, or L; and the substitution at position 78 may be X78Q or Y. In other embodiments, the substitution at position 10 is S10D or N; the substitution at position 53 is G53K, E, or L; and the substitution at position 78 is M / N78Q or Y. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 10, 53, and 78.

[0130] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:8 or SEQ ID NO:18, and comprises at least three substitutions at positions 38, 54, and 78. The substitution at position 38 may be X38Q; the substitution at position 54 may be X54P; and the substitution at position 78 may be X78Q. In other embodiments, the substitution at position 38 is H38Q; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Q. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 38, 54, and 78.

[0131] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:8 or SEQ ID NO:18, and comprises at least three substitutions at positions 10, 38, and 53. The substitution at position 10 may be X10N or D; the substitution at position 38 may be X38Q; and the substitution at position 53 may be X53E, L, or K. In other embodiments, the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; and the substitution at position 53 is G53E, L, or K. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 10, 38, and 53.

[0132] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, and comprises at least three substitutions at positions 10, 53, and 54. The substitution at position 10 may be X10N or D; the substitution at position 53 may be X53L or K; and the substitution at position 54 may be X54P. In other embodiments, the substitution at position 10 is S10N; the substitution at position 53 is G53L; and the substitution at position 54 is S54P. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 10, 53, and 54.

[0133] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, and comprises at least four substitutions at positions 10, 38, 53, and 54. The substitution at position 10 may be X10N or D; the substitution at position 38 may be X38Q; the substitution at position 53 may be X53E or K; and the substitution at position 54 may be X54P. In other embodiments, the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; the substitution at position 53 is G53E or K; and the substitution at position 54 is S54P. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 10, 38, 53, and 54.

[0134] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, and comprises at least four substitutions at positions 10, 53, 54, and 78. The substitution at position 10 can be X10N or D; the substitution at position 53 can be X53L or K; the substitution at position 54 can be X54P; and the substitution at position 78 can be X78Y or Q. In other embodiments, the substitution at position 10 is S10N or D; the substitution at position 53 is G53L or K; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Y or Q. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 10, 53, 54, and 78.

[0135] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:8 or SEQ ID NO:18, and comprises at least four substitutions at positions 10, 38, 53, and 78. The substitution at position 10 may be X10D or N; the substitution at position 38 may be X38Q; the substitution at position 53 may be X53L, E or K; and the substitution at position 78 may be X78Y or Q. In other embodiments, the substitution at position 10 is S10D or N; the substitution at position 38 is H38Q; the substitution at position 53 is G53L, E or K; and the substitution at position 78 is M / N78Y or Q. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 10, 38, 53, and 78.

[0136] In other embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (such as about any of 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to SEQ ID NO:8 or SEQ ID NO:18, and comprises at least five substitutions at positions 10, 38, 53, 54, and 78. The substitution at position 10 may be X10N or D; the substitution at position 38 may be X38Q; the substitution at position 53 may be X53K or L; the substitution at position 54 may be X54P; and the substitution at position 78 may be X78Y or Q. In other embodiments, the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; the substitution at position 53 is G53K or L; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Y or Q. In some embodiments, the variant lectin polypeptide exhibits improvements in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise substitutions at positions 10, 38, 53, 54, and 78.

[0137] In some embodiments, any of the variant lectin proteins disclosed herein or functional fragments thereof are fully functional lectin polypeptides that can naturally dimerize (i.e., form dimers) with other lectin polypeptides. In additional embodiments, the variant lectin proteins disclosed herein or functional fragments thereof are not specifically engineered to prevent the natural formation of dimers in a "monomeric lectin" form (see, e.g., International Patent Application Publication No. WO 2014197650, incorporated herein by reference), which describes a lectin polypeptide that is engineered to be unable to form a dimer with another lectin polypeptide by, for example, inserting up to four amino acids in the dimerization site (e.g., the dimerization site at Ser16 and Gly17 in SEQ ID NOs: 8 and 18; see also Moulaei et al., 2010, Structure, 18: 1104-15 and Moulaei et al., 2015, Retrovirology, 12: 6, incorporated herein by reference).

[0138] B. Variant lectin polypeptide dimer

[0139] Any of the variant lectin polypeptides disclosed herein may also dimerize to form a lectin polypeptide dimer. As used herein, the phrase "polypeptide dimer" refers to two proteins (or functional fragments thereof) bound together by one or more peptide bonds. In some embodiments, the variant lectin polypeptides of the polypeptide dimer are identical, while in other embodiments, each variant may have unique substitutions relative to the other variants in the dimer.

[0140] Optionally, the variant lectin polypeptides of the polypeptide dimer can be separated by a linker. "Linker" or "peptide linker" as used interchangeably herein generally refers to a synthetic amino acid sequence that connects or links two polypeptide sequences (e.g., connects two variant lectin polypeptides). The linker can connect the two amino acid sequences by a peptide bond. In some embodiments, the linker of the present disclosure connects the biologically active portion to the second portion in a linear sequence. In some embodiments, the length of the linker can be 1 to 50 or more amino acids (such as about 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 or more amino acids in length). In some embodiments, the linker comprises one of SEQ ID NOs: 32-57. In other embodiments, the linker comprises one of (GGG)n or (GPG)n, wherein n=1-8 (such as any one of 1, 2, 3, 4, 5, 6, 7 or 8).

[0141] In some embodiments, provided herein are isolated variant lectin polypeptide dimers or functional fragments thereof comprising a polypeptide having the same sequence as SEQ ID NO: 8 and / or SEQ ID NO: NO: 18 Two amino acid sequences that are at least about 60% identical (e.g., any of about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), wherein each variant lectin component of the dimer comprises one or more (e.g., 1, 2, 3, 4, or 5) substitutions at positions 10, 38, 53, 54, and 78. It should be further understood that each variant lectin component of the dimer can have a unique substitution relative to another member. In some embodiments, the variant lectin polypeptide dimer exhibits an improvement in one or more properties, including but not limited to improved thermal stability, improved expression, and / or improved protease stability, compared to a parent lectin protein (e.g., SEQ ID NO: 8 or SEQ ID NO: 18) that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78 and / or is not a polypeptide dimer. In one embodiment, the variant lectin polypeptide dimer exhibits an improvement in thermal stability (e.g., an improvement in thermal stability of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% or more, including any values ​​between these percentages) compared to a parent lectin protein that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78 and / or is not a polypeptide dimer. In another embodiment, the variant lectin exhibits improved expression (e.g., recombinant expression; such as an improvement in expression of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% or more, including any values ​​in between these percentages) compared to a parent lectin protein that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78 and / or is not a polypeptide dimer.In yet another embodiment, the variant lectin exhibits improved protease stability (e.g., an improvement in protease stability of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, or 150% or more, including any values ​​between these percentages, compared to a parent lectin protein that does not comprise one or more substitutions at positions 10, 38, 53, 54, and 78 and / or is not a polypeptide dimer. In some embodiments, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence of one of SEQ ID NOs: 20, 22, 24, 26, 29, or 31. Thermal stability, expression, and protease stability can be assessed by any means known in the art, including those set forth in Examples 2 and 3 herein.

[0142] In some embodiments, each variant lectin polypeptide of the polypeptide dimer disclosed herein is a fully functional lectin polypeptide that is capable of naturally dimerizing (i.e., forming a dimer) with other lectin polypeptides (i.e., in the case of a lectin polypeptide dimer, each member of the dimer can further dimerize with another lectin polypeptide). In additional embodiments, each variant lectin polypeptide of the polypeptide dimer disclosed herein is not specifically engineered to be a "monomeric lectin" form that prevents natural dimer formation (see, e.g., International Patent Application Publication No. WO 2014197650, incorporated herein by reference), which describes lectin polypeptides that are engineered to be unable to form dimers with another lectin polypeptide by, for example, inserting up to four amino acids at the dimerization site (such as the dimerization site at Ser16 and Gly17 in SEQ ID NOs: 8 and 18; see also Moulaei et al., 2010, Structure, 18: 1104-15 and Moulaei et al., 2015, Retrovirology, 12: 6, incorporated herein by reference).

[0143] C. Nucleic acids and vectors

[0144] In another aspect, provided herein is any isolated, recombinant, substantially pure, synthetically derived or non-naturally occurring nucleic acid comprising a nucleotide sequence encoding any variant lectin polypeptide or variant lectin polypeptide dimer disclosed herein and which has at least antiviral activity.

[0145] In addition, of interest are vectors comprising polynucleotides encoding variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein. It will be apparent to the skilled artisan that the vector may be any suitable expression vector and that the choice of vector may vary depending on the type of cell into which the vector is to be inserted. Suitable vectors include pGAPT-PG, pRAX1, pGAMD, pGPT-pyrG1, pC194, pJH101, pE194, and pHP13 (see Harwood and Cutting [eds.], Chapter 3, Molecular Biological Methods for Bacillus, John Wiley & Sons

[1990] ). See also Perego, Integrational Vectors for Genetic Manipulations in Bacillus subtilis, in Sonenshein et al. [eds.] Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology and Molecular Genetics, American Society for Microbiology, Washington (1993), pp. 615-624, and p2JM103BBI.

[0146] Expression vector can be one of any number of vectors or boxes for transforming suitable production hosts known in the art. Typically, vector or box will include sequences for instructing transcription and translation of related genes, selectable markers and sequences for allowing autonomous replication or chromosomal integration. Suitable vectors generally include 5' districts containing genes for transcription initiation control and 3' districts for DNA fragments for controlling transcription termination. Two control regions can be derived from genes homologous to the gene of the production host cell transformed and / or be natural genes for the production host, although such control regions do not need to be derived in this way.

[0147] The DNA fragment that controls transcription termination can also be derived from various genes natural to the preferred production host cell. In certain embodiments, it is optional to include a termination control region. In certain embodiments, the expression vector includes a termination control region derived from a preferred host cell.

[0148] Expression vectors can be included in a production host, particularly in the cells of a microbial production host. The production host cells can be microbial hosts found in the fungal or bacterial families and which grow over a wide range of temperatures, pH values and solvent tolerances. For example, any of bacteria, algae and fungi (such as filamentous fungi and yeasts) can suitably accommodate the expression vector.

[0149] Including an expression vector in the production host cells can be used to express a target protein such that it can be present intracellularly, extracellularly or in a combination of intracellular and extracellular. Compared with methods for recovering proteins produced by intracellular expression, extracellular expression makes it easier to recover the desired protein from the fermentation product.

[0150] The recombinant expression vector can be any vector, such as a plasmid or a virus, which can be conveniently subjected to recombinant DNA procedures and result in the expression of a nucleotide sequence. Vector selection typically depends on the compatibility of the vector with the production host into which the vector is to be introduced. The vector can be a linear or closed circular plasmid. The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can contain any means for ensuring self-replication.

[0151] Alternatively, the vector can be a vector that integrates into the genome when introduced into the production host and replicates with the chromosome into which it has been integrated. Some non-limiting examples of such vectors are provided in the Fungal Genetics Stock Center Catalogue of Strains (FGSC, <www.fgsc.net), additional examples of suitable expression and / or integration vectors are provided in Sambrook et al., (1989) supra, Ausubel (1987) supra, van den Hondel et al. (1991) in Bennett and Lasure (eds.) MORE GENE MANIPULATIONS IN FUNGI, Academic Press, 396 - 428 and U.S. Patent No. 5,874,276.

