Detection method of salmonella

Abstract

To conduct detection of Salmonella, serotype transversely and specifically relative to other strains.SOLUTION: A heat-killed bacterial cell of Salmonella and antigen liquid containing an adjuvant are inoculated in a BALB / c mouse. After the inoculation, splenocytes or lymph node cells of the mouse and P3U1 are fused to prepare a hybridoma, and therefrom, a hybridoma for producing a monoclonal IgG antibody showing Salmonella specificity is selected, whereby three hybridomas for producing superior Salmonella specific antibodies are successfully acquired. Next, these monoclonal antibodies are used to detect Salmonella of a plurality serotypes and other strains according to sandwich ELISA, thereby obtaining a result in which Salmonella is detected serotype transversely, whereas other strains are not detected.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting Salmonella, an antibody used in the method, a kit for use in the method, an antibody for preventing or treating Salmonella infection and a pharmaceutical composition containing the antibody, and a vaccine against Salmonella infection. [Background technology]

[0002] Salmonella (Salmonella enterica) is a gram-negative, facultative anaerobic bacillus belonging to the Enterobacteriaceae family. Salmonella is generally classified into approximately 2,500 serotypes based on the combination of O and H antigens. Among these, the subspecies I (subsp. enterica), which causes gastroenteritis, contains approximately 1,500 serotypes. According to a WHO report, approximately 600,000 people develop bacteremia due to Salmonella annually, of which approximately 80,000 die. In Japan, there are more than 200 cases of Salmonella food poisoning annually, with over 500 cases in some years. In particular, Salmonella is considered a particularly common cause of food poisoning, with mass outbreaks occurring in large facilities such as schools and hospitals.

[0003] The use of antibodies for Salmonella detection is described, for example, in Patent Document 1. Patent Document 1 discloses the use of antibodies that bind to pagC, oppA, sapA, sinI, shdA, stbD, tcfB, topB, yfeN, yijO, yhiD, or yhjD polypeptides to detect Salmonella, particularly S. typhi and S. typhimurium. However, antibodies often have such high antigen specificity that they cannot detect different serotypes of Salmonella. In situations where Salmonella detection is actually required, it is necessary to determine whether or not the sample is Salmonella. Furthermore, since it is unknown which serotype of Salmonella is contained in the sample, the required Salmonella detection antibody does not react only with a specific serotype, but rather reacts across multiple serotypes of Salmonella while not reacting with other bacteria.

[0004] Treatment for Salmonella infections typically involves fluid replacement and intravenous infusion to prevent dehydration and promote bacterial excretion, but these measures are symptomatic and not considered proactive. Antibiotics such as ciprofloxacin, azithromycin, or ceftriaxone may also be used, but these may actually prolong bacterial excretion and are therefore only administered when there is a risk of bacteremia. Furthermore, drug-resistant Salmonella has been increasing in recent years.

[0005] As described above, control of Salmonella is a major public health issue. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. WO2007 / 027936 Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to perform simple, rapid, and highly sensitive detection of Salmonella across serotypes, which is required in various situations such as medical settings, food contamination sites, laboratories, etc. Furthermore, the present invention also provides new active ingredients useful for preventing or treating Salmonella infections, pharmaceuticals containing such active ingredients, and vaccines against Salmonella infections. [Means for solving the problem]

[0008] The present inventors conducted extensive research to solve the above-mentioned problems. First, BALB / c mice were inoculated with an antigen solution containing heat-killed Salmonella bacteria and an adjuvant. After inoculation, the mouse spleen cells or lymph node cells were fused with P3U1 to produce hybridomas. From these, hybridomas producing monoclonal IgG antibodies specific to Salmonella were selected, and three hybridomas producing excellent Salmonella-specific antibodies were successfully isolated.

[0009] These monoclonal antibodies were then evaluated by ELISA to confirm their recognition of the Salmonella-derived oligopeptide-binding protein (Salmonella OppA) and their reactivity against multiple serotypes of Salmonella and other commensal and pathogenic bacteria.The results showed that they detected all tested Salmonella species with high sensitivity, but did not detect bacteria other than Salmonella.

[0010] Furthermore, we found that one of these monoclonal antibodies can protect against Salmonella infection by inhibiting Salmonella growth and energy metabolism, and that this protection can suppress the symptoms caused by Salmonella infection, such as weight loss and even individual death. Furthermore, we found that Salmonella OppA induces antibody production that reacts across serotypes of Salmonella, and therefore, it can be used as a vaccine against Salmonella infection.

[0011] Thus, in a first aspect, the present invention provides a method for cross-serotype detection of Salmonella in a sample (hereinafter abbreviated as the detection method of the present invention), which comprises contacting the sample with one or two antibodies selected from the following group (hereinafter abbreviated as the antibody group of the present invention): 1) L2-1H11 antibody having at least the amino acid sequence of SEQ ID NO: 9 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 12 as the VL CDR3 region; 2) the 15A7 antibody, having at least the amino acid sequence of SEQ ID NO: 19 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 22 as the VL CDR3 region; 3) the 19D5 antibody, having at least the amino acid sequence of SEQ ID NO: 29 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 32 as the VL CDR3 region; and 4) An antibody having one or two amino acid mutations in each of the VH CDR3 region and / or VL CDR3 region of the antibody of 1) to 3) above.

[0012] The antibodies belonging to the antibody group of the present invention are preferably isolated antibodies. The antibodies belonging to the antibody group of the present invention may be polyclonal or monoclonal antibodies. From the viewpoint of preventing erroneous detection due to non-specific reactions, it is preferable to use monoclonal antibodies. However, it is also preferable to use polyclonal antibodies from the viewpoints that they are inexpensive, can be produced in a relatively short time, have high detection sensitivity, and have a high ability to capture target proteins.

[0013] In the antibody of 4) above, the amino acid mutation may be any of substitution, deletion, or insertion of an amino acid residue, but substitution is preferred, and conservative amino acid substitution is particularly preferred from the viewpoint of maintaining immunological specificity.

[0014] The detection method of the present invention is preferably an immunochromatography method using any two antibodies selected from the above-mentioned group of antibodies of the present invention as a labeled antibody and a capture antibody, respectively.

[0015] In further preferred embodiments, the antibodies of the present invention have the following characteristics: 1) an L2-1H11 antibody having at least the amino acid sequence of SEQ ID NO: 7 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 8 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 9 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 10 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 11 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 12 as a VL CDR3 region; 2) the 15A7 antibody, which has at least the amino acid sequence of SEQ ID NO: 17 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 18 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 19 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 20 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 21 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 22 as a VL CDR3 region; 3) a 19D5 antibody having at least the amino acid sequence of SEQ ID NO: 27 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 28 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 29 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 30 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 31 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 32 as a VL CDR3 region; and 4) An antibody having one or two amino acid mutations in each of the VH CDR1 region, VH CDR2 region, VH CDR3 region, VL CDR1 region, VL CDR2 region and / or VL CDR3 region of the antibody of 1) to 3) above.

[0016] In a more preferred embodiment, the antibodies of the present invention have the following characteristics: 1) L2-1H11 antibody having the amino acid sequence of SEQ ID NO: 3 as the VH region and the amino acid sequence of SEQ ID NO: 5 as the VL region; 2) 15A7 antibody having the amino acid sequence of SEQ ID NO: 13 as the VH region and the amino acid sequence of SEQ ID NO: 15 as the VL region; 3) the 19D5 antibody, having the amino acid sequence of SEQ ID NO: 23 as the VH region and the amino acid sequence of SEQ ID NO: 25 as the VL region; and 4) An antibody having a sequence identity of 80% or more with the antibodies of 1) to 3) above.

[0017] In a second aspect, the present invention provides an antibody for serogroup-wide detection of Salmonella, which is any one antibody selected from the above-described group of antibodies of the present invention, or a combination of two or more identical or different antibodies. The antibody combination is, for example, a combination of two identical or different antibodies used for detecting Salmonella by immunochromatography. More preferably, it is a combination of the above-described 1) L2-1H11 antibody or 4) antibodies derived from the L2-1H11 antibody of 1), with the above-described 2) 15A7 antibody or 3) 19D5 antibody, or an antibody derived from the 2) 15A7 antibody or 3) 19D5 antibody of 4). The antibody of the present invention is characterized by recognizing Salmonella OppA.

[0018] In a third aspect, the present invention provides a kit for cross-serotype detection of Salmonella, comprising any one or a combination of antibodies selected from the above-described group of antibodies of the present invention. The kit comprises, for example, the L2-1H11 antibody (described above in 1) immobilized linearly on a solid phase to serve as a capture antibody, and the 15A7 antibody (described above in 2) sensitized with gold colloid to serve as a labeled antibody.

[0019] In a fourth aspect, the present invention provides the L2-1H11 antibody described above in 1) or an antibody derived from the L2-1H11 antibody described above in 4), which is used as an antibody for preventing or treating Salmonella infection.The present invention also provides a pharmaceutical composition for preventing and / or treating Salmonella infection, comprising the antibody as an active ingredient. In a fifth aspect, the present invention provides a Salmonella OppA protein as an active ingredient of a vaccine for Salmonella infection. The present invention also provides a Salmonella infection vaccine, characterized by comprising a Salmonella OppA protein as an active ingredient. [Effects of the Invention]

[0020] The present invention enables the simple (e.g., kit-based detection of Salmonella across multiple serotypes, eliminating the need for a special laboratory), rapid (test time: less than 1 hour, preferably less than 30 minutes, e.g., 15 minutes) and highly sensitive detection of other bacteria in clinical settings. Furthermore, the L2-1H11 antibody described in 1) above reduced the oxygen consumption of Salmonella over time, suggesting that the antibody may be capable of inhibiting Salmonella growth. Furthermore, when the L2-1H11 antibody described in 1) above was administered to mice, preventive and therapeutic effects against Salmonella infection were confirmed. Furthermore, because all of these antibodies recognize the Salmonella OppA protein, the Salmonella OppA protein could be used as a vaccine against Salmonella. [Brief explanation of the drawings]

