IMMUNOGENIC COMPOSITIONS FOR AFRICAN SWINE FEVER VIRUS

Abstract

The present disclosure provides immunogenic compositions or vaccines against African Swine Fever Virus (ASFV), methods of making and using such immunogenic compositions or vaccines, and methods of administering such immunogenic compositions or vaccines. In some forms, a plurality of ASFV antigens are combined together into a live-vectored multivalent immunogenic composition. In some forms, the live-vectored multivalent immunogenic composition is a multicistronic expression cassette.

Claims

1. An immunogenic composition comprising a chimeric gene comprising at least one nucleic acid sequence encoding for a sequence having at least 80% sequence homology with any one or more of SEQ ID NOS: 1-101, and any combination thereof.

2. The immunogenic composition of claim 1, wherein the chimeric gene includes at least two of said nucleic acid sequences.

3. The immunogenic composition of claim 2, wherein said chimeric gene includes a self-cleaving peptide linker between each of the at least two nucleic acid sequences.

4. The immunogenic composition of claim 1, wherein said immunogenic composition is in the form of a multicistronic cassette.

5. The immunogenic composition of claim 1, wherein said chimeric gene is inserted into a vector.

6. The immunogenic composition of claim 5, wherein said vector is selected from the group consisting of an adenovirus, baculovirus, or lentivirus vector.

7. The immunogenic composition of claim 6, wherein said vector is a single-cycle replicon adenovirus or an attenuated bovine parainfluenza virus type 3 genotype c (BPIV3c).

8. The immunogenic composition of claim 1, wherein said chimeric gene is selected from the group consisting of SEQ ID NOS. 102-131.

9. The immunogenic composition of claim 1, further comprising an antigen from another disease-causing organism.

10. The immunogenic composition of claim 9, wherein said another disease-causing organism is selected from the group consisting of Actinobacillus pleuropneumonia; Adenovirus; Alphavirus such as Eastern equine encephalomyelitis viruses; Bordetella bronchiseptica; Brachyspira spp., preferably B. hyodyentheriae; B. piosicoli, Brucella suis, preferably biovars 1, 2, and 3; Classical swine fever virus; Clostridium spp., preferably Cl. difficile, Cl. perfringens types A, B, and C, Cl. novyi, Cl. septicum, Cl. tetani; Coronavirus, preferably Porcine Respiratory Corona virus; Eperythrozoonosis suis; Erysipelothrix rhsiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1, 7 and 14: Hemagglutinating encephalomyelitis virus; Japanese Encephalitis Virus; Lawsonia intracellularis; Leptospira spp.; preferably Leptospira australis; Leptospira canicola; Leptospira grippotyphosa; Leptospira icterohaemorrhagicae; and Leptospira interrogans; Leptospira pomona; Leptospira tarassovi; Mycobacterium spp. preferably M. avium; M. intracellulare; and M. bovis; Mycoplasma hyopneumoniae (M hyo); Pasteurella multocida; Porcine circovirus; Porcine cytomegalovirus; Porcine Parvovirus; Porcine Reproductive and Respiratory Syndrome (PRRS) Virus; Pseudorabies virus; Rotavirus; Salmonella spp.; preferably S. thyhimurium; and S. choleraesuis; Staph. hyicus; Staphylococcus spp. preferably Streptococcus spp., preferably Strep. suis; Swine herpes virus; Swine Influenza Virus; Swine pox virus; Swine pox virus; Vesicular stomatitis virus; Virus of vesicular exanthema of swine; Leptospira hardjo; and/or Mycoplasma hyosynoviae.

11. The immunogenic composition of claim 1, further comprising at least one pharmaceutical-acceptable carrier.

12. A immunogenic composition comprising at least one recombinant sequence selected from the group consisting of sequences having at least 80% sequence identity to one of SEQ ID NOS. 1-101.

13. The immunogenic composition of claim 12, further comprising an antigen from another disease-causing organism.

14. The immunogenic composition of claim 13, wherein said another disease-causing organism is selected from the group consisting of Actinobacillus pleuropneumonia; Adenovirus; Alphavirus such as Eastern equine encephalomyelitis viruses; Bordetella bronchiseptica; Brachyspira spp., preferably B. hyodyentheriae; B. piosicoli, Brucella suis, preferably biovars 1, 2, and 3; Classical swine fever virus; Clostridium spp., preferably Cl. difficile, Cl. perfringens types A, B, and C, Cl. novyi, Cl. septicum, Cl. tetani; Coronavirus, preferably Porcine Respiratory Corona virus; Eperythrozoonosis suis; Erysipelothrix rhsiopathiae; Escherichia coli; Haemophilus parasuis, preferably subtypes 1, 7 and 14: Hemagglutinating encephalomyelitis virus; Japanese Encephalitis Virus; Lawsonia intracellularis; Leptospira spp.; preferably Leptospira australis; Leptospira canicola; Leptospira grippotyphosa; Leptospira icterohaemorrhagicae; and Leptospira interrogans; Leptospira pomona; Leptospira tarassovi; Mycobacterium spp. preferably M. avium; M. intracellulare; and M. bovis; Mycoplasma hyopneumoniae (M hyo); Pasteurella multocida; Porcine circovirus; Porcine cytomegalovirus; Porcine Parvovirus; Porcine Reproductive and Respiratory Syndrome (PRRS) Virus; Pseudorabies virus; Rotavirus; Salmonella spp.; preferably S. thyhimurium; and S. choleraesuis; Staph. hyicus; Staphylococcus spp. preferably Streptococcus spp., preferably Strep. suis; Swine herpes virus; Swine Influenza Virus; Swine pox virus; Swine pox virus; Vesicular stomatitis virus; Virus of vesicular exanthema of swine; Leptospira hardjo; and/or Mycoplasma hyosynoviae.

15. The immunogenic composition of claim 12, further comprising at least one pharmaceutical-acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0087] FIG. 1A is table illustrating the results from ASFV ORF down selection criteria;

[0088] FIG. 1B is flow diagram illustrating the ASFV ORF down selection criteria;

[0089] FIG. 2A is a flow diagram illustrating the design of multicistronic cassettes;

[0090] FIG. 2 B is a set of photographs illustrating the validation of protein expression;

[0091] FIG. 3 is a graph illustrating IFN-γ response to predicted SLA I binding motifs;

[0092] FIG. 4A is graph illustrating RD-Ad primed antibody responses against ASFV antigens p32, p54, pp62, and p′72;

[0093] FIG. 4B is a set of photographs illustrating recognition of ASF virus (Georgia 2007/1) by sera from vaccinees was validated by IFA using ASFV-infected primary porcine macrophages probed with sera from blood collected 1 week post-boost;

[0094] FIG. 5A is a graph illustrating RD-Ad primed IFN-γ responses against ASFV antigen p37 (part of pp220);

[0095] FIG. 5B is a graph illustrating RD-Ad primed IFN-γ responses against ASFV antigen p150 antigen (also part of pp220);

[0096] FIG. 6A is a graph illustrating RD-Ad primed CTL responses to p32, p54, p62, p′72, and FMD [negative control] with the ENABL adjuvant;

[0097] FIG. 6B is a graph illustrating RD-Ad primed CTL responses to p32, p54, p62, p′72, and FMD using the TXO adjuvant;

[0098] FIG. 7A is a graph illustrating survival post-challenge;

[0099] FIG. 7B is a graph illustrating viremia post-challenge;

[0100] FIG. 8A is a dot-blot analysis illustrating protein concentration of 1) p72; 2) p62; 3) p54; 4) p32; and 5) KP1712 chimera at 1:5,000 serum dilution;

[0101] FIG. 8B is a dot-blot analysis illustrating signal detection sensitivity of 4 ng p72, p54, p32, and KP1712 chimera proteins at 1:5,000 serum dilution; and

[0102] FIG. 8C is a dot-blot analysis [using 4 ng protein concentration] illustrating that only KP1712, but not p32, p54, and p72 were detectable at 1:20,000.

DETAILED DESCRIPTION

[0103] The following detailed description and examples set forth preferred materials and procedures used in accordance with the present disclosure. It is to be understood, however, that this description and these examples are provided by way of illustration only, and nothing therein shall be deemed to be a limitation upon the overall scope of the present disclosure.

[0104] “A”, “an”, and “the” include the singular and plural forms thereof unless the context clearly indicates otherwise.

[0105] “Comprising”, “comprises”, “comprise”, “including”, “includes”, “include”, “having”, “has”, and “with” are all defined as being inclusive of the specified components as well as other unspecified components and can be used interchangeably.

[0106] An “immunogenic or immunological composition” refers to a composition of matter that comprises at least one antigen which elicits an immunological response in the host of a cellular and/or antibody-mediated immune response to the composition or vaccine of interest. Usually, an “immunological response” includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or γδ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction in the severity or prevalence of, up to and including a lack of symptoms normally displayed by an infected host, a quicker recovery time and/or a lowered viral titer in the infected host.

[0107] The term “transfected into a viral vector” means, and is used as a synonym for “introducing” or “cloning” a heterologous DNA sequence encoding a desired antigen into a viral vector, such as for example into a single-cycle replicon adenovirus, an attenuated bovine parainfluenza virus type 3 genotype c (BPIV3c), or a conventional vector such as a baculovirus vector. A “transfer vector” means a DNA molecule, that includes at least one origin of replication, the heterologous ASFV DNA sequence that encodes a desired antigen, in the present case of ASFV, DNA sequences which allow the cloning of said heterologous ASFV DNA sequence into the viral vector will be included. Preferably the sequences which allow cloning of the heterologous DNA sequence into the viral vector are flanking the heterologous DNA. Even more preferably, those flanking sequences are at least homologous in parts with sequences of the viral vector. The sequence homology then allows recombination of both molecules, the viral vector, and the transfer vector to generate a recombinant viral construct containing the heterologous DNA sequence encoding a desired antigen.

[0108] “Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalene or squalene oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).

[0109] For example, it is possible to use the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.

[0110] A further instance of an adjuvant is a compound chosen from the polymers of acrylic or methacrylic acid and the copolymers of maleic anhydride and alkenyl derivative. Advantageous adjuvant compounds are the polymers of acrylic or methacrylic acid which are cross-linked, especially with polyalkenyl ethers of sugars or polyalcohols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Persons skilled in the art can also refer to U.S. Pat. No. 2,909,462 which describes such acrylic polymers cross-linked with a polyhydroxylated compound having at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least three hydroxyls being replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals may themselves contain other substituents, such as methyl. The products sold under the name Carbopol; (BF Goodrich, Ohio, USA) are particularly appropriate. They are cross-linked with an allyl sucrose or with allyl pentaerythritol. Among then, there may be mentioned Carbopol 974P, 934P and 971P. Among the copolymers of maleic anhydride and alkenyl derivative, the copolymers EMA (Monsanto) which are copolymers of maleic anhydride and ethylene. The dissolution of these polymers in water leads to an acid solution that will be neutralized, preferably to physiological pH, in order to give the adjuvant solution into which the immunogenic, immunological or vaccine composition itself will be incorporated.

[0111] Further suitable adjuvants include, but are not limited to, the RIM adjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.), SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridine lipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide among many others.

[0112] Preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 100 μg to about 10 mg per dose. Even more preferably, the adjuvant is added in an amount of about 500 μg to about 5 mg per dose. Even more preferably, the adjuvant is added in an amount of about 750 μg to about 2.5 mg per dose. Most preferably, the adjuvant is added in an amount of about 1 mg per dose.

[0113] “Sequence Identity” as it is known in the art refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, namely a reference sequence and a given sequence to be compared with the reference sequence. Sequence identity is determined by comparing the given sequence to the reference sequence after the sequences have been optimally aligned to produce the highest degree of sequence similarity, as determined by the match between strings of such sequences. Upon such alignment, sequence identity is ascertained on a position-by-position basis, e.g., the sequences are “identical” at a particular position if at that position, the nucleotides or amino acid residues are identical. The total number of such position identities is then divided by the total number of nucleotides or residues in the reference sequence to give % sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to, those described in Computational Molecular Biology, Lesk, A. N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods to determine the sequence identity are designed to give the largest match between the sequences tested. Methods to determine sequence identity are codified in publicly available computer programs which determine sequence identity between given sequences. Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs optimally align sequences using default gap weights in order to produce the highest level of sequence identity between the given and reference sequences. As an illustration, by a polynucleotide having a nucleotide sequence having at least, for example, 85%, preferably 90%, even more preferably 95% “sequence identity” to a reference nucleotide sequence, it is intended that the nucleotide sequence of the given polynucleotide is identical to the reference sequence except that the given polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence having at least 85%, preferably 90%, even more preferably 95% identity relative to the reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 15%, preferably 10%, even more preferably 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Analogously, by a polypeptide having a given amino acid sequence having at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity to a reference amino acid sequence, it is intended that the given amino acid sequence of the polypeptide is identical to the reference sequence except that the given polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid alterations per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a given polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or the carboxyl terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in the one or more contiguous groups within the reference sequence. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. However, conservative substitutions are not included as a match when determining sequence identity.

[0114] “Sequence homology”, as used herein, refers to a method of determining the relatedness of two sequences. To determine sequence homology, two or more sequences are optimally aligned, and gaps are introduced if necessary. However, in contrast to “sequence identity”, conservative amino acid substitutions are counted as a match when determining sequence homology. In other words, to obtain a polypeptide or polynucleotide having 95% sequence homology with a reference sequence, 85%, preferably 90%, even more preferably 95% of the amino acid residues or nucleotides in the reference sequence must match or comprise a conservative substitution with another amino acid or nucleotide, or a number of amino acids or nucleotides up to 15%, preferably up to 10%, even more preferably up to 5% of the total amino acid residues or nucleotides, not including conservative substitutions, in the reference sequence may be inserted into the reference sequence. Preferably the homologous sequence comprises at least a stretch of 50, even more preferably 100, even more preferably 250, even more preferably 500 nucleotides.

[0115] A “conservative substitution” refers to the substitution of an amino acid residue or nucleotide with another amino acid residue or nucleotide having similar characteristics or properties including size, hydrophobicity, etc., such that the overall functionality does not change significantly.

[0116] Isolated” means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.

[0117] “Modified polypeptide” of a polypeptide according to the disclosure is understood as designating a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, having at least one modification with respect to the normal sequence. These modifications will especially be able to bear an amino acids at the origin of a specificity, of pathogenicity and/or of virulence, or at the origin of the structural conformation, and of the capacity of membrane insertion of the polypeptide according to the disclosure. It will thus be possible to create polypeptides of equivalent, increased or decreased activity, and of equivalent, narrower, or wider specificity. Among the modified polypeptides, it is necessary to mention the polypeptides in which up to 5 amino acids can be modified, truncated at the N- or C-terminal end, or even deleted or added.

[0118] As is indicated, the modifications of the polypeptide will especially have as objective: to render it capable of modulating, of inhibiting or of inducing the expression of ASFV gene and/or capable of modulating the replication cycle of ASFV in the cell and/or the host organism, of allowing its incorporation into vaccine compositions, and/or of modifying its bioavailability as a compound for therapeutic use.

[0119] The methods allowing said modulations on eukaryotic or prokaryotic cells to be demonstrated are well known to the person skilled in the art. It is likewise well understood that it will be possible to use the nucleotide sequences coding for said modified polypeptides for said modulations, for example through vectors according to the disclosure and described below, in order, for example, to prevent or to treat the pathologies linked to the infection.

