Staphylococcus aureus viable vaccine

Abstract

The invention relates to the field of biological medicine, in particular to the field of animal vaccines. More specifically, the invention relates to a staphylococcus aureus viable vaccine and application thereof.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly to the field of animal vaccines. More particularly, the present invention relates to a live Staphylococcus aureus vaccine and its application. Background of the Invention

[0003] Staphylococcus aureus is a zoonotic pathogen, a common pathogen in livestock farming, causing diseases such as mastitis, skin infections, arthritis, and other conditions. For example, S. aureus-induced mastitis in dairy cows is detrimental to the global dairy industry. Intramammary infections in lactating sheep and goats can lead to economic losses in cheese production. Infection of broiler chickens with S. aureus can lead to joint infections (such as bacterial chondronecrosis with osteomyelitis). In rabbits, S. aureus causes skin lesions and invades the subcutaneous tissue, causing pododermatitis, abscesses, and mastitis.

[0004] Morel's disease is one of the factors hindering the development of the sheep or goat industry. It is caused by Staphylococcus aureus subspecies anaerobius. The clinical manifestations are mainly abscesses near the superficial lymph nodes, mainly located in the superficial neck, subiliac region, parotid gland and mandible. The incubation period is estimated to be 3 weeks. The abscesses often last for 1 to 5 months before rupturing and completely healing. Sheep pustule disease caused by S. aureus subspecies anaerobius was first diagnosed in goats in October 2001. Later, chickens were also found to be infected with S. aureus subspecies anaerobius. O et al.Polish journal of veterinarysciences vol.13,3(2010):437-45.).

[0005] Staphylococcus aureus infection is one of the major factors hindering the development of animal husbandry. In recent decades, antibiotics have been used to improve feed conversion efficiency, treat diseases, and prevent infections in livestock production. In industrial agriculture, antibiotics are often used in animal feed to promote growth, which has led to the development of antimicrobial resistance (AMR). The development of antibiotic-resistant bacteria further exposes farm workers to new resistant strains of bacteria and increases the risk of infection and disease for these workers. Studies have shown that Staphylococcus aureus can be transmitted between different animal species and humans through the food chain, such as pigs, poultry, sheep, goats, and horses. Therefore, there is an urgent need to develop drugs or methods to prevent or treat Staphylococcus aureus infections in livestock.

[0006] Antimicrobial peptides (AMPs) are promising alternatives to antibiotics for the treatment of Staphylococcus aureus infections. For example, Shah P et al. discovered a natural AMP, Polybia MP 1, which exhibits antimicrobial activity against multidrug-resistant Staphylococcus aureus isolated from breast milk of patients with mastitis (Shah P et al. Int J Pept Res Ther. 2022; 28:44). However, the in vivo effects of most AMPs are insufficiently studied.

[0007] Abd El-Hamid et al. studied the inhibitory effects of plant extracts such as carvacrol, linalool, and eugenol on multidrug-resistant and highly virulent MRSA strains (Abd El-Hamid MI et al. Front Cell Infect Microbiol. 2023; 13.). However, plant extracts alone have limited effects and need to be used in combination with antibiotics.

[0008] Methods for treating Staphylococcus aureus infections also include nanoparticles, phages, and antibodies. However, most of these strategies have only been studied in vitro and lack in vivo animal studies to clarify their effects and mechanisms. In addition, the toxicity of these strategies to animals needs to be considered in practice.

[0009] In summary, there is currently no method in the livestock industry that can both circumvent antibiotic resistance and treat Staphylococcus aureus infection. Therefore, preventing Staphylococcus aureus infection is extremely important. Developing an anti-Staphylococcus aureus vaccine for livestock will provide a better solution to the problems of Staphylococcus aureus infection and resistance in the livestock industry. Summary of the Invention

[0011] The present invention provides a live bacterial vaccine for preventing and / or treating Staphylococcus aureus infection in non-human animals, comprising an effective amount of a live Staphylococcus aureus strain, wherein, compared with a corresponding control strain,

[0012] a) the live bacterial strain has reduced Sae two-component system activity, preferably, lacks Sae two-component system activity;

[0013] b) the viable bacterial strain has reduced adenosine synthase A (adsA) activity, preferably, lacks adenosine synthase A (adsA) activity; and / or

[0014] c) the viable bacterial strain has reduced capsule production, preferably, lacks capsule production; and

[0015] The live bacterial strain expresses antigens EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA and mLukB.

[0016] The present invention provides a method for preventing and / or treating Staphylococcus aureus infection in a non-human animal, comprising administering the live bacterial vaccine of the present invention to the non-human animal. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1. Comparative sequencing maps of target gene adsA, CPs, and saePQRS deletions.

[0019] Figure 2 , before immunization, test the IgG levels of experimental animals against clinical isolates.

[0020] Figure 3 , sample collection and disease observation plan in animal experiments.

[0021] Figure 4 , after immunization, detect the IgG level of experimental animals against the candidate vaccine strain. Detailed Description of the Invention

[0023] Before describing various aspects of the present invention, it must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The term "and / or" is intended to cover any combination of items connected by the term, equivalent to listing all combinations individually. For example, "A, B, and / or C" covers "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C." Unless otherwise defined, 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.

[0024] All patent documents and non-patent documents mentioned herein are incorporated by reference in their entirety.

[0025] "Sequence identity" has a recognized meaning in the art, and published techniques can be used to calculate the percentage of sequence identity between two nucleotide sequences or amino acid sequences. Sequence identity can be measured along the entire length of a specified nucleotide sequence or amino acid sequence or along a region of the sequence. In some embodiments, sequence identity is measured along the entire length of a specified nucleotide sequence or amino acid sequence. Many methods for measuring sequence identity are known in the art and can be applied in the present invention.

[0026] In one aspect, the present invention provides a live Staphylococcus aureus vaccine comprising a live Staphylococcus aureus strain, wherein, for example, compared to a corresponding control strain

[0027] i) the live bacterial strain has reduced SAE two-component system activity, preferably, lacks SAE two-component system activity;

[0028] ii) the expression of one or more genes of the sae two-component system in the viable bacterial strain is reduced; and / or

[0029] iii) wherein the viable bacterial strain comprises one or more mutations in one or more genes of the sae two-component system.

[0030] The two-component Sae system has been shown to be essential for evading killing by human neutrophils. This system consists of the histidine kinase sensor SaeS, the response regulator SaeR, and two accessory proteins, SaeP and SaeQ. The sensor protein SaeS detects environmental cues, such as changes in pH or oxygen levels, and then phosphorylates the response regulator SaeR. Phosphorylated SaeR then binds to the promoter regions of target genes, activating or repressing their transcription. The action of the SaeR response regulator and its cognate sensor kinase, SaeS, has been shown to be essential for initiating infection.

[0031] "Sae two-component system activity" includes detection of environmental signals by SaeS, phosphorylation of SaeR, and / or activation or repression of target gene transcription.

[0032] In some embodiments, the expression of one or more genes of the Sae two-component system in the living bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain.

[0033] In some embodiments, the expression of one or more genes selected from SaeS, SaeR, SaeP and SaeQ in the viable bacterial strain is reduced. Preferably, the expression of all SaeS, SaeR, SaeP and SaeQ genes in the viable bacterial strain is reduced. More preferably, none of SaeS, SaeR, SaeP and SaeQ are expressed in the viable bacterial strain.

[0034] The term "gene" herein may refer to a protein coding sequence, but may also encompass expression regulatory elements / sequences, such as promoters, enhancers, and the like.

[0035] As used herein, a "mutation" may be an addition, substitution or deletion of one or more nucleotides.

[0036] The mutation in the gene results in reduced expression of the encoded protein or results in expression of a protein with reduced activity. In some embodiments, the mutation in the gene results in no expression of the encoded protein or expression of an inactive protein. In some embodiments, the mutation is a frameshift mutation, which results in mistranslation of one or more genes.

[0037] The term "mutation" can include the deletion of a gene, such as a complete or partial deletion of a gene. In some embodiments, the viable bacterial strains of the present invention include a deletion of one or more genes. One or more genes can be completely deleted from the strain, such that the protein encoded by the one or more genes is absent from the viable bacterial strains of the present invention. One or more genes can also be partially deleted, such that only one or more inactive truncated proteins are present in the viable bacterial strains of the present invention.

[0038] The mutation of one or more genes can be achieved by various methods known in the art. In some embodiments, mutation is introduced into the viable bacterial strain by genetic engineering. In some embodiments, mutation is not a naturally occurring mutation. For example, mutation (such as deletion) can be achieved by homologous recombination (such as double homologous recombination). In some embodiments, mutation is carried out by directed mutagenesis, for example, by CRISPR, TALEN or ZFN technology.

[0039] In some embodiments, the viable bacterial strain comprises one or more mutations in one or more genes selected from SaeS, SaeR, SaeP and SaeQ. Preferably, all of SaeS, SaeR, SaeP and SaeQ in the viable bacterial strain are mutated.

[0040] In some embodiments, mutation of one or more genes of the Sae two-component system may result in reduced expression or no expression of one or more genes of the Sae two-component system, or reduced expression or no activity of one or more proteins of the Sae two-component system.

[0041] In some embodiments, the mutation comprises the deletion of one or more genes of the Sae two-component system. Preferably, all genes of the Sae two-component system in the viable bacterial strain are completely or partially deleted. More preferably, all genes of the Sae two-component system in the viable bacterial strain are completely deleted.

[0042] In some embodiments, one or more of the SaeS, SaeR, SaeP and SaeQ genes in the live bacterial strain are deleted. Preferably, the SaeS, SaeR, SaeP and SaeQ in the live bacterial strain are all completely or partially deleted. More preferably, the SaeS, SaeR, SaeP and SaeQ in the live bacterial strain are all completely deleted.

[0043] Exemplary protein sequences of SaeP, SaeQ, SaeS, and SaeR of S. aureus (from strain Newman) are set forth in SEQ ID NOs: 34-37, respectively; exemplary coding sequences of SaeP, SaeQ, SaeS, and SaeR of S. aureus (from strain Newman) are set forth in SEQ ID NOs: 38-41, respectively; an exemplary protein sequence of SaeS of S. aureus (from strain JE2) is set forth in SEQ ID NO: 42; and an exemplary coding sequence of SaeS of S. aureus (from strain JE2) is set forth in SEQ ID NO: 43.

[0044] In some embodiments, SaeP comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO:34.

[0045] In some embodiments, SaeQ comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO:35.

[0046] In some embodiments, SaeS comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO:36 or SEQ ID NO:42.

[0047] In some embodiments, SaeR comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% sequence identity to SEQ ID NO:37.

[0048] In some embodiments, when comparing the activity or expression of the Sae two-component system, the "control strain" may refer to a strain of the same species in which the Sae two-component system activity or Sae two-component system gene has not been changed; in some embodiments, when comparing the activity or expression of the Sae two-component system, the "control strain" may also refer to a strain of the same species that does not contain the above-mentioned Sae two-component system gene mutation.

[0049] In some embodiments, the viable bacterial strain also has reduced adenosine synthase A (adsA) activity, preferably lacks adenosine synthase A (adsA) activity, compared to a corresponding control strain.

[0050] In some embodiments, the expression of the adsA gene in the viable bacterial strain is reduced, and preferably, the adsA gene is not expressed.

[0051] In some embodiments, the viable bacterial strain comprises a mutation in the adsA gene. In some embodiments, the mutation results in reduced or no expression of the adsA gene, or reduced or no expression activity of the adsA protein.

[0052] Adenosine synthase A (adsA) is an important virulence factor of Staphylococcus aureus. An exemplary adsA of Staphylococcus aureus comprises the amino acid sequence of SEQ ID NO: 1. However, those skilled in the art are aware that due to polymorphism between species and strains, adsA may have slight differences from SEQ ID NO: 1 while still retaining the same or similar functions.

[0053] In some embodiments, the adsA comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 1.

[0054] In some embodiments, the live bacterial strains of the present invention have adsA activity reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain. In some preferred embodiments, the live bacterial strains of the present invention have no adsA activity. Reduced or absent adsA activity can result in attenuation of the live bacterial strains of the present invention.

[0055] In some embodiments, the viable bacterial strain of the present invention comprises a mutation in the adsA gene encoding adsA. Such a mutation may be an addition, substitution, or deletion of one or more nucleotides.

[0056] In some embodiments, the mutation in the adsA gene results in reduced expression of the adsA protein or results in expression of a mutant adsA protein with reduced activity. In some embodiments, the mutation in the adsA gene results in no expression of the adsA protein or expression of a mutant adsA protein with no activity.

[0057] In some embodiments, the mutation comprises a deletion of the adsA gene, such as a complete or partial deletion of the adsA gene. The adsA gene can be completely deleted from the strain, such that the adsA gene is substantially absent from the viable strain of the present invention. The adsA gene can also be partially deleted, such that only adsA protein with reduced activity or no activity is present in the viable strain of the present invention. In some embodiments, the mutation is a frameshift mutation, which results in mistranslation of the adsA protein. In some embodiments, the mutation in the adsA gene results in the deletion of the portion of adsA responsible for adenosine production.

[0058] In some embodiments, when comparing adsA activity, the "control strain" may refer to a strain of the same species in which the adsA activity or adsA gene has not been changed; in some embodiments, when comparing adsA activity, the "control strain" may also refer to a strain of the same species that does not contain the above-mentioned adsA gene mutation.

[0059] The mutation of the adsA gene can be achieved by various methods known in the art. In some embodiments, the mutation is introduced into the viable bacterial strain by genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, the mutation (e.g., deletion) can be achieved by homologous recombination (e.g., double homologous recombination). In some embodiments, the mutation is achieved by directed mutagenesis, for example, by CRISPR, TALEN, or ZFN technology.

[0060] In some other embodiments, the live bacterial strains of the present invention are derived from a parent live bacterial strain that has been attenuated. For example, the parent live bacterial strain may have reduced or no adenosine synthase A (adsA) activity, for example, compared to a corresponding control strain. For example, the live bacterial strains of the present invention can be obtained by introducing mutations in one or more genes of the Sae two-component system into a parent live bacterial strain that already has reduced or no adenosine synthase A (adsA) activity.

[0061] In some embodiments, the live bacterial strains of the present invention also have reduced capsule production, preferably, lack capsule production, e.g., compared to a corresponding control strain.

[0062] In some embodiments, expression of one or more capsular polysaccharide synthesis genes is reduced in the bacterial strains of the invention, eg, compared to a corresponding control strain.

[0063] In some embodiments, the live bacterial strain comprises one or more mutations in one or more capsular polysaccharide synthesis genes.

[0064] In some embodiments, when comparing capsule production or expression of capsular polysaccharide synthesis genes, the "control strain" may be the parent strain from which the live bacterial strain of the present invention is derived. In some embodiments, the "control strain" may refer to a strain of the same species in which the capsule / capsular polysaccharide production or capsular polysaccharide synthesis genes have not been altered. In some embodiments, the "control strain" may also refer to a strain of the same species that does not contain one or more of the above-mentioned capsular polysaccharide synthesis gene mutations.

[0065] In some embodiments, the amount of capsule production in the viable bacterial strains of the present invention is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the corresponding control strain. In some preferred embodiments, the viable bacterial strains of the present invention do not produce a capsule.

[0066] In some embodiments, the production of capsular polysaccharides in the viable bacterial strains of the present invention is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain. In some preferred embodiments, the viable bacterial strains of the present invention do not produce capsular polysaccharides.

[0067] In some embodiments, the expression of one or more capsular polysaccharide synthesis genes in the viable bacterial strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain.

[0068] In some embodiments, the expression of all capsular polysaccharide synthesis genes is reduced in the living bacterial strain.

[0069] In some embodiments, the expression of all capsular polysaccharide synthesis genes in the live strain is reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to the corresponding control strain. In some preferred embodiments, no capsular polysaccharide synthesis genes are expressed in the live strain.

[0070] As used herein, the term "capsular polysaccharide synthesis gene" refers to any gene involved in the production of capsular polysaccharide in bacteria. A capsular polysaccharide synthesis gene can encode a capsular polysaccharide synthesis protein. A capsular polysaccharide synthesis gene can refer to a protein coding sequence, but can also include expression control elements / sequences, such as promoters, enhancers, and the like.

[0071] In some embodiments, the living strains of the present invention comprise one or more mutations in one or more capsular polysaccharide synthesis genes, which mutations may be additions, substitutions or deletions of one or more nucleotides.

[0072] In some preferred embodiments, all capsular polysaccharide synthesis genes in the viable bacterial strain are mutated. In some embodiments, the mutations in the capsular polysaccharide synthesis genes result in reduced expression of capsular polysaccharide synthesis proteins or expression of capsular polysaccharide synthesis proteins with reduced activity. In some embodiments, the mutations in the capsular polysaccharide synthesis genes result in no expression of capsular polysaccharide synthesis proteins or expression of inactive capsular polysaccharide synthesis proteins. In some embodiments, the mutations are frameshift mutations, resulting in mistranslation of one or more capsular polysaccharide synthesis genes.