[0152] Particularly useful vectors include pTREX, pFB6, pBR322, PUCI8, pUCIOO, and pENTR / D. Suitable plasmids for use in bacterial cells include pBR322 and pUC19, which allow replication in E. coli, and pE194, which allows replication in Bacillus, for example. Briefly, for production in production host cells, reference is made to Sambrook et al., (1989) supra, Ausubel (1987) supra, van den Hondel et al., (1991) in Bennett and Lasure (eds.) MORE GENE MANIPULATIONS IN FUNGI, Academic Press (1991), pp. 76 and 396-428; Nunberg et al., (1984) Mol. Cell Biol. 4:2306-2315; Boel et al., (1984) 30 EMBO 1 3:1581-1585; Finkelstein in BIOTECHNOLOGY OF FILAMENTOUS FUNGI [Biotechnology of Filamentous Fungi], Finkelstein et al., ed. Butterworth-Heinemann, Boston, Massachusetts (1992), Chapter 6; Kinghorn et al. (1992), APPLIED MOLECULAR GENETICS OF FILAMENTOUS FUNGI [Applied Molecular Genetics of Filamentous Fungi], Blackie Academic and Professional, Chapman and Hall, London; Kelley et al., (1985) EMBO [Journal of the European Molecular Biology Association] 1 4:475-479; Penttila et al., (1987) Gene [Gene] 61:155-164; and U.S. Patent No. 5,874,276.

[0153] Lists of suitable vectors can be found in the Fungal Genetics Stock Center strain catalog (FGSC, www at fgsc.net) and the Bacillus Genetics Stock Center (BGSC, Hypertext Transfer Protocol Secure: / / bgsc.org). Suitable vectors include those obtained from, for example, Invitrogen Life Technologies and Promega. Specific vectors suitable for use in fungal host cells include vectors such as pFB6, pBR322, pUC 18, pUC100, pDONTm201, pDONRTm221, pENTRTm, pGEM(D3Z, and pGEM(D4Z).

[0154] The vector system may be a single vector or plasmid or two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.

[0155] The vector may also contain one or more selectable markers to allow easy selection of transformed cells. Selectable markers are genes whose products provide biocide or viral resistance, etc. Examples of selectable markers include markers that confer antimicrobial resistance. Nutritional markers may also be used in the present invention, including those markers known in the art as amdS, argB, and pyr4. In some embodiments, the expression vector also includes a replicon, a gene encoding antibiotic resistance that allows selection of bacteria carrying the recombinant plasmid, and unique restriction sites in non-essential regions of the plasmid that allow insertion of heterologous sequences. The specific antibiotic resistance gene selected is not critical; any of the many resistance genes known in the art are suitable.

[0156] The vector may also contain elements that allow the vector to be stably integrated into the production host genome or the vector to replicate autonomously in the production host independently of the cell genome. For integration into the host cell genome, the vector may rely on a nucleotide sequence encoding an aspartic protease or any other element of the vector for stable integration of the vector into the genome by homologous or nonhomologous recombination.

[0157] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the production host.

[0158] More than one copy of the nucleotide sequence encoding the variant lectin polypeptide or variant lectin polypeptide dimer disclosed herein may be inserted into the production host to increase the production of the variant lectin polypeptide or variant lectin polypeptide dimer. The increase in the number of copies of the nucleotide sequence may be obtained by integrating at least one additional copy of the sequence into the genome of the production host or by including an amplifiable selectable marker gene, and thus the additional copies of the nucleotide sequence may be selected by culturing the production host cell in the presence of an appropriate selection agent.

[0159] A vector comprising a nucleotide sequence encoding a variant lectin polypeptide or variant lectin polypeptide dimer disclosed herein is introduced into a production host such that the vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector. Integration is generally considered an advantage because the nucleotide sequence is more likely to be stably maintained in the production host. As discussed above, integration of the vector into the production host chromosome can occur by homologous or nonhomologous recombination.

[0160] Exemplary vectors include, but are not limited to, pGXT (same as the pTTTpyr2 vector described in published PCT application WO 2015 / 017256). Standard bacterial expression vectors, including bacteriophage X and M13, and plasmids such as pBR322-based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ may also be mentioned. Epitope tags (e.g., c-myc) may also be added to recombinant proteins to provide convenient isolation methods. Examples of suitable expression and / or integration vectors are provided in Sambrook et al., (1989) supra, Bennett and Lasure (eds.) More Gene Manipulations in Fungi, (1991) Academic Press, pp. 70-76 and 396-428, and articles cited therein; U.S. Pat. No. 5,874,276, and in the Fungal Genetics Stock Center Catalogue of Strains (FGSC).

[0161] Useful vectors can be obtained from Promega and Invitrogen. Some specific useful vectors include pBR322, pUC18, pUC100, pDONTm201, pENTRTm, pGEN(11)3Z and pGEN4D4Z. However, other forms of expression vectors that perform equivalent functions and are known or become known in the art may also be used. Thus, a variety of host / expression vector combinations may be used in expressing the DNA sequences disclosed herein. For example, useful expression vectors can be composed of segments of chromosomal, non-chromosomal, and synthetic DNA sequences, such as various known derivatives of 5V40 and known bacterial plasmids (e.g., plasmids from E. coli, including col El, pCR1, pBR322, pMb9, pUC 19, and derivatives thereof), broader host range plasmids (e.g., RP4), phage DNA (e.g., various derivatives of phage λ, such as NM989), as well as other DNA phages (e.g., M13 and filamentous single-stranded DNA phages), yeast plasmids (e.g., 2.mu plasmid or derivatives thereof).

[0162] D. Host cells

[0163] The isolated cells containing DNA constructs or expression vectors are advantageously used as host cells for recombinantly producing variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein. Cells can be conveniently transformed with DNA constructs encoding variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein by integrating the DNA construct (in one or more copies) into the host chromosome. It is generally believed that this integration is advantageous because the DNA sequence is more likely to be stably maintained in the cell. The DNA construct can be integrated into the host chromosome according to conventional methods, for example, by homologous or heterologous recombination. Alternatively, cells can be transformed with expression vectors associated with different types of host cells.

[0164] In certain embodiments, the host cell expresses a heterologous polynucleotide encoding a variant lectin polypeptide or variant lectin polypeptide dimer disclosed herein or a functional variant thereof. In certain aspects, the heterologous polynucleotide encoding the variant lectin polypeptide is an expression cassette introduced into a recombinant cell. In certain embodiments, at least one expression cassette is introduced into a host cell. In other embodiments, at least two expression cassettes are introduced into a host cell. Therefore, in certain aspects, the host cell of the present disclosure comprises one or more variant lectin polypeptide or variant lectin polypeptide dimer expression cassettes introduced therein, wherein the host cell expresses the variant lectin polypeptide or variant lectin polypeptide dimer when cultured under appropriate conditions.

[0165] In certain aspects, the host cell is a Gram-positive bacterial cell and includes Bacillus, Clostridium, and Mollicutes (e.g., including Lactobacillales with Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Oscillospiraceae, Streptococcaceae, and Lactobacillales with Alicyclobacillaceae). cellaceae, Bacillaceae, Caryophanaceae, Listeriaceae, Paenibacillaceae, Planococcaceae, Sporolactobacillaceae, Staphylococcaceae, Thermoactinomycetaceae, Turicibacteraceae, and the Bacillales.

[0166] In certain embodiments, species of the Bacillaceae family include Alkalibacillus, Amphibacillus, Anoxybacillus, Bacillus, Caldalkalibacillus, Cerasilbacillus, Exiguobacterium, Filobacillus, Geobacillus, Gracilibacillus, Halobacillus, Halolactibacillus, Jeotgalibacillus, Lentibaci llus), Marinibacillus, Oceanobacillus, Ornithinibacillus, Paraliobacillus, Paucisalibacillus, Pontibacillus, Pontibacillus, Saccharococcus, Salibacillus, Salinibacillus, Tenuibacillus, Thalassobacillus, Ureibacillus, and Virgibacillus.

[0167] In other embodiments, the Bacillus species cells include, but are not limited to, B. acidiceler, B. acidicola, B. acidocaldarius, B. acidoterrestris, B. aeolus, B. aerius, B. aerophilus, B. agaradhaerens, B. agri, B. aidingensis, B. akib ai), Bacillus alcalophilus, Bacillus algicola, Bacillus alginolyticus, Bacillus alkalidiazo-trophicus, Bacillus alkalinitrilicus, Bacillus alkalitelluris, Bacillus altitudinis, Bacillus alveayuensis, Bacillus alvei, Bacillus amylolyticus, Bacillus thiamine Bacillus aneurinilyticus, Bacillus aneurinolyticus, Bacillus anthracis, Bacillus aquimaris, Bacillus arenosi, Bacillus arseniciselenatis, Bacillus arsenicoselenatis, Bacillus arsenicus, Bacillus arvi, Bacillus asahii, Bacillus atrophaeus, Bacillus aurantii, Bacillus arsenicus, Bacillus arvi, Bacillus asahii, Bacillus atrophaeus, Bacillus aurantii .aurantiacus), Bacillus axarquiensis, Bacillus azotofixans, Bacillus azotoformans, Bacillus badius, Bacillus barbaricus, Bacillus bataviensis, Bacillus beijingensis, Bacillus benzoevorans, Bacillus bogoriensis, Bacillus boroniphilus, Bacillus potsdamiensis.borstelenis), Bacillus butanolivorans, Bacillus carboniphilus, Bacillus cecembensis, Bacillus cellulosilyticus, Bacillus centrosporus, Bacillus chagannorensis, Bacillus chitinolyticus, Bacillus chondroitinus, Bacillus choshinensis, Bacillus cibi, Bacillus circulans, .

[0168] Bacillus clarkii, Bacillus clausii, Bacillus coagulans, Bacillus coahuilensis, Bacillus cohnii, Bacillus curdianolyticus, Bacillus cycloheptanicus, Bacillus decisifrondis, Bacillus decolorationis, Bacillus dipsosauri, Bacillus drentensis, Bacillus edaphicus, Bacillus ehimensis, Bacillus endophyticus, Bacillus farraginis, Bacillus fastidis osus), B. firmus, B. plexus, B. foraminis, B. fordii, B. formosus, B. fortis, B. fumarioli, B. funiculus, B. fusiformis, B. galactophilus, B. galactosidilyticus, B. gelatini, B. gibsonii, B. ginsengi, B. ginsengihumi, B. globisporus, B. globisporus subsp.globisporus), B.globisporus subsp.marinus, B.glucanolyticus, B.gordonae, B.halmapalus, B.haloalkaliphilus, B.halodenitrificans, B.halodurans, B.halophilus, B.hemicellulosilyticus, B.herbersteinensis, B.horikoshihorikoshii), Garden Bacillus (B.horti), Land Bacillus (B.hemi), Hwajintan Bacillus (B.hwajinpoensis), Disease Research Institute Bacillus (B.idriensis), Indian Bacillus (B.indicus), Infantis Bacillus (B.infantis), Lower Bacillus (B.infernus), Abnormal Bacillus (B.insolitus), Isabeliae Bacillus (B.jeotgali), Hot Bacillus (B.kaustophilus), Kobensis Bacillus (B.kobensis), Bacillus koreensis, Bacillus kribbensis, Bacillus krulwichiae, Bacillus laevolacticus, Bacillus larvae, Bacillus laterosporus, Bacillus lautus, Bacillus lehensis, Bacillus lentimorbus, Bacillus lentus, Bacillus litoralis, Bacillus luciferensis, Bacillus macauensis, Bacillus macerans, Bacillus macquariensis, Bacillus machyae, Bacillus malacitus, Bacillus mannanilyticus, Bacillus marinus, Bacillus marisflavi, Bacillus marismortui, Bacillus massiliensis, Bacillus methanolicus, Bacillus miritzii (B.migulanus), B.mojavensis, B.mucilaginosus, B.muralis, B.murimartini, B.mycoides, B.naganoensis, B.nealsonii, B.neidei, B.niabensis, B.niacini, B.novalis, B.odsey (B.odysseyi), B.okhensis, B.okuhidensis, B.oleronius, B.oshimensis, B.pabuli, B.pallidus, B.illeg., B.panaciterrae, B.pantothenticus, B.parabrevis, B.pasteurii, B.patagonianus B.patagoniensis), B.peoriae, B.plakortidis, B.pocheonensis, B.polygoni, B.polymyxa, B.popilliae, B.pseudalcaliphilus, B.pseudofirmus, B.pseudomycoides, B.psychrodurans, Bacillus psychrophilus, Bacillus psychrosaccarolyticus, Bacillus psychrotolerans, Bacillus pulvifaciens, Bacillus pycnus, Bacillus qingdaonensis, Bacillus reuszeri, Bacillus runs, Bacillus safensis, Bacillus salarius, Bacillus salexigens, Bacillus halophilus Bacillus saliphilus, Bacillus schlegelii, Bacillus selenatarsenatis, Bacillus selenitrireducens, Bacillus seohaeanensis, Bacillus shackletonii, Bacillus silvestris, Bacillus simplex, Bacillus siralis, Bacillus smithii, Bacillus soli, Bacillus sonora desertiformis, Bacillus seleni ...sonorensis), B. sphaericus, B. sporothermodurans, B. stearothermophilus, B. stratosphericus, B. subterraneus, B. subtilis subsp.spizizenii, B. subtilis subsp. subsp.subtilis), Taiwan Bacillus (B.taeanensis), Tequilensis Bacillus (B.tequilensis), Thermoantarcticus Bacillus (B.thermantarcticus), Thermoaerophilus Bacillus (B.thermoaerophilus), Thermoamylovorans Bacillus (B.thermoantarcticus), Thermocatenulatus Bacillus (B.thermocloacae), Thermodenitrificans Bacillus (B.thermodenitrificans), Thermoglucosidasius Bacillus (B.thermoleovorans), Thermored Bacillus (B.t hermoruber), B. thermosphaericus, B. thiaminolyticus, B. thioparans, B. thuringiensis, B. tusciae, B. validus, B. vallismortis, B. vedderi, B. velezensis, B. vietnamensis, B. vireti, B. vulcani, B. wakoensis, and B. weihenstephanensis.