[0120] The preceding modified polypeptides can be obtained by using combinatorial chemistry, in which it is possible to systematically vary parts of the polypeptide before testing them on models, cell cultures or microorganisms for example, to select the compounds which are most active or have the properties sought.

[0121] Chemical synthesis likewise has the advantage of being able to use unnatural amino acids, or nonpeptide bonds. Thus, in order to improve the duration of life of the polypeptides according to the disclosure, it may be of interest to use unnatural amino acids, for example in D form, or else amino acid analogs, especially sulfur-containing forms, for example.

[0122] Finally; it will be possible to integrate the structure of the polypeptides according to the disclosure, its specific or modified homologous forms, into chemical structures of polypeptide type or others. Thus, it may be of interest to provide at the N- and C-terminal ends compounds not recognized by the proteases.

EXAMPLES

[0123] The following examples illustrate embodiments of the disclosure. Nothing in these examples should be limiting to the disclosure as these are representative in nature.

Example 1

[0124] This example ranked ASFV ORFs for their ability to bind to SLA alleles.

Materials and Methods

[0125] All the conserved ASFV (Georgia 2007/1) open-reading frames (ORFs) were screened for the presence of peptide motifs that bind strongly to 75 defined SLA I alleles and identified novel targets containing putative CTL epitopes (found on the web at cbs.dtu.dk/services/NetMHCpan/). The targets were ranked based on the number of putative CTL epitopes and binding affinity. As shown in FIG. 1A, ORFs were screened using 75 SLA alleles. The binding affinities of top 3 strong binders [SBs] (example in blue color) were used to calculate mean binding affinity for each ORF. The mean binding affinities from the 75 alleles were used to calculate grand mean binding affinities and the outcome was used to rank the ORFs as illustrated using means from 3 alleles. This ranking criterion is reasonable since it is based on putative epitopes as predicted by their ability to bind to SLA alleles. Three ORFs (B385R, A859L, G1340L) and three SLA alleles (SLA-1*0701; SLA-2*0401; and SLA-3*0503) were used to illustrate the down selection criteria. The algorithm, as described in NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data. Vanessa Jurtz, Sinu Paul, Massimo Andreatta, Paolo Marcatili, Bjoern Peters and Morten Nielsen. The Journal of Immunology (2017) ji1700893; DOI: 10.4049/jimmunol.1700893; or NetMHCpan-3.0: improved prediction of binding to MHC class I molecules integrating information from multiple receptor and peptide length data sets. Morten Nielsen and Massimo Andreatta. Genome Medicine (2016): 8:33; and NetMHCpan, a method for MHC class I binding prediction beyond humans. Ilka Hoof, Bjoern Peters, John Sidney, Lasse Eggers Pedersen, Ole Lund, Soren Buus, and Morten Nielsen. Immunogenetics 61.1 (2009): 1-13] (the teachings and contents of which are incorporated by reference herein) predicts binding of peptides to known MHC molecules using Artificial Neural Networks. All the ASFV open reading frames [ORFs] selected were subjected to this process and shown to contain multiple putative strong MHC I-binding peptide motifs. MHC-1 molecules have a strong preference for binding 9-mer peptides. The output is based on predicted binding affinity and is presented as either strong binder (SB) or weak binder (WB). Some ORFs have several predicted SBs, some have one or two, whereas others have none. However, only peptides with high affinity can compete for binding to the cognate MHC molecule(s). The grand mean affinity is expected to be a representative estimated binding index for each ORF. FIG. 1B illustrates a flow diagram summarizing the down selection work flow. The targets were validated using ASFV convalescent serum followed by in vitro confirmation of the SLA class I-binding motifs using peptides. One hundred ORFs were selected (see list below) and used to design multicistronic expression cassettes separated by an efficient self-cleaving 2A peptide linker (FIG. 2A). The multicistronic expression cassettes were modified to add, in-frame, a HA-tag at the N-termini and a FLAG-tag at the C-termini (FIG. 2A). The amino acid sequences of the multicistronic cassettes were used to design synthetic genes codon-optimized for protein expression in swine cells. Recombinant DNA and SC-Ad expression constructs were generated using the synthetic genes (FIG. 2B). Protein expression by the constructs was evaluated by immunocytometric analysis using anti-HA and anti-FLAG monoclonal antibodies (mAbs), and ASFV-specific convalescent serum was used to validate authenticity of the expressed antigens (FIG. 2B). Protein expression by recombinant SC-Ad was confirmed by immunocytometric analysis of infected cells using anti-FLAG mAb (upper panel) and authenticity was validated by the ASFV convalescent serum (lower panel). Note: SC-Ad 2-4 on upper panel—most of the infected cells lifted off Virus dose was reduced for the lower panel to solve this problem. Data shown is for four constructs. Auto-cleavage of the 2A peptide linker is verifiable using Western blot probed with anti-tag mAbs. Peptides (9-mer) were used to validate the predicted ASFV (Georgia 2007/1) SLA I binding motifs by IFN-γ EliSpot assay using splenocytes from immunized pigs (FIG. 3).

Results and Discussion

[0126] Since peptide presentation by Swine Leukocyte Antigen I (SLA I) is required to prime and expand CTLs, a strategy that utilizes an algorithm based on SLA I-peptide binding is expected to identify putative candidate antigens based on the presence of peptide motifs predicted to bind with high affinity to defined SLA I molecules.

[0127] The flow diagram in FIG. 2A illustrates the design of multicistronic cassettes for the ASFV antigens. Up to seven ORFs were included in a single cassette since the adenovirus vector can accommodate insert size of up to 8 kb. Codon-optimized synthetic genes were first cloned into pCDNA3 mammalian expression vector which has a strong CMV promoter (CMVp) and anti-HA/FLAG mAbs were used to confirm protein expression (data not shown). The photographs in FIG. 2B illustrate the validation of protein expression. The graph in FIG. 3 illustrates IFN-γ response to predicted SLA I binding motifs. Sample data from 3 pools (20 peptides/pool) is shown. Some pigs (e.g. #4) had strong responses against multiple pools.

Example 2

[0128] This example demonstrates RD-Ad primed antibody responses against ASFV antigens, recognition of ASF virus (Georgia 2007/1) by sera from vaccinees, RD-Ad primed IFN-γ responses against ASFV antigens, and RD-Ad primed CTL responses.

Materials and Methods

[0129] To demonstrate RD-Ad primed antibody responses against ASFV antigens, three immunogen formulations were tested. Formulation 1 (pig #s 1-5: 1×10.sup.10 ifu/adjuvant 1); Formulation 2 (pig #s 6-10: 1×10.sup.11 ifu/adjuvant 1); Negative controls (pig #s 11-15: 1×10.sup.11 ifu); and Formulation 3 (pig #s 16-20: 1×10.sup.11 ifu/adjuvant 2). The positive control was ASFV-specific convalescent serum (red star symbol). Antibody endpoint titers were determined by ELISA (data for representative antigens is shown). Results are provided in FIG. 4A.

[0130] To demonstrate and validate recognition of ASF virus (Georgia 2007/1) by sera from vaccinees, IFA was conducted using ASFV-infected primary porcine macrophages probed with sera from blood collected 1 week post-boost. Representative data for the vaccinees and negative controls are shown in FIG. 4B.

[0131] To demonstrate RD-Ad primed IFN-γ responses against ASFV antigens. IFN-γ responses for two representative antigens, p37 and p150, are shown in FIG. 5A and FIG. 5B, respectively. Both of these ASFV antigens are part of the pp220 polyprotein. Treatment groups are as indicated in FIG. 5

[0132] To demonstrate RD-Ad primed CTL responses, ASFV antigen-specific CTL responses were evaluated by standard .sup.51Cr release assays using PBMCs isolated post-boost. CTL activity, at defined effector-target ratios, was determined using .sup.51Chromium-labelled autologous skin fibroblasts (targets) transfected with DNA construct expressing cognate antigen. Fibroblasts transfected with a construct expressing an FMD antigen served as negative control targets. Priming of CTLs was influenced by the type of adjuvant used. FIG. 6A illustrates the RD-Ad ASFV antigen cocktail formulated with ENABL adjuvant (Benchmark Biolabs). FIG. 6B illustrates the RD-Ad ASFV antigen cocktail formulated with TXO adjuvant (Zoetis).

Example 3

[0133] This example determines survival and viremia post-challenge with ASFV.

Materials and Methods

[0134] Pigs were immunized with either RD-Ad ASFV antigen cocktail formulated in TXO adjuvant (T1) or ENABL adjuvant (T2). Negative controls received RD-Ad expressing Luciferase formulated in TXO (C1) or ENABL (C2), respectively. The pigs were challenged with 3 log.sub.10 TCID.sub.50 ASFV [Georgia 2007/1] administered intranasally. Viremia in blood at termination was determined by qPCR.

Results

[0135] Results are provided in FIGS. 7A and 7B. As can be seen, 5/9 pigs from the TI group were alive and healthy 17 days post-challenge compared to 2/10 from the T2 group, 2/5 pigs from the CI and C2. However, the pigs from the T2, C1, and C2 were moribund [based on clinical scores and evaluation by attending veterinarian] and needed to be euthanized.

Example 4

[0136] This example estimates protein concentration.

Materials and Methods

[0137] Protein concentration was estimated using BCA and antigen loads were normalized by SDS-PAGE analysis. Dot blots were probed with convalescent serum. Antigens were titrated and the blot was probed at 1:5,000 serum dilution: 1) p′72; 2) p62; 3) p54; 4) p32; and 5) KP1712 chimera. Results are provided in FIG. 8A. Probing a blot loaded with 4 ng of each antigen showed that PK1712 was better than p32, p54 & p72 at 1:5,000. Results are provided in FIG. 8B. Additionally, KP1712 was the only one detectable at 1:50,000. Notably, p32, p54, and p72 were not detectable at 1:20,000, but KP1712 was still detectable at 1:100,000 serum dilution (data not shown). Results are provided in FIG. 8C.

Results and Discussion

[0138] It is desirable to develop a vaccine and a compatible diagnostic tool capable of differentiating infected from vaccinated animals [DIVA]. In the course of validating ASFV antigens using the ASFV convalescent serum, a chimeric antigen designated KP1712, was recognized more strongly than p32, p54, p′72, and pp62 which have previously been evaluated as diagnostic antigens. The convalescent serum is a valuable probe since it was produced by sequential infection of a pig with a series of tissue culture-adapted and wild-type viruses from genotypes I (DR11, Haiti 81, Lisbon 60, Malawi 83, and UG-61), VIII, and X (13). The serum was gamma-irradiated and provided by DHS S&T. There is a p72-based Lateral Flow Device (LFD) in the market (Ingenasa, Spain) and the OIE's ASFV reference ELISA (INgezim PPA COMPAC) utilizes virus lysate.

[0139] The results indicate that a KP1712-based LFD will likely be a superior diagnostic tool. Notably, this was confirmed by comparing the reactivity of the convalescent serum against normalized amounts of affinity purified antigens (quality control validated by Western Blot) using Dot Blot assay (FIG. 8). The sensitivity limit for the recombinant KP1712 chimera was determined to be <0.24 ng, which is 16 fold lower than for p72 (3.9 ng) and 32 fold lower than p32 and p54 (7.8 ng) (FIG. 8). Therefore, the KP1712 antigen is being used to develop a prototype Lateral Flow Device (LFD) as a highly sensitive tool for ASFV diagnosis using saliva and sera from pigs and wild boars.

[0140] Relevant to development of a subunit vaccine and a compatible DIVA diagnostic tool, the p72 antigen contains antibody and CTL epitopes. In addition, it contains multiple predicted strong SLA I binding motifs. However, the KP1712 antigen has few and weak predicted SLA I binding motifs whose affinities are below cutoff threshold. Consequently, it is rational to retain the p72 antigen in a vaccine and utilize the KP1712 as a diagnostic antigen.

Example 5

[0141] This example prepares representative multicistronic cassettes useful for administration to subjects in need thereof.

Materials and Methods

[0142] The selected ASFV [Georgia 2007/1] polypeptide sequences were used to design multicistronic expression cassettes separated by an efficient self-cleaving 2A peptide linker as illustrated in FIG. 2A using ORF1 [generally genes encoding large antigens: ˜3.5 kb], ORF2 [genes encoding medium size antigens: ˜2 kb], and ORF3 [genes encoding small antigens: ˜1 kb] as an example. The multicistronic cassettes contains 3-7 ORFs and overall chimeric gene size was limited to about 4.5 kb for stability and protein expression efficiency. Each multicistronic polypeptide sequence cassette was modified to add, in-frame, a HA-tag at the N-termini and a FLAG-tag at the C-termini (FIG. 2A). The resultant amino acid sequences of the multicistronic cassettes were used to design synthetic genes codon-optimized for protein expression in swine cells. The synthetic genes were cloned into pCDNA3.1+ mammalian expression vector [outsourced from GenScript], which utilizes CMV promoter to drive protein expression. SC-Ad expression constructs were generated using the synthetic genes (FIG. 2B). Protein expression by the expression constructs was evaluated by immunocytometric analysis using anti-HA and anti-FLAG monoclonal antibodies (mAbs), and ASFV-specific convalescent serum was used to validate authenticity of the expressed antigens (FIG. 2B).