[0021] [Figure 1] The reactivity of L2-1H11 monoclonal IgG antibody against Salmonella and other commensal and pathogenic bacteria was evaluated by ELISA. [Figure 2] Solubilized S. Typhimurium was treated with the L2-1H11 antibody and immunoprecipitation was performed. After SDS-PAGE, the gel was silver stained and subjected to proteome analysis using a mass spectrometer. Western blotting was also performed using the immunoprecipitated sample. The black arrow indicates the molecule recognized by the L2-1H11 antibody. [Figure 3] E. coli was transformed with the OppA plasmid derived from S. Typhimurium to produce recombinant OppA protein, which was then subjected to SDS-PAGE and CBB staining (left). Western blotting using the SDS-PAGE gel confirmed that the recognition molecule for L2-1H11 was OppA (right). [Figure 4]The reactivity of 12 anti-OppA monoclonal antibodies against solubilized Salmonella serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Thompson, S. Schwarzengrund, E. coli DH5α, and recombinant OppA proteins was assessed by ELISA. [Figure 5] The reactivity of 12 anti-OppA monoclonal antibodies against solubilized Salmonella serovar S. Enteritidis was evaluated by sandwich ELISA. The antibodies listed below the horizontal axis are the capture antibodies immobilized on a solid phase, and the bar graphs for each antibody show the reactivity with the labeled antibodies 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11, from left to right. [Figure 6] The reactivity of 12 anti-OppA monoclonal antibodies against solubilized Salmonella serovar S. Typhimurium was evaluated by sandwich ELISA. The antibodies listed below the horizontal axis are the capture antibodies immobilized on a solid phase, and the bar graphs for each antibody show the reactivity with the labeled antibodies 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11, from left to right. [Figure 7] The reactivity of 12 anti-OppA monoclonal antibodies against solubilized Salmonella serovar S. Infantis was evaluated by sandwich ELISA. The antibodies listed below the horizontal axis are the capture antibodies immobilized on a solid phase, and the bar graphs for each antibody show the reactivity with the labeled antibodies 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11, from left to right. [Figure 8]The reactivity of 12 anti-OppA monoclonal antibodies against solubilized Salmonella serovar S. Thompson was evaluated by sandwich ELISA. The antibodies listed below the horizontal axis are the capture antibodies immobilized on a solid phase, and the bar graphs for each antibody show the reactivity with the labeled antibodies 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11, from left to right. [Figure 9] The reactivity of 12 anti-OppA monoclonal antibodies against solubilized Salmonella serovar S. Schwarzengrund was evaluated by sandwich ELISA. The antibodies listed below the horizontal axis are the capture antibodies immobilized on a solid phase, and the bar graphs for each antibody show the reactivity with the labeled antibodies 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11, from left to right. [Figure 10] The reactivity of 12 anti-OppA monoclonal antibodies with solubilized E. coli DH5α was evaluated by sandwich ELISA. The antibodies listed below the horizontal axis are the capture antibodies immobilized on a solid phase. The bars for each antibody show the reactivity with the labeled antibodies, 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11, from left to right. [Figure 11] The reactivity of 12 anti-OppA monoclonal antibodies with solubilized OppA recombinant protein was evaluated by sandwich ELISA. The antibodies listed below the horizontal axis are the capture antibodies immobilized on a solid phase. The bars for each antibody show the reactivity with the labeled antibodies, 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11, from left to right. [Figure 12] Direct ELISA confirmed that purified polyclonal antibodies obtained from the serum of rabbits immunized with the OppA recombinant protein reacted with the OppA recombinant protein. [Figure 13]The L2-1H11 monoclonal antibody and purified polyclonal antibodies derived from rabbit serum were used as solid-phase and detection antibodies in sandwich ELISA, and it was confirmed that the OppA recombinant protein and multiple serotypes of Salmonella (S. Enteritidis, S. Typhimurium, S. Infantis, S. Thompson, S. Schwarzengrund) could be detected. [Figure 14] The 15A7 monoclonal antibody and purified polyclonal antibodies derived from rabbit serum were used as solid-phase and detection antibodies in sandwich ELISA, and it was confirmed that the OppA recombinant protein and multiple serotypes of Salmonella (S. Enteritidis, S. Typhimurium, S. Infantis, S. Thompson, S. Schwarzengrund) could be detected. [Figure 15] The 19D5 monoclonal antibody and purified polyclonal antibodies derived from rabbit serum were used as solid-phase and detection antibodies in sandwich ELISA, and it was confirmed that the OppA recombinant protein and multiple serotypes of Salmonella (S. Enteritidis, S. Typhimurium, S. Infantis, S. Thompson, S. Schwarzengrund) could be detected. [Figure 16] To determine whether the anti-OppA monoclonal antibodies 15A7, 19D5, and L2-1H11 have functionality against Salmonella, we focused on their effect on the energy metabolism of Salmonella after antibody addition and measured oxygen consumption rate (OCR) using a flux analyzer. In the figure, "OppA specific mAb" refers to 15A7 and 19D5. [Figure 17] The supernatant of the fecal homogenate solution obtained from Salmonella-infected mice was inoculated onto Salmonella-Shigella agar medium, and the CFU were counted after overnight incubation. [Figure 18]The intestines were excised from Salmonella-infected mice 2 and 4 days after infection, and the number of neutrophils in the lamina propria was calculated by FACS analysis. [Figure 19] Myeloperoxidase (MPO), a neutrophil-specific enzyme, was measured by ELISA in feces from Salmonella-infected mice. [Figure 20] 1 shows the preventive effect of L2-1H11 antibody against Salmonella infection. [Figure 21] 1 shows the therapeutic effect of L2-1H11 antibody after Salmonella infection. [Figure 22] 1 shows the reactivity of antibodies produced by subcutaneous administration of OppA recombinant protein to OppA and each serotype of Salmonella. [Figure 23] 1 shows the neutralizing activity of antibodies produced by subcutaneous administration of OppA recombinant protein. [Figure 24] 1 shows the survival rate after Salmonella infection in patients receiving subcutaneous administration of OppA recombinant protein. [Figure 25] 1 shows the reactivity of IgA antibodies produced by oral administration of OppA recombinant protein to OppA and each serotype of Salmonella. [Figure 26] 1 shows the neutralizing activity of IgA antibodies produced by oral administration of OppA recombinant protein. [Figure 27] 1 shows the results of a Salmonella infection test after administration of OppA recombinant protein. [Figure 28] 1 shows the survival rate after Salmonella infection in patients receiving subcutaneous administration of OppA recombinant protein. [Figure 29] 1 shows the inhibitory effect of administration of OppA recombinant protein on bacterial translocation after Salmonella infection. [Figure 30] 1 shows the inhibitory effect of administration of OppA recombinant protein on the inflammatory response associated with bacteremia after Salmonella infection. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention is described in detail below. The terms used in this specification are understood according to their general definitions in the fields of microbiology, biochemistry, molecular biology, medicine, and pharmacology. However, for terms specifically explained or defined in this specification, the explanations or definitions in this specification take precedence. Furthermore, the literature cited in this specification is incorporated herein by reference.

[0023] As used herein, Salmonella (Salmonella enterica) refers to a bacterial species of subsp. enterica, which is a non-typhoidal Salmonella, and particularly refers to, but is not limited to, S. Typhimurium, S. Enteritidis, S. Infantis, S. Thompson, S. Schwarzenground, and other species that are often identified as the cause of infectious diseases in Japan. When simply referred to as Salmonella in this specification, it is intended to comprehensively refer to non-typhoidal Salmonella.

[0024] In a first aspect, the present invention relates to a method for detecting Salmonella in a sample by recognizing Salmonella OppA as an antigen using an antibody selected from the group of antibodies of the present invention. The specific technique for this method can be selected from various techniques utilizing antigen-antibody reactions and is not limited to a specific technique. Examples of specific techniques that can be used in the detection method of the present invention include, but are not limited to, immunoprecipitation, Western blotting, ELISA, and immunochromatography.

[0025] Western blotting is a method in which proteins are transferred to a membrane after SDS-PAGE and then a specific protein is detected using an antibody. In the detection method of the present invention, the antibody used is an antibody selected from the group of antibodies of the present invention, the protein to be detected is Salmonella OppA, and the reagents, procedures, and conditions of conventional Western blotting can be used.

[0026] ELISA is a method in which a target antigen or antibody contained in a sample is captured with a specific antibody or antigen and quantitatively detected using an enzyme reaction. ELISA methods include direct, indirect, sandwich, and competitive methods, each of which uses different reagents and specific procedures. However, those skilled in the art can perform ELISA by selecting an antibody selected from the antibody group of the present invention as the antibody to be used and Salmonella OppA as the protein to be detected, and by appropriately selecting other conditions, etc.

[0027] Immunochromatography is understood as an immunoassay method that detects an antigen using a labeled antibody and a capture antibody. Typically, the steps of immunochromatography include contacting a sample with a labeled antibody, applying the sample to a sample application portion of a solid phase, developing the labeled antibody-antigen conjugate in the sample, and capturing the labeled antibody-antigen conjugate with a capture antibody immobilized on a determination portion to form a labeled antibody-antigen-capture antibody conjugate, which is then determined visually or by an appropriate measurement device. Therefore, the detection method of the present invention typically includes: 1) a step of contacting a sample with a labeled antibody; 2) a step of developing the sample contacted with the labeled antibody on a solid phase having a determination portion to which a capture antibody is immobilized; and 3) a step of confirming the determination portion.

[0028] In the detection method of the present invention, the specimen may be any specimen used in food poisoning tests, such as feces, wastewater, beverages, or food, and may be a specimen diluted or suspended in water, a buffer solution, etc. The term "specimen" also encompasses not only specimens or specimen dilutions or suspensions, but also mixtures of these with labeled antibodies.

[0029] In the detection method of the present invention, the labeled antibody is a labeled antibody selected from the group of antibodies of the present invention or an antibody that specifically reacts with the antibody. The labeling method may be any method commonly used in the art, and typically includes, but is not limited to, binding a metal colloid, a fluorescent dye, magnetic latex beads, or the like to the antibody, such as sensitizing the antibody to gold colloid particles as a metal colloid.

[0030] In the detection method of the present invention, the capture antibody is an antibody selected from the group of antibodies of the present invention that binds to Salmonella OppA. For example, in immunochromatography, the capture antibody is typically immobilized on a solid phase on which the sample is developed. The capture antibody captures the antigen bound to the labeled antibody, i.e., the labeled antibody-bound OppA, on the solid phase. Typically, the capture antibody is immobilized on a portion of the solid phase, for example, in a linear fashion perpendicular to the direction of electrophoresis, to form a test area.