[0169] In a specific embodiment, the Bacillus species cell is selected from the group consisting of: Bacillus subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. kluyveri, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. radiata, and B. thuringiensis. As used herein, "Bacillus" includes Bacillus species that have been reclassified, including but not limited to B. stearothermophilus, which is now named "Geobacillus stearothermophilus".

[0170] Other examples of suitable bacterial host organisms include Streptomyces species, such as Streptomyces murinus; lactic acid bacteria species, including Lactococcus species, such as Lactococcus lactis; Lactobacillus species, including Lactobacillus reuteri; Leuconostoc species; Pediococcus species; and Streptococcus species. Alternatively, strains of gram-negative bacterial species belonging to the family Enterobacteriaceae (including Escherichia coli) or Pseudomonadaceae can be selected as host organisms.

[0171] Suitable yeast host organisms can be selected from biotechnology-related yeast species, such as, but not limited to, yeast species, such as Pichia species, Hansenula species or Kluyveromyces, Yarrowinia, Schizosaccharomyces species; or species of the genus Saccharomyces, including Saccharomyces cerevisiae; or species belonging to the genus Schizosaccharomyces, such as, for example, S. pombe species. Methylotrophic yeast species strains Pichia pastoris can be used as host organisms. Alternatively, the host organism can be a Hansenula species.

[0172] In filamentous fungi, suitable host organisms include species of Aspergillus, for example, Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori or Aspergillus nidulans. Alternatively, strains of Fusarium species (for example, Fusarium oxysporum) or strains of Rhizomucor species (such as Rhizomucor miehei) can be used as host organisms. Other suitable strains include Thermomyces and Mucor species. In addition, Trichoderma species can be used as hosts. The glucoamylase expressed by the fungal host cell can be glycosylated, that is, will contain a glycosyl moiety. The glycosylation pattern may be the same or different than that present in the wild-type glucoamylase. The type and / or extent of glycosylation may confer alterations in enzymatic and / or biochemical properties.

[0173] As generally described above and further described in the examples below, certain embodiments of the present disclosure relate to recombinant host cells expressing heterologous variant lectin polypeptides or variant lectin polypeptide dimer proteins, recombinant polynucleotides (e.g., vectors, expression cassettes) encoding heterologous variant lectin polypeptides or variant lectin polypeptide dimer proteins, which are particularly suitable for introduction (e.g., transformation) into host cells (i.e., for expression of heterologous variant lectin polypeptides or variant lectin polypeptide dimer proteins), etc.

[0174] In other embodiments, the host cells of the present disclosure are made to lack the production of one or more natural (endogenous) proteins. In some aspects, the recombinant host cells of the present disclosure comprise deletions or destructions of one or more endogenous genes encoding one or more proteases that are natural to the recombinant cell. For example, in certain embodiments, recombinant host cells that are defective in producing one or more natural (endogenous) proteases can be used to mitigate variant lectin polypeptides or variant lectin polypeptide dimer protein degradation (e.g., in fermentation and / or downstream processing of variant lectin polypeptides or variant lectin polypeptide dimer proteins). It is also contemplated herein that recombinant host cells that are defective in producing one or more natural background proteases or other problematic (natural) background proteins will promote downstream recovery and purification of variant lectin polypeptides or variant lectin polypeptide dimer proteins (e.g., by reducing undesirable host cell background (natural) protein contaminants).

[0175] Therefore, certain embodiments are particularly directed to nucleic acids, polynucleotides (e.g., plasmids, vectors, expression cassettes), regulatory elements, etc. suitable for use in constructing recombinant host cells. Therefore, as shown in the examples and generally described herein, recombinant cells of the present disclosure can be constructed by those skilled in the art using standard and conventional recombinant DNA and molecular cloning techniques well known in the art. Methods for genetically modifying cells include, but are not limited to, (a) introducing, replacing, or removing one or more nucleotides in a gene, or introducing, replacing, or removing one or more nucleotides in a regulatory element required for transcription or translation of a gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) site-specific mutagenesis, and / or (g) random mutagenesis.

[0176] In certain embodiments, the recombinant (modified) cells of the present disclosure can be constructed by reducing or eliminating the expression of a gene using methods well known in the art (e.g., insertion, disruption, substitution or deletion). The portion of the gene to be modified or inactivated can be, for example, a coding region or a regulatory element required for expression of the coding region.

[0177] Examples of such regulatory or control sequences may be promoter sequences or functional portions thereof (i.e., portions sufficient to affect the expression of nucleic acid sequences). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, and the like.

[0178] In certain other embodiments, the modified cells are constructed by gene deletion to eliminate or reduce the expression of a gene.Gene deletion technology enables the partial or complete removal of genes, thereby eliminating their expression, or expressing a non-functional (or reduced activity) protein product.

[0179] In such a method, the deletion of a gene can be accomplished by homologous recombination using a plasmid constructed to continuously contain 5' and 3' regions flanking the gene. For example, continuous 5' and 3' regions can be introduced into cells associated with a second selectable marker at a permissive temperature (e.g., on a temperature-sensitive plasmid (such as pE194)) to allow the plasmid to be established in the cell. The cells are then transferred to a non-permissive temperature to select cells that integrate the plasmid into one of the homologous flanking regions of the chromosome. The selection of plasmid integration is affected by selecting a second selectable marker. After integration, the recombination event at the second homologous flanking region is stimulated by moving the cells to a permissive temperature for several generations without selection. The cells are plated to obtain a single colony, and the colony is checked for loss of two selectable markers.

[0180] Thus, one skilled in the art can readily identify nucleotide regions (suitable for complete or partial deletion) in the coding sequence of a gene and / or in the non-coding sequence of a gene.

[0181] In other embodiments, modified cells are constructed by introducing, substituting, or removing one or more nucleotides in a gene or a regulatory element required for its transcription or translation.

[0182] For example, nucleotides may be inserted or removed to result in the introduction of a stop codon, the removal of a start codon, or a frameshift of an open reading frame. Such modifications may be accomplished by site-directed mutagenesis or PCR-generated mutagenesis according to methods known in the art. Therefore, in certain embodiments, the gene of the present disclosure is inactivated by complete or partial deletion.

[0183] In another embodiment, modified cells are constructed by gene conversion processes. For example, in gene conversion methods, the nucleic acid sequence corresponding to the gene is induced in vitro to produce a defective nucleic acid sequence, and then the defective nucleic acid sequence is transformed into a host cell to produce a defective gene. Through homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may be desirable that the defective gene or gene fragment also encodes a marker that can be used to select a transformant containing the defective gene. For example, the defective gene can be associated with a selectable marker and introduced into a non-replicating or temperature-sensitive plasmid. The selection of plasmid integration is affected by selecting markers under conditions that do not allow plasmid replication. The selection of the second recombination event that causes gene replacement is affected by checking whether the bacterium colony loses the selectable marker and whether the mutated gene is obtained. Alternatively, the defective nucleic acid sequence can contain the insertion, substitution or deletion of one or more nucleotides of the gene, as described below.

[0184] In other embodiments, modified cells are constructed using nucleotide sequences complementary to the nucleotide sequence of the gene by the antisense technology established. More particularly, the expression of the gene of the host cell can be reduced (downregulated) or eliminated by introducing a nucleotide sequence complementary to the nucleotide sequence of the gene, which can be transcribed in the cell and can hybridize with the mRNA produced in the cell. Under conditions where the complementary antisense nucleotide sequence is allowed to hybridize with the mRNA, the amount of the translated protein is therefore reduced or eliminated. Such antisense methods include but are not limited to RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to technicians.

[0185] In other embodiments, modified cells are constructed by random or specific mutagenesis using methods well known in the art (including but not limited to chemical mutagenesis and transposition).Gene modification can be performed by subjecting parental cells to mutagenesis and screening mutant cells whose gene expression has been reduced or eliminated.Specific or random mutagenesis can be performed, for example, by using suitable physical or chemical mutagens, using suitable oligonucleotides, or subjecting DNA sequences to mutagenesis produced by PCR. In addition, mutagenesis can be performed by using any combination of these mutagenesis methods.Examples of physical or chemical mutagens suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When such agents are used, mutagenesis is typically performed by incubating the parental cells to be mutagenized under appropriate conditions in the presence of the selected mutagenizing agent, and selecting for mutant cells that exhibit reduced or no expression of the gene.

[0186] PCT Publication No. WO 2003 / 083125 (incorporated herein by reference) discloses methods for modifying Bacillus cells, such as using PCR fusion to generate Bacillus deletion strains and DNA constructs to bypass E. coli. PCT Publication No. WO 2002 / 14490 (incorporated herein by reference) discloses methods for modifying Bacillus cells, including (1) construction and transformation of an integrating plasmid (pComK), (2) random mutagenesis of coding sequences, signal sequences, and propeptide sequences, (3) homologous recombination, (4) improving transformation efficiency by adding nonhomologous flanks to the transforming DNA, (5) optimizing double crossover integration, (6) site-directed mutagenesis, and (7) markerless deletion.

[0187] Suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., E. coli, Bacillus species) are well known to those skilled in the art. In fact, methods such as transformation, including protoplast transformation and mid-plate collection, transduction, and protoplast fusion, are known and suitable for use in the present disclosure. Transformation methods are particularly suitable for introducing the DNA constructs of the present disclosure into host cells.

[0188] In addition to common methods, in some embodiments, host cells are directly transformed (i.e., before introducing the host cells, no intermediate cells are used to amplify the DNA construct or otherwise process the DNA construct). Introducing the DNA construct into the host cell includes those physical and chemical methods known in the art for introducing DNA into the host cell without inserting into a plasmid or vector. Such methods include but are not limited to calcium chloride precipitation, electroporation, naked DNA, liposomes, etc. In additional embodiments, the DNA construct is co-transformed with a plasmid without inserting into the plasmid. In other embodiments, by methods known in the art, a selective marker is deleted or substantially excised from a modified host strain. In certain embodiments, the carrier is decomposed from the host chromosome, and the flanking region is left on the chromosome, while the intrinsic chromosome region is removed.

[0189] Promoters and promoter sequence regions for expressing genes, open reading frames (ORFs) and / or variant sequences thereof in host cells are generally known to those skilled in the art. Promoter sequences of the present disclosure are generally selected so that they function in host cells. For example, promoters that can be used to drive gene expression in Bacillus cells include, but are not limited to, subtilis alkaline protease (aprE) promoter, α-amylase promoter (amyE) of Bacillus subtilis, α-amylase promoter (amyL) of Bacillus licheniformis, α-amylase promoter of Bacillus amyloliquefaciens, neutral protease (nprE) promoter from Bacillus subtilis, mutant aprE promoter, or any other promoter from Bacillus licheniformis or other related Bacillus. Methods for screening and generating promoter libraries with a range of activities (promoter strengths) in Bacillus cells are described in PCT Publication No. WO 2002 / 14490 (incorporated herein by reference).