Results

[0143] The following multicistronic cassettes were generated:

Polypeptide Sequence Used to Generate Multicistronic Expression Cassettes:

[0144]

TABLE-US-00001 1. p220.1 (SEQ ID NO. 102) MGNRGSSTSSRPLPSSEANIYAKLQDHIQRQTRPFSGGGYFNGGGDKNPVQHIKDYHIDSVSSKA KLRIIEGIIRAIAKIGFKVDTKQPIEDILKDIKKQLPDPRAGSTFVKNAEKQETVCKMIADAINQEFI DLGQDKLIDTTEGAASICRQIVLYINSLTHGLRAEYLDVHGSIENTLENIKLLNDAIKQLHERMVT EVTKAAPNEEVINAVTMIEAVYRRLLNEQNLQINILTNFIDNILTPTQKELDKLQTDEVDIIKLLND TNSVLGTKNFGKVLSYTLCNLGIAASVANKINKALQKVGLKVEQYLQSKNWAEFDKELDLKRF SGLVSAENIAEFEKAVNLLRQTFNERHKILENSCAKKGGDEEKTPLDRRIEAQRLDRKHILMEFL NKSTQAYNDFLENVKKIGIKLVKEIALTPNITRLRDALSRINDMGTIALDLSLIGFYTNAAAREER ETFLTQLTLVKNVLEEISKTDPNFKNLYDSCSRLLQIIDFYTDIVQKKYGGEEDCECTRVGGAALT VEELGLSKAARSQVDLNQAINTFMYYYYVAQIYSNLTHNKQEFQSYEENYATILGDAIAGRLMQ LDTEKNARINSPAVDLARGHVGPNPGGAQEVDWKATVSAIELEYDVKRRFYRALEGLDLYLKNI TKTFVNNIDSIQTVQQMLDGVRIIGRWFTETTGDTLAQVFESFPTSTGNDSNVFTDNAPAGHYYE KVAAEIQQGRSVGTLRPVRASQAKNIRDLIGRSLSNFQALKNIINAFARIGDMLGGEELRQMVPM SPLQIYKTLLEYLQHSALSVGLKNLNQSEIGGQRMALAQTAEEAAQRVYLSTVRVNDALSTRWE TEDVFFTFMLKSMAAKIFIVLGIYDMFERPEPVYKLIPTRMILGGADELEPEVIPEAAELYFRLPRL AEFYQKLFSFRDENVQISMLPELEGIFSGLIRIIFMRPIELINIGDYSETEIRQLIKEINVIYQHFNLEY GEQEATKKALIHFVNEINRRFGVITRTEWEKFQRIVQEARTMNDFGMMNQTNYSILPDEDGYTQ SSQLLPSDRFISPSTQPTPKWRPALYNIDSVDVQTGMLQPNSQWDLVQKFRKQLSEMFEDPSLQQ ELGKISYQELIRQAINELKKEHTDKIQIVSKLIQGSESLADTDVNKIFLFHETVITGLNLLSAIYVLL NNFRNNIKGLDLDTIQKSIIEWLRETQAANVNRANLIDWLGRKHGAISEIRNPGLVIKEINMRLSM VYPDPTTEAAAA 2. p220.2 (SEQ ID NO. 103) GLDLDTIQKSIIEWLRETQAANVNRANLIDWLGRKHGAISEIRNPGLVIKEINMRLSMVYPDPTTE AAAAAQDRNLTTETLFAWIVPYVGIPAGGGVRPEQELAARYLVDNQRIMQLLLTNIFEMTSSFN KMVQVRFPETSTAQVHLDFTGLISLIDSLMADTKYFLDLLRPHIDKNIIQYYENRSNPGSFYWLEE HLIDKLIKPPTDAGGRPLPGGELGLEGVNQIINKTYTLLTKPYNVLQLRGGAQRRDAANIQINNNP QSSERFEQYGRVFSRLVFYDALENNSGLRVEQVALGDFRLSNLIRTNNAQEENTLSYWDNIALRT YANVNDAANNLRRYRLYGSDYGIQNNRSMMMVFNQLIASYITRFYDAPSGKIYLNLINAFANGN FSQAVMEMGYAHPDLARNNNVFGHRGDPTEQSVLLLSLGLILQRLIKDTNRQGLSQHLISTLTEI PIYLKENYRANLPLFNKMFNILISQGELLKQFIQYTNVQLARPNLTALLGANNDSVIYYNNNNVP ATGLSVGQAALRGIGGVFRPNVTLMPLGDAQNNTSDVVRKRLVAVIDGIIRGSHTLADSAMEVL HELTDHPIYLETEEHFIQNYMSRYNKEPLMPFSLSLYYLHDLRIENNEVYDPLLYPNLESGSPEFK LLYGTRKLLGNDPVQLSDMPGVQLIMKNYNETVVAREQITPTRFEHFYTHAIQALRFIINIRSFKT VMMYNENTFGGVNLISENRDDKPIITAGIGMNAVYSLRKTLQDVISFVESSYQEEQINHIHKIVSP KGQTRTLGSNRERERIFNLFDMNIIPINVNALMRSIPLANIYNYDYSFEEIACLMYGISAEKVRSLD TTAPQPDVAEVLNIPNRPPINTREFMLKLLINPYVSVSITQYGNELLSKGNAGYMSRIFRGDNALN MGRPKFLSDQIFNKVLFGSLYPTQFDYDEAGPSLAAGIQRGRERWGHPMSIYINQALHEIVRTIRL AETVRGLRNVIDRNQIIGELNAFRTQLEDTRREVNNLIQTPEIQNNPTPEIIAAIQNWVQQYRGQIT NLIDLIGNAGQANSMINLIQNITPQTAGAQLTALFNIRGLPAPPPRQALQNDIEAMQWFMTMVIN HPPVLIAPFMLLVNNLKEFLNTLERYVYKTPRWLGPGTARIAQPPVGMAPGINMRHHTSYTENS VLTYITEQNREEGPWSIVKQVGVGIQKPTLVQIGKDRFDTRLIRNLIFITNIQRLLRLRLNLELSQFR NVLVSPDHIINPSITEYGFSITGPSETFSDKQYDSDIRIL 3. p72-p15-B602L (SEQ ID NO. 104) MASGGAFCLIANDGKADKIILAQDLLNSRISNIKNVNKSYGKPDPEPTLSQIEETHLVHFNAHFKP YVPVGFEYNKVRPHTGTPTLGNKLTFGIPQYGDFFHDMVGHHILGACHSSWQDAPIQGTSQMGA HGQLQTFPRNGYDWDNQTPLEGAVYTLVDPFGRPIVPGTKNAYRNLVYYCEYPGERLYENVRF DVNGNSLDEYSSDVTTLVRKFCIPGDKMTGYKHLVGQEVSVEGTSGPLLCNIHDLHKPHQSKPIL TDENDTQRTCSHTNPKFLSQHFPENSHNIQTAGKQDITPITDATYLDIRRNVHYSCNGPQTPKYY QPPLALWIKLRFWFNENVNLAIPSVSIPFGERFITIKLASQKDLVNEFPGLFVRQSRFIAGRPSRRNI RFKPWFIPGVINEISLTNNELYINNLFVTPEIHNLFVKRVRFSLIRVHKTQVTHTNNNFIHDEKLMS ALKWPIEYMFIGLKPTWNISDQNPHQHRDWHKFGHVVNAIMQPTHHAEISFQDRDTALPDACSS ISDISPVTYPITLPIIKNISVTAHGINLIDKFPSKFCSSYIPFHYGGNAIKTPDDPGAMMITFALKPREE YQPSGHINVSRAREFYISWDTDYVGSITTADLVVSASAINFLLLQNGSAVLRYSTGSGATNFSLLK QAGDVEENPGPMADFNSPIQYLKEDSRDRTSIGSLEYDENADTMIPSFAAGLEEFEPIPDYDPTTS TSLYSQLTHNMEKIAEEEDSNFLHDTREFTSLVPDEADNKPEDDEESGAKPKKKKHLFPKLSSHK SKGSGATNFSLLKQAGDVEENPGPMAEFNIDELLKNVLEDPSTEISEETLKQLYQRTNPYKQFKN DSRVAFCSFTNLREQYIRRLIMTSFIGYVFKALQEWMPSYSKPTHTTKTLLSELITLVDTLKQETN DVPSESVVNTILSIADSCKTQTQKSKEAKTTIDSFLREHFVFDPNLHAQSAYTCADTNVDTCASM CADTNVDTCASMCADTNVDTCASTCTSTEYTDLADPERIPLHIMQKTLNVPNELQADIDAITQTP QGYRAAAHILQNIELHQSIKHMLENPRAFKPILFNTKITRYLSQHIPPQDTFYKWNYYIEDNYEEL RAATESIYPEKPDLEFAFIIYDVVDSSNQQKVDEFYYKYKDQIFSEVSSIQLGNWTLLGSFKANRE RYNYFNQNNEIIKRILDRHEEDLKIGKEILRNTIYHKKAKNIQETGPDAPGLSIYNSTFHTDSGIKG LLSFKELKNLEKASGNIKKAREYDFIDDCEEKIKQLLSKENLTPDEESELIKTKKQLDNALEMLNV PDDTIRVDNKLEKEILYTKAEL 4. p62-p32-p54-EP153R-p10 (SEQ ID NO. 105) MPSNMKQFCKISVWLQQHDPDLLEIINNLCMLGNLSAAKYKHGVTFIYPKQAKIRDEIKKHAYS NDPSQAIKTLESLILPFYIPTPAEFTGEIGSYTGVKLEVEKTEANKVILKNGEAVLVPAADFKPFPD RRLAVWIMESGSMPLEGPPYKRKKEGGGNDPPVPKHISPYTPRTRIAIEVEKAFDDCMRQNWCS VNNPYLAKSVSLLSFLSLNHPTEFIKVLPLIDFDPLVTFYLLLEPYKTHGDDFLIPETILFGPTGWN GTDLYQSAMLEFKKFFTQITRQTFMDIADSATKEVDVPICYSDPETVHSYANHVRTEILHHNAVN KVTTPNLVVQAYNELEQTNTIRHYGPIFPESTINALRFWKKLWQDEQRFVIHGLHRTLMDQPTYE TSEFAEIVRNLRFSRPGNNYINELNITSPAMYGDKHTTGDIAPNDRFAMLVAFINSTDFLYTAIPEE KVGGNETQTSSLTDLVPTRLHSFLNHNLSKLKILNRAQQTVRNILSNDCLNQLKHYVKHTGKNEI LKLLQEGSGATNFSLLKQAGDVEENPGPMDFILNISMKMEVIFKTDLRSSSQVVFHAGSLYNWFS VEIINSGRIVTTAIKTLLSTVKYDIVKSARIYAGQGYTEHQAQEEWNMILHVLFEEETESSASSENI HEKNDNETNECTSSFETLFEQEPSSEVPKDSKLYMLAQKTVQHIEQYGKAPDFNKVIRAHNFIQTI YGTPLKEEEKEVVRLMVIKLLKKISFYLTYIGSGATNFSLLKQAGDVEENPGPMDSEFFQPVYPR HYGECLSPVTTPSFFSTHMYTILIAIVVLVIIIIVLIYLFSSRKKKAAAIEEEDIQFINPYQDQQWVEV TPQPGTSKPAGATTASVGKPVTGRPATNRPATNKPVTDNPVTDRLVMATGGPAAAPAAASAPA HPAEPYTTVTTQNTASQTMSAIENLRQRNTYTHKDLENSLGSGATNFSLLKQAGDVEENPGPMF SNKKYIGLINKKEGLKKKIDDYSILIIGILIGTNILSLIINIIGEINKPICYQNDDKIFYCPKDWVGYNN VCYYFGNEEKNYNASNYCKQLNSTLTNNTILVNLTKTLNLTKTYNHESNYWVNYSLIKNEV LLRDSGYYKKQKHVSLLYICSKGSGATNFSLLKQAGDVEENPGPMPTKAGTKSTANKKTTKGSS KSGSSRGHTGKTHASSSMHSGMLYKDMVNIARSRGIPIYQNGSRLTKSELEKKIKRSK 5. K205R-A104R-CD2vΔcd-A151R-9GL-K196R-CP80R (SEQ ID NO. 106) MVEPREQFFQDLLSAVDQQMDTVKNDIKDIMKEKTSFMVSFENFIERYDTMEKNIQDLQNKYEE MAANLMTVMTDTKIQLGAIIAQLEILMINGTPLPAKKTTIKEAMPLPSSNTNNEQTSPPASGKTSE TPKKNPTNAMFFTRSEWASSNTFREKFLTPEIQAILDEQFANKTGIERLHAEGLYMWRTQFSDEQ KKMVKEMMKKGSGATNFSLLKQAGDVEENPGPMSTKKKPTITKQELYSLVAADTQLNKALIER IFTSQQKIIQNALKHNQEVIIPPGIKFTVVTVKAKPARQGHNPATGEPIQIKAKPEHKAVKIRALKP VHDMLNGSGATNFSLLKQAGDVEENPGPMIILIFLIFSNIVLSIDYWVSFNKTIILDSNITNDNNDIN GVSWNFFNNSFNTLATCGKAGNFCECSNYSTSIYNITNNCSLTIFPHNDVFDTTYQVVWNQIINY TIKLLTPATPPNITYNCTNFLITCKKNNGTNTNIYLNINDTFVKYTNESILEYNWNNSNINNFTATC IINNTISTSNETTLINCTYLTLSSNYFYTFFKLYYIPLSIIIGITISILLISIITFLGSGATNFSLLKQAGDV EENPGPMNKKIIVMMALLHKEKLIECIYHELENGGTILLLTKNIVVSEISYIGNTYKYFTFNDNHD LISKEDLKGATSKNIAKMIYNWIIKNPQNNKIWSGEPRTQIYFENDLYHTNYNHKCIKDFWNVST SVGPHIFNDRSIWCTKCTSFYPFTNIMSPNIFQGSGATNFSLLKQAGDVEENPGPMLHWGPKYWR SLHLYAIFFSDAPSWKEKYEAIQWILNFIESLPCTRCQHHAFSYLTKNPLTLNNSEDFQYWTFAFH NNVNNRLNKKIISWSEYKNIYEQSILKTIEYGKTDFIGAWSSLGSGATNFSLLKQAGDVEENPGP MNIIRKLKPGTISLVLGPMFAGKTTFLIHCIYMLERLEKKVVFIKSTKNTRDKTIKTHSGIQLRPKQ CKIIESTQLSDVGSLTDIHAVVVDEAHFFDDLITCRTWAEEEKIIILAGLNASFEQKMFPPIVRIFPY CSWVKYIGRTCMKCNQHNACFNVRKNADKTLILAGGSELYVTCCNNCLKNTFIKQLQPIKYGSG ATNFSLLKQAGDVEENPGPMLIPVVCFTCGFPIGTYAAIFDKARTEYIKTKMGGTLPQNIPLDASL QIELKDLITALGIPMRVCCRTHLITTLDYRKYY 6. B438L-R298L-NP419L-K145R (SEQ ID NO. 107) MYHDYASKLLADYRSDPPLWESDLPRHNRYSDNILNSRYCGNKNGAAPVYNEYTNSPEKAEKG LQLSDLRNFSFMLNPQHKNIGYGDAQDLEPYSSIPKNKLFNHFKNHRPAFSTHTENLIRRNVVRT EKKTFPQVASLKGTQKNCLTQPSSLPSLKNPKNSSVPSTRFSEHTKFFSYEDLPKLRTKGTIKHEQ HLGDQMPGQHYNGYIPHKDVYNILCLAHNLPASVEKGIAGRGIPLGNPHVKPNIEQELIKSTSTY