[0031] In the detection method of the present invention, the solid layer may be any solid layer commonly used in antibody-based antigen detection, including, but not limited to, a solid layer made of a microporous material such as glass fiber, nylon, nitrocellulose, polyvinylidene difluoride, cellulose acetate, etc. Furthermore, functionally, the solid layer includes a sample application area, an electrophoresis area, and a determination area.

[0032] In the detection method of the present invention, the method of developing the sample is appropriately selected depending on the solid layer. For example, the development may be based on capillary action in the solid layer or on electrophoresis with the application of an external force such as electricity. Electrophoresis utilizing capillary action in the solid layer is preferred because it requires less equipment and is simpler.

[0033] In the detection method of the present invention, it is of course also possible to detect an antigen in a liquid sample by contacting it with an antibody selected from the group of antibodies of the present invention, without using a solid phase.

[0034] In the detection method of the present invention, the determination or confirmation of the determination portion is appropriately selected depending on the labeled antibody. For example, such determination or confirmation of the determination portion includes not only visual inspection but also measurement methods depending on the label attached to the antibody, such as fluorescence measurement. Visual inspection is preferred because it requires less equipment, but fluorescence measurement, for example, has the advantage of enabling quantitative measurement based on fluorescence intensity.

[0035] OppA, the target of recognition in the detection method of the present invention, is known as an oligopeptide-binding protein in various bacteria, and its structure and function have already been the subject of extensive research (e.g., Ian D. Hiles and Christopher F. Higgins, Peptide uptake by Salmonella typhimurium The periplasmic oligopeptide-binding protein. Eur. J. Biochem. 158, 561-567 (1986); the contents of this document are incorporated herein by reference). The detailed structure of OppA is registered in public databases (such as UniProtKB and GenBank). However, the present inventors performed homology analysis using CLUSTALW on the OppA amino acid sequences derived from five Salmonella serovars (Enteritidis, Typhimurium, Infantis, Thompson, and Schwarzengrund) and E. coli. The homology between the Salmonella OppA amino acid sequences was over 99%, but when comparing the OppA amino acid sequences of the five Salmonella serovars with E. coli, the homology was approximately 84%. For reference, the amino acid sequences of S. Enteritidis and E. coli DH5α are shown below as SEQ ID NOs: 1 and 2, respectively. It should be understood that for all amino acid sequences, the one-letter and three-letter abbreviations are interchangeable.

[0036] S. Enteritidis OppA amino acid sequence (SEQ ID NO: 1): MSNITKKSLIAVGILTALIAASAATAADVPAGVQLADKQTLVRNNGSEVQSLDPHKIEGVPESNVSRDLFEGLLISDVEGHPSPGVAEKWENKDFKVWTFHLRENAKWSDGTPVTAHDFVYSWQRLADPNTASPY ASYLQYGHIANIDIIAGKKPATDLGVKALDHTFEVTLSEPVPYFYKLLVHPSVSPVPKSAVEKFGDKWTQPANIVTNGAYKLKNWVVNERIVLERNPQYWDNDKTVINQVTYLPISSEVTDVNRYRSGEIDMTY NNMPIELFQKLKKEIPNEVRVDPYLCTYYYEINNQKAPFNDVRVRTALKLALDRDIIVNKVKNQGDLPAYSYTPPYTDGAKLVEPEWFKWSQQKRNEEAKKLLAEAGFTADKPLTFDLLYNTSDLHKKLAIAVASI WKKNLGVNVNLENQEWKTFLDTRHQGTFDVARAGWCADYNEPTSFLNTMLSDSSNNTAHYNSPAFDKLIADTLKVADDTQRSELYAKAEQQLDKDSAIVPVYYYVNARLVKPWVGGYTGKDPLDNIYVKNLYIIKH

[0037] OppA amino acid sequence of E. coli DH5α (SEQ ID NO: 2): MTNITKRSLVAAAGVLAALMAGNVALAADVPAGVTLAEKQTLVRNNGSEVQSLDPHKIEGVPESNISRDLFEGLLVSDLDGHPAPGVAESWDNKDAKVWTFHLRKDAKWSDGTPVTAQDFVYSWQRSVDPNTASPY ASYLQYGHIAGIDEILEGKKPITDLGVKAIDDHTLEVTLSEPVPYFYKLLVHPSTSPPVPKAAIEKFGEKWTQPGNIVTNGAYTLKDWVVNERIVLERSPTYWNNAKTVINQVTYLPIASEVTDVNRYRSGEIDMTY NSMPIELFQKLKKEIPDEVHVDPYLCTYYYEINNQKPPFNDVRVRTALKLGMDRDIIVNKVKAQGNMPAYGYTPPYTDGAKLTQPEWFGWSQEKRNEEAKKLLAEAGYTADKPLTINLLYNTSDLHKKLAIAASSL WKKNIGVNVKLVNQEWKTFLDTRHQGTFDVARAGWCADYNEPTSFLNTMLSNSSMNTAHYKSPAFDSIMAETLKVTDEAQRTALYTKAEQQLDKDSAIVPVYYYVNARLVKPWVGGYTGKDPLDNTYTRNMYIVKH

[0038] In the present invention, antibodies may be so-called complete antibodies as well as various structures, such as, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, humanized antibodies, antibody fusions, and fragments of each of these.

[0039] Antibodies are typically tetramers consisting of two pairs of light and heavy chains, with the amino-terminal portion of each chain containing a variable region primarily responsible for antigen recognition. Three loops in the variable region assemble in each V domain of the heavy and light chains (also called VH for the heavy chain and VL for the light chain) to form the antigen-binding site. Each loop is also called a complementarity-determining region (hereinafter also referred to as "CDR"), and this region exhibits the most significant amino acid sequence variation. "Variable" refers to the fact that certain segments of the variable region vary widely among antibody sequences. The variability within the variable region is not uniformly distributed. Instead, V domains consist of relatively invariant structures called framework regions (FRs) of 15 to 30 amino acids separated by short, highly variable regions called "hypervariable regions," each 9 to 15 amino acids long or longer. Each VH and VL consists of three hypervariable regions (also known as "complementarity-determining regions" or "CDRs") and four FRs, which are arranged from the amino terminus to the carboxy terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Among the CDRs, CDR3 is a segment that is particularly important for determining complementarity, followed by the other CDRs. FRs contribute little or nothing to determining complementarity. Therefore, when specifying an antibody in terms of its antigen-binding specificity, it is possible to adequately identify the antibody by specifying only the CDR sequence, for example, the amino acid sequence of CDR3 or the amino acid sequences of CDRs 1 to 3, rather than specifying the entire amino acid sequence of the antibody.

[0040] In one embodiment, the antibody is an antibody fragment. Specific antibody fragments include, but are not limited to, (i) a Fab fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a single variable region (Ward et al., 1989, Nature 341:544-546, incorporated by reference in its entirety); (v) an isolated CDR region; (vi) a F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; and (vii) a single-chain Fv molecule (scFv), in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 2002). al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, incorporated by reference in its entirety); (viii) bispecific single-chain Fvs (WO 03 / 11161, incorporated by reference herein); and (ix) "diabodies" or "triabodies," which are multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods Enzymol. 326:461-479; WO 94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all incorporated by reference in their entirety).

[0041] In some embodiments, the antibodies may be a mixture of different species, e.g., chimeric and / or humanized antibodies, etc. That is, in the present invention, CDR sets may be used with framework and constant regions other than those specifically set forth by sequence herein.

[0042] In general, both "chimeric antibody" and "humanized antibody" refer to antibodies that combine regions from two or more species. For example, a "chimeric antibody" traditionally contains variable region(s) from a mouse (or in some cases, a rat) and constant region(s) from a human. A "humanized antibody" typically refers to a non-human antibody that has variable domain framework regions swapped for sequences found in a human antibody. Generally, in a humanized antibody, the entire antibody, except for the CDRs, is encoded by a polynucleotide of human origin or is identical to such an antibody except within its CDRs. The CDRs, some or all of which are encoded by nucleic acid from a non-human organism, are grafted onto the beta-sheet framework of a human antibody variable region to create an antibody whose specificity is determined by the grafted CDRs. The production of such antibodies is described, for example, in WO92 / 11018, Jones, 1986, Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536, all of which are incorporated herein by reference in their entireties.

[0043] In one embodiment, the antibody is a minibody. A minibody is a minimized antibody-like protein that contains an scFv linked to a CH3 domain (Hu et al., 1996, Cancer Res. 56:3055-3061, incorporated by reference in its entirety). In some cases, the scFv may be linked to an Fc region and may include some or the entire hinge region.

[0044] The antibodies of the present invention are typically isolated or recombinant. "Isolated," as used to describe the various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from the cell or cell culture in which it is expressed. Typically, an isolated polypeptide is prepared by at least one purification step. An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to Salmonella OppA is substantially free of antibodies that specifically bind to antigens other than Salmonella OppA.

[0045] Multiple isolated monoclonal antibodies can be used in combination in the detection method of the present invention, for example, in the case of immunochromatography, by labeling one to form a labeled antibody and immobilizing the other on a solid phase to form a capture antibody.

[0046] An antibody selected from the group of antibodies of the present invention preferably specifically binds to Salmonella OppA. "Specific binding" or "specifically binds," or "specific" for a particular antigen or antigenic fragment, refers to binding that is measurably different from non-specific interactions. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which is typically a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target.

[0047] Specific binding to a particular antigen or antigen fragment may be, for example, at least about 1.0x10 -1 , at least about 5.0x10 -2 or preferably at least about 1.0x10 -2This is demonstrated by an antibody having a Kd (1 / s) for an antigen or antigen fragment of 20, 50, 100, 500, 1,000, or 5,000 times or more higher than a control molecule for the antigen or antigen fragment. Here, Kd refers to the dissociation rate constant of a specific antibody-antigen interaction. Typically, an antibody that specifically binds to an antigen has a Kd that is 20-, 50-, 100-, 500-, 1,000-, or 5,000-fold higher than a control molecule for the antigen or antigen fragment.

[0048] Furthermore, specific binding to a particular antigen or antigen fragment is exhibited by an antibody having an association rate constant Ka (1 / Ms) for the antigen or antigen fragment that is at least 20, 50, 100, 500, 1,000, or 5,000 times higher than that for the antigen or antigen fragment relative to a control, where Ka refers to the association rate of a particular antibody-antigen interaction.