[0190] E. Antiviral composition

[0191] Any of the antiviral variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein for use in the methods disclosed herein can be formulated into a composition (e.g., a pharmaceutical or nutritional composition). In some embodiments, the variant lectin polypeptides or variant lectin polypeptide dimers with antiviral activity described herein can be used to treat and / or prevent diseases associated with viral infection (such as, but not limited to, porcine reproductive and respiratory syndrome (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus, or equine viral arteritis (EVA)). In one embodiment, the present invention provides a composition, preferably a pharmaceutical composition, comprising a protein according to the present invention. The pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0192] The composition can be presented in any form, for example as a tablet, as an injectable fluid or as an infusion fluid, etc. In addition, the composition, protein, nucleotide and / or carrier according to the present invention can be administered via different routes, such as topical, intravenous, rectal, bronchial, nasal or oral. Yet another suitable route of administration is using a duodenal drip.

[0193] In one embodiment, the administration route used is an intravenous route. It is clear to those skilled in the art that an effective amount of a variant lectin polypeptide or variant lectin polypeptide dimer according to the present invention is preferably delivered. Another suitable route is a subcutaneous route. If an intravenous administration route is used, a protein according to the present invention can be applied (at least for a certain amount of time) via continuous infusion.

[0194] The compositions according to the present invention may optionally contain pharmaceutically acceptable excipients, salts, stabilizers, activators, carriers, osmotic agents, propellants, disinfectants, diluents and preservatives. Suitable excipients are well known in the art of pharmaceutical formulations and can be easily found and applied by the skilled person, for example, in Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pennsylvania, 17th edition 1985.

[0195] For oral administration, variant lectin polypeptides or variant lectin polypeptide dimers can be administered, for example, in solid dosage forms such as capsules, tablets (e.g., with enteric coatings) and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. Variant lectin polypeptides or variant lectin polypeptide dimers can be encapsulated in gelatin capsules with inactive ingredients and powdered carriers (e.g., glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum powder, magnesium carbonate, etc.). Examples of additional inactive ingredients that can be added to provide desired color, taste, stability, buffering capacity, dispersibility or other known desired characteristics are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be manufactured as sustained-release products for use in sustained release of drugs over several hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain coloring and flavoring to improve palatability.

[0196] Enteric coatings prevent the release of active compounds from the orally ingestible dosage form. Depending on the composition and / or thickness, enteric coatings withstand gastric acid for a desired time before they begin to disintegrate and allow the variant lectin polypeptide or variant lectin polypeptide dimer to be slowly released in the lower stomach, small intestine or large intestine. Some examples of enteric coatings are disclosed in U.S. Pat. No. 5,225,202 (incorporated by reference). Examples of enteric coatings include beeswax and glyceryl monostearate; beeswax, shellac and cellulose, optionally containing neutral copolymers with polymethacrylates; copolymers of methacrylic acid and methyl methacrylate or neutral copolymers of polymethacrylates containing metal stearates (for references to enteric coatings, see: U.S. Pat. Nos. 4,728,512, 4,794,001, 3,835,221, 2,809,918, 5,225,202, 5,026,560, 4,524,060, 5,536,507). Most enteric coating polymers become soluble at pH 5.5 and above, with maximum solubility at pH values ​​above 6.5. Enteric coating can also include a subcoating and an outer coating step, such as a pharmaceutical composition intended for specific delivery in the lower GI tract, i.e., in the colon (pH 6.4 to 7.0, ileum pH 6.6), in contrast to the pH of the upper intestine, the pH range of the duodenum of the small intestine is 7.7-8 (after the addition of pancreatic juice and bile). The pH differences in the intestine can be used to target enteric-coated variant lectins or variant lectin dimer compositions to specific areas in the digestive tract. It also allows the selection of specific variant lectins or variant lectin dimers that are most active at a specific pH in the intestine.

[0197] Any of the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein can also be administered intranasally. As used herein, "nasal administration" means the same as "intranasal administration", wherein the composition is administered to the inside of the nasal cavity. In some embodiments, the intranasal dosage composition further comprises a mucoadhesive. The mucoadhesive imparts mucoadhesive properties to the composition, so that the composition remains in the nasal cavity and does not drip out of the nasal cavity from the external nostril (nare / nostril) or the back of the throat. Nasal mucociliary clearance is one of the limiting factors for nasal drug delivery because it reduces the time allowed for drug absorption. Therefore, nasal drug absorption can be improved by extending the contact time between the drug and the nasal mucosa. Mucoadhesion refers to the attachment of the composition to the nasal mucosa, involving an interaction between mucin and a mucoadhesive (a synthetic or natural polymer). The sequential events that occur during mucoadhesion include the first step in which the mucoadhesive absorbs water from the nasal mucosa and swells. Then, the mucoadhesive tightly penetrates into the nasal mucosa, thereby confining the formulation to the nasal cavity and enhancing the drug concentration gradient across the epithelium.

[0198] Exemplary mucoadhesives include alginates (e.g., sodium alginate), cellulose and cellulose derivatives (e.g., carboxymethyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, microcrystalline cellulose, combinations thereof, etc.), chitosan, gelling proteins (e.g., gelatin), hydroxyethyl methacrylate, modified starches (e.g., heat-modified starches, etc.), natural gums and polysaccharides (e.g., Acacia, gum Arabic, guar gum, gum Karaya, pectin, tragacanth, combinations thereof, etc.), polyacrylic acids (e.g., CAS No. 9003-01-4 from Lubrizol), 934P), poly(acrylic acid / divinylbenzene), poly(lactic acid), polycarbophil (i.e., polyacrylic acid cross-linked with divinyl glycol), polyvinylpyrrolidone, psyllium seed gum, resins (e.g., Amberlite-200, cation exchange resins based on sulfonic acid exchange groups on a polystyrene matrix, etc.), or combinations thereof. Specific mucoadhesives include microcrystalline cellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, or combinations thereof.

[0199] The composition may optionally further comprise an intranasal formulation excipient, such as a buffer, a flavoring agent, a sweetener, a tonicity agent, an antimicrobial preservative, an antimicrobial preservative synergist, a surfactant, an emulsifier, a solubilizer, an absorption enhancer, or a combination thereof. In some cases, a single compound or material will satisfy two or more of the above general classifications. For example, a compound may simultaneously act as an emulsifier and a surfactant.

[0200] Liquid mucoadhesive intranasal dosage compositions may further include an antimicrobial preservative to prevent the undesirable growth of bacteria, molds, fungi or yeasts. Examples of suitable antimicrobial preservatives include benzyl alcohol, benzalkonium chloride, alkali metal benzoates (e.g. sodium benzoate), alkali metal sorbates (e.g. potassium sorbate), sodium isoascorbate, sodium nitrite, calcium sorbate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), parabens (e.g. lower alkyl esters of parabens), alkali metal salts of parabens, including sodium and potassium salts of methylparaben, ethylparaben, propylparaben or butylparaben, or a combination thereof. Specific antimicrobial preservatives include benzyl alcohol, benzalkonium chloride or a combination thereof.

[0201] The antimicrobial preservative may be present in the liquid mucoadhesive intranasal dosage composition in an amount of about 0.001 to about 1.0% w / v, specifically about 0.01 to about 0.55% w / v, and still more specifically about 0.1 to about 0.3% w / v.

[0202] The liquid mucoadhesive intranasal dosage composition may further optionally include an antimicrobial preservative enhancer, such as ethylenediaminetetraacetic acid (EDTA) or a pharmaceutically acceptable salt thereof (e.g. calcium disodium EDTA). The antimicrobial preservative enhancer may be present in the formulation in an amount of about 0.001 to about 0.1% w / v, particularly about 0.01 to about 0.05% w / v, and more particularly about 0.02 to about 0.04% w / v.

[0203] The liquid mucoadhesive intranasal dosage composition can be isotonic, or isotonic and buffered. The liquid mucoadhesive intranasal dosage formulation can optionally contain a tonicity agent, such as dextrose, glycerol, mannitol, potassium chloride, sodium chloride, or a combination thereof. One of ordinary skill in the art can determine the amount of the tonicity agent without undue experimentation.

[0204] The liquid mucoadhesive intranasal dosage composition may comprise a buffer. Exemplary buffers include citrates, acetates, phosphates (e.g., citric acid, sodium citrate, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof).

[0205] In further embodiments, any of the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein can be formulated to be suitable for topical application or administration. Therefore, the compositions described herein are stable, beautiful and well tolerated by the subject. As non-limiting examples, the compositions described herein can be formulated into solutions, suspensions, gels, hydrogels, creams, emulsions, microemulsions, nanoemulsions, lotions, sprays, ointments, patches, tissue cloths, wipes, soaps, pastes, aerosols and masks suitable for topical use. The variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein can be incorporated into these topical formulations in an amount between 0.01w% and the solubility limit.

[0206] F. Feed and feed additive preparations

[0207] Any of the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein (alone or in combination with at least one direct-fed microorganism) can be encapsulated alone and / or in combination with at least one enzyme for use in animal feed or premixes. In addition, the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein (alone or in combination with at least one direct-fed microorganism) alone and / or in combination with at least one protease, amylase, xylanase, β-glucosidase and / or phytase, whether encapsulated or not, can be in the form of granules.

[0208] Animal feeds may include plant materials such as corn, wheat, sorghum, soybean, canola, sunflower, or mixtures of any of these plant materials or plant protein sources for poultry, swine, ruminants, aquaculture, and pets. The terms "animal feed", "feedstock" and "fodder" are used interchangeably and may comprise one or more feed materials selected from the group consisting of: a) cereals, such as small grains (e.g., wheat, barley, rye, oats and combinations thereof) and / or large grains, such as maize or sorghum; b) by-products from cereals, such as corn gluten meal, distillers dried grains with solubles (DDGS), particularly corn-based distillers dried grains with solubles (cDDGS), wheat bran, semolina, wheat short meal, rice bran, rice husks, oat hulls, palm kernels and citrus pulp; c) proteins obtained from sources such as soy, sunflower, peanut, lupine, pea, bean, cotton, canola, fish meal, dried plasma protein, meat and bone meal, potato protein, whey, copra, sesame; d) oils and fats obtained from plant and animal sources; and / or e) minerals and vitamins.

[0209] When used as or for preparing feed (such as functional feed), the variant lectin of the present invention or a feed additive composition containing the variant lectin can be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, a nutritionally active ingredient. For example, at least one component selected from the group consisting of proteins, peptides, sucrose, lactose, sorbitol, glycerol, propylene glycol, sodium chloride, sodium sulfate, sodium acetate, sodium citrate, sodium formate, sodium sorbate, potassium chloride, potassium sulfate, potassium acetate, potassium citrate, potassium formate, potassium acetate, potassium sorbate, magnesium chloride, magnesium sulfate, magnesium acetate, magnesium citrate, magnesium formate, magnesium sorbate, sodium metabisulfite, methylparaben and propylparaben. In some embodiments, the variant lectin or a feed additive composition containing the variant lectin is fused to a carrier molecule, such as any of those disclosed herein.

[0210] In one embodiment, the variant lectin of the present invention or a composition containing the variant lectin is mixed with a feed component to form a feed. As used herein, "feed component" means all or part of a feed. A part of a feed may mean one component of a feed or more than one (e.g., 2 or 3 or 4 or more) components of a feed. In one embodiment, the term "feed component" encompasses a premix or a premix ingredient.