[0073] In some embodiments, the mutation comprises a deletion of a capsular polysaccharide synthesis gene, for example, a complete or partial deletion of a capsular polysaccharide synthesis gene. In some embodiments, the viable bacterial strains of the present invention comprise a deletion of one or more capsular polysaccharide synthesis genes. One or more capsular polysaccharide synthesis genes may be completely deleted from the strain so that one or more capsular polysaccharide synthesis proteins are not present in the viable bacterial strains of the present invention. One or more capsular polysaccharide synthesis genes may also be partially deleted so that only one or more inactive truncated capsular polysaccharide synthesis proteins are present in the viable bacterial strains of the present invention. In some preferred embodiments, all capsular polysaccharide synthesis genes in the viable bacterial strains of the present invention are completely or partially deleted. In some preferred embodiments, all capsular polysaccharide synthesis genes in the viable bacterial strains of the present invention are completely deleted.

[0074] The mutation of one or more capsular polysaccharide synthesis genes can be achieved by various methods known in the art. In some embodiments, the mutation is introduced into the viable bacterial strain by genetic engineering. In some embodiments, the mutation is not a naturally occurring mutation. For example, the mutation (e.g., deletion) can be achieved by homologous recombination (e.g., double homologous recombination). In some embodiments, the mutation is carried out by directed mutagenesis, for example, by CRISPR, TALEN or ZFN technology.

[0075] Serotype 5 or serotype 8 Staphylococcus aureus contains 16 capsular polysaccharide synthesis genes clustered in its genome: capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP. Exemplary amino acid sequences of capA to capP of serotype 5 Staphylococcus aureus are shown in SEQ ID NOs: 2-17, respectively. Exemplary coding sequences of capA to capP of serotype 5 Staphylococcus aureus are shown in SEQ ID NOs: 18-33, respectively. Therefore, those skilled in the art can identify capsular polysaccharide synthesis genes / gene clusters of other serotypes or isolates and perform the above-mentioned mutations.

[0076] In some embodiments, the one or more capsular polysaccharide synthesis genes encode one or more capsular polysaccharide synthesis proteins having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to any one of SEQ ID NOs: 2-17.

[0077] In some embodiments, capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP are all deleted in the viable bacterial strain, preferably completely deleted.

[0078] In some embodiments, the live bacterial strain has reduced virulence.

[0079] For example, the virulence of the live bacterial strains of the present invention can be reduced by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more compared to a corresponding control strain.

[0080] In some embodiments, the live bacterial strain has increased immunogenicity. Immunogenicity can refer to the ability to elicit an immune response (e.g., an antibody-mediated immune response) in a host.

[0081] For example, the immunogenicity of the live bacterial strains of the invention can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, or more, compared to a corresponding control strain.

[0082] In some embodiments, the "control strain" may be the parent strain from which the live bacterial strain of the present invention is derived. In some embodiments, the "control strain" may refer to a strain of the same species whose Sae two-component system, adsA activity, and / or capsule / capsular polysaccharide production are unchanged. In some embodiments, the "control strain" may also refer to a strain of the same species that does not contain a mutation in one or more of the above-mentioned Sae two-component system genes, adsA genes, and / or capsular polysaccharide synthesis genes.

[0083] The viable bacterial strain may be derived from a parent strain, which is a wild-type strain of the same species. In some embodiments, the wild-type strain may be a strain that has not been genetically engineered. In some embodiments, the wild-type strain may be a strain that has not been genetically engineered for the Sae two-component system gene, the adsA gene, and / or the capsular polysaccharide synthesis gene. In some embodiments, the wild-type strain may be a clinical isolate.

[0084] The live bacterial strain may be derived from a parent strain that already has low toxicity, for example, the parent strain is an attenuated strain. The live bacterial strain of the present invention may contain other modifications that may result in attenuation.

[0085] In some embodiments, the live bacterial strain can be derived from any isolate of Staphylococcus aureus.

[0086] In some embodiments, the live bacterial strains of the present invention may be derived from strains of various serotypes. Generally speaking, the serotype of a strain is determined by the capsular polysaccharide produced by the strain.

[0087] In some embodiments, the live Staphylococcus aureus strain is derived from various serotypes of Staphylococcus aureus, including but not limited to serotype 5, serotype 8, etc. In some specific embodiments, the live Staphylococcus aureus strain of the present invention is derived from serotype 5 Staphylococcus aureus. In some specific embodiments, the live Staphylococcus aureus strain of the present invention is derived from serotype 8 Staphylococcus aureus.

[0088] In some embodiments, the live Staphylococcus aureus strain is derived from Staphylococcus aureus Newman, USA300, JE2, or ATCC29213. In some preferred embodiments, the live Staphylococcus aureus strain of the present invention is derived from Staphylococcus aureus Newman. Staphylococcus aureus Newman is a serotype 5 strain commercially available from the National Collection of Type Cultures (NCTC) in the United Kingdom under the accession number NCTC 8178.

[0089] In some embodiments, the live Staphylococcus aureus strain, for example, compared to a corresponding control strain,

[0090] a) the viable bacterial strain has reduced activity of the Sae two-component system, preferably, lacks Sae two-component system activity; the expression of one or more genes of the Sae two-component system in the viable bacterial strain is reduced; and / or the viable bacterial strain comprises one or more mutations in one or more genes of the Sae two-component system; and

[0091] b) the viable bacterial strain has reduced adenosine synthase A (adsA) activity, preferably lacks adenosine synthase A (adsA) activity, for example, expression of the adsA gene in the viable bacterial strain is reduced compared to a corresponding control strain; and / or the viable bacterial strain comprises a mutation in the adsA gene. The terms herein have the definitions set forth above.

[0092] In some embodiments, the live Staphylococcus aureus strain, for example, compared to a corresponding control strain,

[0093] a) the Sae two-component system activity of the live bacterial strain is reduced, preferably, lacks Sae two-component system activity;

[0094] The expression of one or more genes of the Sae two-component system in the viable bacterial strain is reduced; and / or the viable bacterial strain comprises one or more mutations in one or more genes of the Sae two-component system; and

[0095] b) the viable bacterial strain has reduced capsule production, preferably, lacks capsule production, for example, expression of one or more capsular polysaccharide synthesis genes is reduced in the viable bacterial strain of the invention compared to a corresponding control strain; and / or the viable bacterial strain comprises one or more mutations in one or more capsular polysaccharide synthesis genes. These terms have the meanings set forth above.

[0096] In some embodiments, the live Staphylococcus aureus strain, for example, compared to a corresponding control strain,

[0097] a) the viable bacterial strain has reduced adenosine synthase A (adsA) activity, preferably lacks adenosine synthase A (adsA) activity; expression of the adsA gene in the viable bacterial strain is reduced; and / or the viable bacterial strain comprises a mutation in the adsA gene; and

[0098] b) the viable bacterial strain has reduced capsule production, preferably, lacks capsule production; the viable bacterial strain has reduced expression of one or more capsular polysaccharide synthesis genes; and / or the viable bacterial strain contains one or more mutations in one or more capsular polysaccharide synthesis genes. These terms have the meanings set forth above.

[0099] In some embodiments, the live Staphylococcus aureus strain, for example, compared to a corresponding control strain,

[0100] a) the viable bacterial strain has reduced Sae two-component system activity, preferably, lacks Sae two-component system activity; the expression of one or more genes of the Sae two-component system in the viable bacterial strain is reduced; and / or the viable bacterial strain comprises one or more mutations in one or more genes of the Sae two-component system; and

[0101] b) the viable bacterial strain has reduced adenosine synthase A (adsA) activity, preferably, lacks adenosine synthase A (adsA) activity; expression of the adsA gene in the viable bacterial strain is reduced; and / or the viable bacterial strain comprises a mutation in the adsA gene; and

[0102] c) the viable bacterial strain has reduced capsule production, preferably, lacks capsule production; the expression of one or more capsular polysaccharide synthesis genes in the viable bacterial strain is reduced; and / or the viable bacterial strain contains one or more mutations in one or more capsular polysaccharide synthesis genes. These terms have the meanings set forth above.

[0103] In some embodiments, the viable bacterial strain can express (e.g., overexpress) a protein of interest. In some embodiments, the viable bacterial strain can comprise a coding sequence for a protein of interest (e.g., introduced), thereby being able to express (e.g., overexpress) the protein of interest.

[0104] In some embodiments, the protein of interest can be an endogenous protein, ie, a protein of Staphylococcus aureus. In some embodiments, the protein of interest can be an exogenous protein, ie, a protein of a species different from Staphylococcus aureus.

[0105] In some embodiments, the coding sequence of the protein of interest is introduced into the viable bacterial strain, for example, via a nucleic acid expression construct. In some embodiments, the introduced coding sequence of the protein of interest is integrated into the genome of the viable bacterial strain.

[0106] As used herein, an "expression construct" refers to a vector, such as a recombinant vector, that is suitable for expressing a nucleotide sequence of interest in a host cell. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence and / or the RNA translation of the nucleotide sequence into a precursor or mature protein. An "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be RNA (e.g., mRNA) that is capable of translation.

[0107] In some embodiments, the protein of interest can be expressed and displayed on the cell surface of the living bacterial strain; in some embodiments, the protein of interest can be expressed and secreted outside the cells of the living bacterial strain.

[0108] In some preferred embodiments, the protein of interest is an antigenic protein, and expressing or displaying the antigenic protein can further improve the immunogenicity of the live bacterial strain.

[0109] Exemplary antigenic proteins include, but are not limited to, EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, mLukB, etc., or any combination thereof.

[0110] In some embodiments, the viable bacterial strain expresses (e.g., overexpresses) EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA, and mLukB.

[0111] EsxA may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 44. EsxB may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 46. mLukS-PV may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 48. mLukF-PV may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 50. mHla may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 52. mSpA may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 54. mTSST1 may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 56. mSEB may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 58. mLukA may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 60. mLukB may comprise an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 99%, or even 100% sequence identity to SEQ ID NO: 62.

[0112] In some embodiments, the vaccine may also include an adjuvant. As used herein, "adjuvant" refers to an additional component in a vaccine used to enhance the immune response, or an auxiliary molecule added to a vaccine or produced by the body after being induced by these additional components, such as, but not limited to, interferons, interleukins, or growth factors. "Adjuvants" as used herein may include aluminum hydroxide and aluminum phosphate, saponins, water-in-oil emulsions, and water-in-oil-in-water emulsions.

[0113] In some embodiments, the vaccine may further comprise a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, saline solutions (e.g., Ringer's solution), alcohol, oils, gelatin, carbohydrates (e.g., lactose, amylose, or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and colorants.

[0114] In some embodiments, the vaccine is administered intramuscularly, intraperitoneally, subcutaneously, orally, or intranasally. In some embodiments, the vaccine is formulated for intramuscular, intraperitoneal, subcutaneous, oral, or intranasal administration. In one embodiment, the vaccine is not for intravenous administration. In some embodiments, the vaccine is in a lyophilized form that can be reconstituted before use.

[0115] In some embodiments, the live Staphylococcus aureus vaccine of the present invention is used to prevent and / or treat bacterial infections in non-human animals. In other words, the vaccine is a veterinary vaccine.

[0116] The non-human animals include, but are not limited to, sheep, goats, pigs, cows, horses, donkeys, cats, dogs, chickens, ducks, geese, etc. In some preferred embodiments, the non-human animals are sheep or goats.

[0117] As used herein, preventing and / or treating a bacterial infection also includes preventing and / or treating a disease or clinical signs or symptoms caused by a bacterial infection.

[0118] In some embodiments, the bacterial infection is a Staphylococcus aureus infection. In some preferred embodiments, the bacterial infection is an anaerobic Staphylococcus aureus (S. aureus subspecies anaerobius) infection. For example, the infection is caused by anaerobic Staphylococcus aureus strains MVF-7 (ATCC 35844), ST1464, Polonia 123. Su-1, Su-4, Po-3, DK-3, It-4, MVF-9, MVF-16, MVF-17-II, MVF-29, MVF-30, MVF-39, MVF-39+, MVF-49, MVF-58, MVF-71, MVF-80, MVF-91, MVF-205, Infections caused by MVF-212, MVF-220, MVF-228, MVF-302, MVF-401, MVF-502, 39+1, 39+2, 43+, 502R+1, 502R+2, 7R0+1, It4R+, MVF43, SU4+, MVF-7+, RDKA-84, MVF84.

[0119] In some embodiments, preventing and / or treating bacterial infection also includes preventing and / or treating mastitis, arthritis, skin lesions, etc. caused by bacterial infection. In some specific embodiments, preventing and / or treating bacterial infection also includes preventing and / or treating Morel's disease.

[0120] In some embodiments, the vaccine comprises an effective amount of the live Staphylococcus aureus strain.

[0121] As used herein, an "effective amount" refers to an amount of a substance, compound, material, or composition containing a compound (e.g., a live bacterial strain of the present invention) that is sufficient to produce at least a prophylactic or therapeutic effect upon administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, delay, or partially alleviate the symptoms of a disease or condition (e.g., a bacterial infection).

[0122] In another aspect, the present invention provides a method for preventing and / or treating bacterial infection in a non-human animal, comprising administering to the non-human animal an effective amount of the live Staphylococcus aureus vaccine of the present invention.

[0123] The non-human animals include, but are not limited to, sheep, goats, pigs, cows, horses, donkeys, rabbits, cats, dogs, chickens, ducks, geese, etc. In some preferred embodiments, the non-human animals are sheep or goats.

[0124] In some embodiments, the bacterial infection is a Staphylococcus aureus infection. In some preferred embodiments, the bacterial infection is an anaerobic Staphylococcus aureus (S. aureus subspecies anaerobius) infection. For example, the infection is caused by anaerobic Staphylococcus aureus strains MVF-7 (ATCC 35844), ST1464, Polonia Infections caused by strains 123, Su-1, Su-4, Po-3, DK-3, It-4, MVF-9, MVF-16, MVF-17-II, MVF-29, MVF-30, MVF-39, MVF-39+, MVF-49, MVF-58, MVF-71, MVF-80, MVF-91, MVF-205, MVF-212, MVF-220, MVF-228, MVF-302, MVF-401, MVF-502, 39+1, 39+2, 43+, 502R+1, 502R+2, 7R0+1, It4R+, MVF43, SU4+, MVF-7+, RDKA-84, MVF84 or their homologous strains.

[0125] In some embodiments, preventing and / or treating bacterial infection also includes preventing and / or treating mastitis, arthritis, skin lesions, etc. caused by bacterial infection. In some specific embodiments, preventing and / or treating bacterial infection also includes preventing and / or treating Morel's disease. Example

[0126] The present invention can be further understood by referring to the specific embodiments listed herein. However, these embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Obviously, various modifications and variations can be made to the present invention without departing from the spirit of the present invention, and therefore, such modifications and variations also fall within the scope of the present invention.

[0127] Example 1: Construction of Staphylococcus aureus strain NMΔadsAΔCPΔsaePQRS

[0128] Staphylococcus aureus strain Newman (NCTC 8178, serotype 5) was cultured in brain heart infusion (BHI) at 37°C. Homologous recombination was used to sequentially delete the target genes adsA, CPs (capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO, and capP), and saePQRS (SaeS, SaeR, SaeP, and SaeQ genes). Sequencing comparisons used to verify the deletion of the target genes adsA, CPs, and saePQRS are shown in Figure 1. The sequences of the target genes are provided at the end of this article or in the sequence listing.

[0129] The specific construction process is:

[0130] Using the corresponding primers listed in Table 1, PCR amplify the upstream and downstream (5'- and 3'-) flanking regions of the target knockout genes, adsA, CPs, and saePQRS, from chromosomal DNA of the Newman strain. PCR products from the upstream and downstream homology arms of each target knockout gene were then combined to generate in-frame deletion fragments of the target knockout gene. The in-frame deletion fragments were cloned into the plasmid pIMAY* to generate the corresponding recombinant plasmids. The recombinant plasmids were introduced into DH5a, transformed into Escherichia coli DC10B, and then transformed into the parent strain (wild-type Newman or a strain with other target genes knocked out). Using homologous recombination, a two-step screening process was performed to identify strains with successful knockout of the target gene. The two-step screening process was as follows: First, transformants were cultured at 37°C in BHI containing 10 μg / ml chloramphenicol for 2-3 days. The clones were PCR screened using the specific primers in Table 1 to select the first recombinant strain (the transformation plasmid was integrated into the genome). The selected first recombinant strain was then passaged 5-7 times in BHI at 28°C without antibiotics and screened on a plate containing PCPA (4-Chloro-DL-phenylalanine, DL-4-chlorophenylalanine) for secondary recombination and plasmid loss. Colonies sensitive to chloramphenicol were then screened, which were the second recombinant strains. Using the primers in Table 1, PCR was performed on the chromosomal DNA of the second recombinant strain to confirm the successful knockout of the target gene. According to the above method, adsA, CPs, and saePQRS were knocked out in sequence, and the strain NMΔadsAΔCPΔsaePQRS was finally obtained.