[0049] Furthermore, the binding characteristics of a specific antibody to an antigen or antigen fragment that specifically binds to the antibody are determined as the equilibrium dissociation constant KD (M) by Kd / Ka. The equilibrium dissociation constant is also called affinity. High binding characteristics are expressed as a low KD value. For example, the antibody used in the detection method of the present invention has an affinity of 5.0 x 10 for Salmonella OppA at 25°C. -9 M or less, preferably 1.0x10 -9 M or less, more preferably 5.0x10 -10 KD of 1.0 x 10 at 37°C or less -9 M or less, preferably 5.0x10 -10 M or less, more preferably 1.0x10 -10 Has a KD of M or less.

[0050] The binding rate constant, dissociation rate constant, and equilibrium dissociation constant between an antibody and an antigen can all be easily determined by techniques known in the art, including, but not limited to, the so-called surface plasmon resonance method, which includes an antigen cross-linking method, an antibody cross-linking method, and a capture method.

[0051] In a second aspect, the present invention provides a group of antibodies that recognize Salmonella OppA, preferably antibodies that specifically bind to Salmonella OppA, for use in the above-described methods of the present invention. The antibodies included in the group of antibodies of the present invention can be used alone or in combination of two or more. Furthermore, any one antibody selected from the group of antibodies of the present invention can be used in combination with another antibody from the group of antibodies of the present invention, such as another antibody that binds to Salmonella OppA. Here, an antibody that recognizes OppA refers to an antibody that tests positive in an antigen-antibody reaction with OppA protein. Any method known in the art can be used to obtain and react with OppA protein. For example, molecules that bind to the antibody can be purified by immunoprecipitation, and then candidate proteins can be identified by proteomic analysis. The candidate proteins can then be prepared as OppA recombinant proteins and their reactivity with the antibody can be confirmed. Specifically, the methods described in Examples 1-3 of the present specification can be used.

[0052] In the most preferred embodiment, antibody 1) L2-1H11 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 3 and a VL having the amino acid sequence shown in SEQ ID NO: 5. The gene encoding the VH of antibody 1) has the nucleotide sequence shown in SEQ ID NO: 4, and the gene encoding the VL of antibody 1) is shown in SEQ ID NO: 6. The amino acid sequence of antibody 1) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0053] Amino acid sequence of VH of antibody 1) (SEQ ID NO: 3): QVQMKESGPGLVAPSQSLSITCTVSGFSLTSYGVNWVRQPPGKGLEWLGVIWGDGSTNYHSALISRLNISKDNSKSQVFLKLNSLQTDDTATYYCAKRGIYDGSFAYWGQGTLVTVSA

[0054] Nucleotide sequence of the gene encoding VH of antibody 1) (SEQ ID NO: 4): CAGGTGCAAATGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCAGGGTTCTCATTAACCAGTTATGGTGTAAACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTGACGGGAGCACAAATTAT CATTCAGCTCTCATATCCAGACTAAACATCAGCAAGGATAACTCCAAGAGCCAAGTTTTCTTAAAACTGAACAGTCTGCAAACTGATGACACAGCCACGTACTACTGTGCCAAACGGGGAATCTATGATGGTTCGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

[0055] Amino acid sequence of VL of antibody 1) (SEQ ID NO: 5): DIVLTQSPAIMSASPGEKVTMTCSASSSVTYMHWYQQKSGTSPKRWIYETSKLASGVPARFSGSGSGTSYSLRISSMEAEDAATYYCQQWSRIPPTFGGGTKLEIK

[0056] Nucleotide sequence of the gene encoding VL of antibody 1) (SEQ ID NO: 6): GACATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAACTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCAAGAGATGGATTTATGAAACATCCAAACTG GCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCAGAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAATGGAGTAGGATCCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

[0057] Antibody 1) was further analyzed in detail, and CDRs 1 to 3 of VH and VL were identified. Antibody 1) of the present invention has an amino acid sequence in which VH CDR1 is represented by SEQ ID NO: 7, VH CDR2 is represented by SEQ ID NO: 8, and VH CDR3 is represented by SEQ ID NO: 9, and an amino acid sequence in which VL CDR1 is represented by SEQ ID NO: 10, VL CDR2 is represented by SEQ ID NO: 11, and VL CDR3 is represented by SEQ ID NO: 12. The amino acid sequences of CDRs 1 to 3 of VH and VL are specifically shown below.

[0058] VH CDR1 amino acid sequence of Antibody 1) (SEQ ID NO:7): GFSLTSYGVN VH CDR2 amino acid sequence of Antibody 1) (SEQ ID NO: 8): VIWGDG STNYHSALIS VH CDR3 amino acid sequence of Antibody 1) (SEQ ID NO: 9): RGIYDGSFAY Antibody 1) VL CDR1 amino acid sequence (SEQ ID NO: 10): SASSSVTYMH VL CDR2 amino acid sequence of antibody 1) (SEQ ID NO: 11): ETSKLAS VL CDR3 amino acid sequence of antibody 1) (SEQ ID NO: 12): QQWSRIPPT

[0059] In the most preferred embodiment, antibody 2) 15A7 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 13 and a VL having the amino acid sequence shown in SEQ ID NO: 15. The gene encoding the VH of antibody 2) has the nucleotide sequence shown in SEQ ID NO: 14, and the gene encoding the VL of antibody 2) is shown in SEQ ID NO: 16. The amino acid sequence of antibody 2) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0060] Amino acid sequence of VH of antibody 2) (SEQ ID NO: 13): QVQLQQSGAVLVRPGASVKISCKAFGYTFTNHHINWVKQRPGQGLDWIGYINPYNDYTNYNQKFKGKATLTVDKSSSTAYMELSSLTSEDSAVYYCARSYGDAMDYWGQGTSVTVSS

[0061] Nucleotide sequence of the gene encoding VH of antibody 2) (SEQ ID NO: 14): CAGGTCCAACTGCAGCAGTCTGGGGCTGTACTGGTGAGGCCTGGGGCCTCAGTGAAGATTTCCTGCAAGGCTTTTGGCTACACCTTCACAAACCATCATATAAACTGGGTGAAACAGAGGCCTGGACAGGGCCTGGACTGGATTGGATATATTAATCCTTATAATGATTATACTA ATTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTATATGGAGCTTAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATCCTACGGAGATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0062] Amino acid sequence of VL of antibody 2) (SEQ ID NO: 15): DIVLTQSPATLSVTPGDRVSLSCRASQSISAYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQSGHSFPLTFGAGTKLELK

[0063] Nucleotide sequence of the gene encoding VL of antibody 2) (SEQ ID NO: 16): GACATTGTGCTGACCCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAGATAGAGTCTCTCTTTCCTGCAGGGCCAGCCAGAGTATTAGCGCCTACTTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAATATGCTTCCCAAT CCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGTCAGATTTCACTCTCAGTATCAACAGTGTGGAACCTGAAGATGTTGGAGTGTATTACTGTCAAAGTGGTCACAGCTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA

[0064] Antibody 2) was further analyzed in detail, and CDR1 to CDR3 of VH and VL were identified. Antibody 2) of the present invention has an amino acid sequence in which VH CDR1 is represented by SEQ ID NO: 17, VH CDR2 is represented by SEQ ID NO: 18, and VH CDR3 is represented by SEQ ID NO: 19, and an amino acid sequence in which VL CDR1 is represented by SEQ ID NO: 20, VL CDR2 is represented by SEQ ID NO: 21, and VL CDR3 is represented by SEQ ID NO: 22. The amino acid sequences of CDR1 to CDR3 of VH and VL are specifically shown below.

[0065] VH CDR1 amino acid sequence of antibody 2) (SEQ ID NO: 17): GYTFTNHHIN VH CDR2 amino acid sequence of antibody 2) (SEQ ID NO: 18): YINPYNDYTNYNQKFKG VH CDR3 amino acid sequence of antibody 2) (SEQ ID NO: 19): SYGDAMDY Antibody 2) VL CDR1 amino acid sequence (SEQ ID NO: 20): RASQSISAYLH VL CDR2 amino acid sequence of antibody 2) (SEQ ID NO: 21): YASQSIS VL CDR3 amino acid sequence of antibody 2) (SEQ ID NO: 22): QSGHSFPLT

[0066] In the most preferred embodiment, antibody 3) 19D5 of the present invention has a VH having the amino acid sequence shown in SEQ ID NO: 23 and a VL having the amino acid sequence shown in SEQ ID NO: 25. The gene encoding the VH of antibody 3) has the nucleotide sequence shown in SEQ ID NO: 24, and the gene encoding the VL of antibody 3) is shown in SEQ ID NO: 26. The amino acid sequence of antibody 3) and the nucleotide sequence of the gene encoding it are specifically shown below.

[0067] Amino acid sequence of VH of antibody 3) (SEQ ID NO: 23): QVQMKESGPGLVQPSQSLSITCTVSGFSLTRYSVHWVRQSPGKGLEWLGAIWRGKSTDYNAAFISRLSISKDNSKSQVFFKMNSLQADDTAIYYCARKRDGYAMDYWGQGTSVTVSS

[0068] Nucleotide sequence of the gene encoding VH of antibody 3) (SEQ ID NO: 24): CAGGTGCAAATGAAGGAGTCAGGACCTGGCCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAGATATAGTGTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGCGATATGGAGGGGTAAAAGCACAGACT ATAATGCAGCTTTCATATCCAGACTGAGCATCAGCAAGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGTCTGCAAGCTGATGACACAGCCATATATTACTGTGCCAGAAAGAGGGACGGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA

[0069] Amino acid sequence of VL of antibody 3) (SEQ ID NO: 25): DVVVTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHIPWTFGGGTTLEIQ

[0070] Nucleotide sequence of the gene encoding VL of antibody 3) (SEQ ID NO: 26): GATGTTGTGGTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTCCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACACATATTCCGTGGACGTTCGGTGGAGGCACCACGCTGGAAATCCAA

[0071] Antibody 3) was further analyzed in detail, and CDR1 to 3 of VH and VL were identified. Antibody 3) of the present invention has an amino acid sequence in which VH CDR1 is represented by SEQ ID NO: 27, VH CDR2 is represented by SEQ ID NO: 28, and VH CDR3 is represented by SEQ ID NO: 29, and an amino acid sequence in which VL CDR1 is represented by SEQ ID NO: 30, VL CDR2 is represented by SEQ ID NO: 31, and VL CDR3 is represented by SEQ ID NO: 32. The amino acid sequences of CDR1 to 3 of VH and VL are specifically shown below.