[0211] Preferably, the feed can be fodder or a premix thereof, a compound feed or a premix thereof. The feed additive composition according to the present invention can be mixed with a compound feed, a compound feed component or mixed into a premix of a compound feed or mixed into fodder, a fodder component or a premix of fodder. As used herein, the term "fodder" means any food provided to an animal (rather than the animal having to forage for itself). Fodder encompasses plants that have been cut. In addition, fodder includes silage, compressed and pelleted feed, oil and mixed rations, and also includes sprouted grains and beans. The fodder can be obtained from one or more plants selected from the group consisting of corn (maize), alfalfa (Medicago truncatula), barley, Lotus japonica, Brassica juncea, Chau moellier, kale, rapeseed (canola), rutabaga (Swede), radish, clover, hybrid clover, red clover, underground clover, white clover, fescue, brome grass, millet, oats, sorghum, soybeans, trees (tree clippings for tree hay), wheat, and legumes.

[0212] The term "compound feed" means a commercial feed in the form of meal, pellets, nuts, cakes or crumbs. Compound feeds can be blended from a variety of raw materials and additives. These blends are formulated according to the specific needs of the target animal. Compound feeds can be complete feeds that provide all the daily nutrients required, concentrates that provide part of the ration (protein, energy), or supplements that only provide additional micronutrients (such as minerals and vitamins). The main ingredients used in compound feeds are feed grains, which include corn, wheat, canola meal, rapeseed meal, lupins, soybeans, sorghum, oats and barley.

[0213] Suitably, a "premix" as referred to herein may be a composition consisting of minor ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products and other essential ingredients. A premix is ​​typically a composition suitable for blending into a commercial ration.

[0214] As used herein, the term "contacting" refers to the indirect or direct application of a variant lectin or a composition containing a variant lectin (or a composition comprising a variant lectin polypeptide or a variant lectin polypeptide dimer as described herein) to a product (e.g., a feed). Examples of application methods that can be used include, but are not limited to: treating the product in a material comprising a feed additive composition, applying directly by mixing the feed additive composition with the product, spraying the feed additive composition onto the surface of the product, or immersing the product in a formulation of the feed additive composition. In one embodiment, the feed additive composition of the present invention is preferably mixed with a product (e.g., a feedstock). Alternatively, the feed additive composition may be included in an emulsion or an original component of the feedstock.

[0215] It is also possible that the variant lectin polypeptide or variant lectin polypeptide dimer described herein can be homogenized to produce a powder. In an alternative embodiment, the variant lectin polypeptide or variant lectin polypeptide dimer as described herein (or a composition comprising the variant lectin polypeptide or variant lectin polypeptide dimer as described herein) can be formulated into particles as described in (referred to as TPT particles) or in WO1997 / 016076 or WO1992 / 012645, which are incorporated herein by reference. "TPT" means thermal protection technology. On the other hand, when the feed additive composition is formulated into particles, the particles comprise a hydration barrier salt coated on a protein core. The advantages of such a salt coating are improved heat resistance, improved storage stability and protection from other feed additives that would otherwise adversely affect the enzyme. Preferably, the salt used for the salt coating has a water activity greater than 0.25 or a constant humidity greater than 60% at 20°C. In some embodiments, the salt coating comprises Na 2 SO 4 .

[0216] The method of preparing a variant lectin polypeptide or variant lectin polypeptide dimer as described herein may also include a further step of granulating the powder. The powder may be mixed with other components known in the art. The powder or a mixture comprising the powder may be forced through a die and the resulting strands cut into suitable pellets of varying lengths.

[0217] Optionally, the granulation step may include a steam treatment or conditioning stage before forming the pellets. The mixture comprising the powder may be placed in a conditioner, such as a mixer with steam injection. The mixture is heated in a conditioner to a specified temperature, for example from 60°C-100°C, and a typical temperature will be 70°C, 80°C, 85°C, 90°C or 95°C. The residence time may vary from a few seconds to a few minutes or even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour. It should be understood that the variant lectin polypeptide or variant lectin polypeptide dimer as described herein is suitable for addition to any suitable feed material.

[0218] It will be appreciated by the skilled person that different animals require different feeds, and even the same animal may require different feeds, depending on the purpose of raising the animal. Optionally, the feed may also contain additional minerals (such as, for example, calcium) and / or additional vitamins. In some embodiments, the feed is a corn soybean meal mixture.

[0219] Feedstock is typically produced in a feed mill where the raw material is first ground to a suitable particle size and then mixed with appropriate additives. The feedstock can then be produced as a paste or pellets; the latter typically involves a process whereby the temperature is raised to a target level and the feed is then passed through a die to produce pellets of a specific size. The pellets are allowed to cool. Subsequently, liquid additives such as fats and enzymes may be added. Production of the feedstock may also involve additional steps including extrusion or puffing prior to pelleting, particularly by suitable techniques that may include at least the use of steam.

[0220] The feed may be a feed for monogastric animals such as poultry (e.g. broilers, laying hens, broiler breeders, hens, turkeys, ducks, geese, waterfowl) and swine (all age categories); ruminants such as cattle (e.g. dairy cows or bulls (including calves)), horses, sheep, pets (e.g. dogs, cats) or fish (e.g. stomachless fish, stomached fish, freshwater fish such as salmon, cod, trout and carp (e.g. koi fish), marine fish (e.g. sea bass); and crustaceans such as shrimp, mussels and scallops).

[0221] The feed additive composition and / or the feed comprising it can be used in any suitable form. The feed additive composition can be used in the form of a solid or liquid formulation or a substitute thereof. Examples of solid formulations include powders, pastes, bolus, capsules, pellets, tablets, powders and granules, which can be wettable, spray-dried or freeze-dried. Examples of liquid formulations include, but are not limited to, aqueous, organic or aqueous-organic solutions, suspensions and emulsions. In some applications, the feed additive composition can be mixed with feed or applied in drinking water. A feed additive composition comprises mixing a variant lectin polypeptide or a variant lectin polypeptide dimer as described herein with a carrier, diluent or excipient acceptable to feed, and (optionally) packaging.

[0222] The feed and / or feed additive composition may be mixed with at least one mineral and / or at least one vitamin. The composition derived therefrom may be referred to herein as a premix. The feed may comprise at least 0.0001% by weight of a feed additive. Suitably, the feed may comprise at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100%; at least 0.020%; at least 0.100%; at least 0.200%; at least 0.250%; at least 0.500% by weight of a feed additive.

[0223] Preferably, the food or feed additive composition may further comprise at least one physiologically acceptable carrier. The physiologically acceptable carrier is preferably selected from at least one of the following: maltodextrin, limestone (calcium carbonate), cyclodextrin, wheat or wheat components, sucrose, starch, Na 2 S0 4 , talc, PVA, and mixtures thereof. In another embodiment, the food or feed additive may further comprise a metal ion chelator. The metal ion chelator may be selected from EDTA or citric acid.

[0224] Dry powders or granules can be prepared by means known to those skilled in the art, such as high shear granulation, drum granulation, extrusion, spheronization, fluidized bed agglomeration, fluidized bed spray drying.

[0225] III. Methods

[0226] A. Cell culture methods

[0227] In certain embodiments, the present disclosure provides recombinant cells capable of producing a target protein (e.g., a variant lectin polypeptide or variant lectin polypeptide dimer as described herein). More particularly, certain embodiments are host cells modified (recombined) with relevant genes expressing heterologous variant lectin polypeptides or variant lectin polypeptide dimers as described herein. Therefore, specific embodiments relate to host cells cultured (fermented) for producing variant lectin proteins. Typically, host cells are fermented using fermentation methods well known in the art.

[0228] In certain embodiments, these cells are grown under batch or continuous fermentation conditions.Classic batch fermentation is a closed system, wherein the composition of the culture medium is set at the beginning of fermentation, and the composition does not change during fermentation.At the beginning of fermentation, the desired organism is inoculated into the culture medium.In this method, fermentation is allowed to occur without adding any component to the system.Typically, batch fermentation meets the qualification of "batch" about adding a carbon source, and control factors (such as pH and oxygen concentration) are often attempted.The metabolites and biomass composition of the batch system are constantly changing until the fermentation stops.In batch culture, cells progress to the high growth logarithmic phase through the static lag phase, and finally enter the stable phase where the growth rate decreases or stops.If not treated, the cells in the stationary phase eventually die.Usually, the cells in the logarithmic phase are responsible for the mass production of products.

[0229] A suitable variation of the standard batch system is the "fed-batch fermentation" system. In this variation of a typical batch system, substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression may inhibit the metabolism of the cell and when it is desirable to have a limited amount of substrate in the culture medium. Measurement of actual substrate concentration in a fed-batch system is difficult and therefore based on measurable factors such as pH, dissolved oxygen, and waste gases such as CO 2 ) is estimated by changes in the partial pressure of ). Batch and fed-batch fermentations are commonly used and well known in the art.

[0230] Continuous fermentation is an open system, wherein the fermentation medium of limitation is continuously added to the bioreactor, and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation usually maintains the culture at a constant high density, wherein the cell is mainly in the logarithmic phase growth. Continuous fermentation allows one or more factors affecting cell growth and / or product concentration to be regulated. For example, in one embodiment, limiting nutrients (such as carbon source or nitrogen source) are maintained at a fixed rate, and all other parameters are allowed to be regulated. In other systems, many factors affecting growth can be constantly changed, and the cell concentration measured by the turbidity of the culture medium remains unchanged. Continuous systems strive to maintain steady-state growth conditions. Therefore, the cell loss caused by extracting the culture medium should be balanced with the cell growth rate in the fermentation. The method for adjusting the nutrients and growth factors used for continuous fermentation technology and the technology for maximizing the product formation rate are well known in the field of industrial microbiology.

[0231] Cultivation / fermentation is typically accomplished in a growth medium comprising an aqueous mineral salts medium, organic growth factors, carbon and energy sources, molecular oxygen, and of course a starting inoculum of the microbial host to be used.

[0232] In addition to the carbon and energy sources, oxygen, assimilable nitrogen and the microbial inoculum, it is necessary to supply the appropriate amounts of mineral nutrients in the right proportions to ensure proper microbial growth, maximize the assimilation of the carbon and energy sources by the cells during microbial conversion, and obtain maximum cell yield and maximum cell density in the fermentation medium.

[0233] The composition of the aqueous mineral medium may vary within wide limits, depending in part on the microorganisms and substrates used, as is known in the art. In addition to nitrogen, these mineral media should also include suitable amounts of phosphorus, magnesium, calcium, potassium, sulfur and sodium in suitable soluble assimilable ionic forms and combinations, and preferably certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron and iodine, as well as others, also in suitable soluble assimilable forms, should also be present, all as known in the art.

[0234] The fermentation reaction is an aerobic process in which the required molecular oxygen is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air, or even substantially pure molecular oxygen, as long as the contents of the fermentation vessel are maintained at a suitable oxygen partial pressure effective to aid the growth of the microbial species in an active manner.

[0235] The fermentation temperature may vary somewhat, but for most host cells the temperature will generally be in the range of about 20°C to 40°C.

[0236] Microorganisms also need assimilable nitrogen sources. Assimilable nitrogen sources can be any nitrogen-containing compound or can release nitrogen in a form suitable for microorganisms to use for metabolism. Although various organic nitrogen source compounds such as protein hydrolysates can be used, cheap nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea and various ammonium salts (such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride or various other ammonia compounds) can usually be used. Ammonia itself is convenient for large-scale operation, and can be used by bubbling through aqueous fermentation (fermentation medium) in a suitable amount. At the same time, such ammonia can also be used to help control pH.

[0237] The pH range in the aqueous microbial ferment (fermentation mixture) should be within the exemplary range of about 2.0 to 8.0. The preference for the microbial pH range depends to some extent on the culture medium employed and the specific microorganism, and thus varies slightly with changes in the culture medium, as can be readily determined by those skilled in the art.

[0238] In some aspects, fermentation is carried out in a manner that the carbon-containing substrate can be controlled as a limiting factor, thereby providing cells with a good conversion of the carbon-containing substrate and avoiding these cells to be polluted by the unconverted substrate of a basic amount. The latter situation is not a problem for water-soluble substrates, because any remaining trace materials can be easily washed off. However, this may be a problem under the situation of non-water-soluble substrates, and the product treatment step that needs to be increased is such as a suitable washing step.