[0131] Table 1. Construction of Newman strains for knockout of target genes adsA, CPs, and saePQRS and primers used to verify successful gene knockout

[0132]

[0133]

[0134] Example 2: Construction of Staphylococcus aureus strains expressing antigens

[0135] In order to improve the protective efficacy and coverage of the strain as a vaccine, the antigens EsxA (amino acid sequence shown in SEQ ID NO: 44, nucleotide sequence shown in SEQ ID NO: 45), EsxB (amino acid sequence shown in SEQ ID NO: 46, nucleotide sequence shown in SEQ ID NO: 47), mLukS-PV (amino acid sequence shown in SEQ ID NO: 48, nucleotide sequence shown in SEQ ID NO: 49), mLukF-PV (amino acid sequence shown in SEQ ID NO: 50, nucleotide sequence shown in SEQ ID NO: 51), mHla (amino acid sequence shown in SEQ ID NO: 52, nucleotide sequence shown in SEQ ID NO: 53), mSpA (amino acid sequence shown in SEQ ID NO: 54, nucleotide sequence shown in SEQ ID NO: 55), mTSST1 (amino acid sequence shown in SEQ ID NO: 56, nucleotide sequence shown in SEQ ID NO: 57), mSEB (amino acid sequence shown in SEQ ID NO: 58), and mHla (amino acid sequence shown in SEQ ID NO: 59, nucleotide sequence shown in SEQ ID NO: 60). ID NO:58, the nucleotide sequence is shown in SEQ ID NO:59), mLukA (amino acid sequence is shown in SEQ ID NO:60, the nucleotide sequence is shown in SEQ ID NO:61), and mLukB (amino acid sequence is shown in SEQ ID NO:62, the nucleotide sequence is shown in SEQ ID NO:63), thereby obtaining a strain with the genotype of NewmanΔCPΔsaePQRSΔhla::EsxAB-mLukSFΔspa::mHla-mSpAΔadsA::mTSST1-mSEBΔlukAB::mLukAB.

[0136] Example 3: Animal Experiment

[0137] In Northwest China, as summer temperatures rise, outbreaks of impetigo occur in mutton farms. The incidence decreases with cooler weather. Impetigo is more likely to occur if animals experience trauma (e.g., shearing). Bacteria isolated from the tissue fluid of infected sheep on mutton farms were cultured, identified using 16S sequencing, and sequenced for multiple key genes. The pathogenic strain was confirmed to be highly homologous to the anaerobic Staphylococcus aureus MVF-7 (ATCC 35844).

[0138] 1) Screening of experimental animals

[0139] To conduct the experiment, we first needed to select sheep with a clear background and no obvious S. aureus infection for the experimental group. This experiment was conducted at a mutton farm in Northwest China. Lambs under one year old were selected from the farm, requiring a clean environment, a clear background, and no visible abscesses. A total of 104 healthy sheep were selected.

[0140] Because pustular disease has a long course and can harbor latent infection, or animals may have been exposed to S. aureus but not developed the disease, serum IgG pre-existing against S. aureus was tested during the screening process. All 104 healthy sheep were numbered and blood was collected 13 days before immunization to test for specific antibodies against the S. aureus strain isolated from the sheep. A total of 60 animals with low antibody responses were selected and officially entered the experimental group. The antibody AUCs for these 60 animals were all below 2.0.

[0141] 2) Experimental design and animal grouping

[0142] Experimental Design: This experiment was divided into two groups, including an immunization group and a control group, with 30 animals in each group, injected with the candidate vaccine strain (obtained in Example 2) and normal saline respectively. See the table below for details:

[0143]

[0144] All 60 animals screened were randomly divided into groups. The median and distribution of antibody levels of the animals in the final group were basically consistent, indicating that the background of the two groups of animals was similar ( Figure 2 ).

[0145] 3) Sample collection and disease observation plan

[0146] This experiment conducted the following two observations: Figure 3 ): After immunization, continue to observe the health of the animals and abnormalities at the injection site, and record any other abnormal reactions to study the safety of the vaccine in sheep; after immunization, observe and record the incidence of impetigo in animals at irregular intervals, including the appearance, recovery, and disappearance of pustules, to study the effectiveness of the vaccine against impetigo in sheep.

[0147] This experiment involved two sample collections: Figure 3 ):

[0148] On the day of immunization, serum was collected on day 0 to test the antibody levels of the animals in the group as a control for immunogenicity studies; after immunization, serum was collected on day 29 to test the antibody levels to study the immunogenicity of the vaccine in sheep.

[0149] At the end of the experiment, one animal in the vaccine group died of other diseases and one animal lost its ear tag, resulting in two animals being excluded, bringing the number of animals included in the subsequent analysis to 28. One animal in the control group lost its ear tag, resulting in the number of animals included in the subsequent analysis to 29.

[0150] 4) Experimental results

[0151] Security:

[0152] After immunization, the injection site of the vaccine group was observed and no obvious redness or swelling was observed. The animals were in good overall condition, with no signs of fatigue or recumbency. One animal in the immunization group died on day 29, which was confirmed to be caused by nasal obstruction after autopsy. No lesions or abnormalities were found in other tissues, and no evidence was found that the animal's death was related to vaccine administration. This preliminarily demonstrates the safety of the vaccine in sheep. Immunogenicity:

[0153] The serum samples from the two groups on day 0 (before immunization) and day 29 (after immunization) were simultaneously subjected to whole-bacteria ELISA against the vaccine strain to detect specific IgG antibodies. The data showed that on day 29 after immunization, there was a significant difference in the antibody levels between the vaccine group and the control group, indicating that the vaccine was able to induce an effective antibody response in the sheep ( Figure 4 ).

[0154] Validity:

[0155] Throughout the observation period, animals were individually examined at all observation points, and their incidence was counted. The first occurrence of abscess infection in an animal was counted as a primary case. Recurrent infection after recovery from the initial bout was counted as a recurrent case. Finally, the number of observed cases was counted, and the number of animals completing the study was used as the baseline for calculating the incidence rate within the group and the final vaccine effectiveness.

[0156] Incidence rate within a group = number of cases within the group / calculation base

[0157] Vaccine effectiveness = 1-(incidence rate in vaccine group / incidence rate in control group)

[0158] The experimental results showed that at the end point (day 143), the vaccine candidate was 65.5% effective against the first episode of impetigo caused by S. aureus (Table 2). The vaccine candidate was 100% effective against recurrent episodes of impetigo caused by S. aureus (Table 3). This indicates that the vaccine candidate is effective in preventing impetigo in sheep, providing long-term protection for more than four months and effectively preventing recurrent episodes.

[0159] Table 2. Vaccine effectiveness at first onset of disease

[0160]

[0161] Table 3. Vaccine effectiveness against recurrent disease

[0162]

[0163]

[0164] Partial sequence information:

[0165] SEQ ID NO 1adsAamino acid sequence

[0166] MKALLLKTSVWLVLLFSAMGLWQVSSAAEQHTPMKAHAVTTIDKATTDRQLVLPTKEAAHQSGE

[0167] EAATNVSASAQGTADDTNNKVTSNAPSNKPSTAVSTTVNETHDVDAQQASTQKPTQSATFKLSNA

[0168] KTASLSPRMFAANAPQTTTHKILHTNDIHGRLAEEKGRVIGMAKLKTVKEQEKPDLILDAGDAFQG

[0169] LPLSNQSKGEEMAKAMNAVGYDAMAVGNHEFDFGYDQLKKLEGMLDFPMLSTNVYKDGKRAF

[0170] KPSTIVTKNGIRYGIIGVTTPETKTKTRPEGIKGVEFRDPLQSVTAEMMRIYKDVDTFVVISHLGIDP

[0171] STQETWRGDYLVKQLSQNPQLKKRITVIDGHSHTVLQNGQIYNNDALAQTGTALANIGKVTFNYR

[0172] NGEVSNIKPSLINVKDVENVTPNKALAEQINQADQTFRAQTAEVIIPNNTIDFKGERDDVRTRETNL

[0173] GNAITDAMEAYGVKNFSKKTDFAVTNGGGIRASIAKGKVTRYDLISVLPFGNTIAQIDVKGSDVWT

[0174] AFEHSLGAPTQKDGKTVLTANGGLLHISDSIRVYYDMNKPSGKRINAIQILNKETGKFENIDLKRV

[0175] YHVTMNDFTASGGDGYSMFGGPREEGISLDQVLASYLKTANLAKYDTTEPQRMLLGKPAVSEQPA

[0176] KGQQGSKGSESGKDTQPIGKDKVMNPAKQPATGKVVLLPTHRGTVSSGAEGSDCALEGTAVSSKS

[0177] GKQLTKMSASKGSGHEKQLPKTGTNQSSSPAAIFVLVAGIGLIATVRRRKASSEQ ID NO 2capA(NWMN_0095):

[0178] MESTLELTKIKEVLQKNLKILIILPLLFLIISAIVTFFVLSPKYQANTQILVNQTKDGNPQFMAQEVQS

[0179] NIQLVNTYKEIVKSPRILDEVSKDLNDKYSPSKLSSMLTITNQENTQLINIQVKSGHKQDSEKIANSF

[0180] AKVTSKQIPKIMSVDNVSILSKADGTAVKVAPKTVVNLIGAFFLGLVVALIYIFFKVIFDKRIKDEEDVEKEGLPLVLGSIQKFN*

[0181] SEQ ID NO 3capB(NWMN_0096):

[0182] MLLMSKKENTTTTTLFVYEKPKSTISEKFRGIRSNIMFSKANGEVKRLLVTSEKPGAGKSTVVSNVAI

[0183] TYAQAGYKTLVIDGDMRKPTQNYIFNEQNNNGLSSLIIGRTTMSEAITSTEIENLDLLTAGPVPPNPS

[0184] ELIGSERFKELVDLFNKRYDIIIVDTPPVNTVTDAQLYARAIKDSLLVIDSEKNDKNEVKKAKALMEKAGSNILGVILNKTKVDKSSSYYHYYGDE*

[0185] SEQ ID NO 4capC(NWMN_0097):

[0186] MIDIHNHILPNIDDGPTNETEMMDLLKQATTQGVTEIIVTSHHLHPRYTTPIEKVKSCLNHIESLEEV

[0187] QALNLKFYYGQEIRITDQILNDIDRKVINGINDSRYLLIEFPSNEVPHYTDQLFFELQSKGFVPIIAHP

[0188] ERNKAISQNLDILYDLINKGALSQVTTASLAGISGKKIRKLAIQMIENNLTHFIGSDAHNTEIRPFLMKDLFNDKKLRDYYEDMNGFISNAKLVVDDKKIPKRMPQQDYKQKRWFGL*

[0189] SEQ ID NO 5capD(NWMN_0098):

[0190] MRGFMAHLSVKLRLLILALIDSLIVTFSVFVSYYILEPYFKTYSVKLLILAAISLFISHHISAFIFNMYH

[0191] RAWEYASVSELILIVKAVTTSIVITMVVVTIVTGNRPFFRLYLITWMMHLILIGGSRLFWRIYRKYLG

[0192] GKSFNKKPTLVVGAGQAGSMLIRQMLKSDEMKLEPVLAVDDDEHKRNITITEGVKVQGKIADIPEL

[0193] VRKYKIKKIIIAIPTIGQERLKEINNICHMDGVELLKMPNIEDVMSGELEVNQLKKVEDLLGRDP

[0194] VELDMDMMISNELTNKTILVTGAGGSIGSEICRQVCNFYPERIILLGHGENSIYLINRELRNRFGKNVDI

[0195] VPIIADVQNRARMFEIMETYKPYAVYHAAAHKHVPLMEDNPEEAVRNNILGTKNTAEAAKNAEVK

[0196] KFVMISTDKAVNPPNVMGASKRIAEMIIQSLNDETHRTNFVAVRFGNVLGSRGSVIPLFKSQIEEGGP

[0197] VTVTHPEMTRYFMTIPEASRLVLQAGALAEGGEVFVLDMGEPVKIVDLARNLIKLSGKKEDDIRIT

[0198] YTGIRPGEKMFEELMNKDEVHPEQVFEKIYRGKVQHMKCNEVEAIIQDIVNDFSKEKIINYANGKKGDNYVR*

[0199] SEQ ID NO 6capE(NWMN_0099):

[0200] MFDDKILLITGGTGSFGNAVMKQFLDSNIKEIRIFSRDEKKQDDIRKKYNNSKLKFYIGDVRDSQSV

[0201] ETAMRDVDYVFHAAALKQVPSCEFFPVEAVKTNIIGTENVLQSAIHQNVKKVICLSTDKAAYPINA

[0202] MGISKAMMEKVFVAKSRNIRSEQTLICGTRYGNVMASRGSVIPLFIDKIKAGEPLTITDPDMTRFLM

[0203] SLEDAVELVVHAFKHAETGDIMVQKAPSSTVGDLATALLELFEADNAIEIIGTRHGEKKAETLLTRE

[0204] EYAQCEDMGDYFRVPADSRDLNYSNYVETGNEKITQSYEYNSDNTHILTVEEIKEKLLTLEYVRNELNDYKASMR*

[0205] SEQ ID NO 7 capF(NWMN_0100):

[0206] MNIVITGAKGFVGKNLKADLTSTTDHHIFEVHRQTKEEELESALLKADFVVHLAGVNRPEHDKEFS

[0207] LGNVSYLDHVLDILTRNTKKPAILLSSSIQATQDNPYGESKLQGEQLLREYAEEYGNTVYIYRWPNL

[0208] FGKWCKPNYNSVIATFCYKIARNEEIQVNDRNVELTLNYVDDIVAEIKRAIEGTPTIENGVPTVPNV

[0209] FKVTLGEIVDLLYKFKQSRLDRTLPKLDNLFEKDLYSTYLSYLPSTDFSYPLLMNVDDRGSFTEFIK

[0210] TPDRGQVSVNISKPGITKGNWHWHHTKNEKFLVVSGKGVIRFRHVNDDEIIEYYVSGDKLEVVDIPVGYTHNIENLGDTDMVTIMWVNEMFDPNQPDTYFLEV*

[0211] SEQ ID NO 8 capG(NWMN_0101):

[0212] MEKLKLMTIVGTRPEIIRLSSTIKADQYFNQILVHTGQNYDYTLNQIFFDDLELRQPDHYLEAVGS

[0213] NLGETMGNIIAKTYDVLLREQPDALLILGDTNSCLAAVSAKRLKIPVFHMEAGNRCFDQNVPEEIN

[0214] RKIVDHVSDVNLPYTEHSRRYLLDEGFNKANIFVTGSPMTEVIEAHRDKINHSDVLNKLGLEPQQY

[0215] ILVSAHREENIDNEKNFKSLMNAINDIAKKYKMPVIYSTHPRSWKKIEESKFEFDPLVKQLKPFGFF

[0216] DYNALQKDAFVVLSDSGTLSEESSILKFPGVLIRTSTERPEVLDKGTVIVGGITYNNLIQSVELAREMQNNNEPMIDAIDYKDTNVSTKVVKIIQSYKDIINRNTWRK*

[0217] SEQ ID NO 9 capH(NWMN_0102):

[0218] MRIAIEKIIGLLKNQSSKESNVKIHRLAYITNSKFDGNNYIDRWCKIRNSHIGEYSYIGFGSDFNNVE

[0219] VGRYCSISSDVKIGLGKHPTHFFSSSPIFYSNNNPFNIKQKFIDFNDQPSRTTIKNDVWIGANVIIMDG

[0220] LTINTGAVIAAGSVVTKNVGAYEVVGGVPAKVIKKRFDNKTIEKLLESKWWEKTPDKLKGFSVEYLNKKDT*

[0221] SEQ ID NO 10 capI(NWMN_0103):

[0222] MRILNIVSSNIVQDPRVLKQIETIKGVTDDYKIVGMNNSQATNKRLENLDCNYRLLGSKVDPKNILS

[0223] KLIKRIRFATGVIREIKAYKPDVIHANDFDVLLMVYLSNYKKANIVYDAHEIYAKNAFINKVPLISKF

[0224] VESIEKHIVKHRVNAFVTVSHAAKEYYQSKGYKKEANVITNAPILNDSREFKEIENFKEIVYQGQIV

[0225] MDRGYEEFIIASSAFKQNAPSFIIRGFGPHEEVIKELISYNPENIRLDKPVEVKELVDKLAESNVGVV

[0226] LTKPVSINFEYTVSNKIFECIHAGLPVILSPVKEHIYLNEKYKFGIVLKEVTPLEIEKAVRKLRDNHDLFNHLRQNAIKASKILNWQIESERLVELYKF*

[0227] SEQ ID NO 11 capJ(NWMN_0104):