[0072] VH CDR1 amino acid sequence of antibody 3) (SEQ ID NO: 27): GFSLTRYSVH VH CDR2 amino acid sequence of antibody 3) (SEQ ID NO: 28): AIWRGKSTDYNAAFIS VH CDR3 amino acid sequence of antibody 3) (SEQ ID NO: 29): KRDGYAMDY Antibody 3) VL CDR1 amino acid sequence (SEQ ID NO: 30): RSSQSLVHSNGNTYLH Antibody 3) VL CDR2 amino acid sequence (SEQ ID NO: 31): KVSNRFS VL CDR3 amino acid sequence of antibody 3) (SEQ ID NO: 32): SQSTHIPWT

[0073] The present invention also provides mutant antibodies, i.e., the antibodies of the present invention may have a number of modifications, including, but not limited to, amino acid modifications in the CDRs (affinity maturation), amino acid modifications in the Fc region, glycosylation variants, and other types of covalent modifications.

[0074] As used herein, a "variant" refers to a polypeptide sequence that differs from that of a parent polypeptide by at least one amino acid modification, which may include substitutions, insertions, and deletions, with substitutions, especially conservative substitutions, being preferred in many cases.

[0075] Generally, as described herein, variants may include any number of modifications as long as the functionality of the antibody is still present, i.e., in the case of amino acid variants made in the CDRs of any of the antibodies of the invention, for example, the antibody should still specifically bind to Salmonella OppA.

[0076] In general, however, the goal in most cases is to alter function with the minimum number of modifications, so that 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions are typically utilized. In some cases, 1-5 modifications are present, and many embodiments also recognize the use of 1-2, 1-3, and 1-4 modifications.

[0077] It should be noted that the number of amino acid modifications may be within the range of a functional domain: for example, it may be desirable to have 1 to 5 modifications in the Fc region of a wild-type or variant protein, e.g., 1 to 5 modifications in the Fv region. Preferably, the variant polypeptide sequence has at least about 80%, 85%, 90%, 95%, or up to 98 or 99% identity with the parent sequence (e.g., the variable region, constant region, and / or heavy and light chain sequences of any antibody selected from the group of antibodies of the present invention). It should be noted that the percentage of identity is determined by the number of amino acids, although this depends on the sequence size.

[0078] As used herein, "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a specific position in a parent polypeptide sequence with another amino acid. For example, the substitution S100A refers to a mutant polypeptide in which the serine at position 100 is replaced with an alanine. As used herein, "amino acid insertion" or "insertion" refers to the addition of an amino acid at a specific position in a parent polypeptide sequence. As used herein, "amino acid deletion" or "deletion" refers to the removal of an amino acid at a specific position in a parent polypeptide sequence.

[0079] As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to generate a variant. Typically, the parent polypeptide herein is any antibody selected from the group of antibodies of the present invention. The parent polypeptide may refer to the polypeptide itself, a composition comprising the parent polypeptide, or the amino acid sequence encoding it. Thus, as used herein, "parent Fc polypeptide" refers to an Fc polypeptide that is modified to generate a variant, and as used herein, "parent antibody" refers to an antibody that is modified to generate a variant antibody.

[0080] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence or nucleotide sequence found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc., has an amino acid sequence or nucleotide sequence that has not been intentionally modified.

[0081] As used herein, a "variant Fc region" refers to an Fc sequence that differs from that of a wild-type Fc sequence by virtue of at least one amino acid modification. An Fc variant may refer to the Fc polypeptide itself, a composition comprising the Fc variant polypeptide, or the amino acid sequence.

[0082] In some embodiments, one or more amino acid modifications are made in one or more CDRs of antibody.Generally, only one or two amino acids are substituted in any single CDR, and usually 3, 4, 5, 6, 7, 8, 9 or 10 or more changes are not made in a set of CDRs.However, in any CDR, any combination of no substitution, one or two, or three or more substitutions can be made, and any other substitutions can be independently and optionally combined.

[0083] In some cases, amino acid modifications in the CDRs are referred to as "affinity maturation." An "affinity matured" antibody has one or more changes in one or more CDRs that result in improved affinity of the antibody for the antigen compared to a parent antibody that does not have those changes. In some cases, although rare, it may be desirable to reduce the affinity of an antibody for its antigen, but this is generally not preferred.

[0084] Affinity maturation can be performed to increase the binding affinity of an antibody for an antigen by at least about 10%, 50%, 100%, 150%, or 1-5 times compared to the "parent" antibody. Preferred affinity-matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by known procedures. See, for example, Marks et al., 1992, Biotechnology 10:779-783, which describes affinity maturation by heavy chain variable (VH) and light chain variable (VL) domain shuffling. Methods for random mutagenesis of CDR and / or framework residues are described, for example, in Barbas, et al. 1994, Proc. Nat. Acad. Sci. USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, J. Immunol. 155:1994-2004; Jackson et al., 1995, J. Immunol. 154(7):3310-9; and Hawkins et al., 1992, J. Mol. Biol. 226:889-896.

[0085] Alternatively, amino acid modifications can be made in one or more CDRs of an antibody of the invention that are "silent" or "conservative," e.g., that do not significantly alter the affinity of the antibody for antigen. These can be made for a variety of reasons, including to optimize expression (which can be made to a nucleic acid encoding an antibody of the invention).

[0086] It is well known in the art that a certain amino acid can be substituted with another amino acid having a similar hydrophobicity index and still produce a protein having a similar biological function (e.g., a protein equivalent in enzymatic activity; hereinafter, "bioisequence"). In such amino acid substitutions, the hydrophobicity index is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5. It is understood in the art that such amino acid substitutions based on hydrophobicity are efficient. The hydrophilicity index is also taken into consideration in creating bioisosteres. As described in U.S. Patent No. 4,554,101, the following hydrophilicity indices are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In such amino acid substitutions, the hydrophilicity index is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0087] Therefore, in the present invention, polypeptides may be subjected to mutations or conservative substitutions. Such mutations may be substitutions, insertions, or deletions. Methods for such mutations are well known in the art and are not limited to any particular method. A conservative substitution refers to a substitution in which the hydrophilicity index and / or hydrophobicity index of the original amino acid and the substituted amino acid are similar as described above, and is a typical example of a silent modification. Examples of such conservative substitutions are well known to those skilled in the art, and include, but are not limited to, substitutions within the following groups: arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; valine, leucine, and isoleucine.

[0088] Thus, variant CDRs and antibodies are included within the definition of the CDRs and antibodies of the invention; i.e., the antibodies of the invention may comprise amino acid modifications in one or more CDRs. Furthermore, as outlined below, amino acid modifications may optionally be made independently in any region outside the CDRs (including framework and constant regions).

[0089] In some embodiments, mutant antibodies specific for Salmonella OppA are described. These antibodies consist of six CDRs, each of which independently differs from the amino acid sequences listed above by 0, 1, or 2 amino acid substitutions. In contrast, the FR region can be mutated more extensively, for example, to achieve approximately 80%, 85%, or 90% identity with the parent FR region sequence, because the amino acid sequence does not significantly affect the binding specificity of the antibody.

[0090] In addition to the modifications outlined above, other modifications can be made. For example, the molecule can be stabilized by the incorporation of disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14:1239-1245, incorporated by reference in its entirety). Furthermore, there are a variety of covalent modifications of antibodies that can be made, as outlined below.

[0091] Covalent modifications of antibodies are included within the scope of this invention and are usually, but not always, done post-translationally. For example, several types of covalent antibody modifications are introduced into the molecule by reacting specific amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or with the N- or C-terminal residues.

[0092] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues may also be derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole, and the like.

[0093] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0094] Lysinyl and amino-terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with this agent has the effect of reversing the charge of the lysinyl residue. Other agents suitable for derivatizing α-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydrides; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and aminotransferase-catalyzed reactions with glyoxylic acid.

[0095] Arginyl residues are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed under alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with lysine groups and the arginine epsilon-amino group.

[0096] The specific modification of tyrosyl residues, where there is particular interest in introducing spectral labels into tyrosyl residues, may be made by reaction with aromatic diozonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated with 125I or 131I to prepare labeled proteins for use in radioimmunoassay, the chloramine T method (described above as suitable).

[0097] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N-R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide and 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Further, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0098] Derivatization with bifunctional agents is useful for crosslinking antibodies to water-insoluble support matrices or surfaces, for use in a variety of methods in addition to those described below. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homobifunctional imidoesters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate)), and bifunctional maleimides (such as bis-N-maleimido-1,8-octane). Derivatization agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate produce photoactivatable intermediates that can form crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices and reactive substrates such as cynomolgusogen bromide-activated carbohydrates (as described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440, all of which are incorporated by reference) are used for protein immobilization.

[0099] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Both forms of these residues are within the scope of this invention.

[0100] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (Tecreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86

[1983] , incorporated by reference in its entirety), acetylation of N-terminal amines, and amidation of C-terminal carboxyl groups.

[0101] Additionally, as will be appreciated by those skilled in the art, labels (including fluorescent, enzymatic, magnetic, radioactive, etc.) can all be added to antibodies (and other compositions of the invention).

[0102] The present invention provides numerous antibodies, each with a specific set of CDRs (including some amino acid substitutions, as outlined above). As outlined above, antibodies can be defined by a set of six CDRs, a variable region, or full-length heavy and light chains (including constant regions). Furthermore, amino acid substitutions can be made, as outlined above. Generally, in the context of changes within a CDR, amino acid modifications are typically described in terms of the number of amino acid modifications that can be made due to the relatively short length of the CDRs. While this also applies to discussing the number of amino acid modifications that can be introduced into a variable, constant, or full-length sequence, it is also appropriate to define these changes in terms of "% identity" in addition to the number of changes. Thus, as described herein, antibodies encompassed within the present invention are 80, 85, 90, 95, 98, or 99% identical to the sequences set forth in the SEQ ID NOs listed herein.