TDVPMLGPLPPKDSQHGREYQEFSANRHMLQVSNILHSVFANHSIKPQILEDIPVLNAQLTSIKPV SPFLNKAYQTHYMENIVTLVPRFKSIANYSSPIPNYSKRNSGQAEYFDTSKQTISRHNNYIPKYTG GIGDSKLDSTFPKDFNASSVPLTSAEKDHSLRGDNSACCISSISPSLGSGATNFSLLKQAGDVEENP GPMSRPEQQLKKMLKNPQAQYAFYPTAKVERISTTQHMYFIATRPMFEGGRNNVFLGHQVGQPI IFKYVSKKEIPGNEVIVLKALQDTPGVIKLIEYTENAMYHILIIEYIPNSVDLLHYHYFKKLEETEA KKIIFQLILIIQNIYEKGFIHGDIKDENLIIDINQKIIKVIDFGSAVRLDETRPQYNMFGTWEYVCPEF YYYGYYYQLPLTVWTIGMVAVNLFRFRAENFYLNDILKRENYIPENISETGKQFITECLTINENKR LSFKSLVSHPWFKGLKKEIQPISELGVDYKNVITGSGATNFSLLKQAGDVEENPGPMLNQFPGQY SNNIFCFPPIESETKSGKKASWIICVQVVQHNTIIPITDEMFSTDVKDAVAEIFTKFFVEEGAVRISK MTRVTEGKNLGKKNATTVVHQAFKDALSKYNRHARQKRGAHTNRGIIPPMLVKYFNIIPKTFFE EETDPIVQRKRNGVRAVACQQGDGSILLYSRTKKEFLGLDNIKKELKQLYLFIDVRVYLDGELYL HRKPLQWIAGQANAKTDSSELHFYVFDCFWSDQLQMPSNKRQQLLTNIFKQKEDLTFIHQVENF SVKNVDEALRLKAQFIKEGYEGAIVRNANGPYEPGYNNYHSAHLAKLKPLLDAEFILVDYTQGK KGKDLGAILWVCELPNKKRFVVTPKHLTYADRYALFQKLTPALFKKHLYGKELTVEYAELSPKT GIPLQARAVGFREPINVLEIIGSGATNFSLLKQAGDVEENPGPMDHYLKKLQDIYTKLEGHPFLFS PSKTNEKEFITLLNQALASTQLYRSIQQLFLTMYKLDPIGFINYIKTSKQEYLCLLINPKLVTKFLKI TSFKIYINFRLKTFYISPNKYNNFYTAPSEEKTNHLLKEEKTWAKIVEEGGEES 7. B385R-F334L-CP312R-H108R-F165R (SEQ ID NO. 108) MDEIINKYQAVEKLFKEIQQGLAAYDQYKTLISEMMHYNNHIKQEYFNFLMIISPYLIRAHSGETL RNKVNNEIKRLILVENINTKISKTLVSVNFLLQKKLSTDGVKTKNMWCTNNPMLQVKTAHNLFK QLCDTQSKTQWVQTLKYKECKYCHTDMVFNTTQFGLQCPNCGCIQELMGTIFDETHFYNHDGQ KAKSGIFNPNRHYRFWIEHILGRNPEQELGTKQDPCGTKVLQQLKKIIKRDNKCIALLTVENIRKM LKEINRTDLNNCVSLILRKLTGVGPPQISESILLRGEYIFTEAIKIREKVCKKGRINRNYYPYYIYKIF DAILPPNDTTNRRILQYIHLQGNDTLANNDSEWESICMELPEIKWKPTDRTHCVHFFGSGATNFSL LKQAGDVEENPGPMLIFISNMEELLIENSQRFTIFPIQHPECWNWYKKLESLTWTAQEVDMCKDI DDWEAMPKPQREFYKQILAFFVVADEIVIENLLTNFMREIKVKEVLYFYTMQAAQECVHSEAYSI QVKTLIPDEKEQQRIFSGIEKHPIIKKMAQWVRQWMDPDRNTLGERLVGFAAVEGILFQNHFVAI QFLKEQNIMPGLVSYNEFISRDEGMHCSFACFLISNYVYNIPEEKIIHKILKEAVELVDEFINYAFD KARGRVPGFSKEMLFQYIRYFTDNLCFMMQCKSIYKVGNPFPQMTKFFLNEVEKTNFFELRPTQ YQNCVKDDAFAFKLFLNDDDFGSGATNFSLLKQAGDVEENPGPMTTHIFHADDLLQALQQAKA EKNFSSVFSLDWDKLRTAKRNTTVKYVTVNVIVKGKKAPLMFNFQNEKHVGTIPPSTDEEVIRM NAENPKFLVKKRDRDPCLQFNKYKISPPLEDDGLTVKKNEQGEEIYPGDEEKSKLFQIIELLEEAF EDAVQKGPEAMKTKHVIKLIQRKISNSAVKNADKPLPNPIARIRIKINPATSILTPILLDKNKPITLQ NGKTSFEELKDEDGVKANPDNIHKLIESHSIHDGIINARSICISNMGISFPLCLEMGVVKVFEKNNG IDVNSIYGSDDISTLVNQIAIAGSGATNFSLLKQAGDVEENPGPMVNLFPVFTLIVIITILITTRELST TMLIVSLVTDYIIINTQYTEQQHENNTFFMPQKNSFNESYNKDKKSNIHIPYQWLAPELKEAESKY WWGNYDPHSEPVLAGASGSGATNFSLLKQAGDVEENPGPMVNPNKRIMNKKSKQASISSILNFF FFYIMEYFVAVDNETSLGVFTSIEQCEETMKQYPGLHYVVFKYMCPADAENTDVVYLIPSLTLHT PMFVDHCPNRTKQARHVLKKINLVFEEESIENWKVSVNTVFPHVHNRLSAPKLSIDEANEAVEKF LIQAGRLMSL 8. F778R-S273R-MGF100.1L_B66L (SEQ ID NO. 109) METFFIETLASDVYGKALNVDLDRLSQAQVKYTLQELISYCSALTILHYDYSTLAARLSVYQLHQ STASSFSKAVRLQAAQSCSRLSPQFVDVVYKYKAIFDSYIDYSRDYKLSLLGIETMKNSYLLKNK DGVIMERPQDAYMRVAIMIYGMGRVVNMKMILLTYDLLSQHVITHASPTMFNAGTKKPQLSSC FLLNVNDNLENLYDMVKTAGIISGGGGGIGLCLSGIRAKNSFISGSGLKSNGIQNYIVLQNASQCY ANQGGLRPGAYAVYLELWHQDIFTFLQMPRLKGQMAEQRLNAPNLKYGLWVPDLFMEILEDQI HNRGDGKWYLFSPDQAPNLHKVFDLERSQHENAHREFKKLYYQYVAEKRYTGVTTAKEIIKEW FKTVVQVGNPYIGFKDAINRKSNLSHVGTITNSNLCIEVTIPCWEGDKAEQGVCNLAAVNLAAFI RENGYDYRGLIEASGNVTENLDNIIDNGYYPTEATRRSNMRHRPIGIGVFGLADVFASLKMKFGS PEAIAMDEAIHAALYYGAMRRSIELAKEKGSHPSFPGSAASKGLLQPDLWVRCGDLVSSWEERV AQTTQGVLTPKRWSQLRLAAMQGLRNGYVTALMPTATSSNSTGKNECFEPFTSNLYTRRTLSGE FIVLNKYLIDDLKEINLWTEAIQQQLLNAGGSIQHILDIPAEIRDRYKTSREMNQKILTKHAAARN PFVSQSMSLNYYFYEPELSQVLTVLVLGWKKGLTTGSYYCHFSPGAGTQKKIIRNSEKACNADC EACLLGSGATNFSLLKQAGDVEENPGPMSILEKITSSPSECAEHLTNKDSCLSKKIQKELTSFLEK KETLGCDSESCVITHPAVKAYAQQKGLDLSKELETRFKAPGPRNNTGLLTNFNIDETLQRWAIKY TKFFNCPFSMMDFERVHYKFNQVDMVKVYKGEELQYVEGKVVKRPCNTFGCVLNTDFSTGTG KHWVAIFVDMRGDCWSIEYFNSTGNSPPGPVIRWMERVKQQLLKIHHTVKTLAVTNIRHQRSQT ECGPYSLFYIRARLDNVSYAHFISARITDEDMYKFRTHLFRIAGSGATNFSLLKQAGDVEENPGP MGNKESKYLEMCSEEAWLNIPNIFKCIFIRKLFYNKWLKYQEKKLKKSLKLLSFYHPKKDFVGIR DMLQMAPGGSYFITDNMTEEFLMLVVKHPEDGSAEFTKLCLKGSCIVIDGYYYDNLHIFISETPDI YKYPLIRYDRMDIKRALILFLLFLVVLSNAFVDYIISNFNHAVTCRKPTYFGIVLQGIFLVILFSIVD YLINENIL 9. NP868R-H339R (SEQ ID NO. 110) MASLDNLVARYQRCFNDQSLKNSTIELEIRFQQINFLLFKTVYEALVAQEIPSTISHSIRCIKKVHH ENHCREKILPSENLYFKKQPLMFFKFSEPASLGCKVSLAIEQPIRKFILDSSILVRLKNRTTFRVSEL WKIELTIVKQLMGSEVSAKLAAFKTLLFDTPEQQTTKNMMTLINPDDEYLYEIEIEYTGKPESLTA ADVIKIKNTVLTLISPNHLMLTAYHQAIEFIASHILSSEILLARIKSGKWGLKRLLPQVKSMTKADY MKFYPPVGYYVTDKADGIRGIAVIQDTQIYVVADQLYSLGTTGIEPLKPTILDGEFMPEKKEFYGF DVIMYEGNLLTQQGFETRIESLSKGIKVLQAFNIKAEMKPFISLTSADPNVLLKNFESIFKKKTRPY SIDGIILVEPGNSYLNTNTFKWKPTWDNTLDFLVRKCPESLNVPEYAPKKGFSLHLLFVGISGELF KKLALNWCPGYTKLFPVTQRNQNYFPVQFQPSDFPLAFLYYHPDTSSFSNIDGKVLEMRCLKREI NHVSWEIVKIREDRQQDLKTGGYFGNDFKTAELTWLNYMDPFSFEELAKGPSGMYFAGAKTGI YRAQTALISFIKQEIIQKISHQSWVIDLGIGKGQDLGRYLDAGIRHLVGIDKDQTALAELVYRKFS HATTRQHKHATNIYVLHQDLAEPAKEISEKVHQIYGFPKEGASSIVSNLFIHYLMKNTQQVENLA VLCHKLLQPGGMVWFTTMLGEQVLELLHENRIELNEVWEARENEVVKFAIKRLFKEDILQETGQ EIGVLLPFSNGDFYNEYLVNTAFLIKIFKHHGFSLVQKQSFKDWIPEFQNFSKSLYKILTEADKTW TSLFGFICLRKNGSGATNFSLLKQAGDVEENPGPMAGRVKIKQKELIDSTVKNKNVMNLFHEIIG SKGNINFSVVWPKFKKIKQSVYDYISTLSVLEKANVMQNFEADKKLLELFVQKLWAAYEGYFK YPEIEKYEVEGQVNFNLVPQCVLEKFSQLYRIRINSELVTLILNSCAFMSKYNDYILKKDPYILTIT PGLCFSPIPNFEDLNFKHLYNSDKNSQHDKEFIMFILYKLYTAALGVYNAISIPDIDVEDLENIILSS VSQIKKQIPRCKDAFNKIESSVHLLRKNFNTYYSDYVGSGYNPTIIMEQYIKDISQDSKNISPRISY QFRTIIKYYRDMIATRHQTMDPQVLNLVKHVEKKLDMLDREKN 10. I329L-A224L-MGF505.6R-B175L (SEQ ID NO. 111) MLRVFIFFVFLGSGLTGRIKPQVTCKYFISENNTWYKYNVTILNSSIVLPAYNTIPSNAAGISCTCH DIDYLQKNNISIHYNTSILKTFQDIRIIRCGMKNISEIAGGFGKELKFLDLRYNDLQVIDYNILRKLI RSNTPTYLYYNNLMCGKRNCPLYYFLLKQEQTYLKRLPQFFLRRINFSNNNTFLYHFLSCGNKPG HEFLEYQTKYCRTKFPEINITVNQLIAKKNTERYKSCYPLVFISILCSCISFLFLFICLLRSICKKYSC TKQDKSSHNYIPLIPSYTFSLKKHRHPETAVVEDHTTSANSPIVYIPTTEEKKVSCSRRKGSGATNF SLLKQAGDVEENPGPMFPKINTIDPYISLRLFEVKPKYVGYSSIDARNQSFAIHDIKNYEKFSNAGL FYTSPTEITCYCCGMKFCNWLYEKHPLQVHAFWSRNCGFMRATLGIIGLKKMIDSYNDYYNNEV FVKHQNRVYTHKRLEDMGFSKPFMRFILANAFIPPYRKYIHKIILNERYFTFKFAAHLLSFHKVNL DNQTTYCMTCGIEPIKKDENFCNACKTLNYKHYKTLNFSVKLGSGATNFSLLKQAGDVEENPGP MFSLQDLCRKNIFFLPNDFSKHTLQWLGLYWKEHGSVHRAEKDSIMIQNELVLSINDALQLAGE EGDTDVVQLLLLWEGNLHYAIIGALKTEKYNLICEYHSQIQDWHILLPMIQDPETFEKCHDLSLG CDFICLLQHAVKYNMLSILVKYKEDLLNARIRHRIQSLFVLACENRRIEIIDWIGQNLPIPEPDAIFS IAVATRDLELFSLGYKIIFDYMQRQGIIQLTNGVRMVVLNRHISMAIDNGLLPFVLETLKHGGNIH RALSYAVTHNRRKILDYLIRQKNIAPNTIERLLYLAVKNQSSRKTLNLLLSYINYKVKNVKKLVE HVVNEKSTLVLKILLEKKENLVDAVLTRLVKHSTYFQVREFIQEFSISPEKFIKIAVREKKNVLIEA ISEDIWENPTERITYLKQIVHTIKYESGRRFLVDIIHSIYQSYSLKHEDILKLATFYVKHNAITHFKD LCKYLWLNRGTESKKLFLECLEIADEKEFPDIKSIVSEYINYLFTAGAITKEEIMQAYDALEGSGA TNFSLLKQAGDVEENPGPMETNCPNILYLSGITIEECLQSKKTATDTLNTNDDEAEVEKKLPSVFT TVSKWVTHSSFKCWTCHLYFKTVPKFVPTYMRENERGEIEMGVLGNFCSFSCAASYVDVHYTEP KRWEARELLNMLYRFFTSQWISYIKPAPSYTMRKEYGGKLSEEAFISELHTLEESISSKHIFI 11. C475L-B354L-D345L-H124R (SEQ ID NO. 112) MTSLLKTDFNVSKYRLIAQKREANAVEIEAALEVVREFIIKKKLILYGGIAIDYALHLKGSSIYPEG ERPDFDMFSPNHVEDAYELADILYEKGFKQVGTVRAIHVQTMRVRTDFVWVADLSYMPPNIFNT IPTLTYKNLKIIHPDYQRAGLHLAFCFPFDNPPREDVFSRFKKDLQRYNLIEKYYPIPVVPVKSTYE SKTFSIPFKQVAIHGFAAYALLYQTLNELRITCKVPEWKTEFPQPSYSYHKNDKNITLTVDMPKA YPALVLATYNPEEVIKEMGLHLTEICEPYMDYSPPIFKTNDIHFFSTMFKELAISIIQDNLIVVSPQY LLLYFLYGAFATPADKSLFLFYYNATLWILEKADSLLNIIQKQTSPEEFTRFANTSPFVLTTRVLSC