[0228] MKFFVLCAIISMNIFIVISTFTKEVLGFPIEPVYYSTMVGIALITTVFAIYKIIVTQEIPRGLILLIAICLLY

[0229] LAFYYFSPDKEEKLAKNNILFFLTWAVPAAISGIYIKYINKATVERFFKLVFFIFSVSFIFVILIPKLTGEI

[0230] PSYINFGLMNYQNASYLSAFTAGLGIYFIMKGSVKHKWIYVLFTIIDIPIVFIPGGRGGAILLILYGLF

[0231] AFILITFKRGIPIAVKSIMYIFALSISSVLIYFLFTKGSNTRTFSYLQGGTLNLEGTSGRGPIYEKGIYFIQ

[0232] QSSLLGYGPFNYYKLIGNIPHNIIIELILSFGLLGFFIIMICILLLVYKMIRNYDPNTIDLLVMFIAIYPITLLMFSSNYLVVSEFWFVLFYFITKGRRHHG*

[0233] SEQ ID NO 12 capK(NWMN_0105):

[0234] MAKKVFIMDSVKTIIGTLLIALGLQFLAYPIINQRVGNEAFGSILTIYTIITITSVVLGNTLNNIRLINM

[0235] NLYKSNHYYWKFASILLISILIESIALIIVFLYFFNLNIIDIIFLILLNILMCLRIYLNVFFRMTLKYNQILY

[0236] IALIQFLGLLIGLFLYYLTQNWIVCFITSELFATIYTLVKLRGLTIGEYQSEDNNVVKDYVMLLSTNSL

[0237] NNLNLYLDRLILLPIIGGTAVTISFLSTFIGKMLATFLYPINNVVLSYISVNESDNIKKQYLKTNLIAIA

[0238] ALCLVMIICYPITIIIVSLLYNIDSSLYSKFIILGNIGVLFNAVSIMIQTLNTKHASITLQANYMTLHTITFIFITILMTIAFGLNGFFWTTLFSNIIKYVILNIIGLKSKFINKKDVD*

[0239] SEQ ID NO 13 capL(NWMN_0106):

[0240] MSEKKILILCQYFYPEYVSSATLPTQLAEDLIANHINVDVMCGWPYEYSNHKQVSKTEMHRGIRIR

[0241] RLKYSRFNNKSKVGRIINFFSLFSKFVINIPKMLKYDQILVYSNPPILPLIPDVLHRLLKKKYSFVVYD

[0242] IAPDNAIKTGATRPGSMIDKLMRYINRHVYKNAENVIVLGTEMKNYLLNHQISKNADNIHVIPNWY

[0243] DMRQLQDNRIYNDTFKAYREQYDKILLYSGNMGQLQDMETLISFLKLNKDQSQTLTILCGHGKKF

[0244] ADVKTAIEDHRIENVKMFEFLTGTDYADVLKIADVCIASLIKEGVGLGVPSKNYGYLAAKKALVLI

[0245] MDKQSDIVQHVEQYDAGIQIDNGDAHAIYNFINTHSSKELHEMGERAHQLFKDKYTREINTMKYYNLLK*

[0246] SEQ ID NO 14 capM(NWMN_0107):

[0247] MKRLFDVVSSIYGLVVLSPILLITALLIKMESPGPAIFKQKRPTINNELFNIYKFRSMKIDTPNVATDL

[0248] MDSTSYITKTGKVIRKTSIDELPQLLNVLKGEMSIVGPRPALYNQYELIEKRTKANVHTIRPGVTGLAQVMGRDDITDDQKVAYDHYYLTHQSMMLDMYIIYKTIKNIVTSEGVHH*

[0249] SEQ ID NO 15 capN(NWMN_0108):

[0250] MRKNILITGVHGYIGNALKDKLIEQGHQVDQINVRNQLWKSTSFKDYDVLIHTAALVHNNSPQAR

[0251] LSDYMQVNMLLTKQLAQKAKAEDVKQFIFMSTMAVYGKEHVGKSDQVDTQTPMNPTTNYGIS

[0252] KKFAEQALQELISDSFKVAIVRPPMIYGAHCPGNFQRLMQLSKRLPIIPNINNQRSALYIKHLTAFIDQ

[0253] LISLEVTGVYHPQDSFYFDTSSVMYEIRRQSHRKTVLINMPSMLNKYFNKLSVFRKLFGNLIYSNTLYENNNALEIIPGKMSLVIADMETTTKDKA*

[0254] SEQ ID NO 16 capO(NWMN_0109):

[0255] MKLTVVGLGYIGLPTSIMFAKHGVLDVLGVDINQQTIDKLQSGQISIEEPGLQEVYEEVLSSGKLKVS

[0256] TTPDASDVFIIAVPTPNNDDQYRSCDISLVMRALDSILSFLEKGNTIIVESTIAKPTMDDFVKPVIENL

[0257] GFTIGEDIYLVHCPERVLPGKILEELVHNNRIIGGVTEACIEAGKRVYRTFVQGEMIETDARTAEMSK

[0258] LMENTYRDVNIALANELTKICNNLNINVLDVIEMANKHPRVNIHQPGPGVGGHCLAVDPPYFIIAKD

[0259] PENAKLIQTGREINNSMPAYVVDTTKQIIKVLSGNKVTVFGLTYKGDVDDIRESPAFDIYELLNQEP

[0260] GIRL YDPHVELDFVEHDMSHAVKDASLVLILSDHSEFKNLSDSHFDKMKHKVIFDTKNVVKSSFEDVSYYNYGNIFNFIDK*

[0261] SEQ ID NO 17 capP(NWMN_0110):

[0262] MCLNFREDNVMKKIMVIFGTRPEAIKMAPLVKEIDNHGNFEANIVITAQHRDMLDSVLSIFDIQAD

[0263] HDLNIMQDQQTLAGLTANALAKLDSIINEEQPDMILVHGDTTTTFVGSLAAFYHQIPVGHVEAGLR

[0264] THQKYSPPFPEELNRVMVSNIAELNFAPTVIAAKNLLFENKDKERIFITGNTVIDALSTTVQNDFVSTII

[0265] NKHKGKKVVLLTAHRRENIGEPMHQIFKAVRDLADEYKDVVFIYPMHRNPKVRAIAEKYLSGRNR

[0266] IELIEPLDAIEFHNFTNQSYLVLTDSGGIQEEAPTFGKPVLVLRNHTERPEGVEAGTSRVIGTDYDNIVRNVKQLIEDDEAYQRMSQANNPYGDGQASRRICEAIEYYFGLRTDKPDEFVPLRHK*

[0267] SEQ ID NO 18 capA(NWMN_0095):

[0268] ATGGAAAGTACATTAGAATTAACAAAAATTAAAGAAGTATTACAAAAAAACTTGAAGATTTTAA

[0269] TTATTTTACCGCTATTATTTTTAATTATTAGCGCTATTGTTACATTTTTCGTCTTATCACCTAAATAT

[0270] CAAGCTAATACTCAAATTTTAGTGAATCAAACTAAGGTGACAATCCTCAGTTTATGGCGCAAG

[0271] AGGTTCAAAGTAATATTCAACTTGTAAATACGTATAAAGAAATTGTTAAAAGTCCTAGAATTTTA

[0272] GATGAGGTGTCAAAGGACTTAAATGATAAGTATTCACCATCTAAATTGTCGAGTATGTTGACAAT

[0273] TACAAACCAAGAAAATACGCAACTTATCAACATCCAAGTTAAAAGTGGTCATAAACAAGATTCG

[0274] GAAAAAAATTGCGAATAGCTTCGCTAAAGTTACAAGTAAACAAATTCCGAAGATTATGAGTGTGG

[0275] ATAACGTATCAATTTTATCTAAAGCAGACGGTACAGCAGTAAAGTCGCACCAAAAACTGTAGT

[0276] GAATCTAATCGGTGCATTCTTTTTAGGATTAGTTGTCGCGCTTATATATATCTTCTTCAAAGTAATT

[0277] TTCGATAAGCGAATTAAAGATGAAGAAGATGTAGAGAAAGAATTAGGATTGCCTGTATTGGGTT

[0278] CAATTCAAAAATTTAATTAA

[0279] SEQ ID NO 19 capB(NWMN_0096):

[0280] TTGCTACTATGTCAAAAAAGGAAAATACGACAACAACTATTTGTATATGAAAAACCAAAAT

[0281] CAACAATTAGTGAAAAGTTTCGAGGTATACGTTCAAACATCATGTTTTCAAAAGCAAATGGTGA

[0282] AGTAAAGCGCTTATTGGTTACTTCTGAAAAGCCCTGGTGCAGGTAAAGTACAGTTGTATCGAAT

[0283] GTAGCGATTACTTATGCACAAGCAGGCTATAAGACATTAGTTATTGATGGCGATATGCGTAAGCC

[0284] AACACAAAACTATATTTTTAATGAGCAAAATAATGGACTATCAAGCTTAATCATTGGTCGAA

[0285] CGACTATGTCAGAAGCAATTACGTCGACAGAAATTGAAAATTTAGATTTGCTAACAGCTGGCCC

[0286] TGTACCTCCAAATCCATCTGAGTTAATTGGGTCTGAAAGGTTCAAAGAATTAGTTGATCTGTTTA

[0287] ATAAACGTTACGACATTATTTGTCGATACACCGCCAGTTAATACTGTGACTGATGCACAACTAT

[0288] ATGCGCGTGCTATTAAAGATAGTCTGTTAGTAATTGATAGTGAAAAAAATGATAAAAATGAAGTT

[0289] AAAAAAGCAAAAGCACTTATGGAAAAAGCAGGCAGTAACATTCTAGGTGTCATTTTGAACAAG

[0290] ACAAAGGTCGATAAATCTTCTAGTTATTCACTTATTATGGAGATGATASEQ ID NO 20 capC(NWMN_0097):

[0291] ATGATTGATATTCATAACCATATTGCCTAATCGATGACGGTCCGACAATGAAACAGAGATG

[0292] ATGGATCTTTTAAAAAACAAGCAWEACHAGGTGTTASUMMERATCATTGTAACATCACCACT

[0293] TACATCCTCGATACCACCTATAGAAAGTGAAATCATGTTTAAACCATTGAAGCTTA

[0294] REPEAT CHAPTERAAATCTAAAGTTTTATGGTATCAAATATATTACCCAAAA

[0295] TCCTTAATTATTGATCGAAAAGTTATTACGGTATATGATTCACGCTATTTACTATATTATTAT

[0296] TCCATCAATGAAGTTCCACACTATACTGATCAATTATTTTTCGAATTACAGAGTAAAGGCTTTG

[0297] TACCGATTATTGCACATCCAGAGCGGATAAAAGCATAAGTCAAAACCTTGACATACTTACGAT

[0298] TTAATTAACAAAGGTGCTTTAAGTCAAGTGACAACGGCGTCATTAGCGGGTATTTCCGGTAAAA

[0299] AATTAGAAAATTAGCAATTCAAATGATTGAAACAATCTGACACATTTCATCGGTTCAGATGC

[0300] GCATAACAGAAATCACCGTTCTTAATGAGACTTATTATTAGAATTACTTACTGATT

[0301] ATTATGAAGATATGAACGGATTTATTAGTAATGCGAAGTTAGTTGTTGATGATAAAAAAAATTCCTA

[0302] AACGAATGCCACAACAAGATTATAAACAGAAAAGATGGTTTGGGTTATAASEQ ID NO 21 capD(NWMN_0098):

[0303] ATGAGGGGTTTTATGGCACATTTATCTGTGAAATTGCGGCTTTTAATACTAGCATTAATCGATTCA

[0304] CTGATAGTGACATTTTCAGTATTCGTAAGTTATTACATTTTAGAACCGTATTTCAAAACATATTCT

[0305] GTCAAATTATTAATATTGGCAGCTATATCACTATTCATATCGCATCATATTTCAGCATTTATTTTTAA

[0306] TATGTATCATCGAGCGTGGGAATATGCCAGTGTGAGTGAATTGATTTTAATTGTTAAAGCTGTGA

[0307] CGACATCTATCGTTATTACGATGGTGGTCGTGACAATTGTTACAGGCAATAGACCGTTTTTTAGA

[0308] TTGTATTTAATTACTTGGATGATGCACTTGATTTTAATAGGTGGCTCAAGGTTATTTTGGGCGTATTT

[0309] ATCGGAAATACCTTGGAGGTAAGTCATTTAATAAGAAGCCAACTTTAGTTGTTGGTGCTGGTCA

[0310] AGCAGGTTCAATGCTGATTAGACAAATGTTGAAAAGTGACGAAATGAAACTTGAACCGGTATT

[0311] AGCAGTCGATGATGACGAACATAAACGCAATATCACAATTACTGAGGGTGAAAAGTCCAAGGT

[0312] AAAATTGCGGATATTCCAGAACTAGTGAGGAAATATAAGATTAAAAAAATCATCATTGCAATTCC

[0313] AACTATTGGTCAAGAGCGTTTGAAAGAAATTAATAATATTTGCCATATGGATGGCGTTGAGTTAT

[0314] TGAAAATGCCAAATATAGAAGACGTCATGTCTGGTGAGTTAGAAGTGAACCAACTTAAAAAAG

[0315] TTGAAGTAGAAGATTTACTAGGCAGAGATCCTGTTGAATTAGATATGGATATGATATCAAATGAA

[0316] TTGACGAATAAAACTATTTTAGTTACGGGTGCAGGTGGTTCAATAGGATCAGAAATTTGTAGAC

[0317] AAGTTTGTAATTTCTATCCAGAACGTATTATTCTACTTGGCCATGGTGAAAACAGTATTTTATTTAA

[0318] TCAATCGTGAATTGCGAAATCGCTTCGGAAAAAATGTTGATATCGTTCCTATTATAGCGGATGTG

[0319] CAAATAGAGCGCGTATGTTTGAAATTATGGAAACGTATAAACCATACGCAGTTTATCATGCAGC

[0320] AGCACACAAGCACGTGCCCGTTAATGGAAGACAACCCTGAAGAAGCAGTACGTAATAATATTTTA

[0321] GGTACGAAAATACTGCTGAAGCTGCTAAAAATGCAGAGGTAAAGAAATTCGTTATGATTTCTA

[0322] CGGATAAAGCCGTTAATCCGCCTAATGTCATGGGAGCTTCAAAGCGAATTGCAGAAATGATTATT

[0323] CAAAGTTTAAATGATGAAACGCATCGAACAAATTTGTTGCAGTGAGATTTGGTAATGTACTTG

[0324] GATCGAGAGGATCTGTGATTCCACTTTTCAAAAGTCAAATTGAAGAAGGTGGGCCAGTTACTGT

[0325] GACACATCCTGAAATGACACGTTACTTTATGACAATTCCTGAAGCTTCTAGACTAGTTTTGCAGG

[0326] CAGGGGCATTAGCAGAAGGTGGCGAAGTATTTGTGCTAGATATGGGAGAACCAGTGAAAATTG

[0327] TAGATTTGGCACGTAATTTAAGCTAAGTGGTAAAAAAGAAGACGACATACGCATTACTTAT

[0328] ACAGGGATTAGACCCGGCGAAAAAATGTTTGAAGAGCTTATGAATAAAGATGAGGTTCATTCCTG

[0329] AACAAGTATTTGAAAAAATTTTATCGTGGCAAAGTACAACATATGAAATGTAATGAAGTTGAAGC

[0330] GATTATTCAAGACATCGTCAATGACTTTTAAAGAAAAAATTATTATGCCAATGGCAAAA

[0331] AGGGAGATAATTTATGTTCGATGA

[0332] SEQ ID NO 22 capE(NWMN_0099):