[0103] The present invention further provides methods for producing any antibody selected from the group of antibodies of the present invention. These methods include culturing host cells containing one or more isolated nucleic acids encoding an antibody of the present invention. As will be understood by those skilled in the art, this can be performed in a variety of ways depending on the characteristics of the antibody. In some embodiments, when the antibody of the present invention is a full-length conventional antibody, e.g., a heavy chain variable region and a light chain variable region, the method is performed under conditions that allow the antibody to be produced and isolated.

[0104] Generally, nucleic acids encoding the antibodies of the present invention are provided. Such polynucleotides encode both the variable and constant regions of the heavy and light chains, respectively, although other combinations are contemplated by the present invention in accordance with the compositions described herein. The present invention also contemplates oligonucleotide fragments derived from the disclosed polynucleotides and nucleic acid sequences complementary to these polynucleotides.

[0105] Polynucleotides may be in the form of RNA or DNA. Polynucleotides in the form of DNA, cDNA, genomic DNA, nucleic acid analogs, and synthetic DNA are within the scope of the present invention. The DNA may be double-stranded or single-stranded, and if single-stranded, may be the coding (sense) strand or non-coding (antisense) strand. The coding sequence encoding a polypeptide may be identical to the coding sequence provided herein, or it may be a different coding sequence that, as a result of redundancy or degeneracy in the genetic code, encodes the same polypeptide as the DNA provided herein.

[0106] In some embodiments, one or more nucleic acids encoding an antibody of the present invention are incorporated into an expression vector, which can be designed to be extrachromosomal or integrated into the genome of the host cell into which it is introduced. Expression vectors can contain any number of appropriate regulatory sequences (including, but not limited to, transcriptional and translational regulatory sequences, promoters, ribosomal binding sites, enhancers, origins of replication, etc.) or other elements (such as selection genes), all operably linked as is well known in the art. In some cases, two nucleic acids can be used, each in a different expression vector (e.g., the heavy chain in a first expression vector and the light chain in a second expression vector) or in the same expression vector. It will be understood by those skilled in the art that the design of one or more expression vectors, including the selection of regulatory sequences, can depend on factors such as the choice of host cell and the desired level of protein expression.

[0107] Generally, nucleic acids and / or expression vectors are introduced into a suitable host cell such that one or more nucleic acids are operably linked to one or more expression control elements (e.g., in a vector, in a construct produced by a cellular process, integrated into the genome of the host cell) using any method appropriate for the host cell selected (e.g., transformation, transfection, electroporation, infection, etc.) to produce a recombinant host cell. The resulting recombinant host cell can be maintained under conditions appropriate for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in media supplemented with appropriate salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing one or more encoded polypeptides. In some cases, the heavy chain is produced in one cell and the light chain is produced in another cell.

[0108] Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, VA, including, but not limited to, Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and numerous other cell lines. Non-mammalian cells, including, but not limited to, bacteria, yeast, insects, and plants, can also be used to express recombinant antibodies. In some embodiments, antibodies can be produced in transgenic animals, such as cows and chickens.

[0109] General methods of antibody molecular biology, expression, purification and screening are described, for example, in Antibody Engineering, edited by Kontermann & Dubel, Springer, Heidelberg, 2001 and 2010; Hayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76; and Morrison, S. (1985) Science 229:1202.

[0110] The present invention also provides an immunochromatography kit for detecting cholera toxin, comprising any one antibody or combination of antibodies selected from the antibody group of the present invention. One embodiment of this kit comprises a detection section, on a matrix made of a porous material such as a nitrocellulose membrane, where a capture antibody is immobilized linearly, and a labeled reagent zone, upstream of the detection section, where a labeled detection antibody (labeled antibody) is carried. Typically, the labeled reagent zone is composed of a porous pad carrying the labeled detection antibody. A developer tank containing a developer solution is provided at the upstream end of the matrix. Furthermore, typically, downstream of the detection zone, a development confirmation section, on which an anti-labeled antibody is immobilized linearly to confirm the development of the labeled antibody, and further downstream of that, a developer absorption section, equipped with a porous absorbent pad for absorbing the developer solution, is provided. Furthermore, if the label is an enzyme label, a substrate zone carrying a substrate for the labeling enzyme is provided upstream of the labeled reagent zone.

[0111] During use, a sample is added to the labeled reagent zone, and the developer pad is inserted into the developer tank by applying pressure to the pusher and moving the protrusion, and developer is supplied to the matrix through the developer pad. As the developer passes through the substrate zone, the substrate is eluted into the developer, causing the developer containing the substrate to flow. As the developer passes through the labeled reagent zone, the labeled antibody and sample are eluted into the developer, causing the developer containing the substrate, labeled antibody, and sample to flow. If the sample contains Salmonella OppA, the Salmonella OppA and the labeled antibody bind via an antigen-antibody reaction. When they reach the detection zone, the immobilized antibody binds to the Salmonella OppA via an antigen-antibody reaction in the detection zone. As a result, the labeled antibody is immobilized in the detection zone via the Salmonella OppA. By measuring the label in the detection zone, Salmonella OppA is detected. If the sample does not contain Salmonella OppA, the labeled antibody does not immobilize in the detection zone and moves further downstream, resulting in no detection of the label in the detection zone. In addition, since the anti-labeled antibody is immobilized in the developer confirmation section downstream of the detection zone, the labeled antibody is fixed to the developer confirmation section. Therefore, if the label is detected in the developer confirmation section, it means that the developer has been properly developed. The developer is finally absorbed by the absorbent pad downstream.

[0112] The present invention also provides a kit for use in the method of the present invention, which comprises at least the monoclonal antibody of the present invention or a combination thereof.

[0113] When using a method that uses the sandwich method as the detection principle, at least one of the immobilized antigen capture antibody and the detection antibody, preferably both, is the monoclonal antibody of the present invention. Furthermore, standard specimen reagents (various concentrations), control reagents, sample dilutions, dilution cartridges, washing solutions, etc. can be combined. When an enzyme label is used, a substrate or reaction stop solution necessary for detecting the label can be included. When the detection antibody is not labeled, for example, a labeled substance that binds to the detection antibody can be included in the kit.

[0114] When immunochromatography is used as the sandwich method, the kit can include a device equipped with a membrane carrier on which an antigen-capturing antibody is immobilized in the detection zone and a pad carrying a labeled detection antibody. The device can also include other components suitable for immunochromatography, such as a developer pad and an absorbent pad.

[0115] The kit of the present invention may further include instructions for use of the kit.

[0116] In a further aspect, the present invention provides the L2-1H11 antibody or its variant described above in 1) as an antibody for preventing or treating Salmonella infection. All antibodies belonging to the antibody group of the present invention bind to Salmonella OppA, but surprisingly, only the L2-1H11 antibody described above in 1) reduced the oxygen consumption of Salmonella after antibody administration. Subsequent in vivo studies using mice confirmed that administration of the L2-1H11 antibody described above in 1) reduced the number of Salmonella bacteria in feces and also prevented the weight loss and subsequent death often observed after Salmonella infection. Based on these findings, the present invention provides the L2-1H11 antibody or its variant described above in 1) that can be used not only as an antibody for detecting Salmonella OppA but also as an active ingredient for inhibiting Salmonella growth and energy metabolism.

[0117] Therefore, the present invention further provides a pharmaceutical composition for preventing or treating Salmonella infections (hereinafter referred to as the pharmaceutical composition of the present invention), which comprises the above-mentioned 1) L2-1H11 antibody or a variant thereof as an active ingredient.

[0118] As used herein, Salmonella infection refers not only to Salmonella infection itself but also to symptoms and complications caused by Salmonella infection, typically including, but not limited to, acute enteritis such as abdominal pain and diarrhea, vomiting, nausea, fever, and weight loss, as well as bacteremia, liver damage, and death. Prevention of Salmonella infection includes not only preventing or reducing Salmonella infection itself, but also one or more of the following effects: eliminating or reducing the risk of symptoms and complications caused by Salmonella infection, reducing their severity, or delaying the onset of the disease. Treatment of Salmonella infection refers not only to reducing the number of Salmonella bacteria infecting a subject and halting or suppressing bacterial growth, but also to improving one or more of the symptoms and complications caused by Salmonella infection.

[0119] The pharmaceutical compositions of the present invention contain an effective amount of the L2-1H11 antibody or variant thereof described in 1) above. Such an effective amount is understood to be an amount sufficient to directly or indirectly achieve preventive or therapeutic treatment against Salmonella infection, and the specific amount can be appropriately determined by one skilled in the art in light of various factors, such as the route of administration, the dosage form and excipients used, the number and frequency of administrations, the severity of symptoms, and the age and weight of the subject. As will be understood in clinical situations, an effective dosage of the pharmaceutical composition may be achieved alone or in combination with another drug, compound, or pharmaceutical composition.

[0120] The route of administration of such a pharmaceutical composition can be appropriately selected from routes commonly used for administering pharmaceuticals, and includes, for example, oral or enteral administration, or parenteral administration such as intravenous, intramuscular, or subcutaneous administration.Typical examples include any route that can deliver the drug to the intestinal epithelium where Salmonella grows, such as oral or enteral administration, or intravenous administration, which can rapidly deliver the drug to the whole body.Subjects to which the pharmaceutical composition can be administered include mammals such as humans, monkeys, dogs, cows, pigs, horses, and mice, and birds such as chickens, ducks, and turkeys, and preferably humans.

[0121] Such pharmaceutical compositions can be prepared according to methods commonly used in the field of pharmacy. Typically, when formulated as a liquid preparation for oral or enteral administration, water or oil is used as the solvent, and additives such as antioxidants, inorganic salts, pH adjusters, preservatives, stabilizers, surfactants, taste adjusters, thickeners, and isotonicity agents may be included. When formulated as a suppository for enteral administration, oils and fats are used as the base, and additives such as preservatives, stabilizers, surfactants, fragrances, and colorants may be included. When preparing an injection solution for intravenous administration, an aqueous solvent such as, but not limited to, sterile water or sterile isotonic saline is suitable.