SQERCTFSPAYRISLANDVQQSQLPLPKTHFLSNSLPDVSTLPYNYYPGKGKDRPTNFSYEKNLLF NIGGKCTPSAMGSGATNFSLLKQAGDVEENPGPMALTTHSGKLIPELQFKAHHFIDKTTVLYGPS KTGKTVYVKHIMKILQPHIEQILVVAPSEPSNRSYEGFVHPTLIHYRLWLADKQKKNDNKGAERF LEAIWQRQTMMSSIYSRVNNIDMLKTLYHKLPIDIQQKENKNIAKVECLKAEQTDQKKEEKITSL YQQLLKKIIIQNIHMYKNLCLTEDEKFTLNYINLNPRLLLILDDCAAELHPLFTKEIFKKFFYQNRH CFISMIICCQDDTDLPANLRKNAFVSIFTNASICMSNFSRQSNRYSKQDKEYVEEISHIVFKGYRKL VYIREDEHRQHFYHSTVPLPTAFSFGSKALLKLCKAVYSKEVVIDKSNPYWSKFRLNFGSGATNF SLLKQAGDVEENPGPMETFVRLFKDSPQQRSDAWHAIRRTQVGGSDLASVLGLNPYKSYYITLA EKANLFKKNLNRAACSWGTLFERVSKDLLELFCQTTVIGDNIHIDGTYLGYPGHSNSPDGFCHLT LGYTQQSWEIKTIFNNVRYEATKRIPVLVEIKSPFNRKIKNSVPSYYMPQIQSGLALSPPISMGIYV EAMFRVCGIHQLGSNNETNTDIHPPESMLPLAWGIITICSTQEHTEAPQDFGTLDAETFRQLLETL YQKDQYTIHYSMPYETACPEMPNVVGYFGWKVFIFQIIPVMKHPQFLKDKYPIIQQFLRDLHTIK ASPSPMEMYEKICCSEESALSTEDIDNFTDMLTGSGATNFSLLKQAGDVEENPGPMNLEYVQVV QKFNQVLLELTKKVCTVVGGSKPTYWYHHIRRVCSECPSMPMSMIGPYLNVYKAQILTRDKNFF MNFDPAHNEYTFIIQKLKEAARNMPEDELEQYWVKLLFLLKSYIKCKPFIN 12. C315R-C147L-MGF505.7R-MGF300.IL (SEQ ID NO. 113) MDALLKEIEKLSQPSLQKENNDVCDLCFMQMKKISNYQLLCEECGQLKDWFEPEYNEKFTVYSR LKIVGANSSYHQRDLDKANSSDYSSLQFHHILEELKSLNVKYMDAGQKPFPIQVLKETAHSYNQ VQQHRVIRSITKLQILASILRSICLKLNIACTVADAARFTQLNTKGISRGMDLLRSLFVDNKITLNV DLNPIDSFINSTYSALQIKQIHQELQEENVYNLKEIVKSFILYADEKNIGVDLNRRTVVIATMYNVL RRAYYPIEIDTVVYQCKIRKNTITRALKMYEDYYSHFKSLYEQYHLNAAKKLIGSGATNFSLLKQ AGDVEENPGPMADNDNEDLIMDDLVEEYVETEEENLVDSEEESEDKDEIVESPSICEGFVQASSQ TLVIIPDNERITSNVLTTFEATRLVAVRAQQLAINGSTMLKKKYSSPIDIAKQELFNRKIPLLVMRC IKVTPEGQKIVEIWNPREMGIPLLDGSGATNFSLLKQAGDVEENPGPMFSLQDLCRKNTFFLPSDF SKHTLHLLGLYWKGHGSIQRIKNDGVLIEHDLTLSINEALILAGEEGNNEVVKLLLLWEGNLHYA IIGALRTENYNLVCEYHSQIQDWHVLLPLIQDPETFEKCHDLSLECDLSCLLQHAVKYNMLSILV KYKEDLLNVLFRQQIQGLFILACENRKLEILTWMGQNLPIPDPEPIFSIAVVTKDLEMFSLGYKIVF EYMENQGLHLTQVVRMVMLNHHFGMVINKGLLPFVLEILNYGGNVNRALSYAVTQNKRKILD HVVRQKNIPHKTIERMLHLAVKKHAPRKTLNLLLSYINYKVKNVKKLLEHVVKYNSTLVIRLLL EKKKNLLDATLTRYVKDSTYFQVKEFMQDFSISPEKFIKIAVREKRNVLIKGISEDIWENPAERIR NLKQIVCTIKYESGRQFLINIIHTIYQSYSLKPEEILKLATFYVKHNATTHFKDLCKYLWLNRRTE SKKLFLECLEIADKKEFPDIKSIVSEYINYLFTAGAITKEEIMQAYALEYAMYGSGATNFSLLKQA GDVEENPGPMVSLTTCCLKNIVNQHAYVENTVLLYHLGLRWNCKTLYQCTQCNGVNYTNSHS DQCKNKDLFLIKVIVKKNLAVARTLLSWGASPEYARLFCRNTEEEQALNVQHVADVPSSKILER LTMSYKGNDEQLLITFYLLNLSTNFSTNLREQVRFKIVSYIICDLAIHQTFKIFYAKNYSLSTLYCIF LAIYYKLYTALRKMVKIYPGLKSFAYLTGFMFDDETVMETYNSTDDEISECKNRIITIKGYYGNIH CRSDIDHMYAFSQNNFW 13. MGF360.6L-MGF360.12L-MGF300.4L-D205R (SEQ ID NO. 114) MNSLQVLTKKVLIENKAFSNYHEDDIFILQQLGLWWENGPIGFCKQCKMVTSGSMSCSDVDSYE LDRALVRAVKKNQTDLIKLFVLWGANINYGIICAKTERTKVLCIQLGADPKFLDVGLYNMFVDLI KQQKVLLAIDIYYDNISILDSFDSHDFYVLIDFIYNRFILNLDEKEKMIKNTYVLKFWFKIAIEFNLI KPIRFLSKKFPHLDYWRLKTAVYLGNVDEIHHAYFQENIRLDPNDMMSLACMYPQNKLGIYYCF ALGANINTALETLIGFINHEVNREITFFSNYGIWSNVHFCISLGANPYTKKIQETLLRQEKNVMMK LLFKKGLLSPHSILHKKILEPSEVRKIISTYEYTETFHSFSSLRDNLRGSGATNFSLLKQAGDVEENP GPMLPSLQSLTKKVLAGQCVPTNQHYLLKCYDLWWHDAPITFDHNLRLIKSAGIKEGLNLNTAL VKAVRENNYNLIKLFAEWGADINYGLVSVNTEHTWDLCRELGAKETLNEEEILQIFIDLKFHKTS SNIILCHEVFSNNPILQKVNNIKMRIEIFWELRELIVKTDLLNNEFSLSTLLLKYWYAIAIRYNLKE AIQYFYQKYTHLNTWRLTCALCFNNVFDLHEAYEKDKIHMDIEEMMRIACIKDHNLSTMYYCY VLGANINQAMLSSIQYYNIENMFFCIDLGADVFEEGTTALGEGYELIKNILSLKIYSPATTPLPKST DPEIIDHALKNYVSKNMMIFLTYDLRGSGATNFSLLKQAGDVEENPGPMQSLFNIALKALTLKNH VEFLKRDKEVLTRLGLCCKNYDLIHKCSECGNICPNGQQHGTCININYLLIYAVKRDNYMLAYR LLCWGANEKFAHYFRRPLPNLKPLLPKKELTPKDIKQLAYEHFHSDSELITVFEVFRKSRNINDCL EFFYKKNIEFEIYFARLYVYSKTFYRKSWYWFCIFMAVKHSMEHALKKITKTYIPTFYNKTTLPL VLFLSACFYENVEWMKNFFYKANKKIQQKMLSYGMEWAATHGKVRTFVCCYTLGGTASLKM YQKAYQNERYMIMALCSYLGNIQINNPWDSLNPYMMVQNKEKFLPLKFSEETQYFYIGSGATNF SLLKQAGDVEENPGPMDTAMQLKTSIGLITCRMNTQNNQIETILVQKRYSLAFSEFIHCHYSINAN QGHLIKMFNNMTINERLLVKTLDFDRMWYHIWIETPVYELYHKKYQKFRKNWLLPDNGKKLIS LINQAKGSGTLLWEIPKGKPKEDESDLTCAIREFEEETGITREYYQILPEFKKSMSYFDGKTEYKHI YFLAMLCKSLEEPNMNLSLQYENRIAEISKISWQNMEAVRFISKRQSLNLEPIIGPAFNFIKNYLRY KH 14. MGF360.4L-MGF360.15R-A238L-H240R-B125R (SEQ ID NO. 115) MNSLQVLTKKVLIETKAFSNYHEDDIFILQQLGLWWENGPIGFCKQCKMVTGGSMSCSDVDSYE LDRALVKAVKENQTDLIKLFVLWGADINFGIMCAKTKQTKDLCIQLGANPEFLDVGLYNMFVY LVKRKKVLLAIEIYYDNILILDSFNSNDFHLLIDFVYNRFILYLDEKEEEMTRNTLVLKFWYKFAI DFNLTKPIHYLSKKFPHLDLWRLQTAIYLGNIDEVHHAYFQENIRLGLNVMMFLACARPGNKLGI YYCFALGADLDRALERLISFNSINREINRKIRGEKRLCIEGSYLSNVYFCIGLGANPYTKKIQEIIKQ KHSNIMILLFSKKKILSPHSVLQNKILDPSDVHKMISTYKNTESFYPFSSLAVKLIQQANIGSGATN FSLLKQAGDVEENPGPMNKIFLNITETINMVLIEFLTGFFYLYGKRLFSISKVMDMICLDYYTIIPA PLAMMLAARLKNYDLMKRLHEWEISIDYALLVVDDVPSIDYCLSLGARSPTRAQKRELLRDNTF NPVYKYLMNCSGFPTKREKNIPCDVQCERLQKNIIKELVFNCSVLLEMVLHTEREYAYALHCAA KHNQLPILMYCWQQSTDAESILLKTCCSDKNINCFNYCILYGGAQNLDAAMVEAAKHDARMLI NYCVMLGGRSLNEAKETAAMFGHIECAQHCFKLQSYVVDTSNTDDTDGSGATNFSLLKQAGDV EENPGPMFPEREIENLFVKWIKKHIRNGNLTLFEEFFKTDPWIVNRCDKNGSSVFMWICIYGRIDF LKFLFEQESYPGEIINPHRRDKDGNSALHYLAEKKNHLILEGVLGYFGKNGTRICLPNFNGMTPV MKAAIRGRSLNMLSLIKFGADPTQKDYHRGFTAWDWAVFTGNMELVKSLNHDYQNLSTCISLF TSWMFSTGGLRKSPKLLLLAVNIMSMKNFLNRIQSSCVGSGATNFSLLKQAGDVEENPGPMAAN IIATRAVPKMASKKEHQYCLLDSQEKRHGHYPFSFELKPYGQTGANIIGVQGSLTHVIKMTVFPF MIPFPLQKTHIDDFIGGRIYLFFKELDMQAVSDVNGMQYHFEFKVVPVSPNQVELLPVNNKYKFT YAIPVVQYLTPIFYDLSGPLDFPLDTLSVHVDILSNHIQLPIQNHNLTTGDRVFISGYKHLQTIELCK NNKIFIKNIPPLSSEKIKLYILKNRIRIPLYFKSLKTSKGSGATNFSLLKQAGDVEENPGPMAVYAK DLDNNKELNQKLINDQLKIIDTLLLAEKKNFLVYELPAPFDFSSGDPLASQRDIYYAIIKSLEERGF TVKICMKGDRALLFITWKKIQSI 15. NP1450L (SEQ ID NO. 116) MKFYYVFSKNMEAGYAEIAAVQFNIAGDNDHKRQGVMEVTISNLFEGTLPAEGGIYDARMGTTDH HYKCITCSHQRKQCMGHPGILQMHAPVLQPLFIAEIRRWLRVICLNCGAPIVDLKRYEHLIRPKRLIE AASSQTEGKQCYVCKAVHPKIVKDSEDYFTFWADQQGKIDKLYPQIIREIFSRVTYDTVVKLGRSKN SHPEKLVLKAIQIPPISIRPGIRLGIGSGPQSFFIDINNVIQYLVRKNLLIPKDLQIVRGQKIPLNIDRNLQT IQQLYYNFLLDSVSTTATQGGTGKRGIVMGARPAPSIMRRLPRKEGRIRKSLLGSQVWSISRSTICGN SDLHLDEVGYPISFARTLQVAETVQHYNINRLMPYFLNGKRQYPGCSRVYKQITQSVHDIEGLKQDF RLEVGDILYRDVVTGDVAFFNRQPSLERSSIGVHRIVVLENPKISTFQMNVSACAWYNADFDGDQM NLWVPWSVMSRVEAELLCSVRNWFISTKSSGPVNGQVQDSTVGSFLLTRTNTPMGKNVMNKLHA MGLFQTTQTDPPCFANYSPTDLLDGKSVVSMLLRQTPINYQRAPTWYSEVYAPYMHYNKQDISTQI RNGELIEGVLDKKAVGAGSSGGIYHLISRRYGPQQALKMIFATQQLALNYVRNAGFTVSTADMLLT PEAHQEVQEIINELLLESEEINNRLLHGDIMPPIGLTTHDFYEKLQLNALKFPDRILKPIMNSINPETNG LFQMVATGAKGSNPNMIHIMAGIGQIEINTQRIQPQFSFGRTLVYYPRFALEAQAYGFICNSYIAGLT SPEFIFGEMNGRFDLINKALSTSSTGYANRKAIFGLQSCIVDYYRRVSIDTRLVQQLYGEDGLDARQL ETVRFETTMLSDQELEDKFKYTGIQSPLFEEEFSRLKKDRDKYRQIFLNVENFNFSQLLTDVRQVPVN VASIVKNILLSSTSGVLPFDEKSILQKYAMVKTFCKNLPYVFINNIQERLQTPIPVYLKRAASLMRMLI RIELATVKTLNITCEQMSAILDLIRLQYTQSLINYGEAVGILAAQSVSEPLTQYMLDSIIHRSVAGGTN KSGIVRPQEIFSAKPVEAEQSSEMLLRLKNPEVETNKTYAQEIANSIELITFERLILQWHLLYETYSST KKNVMYPDFASDVEWMTDFLENHPLLQPPEDIANWCIRLELNKTIMILKSISLESIINSLRAKHPNTY IMHSVENTASGIPIIIRIYLRESAFRRSTNTRMATDEKIAVNVVDKLLNSTIRGIPGIKNANVVKLMRH RVDAQGKLVRLDNIYAIKTNGTNIFGAMLDDNIDPYTIVSSSIGDTMELYGIEAARQKIISEIRTVMG DKGPNHRHLLMYADLMTRTGQVTSLEKAGLNAREPSNVLLRMALSSPVQVLTDAAVDSAVNPIYG IAAPTLMGSVPRIGTMYSDIIMDEKYITENYKSVDSLIDML 16. G1340L (SEQ ID NO. 117) MDFQNDFLTNPLRVTLYNPVENEYTKTFIFLGSVPANVLQACRKDLQRTPKDKEILQNFYGEDWEK KLSQYVVGGDSDDLDEFEKLFVEDRGEETNVMMPEIETMYSEYSIFPEDTFKDIREKIYVATGIPPYR QHIFFFQNNALQVTYRLLLSGSGVALDIRDYKKEFQQVGGVNIDASMESQKDELYVEALDSFQLIK NIHHIFVADLNTLVAPMRRQISIAIEDNYQFDLLYYGLIMKYWPLLSPDAFKLLVQSPLQMEKQYPA LSPSLTSLKKRLLLEQKLINFTYARAQQVIAKYEGNRLTRGTLAVTSAMIKISPLVNIQINVRNVFDLF