[0333] ATGTTCGATGACAAAATTTTTATTACTGGGGGCACAGGATCATTCGGTAATGCTGTTATGAA

[0334] ACAGTTTTTAGATTCTAATATTAAAGAAATTCGTATTTTTTCACGCGATGAGAAAAAACAAGATG

[0335] ACATTCGAAAAAAATATAATAATTCAAAATTAAAGTTCTACATTGGTGATGTGCGTGATAGTCAA

[0336] AGTGTAGAAACAGCAATGCGAGATGTTGATTACGTATTCCATGCAGCAGCTTTAAAACAAGTGC

[0337] CGTCATGTGAATTCTTTCCAGTTGAGGCAGTGAAGACAAATATTATTGGTACAGAAAATGTCTTA

[0338] CAAAGTGCTATTCATCAAAATGTTAAAAAAGTCATATGTTTATCTACAGATAAGGCAGCGTATCC

[0339] TATTAATGCTATGGGTATTTCAAAAGCAATGATGGAAAAAGTATTCGTAGCCAAATCAAGAAATA

[0340] TTCGTAGTGAACAAACGCTTATTTGTGGTACAAGATACGGTAATGTGATGGCTTCAAGAGGATC

[0341] AGTAATACCTTTGTTTATCGACAAAATCAAAGCTGGAGAACCTTTAACGATTACAGATCCTGATA

[0342] TGACAAGATTTTTAATGAGCTTAGAAGATGCGGTAGAACTAGTTGTTCATGCATTTAAGCATGCA

[0343] GAGACAGGAGATATTATGGTTCAAAAAGCACCAAGCTCAACGGTAGGGGATCTTGCGACCGCA

[0344] TTATTAGAATTGTTTGAAGCTGATAATGCAATTGAAATCATTGGTACGCGACATGGAGAGAAAA

[0345] AAGCAGAAACATTGTTGACGAGAGAAGAATACGCACAATGTGAAGATATGGGTGATTATTTTAG

[0346] AGTGCCGGCAGACTCCAGAGATTTAAATTATAGTAATTATGTTGAAACCGGTAACGAAAAGATT

[0347] ACGCAATCTTATGAATATAACTCCGATAATACACATATTTTAACGGTGGAAGAGATAAAAGAAAA

[0348] ACTTTTAACACTAGAATATGTTAGAAACGAATTGAATGATTATAAAGCTTCAATGAGATAGSEQ IDNO 23 capF(NWMN_0100):

[0349] TTGAATATTGTAATTACAGGAGCAAAAGGTTTTGTAGGAAAAAACTTGAAAGCAGATTTAACTT

[0350] CAACGACAGATCATCATATTTTCGAAGTACATCGACAAACTAAAGAGGAAGAATTAGAGTCAGC

[0351] ATTGTTGAAAGCAGACTTTGTCGTGCATTTAGCGGGTGTTAATCGACCTGAACATGACAAAGAA

[0352] TTCAGCTTAGGAAACGTGAGTTATTTAGATCATGTACTTGATATATTAACTAGAAATACGAAAAA

[0353] GCCAGCGATATTATTATCGTCTTCAATACAAGCAACACAAGATAATCCTTATGGTGAGAGTAAGT

[0354] TGCAAGGGGAACAGCTATTAAGAGAGTATGCCGAAGAGTATGGCAATACGGTTTATATTTATCGC

[0355] TGGCCAAATTTATTCGGCAAGTGGTGTAAGCCGAATTATAACTCAGTGATAGCAACATTTTGTTA

[0356] CAAAATTGCACGTAACGAAGAGATTCAAGTTAATGATCGGAATGTTGAACTAACGCTAAACTAC

[0357] GTGGATGATATCGTCGCTGAAATAAAGCGTGCTATTGAAGGAACTCCAACGATTGAAATGGTG

[0358] TACCTACAGTACCAAACGTATTTAAAGTGACATTGGGAGAAATTGTAGATTTATTATACAAGTTC

[0359] AACAGTCACGTCTCGATCGAACATTGCCGAAATTAGATAACTTGTTTGAAAAGATTTGTATA

[0360] GTACGTATTTAAGCTATCTACCTAGTACAGACTTTAGTTATTCCCTTACTTATGAATGTGGATGATA

[0361] GGGGTTCTTTTACAGAATTTATAAAAACACCGGATCGTGGTCAAGTTTTCTGTAAATATTTCTAAA

[0362] CCAGGTATTACTAAAGGTAATCACTGGCATCATACTAAAAACGAAAAATTTCTAGTCGTATCAGG

[0363] TAAAGGGGTAATTCGTTTTAGACATGTTAATGATGATGAAATCATTGAATATTATGTTTCTGGCGA

[0364] CAATTAGAAGTTGTAGACATACCAGTAGGATACACACATAATATTGAAAATTTAGGCGACACA

[0365] GATATGGTAACTATTATGTGGGTGAATGAAATGTTTGATCCAAATCAGCCAGATACGTATTTCTTG

[0366] GAGGTATAG

[0367] SEQ ID NO 24 capG(NWMN_0101):

[0368] ATGGAAAAACTGAAATTAATGACAATAGTTGGTACAAGGCCTGAAATCATTCGTTTATCATCAAC

[0369] GATTAAAGCATGTGATCAATATTTTAATCAGATATTAGTACACACTGGTCAAAATTATTGATTATAC

[0370] ATTGAATCAAATTTTCTTTGATGATTTGGAATTAAGACAACCGGACCACTACTTAGGCAGTTG

[0371] GAAGTAACCTTGGAGAAACGATGGGGAATATTTGCGAAGACATATGTGTTTTATTACGCGAA

[0372] CAACCAGATGCACTTTTAATTCTTGGTGATACAAATAGTTGTTTAGCAGCAGTATCTGCTAAACG

[0373] ATTAAAGATTCCTGTGTTCCACATGGAAGCGGGTAATAGATGCTTTGATCAGAATGTACCTGAAG

[0374] AAATCAATCGTAAAATTGTTGACCATGTCAGTGATGTGAATCTACCTTACGGAACATAGCAGA

[0375] CGTTATTTATTAGATGAAGGCTTCAATAAAGCGAATATCTTTGTGACAGGATCACCGATGACAGA

[0376] AGTGATAGAAGCGCATCGAGATAAAATTAATCAGTGACGTTTTAAATAAACTAGGATTAA

[0377] CCGCAACAATACATTTTAGTCTGCGCATAGAGAAGAGAATATCGATAATGAAAAGAATTTTAA

[0378] ATCATTAATGAATGCGATAAATGATATTGCCAAAAAGTATAAAATGCCTGTGATTTATTCAACGCA

[0379] TCCAAGAAGTTGGAAGAAAATTGAAGAAAGTAAATTTGAATTTGATCCATTAGTTAAACAGTTA

[0380] AAGCCATTTGGTTTCTTTGATTATAATGCATTGCAAAAGATGCATTTGTTGTGCTATCAGATAGT

[0381] GGAACATTGTCAGAAGAGTCGTCTATTTTGAAGTTCCCTGGTGTCCTTATTCGAACTTCCACAG

[0382] AAAGACCGGAAGTACTAGATAAAGGTACGGTTATTGTAGGTGGTATTACCTATAACAATCTAATC

[0383] CAATCCGTTGAACTAGCAAGAGAGATGCAAAACAATAACGAACCGATGATTGATGCTATTGATT

[0384] ATAAAGACACTAACGTTTCGACAAAGGTAGTTAAAATTATTCAAAGCTATAAAGATATTATCAAT

[0385] CGAAATACTTGGAGGAAATGA

[0386] SEQ ID NO 25 capH(NWMN_0102):

[0387] ATGAGGATAGCGATTGAAAAGATAATTGGTTTGCTGAAAAACCAGTCCTCTCAAAGAATCGAATG

[0388] TTAAGATTCATCGCTTGGCGTATATTACAAACTCAAAATTTGATGGCAATAACTATATAGATAGAT

[0389] GGTGTAAAATCAGGAATTCTCACATTGGTGAATACAGTTATATTGGATTTGGTAGTGATTTTAATA

[0390] ATGTAGAAGTAGGAAGATATTGTTCGATATCTTCGGATGTAAAAATTGGGTTAGGAACATCCT

[0391] ACACACTTTTTTAGCTCATCACCGATTTTTTATTCTAATAATAATCCATTTAACATAAAGCAAAAG

[0392] TTTATAGACTTTAATGACCAACCAAGCCGTACAACAATTAAAATGATGTGTGGATTGGTGCAA

[0393] ATGTAATTATTATGGATGGTTTAACAATAAATACTGGTGCAGTCATAGCAGCCGGCTCAGTTGTTA

[0394] CTAAAAATGTAGGAGCATATGAGGTTGTTGGTGGTGTTCCTGCAAGGTGATTAAGAAGCGATT

[0395] TGACAATAAAACAATTGAAAAACTTTTGGAAAGCAAGTGGTGGGAGAAAACGCCTGACAAAC

[0396] TAAAAGGATTTTCGGTTGAATATTTAAATAAAAGGATACTTAA

[0397] SEQ ID NO 26 capI(NWMN_0103):

[0398] ATGAGAATTTTTAAATATTGTATCGAGTAATATTGTTCAAGACCCAAGGGTACTTAAACAAATAGA

[0399] AACAATTAAAGGCGTTACGGATGATTATAAATTGTTGGAATGAATAATTCACAAGCTACTAATA

[0400] AGCGATTGGAAAATTTAGATTGTAATTATCGTTTGTTAGGTAGCAAGGTAGATCCAAAAAATATT

[0401] CTTTCTAAATTAATTAAGCGTATAAGATTTGCAACAGGTGTTATCCGAGAAATTAAGCTTATAA

[0402] ACCTGACGTGATTCATGCAAATGATTTCGACGTATTATTAATGGTCTATTTAAGCAATTATAAAAA

[0403] AGCTAATATTGTTTATGATGCGCATGAAATATATGCGAAAAATGCCTTTATTAATAAAGTTCCACT

[0404] TATTTCAAAGTTTGTAGAAAGTATAGAAAACACATAGTAAAAACATCGTGTTAATGCCTTCGTAA

[0405] CAGTAAGTCATGCAGCAAAAGAATATTATCAATCTAAAGGATATAAGAAGGAGCGAATGTTAT

[0406] TACGAATGCACCTATTTTAAATGATAGCAGAGAATTTAAAGAAATCGAAAACTTTAAAGAAATT

[0407] GTATATCAAGGTCAAATTGTAATGGACAGGGATATGAAGAGTTTATTATTGCTTCATCAGCTTTT

[0408] AAACAAAATGCTCCTTCATTCATAATTCGAGGGTTTGGTCCGCATGAAGAAGTGATAAAAGAAC

[0409] TGATTAGTTATAACCCGGAAAATATTAGGTTGGATAAACCAGTTGAAGTAAAAAATTGGTTGAT

[0410] AAGTTAGCAGAAAGTAATGTTGGTGTTGTCTTGACGAAACCAGTATCTATTAATTTTGAATATAC

[0411] AGTATCTAATAAAATTTTTGAATGTATACATGCTGGTTTACCAGTAATTTTATCTCCTGTTCAAAGA

[0412] GCATATTTATCTCAATGAAAAATAAATTTGGCATTGTTCTAAGGAAGTTACGCCGTTAGAAA

[0413] TTGAAAAGGCGTTAGAAAAATTAAGAGATAATCACGATTTGTTTAATCATTTACGTCAAAATGCA

[0414] ATTAAGGCGTCTAAAATTTTGAATTGGCAAATAGAGAGTGAACGGTTTAGTAGAATTATATAAATT

[0415] TTAA

[0416] SEQ ID NO 27 capJ(NWMN_0104):

[0417] ATGAAATTTTTTGTACTTTGTGCAATTATCAGCATGAACATATTTATAGTAATCTCTACATTTACTA

[0418] AAGAAGTATTAGGGTTCCCTATATAGAGCCGGTGTATTACTCAACCATGGTTGGTATAGCATTAATT

[0419] ACTACGGTGTTTGCTATTTATAAGATAATTGTCACCCAAGAAATTCCGCGAGGGTTAATATTATTA

[0420] ATTGCTATATGTTTGCTTTATCTAGCTTTTTATTATTTTTCACCAGATAAGGAAGAGAAACTAGCT

[0421] AAAAATAATATTCTATTCTTTTTAACATGGGCAGTTCCAGCGCAATTAGTGGTATTTATATTAAAA

[0422] TATATAAACAAGGCTACGGTAGAAAGATTTTTTAAATTAGTATTTTCATATTTTCTGTTTCATTTA

[0423] TTTTTGTAATTTTTAATACCAAAACTTACAGGTGAGATACCTAGCTATATCAATTTTGGACTTATGA

[0424] ACTATCAAAACGCTTCGTACCTTTCAGCATTTACTGCCGGATTAGGCATTTATTTCATTATGAAAG

[0425] GTTCAGTTAAACATAAGTGGATATATGTTCTATTTACAATAATTGATATCCCTATTGTGTTTATACC

[0426] AGGAGGGCGTGGAGGTGCTATTTTATTAATTCTTTACGGCTTATTTGCATTTATACTTATTACGTTT

[0427] AAAAGAGGAATACCTATCGCAGTAAAAAGCATTATGTATATTTTTGCATTAAGCATATCTAGTGTA

[0428] TTGATTTACTTTCTTTTTACAAAAGGTTCGAATACTAGAACATTTTCATATCTACAAGGTGGAAC

[0429] ACTTAATTTAGAAGGTACTTCTGGAAGAGGACCGATTTATGAAAAAGGTATTTACTTTATTCAAC

[0430] AAAGTTCGTTATTAGGCTATGGGCCATTTAACTATTATAAACTAATCGGAAATATACCACATAACA

[0431] TCATTATTGAGTTGATTCTATCATTTGGCTTATTAGGGTTTTTTATCATAATGATTTGCATTTTGCTA

[0432] CTAGTTTATAAAATGATTAGGAACTATGATCCAAACACTATAGATTTACTCGTTATGTTTATAGCA

[0433] ATCTATCCAATCACATTATTAATGTTTAGTTCAAATTATTTAGTTGTAAGTGAATTTTGGTTTGTGT

[0434] TGTTCTATTTTATTACAAAAGGACGGCGTCATCATGGCTAA

[0435] SEQ ID NO 28 capK(NWMN_0105):

[0436] ATGGCTAAGAAAGTTTTTATTATGGATAGCGTAAAGACAATAATTGGTACGTTGCTTATAGCTTTA

[0437] GGATTACAATTTTTAGCTTATCCAATTATTAATCAACGAGTAGGTAATGAAGCGTTCGGTTCTATT

[0438] TTAACGATTTATACAATAATAACAATCACGAGTGTTGTATTAGGCAATACGCTTAACAATATACGA

[0439] TTGATTAATATGAATCTATACAAATCCAATCATTACTACTGGAATTTGCATCGATACTTTTAATCT

[0440] CAATTTCGATTGAGAGTATAGCTTTAATTATTGTATTTCTTTACTTTTTTAATTTGAACATCATCGA

[0441] TATTATCTTTTTAATTCTACTTAATATTTTAATGTGTTTAAGGATTTATCTGAATGTATTTTTTAGGA

[0442] TGACTTTAAAATATAATCAGATTTTGTATATTGCTCTTATTCAATTTTTAGGTTTGCTGATAGGACT

[0443] ATTTCTATATTATTTAACCCAAAACTGGATTGTTTGTTTTATTACCAGTGAATTGTTTGCAACGAT

[0444] ATATACATTGGTTAAATTACGGGGATTAACTATAGGCGAGTATCAAAGTGAAGATAATAATGTGG

[0445] TAAAAGATTATGTGATGCTACTGAGTACAAATAGCCTTAATAATTTGAATCTCTATTTAGATAGAT

[0446] TAATCTTATTACCAATTATAGGTGGAACAGCTGTAACTATATCATTTCTTTCAACATTTATTGGGA

[0447] AAATGTTAGCTACATTTCTATATCCGATTAATAATGTAGTACTTTCATATATTTCTGTAAATGAAAG

[0448] TGACAATATAAAGAAGCAATATTTGAAAACTAATCTAATTGCTATAGCTGCCCTATGTTTAGTCAT

[0449] GATTATATGTTATTCCAATTACAATAATTATTGTCTCTTTACTGTATAACATTGATTCAAGTTTATATT

[0450] CGAAGTTTATTATTTTAGGTAATATAGGTGTTTTATTCAATGCAGTGAGTATTATGATCCAAACTTT

[0451] AAATACAAAACACGCATCAATAACATTACAAGCGAATTATATGACGCTTCACACGATTACATTTA

[0452] TATTCATAACTATTTTAATGACAATTGCGTTTGGTCTAAATGGATTCTTTTGGACAACGCTGTTCA

[0453] GCAACATTATTAAGTATGTGATTTTAAATATTATAGGTTTAAAGTCTAAATTCATTAATAAAAAGG

[0454] ACGTCGATTAG

[0455] SEQ ID NO 29 capL(NWMN_0106):