[0122] In a further aspect, the present invention proposes the use of the Salmonella OppA protein as a vaccine against Salmonella, since all antibodies belonging to the antibody group of the present invention bind to Salmonella OppA. The Salmonella OppA protein itself can be used as an antigen in a vaccine against Salmonella. Specifically, when a recombinant OppA protein was prepared and administered to mice, antibodies reactive to Salmonella across serotypes were produced. Surprisingly, inhibition of Salmonella growth, reduction in individual deaths, and suppression of various symptoms of Salmonella infection were confirmed.

[0123] Therefore, in a further aspect, the present invention provides a Salmonella vaccine comprising, as an antigen, the Salmonella OppA protein of SEQ ID NO: 1 or a variant thereof, for example, a protein having at least 90%, preferably at least 95%, and more preferably at least 99% sequence identity to the Salmonella OppA protein of SEQ ID NO: 1. The term "vaccine" as used herein refers to a pharmaceutical composition intended to suppress or reduce pathogen infection itself or alleviate symptoms caused by infection by inducing immunity using the antigen. Therefore, the above description of the pharmaceutical composition can also be applied to this Salmonella vaccine. The vaccine is intended primarily to be administered prophylactically before infection with Salmonella, but it can also be administered after infection. [Example]

[0124] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0125] 1. Antibody Production 1-1.Cell fusion 1.0x10 heat-killed cells of S. Enteritidis (RIMD ID: 1933006) or S. Typhimurium (RIMD ID: 1985055) 8 CFU / mouse were suspended in phosphate-buffered saline (PBS, pH 7.4) and mixed with Sigma adjuvant system (Sigma-Aldrich). The suspension was subcutaneously administered twice weekly to BALB / C mice (female, 8 weeks old) purchased from CLEA Japan. One week after the second subcutaneous administration, the spleen and inguinal / popliteal lymph nodes were collected from the mice. The spleen and inguinal / popliteal lymph nodes were crushed and passed through a 100 μm cell strainer (CORNING). After hemolysis using hemolysis buffer, immune cells were prepared. The cells were then fused with P3U1 myeloma cells using polyethylene glycol 1500 (Roche) to establish 12 hybridomas. The 12 hybridomas were numbered as follows: 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11.

[0126] 1-2. Primary screening (ELISA) To examine the Salmonella specificity of the antibodies in the culture supernatant of each hybridoma (designated by the hybridoma number), ELISA was performed as follows. Hybridomas were suspended in DMEM high-glucose medium (Nacalai Tesque) containing HAT (MP Biomedicals) (composition: 10% fetal bovine serum; Gibco; 1% penicillin-streptomycin; Nacalai Tesque; 1% 100 mM sodium pyruvate solution, 1% MEM non-essential amino acid solution; Nacalai Tesque; 0.1% 2-mercaptoethanol; Gibco; 10 ng / ml recombinant murine IL-6; PEPROTE). 100 μl of suspension was seeded into a 96-well flat-bottom plate (Sumitomo Bakelite) at 37°C in a 10% CO2 incubator for 10–14 days. Heat-killed S. Enteritidis and S. Typhimurium cells suspended in PBS were seeded at 0.1 mg / well on a 96-well immunoplate (Thermo Fisher Scientific) and incubated overnight at 4°C. 1% bovine serum albumin (BSA; Nacalai Tesque) dissolved in PBS was seeded and blocked for 2 hours at room temperature (25°C). After washing three times with 0.05% Tween 20-PBS, hybridoma culture supernatant was added at 50 μl / well and incubated for 2 hours at room temperature (25°C). After washing three times with 0.05% Tween 20-PBS, HRP-labeled goat anti-mouse IgG antibody was added at a 1:4,000 dilution in 1% bovine serum albumin-0.05% Tween 20-PBS solution and seeded at 100 μl / well and incubated for 1 hour at room temperature (25°C). Salmonella-specific antibodies were detected by adding 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Southern Biotech) to the 96-well plate and incubating for 2 min. 0.5 M HCl was added to the 96-well plate, and the absorbance at 450 nm (OD ) was measured using an iMark microplate reader (Bio-Rad). 450) was measured. The L2-1H11 hybridoma was suspended in DMEM high-glucose medium containing HT (MP Biomedicals) (similar composition to HAT medium) and subjected to limiting dilution. The cells were then seeded into flat-bottom 96-well plates (Sumitomo Bakelite) for suspension cell cloning. The results for the L2-1H11 monoclonal IgG antibody, which showed the best results based on absorbance, are shown in Figure 1. The L2-1H11 antibody exhibited cross-reactivity with the Salmonella species S. Enteritidis, S. Typhimurium, S. Infantis, S. Thompson, and S. Schwarzengrund, but no cross-reactivity with other non-Salmonella bacteria was observed.

[0127] 1-3. Identification of the molecule recognized by the L2-1H11 antibody S. Typhimurium was suspended in B-PER Complete Bacterial Extraction Reagent (Thermo Fisher Scientific) supplemented with 0.5M EDTA (Nacalai Tesque) to a final concentration of 1 mM and incubated at room temperature (25°C) for 15 minutes. After centrifugation at 6,000g for 20 minutes at room temperature (25°C), the supernatant was collected and mixed with L2-1H11 antibody and Protein G Sepharose (Nacalai Tesque). The mixture was incubated at 4°C for 1 hour for immunoprecipitation. After centrifugation at 12,000g for 20 seconds at 4°C, the sample was subjected to SDS-PAGE using a NuPAGE electrophoresis system (Life Technologies) with 4%-12% Bis-Tris gel in MES buffer. SeeBlue Plus2 Pre-stained Standard (Thermo Fisher Scientific) was used as a molecular weight marker. After silver staining with the Pierce Silver Stain Kit (Thermo Fisher Scientific), the samples were digested in-gel with trypsin to generate peptide fragments, followed by proteome analysis using a mass spectrometer. Western blotting was also performed using the immunoprecipitated samples. The results are shown in Figure 2. The L2-1H11 antibody was found to recognize a polypeptide with a molecular weight of approximately 56 kDa. Based on this molecular size, we speculate that the L2-1H11 antibody recognizes an oligopeptide-binding protein (OppA).

[0128] Next, DNA amplification by PCR was performed using the following primers to obtain the OppA fragment; Forward 5'GGG CATAT GGGCAGACGTGCCCGCCGGCGTTCAG 3' (SEQ ID NO: 33) NdeI region is underlined, Reverse 5' GG GGATCC TTAATGTTTGATAATATATAAGTTTTTC 3' (SEQ ID NO: 34) The BamHI region is underlined. The PCR products of pET16b (Novagen) and OppA were digested with NdeI (NEB) and BamHI (NEB), respectively. The resulting OppA fragment was inserted into pET16b and ligated using Ligation High Ver. 2 (TOYOBO) to generate the pET16b-OppA plasmid. To obtain recombinant OppA protein, the pET16b-OppA plasmid was transformed into E. coli strain BL21(DE3). Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (Nacalai Tesque). The culture pellet was suspended in buffer A (10 mM Tris-HCl [pH 8.0], 400 mM NaCl, 5 mM MgCl2, 0.1 mM PMSF, 1 mM 2-mercaptoethanol, and 10% glycerol) and sonicated three times for 1 min. After centrifugation at 17,800 g for 15 minutes at 4°C, the supernatant was filtered through a 0.45 μm filter (Merck Millipore). Protein purification was performed using a HiTrap HP column (Cytiva) attached to an AKTAprime plus (Cytiva) or equivalent NGC Chromatography Systems (Bio-Rad). OppA was eluted with Buffer A containing 100–500 mM imidazole. The eluted protein was loaded onto a PD-10 column (Cytiva) and PBS was replaced. The concentration of the purified protein was measured using a BCA protein assay kit (ThermoFisher Scientific). The purity of the eluted protein was confirmed by SDS-PAGE (Life Technologies) using a NuPAGE electrophoresis system (Life Technologies) with 2 μg of OppA recombinant protein as a sample, a 4%-12% Bis-Tris gel, and MES buffer. After SDS-PAGE, the 4%-12% Bis-Tris gel was stained with Coomassie Brilliant Blue using EzStainAQua (ATTO), and a specific OppA band was detected.

[0129] The resulting OppA recombinant protein (2 μg) and immunoprecipitated samples were subjected to SDS-PAGE using a NuPAGE electrophoresis system (Life Technologies) with a 4%-12% Bis-Tris gel and MES buffer. SeeBlue Plus2 Pre-Stained Standard (Thermo Fisher Scientific) was used as a molecular weight marker. The gel was transferred to a PVDF membrane (EMD Millipore Corporation), which was then blocked overnight at 4°C in 4% skim milk dissolved in 0.05% Tween 20-PBS. The primary antibody, L2-1H11 monoclonal antibody (40 ng / ml), and the secondary antibody, HRP-conjugated anti-mouse IgG (x5000) (Southern Biotech), were diluted with Can Get Signal Solution (TOYOBO) and applied to the membrane. Each antibody was incubated at room temperature (25°C) for 1 hour. After washing with 0.05% Tween 20-PBS, the bands of the target proteins were detected using HRP chemiluminescence as an indicator using an ImageQuant LAS 4000 (FUJIFILM). The results are shown in Figure 3.

[0130] 1-4. Secondary screening (ELISA) As in the primary screening, the reactivities of the 2H11, 20A12, 9B3, 9A11, 2G1, 2E9, 4E9, 2H10, 6D8, 15A7, 19D5, and L2-1H11 antibodies against S. Enteritidis (RIMDID: 1933006), S. Typhimurium (RIMDID: 1985055), S. Infantis (RIMDID: 1905012), S. Thompson (RIMDID: 1982025), S. Schwarzengrund (RIMDID: 3178001), E. coli DH5α (Competent Quick DH5α; Toyobo), and recombinant OppA proteins were confirmed by ELISA. The results are shown in Figure 4. It was confirmed that 15A7, 19D5, and L2-1H11 showed good reactivity with the five serotypes of Salmonella and the OppA recombinant protein, but did not show any reactivity with E. coli DH5α. Therefore, these three antibodies were selected as candidate antibodies.