PATPDIPQLVVFFYSKTGPTVVSKHHITSTEPEKFSNKTFRVPTIILIRFINKKAFILTIQNNGHYFIESN WSENERHDFNSVVSTLNNFINPIIHTINDMGPAAFPRGGSLPLPSNEDIQISISSMSVSTFWPYTLSSKG FTELKSRWREYEQAGIISVRGLQQTGVYNFLFKKGIYSYDPHEIERMIIISSGPGRKMDINVALLQNT YAYLFDTNVAARWETIYGGRNIRIYHRVTDIKIEMFNITQEEFNYLWVYLFVFLDNLITGPDKILVNK LSQLHDKQQGKGASQLRALQEQDPDLYDLRKYDTQATVYSVLCQHPRPPVIYSEAEVKSMPPAKR KELVKYWNFTEGVPAYYSCPHPDYPHLSLLEGRHPLNYCLPCCQKTKALLGTKRFYINNTCLTKHT FVEQDLEDLNTQTSRHTLSYGKKIPVNRIAFLPHQIADELFLNTIKEPDIFCIVGVEQTMLGISNAGLF YSLARILDLAPKALAIEIAKAANTPQYYILGNGAGNMFSSGAELANLILQTFVEQKNQLLQWDTTW QDIFLDLVAICYDLHCVFFKDKQGDIGFEVSPSTIQKILSPSKKIAIIFDTDEGIYPMAITQQKRFLKNSE AQYIFTEDDPVMEVIQSMSEFMCKDNWWDIHDVKNIPGYTVGKKLINRHNFCYALLIDSDTDRPIYF PIRLSSYIHDDIPIDFDLRPTQIASFEETWKFITLFNKQYKQYEIVPSAVLQNIKKEFVGFLSEGKTGLY FYYAPTQTLPATLEKLPIATLTIDPRDIDQAILYPLEEPYPQQNKANKAFYINHLYKFLLIEFFDVLYG LQSNSTRKHIENLFQKTDFQKITSVTEFYTKLSDFVDLNDIHTIKHILETTDAEHALKVLQKNIFNFDY TLLSPLQSYTYDELCQHLKKLLTPRIEFYEDIETIDRGLINIYTSCQYSTLNQPQCKKKRLRIPVNHFE NYIHILAADILNPLKHSTLLLTGLGVIDDLQFILRPQEIISVKNKF 17. M1249L-A118R-I73R (SEQ ID NO. 118) MEEVITIAQIVHRGTDILSLNNEEIEALVDEIYSTLKGSNDIKNIRLIDFLFTLKDFVNHVRAEQSKLPD LSMPIEAYIRQLLVDPDVVPIVSEKKKELRVRPSTRKEIFLINGTHLAVPAEAPIEIYGLKLRLKTFSPQ CFMRMAEIGSFSPETLGYVASGANLTNFIRVFMKCVDQETWKKNGEGVVVTTKENIIQFTHQYIEL YKFLRSGGHSWLINRLAEEMVHRKLDREDQGSHISNIVETEEIEPEENIKRVIFFLKELSTMYSVSPVF TSGYMPLLYDLYRAGYLEVLWNPVEQKFLQHAEQREKEQMILQQVDMKLTEVITQARQYFKIMEE KIGRVQSDAIREILTMEGKVDDPNSILQEVIKACGKQEAELITTEYLNIKKQWELQEKNACAHLKLV KQLRSGLQYAELLKVLESIRVLYKEKNNTTNWNLCKACGFKLLCPHVDMLIQLQAAEASYDTMRT KLMKFSGINKEKENNQGLIYSYFCKICGEELAHFIQEDRTADVGIIGDLNSKLRVFIWQETMKACTFI HFGKLVDVKQFANIAVNVCLPLVYSIENIKKEEDYDPLTQLYAVIYIYAYILNLIYSSQKNKEFLTITI HGMKADSSLNAYVTFLLEKMMQQYSGIINQLSEITDQW1ANNFREAFKKIIHQNGLQGLSVQDDTK VLLTEILLDPMYDYAATVARIDGSIPMHKPRTPKEAEYEFKTVIGRTPAELLSQKEFYDKIYTSKYRP DFTQLTRLNDIYFQEESLRVWWGGRDEEKTSTLIYLRAYELFLKYLQNAPNFNSELAEFKTYENAY GEQKALLAQQGFYNIFDPNTGRADQRTRLFEYKRLPISTLYDERGLPHKWTIYVYKAVDSSQKPAEI EVTRKDVIKKIDNHYALADLRCSVCHVLQHEVGQLNIKKVQTALKASLEFNTFYAFYESRCPKGGL HDFQDKKCVKCGLFTYIIYDHLSQPELVHDYYNNYKDQYDKEKMSIRSIQIKKDMTTPSTETQPKPP QEPWTFDYGKIIKTAKILDISPAVIEAIGAMEGRSYADIREGQGAPPPPTSMDDPRLMAVDSAVRIFL YNYNCLRHVSTFNKPPIHVERLVKHLSYEEKEDLEKVLPNVVNEYHTTFKHLRVTDPASALLYSIEF LCISFLTLYEIKEPSWVVNIVREFALTELNTIIQSEKLLSKPGAFNFMIFGEDFVCSGEDSSMDDISAYS SPGLFGEDIIDRLDDPFSIEDVDISLDVLDNLAPQMHSNVSFNFIACVLFPTPLIPSMAMSIPRMINKRK KRIQFLTFLTNLFLYNIVQHCISGIMETQKLISMVKEALEKYQYPLTAKNIKVVIQKEHNVVLPTGSIN SILYSNSELFEKIDKTNTIYPPLWIRKN 18. EP1242L-I9R-C62L (SEQ ID NO. 119) MEPLRPQITYGPIETVDNEELTEADMLSFISAAVNSTGLIGYNIKSFDDLMDNGIPQIVKQMFNVDIT YKDQRDHTEIDKLRESVQIQFNFTDVNIERPQHRNYSQGNKINLLPNKARLCGLSYSGPVNLAAEVI LTAHYSNGRQEVKRASIPPFQVSTFPIMRGSNRCHTHHLSKTAKKEIGEDPNEPGGYFIARGGEWVV DLLENIRFNTLHIHYHTMQQGNNEIIRGEFISQPGGAFENSSQIIIRYMTTGAMEINSTKFSKLRIPWY LIFRMFGMTGDDSIIEQVVFDLESNSLVNTFMIEILEKSIHVLDPIFQPVQHELNREKIIQFLSEKVSKF VSNPSAYKSDENAVQYLNERQLTILDKILLPHMGQTADTRVRKLRFLGLLIHKILLVIMNVFPPTDR DSYRTKRVHGSGVSLAKAFKAIFNTSVIAPIINGFKELLKQTAFEELTQRNIIEAFSAALSKNTASDLN RSMEQSIISGNKTIMVRQRPIVNRVSTQSLERKNLLNTISALRTVNTHNTTNASKQTERADMMRRVH ASYPGYICVAQSADTGEKVGMSKQLAITANVCTAGEVLSLKQRLLSDPAIQQLADVSNKDIVRKGL ARVFINGEWIGCCTNAFELAQRYRMLRREGKVVHPHTTIYWDSMVDEVEFWLDVGRLTRPLLIVD NNIEKYNQACYKAAEARKKGDKDWEKHKIPFIQNTRFTPQMAKDILAGTLTLEDLVAQGICEFITPE EAENCLVAFSIIELRKHKFIDVTRRFTHVDVPQAILGLAALVSPYANCTQPARVTYETNQGRQTGGW YCFSWPYRVDMNRFFQFYNEMPLVKTIAHNYVIPNGLNTIVAYMIYGGYNQEDSVIVSQSFIDRGGF AGTFYREEKVELESDIESFGKPDPLITKNLKPGANYEKLVDGFVPVGTVVKKGDIIIGKVAKIRGEKD ELNKYIDRSVMYGFDEPAVVDAVMRPHGPNDEIFGLMRLRYERNLNIGDKMSSRSGNKGIAALALP TSDMPFTEDGLQPDLIVNPHSFIPSRMTNGQMIETTVGLANALQGVVTDGTAFLPINVQLLSERLAQE GLRFNGCQKMFNGQTGEYFDAAIFIGPTYHQRLQKFYLDDRYAVASYGPTDALTGQPLDGKRSHG GLRLGEMEHWVLTAQGAMQTIIEKSFIDDSDGCISYICRNCGEPAIYNASHPIYKCMNCDVQADIGM VDSRRSSIVFQHEMRAANVNITSVLSPRVFQPAMGTFSVTASAKNDNAVCKYLKEPMVENKNYKNI LRNEFIDKKNLNDALRQHITVHNPVVDWCNNYSTFSSQDFEEYKIYIHSDLMDGRPSPKKTWCVILM NWGSISSGTPGLFVESIRNTPSVVKINVIFLKVISNTAVSVFWRDRRIRFESDWLNSYFQK 19. G1211R-I7L-L83L (SEQ ID NO. 120) MISIMDRSEIVARENPVITQRVTNLLQTNAPLLFMPIDIHEVRYGAYTLFMYGSLENGYKAEVRIENI PVFFDVQIEFNDTNQLFLKSLLTAENIVYERLETLTQRPVMGYREKEKEFAPYIRIFFKSLYERRKAIT YLNNMGYNTAADDTTCYYRMVSRELKLPLTSWIQLQHYSYEPRGLVHRFSVTPEDLVSYQNDGPT DHSIVMAYDIETYSPVKGTVPDPNQANDVVFMICMRIFWIHSTEPLASTCITMAPCKKSSEWTTILCS SEKNLLLSFAEQFSRWAPDICTGFNDSRYDWPFIVEKSMQHGILEEIFNKMSLFWHQKLDTILKCYY VKEKRVKISAEKSIISSFLHTPGCLPIDVRNMCMQLYPKAEKTSLKAFLENCGLDSKVDLPYHLMWK YYETRDSEKIADVAYYCIIDAQRCQDLLVRHNVIPDRREVGILSYTSLYDCIYYAGGHKVCNMLIAY AIHDEYGRIACSTIARGKREHGKYPGAFVIDPVKGLEQDKPTTGLDFASLYPSLIMAYNFSPEKFVAS RDEANSLMAKGESLHYVSFHFNNRLVEGWFVRHNNVPDKMGLYPKVLIDLLNKRTALKQELKKL GEKKECIHESFIPGFKELQFRHAMVDAKQKALKIFMNTFYGEAGNNLSPFFLLPLAGGVTSSGQYNL KLVYNFVINKGYGIKYGDTDSLYITCPDSLYTEVTDAYLNSQKTIKHYEQLCHEKVLLSMKAMSTL CAEVNEYLRQDNGTSYLRMAYEEVLFPVCFTGKKKYYGIAHVNTPNFNTKELFIRGIDIIKQGQTKL TKTIGTRIMEESMKLRRPEDHRPPLIEIVKTVLKDAVVNMKQWNFEDFIQTDAWRPDKDNKAVQIF MSRMHARREQLKKHGAAASQFAEPEPGERFSYVIVEKQVQFDIQGHRTDSSRKGDKMEYVSEAKA KNLPIDILFYINNYVLGLCARFINENEEFQPPDNVSNKDEYAQRRAKSYLQKFVQSIHPKDKSVIKQG NVHRQCYKYIHQEIKKKIGIFADLYKEFFNNTTNPIESFIQSTQFMIQYFDGEQKVNHSMKKMVEQH ATASNRAGKPAGNPAGNALMRAIFTQLITEEKKIVQALYNKGDAIFIDLLTYIINNINYKIATFQTKQ MLTFEFSSTHVELLLKLNKTWLILAGIHVAKKHLQAFLDSYNNESPSRTFIQQAIEEECGSIKPSCYDF ISMGNHPIKQDMKTCDYYHGEKKLKYMRRMLYNEPLGTYAVSSLFGCDMIVLTWNCTISGRTLHR RIHTRFGQYYHNNCYYTKIDDIIGDYPDTFYRPLYRYKPMDTSLKNNDGALEADNKNYQDYRAEP DKTNDVLDVTKYNSMVDCCHKNYSTFTSEWYINERKYNDVPEGPKKAVVHRCTIL 20. P1192R-EP152R-E66L (SEQ ID NO. 121) MEAFEISDFKEHAKKKSMWAGALNKVTISGLMGVFTEDEDLMALPIHRDHCPALLKIFDEIIVNATD HERACHNKTKKVTYIKISFDKGVFSCENDGPGIPIAKHEQASLIAKRDVYVPEVASCHFLAGTNINK AKDCIKGGTNGVGLKLAMVHSQWAILTTADGAQKYVQHINQRLDIIEPPTITPSREMFTRIELMPVY QELGYAEPLSETEQADLSAWIYLRACQCAAYVGKGTTIYYNDKPCRTGSVMALAKMYTLLSAPNS TIHTATIKADAKPYSLHPLQVAAVVSPKFKKFEHVSVINGVNCVKGEHVTFLKKTINEMVVKKFQQ TIKDKNRKTTLRDSCSNIFIVIVGSIPGIEWTGQRKDELSIAENVFKTHYSIPSSFLTSMTKSIVDILLQSI SKKDNHKQVDVDKYIRARNAGGKRAQDCMLLAAEGDSALSLLRTGLTLGKSNPSGPSFDFCGMIS LGGVIMNACKKVTNITTDSGETIMVRNEQLTNNKVLQGIVQVLGLDFNCHYKTQEERAKLRYGCIV ACVDQDLDGCGKILGLLLAYFHLFWPQLIIHGFVKRLLTPLIRVYEKGKTMPVEFYYEQEFDAWAK KQTSLANHTVKYYKGLAAHDTHEVKSMFKHFDNMVYTFTLDDSAKELFHIYFGGESELRKRELCT GVVPLTETQTQSIHSVRRIPCSLHLQVDTKAYKLDAIERQIPNFLDGMIRARRKILAGGVKCFASNN RERKVFQFGGYVADHMFYHHGDMSLNTSIIKAAQYYPGSSHLYPVFIGIGSFGSRHLGGKDAGSPR YISVQLASEFIKTMFPAEDSWLLPYVFEDGQRAEPEYYVPVLPLAIMEYGANPSEGWKYTTWARQL EDILALVRAYVDKDNPKHELLHYAIKHKITILPLRPSNYNFKGHLKRFGQYYYSYGTYVISEQRNIITI TELPLRVPTVAYIESIKKSSNRMTFIEEIIDYSSSETIEILVKLKPNSLNRIVEEFKETEEQDSIENFLRLR NCLHSHLNFVKPKGGIIEFNTYYEILYAWLPYRRELYQKRLMREHAVLKLRIIMETAIVRYINESAEL NLSHYEDEKEASRILSEHGFPPLNHTLIISPEFASIEELNQKALQGCYTYILSLQARELLIAAKTRRVEK IKKMQARLDKVEQLLQESPFPGASVWLEEIDAVEKAIIKGRNTQWKFHMYSILIACLVLLLCLVIYV GHRADHARKYLEGMWHGDPVFLKQSGLQSFYLYIQPDHTCFFSIVNKNGEKLMETKIPCTITNKIY MFFKPIFEFHVVMEDIHSYFPKQFNFLLDSTEGKLILENNHVIYAVLYKDNFATALGKTVEKYITQN MHISIITRYTLKYIYMHLTHNHILFTYIMRIYLIKHNHMLFTTHVYTYIM 21. D1133L-E165R-C122R (SEQ ID NO. 122) MAYPELDAADFLQQLARRKEFKSLISPPVDQKELIRDLRAHFVQIGGPGCEKGGRAFFPCDPYASPF PSIKGLQLHNAQLFVQNFQNPNTPYSRLLLNWQTGTGKSIAAIAIARQFMNHYMNFIENAPWIFVVG FIRAIIQTEMLRRPELGFVSYKEVAELHRLLHIAKQSGSTTSVESRHLNGFVSTLKRRLTDRNRGGFF