[0456] ATGAGTGAAAAAAAAGATTTTGATTTTATGTCAGTATTTTTATCCGGAATATGTATCTTCTGCGACG

[0457] TTACCAACTCAATTGGCGGAAGATTTAATTGCGAATCACATTAATGTCGATGTCATGTGTGGATG

[0458] GCCATATGAATATAGTAATCATAAACAGGTTTCTAACCGAGATGCATCGTGGTATTCGCATTC

[0459] GACGTCTCAAGTATTCGAGGTTTAATAACAAAAGTAAGGTTGGAAGGATCATCAATTTCTTTAG

[0460] TTTATTTTCAAAATTCGTGATTAATATACCTAAAATGTTGAAATATGATCAGATTCTTGTTTACTCT

[0461] AATCCACCCAATCTTGCCATTAATACCAGACGTTTTACACAGACTGCTTAAGAAAAAATATTCTTT

[0462] TGTGGTGTATGATATAGCACCTGATAATGCGATTAAGACAGGTGCAACTCGTCCAGGTAGCATGA

[0463] TTGATAAGCTGATGCGTTACATTAATAGACATGTCTACAAGAATGCTGAATGTCATTGTCCTT

[0464] GGTACGGAAATGAAAAACTACTTACTAAATCATCAAATTTCTAAAAATGCTGACAATATCCATGT

[0465] GATTCCTAACTGGTATGACATGCGTCAATTACAAGACAATCGTATCTATAATGACACATTTAAAG

[0466] CTTACCGTGAGCAATACGACAAAATTTTATTGTATAGCGGTAATATGGGGCAGTTACAGGATATG

[0467] GAGACACTTATCTCATTTTTAAAATTAAATAAGGATCAGTCTCAAACGTTAACAATACTTTGTGG

[0468] TCATGGTAAGAAATTTGCAGATGTCAAAACGGCAATAGAAGACCATCGTATTGAAAATGTTAAA

[0469] ATGTTTGAGTTTTTAACAGGTACAGACTATGCTGACGTATTAAAAATTGCGGATGTATGTATTGC

[0470] ATCGCTGATTAAAGAAGGCGTCGGTTTAGGCGTGCCGAGCAAGAATTATGGCTATCTTGCAGCT

[0471] AAGAAAGCGTTGGTACTCATCATGGATAAGCAATCTGATATCGTTCAACATGTTGAACAATATGA

[0472] TCGGGTATCCAAATTGATAATGGCGATGCACATGCCATTTATAACTTCATCAACACTCACTCGA

[0473] GTAAGGAATTGCACGAGATGGGTGAGCGCGCACATCAACTGTTTAAAGATAAATATACGAGAG

[0474] AAATTAATACTATGAAGTATTACAATCTGTTGAAGTGA

[0475] SEQ ID NO 30 capM(NWMN_0107):

[0476] ATGAAGCGATTATTCGATGTAGTGAGTTCAATATATGGTTTAGTAGTTTTAAGTCCGATTCTGTTA

[0477] ATTACAGCATTACTAATTAAAATGGAATCACCTGGACCAGCCATTTTTCAAACAAAAAAGACCGA

[0478] CGATTAATAATGAATTGTTTAATATTTATAAGTTTAGATCAATGAAAATAGACACACCTAATGTTG

[0479] CAACTGATTTAATGGATTCAACATCGTATATAACAAAGACAGGGAAGGTCATTCGTAAGACCTC

[0480] TATTGATGAATTGCCACAATTATTGAATGTTTTAAAAGGAGAAATGTCAATTGTAGGTCCTAGAC

[0481] CAGCGCTTTATAATCATACGAATTAATCGAAAAACGTACAAAAGCGAACGTGCATACGATTAG

[0482] ACCAGGTGTGACAGGACTAGCTCAAGTGATGGGGGAGAGATGATATCACTGATGATCAAAGT

[0483] AGCGTATGATCATTATTACTTAACACATCAATCTATGATGCTTGATATGTATATCATATATAAAACA

[0484] ATTAAAAATATCGTTACTTCAGAAGGTGTGCATCACTAA

[0485] SEQ ID NO 31 capN(NWMN_0108):

[0486] ATGAGAAAAAATATTTTAATTACAGGCGTACATGGATATATCGGTAATGCTTTAAAAGATAAGCTT

[0487] ATTGAACAAGGACATCAAGTAGATCAAATTAATGTTAGGAATCAATTATGGAAGTCGACCTCGT

[0488] TCAAAGATTATGATGTTTAATTCATACAGCAGCTTTGGTTCAACAACAATTCACCTCAAGCAAGG

[0489] CTATCTGATTATATGCAAGTGAATATGTTGCTGACGAAACAATTGGCACAAAAGGCTAAAGCTG

[0490] AAGACGTTAAACAATTTATTTTTATGAGTACTATGGCAGTTTATGGAAAAGAAGGTCATGTTGGT

[0491] AAATCAGATCAAGTTGATACACAAACACCAATGAACCCTACGACCAACTATGGTATTTCCAAAA

[0492] AGTTCGCTGAACAAGCATTACAAGAATTGATTAGTGATTCGTTTAAAGTAGCAATTGTGAGACC

[0493] ACCAATGATTTATGGTGCACATTGCCCAGGAAATTTCCAACGGTTAATGCAATTGTCAAAGCGAT

[0494] TGCCAATCATTCCCAATATTAACAATCAGCGCAGTGCATTATATATTAAACATCTGACAGCATTTA

[0495] TTGATCAATTAATATCATTAGAAGTGACAGGTGTGTACCATCCTCAAGATAGTTTTTACTTTGATA

[0496] CATCGTCAGTAATGTATGAAATACGTCGCCAATCACATCGTAAAACGGTATTGATCAACATGCCT

[0497] TCAATGCTAAATAAGTATTTTAATAAGTTGTCGGTCTTTAGAAAATTATTCGGCAATTTAATATAC

[0498] AGCAATACGTTATATGAAAATAATAATGCACTTGAAATTATTCCTGGAAAAATGTCACTTGTTATT

[0499] GCGGACATCATGGATGAAACGACAACCAAAGATAAGGCATAA

[0500] SEQ ID NO 32 capO(NWMN_0109):

[0501] ATGAAGTTAACAGTAGTTGGCTTAGGTTATATTGGTTTACCAACATCAATTATGTTTGCAAAACA

[0502] TGGCGTCGATGTGCTTGGTGTTGATATTAATCAGCAAACGATTGATAAGTTACAAAGTGGTCAA

[0503] ATTAGTATTGAAGAACCTGGATTACAAGAGAGGTTATGAAGAGGTACTGTCATCGGGAAAATTGA

[0504] AGGTATCTACAACGCCAGATGCATCTGATGTTTTTATCATTGCCGTTCCGACGCCGAATAATGAT

[0505] GATCAGTACCGGTCATGTGACATTTCGCTAGTTATGCGTGCATTAGATAGTATTTTTTATCATTTTTAG

[0506] AAAAAGGAAATACCATTATTGTAGAGTCGACAATTGCGCCTAAAACGATGGATGATTTTGTAAA

[0507] ACCAGTCATTGAAAATTTAGGGTTTACAATAGGTGAAGATATTTATTTAGTGCATTGTCCAGAAC

[0508] GTGTACTGCCAGGAAAAATTTTAGAAGAATTAGTTCATAACAATCGTATCATTGGCGGTGTGACT

[0509] GAAGCTTGTATTGAAGCGGGTAAACGTGTCTATCGCACATTCGTTCAGGGAGAAATGATTGAAA

[0510] CAGATGCACGTACTGCTGAAATGAGTAAGCTAATGGAAAACACATATAGAGACGTGAACATTGC

[0511] TTTAGCTAATGAATTAACAAAAATTTGCAATAACTTAAATATTAATGTATTAGATGTGATTGAAAT

[0512] GGCAAACAAACATCCGCGTGTTAACATCCATCAGCCTGGTCCAGGTGTAGGCGGTCATTGTTTA

[0513] GCTGTTGATCCGTACTTTATTATTGCTAAAGACCCTGAAAATGCAAAGTTAATTCAAACTGGACG

[0514] TGAAATTAATAATTCAATGCCGGCCTATGTTGTTGATACAACGAAGCAAATCATCAAAGTGTTGA

[0515] GCGGGAATAAAGTCACAGTATTTGGTTTAACTTATAAAGGTGATGTTGATGATATAAGAGAATCA

[0516] CCAGCATTTGATATTTATGAGCTATTAAATCAAGAACCAGACATAGAAGTATGTGCTTATGATCCA

[0517] CATGTTGAATTAGATTTTGTGGAACATGATATGTCACATGCTGTCAAAGACGCATCGCTAGTATT

[0518] GATTTTAAGTGACCACTCAGAATTTAAAAATTTATCGGACAGTCATTTTGATAAAATGAAGCATA

[0519] AAGTGATTTTTGATACAAAAAATGTTGTGAAATCATCATTTGAAGATGTATCGTATTATAATTATG

[0520] GCAATATATTTAATTTTATCGACAAAATAA

[0521] SEQ ID NO 33 capP(NWMN_0110):

[0522] ATGTGTTTGAACTTCAGAGAGGATAATGTTATGAAAAAAATTATGGTTATTTTCGGTACGAGACC

[0523] CGAAGCAATAAATTGGCACCATTAGTAAGAAGAATTGATCATAATGGGAACTTTGAAGCGAAC

[0524] ATTGTGATTACAGCACAACATAGAGATATGTTAGATAGTGTGTTAAGTATATTTGATATTCAAGCT

[0525] GATCATGATTTAAATATTATGCAAGATCAACAAACATTAGCAGGCCTTACGGCGAATGCACTTGC

[0526] TAAACTTGATAGCATCATTAATGAGGAACAACCGGATATGATTTTAGTACATGGTGATACTACAA

[0527] CGACTTTTGTAGGAAGTTTGGCAGCATTTTATCATCAAATTCCGGTCGGACATGTAGAAGCTGG

[0528] ACTTCGAACACATCAGAAATACTCACCATTTCCTGAAGAGTTAAATCGAGTCATGGTAAGTAAT

[0529] ATTGCTGAATTGAATTTTGCGCCAACAGTAATTGCAGCTAAAAATTTACTTTTTGAAAACAAAG

[0530] ACAAAGAGCGTATCTTTATTACTGGAAATACAGTTATTGACGCATTGTCAACAACAGTTCAAAAT

[0531] GATTTTGTTTCAACGATTATTAATAAACATAAAGGCAAGAAAGTTGTTTTACTAACAGCGCATCG

[0532] TCGTGAAAATATTGGGGAACCGATGCATCAGATTTTTAAAGCAGTAAGAGATTTGGCAGATGAA

[0533] TATAAAGATGTTGTCTTCATTTATCCAATGCATCGTAATCCAAAGGTAAGAGCGATTGCCGAAAA

[0534] ATATTTATCTGGGAGAAATCGGATTGAATTAATTGAGCCATTAGATGCGATTGAGTTCCATAATTT

[0535] TACAAATCAATCGTACCTCGTGCTGACAGATTCTGGTGGTATTCAAGAGGAGGCTCCTACATTTG

[0536] GAAAACCTGTGTTGGTATTAAGGAATCATACAGAGCGTCCCGAAGGCGTTGAGGCGGGAACAT

[0537] CGAGAGTAATTGGCACAGATTATGACAATATTGTTCGAAATGTGAAACAATTGATTGAGGATGAT

[0538] GAAGCGTATCAACGTATGAGTCAAGCGAATAATCCATATGGTGATGGACAAGCATCACGACGTA

[0539] TTTGTGAAGCAATAGAATATTATTTTGGATTGCGCACAGACAAGCCGGATGAATTCGTACCTTTA

[0540] CGTCACAAATAASEQ ID NO 34 saeP amino acid sequence(Newman)

[0541] MNTKYFLAAGAVITTLALGACGNSNSQDQGNKTEQKTKSEDSNVKTDKTKHLTGTFSSKNGETVE

[0542] GKAEIKNGKLMLTNYKSSKGPDLYVYLTKNGDIKNGKEIAMVDYDKEKQTFDLKNVDLSKYDEVTIYCKKAHVIFGGAKLK*

[0543] SEQ ID NO 35 saeQ amino acid sequence(Newman)

[0544] MNKLLLLVTFIIRVGSGIVMLMQGYEKLTGGFTLKGLVPVIANNTDSPEWYKWFFANIVAHTTSLF

[0545] DIVVPLGEIAIGLGLIFGVFAYAASFFGAFVMINYILADMIFTYPLQLTFFILLLMSHSLLKQISLKEIINYFRGRKNRGEKIDDPLTDRG*

[0546] SEQ ID NO 36 saeS amino acid sequence(Newman)

[0547] MVLSIRSQIIIGVVSSIPLTSTILAIILMWFNGHMTLTLTLTTIITSCLTLLICSIFINPLIQKIKQFNIKT

[0548] KQFANGNYASNDKTFNSPKEIYELNQSFNKMASEITQQMNQIKSEQQEKTELIQNLAHDLKTPLASI

[0549] ISYSEGLRDGIITKDHEIKESYDILIKQANRLSTLFDDMTHIITLNTGKTYPPELIQLDQLLVSILQPYE

[0550] QRIKHENRTLEVNFCNEIDAFYRTPLERILTNLLDNALKFSNVGSRIDINISENEDQDTIDIAISDE

[0551] GIGIIPELQERIFERTFRVENSRNTKTGGSGLGLYIANELAQNNAKISVSSDIDVGTTMTVTLHKLDITS*

[0552] SEQ ID NO 37 saeR amino acid sequence(Newman)

[0553] MTHLLIVDDEQDIVDICQTYFEYEGYKVTTTTSGKEAISLLSNDIDIMVLDIMMPEVNGYDIVKEM

[0554] KRQKLDIPFIYLTAKTQEHDTIALTLGADDYVKKPFSPRELVLRINNLLTRMKKYHHQPVEQLSFD

[0555] ELTLINLSKVVTVNGHEVPMRIKEFELLWYLASRENEVISKSELLEKVWGYDYYEDANTVNVHIHRIREKLEKESFTTTITITTVWGLGYKFERSR*

[0556] SEQ ID NO 38 saeP nucleotide sequence(Newman)

[0557] ATGAATACAAAATTTTTTAGCAGCTGGTGCTGTTATCACAACATTAGCTTTAGGTGCTTGTGG

[0558] TAATTCTAATTCACAAGATCAAGGTAACAAAACTGAACAAAAAACAAAGTCAGAAGATAGCAA

[0559] TGTTAAAACTGATAAAACAAAACACCTAACAGGTACATTCAGTTCTAAAAACGGTGAAACTGT

[0560] TGAAGGTAAAGCTGAGATTAAAAATGGTAAATTAATGCTTACTAACTACAAATCATCAAAAGGT

[0561] CCAGATTTATACGTCTACCTAACAAAAAATGGCGACATTAAAAACGGTAAAGAAATCGCAATGG

[0562] TTGACTACGATAAAGAAAAACAAACATTTGATCTTAAAAATGTAGATTTAAGCAAATATGATGA

[0563] AGTAACAATCTATTGTAAAAAAGCTCACGTCATCTTCGGCGGCGCTAAATTAAAATAASEQ ID NO39 saeQ nucleotide sequence(Newman)

[0564] ATGAATAAATTATTGCTACTCGTTACATTTATCATTCGTGTGGGTTCAGGTATTGTTATGTTAATGC

[0565] AAGGCTACGAAAAATTAACGGGCGGATTTACGCTGAAAGGTTTAGTACCAGTCATCGCTAACAA

[0566] TACTGATTCACCAGAGTGGTATAAGTGGTTTTTCGCAAATATAGTTGCACATACGACGTCATTATT

[0567] TGATATTGTTGTCCCACTCGGAGAGATTGCAATTGGATTAGGTTTAATTTTTGGAGTTTTTGCATA

[0568] TGCTGCTAGTTTCTTTGGAGCCTTTGTTATGATAAATTATATCTTAGCAGATATGATATTTACGTAT

[0569] CCTCTTCAATTAACTTTCTTTATCCTTTTACTAATGAGTCACTCATTGTTAAAACAGATTTCACTT

[0570] AAAGAAATCATTAATTACTTTAGAGGTCGTAAGAACAGAGGTGAAAAAAATAGATGACCCACTTA

[0571] CTGATCGTGGATGASEQ ID NO 40 saeS nucleotide sequence(Newman)