[0239] As mentioned above, the time to reach this level is not critical and may vary with the specific microorganism and fermentation process being performed. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether the desired carbon source level has been reached.

[0240] If desired, part or all of the carbon source and energy material and / or part of the assimilable nitrogen source (such as ammonia) may be added to the aqueous mineral culture medium before feeding the aqueous mineral culture medium to the fermentor.

[0241] Each stream introduced into the reactor is preferably controlled at a predetermined rate, or in response to demand which can be determined by monitoring, for example, the concentration of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gas from the fermentor, cell density measurable by dry cell weight, light transmittance, etc. The feed rates of the various materials can be varied to obtain the fastest possible cell growth rate consistent with efficient utilization of carbon and energy sources, and to obtain the highest possible microbial cell yield relative to substrate changes.

[0242] In batch operation or preferably fed-batch operation, all equipment, reactors or fermentation apparatus, vessels or containers, piping, associated circulation or cooling equipment, etc. are initially sterilized, usually by the use of steam, such as at about 121° C. for at least about 15 minutes. The sterilized reactor is then inoculated with a culture of the selected microorganism in the presence of all required nutrients, including oxygen and a carbon-containing substrate. The type of fermentor used is not critical.

[0243] B. Protein recovery

[0244] The present disclosure further describes and exemplifies particularly suitable processes (methods) for harvesting, clarifying, recovering, purifying, etc. fermentation broths in which one or more variant lectin polypeptides or variant lectin polypeptide dimer proteins have been produced. Therefore, certain embodiments are particularly directed to collecting broth at the end of fermentation, harvesting the collected broth, recovering one or more variant lectin polypeptides or variant lectin polypeptide dimer proteins from the harvested broth (e.g., such as clarifying the harvested broth, concentrating the clarified broth, purifying the clarified broth concentrate, etc.). In some aspects, the purified protein preparation is derived from the fermentation broth collected and harvested as described herein.

[0245] Certain other aspects of the present disclosure provide, inter alia, novel methods for recovering and optionally purifying recombinantly produced proteins (such as variant lectin polypeptides or variant lectin polypeptide dimer proteins) obtained from recombinant cells (e.g., recombinant Gram-negative cells, recombinant Gram-positive cells, recombinant plant (e.g., tobacco) cells, recombinant fungal cells, etc.) expressing the recombinantly produced proteins. Certain other aspects of the present disclosure provide, inter alia, novel methods for recovering and optionally purifying variant lectins or variant lectin dimers obtained from naturally occurring sources.

[0246] Therefore, in certain aspects, variant lectin polypeptides or variant lectin polypeptide dimer protein preparations are recovered according to the compositions and methods of the present disclosure. In other aspects, variant lectin polypeptides or variant lectin polypeptide dimer preparations are recovered and purified according to the methods of the present disclosure. As used herein, the terms "purified", "isolated" or "enriched" with respect to proteins mean that variant lectin polypeptides or variant lectin polypeptide dimers are converted from a less pure state by separating them from some or all of the contaminants with which they are associated. Contaminants include, but are not limited to, microbial cells, metabolites, solvents, chemicals, colors, inactive forms of target variant lectins or variant lectin dimers, aggregates, processing aids, inhibitors, fermentation media, cell debris, nucleic acids, proteins other than target variant lectins or variant lectin dimer proteins, host cell proteins, cross-contaminants from production equipment, and the like.

[0247] Therefore, in the context of "purified variant lectin polypeptide or variant lectin polypeptide dimer" as used herein, purification can be accomplished by separation techniques recognized in the art, including but not limited to ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salt precipitation, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove whole cells, cell debris, impurities, foreign proteins, or enzymes that are not desired in the final composition.

[0248] Components that provide additional benefits, such as activators, anti-inhibitors, desired ions, compounds that control pH, or other enzymes or chemicals, may then be further added to the purified or isolated variant lectin polypeptide or variant lectin polypeptide dimer composition.

[0249] As used herein, variant lectin "purity" is a relative term and is not intended to be limiting when used in phrases such as "the recovered variant lectin has a higher purity, the same purity, or a lower purity than before the recovery process." For example, the relative "purity" of a protein before and after a recovery process can be determined using methods known in the art, including but not limited to general quantitative methods (e.g., Bradford assay, UV-Vis, activity assay), electrophoresis analysis (SDS-PAGE), analytical HPLC, mass spectrometry, hydrophobic interaction chromatography, and the like.

[0250] Thus, according to certain aspects, variant lectin polypeptides or variant lectin polypeptide dimer protein preparations are recovered from the fermentation broth, wherein these recovered variant lectin preparations have a higher purity after undergoing one or more recovery processes described herein. For example, the fermentation broth (e.g., the whole broth at the end of fermentation) can be subjected to one or more protein recovery processes, including but not limited to broth conditioning processes, broth clarification processes, protein enrichment and / or protein purification processes (e.g., protein concentration, filtration, precipitation, crystallization, crystal separation, crystal sludge dissolution processes, etc.), buffer exchange processes, sterile filtration processes, etc. In certain aspects, the fermentation broth is subjected to a broth treatment (broth conditioning) process to improve subsequent broth processing characteristics.

[0251] In certain embodiments, such as when host cells have been constructed for variant lectin expression of intracellular variant lectin dimers, the fermentation broth is subjected to a cell lysis process. For example, cell lysis processes include, but are not limited to, enzyme treatment (e.g., lysozyme, proteinase K treatment), chemical means (e.g., ionic liquids), physical means (e.g., French press, ultrasound), simply keeping the culture without feed, etc. Therefore, in certain preferred embodiments, the broth lysis process releases the variant lectin into the (lysed) cell broth.

[0252] Therefore, as described herein, the methods / processes disclosed herein are not intended to be limiting, as a skilled artisan can readily adapt or modify one or more of the compositions and / or methods disclosed herein for use in recovering a specific variant lectin and / or combination thereof of a variant lectin dimer protein.

[0253] C. Methods for treating or preventing viral infections

[0254] Also provided herein are methods of treating or preventing viral infection in an animal in need thereof by administering to the animal one or more of the variant lectin polypeptides or variant lectin polypeptide dimers disclosed herein or a composition containing one or more of these variant lectin polypeptides or variant lectin polypeptide dimers.

[0255] In one embodiment, the variant lectin protein or a functional fragment thereof comprises an amino acid sequence that is at least about 60% identical (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to SEQ ID NO: 8 or SEQ ID NO: 18, wherein the variant lectin comprises one or more (e.g., 1, 2, 3, 4 or 5) substitutions at positions 10, 38, 53, 54 and 78. In some embodiments, the variant lectin polypeptide is administered to an animal (such as, for example, a pig or a horse) by oral, nasal and / or topical administration. The variant lectin protein or a functional fragment thereof can exhibit antiviral activity against infection with one or more of porcine reproductive and respiratory syndrome (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus, or equine viral arteritis (EVA).

[0256] In another embodiment, the variant lectin polypeptide dimer or its functional fragment comprises the polypeptide of SEQ ID NO: 8 and / or SEQ ID NO: NO:18 Two amino acid sequences that are at least about 60% identical (e.g., any of about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical), wherein each variant lectin component of the dimer comprises one or more (e.g., 1, 2, 3, 4, or 5) substitutions at positions 10, 38, 53, 54, and 78. In separate embodiments, each variant lectin component of the dimer may be identical or may have unique substitutions relative to another member. In some embodiments, the variant lectin polypeptide is administered to an animal (such as, for example, a pig or a horse) by oral, nasal and / or topical administration. The variant lectin polypeptide dimer or a functional fragment thereof may exhibit antiviral activity against infection with one or more of porcine reproductive and respiratory syndrome (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus, or equine viral arteritis (EVA).

[0257] The invention may be further understood by reference to the following examples, which are provided by way of illustration and not limitation.

[0258] Examples

[0259] Example 1: Engineering Greifsner to Improve One or More of Expression, Stability, Solubility, Isoelectric Point, or Binding indivual

[0260] This example describes the creation of a library for engineering Grefsen to improve one or more of expression, stability, solubility, isoelectric point, or binding.

[0261] Library creation: Based on the sequence of SEQ ID NO:8, positions 10, 31, 36, 38, 48, 49, 53, 54, 78 and 83 were selected for saturation library creation. Eight of these positions have never been engineered, and at position 78, only substitutions replacing the native M with Q have been thoroughly investigated. Other substitutions may be as good or even better than Q at this position. In addition, in proteins produced by natural algae, position 31 is occupied by non-natural amino acids. In the recombinant version of Grefson, it has been replaced by alanine, but other replacement amino acids may also be preferred.

[0262] The Griffson library was constructed as follows. A first DNA fragment comprising a (5′) aprE gene flanking region (5′ aprE gene FR) comprising Bacillus subtilis aprE homology (SEQ ID NO: 16) was operably linked to a polynucleotide construct comprising a Bacillus subtilis rrnI-p2 promoter region DNA sequence (SEQ ID NO: 2; SEQ ID NO: 40 from WO2020112609), which was operably linked to a DNA sequence of the 5′ untranslated region of Bacillus subtilis aprE (5′-UTR; SEQ ID NO: 3), which was operably linked to a DNA sequence encoding a Bacillus subtilis AprE signal sequence (SEQ ID NO: 4), which was operably linked to a DNA sequence encoding a mature Trichoderma sp. variant sequence, which was operably linked to a Bacillus amyloliquefaciens Bpn terminator (SEQ ID NO: 6), which was operably linked to a selectable marker (SEQ ID NO: 7). NO:5), the selectable marker is operably linked to the (3') aprE gene flanking region (3' aprE gene FR) (SEQ ID NO:15).

[0263] More particularly, these DNA fragments are assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes comprising one or more mature sequence modifications as described herein. For example, the subtilis strain comprising the variant mature region sequence is constructed by integrating the DNA fragment (as described above) into the genome, wherein these fragments contain mature sequence variants. The variant expression cassette is developed into a 5.7kb linear DNA fragment and used to transform competent subtilis cells, wherein the transformation mixture is plated onto a LA plate containing 5ppm kanamycin and incubated overnight at 37°C. Pick a single colony and grow it in Luria broth at 37°C under antibiotic selection.

[0264] For variant assay experiments, transformed cells were grown in 96-well MTPs in semi-defined medium (enriched medium based on MOPS buffer) at 37°C, 270 rpm, 80% humidity in a shaking incubator for 2 days before harvesting.

[0265] Construction of linked dimers of Gravesin M78 (GGG, GTG, GPG): Linked dimers of Gravesin M78 (SEQ ID NO: 7) were constructed by overlapping PCR using oligonucleotides to incorporate linkers GGG or GTG or GPG, thereby generating dimer nucleotide sequences (SEQ ID NOs: 19, 28, 30).

[0266] A first DNA fragment comprising a (5′) aprE gene flanking region (5′ aprE gene FR) comprising Bacillus subtilis aprE homology (SEQ ID NO: 16) is operably linked to a polynucleotide construct comprising a Bacillus subtilis rrnI-p2 promoter region DNA sequence (SEQ ID NO: 2) operably linked to a DNA sequence of the Bacillus subtilis aprE 5′ untranslated region (5′-UTR; SEQ ID NO: 3), which in some cases is operably linked to a DNA encoding a Bacillus subtilis AprE signal sequence (SEQ ID NO: 4), which is operably linked to a DNA sequence encoding a mature Trichoderma sp. M78 dimer sequence (SEQ ID NO: 19 or 28 or 30), which is operably linked to a Bacillus amyloliquefaciens Bpn terminator (SEQ ID NO: 6), which is operably linked to a selectable marker (SEQ ID NO: 7). NO:29), the selectable marker is operably linked to the (3') aprE gene flanking region (3' aprE gene FR) (SEQ ID NO:15).