QFYGYKEFASKLFNITSKGEEKNFDVLSLFHRSDEAEDTLNENDISQFVQKISEAETNGLIRVNQKIM EQLRGGLLIADEIHNVYNIQERNNYGIALQYVLDAFPPHQAPRAVFMSATPVTGSVMEYVDLLNLL VPRHELPNGQPLQRQQLFDSSGHSVKWKKDALALVERLSTGRVSFLLDTNTNFYPERIFAGKMLSY KDETLPYLHFIECPMSEYQLETLKQLGPDPKISSNAYSIYDMVFPNPKFSKQTEPKAYGLFNSTETPT ALSMASTDWLLENGVQIIEPSRRAPFNVSGSFLSLQPPTHISGLAFYSGKYTQMMKDILSIIRQGRGKI LIYHNRVRMSGVLILQEILQSNGILNEVSSPVGTTRCSICAAIRDEHTHSDHQFIPVRFTILHSEIEPAVR ERSLALFNASSNLEGHQLRILIGSKVIVEGLNFQAVRYEMIMSLPLDIPRLIQVFGRVVRKNSHMELP PSERNVTIYLYVSTTPDGGPELAKYAQKLKEYILIQEGDKALRKHAIDGFTNQIKIDKPMLESLPLSPS ITPANVGATVLNTFEAYGYGEQEVKTISNIIISLFMARPVWTYSELWKAVSTPKLIQGMDNKLFSED NFALALISLCYSKNQCKELWIQNRLCTIMHVPAKPEHLYVAAVLNHKKEPVLDIETYIRDFQPPAMH SIRITKYLEHSQTKEPFQVLYEKFQKDFQDEPMEQVLIHYPASFHYTMLEALIIDNLAGMGALVEVY KKFFIAFSKKDIQPFPDIFKIISHVPGDDNTLVGYATEDSVRLITSREDKTWHEIPLYMLNINVKRKEN DIVIGYMESKGKALKFKIRPPIQVLKKNEITDIRMLNRGAVCETRGREEQQKIADQLGISLNLTKISAI KLCLLIRNNLLQKEMEARNQPNGMQDGIRWFYLFNDKMPSLVHTSMATNFFIQPITEEAEAYYPPS VITNKRKDLGVDVYCCSDLVLQPGLNIVRLHIKVACEHMGKKCGFKIMARSSMCTHERLLILANGI GLIDPGYVGELMLKIINLGDTPVQIWAKECLVQLVAQGDHVPDHINILKRNQIFPLFAPTPRGEGRFG STGEAGIMRTMKICKACSSCMVRTYVDGNIIFRCSCGESVQGDSQNLLVSSKVYHTGEMEDKYKIFI KNAPFDPTNCQIKKDCPNCHLDYLTQICIGSQKIIILVCRCGYMSNRG 22. F1055L-E146L-I8L (SEQ ID NO. 123) MQETFKFLRCNSQGEAVEDKYSLETLKNHFVVRDEYNNLFRVFSSRDDFWEWEAAQPFEQKCFHE VVFGFLPQRLKFDIDFPVNKSYSDDNVDDNDNVEDNVYDILDMIINVIMDVFYETYSLPYNINLTRE QILLTDSIGLNKKRELKYSFHIILYTYSVLNNNEAKVFTSKVLENLPKHVYPFVDPQVNKSIQNFRIIG SHKKGSMRVKMFNEELAEVFETSTTIKKSDTLIATPFETTCLPCIFTNVKETTPSSCDTIQQSELEEVL KFAGTLCKNHCFLRVHKNLVLFKRTSPSYCEICKRMHDKDNTLILRVTGNKVYQHCRHDNKHSLL MGSLSGTNNFVETYVDQVMTKSIEVHESILFEELPDTQKHIYDESSMREYERVPTLVVKAQMKIGKT VQLRNYLQKYYGNNSISKQQTIRFVTFRQIFSKNIQSRLPNFTLYSEVTGDLDSYERVIIQVESLFRLT STAEPVDLLILDEVESIFNQFNSGLHKYFAPSFAIFMWMLETANYVICLDANLGNRTYNILQRFRGD VPIFFHWNQYKRAQHDTYYFTSSRETWLNNLLKDLLEDKKIVIPTNSLMEARLLQSFIQKKFPEKKI GFYSSKSTAHERESHFNNVSYYWGLVDILIYTPTISAGVSYEDKRFDVLYGFFNNMSCDVETCCQM LGRVRELKSKCYKICLQGKQNYYPETIEDIEMFTLQKRDTLFQTISNHQLSFTYSKETGRPIYYKTPY YFILWLETMRIQHLSKNHFITRFINQIADTGAKVFILTGEKLETVKQYTSIKMEIKHQDYVNIASAETI DANKALQIKQNLKEGITVDQQDLFAYEKYKLLEFYAWHGHKITPKFVEQYNSFMTKQNYTGRVQI SRGKTVYESLTMLQTQELNFHQWAMQHAEHHDLQFNYSFQSHMYAIMLLTKCGFKCVQDPNILTN EQLMTKLVDEFVQYDLSAISFEFKLKKPNKTDPQTILKFINKVLGLRYGLKIHHNKGNYYIKNTKAG SLIPFVRQQIKQSPCVVSNLLPITETSSAKEETSPIKEETFTETMGGTTDFVLSITIVLVILIIIAFIWYNFT GWSPFKYSKGNTVTFKTPDESSIAYMRFRNCVFTFTDPKGSLHSIDVTEVLNNMAKGFRDAQNPPSS FTLGGHCQAPLNAFSFVLPGVNDRATVATADEAKKWENCDATLTGLQRIIMGNRLIKKDLKKCEY YYGEQQNLKQIWRLLFNEPLGTYVVSSFLKKNYVVISFSCPTNTRIMHLRINICYDLYCINGEYYEKI DDFIRLYPHIFYRPLYRYKS 23. B962L-H233R-H171R (SEQ ID NO. 124) MGKPILLEPGHLYNVPAEHKNDVPIHYIITWIKQRLPEFGGAIPTSLADRVLIIKSRTGSGKSTALPVH VFRILRNENTHSFQKYLGRSVICTQPRVLTAVTLAKDIGASTHYPDMILGQTVGYQTKPLTEKPNRG LIYATAGVLLAQLHTMTDDEIASRYAFMIIDEAHERALGIDLMLMYIKSMLQRMLQRGSIGALRIPF VILTSATIDTHKYSTYFGIGKENIILVEGRQYGVETHWPLYNTNNYIKTACETALTIHKENIHDRPTEA DILIFMPGMAEIRFLSMLLNNANMDLAKEKLPLMLILPIDSEAIAQENEAYLGLKAEIKNLWVKNPL TAKVEKPLRRVIVSTVVAETGLTIETLKYVIDPGWNRSIETYYPEWAGGLITRPAAQSRIEQRKGRVG RVFPGHFYPLYTKHVFEQIPVQQYPEIITEGPGAIFLSIVVETIKKNKEGVFKAEEIDMLDPPPTDALAS AIERAIVAGLLTRGEKGLQLTQLGDIASRFSFLSIEEARMCFSGYFWQAAISDIATILAVVSVADKKLT NLLDSKQRNGAMLAEAVLAGIPPFLQNIDNAYTNIHLLLADDLLEGLFIFEGFQHAIVYFINNKVNN VAKHLREWCEKKMLKYSSMVQILARREDILNELAIVGLNPFHQWQNRLASANAETFLKRVCTLKQ CMYEAYRLNCFCYDEHRLLYTGRNGIHFSYHDAVIKNPSCIVTPRIMLSPVSKQYMEWRLEPSFVSV LDGFVNVDINFLLPRQEIPNILGGVENEEEEPPLPIQVFLHKYVKTHFHFSGKSFKELKMKPSQMIKFP ETTLINMIPDIPKNVVQTYLEINVCHRYSFKRLIYCETFYTDMDDVQHENSVELIGLPMAAHHLTIND FNKLYFILLKPDGFLIMYDLHQGQEAFWLHSLQDALGHHTIRRDMDFHTIPEWETIFKECGFTPIFSK QPSEHELFIVFKKMILIASPFSLAHLEYLHTWHVTIKNIAQQHGLDIKVAIVVSTSHLNNFLPISGALNI ECITFPSCGIKEIDLLWARIKLFQHYCAIGARLLWLVSADIRPPVSAWPAIADSLKKGADAVVIPYPSR WNNLIPTVIKEIVVHQKKCLVAVDARHLDTDTQIVGAGMGCIVLTLKALMVRLSIGKQPVKILWPD LHGTAEGIPLEGVEVGWFLNAYAFIKLNIRCLGADHIAQHLTMVVYDLLVSLSKESIDVLRFVEANL AAFNQQYIFFNIQRKNSITTPLLITPQQEKISQIVEFLMDEYNKNNRRPSGPPREQPMFIPLLPYQQSSD EQPMMPYQQPPGNDDQPYEQIYHKKHASQQVNTELNDYYQHILALGDEDKGMDSMLKLPEKAKR DSDDEDDMFSIKN 24. C962R-MGF505-3R-C147L (SEQ ID NO. 125) MREESWEDHDTIQLTAQRKYLAEVQALETLLTRELSVFLTEPGSKKTNIINRITGKTYALPSTELLRL YEHLEQCRKQGALMYFLERQGTYSGLMLDYDLKLNTNAVPPLEPPALSRLCHRIFVHIKNSSVLPE GSHKIHFFFTLKPEVVQGKYGFHVLIPGLKLAASTKKSIIGSLQHDATVQKILHEQGVTNPESCLDPH SASVPSLLYGSSKLNHKPYQLKTGFELVFDSSDPDYIPIHQIKNLESYNLVSELSLTNEQGSLVRPVYC AADIAAEKEEEIPTEDHSLSILMLHDPEARYLHKILNLLPPEYYVEYPLWSNVVFALANTSANYRPLA EWFSQKCPEKWNTGGKEKLEKLWNDASHHTEKKITKRSIMYWAHKHAPQQYKEIVEQGYFSILAE YVYSYNGMLEHYMIAKVIYAMMGNKFVVDVDSNGKYVWFEFVLPGQPMNQGEIWKWRKEVNP DELHIYISENFSRVMDRITEHIKYHLSQPHESNILNYYKKLLKAFERSKSKIFNDSFKKGVIRQAEFLF RQRSFIQTLDTNPHLLGVGNGVLSIETIPAKLINHFHEHPIHQYTHICYVPFNPENPWTKLLLNALQDII PELDARLWIMFYLSTAIFRGLKEALMLLWLGGGCNGKTFLMRLVAMVLGDHYASKLNISLLTSCRE TAEKPNSAFMRLKGRGYGYFEETNKSEVLNTSRLKEMVNPGDVTARELNQKQESFQMTATMVAA SNYNFIIDTTDHGTWRRLRHYRSKVKFCHNPDPSNPYEKKEDPRFIHEYIMDPDCQNAFFSILVYFW EKLQKEYNGQIKKVFCPTIESETEAYRKSQDTLHRFITERVVESPSAETVYNLSEVVTAYAEWYNTN INVKRHIALELSQELENSVLEKYLQWSPNKTRILKGCRILHKFETLQPGESYIGVSTAGTLLNTPICEP KNKWWEWSPNPSAPPEKEASAPTPMSSSLQELCRKKLPDCILPEFFDDYVLQLLGLHWQDHGSLQR IEKNQILVQQEPIHINEALKVAASEGNYEIVELLLSWEADPRYAVVGALESKYYDLVYKYYDQVKD CHDILPLIQNPETFERCHELNSTCSLKCLFKHAVINDMLPILQKYTDYLDRWEYCSQMLFELACSKK KYEMVVWIEGVLGVGKVTSLFTIAISNRDLQLYSLGYSIILENLYSCGQDPKFLLNHFLRDVSIKGLL PFVIKTIEYGGSKEIAITLAKKYQHKHILKYFETWESMADNDNEDLIMDDLVEEYVETEEENLVDSE EESEDKDEIVESPSICEGFVQASSQTLVIIPDNERITSNVLTTFEATRLVAVRAQQLAINGSTMLKKKY SSPIDIAKQELFNRKIPLLVMRCIKVTPEGQKIVEIWNPREMGIPLLD 25. A859L-B318L-B169L (SEQ ID NO. 126) MCAGFYVAVHPWLEAQSLHKVGHTGNLAARLHDGSYTTCFTDEWKYCFTLETSTKKDAQKIEAG VLYCAQFFRVKNKELVCLLPEKIKQLAEDVANCLDISYTLCDSPAYEMNDSTIVVEPSLPSDPLISKE KLRHLVITPVEDEHFADDVLFFSTDETRTAIEDRLYQKEAANMGYQELRRSGRAILQMACRCGKTR VAYLILSNYLQGKVLYLVPGLSLLRQTLEKLYQYGISLKNVLLVGSDQTRIVLNHDNIEMTTNPVFI AKRIREAPSLLVIATYQSSTLLVDDFDLIISDECHRICGEWETRPFTHVLLNFKKGHRLFLTATPRYDT PLSMKNRELFGGVAFRYYLREGIEAGYVNDFELQMVAAPKLAHQPSTKEETTKQIIVKQIIMALAYL KTNIPAPKMLVFTRDIKQAKELYAALVDQGVYALIAHSTLPRQVILKTFTEFCSSKEPVILLNCRLFQ EGVEVPELNAVFFAAPRHSPRDIIQSICRPLNKQVQKPHATIFLPLEVNTENVCLDRFSSIIPFADALAS EDPRFYEHLLNPSEVAYPINWIGAHGSVSELLQLARHAIRYGTQGKIDRLTRSERLPWKAAFAELKR TVEICCRYPKINDGFHFGGATLRFDTWYKWVIKSYLQYKNKEPSSLEPYQVSDLESLQDWTTRGVG GPYPWEESMAFLETWLAQNKGELVAIDIHQGGWIGLDATPMERLSGVLTTVSQRDGRSYGKNKKL RPKKGFMIPPQQAQDLDRIFGKHNLKWRKDRVNGFLKEDEHGNYTGEPTCIQEAYRTFKEYVKTNP EYIEKYWPGYAKGKHKHQELPHIWEKGLAPPRYKAFKDGNKQLIQRSPKKKDIKNMLHLIYISIIVV LIIILISYTRKPKYFRITAPRSVALFHGIHPLNPKNYKTFSEEFETILNNAIEDGDFKGQLTEPCSYALRG GKYIRPIILMEIVRACQLQHSFGAPIYPAEAALAAEYFHVASLIIDDMPSFDNDVKRRNKDTVWARF GVAKAQMSALALTMQGFQNICRQIDWIKEHCPRFPDPNQLGALLCTFVSHSLNSAGSGQLVDTPEK TIPFFKIAFIMGWVLGTGSVEDIGMIERAAHCFGHAFQLADDIKDHDTDTGWNYAKIHGKQKTFDD VAQSLQECKKILHGKKIFTSIWNEIFQKVINVALGTMNVDFIAGINNLGEKIYTCEPFKTSFQNPFIVA LIITAVVLVVFFAICNPPVDKKRKTKTAIYVYICIVALLFLHYYVLNHQLNDIYNKSNMDVIVSSIHD KYKGGDEIIPPISPPSVSNELEEDQPKKIPAGPKPADSKPVSLPDSKPLVPLQEVIMPSQYNN 26. C717R-H359L-F317L (SEQ ID NO. 127) MTKLAQWMFEQYVKDLNLKNRGSPSFRKWLTLQPSLLRYSGVMRANAFDILKYGYPMQQSGYTV ATLEIHFKNIRSSFANIYWNRDSEEPEYVCCCATYQSFIDGEYRYRFVWYQPFIEAYNAIEAALDPLE TTILNLIAARDLDFVVHIFPYNKGHEDYLASTQLILKIFIATLLMDILRIKDNTLDVHLNSDYIIVMERL