[0572] ATGGTGTTATCAATTAGAAGTCAATTATTGGCGTCGTATCGAGTATACCATTAACTTCAACT

[0573] ATTTTAGCAATTGCATATATTTTAATGTGGTTTAACGGCCATATGACACTAACTTTGACCTTAACG

[0574] ACAATAATTACAAGCTGTTTAACCTTATTAATATGTAGTATTTTTATTAATCCACTTATACAAAAAA

[0575] TTAAGCAGTTTAATATAAACTAAGCAATTTGCTAACGGAAATTACGCAAGCAATGATAAAAC

[0576] GTTTAATTCACCAAAAGAAATTTATGAATTAAATCAATCTTTTAATAAAATGGCTTCTGAAATTAC

[0577] GCAACAAATGAATCAAATTAAATCCGAACAACAAGAAAAAACAGAACTGATTCAAAACTTAGC

[0578] CCATGATTTAAAACACCTTTAGCAAGCATTATTTCATATTCTGAAGGACTACGTGATGGTATAT

[0579] CACTAAGGATCATGAGATTAAAGAGTCATACGACATATTAATTAAACAAGCAAACAGATTATCAA

[0580] CATTATTTGATGATATGACTCATATTATCACTTTAAATACAGGTAAAACATATCCCCCAGAATTAAT

[0581] ACAACTAGACCAATTACTTGTATCAATATTGCAACCATATGAGCAACGTATCAAACATGAAAACC

[0582] GCACATTAGAAGTGAATTTCTGTAACGAAATTGATGCATTTTATCAATATCGAACGCCACTTGAG

[0583] CGTATTTTAACAAACTTACTTGATAATGCGCTAAAATTTTCAAATGTTGGTAGTCGCATTGATATT

[0584] AATATTAGTGAAAACGAAGATCAAGATACTATCGACATTGCTATTAGCGATGAAGGTATTGGCAT

[0585] TATACCAGAACTACAAGAACGTATATTCGAACGTACATTCAGAGTAGAAAACTCTCGTAATACA

[0586] AAAACGGGTGGTTCTGGATTAGGCTTATATATAGCTAATGAACTCGCGCAACAAAATAACGCAA

[0587] AAATCAGTGTAAGCAGTGATATAGATGTAGGAACTACGATGACTGTAACATTACACAAATTAGA

[0588] CATTACGTCATAASEQ ID NO 41 saeR nucleotide sequence(Newman)

[0589] ATGACCCACTTACTGATCGTGGATGATGAACAAGACATTGTAGACATTTGTCAAACCTATTTTGA

[0590] ATATGAAGGTTACAAAGTAACAACGACAACTAGCGGTAAAGAAGCAATTTCTTTACTATCAAAT

[0591] GATATTGATATCATGGTACTTGATATCATGATGCCAGAAGTTAATGGTTACGACATTGTCAAAGAA

[0592] ATGAAAAGGCAAAAATTAGATATCCCCTTTATCTATTTAACTGCCAAACACAAGAACATGATAC

[0593] CATTTACGCCTTAACTTTAGGTGCAGATGACTATGTCAAAAAAACCATTTAGTCCAAGGGAACTC

[0594] GTTTTACGTATTAATAATTTACTTACAAGAATGAAGAAATACCATCATCAACCAGTTGAACAACT

[0595] GTCGTTTGATGAATTAACACTTATTAACTTAAGTAAAGTTGTGACTGTAAATGGTCACGAAGTCC

[0596] CTATGCGTATTAAGGAATTTGAGTTATTGTGGTATTTAGCTTCTAGAGAAGAAAATGAAGTTATTTCTA

[0597] AATCAGAATTACTTGAAAAAGTTTGGGGATATGACTATTACGAAGATGCTAATACCGTGAATGTC

[0598] CATATACACCGTATTAGAGAAAAATTAGAAAAGAGAGCTTTACAACATATACCATCACAACTGT

[0599] ATGGGGATTAGGATATAAATTTGAAAGGAGCCGATAA

[0600] SEQ ID NO:42 saeS amino acid sequence(JE2)

[0601] MVLSIRSQIIIGVVSSILLTSTILAIAYILMWFNGHMTLTLTTTTIITSCLTLLICSIFINPLIQKIKQFNIKT

[0602] KQFANGNYASNDKTFNSPKEIYELNQSFNKMASEITQQMNQIKSEQQEKTELIQNLAHDLKTPLASI

[0603] ISYSEGLRDGIITKDHEIKESYDILIKQANRLSLTFDDMTHIITLNTGKTYPPELIQLDQLLVSILQPYE

[0604] QRIKHENRTLEVNFCNEIDAFYQYRTPLERILTNLLDNALKFSNVGSRIDINISENEDQDTIDIAISDE

[0605] GIGIIPELQERIFERTFRVENSRNTKTGGSGLGLYIANELAQQNNAKISVSSDIDVGTTMTVTLHKLDITS*

[0606] SEQ ID NO:43 saS nucleotide sequence (JE2)

[0607] ATGGTGTTATCAAATTGAAGTCCAAATCATTATTGGCGTCGTATCGAGTATACTATTAACTTCAACT

[0608] ATTTTAGCAATTGCATATATTTAATGTGGTTTAACGGCCATATGACACTAACTTTGACCTTAACG

[0609] ACAATAATTACAAGCTGTTTAACCTTTATTAATATGTAGTATTTTTATTAATCCACTTATACAAAAAA

[0610] TTAAGCAGTTTAATATAAAAACTAAGCAATTTGCTAACGGAAATTACGCAAGCAATGATAAAAC

[0611] GTTTAATTCACCAAAAGAAATTTATGAATTAAATCAATCTTTTAATAAAATGGCTTCTGAAATTAC

[0612] GCAACAAATGAATCAAATTAAATCCGAACAACAAGAAAAAACAGAACTGATTCAAAACTTAGC

[0613] CCATGATTTAAAAAACCCTTTAGCAAGCATTATTTCATATTCTGAAGGACTACGTGATGGTATAAT

[0614] CACTAAGGATCATGAGATTAAAGAGTCATACGACATATTAATTAACAAGCAAACAGATTATCAA

[0615] CATTATTTGATGATATGACTCATATTATCACTTTAAATACAGGTAAAACATATCCCCCCAGAATTAAT

[0616] ACAACTAGACCAATTACTTGTATCAATATTGCAACCATATGAGCAACGTATCAAACATGAAAACC

[0617] GCACATTAGAAGTGAATTTCTGTAACGAAATTGATGCATTTTATCAATATCGAACGCCACTTGAG

[0618] CGTATTTTAACAAACTTACTTGATAATGCGCTAAAATTTTCAAATGTTGGTAGTCGCATTGATATT

[0619] AATATTAGTGAAAACGAAGATCAAGATACTATCGACATTGCTATTAGCGATGAAGGTATTGGCAT

[0620] TATACCAGACTACAAGAACGTATATTCGAACGTACATTCAGAGTAGAAACTCTCGTAATACA

[0621] AAAACGGGTGGTTCTGGATTAGGCTTATATATAGCTAATGAACTCGCGCAACAAAATAACGCAA

[0622] AAATCAGTGTAAGCAGTGATATAGATGTAGGAACTACGATGACTGTAACATTACACAAATTAGA

[0623] CATTACGTCATAA

[0624] SEQ ID NO:44 EsxAnucleotide sequence(codon optimized):

[0625] ATGGCGATGATAAAGATGTCACCGGAGGAGATTCGTGCTAAATCACAATCTTACGGTCAGGGAT

[0626] CAGATCAGATAAGACAAATCTTGTCAGATTTGACTAGAGCGCAAGGTGAGATCGCGGCGAATT

[0627] GGGAAGGCCAGGCGTTCTCTAGATTTGAGGAGCAGTTCCAACAGTTATCTCCAAAAGTAGAAA

[0628] AGTTCGCTCAGTTATTGGAGGAGATCAAGCAACAGTTAAATTCTACAGCAGACGCGGTTCAAG

[0629] AACAGGATCAGCAGTTATCAAACAATTTCGGATTGCAGTAA

[0630] SEQ ID NO:45 EsxAamino acid sequence from Newman:

[0631] MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLNSTADAVQEQDQQLSNNFGLQ*

[0632] E SEQ ID NO:46 EsxB nucleotide sequence(codon optimized):

[0633] ATGGGAGGTTACAAAGGTATCAAGGCGGATGGCGGTAAGGTAGATCAGGCGAAGCAGTTGGCG

[0634] GCTAAGACAGCAAAGGATATAGAGGCGTGTCAGAAGCAGACTCAACAATTAGCGGAATATATTG

[0635] AGGGCTCTGACTGGGAGGGTCAGTTCGCTAATAAGGTAAAGGATGTTTTGTTGATTATGGCAAA

[0636] ATTTCAGGAAGAGTTGGTCCAGCCGATGGCGGATCACCAGAAGGCAATTGATAATTTGTCACAA

[0637] AATTTAGCAAAATACGACACATTATCAATCAAACAAGGCTTGGATAGAGTAAATCCATGA

[0638] SEQ ID NO:47 EsxB amino acid sequence from Newman:

[0639] MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP*

[0640] SEQ ID NO:48 mLukS-PV nucleotide sequence:

[0641] atggtcaaaaaaagactattagctgcaacattgtcgttaggaataatcactcctattgctacttcgtttcatgaatctaaagctgataacaatattgagaatattggt

[0642] gatggcgctgaggtagtcaaaagaacagaagatacaagtagcgataagtggggggtcTTTcaaaatattcagtttgattttgttaaagataaaaagtataac

[0643] aaagacgctttgattttaaaaatgcaaggttttatcaattcaaagactacttattacaattacaaaaacacagatcatataaaagcaatgGCTtggcctttccaa

[0644] tacaatattggtctcaaaacaaatgaccccaatgtagatttaataaattatctacctGCTaataaaatagattcagtaaatgttagtcaaacattaggttataaca

[0645] taggtggtaattttaatagtggtccatcaacaggaggtaatggttcatttaattattcaaaaacaattagttataatcaacaaaactatatcagtgaagtagaacgt

[0646] caaaattcaaaaagtgttcaatggggaataaaagctaattcatttatcacatcattaggtaaaatgtctggacatgatccaaatttatttgttggatataaaccaG

[0647] CAagtcaaaatccgagagactattttgttccagacaatgaattacccccattagtacacagtggtttcaatcctGCTtttattgcaactgtttctcatgaaaaa

[0648] ggctcaggagatacaagtgaatttgaaataacgtatggcagaaatatggatgttactcatgctactagaagaacaGCTcactatggcaatagtGCAttag

[0649] aaggatctagaatacacaacgcatttgtaaacagaaattacacagttaaatatgaagtgaactggaaactcatgaaattaaagtgaaaggacataattgaSEQ ID NO:49 mLukS-PV amino acid sequence:

[0650] MVKKRLLAATLSLGIITPIATSFHESKADNNIENIGDGAEVVKRTEDTSSDKWGVFQNIQFDFVKDK

[0651] KYNKDALILKMQGFINSKTTYYNYKNTDHIKAMAWPFQYNIGLKTNDPNVDLINYLPANKIDSVN

[0652] VSQTLGYNIGGNFNSGPSTGGNGSFNYSKTISYNQQNYISEVERQNSKSVQWGIKANSFITSLGKM

[0653] SGHDPNLFVGYKPASQNPRDYFVPDNELPPLVHSGFNPAFIATVSHEKGSGDTSEFEITYGRNMDVTHATRRTAHYGNSALEGSRIHNAFVNRNYTVKYEVNWKTHEIKVKGHN*

[0654] SEQ ID NO:50 mLukF-PV nucleotide sequence:

[0655] atgaaaaaaatagtcaaatcatcagttgttacatcaattgcattgcttttgctatccaatacagttgatgcagctcaacatatcacacctgtaagtgagaaaaagg

[0656] ttgatgataaaattactttgtacaaaacaactgcaacatcagattccgataagttaaaaatttctcagattttaacttttaattttattaaagataaaagttatgataaa

[0657] gatacattaatactcaaagctgctggaaacatttattctggctatacaaagccaaatccaaaagacactattagttctcaattttattggggttctaagtacaacatt

[0658] tcaattaattcagattctaatgactcagtaaacgttgtagattatgcacctGCTaatcaaaatgaagaatttcaagtacaacaaacggtaggttattcttatggtg

[0659] gaGCAattaatatctctaacggcttatcaggtggaggtaatggttcaaaatctttttcagagacaattaactataaacaaGCTagctatagaactagcttag

[0660] ataaaagaactaatttcaaaaaaattggttgggatgttgaagcacataaaattatgaataatggttggggaccatatggcagagatagttatcattcaacttatgg

[0661] taatgaaatgtttttaggctcaagacaaagcaacttaaatgctggacaaacttcttggaatatcacaaaatgccagtgttatccagaggtaacttcaatccaga

[0662] atttattggtgtcctatctcgaaaacaaaacgctgcaaaaaaatcgaaaattactgttacttatcaaagagaaatggatagatatacaaacttttggaatcaacttc

[0663] actggataggtaataattataaagatgaaaaatagagcaactcatacatcaatttatgaagttgattgggaaaatcatacagttaaattaatagatactcaatctaa

[0664] ggaaaaaaatcctatgagctaa

[0665] SEQ ID NO:51 mLukF-PV amino acid sequence:

[0666] MKKIVKSSVVTSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTATSDSDKLKISQILTFNFIKDKS

[0667] YDKDTLILKAAGNIYSGYTKPNPKDTISSQFYWGSKYNISINSDSNDSVNVVDYAPANQNEEFQVQ

[0668] QTVGYSYGGAINISNGLSGGGNGSKSFSETINYKQASYRTSLDKRTNFKKIGWDVEAHKIMNNGW

[0669] GPYGRDSYHSTYGNEMFLGSRQSNLNAGQNFLEYHKMPVLSRGNFNPEFIGVLSRKQNAAKKSKITVTYQREMDRYTNFWNQLHWIGNNYKDENRATTSYEVDWENHTVKLIDTQSKEKNPMS*

[0670] SEQ ID NO:52 mHla nucleotide sequence:

[0671] atgaaaacacgtatgtcagctcagtaacaaacactattgtaggttccatattaatgaatcctgtcgctaatgccgcagattctgatattaatattaaactag

[0672] gtactacagatattggaagcaatactacagtaaaaacaggtgatttagtcacttatgataaagaaaatggcatgctaaaaaagtattttatagttttatcgatgat

[0673] aaaaatctaaataaaaactgctagttattagaacgaaaggtaccattgctggtcaatatagagtttatagcgaagaaggtgctaaaaaagtggtttagcctg

[0674] gccttcagcctttaaggtacagttgcaactacctgaaatgaagtagctcaaatatctgattactatccaagaaattcgattgatacaaaagagtatatgagtactt

[0675] taacttatggattcaacggtaatgttactggtgatgatacaggaaaaattggcggccttattggtgcaaatgtcgattggtctaacactgaaatatgttcaacct

[0676] gatttcaaaacaattttagagagcccaactgataaaaaagtaggctggaaagtgatatttaacaatatggtgaatcaaaattggggaccatatgatagagattct

[0677] tggaacccggtatatggcaatcaacttttcatgaaaactagaaatgctctatgaaagcagcagataacttccttgatcctaaaagcaagttctcttattatctt

[0678] cagggttttcaccagacttcgctacagttattacttatggatagaaaagcatccaaacaaaaatagatgtaatacgaacgagttcgtgatgactac

[0679] caattgctatggacttcaaaattggaaggtaccaataaagataaatggatagatcgttcttcagaaagatataaaatcgattgggaaaaagaagaaat

[0680] gacaataa

[0681] SEQ ID NO:53 mHla amino acid sequence:

[0682] MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKLGTTDIGSNTTVKTGDLVTYDKENGMLKKVFY

[0683] SFIDDKNLNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYPRNSI

[0684] DTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGLTKYVQPDFKTILESPTDKKVGWKVIFNN

[0685] MVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLLWTSTNWKGTNTKDQWIDRSSERYKIDWEKEEMTN*

[0686] SEQ ID NO:54 mSpA nucleotide sequence:

[0687] ttgaaaaagaaaaacatttattcaattcgtaaactaggtgtaggtattgcatctgtaactttaggtacattacttatatctggtggcgtaacacctgctgcaaatgct

[0688] gcgcaacacgatgaagctAAAAAAaatgctttttatcaagtcttaaatatgcctaacttaaatgctgatcaacgcaatggttttatccaaagccttaaaGC

[0689] AGCAccaagccaaagtgctaacgttttaggtgaagctcaaaaacttaatgactctcaagctccaaaagctgatgcgAAAAAAaataacttcaacaa

[0690] agataaaaaaagcgccttctatgaaatcttgaacatgcctaacttaaacgaagcgcaacgtaacggcttcattcaaagtcttaaaGCAGCAccaagcca

[0691] aagcactaacgttttaggtgaagctaaaaaattaaacgaatctcaagcaccgaaagctgataacaatttcaacaaagaaAAAAaatgctttctatgaa

[0692] atcttgaatatgcctaacttaaacgaagaacaacgcaatggtttcatccaaagcttaaaaGCAGCAccaagccaaagtgctaacctattgtcagaagcta

[0693] aaaagttaaatgaatctcaagcaccgaaagcggataacaaattcaacaaagaaAAAAAAaatgctttctatgaaatcttacatttacctaacttaaacga

[0694] agaacaacgcaatggtttcatccaaagcctaaaaGCAGCAccaagccaaagcgctaaccttttagcagaagctaaaaagctaaatgatgctcaagca

[0695] ccaaaagctgacaacaaattcaacaaagaaAAAAAAaatgctttctatgaaattttacatttacctaacttaactgaagaacaacgtaacggcttcatcca