[0267] These DNA fragments are assembled using standard molecular biology techniques. The dimer sequence is constructed as a 6kb linear DNA fragment expression cassette and then used to transform competent Bacillus subtilis 9 protease deletion cells. The nine proteases missing in this strain are aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and nprB. The transformation mixture is plated on a prototrophic selective LA plate and incubated overnight at 37°C. Pick a single colony and grow it in Luria broth at 37°C under prototrophic selection. The transformed cells are grown in a 24-well MTP in a medium (a semi-defined medium enriched based on MOPS buffer, urea as the main nitrogen source, maltodextrin as the main carbon source, supplemented with 3% soytone for robust cell growth, containing antibiotic selection), at 37°C, 250rpm, 80% humidity, in a shaking incubator for 48 hours. Intracellular samples are analyzed by SDS-PAGE gel.

[0268] Construction of dimers of Gravesone variant S10N / G053K / M78Y: A linker library connecting two monomers of Gravesone variant S10N / G053K / M78Y (SEQ ID NO.13) was designed and ordered for synthesis. The library includes 27 linker sequences (SEQ ID NO:32 to 57). The construction of strains expressing dimers of the Gravesone variant sequence (SEQ ID NO.13) was carried out in a similar manner as described above.

[0269] These DNA fragments are assembled using standard molecular biology techniques. The dimer sequence is constructed as a 6 kb linear DNA fragment expression cassette and then used to transform competent Bacillus subtilis 5 protease deletion cells. The 5 proteases missing in this strain are aprE, nprE, epr, ispA, bpr. The transformed cells are grown in a 24-well MTP in a medium (an enriched semi-defined medium based on MOPS buffer, urea as the main nitrogen source, maltodextrin as the main carbon source, supplemented with 3% soytone for robust cell growth, containing antibiotic selection) at 37 ° C, 250 rpm, 80% humidity, in a shaking incubator for 48 hours. Intracellular samples are analyzed by SDS-PAGE gel.

[0270] Example 2: Measurement of Performance Index (PI) and Melting Point of Gravesson Variants

[0271] Measurement of library variant expression using UPLC: To generate Gravesson samples for screening, transformed Bacillus subtilis cells were grown in semi-defined medium at 37°C for 68 hours in 96-well microtiter plates (MTP, manufactured). 30ul of 3N H2SO4 was added to each well (350ul) and the resulting culture broth was brought to pH 2. The culture was harvested by centrifugation at 3000g for 15min and the culture was centrifuged by The culture supernatant was filtered through filter plates (EMD Millipore, Billerica, MA, USA).

[0272] Typically, 280 μl of filtered culture broth was neutralized by adding 150 μl of 1.5 N sodium citrate (pH 9) to a resulting pH of pH 6.5. The neutralized samples were plated in a new 96-well plate (NUNC, 267245) in MilliQ H 2 The GRFT concentration of the sample was determined by HPLC. The GRFT concentration of the sample was further diluted 5 times in 1% PBS. 10 μl of this diluted sample was loaded onto UPLC for quantification. Protein components were separated using Zorbax 300SB-C3 columns (Agilent), and a linear gradient of 0.1% trifluoroacetic acid (buffer A) in water and 0.1% trifluoroacetic acid (buffer B) in acetonitrile was run, and the concentration of GRFT was determined by detection at 220 nm on UHPLC. The GRFT concentration of the sample was calculated using a standard curve of the purified reference wild-type GRFT. The expression performance index (PI) was calculated by dividing the variant expression by the N-GRFT (M078N) expression. Table 1 summarizes the PI expression of the variant.

[0273] Measurement of thermal melting points of Gravesin variants using Sypro orange dye binding: 15 μl of neutralized MTP Gravesin samples were mixed with 5 μl of 125-fold diluted Sypro orange (Thermo Fisher, S6650), which were mixed in Roche 384-well qPCR plates (Thermo Fisher, 4309849). The plates were sealed with Roche seals and rotated rapidly. Tm was measured using a Roche 480 light cycler. Fluorescence changes were recorded at a scan rate of about 1C / min, data were exported and processed using R scripts. The transitions corresponding to the thermal melting temperatures of Gravesin were compared. The PI of the Tm of the variant was calculated by subtracting N-GRFT (M078N) Tm from the Tm value of each variant. Table 1 lists this PI Tm for all tested variants.

[0274] Table 1: Tm performance index (PI) and expression relative to M078N for all tested variants are listed. Sequence numbering is based on SEQ ID NO:8.

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285] Example 3: Evaluation of the stability of Gravesin protease

[0286] The selected variants were grown in shake flasks and tested for protease stability in the presence of protease FNA (BPN'Y217L variant subtilisin from Bacillus amyloliquefaciens) or pepsin. For FNA (in-house purified enzyme) treatment, 10ppm FNA and 100ul filtered shake flask samples were incubated at pH 6.5 for 42 hours to 7 days. At the end of the incubation, the samples were run on SDS-PAGE gels, and the intensity of the Gravesson bands was quantitatively compared with the control without FNA treatment. For pepsin treatment (Sigma, P7125-100 g), 8ppm pepsin and 100μl rough Gravesson samples were incubated at pH 2 for 22 hours to 7 days, and at 22 hours or 7 days, the samples were run on SDS-PAGE to perform Gravesson quantification. The band intensities of the controls without FNA or pepsin treatment were compared. Table 2 summarizes the data.

[0287] Table 2: Protease stability assay results for selected variants

[0288]

[0289] A panel of combinatorial variants were grown in shake flasks. They were selected to compare their stability in the presence of the protease FNA or LAS (LAS is linear alkylbenzene sulfonate, a surfactant), or a combination of FNA and LAS. For FNA or surfactant treatment, 10 ppm of FNA in buffer or 0.05% LAS or 0.05% LAS alone was incubated with 100 ul of semi-purified sample at pH 6.5 for 5 days. At the end of the incubation, the samples were run on an SDS-PAGE gel and compared to an untreated control. The SDS-PAGE gel is shown in Figure 2. Figure 1 .

[0290] Example 4: Cytotoxicity testing of Gravesin variants

[0291] 103-104 cells (MARC-145 cell line) were seeded in a 96-well plate. GRFT at different concentrations (10ug-200μg) was added to the wells and incubated overnight. MTT solution was added and incubated at 37°C for 3 hours. 100ul MTT assay solvent was added and incubated in the dark for 15-20min. OD was measured at 590nm. Cells without GRFT were used as controls (Li et al., Arch Virol. [Virology Archives] 2018; 163(12): 3317-3325).

[0292] PRRSV virus was incubated with different concentrations of GRFT at 37°C and then added to target cells and incubated for 1 hour. After 1 hour, unbound virus was washed off, fresh medium was added and further incubated for 24 hours or 48 hours. Infected cells were stained with 0.1% methylene blue solution and plaques were counted manually to establish PFU / ml (plaque forming units / ml) or TCID 50 was calculated by the Reed and Muench method (Lei et al., On the Calculation of TCID 50 for Quantitation ofVirus Infectivity.[Calculate TCID 50 To quantify viral infectivity] Virol. Sin. [Chinese Virology] (2020)).

[0293] To estimate viral replication, infected cells were lysed for RNA isolation and QPCR was performed to quantify viral RNA (Li et al., Arch Virol. 2018; 163(12): 3317-3325).

[0294] like Figure 2 As shown, nine GRFT variants were tested for their antiviral efficacy against the MARC-145 cell line challenged with low viral load PRRSV. Single variants (M78N GRFT, G53K GRFT, G53L GRFT) and combination variants (GRFT S10D, H38Q, G53K, M78Y; GRFT S10D, H38Q, G53K, S54P; GRFT S10N, G35K, N78Y; GRFT S10D, H38Q, G53E) showed higher percentages of viral inhibition.

[0295] like Figure 3As shown, nine GRFT variants were tested for their antiviral efficacy against MARC-145 cell lines challenged with high viral loads of PRRSV. Single variants (M78N GRFT, G53K GRFT, G53L GRFT) and combination variants (GRFT S10D, H38Q, G53K, M78Y; GRFT S10D, H38Q, G53K, S54P; GRFT S10N, G35K, N78Y; GRFT S10D, H38Q, G53E) showed higher percentages of viral inhibition.

[0296] Figure 4 Depicted are SDS-PAGE gels of intracellular samples from 9 protease deletion strains of Bacillus subtilis that produce Grefsen M78 monomers and dimers with GGG, GPG, GTG linkers. Lane 1 is See Blue Plus 2 molecular weight ladder; lane 2 is the negative control Bacillus subtilis 9 protease deletion strain; lanes 3, 8, 10, and 12 show Grefsen M78 monomers; lanes 4-5 are Grefsen M78 GGG dimers; lanes 6-7 are Grefsen M78 GPG dimers, and lane 9 is Grefsen M78 GTG dimers.

[0297] Example 5: Efficacy of Graveson Nasal Spray against PRRSV-2 (PRRSV 1-7-4L1A isolate) virus

[0298] Fifty-six pigs (21 days old; mixed sex, 1:1) were purchased and delivered to the animal facility at -7 days post-challenge (-7 DPC). The pigs were screened to verify that they were virologically negative (by PCR testing) for PRRSV, swine influenza virus A, porcine circovirus 2 and 3, and serologically negative (by ELISA testing) for PRRSV.

[0299] The pigs were grouped by weight and then randomly divided into 4 groups: (1) NT / NC - untreated unchallenged pigs; (2) NT / C - untreated challenged pigs; (3) LDT / C - challenged pigs receiving low doses of Greifsern; and (4) HDT / C - challenged pigs receiving high doses of Greifsern, with 17 pigs per group, each in one room (Table 3). The Greifsern molecule used in this experiment corresponds to the dimer of SEQ ID NO: 26. Each pig was implanted with a microchip to monitor body temperature.

[0300] Table 3: Experimental design to study the effect of graversun administration on PRRSV-2 viral infection.

[0301]

[0302] 1 Treatment groups: NT / NC—untreated, non-challenged pigs; NT / C—untreated, challenged pigs;

[0303] LDT / C - challenged pigs receiving low dose of graifene; HDT / C - challenged pigs receiving high dose of graifene

[0304] 2 The challenge virus was PRRSV 1-7-4L1A isolate. 5 TCID50 / pig. 4ml IN (2ml / nostril)

[0305] From -3 to 10 DPC, groups 1 and 2 received mock treatment with 1% Methocel E3 in phosphate buffered saline (PBS) via intranasal spray, while groups 3 and 4 received the corresponding doses of antiviral compounds via intranasal spray twice daily. At 0 DPC, group 1 received virus-negative medium as an unchallenged control, while groups 2 to 4 were inoculated with 10 5 The pigs were challenged with PRRSV 1-7-4L1A isolate at a dose of TCID50 / pig. All pigs were euthanized and necropsied at 42 DPC.

[0306] Pigs were monitored daily for clinical signs, including lethargy and anorexia. Microchip temperature was recorded once every morning. Pigs were weighed at -7, -3, 0, 10, 14, 21, 28, 35, and 42 DPC to calculate average daily gain (ADG). Individual serum samples were collected at -4, 0, 1, 2, 4, 7, 10, 14, 21, 28, 35, and 42 DPC.

[0307] At the end of the study (42 DPC) or when a pig died during the trial, necropsy was performed to sample tissue from all pigs. At necropsy, gross lung lesions were scored. A section of lung tissue was also placed in 10% formalin for histopathological evaluation.

[0308] Serum samples were tested by quantitative PRRSV real-time RT-PCR to determine viremia levels. Formalin-fixed lung tissues were examined for histopathological lesions and immunohistochemical staining.

[0309] Clinical signs, body temperature, average daily weight gain, virus levels in serum (viremia), gross lung lesion scores, and microscopic lung lesion scores were compared between the groups to evaluate the efficacy of the treatment.

[0310] For all outcome measures, data were analyzed per pig (except oral solution). Data were analyzed by ANOVA using the FitModel platform of JMP 14.0. All viral load data were analyzed on a log-transformed scale. Differences between treatment means were determined using the Tukey test. A probability of P < 0.05 was considered significant, and 0.05 ≤ P < 0.10 was considered a trend.