WPHIKDAIEHFFEAHKDLLGYLIAFRNGGNFAGSLRPSCGQKIVPLTIREVLQMNDINLAVWREVFI MQECSDLVINGIAPCFPIFNTWTYLQGINQIFFENTSLQEKFKKDFIARELSKEIIKGQKTLNDKEFKK LSLHQIQYMESFLLMSDVAIMITTEYVGYTLQSLPGIISRSSYLSPIVKNILMDEDSFMSLLFDLCYGA YVLHKKENVIHADLHLNNMTYYHFNPTSFTDRNKPGKYTLKVKNPVIAFITGPKVETETYVFKHID GFGCIIDFSRAIMGPNHAIKLERQYGLAFVNTFYRNQSEHILKVLRYYFPEMLTNRENEIQGVILSNF NFFFNSITAIDFYAIARNLRSMLSLDYLHTSEVKRNVEISQTFLDTCQFLEEKAVEFLFKNLHTVLSGK PVEKTAGDVLLPIVFKKFLYPNIPKNILRSFTVIDVYNYNMKRYSGKAIQTFPPWAQTKEILTHAEGR TFEDIFPRGELVFKKAYAENNHLDKILQRIREQLANENLMEKIFQNVEIKPFLIDFSNLFIKNAAKKLF QLEEQLPLVPVNVVMDFKGISRAAVHGLSRVLQDEIPNYMLDIKPGGYKIEDSTDLFMTEQFIRNRI NFIPIYAKNETLVFALRSLNNSCEVKTIYSRDLIQVAGPKLKYPIFNPTFEIGFLQPGKSLIIEDIYIKKGI GRKHAAFNLAVKTHFSHLDIEQYPTDKKEYMALSGYKQSSMTSDPRHHRLGLCFPAVPLPHINQAV RTYLKNACRIIIGRIQSIQKIYENFEEPQPELVLFSMDEEKTKAIMKDETHTIGNLLKTYIYEMIPDISF VGYQCVPHKQEMVLTIIHKASQEDLITLLEKSIQNIIQTFQILEKNVDELIAMVETQMDKLGFLLNHI GKQVTTKVLSNAHITQTMKEIILENHSVDGGAAKNVSKGKSSPKEKKHWTEFESWEQLSKSKRSFK EYWAERNEIVNTLLLNWDNVRGAIKKFLDDDREWCGRINMINGVPEIVEIIPSPYRAGENIYFGSEA MMPADIYSRVANKPAMFVFHTHPNLGSCCGGMPSICDISTTLRYLLMGWTAGHLIISSNQVGMLTV DKRIIVDLWANENPRWLMAQKILDIFMMLTSRRSLVNPWTLRDLKKILQDYGIEYIIFPSNDFFIYED ERLLMFSKKWTNFFTLHELLDDLETTETKASSTT 27. MGF505-10R-B475L-MGF360-14L (SEQ ID NO. 128) MFSLQELCRKNIYILPYPLAKHVLQQLGLYWKGHGSLQRIGDDHVLLQQDLIFSINEALRMAGEEG NNEVVKLLLLWEGNLHYAIIGALEGDRYDLIHKYYDQIGDCHKILPLIQDPQIFEKCHELSNSCNIRC LLEHAVKFIDMLSILQKHKEQIRLHMALTQILFELACHERKNDIIRWIGYSLHIHHLETIFDVAFAHKN LSLYVLGYELLMHKVNTEAAYIELPNLLSYHLRTAAAGGLLNFMLETIKHGGYLDKTVLSAAIRYK HRKIVAHFIHQVPRKTVKKLLLYAVQARAPKKTLNLLLSSLNYSVHTITKQLVHNVVIYSSTLIVKLL LMRRKNKLNLVDAVLARLVKYSTYTDIVQFMGEFSVSPERVIKMAARESRTFLIEMISKAAWGNHP QTLIHHLKQLTNTMKPQSGKDHIIYTIHYIYLNSNMLVAEEEKNIFKLAKFYANHNAVNRFKQICED YYILDARFKTLILECFEIAVQKNYPRIANIVDDYIRFLFYRGNITEEEIREAYSLKDAEVYVDLKWLQ QGEMVMDQEESHVISIFETLGAYFINIFYNFLYKNALYKKHSIVTEYQYQVKGYILGVKQNKKLYE KMLDSFYKYFCNITQINSKTLNFSNFVTTIVDSFIPKEYSQSISLEKKESILELLLCDYISNLGTFITTEK MLPFIIKNRKENYHKVTKEMQDYSLTFLLKKRMELYNKFLRKQAYVEPETELEETYARLSSYNRSL LHQIEELTSENKSLLADLSTLRKKYEKRQSEYRRLVQLLYQQIQRSSTSKSSYPLTKFIETLPSEHFSN EEYQKETPADQKEVVEMELLRKQELLTSQELTSKSPNNYPVPHSRTIVSKPLDNYPVPRSRTTTKIDF DNSLQNQELHTKNGFSEKDIVEFGQDKPEEENILAIDQDKPEEETTLAIKQDISEEDNIFAIDQDKPEFN QDTPEFKEAVLDIKENILEEENQDEPIVQNPFLENFWKPEQKTFNQSGLFEESSNFSNDWSGGDVTLN FSMLSLQTLAKKVVACNYLSSDYDYTLQRFGLWWDLGPIHLCNNCKQVFSYKHLQCFSEDDLCLE AALVKAVKSDNLELIRLFVDWGANPEYGLIRVPAVYLKRLCAELGGLTPVSEPRLLEILKEVARLKS CAGVLLGYDMFCHNPLLETVTRTTLDTVTYTCSNIPLTGDTAHHLLTKFWFALALRHNFTKAIHYF YKRHKNHLYWRVACSLYFNNIFDIHELCREKEICISPNLMMKFACLREKNYAAIYYCHRLGASLDY GMNLSIYNNNTLNMFFCIDLGAADFDRAQLIAFIKAYMYNLSNIFLVKQLFSRDVTLVLDVTEPQEIY DMLKTYTSKNLKRAEEYLTAHPEIIVID 28. MGF505-2R-B407L-MGF360-13L-E111R (SEQ ID NO. 129) MFSLQDLCRKHLFILPDVFGEHVLQRLGLYWRCHGSLQRIGDDHILIRRDLILSTNEALRMAGEEGN NEVVKLLLLWKGNLHYAVIGALQGDQYDLIHKYENQIGDFHFILPLIQDANTFEKCHALERFCGVS CLLKHATKYNMLPILQKYQEELSMRAYLHETLFELACLWQRYDVLKWIEQTIHVYDLKIMFNIAIS KRDLTMYSLGYIFLFDRGNTEATLLTQHLKKTAAKGLLHFVLETLKYGGNIDTVLTQAVKYNIIRK LLDYFLRQLPRKHIEKLLLLAVQEKASKKTLNLLLSHLNYSVKRIKKLPRYVIEYESTLVIKILLKKR VNLIDAMLEKMVRYFSATKVRTIMDELSISPERVIKMAIQKMRTDIVIHTSYVWEDDLERLTRLKN MVYTIKYEHGKKMLIKVMHGIYKNLLYGEREKVMFYLAKLYVAQNAATQFRDICKDCYKLDVAR FKPRFKQLILDCLEIITKKSCYSILEILEKHIISLFTMKVMTEEEKNLCLEILYKVIHYKTIQCMEDTTFL EGANLAGITTLMNNLHINEQANLEELEKQVMGKQQSFPTDHFDEELNGLAKSLGINFNDPEFSLDSP HSIISKKPSGRGGDKVHGGIRRDSVCTDSICSDSVCSGSIRSGSIRSGSIRNGSIRSGSVRDGSIRNGSVR SGKTRRGLACNSSSRNDRGYSLSTHRKKYAESEASQKTAISKRDRKNHYAESEYSEKSIKPSTKQVD RLINHLRSNGDPNSFYKKDFIDYERKTKLVKLEKINMLLTYLGNEQISTDDIKIPTIDSSMQEIDDVIE MLTLRNVGIRYSSIAEEILIGLARGLEIVFDGTREIPFLNYRPDYTGLHNTFMIKLFKMRYETSQVVGN LVQNMSPLSKICLELGPSLLLYPALIRTKHKASEDLYNLLQKGPEDPFTAYNEIHETLKKNNKMSLPL SLQTLVKKTIASQCLSIDEHCILKYCGLWWHDAPLKLCMDRGRIQIKSGFLGEDIDLRVALIIAVKEN NYSLIKLFTEWGANINYGLLSINTKHIRELCRQLGAKETLEDNDIFRIFTRIMHNKTSGSIILCHEIFMN NPILENKFVIQLRGLIYKRLWGLIEIKETDELNGLLVKYWYAKAVQYDCKDAICFLDEKYTDLNEW RLKCLLYYNKIYELHEMYHKENIQIDVHDMICLASTKDNNPLTIYYCYALGGNINQAMLTSVQYYN IGNIFFCIDLGGNAFEEGRAIAEQKGYNFLSHSLALDIYSSDASLPLNLKDPEETSSLLKDYKSKNLSII WEYSHNILMSFSECPLVISACKKFLQKRITIENEALINALITALAQTSTLNDLCLLPIQTYLLSYKNAFE WIHFVCIAITTILDNKYNWKDCTVDINYIFLHVTYIYNIKTKEYLDYCS 29. K421R-EP424R-D339L-S183L (SEQ ID NO. 130) MYTHVDVVGIAEASAALYVQKDRDRYLDVLTTIENFIYQHKCIITGESAHLLFLKKNIYLYEFYSNN VAEHSKALATLLYKLDPEYLTRYTVLITKIPNHWYVINVDQREFVRLYAIPAVKQHLPIPILPFYCTS ALTQQELFCLGPELQLIQIYSKLCNPNFVEEWPTLLDYEKSMRMLFLEQFPQRLEMTGGKKEEKEK HESIIKKIILEMVSTRQRIVVGGYIQKNLYNHVLKNRNRLQLITSLNIYEEKDIIQQFCDSNGLKIKIRIN NPLLPTNPELRRLTIYFNHNNDDDQSYLIVDMYNTGSYELVPTNQINTLDGSFLIGTPFVQARFLLVEI WVLMLIAQQTKKDTKKIIQFFINQYEMLMNSPWPSMEALFPSSSKRYLGNYVDPNALIKWAQLKLK RIPPFYPGKPDEESCMSNYYYYYGGGRYDWLKTVEPTNFLKIGLPYQAFIPLHLQHQATTPPSILEKF KRADILLNEVKAEMDPLMLQPETEKKLFQILSSIDMFKGLRKKVEFTYNAQIVTNAWLKMYELLNT MNFNNTSQAFCNCELPGGFISAINHFNYTMMHYPTFNWVASSLYPSSETDALEDHYGLYQCNPDN WLMQSPLLKKNIDYNNGDVTIASNVKNLALRATQRLTPIHLYTADGGINVGFIDYNKQEELNLKLH FGQALTGLLSLSKGGNMILKHYTLNHAFTLSLICVFSHFFEELYITKPTSSRPTNSETYIVGKNRLRLF TPKEEQVLLKRLEFFNDTPLVDLSLYQNLLESVYFAVETIHLKQQIEFLNFGMKCYRHFYNKIKLLN DYLAPKKKIFQDRWRVLNKLYVLEKKHKLKLCASMIDQKIFETTLNIDDPTNFCTNVEAHLLKELE NIYVGKCFKNSFILNITGVIQRSPCFIMRTNNSGRGYMHVRFSAVVSYLNAFDLIAAVKIIKNDSNIIL GESLLTEPVTIVIPSSESQNNVAEVGQIVPVQLANSSVYYIPGRQQASATGSIFIPKHTFSVYHVQEEL TQEQALNLTKLVNIIEMLLESRSKKDFKQICFFEKLYYTYSISSDEILDLKIWKGPKGKEMSRLKPCN VLSFLYDALKNKNSSLGFWARPPNLLKSSPLAYQQDQNSFNATELPIICSAEVMFVTLLKEIINYLQFI NDLCDTFNNEQLIKRHENIWMLIEQRKIGHDFMSVVVGGVEYSLNNWARYEIKRRAAELESVNYYP HCEYIMPEDIVVSILGSKPNCPFLEALKRFHDFLKKRRIIFKGEYLVIPWMGAQDVADMIHHVENRIN LDHLEDLAHMLKLITYHRSFDTCINQAFEHLYAFKFPDANIETHELKHIRQLEKKMYGYILRLEKLQ TVLTFYIEFLLKQV 30. EP296R-B263R-C257L-I243L-A179L-B117L (SEQ ID NO. 131) MFGAFVSHRLWSDSGCTTTCITNSIANYVAFGEQIGFPFKSAQVFIAGPRKAVINIQEDDKVELLKMI VKHNLWVVAHGTYLDVPWSRKSAFVTHFIQQELLICKEVGIKGLVLHLGAVEPELIMEGLKKIKPV EGVVIYLETPHNKHHTYKYSTIEQIKELFLRIRNTRLKQIGLCIDTAHIWSSGVNISSYNDAGQWLRSL ENIHSVIPPSHIMFHLNDAATECGSGIDRHASLFEGMIWKSYSHKIKQSGLYCFVEYVTRHQCPAILE RNLGSSMQLQTALTAEFTTLKSLLKMEDETELCFRSNKVTRLEMFVCTYGGKITSLACSHMELIKM LQIAEPVKALNCNFGHQCLPGYESLIKTPKKTKNMLRRPRKTEGDGTCFNSAIEASILFKDKMYKLK CFPSTGEIQVPGVIFPDFEDGKNIIQQWVDFLQHQPIEKKIQIIEFKTIMINFKFQINPVSPRVIIHLKKFA ALLEHIPTPYPIREIKPPLEDSKVSAKFMVSPGKKVRINVFLKGKINILGCNTKESAETIYTFLKDLISV HWQEILCVLPVPDMYSVCDVVRDAVAQSHLCACPNDKLPQCKGVTKAPPKCSVFHVAKLQDTKF KWKYTLDPLKAQKLSQIDKDIEKDAITLKLIYGIELSPEDLEWWKMQRCLINKKTGAKGGQFANKY LERQDLELLGYSPTPIIGGDFMFTALPDKVLRTIPVAWDRFLNPAMMIFFLIILLCVILGIFYVLVRNTL RRKQKSKQHQMEIKRFIKEKEQDPYIHTSFESWPADPNKEWKDLIPVYEAQGYCMADYRKKLGMP PGPNCMKMHIARDSIVFLLNKYLQNTILTNKIEQECFLQADTPKKYLQYIKPFLINCMTKNITTDLVM KDSKRLEPYIILEMRDIIQMMFFRTLQKHMFFKEHTDLCTEYAQKIEASCYHYTYQQQEKTFLEEYS TRCGTINHIINCEKKSHQQQDNDALNKLISGELKPEAIGSMTFAELCPSAALKEKTEITLRSQQKVAE KTSQLYKCPNCKQRMCTYREVQIRALDEPSTIFCTCKKCGHEFIGMEGEELIYHNIINEILVGYIKYYI NDISEHELSPYQQQIKKILTYYDECLNKQVTITFSLTSVQEIKTQFTGVVTELFKDLINWGRICGFIVFS AKMAKYCKDANNHLESTVITTAYNFMKHNLLPWMISHGGQEEFLAFSLHSDMYSVIFNIKYFLSKF CNHMFFRSCVQLLRNCNLIMGYTIQLDKDGDYCWDEDPTHHDPYMQANATSHVATSYATTSHAA TPHAAAHHTFHEPFIKLNLTDKNIFNGLGFILIVIFIYLLLITLQQMLTRHIYNTVQHCVKAHLDSKNL Q

[0145] Such multicistronic cassettes can be administered to animals as described above.