[0696] aagccttaaaGCAGCAccttcagtgagcaaagaaattttagcagaagctaaaaagctaaacgatgctcaagcaccaaaagaggaagacaataacaa

[0697] gcctggcaaagaagacaataacaagcctggcaaagaagacaacaacaagcctggtaaagaagacaacaacaagcctggcaaagaagacggcaacaa

[0698] gcctggtaaagaagacaacaaaaaacctggtaaagaagatggcaacaagcctggtaaagaagacaacaaaaaacctggtaaagaagacggcaacaag

[0699] cctggcaaagaagatggcaacaaacctggtaaagaagatggtaacggagtacatgtcgttaaacctggtgatacagtaaatgacattgcaaaagcaaacg

[0700] gcactactgctgacaaaattgcagcagataacaaattagctgataaaaacatgatcaaacctggtcaagaacttgttgttgataagaagcaaccagcaaacc

[0701] atgcagatgctaacaaagctcaagcattaccagaaactggtgaagaaaatccattcatcggtacaactgtatttggtggattatcattagccttaggtgcagcg

[0702] ttattagctggacgtcgtcgcgaactataa

[0703] SEQ ID NO:55 mSpA amino acid sequence:

[0704] MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAKKNAFYQVLNMPNLNADQRNGFI

[0705] QSLKAAPSQSANVLGEAQKLNDSQAPKADAKKNNFNKDKKSAFYEILNMPNLNEAQRNGFIQSL

[0706] KAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNAFYEILNMPNLNEEQRNGFIQSLKAAPSQS

[0707] ANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSLKAAPSQSANLLAEA

[0708] KKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILAEAKKLNDAQ

[0709] APKEEDNNKPGKEDNNKPGKEDNNKPGKEDNNKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDN

[0710] KKPGKEDGNKPGKEDGNKPGKEDGNGVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELVVDKKQPANHADANKAQALPETGEENPFIGTTVFGGLSLALGAALLAGRRREL*

[0711] SEQ ID NO:56 mTSST1 nucleotide sequence:

[0712] ATGAATAAAAAATTACTAATGAATTTTTTTATCGTAAGCCCTTTGTTGCTTGCGACAATCGCTACA

[0713] GATTTTACCCCTGTTCCCTTATCATCTAATCAAATAATCAAAACTGCAAAAGCATCTACAAATGA

[0714] CAATATTAAGGATTTGTTGGATTGGTATTCAAGTGGCTCTGATACGTTTACAAACTCTGAGGTATT

[0715] GGCTAACAGTAGAGGCTCAATGCGAATTAAAAATACGGACGGTTCAATTAGTTTGATAGCGTTT

[0716] CCATCTCCTTACTACTCTCCGGCATTCACTAAGGGCGAGAAGGTTGACTTAAATACTAAAAGAA

[0717] CGAAGAAGTCAGCACACGTCTGAAGGTACATACATACATTTCCAGATCAGTGGTGTGACTAA

[0718] CACTGAAAAATTGCCAACACTATTGAATTACCTTTGAAAGTTAAAGTGCATGGTAAGGATAGT

[0719] CCATTAAAGTATTGGCCAAAGTTCGACAAAAGCAGTTAGCGATTAGTACATTGGACTTCGAAA

[0720] TCCGTCATCAGTTAACTCAGATTCATGGTTTGTACCGTAGTTCTGACAAGACGGGAGGCTACTG

[0721] GAAAATCACTATGAATGACGGTTCAACTTATCAGAGTGATTTAAGTAAAAAATTCGAGTATAATA

[0722] CGGAGAAACCACCTATAATAGACGAAATCAAAACAATCGAAGCTGAGATAATTAA

[0723] SEQ ID NO:57 mTSST1 amino acid sequence:

[0724] MNKKLLMNFFIVSPLLLATIATDFTPVPLSSNQIIKTAKASTNDNIKDLLDWYSSGSDTFTNSEVLAN

[0725] SRGSMRIKNTDGSISLIAFPSPYYSPAFTKGEKVDLNTKRTKKSQHTSEGTYIHFQISGVTNTEKLPT

[0726] PIELPLKVKVHGKDSPLKYWPKFDKKQLAISTLDFEIRHQLTQIHGLYRSSDKTGGYWKITMNDGSTYQSDLSKKFEYNTEKPPINIDEIKTIEAEIN*

[0727] SEQ ID NO:58 mSEB nucleotide sequence:

[0728] ATGTATAAGAGATTATTTATTTCACATGTAATTTTGATATTCGCACTGATATTAGTTATTTCTACACC

[0729] CAACGTTTTAGCAGAGAGTCAGCCGGACCCGAAGCCTGACGAGTTGCATAAGAGTTCTAAGTT

[0730] TACTGGTTTAATGGAAAACATGAAGGTGTTGTACGATGATAATCATGTCTCAGCAATCAATGTGA

[0731] AGAGTATAGATCAATTCCGATATTTCGACTTGATTTATTCAATTAAGGATACAAAGTTAGGAAATT

[0732] ACGATAATGTACGAGTAGAGTTCAAAAATAAAGACTTGGCGGACAAGTACAAGGACAAGTACG

[0733] TAGACGTTTTTGGTGCAAATGCGTACTACCAGTGCGCTTTCTCAAAAGACAAATGATATTAAT

[0734] TCTCACCAGACTGATAAGCGTAAAACGTGTATGTATGGTGGCGTTACAGAGCATAACGGCAATC

[0735] AATTGGACAAGTACAGATCTATAACTGTAAGAGTGTTTGAAGATGGCAAGAACTTATTGAGTTT

[0736] CGATGTTCAAACTAACAAAAAAAAGGTTACAGCTCAAGAATTAGACTACTTGACTAGACACTAT

[0737] TTGGTTAAGAATAAAAAGTTGTACGAGTTCAACAACTCACCATACGAGACTGGCTACATTAAGT

[0738] TTATTGAAAATGAGAACTCTTTTTGGTACGATATGATGCCTGCACCGGGCGATAAATTCGACCAG

[0739] TCTAAGTATTTAATGATGTATAATGACAACAAGATGGTGGACAGTAAGGACGTTAAGATCGAAG

[0740] TGTACTTGACTACTAAGAAGAAGTGA

[0741] SEQ ID NO:59 mSEB amino acid sequence:

[0742] MYKRLFISHVILIFALILVISTPNVLAESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSI

[0743] DQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANAYYQCAFSKKTNDINSH

[0744] QTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLV

[0745] KNKKLYEFNNSPYETGYIKFIENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK*

[0746] SEQ ID NO:60 mLukA nucleotide sequence:

[0747] atgaaaaataaaaaacgtgttttaatagcgtcatcattatcatgtgcaattttattgttatcagcagcaacgactcaagcaaattcagctcataaagactctcaaga

[0748] ccaaaataagaaagaacatgttgataagtctcaacaaaaagacaaacgtaatgttactaataaagataaaaattcaacagcaccggatgcaattgggaaaaa

[0749] cggtaaaatcacaaaacgaactgaaacagtatatgatgagaaaacaaatatactccaaaatttacaattcgactttatcgatgatccaacttatgacaagaatgt

[0750] attacttgttaaaaaacaaggctcaattcattcaaatttaaagtttgaatctcataaagaagaaaaaaattcaaattggttaaagtatccaagtgagtaccatgtag

[0751] attttcaagtaaaaagaaatcgtaaaactgaaatattagaccaattgccgaaaaataaaatttcaactgcaaaagtagacagtacattttcatatagctcaggtg

[0752] gtaaattcgattcaacaaaaggtattggacgaacttcatcaaatagctactccaaaacgattagttataatcagcaaaattatgacacaattgccagcggtaaaa

[0753] ataataactggcatgtacactggtcagttattgcgaatgacttgaagtatggtggagaagtgaaaaatagaaatgatgaattattattctatagaaatacgagaa

[0754] ttgctactgtagaaaaccctgaactaagctttgcttcaaaatatagatacccagcattagtaagaagtggctttaatccagaatttttaacttatttatctaatgaaa

[0755] agtcaaatgagaaaacgcaatttgaagtaacatacacacgaaatcaagatattttgaaaaacagacctggaatacattatgcacctccaattttagaaaaaaat

[0756] aaagatggtcaaagattaattgtcacttatgaagttgattggaaaaataaaacagttaaagtcgttgataaatattctgatgacaataaaccttataaagaaggat

[0757] aa

[0758] SEQ ID NO:61 mLukA amino acid sequence:

[0759] MKNKKRVLIASSLSCAILLLSAATTQANSAHKDSQDQNKKEHVDKSQQKDKRNVTNKDKNSTAP

[0760] DAIGKNGKITKRTETVYDEKTNILQNLQFDFIDDPTYDKNVLLVKKQGSIHSNLKFESHKEEKNSN

[0761] WLKYPSEYHVDFQVKRNRKTEILDQLPKNKISTAKVDSTFSYSSGGKFDSTKGIGRTSSNSYSKTIS

[0762] YNQQNYDTIASGKNNNWHVHWSVIANDLKYGGEVKNRNDELLFYRNTRIATVENPELSFASKYR

[0763] YPALVRSGFNPEFLTYLSNEKSNEKTQFEVTYTRNQDILKNRPGIHYAPPILEKNKDGQRLIVTYEVDWKNKTVKVVDKYSDDNKPYKEG*

[0764] SEQ ID NO:62 mLukB nucleotide sequence:

[0765] atgattaaacaactatgtaaaaatatcacaatttgtacgttagcactatcgactactttcactgtattaccagctacttcatttgcaaagattaattctgaaatcaaac

[0766] aagtttctgagaagaatcttgatggtgatactaaaatgtatacagagacagctacaacaagtgatagtcaaaaaaatattactcaaagcttacaatttaatttctta

[0767] actgaacctaattgataaaagaaacagtatttattaaagcaaaaggtacaattggtagtggtttgagaattttagacccaaatggttattggaatagtacattaag

[0768] atggcctggatcttattcagtttcaattcaaaatgttgatgacaacaacaatacaaatgtgactgactttgcaccaaaaaatcaggatgaatcaagagaagttaa

[0769] atatacgtatggttataaaacaggtggagatttttcgattaatcgtggaggcttaactggaaatattacaaaagagagtaattattcagagacgattagttatcaa

[0770] caaccatcatatcgtacattacttgatcaatctacgtcacataaaggtgtaggttggaaagtagaagcacatttgataaataatatgggacatgaccatacgag

[0771] acaattaactaatgatagtgataatagaactaaaagtgaaattttttctttaacacgaaatggaaatttatgggcgaaagataatttcacacctaaagacaaaatg

[0772] cctgtaactgtgtctgaagggtttaatccagaatttttagctgttatgtcacatgataaaaaagacaaaggtaaatcacaatttgttgttcattataaaagatcaatg

[0773] gatgagtttaaaatagattggaatcgccatggtttctggggctattggtctggtgaaaaccatgtagataaaaaagaagaaaaattatcagcattatatgaagtt

[0774] gattggaagacacataatgtgaagtttgtaaaagtacttaatgataatgaaaagaaataa

[0775] SEQ ID NO:63 mLukB amino acid sequence:

[0776] MIKQLCKNITICTLALSTTFTVLPATSFAKINSEIKQVSEKNLDGDTKMYTETATTSDSQKNITQSLQF

[0777] NFLTEPNYDKETVFIKAKGTIGSGLRILDPNGYWNSTLRWPGSYSVSIQNVDDNNNTNVTDFAPKN

[0778] QDESREVKYTYGYKTGGDFSINRGGLTGNITKESNYSETISYQQPSYRTLLDQSTSHKGVGWKVEA

[0779] HLINNMGHDHTRQLTNDSDNRTKSEIFSLTRNGNLWAKDNFTPKDKMPVTVSEGFNPEFLAVMSH

[0780] DKKDKGKSQFVVHYKRSMDEFKIDWNRHGFWGYWSGENHVDKKEEKLSALYEVDWKTHNVKFVKVLNDNEKK*。

Claims

1. A live bacterial vaccine for preventing and / or treating Staphylococcus aureus infection in non-human animals, comprising an effective amount of a live strain of Staphylococcus aureus, wherein: Compared with the corresponding control strains, a) the live bacterial strain has reduced Sae two-component system activity, preferably, lacks Sae two-component system activity; b) the live bacterial strain has reduced adenosine synthase A (adsA) activity, preferably, lacks adenosine synthase A (adsA) activity; and c) the live bacterial strain has reduced capsule production, preferably, lacks capsule production; Wherein, the live bacterial strain expresses antigens EsxA, EsxB, mLukS-PV, mLukF-PV, mHla, mSpA, mTSST1, mSEB, mLukA and mLukB.

2. The live bacterial vaccine according to claim 1, wherein a) the expression of one or more genes, preferably all genes, of the Sae two-component system in the live bacterial strain is reduced or not expressed; b) the expression of adsA gene in the live bacterial strain is reduced or not expressed; and c) the expression of one or more capsular polysaccharide synthesis genes, preferably all capsular polysaccharide synthesis genes in the live bacterial strain is reduced or not expressed.

3. The live bacterial vaccine according to claim 1 or 2, wherein a) the viable bacterial strain comprises one or more mutations in one or more genes, preferably all genes, of the sae two-component system; b) the viable bacterial strain comprises a mutation in the adsA gene; and c) The viable bacterial strain contains one or more mutations in one or more capsular polysaccharide synthesis genes, preferably all capsular polysaccharide synthesis genes.

4. The live bacterial vaccine according to claim 3, wherein the mutation is a complete or partial deletion of a gene.

5. The live bacteria vaccine according to claim 4, wherein in the live bacteria strain, the SaeS, SaeR, SaeP and SaeQ genes of the Sae two-component system are completely or partially deleted, the adsA gene is completely or partially deleted, and the capsular polysaccharide synthesis genes capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO and capP genes are completely or partially deleted.

6. The live bacterial vaccine according to claim 4, wherein the SaeS, SaeR, SaeP and SaeQ genes respectively encode the amino acid sequences shown in SEQ ID NOs: 34-37 or amino acid sequences having at least 75% sequence identity thereto; the adsA gene encodes the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least 75% sequence identity thereto; and the capsular polysaccharide synthesis genes capA, capB, capC, capD, capE, capF, capG, capH, capI, capJ, capK, capL, capM, capN, capO and capP genes respectively encode the amino acid sequences shown in SEQ ID NOs: 2-17 or amino acid sequences having at least 75% sequence identity thereto.

7. The live bacterial vaccine according to any one of claims 1 to 6, wherein EsxA comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:44; EsxB comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:46; mLukS-PV comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:48; mLukF-PV comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:50; mHla comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:52; mSpA comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:54; mTSST1 comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:56; mSEB comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:58; mLukA comprises an amino acid sequence having at least 85% 100% sequence identity to SEQ ID NO:60; and mLukB comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

62.

8. The live bacterial vaccine according to any one of claims 1 to 7, wherein the live Staphylococcus aureus strain is derived from the Staphylococcus aureus Newman strain, the USA300 strain, the JE2 strain or the ATCC29213 strain, preferably from the Staphylococcus aureus Newman strain.

9. The live bacterial vaccine according to any one of claims 1 to 8, further comprising an adjuvant and / or a pharmaceutically acceptable carrier.

10. The live bacterial vaccine according to any one of claims 1 to 9, which is administered subcutaneously, intramuscularly, intraperitoneally, orally or intranasally, preferably subcutaneously.

11. The live bacterial vaccine according to any one of claims 1 to 10, wherein the non-human animal is selected from sheep, goats, pigs, cattle, horses, donkeys, cats, dogs, chickens, ducks, and geese, preferably, the non-human animal is a sheep or a goat.

12. The live bacterial vaccine according to any one of claims 1 to 11, wherein the Staphylococcus aureus infection is an anaerobic Staphylococcus aureus (S. aureus subspecies anaerobius) infection; for example, the infection is caused by anaerobic Staphylococcus aureus strains MVF-7 (ATCC 35844), ST1464, Polonia 123. Su-1, Su-4, Po-3, DK-3, It-4, MVF-9, MVF-16, MVF-17-II, MVF-29, MVF-30, MVF-39, MVF-39+, MVF-49, MVF-58, MVF-71, MVF-80, MVF-91, MVF-205, MVF -212, MVF-220, MVF-228, MVF-302, MVF-401, MVF-502, 39+1, 39+2, 43+, 502R+1, 502R+2, 7R0+1, It4R+, MVF43, SU4+, MVF-7+, RDKA-84, MVF84 or its homologous strains.

13. The live bacterial vaccine according to any one of claims 1 to 12, wherein the vaccine is used to prevent and / or treat mastitis, arthritis, or skin lesions caused by Staphylococcus aureus infection.

14. The live bacterial vaccine according to any one of claims 1 to 12, wherein the vaccine is used to prevent and / or treat Morel's disease.

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