[0311] Preparation of Gravesin nasal spray: 50g Methocel E3 Premium LV HP MC is dissolved in a total of 500ml of PBS to obtain a 10% (w / v) Methocel E3 solution. Purified Gravesin (KF474; corresponding to SEQ ID NO: 26) is diluted to 16.7g / L (high dose) and 8.35g / L (low dose) respectively. A portion of 10% Methocel E3 is added to nine portions of each diluted Gravesin sample, which results in a final concentration of 1% Methocel E3 in PBS with 15g / L Gravesin or 7.5g / L Gravesin in the final formulation. They are fully mixed, filtered through a 0.22um filter, then aliquoted and stored at -80°C.

[0312] result

[0313] The PRRSV challenge model successfully established viremia in pigs, as evidenced by increased body temperature in the challenged group ( Figure 5 In addition, pigs in the HDT / C group returned to normothermia as early as 14 days PI (DPI), compared with 23 days PI in the NT / C group.

[0314] Mortality was reduced in the Gravesin treated groups compared to untreated challenged pigs (12% and 6% reduction in the LDT / C and HDT / C groups, respectively, compared to NT / C) (Table 4). No mortality was observed in unchallenged untreated pigs (NT / NC).

[0315] Table 4: Mortality of pigs during the 42 days after challenge

[0316] <![CDATA[Group 1 > Mortality rate (number of deaths per group) NT / NC 0 / 5 NT / C 4 / 17(23.6％) LDT / C 2 / 17(11.8％) HDT / C 3 / 17(17.6％)

[0317] 1 Treatment groups: NT / NC - untreated unchallenged pigs; NT / C - untreated challenged pigs; LDT / C - challenged pigs receiving low dose of greifsin; HDT / C - challenged pigs receiving high dose of greifsin

[0318] Compared with the NT / C group, the serum viral loads in the HDT / C and LDT / C groups were significantly lower on days 2 and 4 after challenge (p = 0.005, p = 0.001), and tended to be lower on days 7 and 10 after challenge (p = 0.1) ( Figure 6 ).

[0319] At the end of the study, both grefsen-treated groups had less gross lung lesions (tissue damage) compared to untreated challenged pigs ( Figure 7), and relatively mild / moderate interstitial pneumonia (microscopic lesions) were observed in the lungs of Graveson-treated pigs compared with untreated challenged pigs ( Figure 8 ).

[0320] Regarding growth performance, high-dose graversun treatment resulted in numerically higher final body weights and a trend toward higher average daily gain ( Fig. 9 and Fig.10 ).

[0321] In conclusion, this example demonstrates that graversun is an effective antiviral drug against PRRSV virus as it reduces plasma viral load, reduces lung lesions and leads to better recovery from infection through higher weight gain.

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Claims

1. A non-naturally occurring variant lectin polypeptide or a functional fragment thereof, comprising an amino acid sequence at least about 60% identical to SEQ ID NO: 8 or SEQ ID NO: 18 and comprising one or more substitutions at positions 10, 38, 53, 54 and 78.

2. The polypeptide of claim 1, comprising at least one substitution at position 10.

3. The polypeptide of claim 2, wherein the substitution at position 10 is selected from the group consisting of X10D, X10E, X10G, X10H, X10N, X10P, X10Q and X10T.

4. The polypeptide of claim 3, wherein the substitution at position 10 is selected from the group consisting of S10D, S10E, S10G, S10H, S10N, S10P, S10Q and S10T.

5. The polypeptide of any one of claims 1 to 4, comprising at least one substitution at position 38.

6. The polypeptide of claim 5, wherein the substitution at position 38 is selected from the group consisting of: X38P and X38Q.

7. The polypeptide of claim 6, wherein the substitution at position A31 is selected from the group consisting of: H38P and H38Q.

8. The polypeptide of any one of claims 1 to 7, comprising at least one substitution at position 53.

9. The polypeptide of claim 8, wherein the substitution at position 53 is selected from the group consisting of X53A, X53C, X53D, X53E, X53H, X53K, X53L, X53M, X53Q, X53R, X53T and X53V.

10. The polypeptide of claim 9, wherein the substitution at position 53 is selected from the group consisting of G53A, G53C, G53D, G53E, G53H, G53K, G53L, G53M, G53Q, G53R, G53T and G53V.

11. The polypeptide of any one of claims 1-10, comprising at least one substitution at position 54.

12. The polypeptide of claim 11, wherein the substitution at position 54 is selected from the group consisting of: X54P and X54T.

13. The polypeptide of claim 12, wherein the substitution at position 54 is selected from the group consisting of: S54P and S54T.

14. The polypeptide of any one of claims 1-13, comprising at least one substitution at position 78.

15. The polypeptide of claim 14, wherein the substitution at position 78 is selected from the group consisting of: X78W, X78H, X78Q and X78Y.

16. The polypeptide of claim 15, wherein the substitution at position 78 is selected from the group consisting of: M / N78W, M / N78H, M / N78Q and M / N78Y.

17. The polypeptide of any one of claims 1-16, wherein the polypeptide comprises at least two substitutions at positions 53 and 78.

18. The polypeptide of claim 17, wherein the substitution at position 53 is X53E, K or L; and the substitution at position 78 is X78N or Y.

19. The polypeptide of claim 18, wherein the substitution at position 53 is G53E or K; and the substitution at position 78 is M / N78Y.

20. The polypeptide of any one of claims 1-19, wherein the polypeptide comprises at least three substitutions at positions 10, 53 and 78.

21. The polypeptide of claim 20, wherein the substitution at position 10 is X10D or N; the substitution at position 53 is X53K, E, or L; and the substitution at position 78 is X78Q or Y.

22. The polypeptide of claim 21, wherein the substitution at position 10 is S10D or N; the substitution at position 53 is G53K, E, or L; and the substitution at position 78 is M / N78Q or Y.

23. The polypeptide of any one of claims 1-19, wherein the polypeptide comprises at least three substitutions at positions 38, 54 and 78.

24. The polypeptide of claim 23, wherein the substitution at position 38 is X38Q; the substitution at position 54 is X54P; and the substitution at position 78 is X78Q.

25. The polypeptide of claim 24, wherein the substitution at position 38 is H38Q; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Q.

26. The polypeptide of any one of claims 1-19, wherein the polypeptide comprises at least three substitutions at positions 10, 38 and 53.

27. The polypeptide of claim 27, wherein the substitution at position 10 is X10N or D; the substitution at position 38 is X38Q; and the substitution at position 53 is X53E, L or K.

28. The polypeptide of claim 28, wherein the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; and the substitution at position 53 is G53E, L or K.

29. The polypeptide of any one of claims 1-19, wherein the polypeptide comprises at least three substitutions at positions 10, 53 and 54.

30. The polypeptide of claim 27, wherein the substitution at position 10 is X10N or D; the substitution at position 53 is X53L or K; and the substitution at position 54 is X54P.

31. The polypeptide of claim 27, wherein the substitution at position 10 is S10N; the substitution at position 53 is G53L; and the substitution at position 54 is S54P.

32. The polypeptide of any one of claims 1-31, wherein the polypeptide comprises at least four substitutions at positions 10, 38, 53 and 54.

33. The polypeptide of claim 32, wherein the substitution at position 10 is X10N or D; the substitution at position 38 is X38Q; the substitution at position 53 is X53E or K; and the substitution at position 54 is X54P.

34. The polypeptide of claim 33, wherein the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; the substitution at position 53 is G53E or K; and the substitution at position 54 is S54P.

35. The polypeptide of any one of claims 1-34, wherein the polypeptide comprises at least four substitutions at positions 10, 53, 54 and 78.

36. The polypeptide of claim 35, wherein the substitution at position 10 is X10N or D; the substitution at position 53 is X53L or K; the substitution at position 54 is X54P; and the substitution at position 78 is X78Y or Q.

37. The polypeptide of claim 36, wherein the substitution at position 10 is S10N or D; the substitution at position 53 is G53L or K; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Y or Q.

38. The polypeptide of any one of claims 1-37, wherein the polypeptide comprises at least four substitutions at positions 10, 38, 53 and 78.

39. The polypeptide of claim 38, wherein the substitution at position 10 is X10D or N; the substitution at position 38 is X38Q; the substitution at position 53 is X53L, E or K; and the substitution at position 78 is X78Y or Q.

40. The polypeptide of claim 39, wherein the substitution at position 10 is S10D or N; the substitution at position 38 is H38Q; the substitution at position 53 is G53L, E or K; and the substitution at position 78 is M / N78Y or Q.

41. The polypeptide of any one of claims 1-40, wherein the polypeptide comprises at least five substitutions at positions 10, 38, 53, 54 and 78.

42. The polypeptide of claim 41, wherein the substitution at position 10 is X10N or D; the substitution at position 38 is X38Q; the substitution at position 53 is X53K or L; the substitution at position 54 is X54P; and the substitution at position 78 is X78Y or Q.

43. The polypeptide of claim 41, wherein the substitution at position 10 is S10N or D; the substitution at position 38 is H38Q; the substitution at position 53 is G53K or L; the substitution at position 54 is S54P; and the substitution at position 78 is M / N78Y or Q.

44. The polypeptide of any one of claims 1-43, wherein the variant lectin polypeptide or a functional fragment thereof is capable of native dimerization with other lectin polypeptides.

45. A polypeptide dimer comprising two polypeptides or functional fragments thereof, wherein the polypeptides or functional fragments thereof comprise an amino acid sequence that is at least about 60% identical to SEQ ID NO: 8 or SEQ ID NO:

18.

46. ​​A variant lectin polypeptide dimer comprising two polypeptides or functional fragments thereof, wherein the polypeptides or functional fragments thereof comprise any one of the polypeptides according to claims 1-44.

47. A polypeptide dimer as described in claim 45 or claim 46, wherein the two polypeptides in the dimer are identical.

48. The polypeptide dimer of any one of claims 45-47, wherein the two polypeptides in the dimer are separated by a linker amino acid sequence.

49. The polypeptide dimer of claim 48, wherein the linker has a length of between 2 and 50 amino acids.

50. The polypeptide dimer of claim 48 or claim 49, wherein the linker comprises the amino acid sequence (GTG) n , where n=1-7.

51. The polypeptide dimer of claim 48 or claim 49, wherein the linker comprises (i) any one of SEQ ID NOs: 32-57; or (ii) (GGG) n or (GPG) n , where n=1-7.

52. The polypeptide dimer of any one of claims 45-51, wherein each polypeptide of the polypeptide dimer is capable of dimerizing with Gravesin protein.

53. A polypeptide dimer as described in any one of claims 45-52, wherein the dimer comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:29 and SEQ ID NO:

31.

54. The polypeptide of any one of claims 1-53, wherein the polypeptide has antiviral activity.

55. A nucleic acid encoding the polypeptide of any one of claims 1-54.

56. A vector comprising the nucleic acid of claim 55.

57. A recombinant host cell comprising the nucleic acid of claim 55 or the vector of claim 56.

58. The host cell of claim 56, wherein the host cell is a fungal cell, an algal cell, a plant cell, a bacterial cell, or a yeast cell.

59. The host cell of claim 58, wherein the host cell is a Bacillus subtilis cell.

60. A pharmaceutical composition comprising a therapeutically effective amount of the polypeptide of any one of claims 1-53 and a pharmaceutically acceptable excipient.

61. The pharmaceutical composition of claim 60, wherein the composition is formulated for oral, nasal and / or topical administration to an animal.

62. A method for treating or preventing a viral infection in an animal, the method comprising administering to the animal a polypeptide according to any one of claims 1 to 54 or a pharmaceutical composition according to any one of claims 59 to 61.

63. The method of claim 62, wherein the polypeptide or pharmaceutical composition is administered to the animal orally, nasally and / or topically.

64. The method of claim 62 or claim 63, wherein the animal is a pig or a horse.

65. The method of claim 64, wherein the pig is a sow, a gilt, a boar, a suckling pig, a weaned pig and / or a finishing pig.

66. The method of any one of claims 62-65, wherein the viral infection comprises porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus, or equine viral arteritis (EVA) virus.

67. A method for producing a polypeptide, the method comprising culturing the host cell of any one of claims 57-59 in a suitable culture medium under conditions suitable for expression of the polypeptide.

68. The method of claim 67, further comprising purifying the polypeptide.

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