[0131] Sandwich ELISA Various combinations of the 12 antibodies obtained in 1-2 above were used to perform sandwich ELISA tests to detect S. Enteritidis (RIMD ID: 1933006), S. Typhimurium (RIMD ID: 1985055), S. Infantis (RIMD ID: 1905012), S. Thompson (RIMD ID: 1982025), S. Schwarzengrund (RIMD ID: 3178001), E. coli DH5α (Competent Quick DH5α; Toyobo), and recombinant OppA proteins. One of the 12 antibodies (solid-phase antibody) was plated onto a 96-well immunoplate (Thermo Fisher Scientific) at 0.2 μg / well in PBS and incubated overnight at 4°C. After removing the solid phase antibody solution, 200 μl / well of 1% BSA-PBS was plated and incubated at room temperature (25° C.) for 2 hours. After washing with 0.05% Tween 20-PBS, OppA antigen (0.5 μg / well) diluted with 1% BSA-0.05% Tween 20-PBS was added. S. Enteritidis (RIMD ID: 1933006), S. Typhimurium (RIMD ID: 1985055), S. Infantis (RIMD ID: 1905012), S. Thompson (RIMD ID: 1982025), S. Schwarzengrund (RIMD ID: 3178001), and E. coli DH5α (Competent Quick DH5α; Toyobo) were cultured in LB medium and solubilized in 1% NP-40 PBS (0.1 mg / well). The plates were then incubated at 37°C for 90 minutes. After washing with 0.05% Tween 20-PBS, the 12 types of OppA-specific monoclonal antibodies (detection antibodies) labeled with HRP using an HRP conjugation kit (Abcam) were diluted with 1% BSA-0.05% Tween 20-PBS to 0.05 μg / well and added to the wells. The plates were then incubated at room temperature (25°C) for 2 hours.After washing with 0.05% Tween 20-PBS, 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Southern Biotech) was added to the 96-well plate and incubated for 2 min. 0.5 M HCl was added to the 96-well plate, and the absorbance at 450 nm (OD ) was measured using an iMark microplate reader (Bio-Rad). 450 The results are shown in Figures 5 to 11.

[0132] Although antibody combinations other than 15A7, 19D5, and L2-1H11 occasionally reacted with some serotypes of Salmonella, only two of the 15A7, 19D5, and L2-1H11 antibodies consistently reacted with the five Salmonella serotypes tested and the OppA recombinant protein. Furthermore, no antibody reacted with E. coli DH5α.

[0133] 1-6. Direct ELISA and sandwich ELISA using polyclonal antibodies Recombinant OppA protein was produced as described in 1-3 above, and the purified protein concentration was measured. Female rabbits (10-12 weeks old) were pre-fed for at least two weeks and then immunized subcutaneously with 400 μg of recombinant OppA protein on day 0. The rabbits were then boosted subcutaneously with 200 μg of recombinant OppA protein on days 14, 28, 42, and 49. On day 56, exsanguination was performed and serum was collected. A portion of the serum collected on day 56 was used for antibody purification using a Protein A column, and the purified antibody was dissolved in 50% glycerol in PBS.

[0134] OppA (5 μg / mL) diluted with PBS was seeded at 100 μL / well on a 96-well immunoplate (Thermo Fisher Scientific) and incubated overnight at 4°C. 1% bovine serum albumin (Nacalai Tesque) dissolved in PBS was seeded and blocked for 2 hours at room temperature (25°C). After washing three times with 0.05% Tween 20-PBS, 100 μL / well of polyclonal antibody (4 mg / mL in PBS) purified from serum derived from rabbits immunized with OppA recombinant was added and incubated for 2 hours at room temperature (25°C). After washing three times with 0.05% Tween 20-PBS, 100 μl of HRP-conjugated goat anti-mouse IgG antibody was added to the wells at a 4000-fold dilution in 1% bovine serum albumin-0.05% Tween 20-PBS solution and plated at 100 μl per well. The plate was incubated at room temperature (25°C) for 1 hour. OppA recombinant protein or Salmonella-specific antibodies were detected by adding 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Southern Biotech) to the 96-well plate and incubating for 2 minutes. The reaction was stopped by adding 0.5 M HCl to the 96-well plate, and the absorbance at 450 nm (OD ) was measured using an iMark microplate reader (Bio-Rad). 450 ) was measured. The results are shown in Figure 12. It was confirmed that the purified polyclonal antibody obtained from the serum derived from the rabbit immunized with the OppA recombinant protein reacted with the OppA recombinant protein.

[0135] One of six antibodies (solid-phase antibodies), including three polyclonal antibodies purified from serum collected from immunized rabbits and the monoclonal antibodies L2-1H11, 15A7, and 19D5, was diluted with PBS and plated onto a 96-well immunoplate (Thermo Fisher Scientific) at 0.2 μg / well and incubated overnight at 4°C. After removing the solid-phase antibody solution, 200 μL / well of 1% BSA-PBS was plated and incubated at room temperature (25°C) for 2 hours. After washing with 0.05% Tween 20-PBS, OppA antigen (0.5 μg / well) diluted with 1% BSA-0.05% Tween 20-PBS was added to the wells. S. Enteritidis (RIMD ID: 1933006), S. Typhimurium (RIMD ID: 1985055), S. Infantis (RIMD ID: 1905012), S. Thompson (RIMD ID: 1982025), S. Schwarzengrund (RIMD ID: 3178001), and E. coli DH5α (Competent Quick DH5α; Toyobo) were cultured in LB medium and solubilized in 1% NP-40 in PBS (0.1 mg / well). The wells were then incubated at 37°C for 90 minutes. After washing with 0.05% Tween 20-PBS, one of six antibodies (detection antibodies), including three polyclonal antibodies and monoclonal antibodies L2-1H11, 15A7, and 19D5, was diluted with 1% BSA-0.05% Tween 20-PBS to 0.05 μg / well and added to the wells. The plate was then incubated for 2 hours at room temperature (25°C). After washing with 0.05% Tween 20-PBS, 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Southern Biotech) was added to the 96-well plate and incubated for 2 minutes. 0.5M HCl was added to the 96-well plate, and the absorbance at 450 nm (OD ) was measured using an iMark microplate reader (Bio-Rad). 450) were measured. The results are shown in Figures 13 to 15. Sandwich ELISA using any combination of the three monoclonal antibodies and purified rabbit serum-derived polyclonal antibodies was able to detect the OppA recombinant protein and multiple serotypes of Salmonella.

[0136] 2. Amino acid and gene sequence analysis Hybridomas 15A7, 19D5, and L2-1H11 (10 6 After RNA was extracted from the cells, cDNA was synthesized. Using the cDNA as a template, VH and VL were amplified and cloned using degenerate primers, and the VH and VL gene sequences were analyzed. The results are shown in the sequence tables.

[0137] 3.Flux analysis S. Typhimurium was cultured in LB medium at 37°C with shaking at 200 rpm, and 1.0 x 10 8 The cells were suspended in PBS at a concentration of CFU / ml and incubated with 15A7, 19D5, L2-1H11, or isotype control antibody at 37°C for 1 hour. 100 μl of the suspension was then seeded onto a Seahorse 24-well plate (Agilent Technologies) coated with 15 μg / ml Cell-Tak (CORNING) and centrifuged at 1,400 xg and 25°C. 400 μl of LB liquid medium (Nacalai Tesque) preheated to 37°C was then added, and oxygen consumption (OCR) was measured using a Seahorse Bioscience XF24 extracellular flux analyzer (Agilent Technologies). Data were analyzed using Xfe Wave software (Agilent Technologies). The results are shown in Figure 16. Although a time-dependent decrease in oxygen consumption was observed after the addition of the L2-1H11 antibody, no decrease in oxygen consumption was observed in the 15A7 and 19D5 antibodies, and oxygen consumption increased over time, similar to the isotype control antibody group. Therefore, it was demonstrated that only the L2-1H11 antibody has the function of suppressing the energy metabolism of S. Typhimurium.

[0138] 4. In vivo testing 4-1. Oral administration test on mice BALB / C mice (female, 8 weeks old) were orally administered filter-sterilized streptomycin and then inoculated with S. Typhimurium (1.0x10 6 Mice were infected with S. Typhimurium by oral administration of 100 μg of L2-1H11 antibody, PBS (mock group), or an isotype control antibody (each at 100 μg). Feces were collected daily from infected mice and suspended in LB liquid medium (Nacalai Tesque) at 100 mg / ml. The suspension was vortexed at 4°C for 10 minutes, and the supernatant was plated on Salmonella-Shigella agar medium (Becton Dickinson). After overnight incubation at 37°C, the number of colonies that emerged was counted. The results are shown in Figure 17. Fecal counts of S. Typhimurium-infected mice conjugated with the L2-1H11 antibody were found to be reduced.

[0139] Peyer's patches were removed from the small intestine of S. Typhimurium-infected mice, then opened longitudinally, thoroughly washed with ice-cold PBS, cut into approximately 2 cm sections, and incubated in 0.5 mM EDTA (Nacalai Tesque) at 37°C for 15 minutes to remove intestinal epithelial cells and intraepithelial lymphocytes. The sections were then cut into small pieces using scissors and incubated twice with RPMI 1640 medium (Sigma-Aldrich) containing 2% newborn calf serum (NCS) (Equitech-Bio) and 0.5 mg / mL collagenase (FUJIFILM Wako Pure Chemicals) at 37°C for 15 minutes. The cell suspension was filtered through a 100 μm cell strainer (CORNING) and subjected to density gradient centrifugation (820 g, 20 minutes, 20°C) to obtain lymphocytes from the small intestinal lamina propria as a cell layer between 40% and 75% Percoll solution (Cytiva). The cells were stained with anti-CD16 / 32 monoclonal antibody (TruStain fcX; BioLegend) to prevent nonspecific staining, and with 7-AAD (BioLegend) to detect dead cells, and then incubated for 15 minutes at room temperature. Neutrophils in the small intestinal lamina propria were stained with FITC anti-mouse Ly6G (clone: ​​1A8, BioLegend), APC-anti-CD45 (clone: ​​30-F11, BioLegend), and APC-Cy7-anti-CD11b (clone: ​​M1 / 70, BioLegend) and incubated for 30 minutes at 4°C. After washing with 2% NCS-PBS, the cells were analyzed using a MACSQuant (Miltenyi Biotech) and the data were analyzed using FlowJo 9.9 (Tree Star). The results are shown in Figure 18. The number of neutrophils in the small intestinal lamina propria was reduced in the L2-1H11 antibody-treated group compared to the isotype control group.

[0140] Frozen fecal samples collected daily from S. Typhimurium-infected mice were suspended at 100 mg / ml in 5 mM EDTA buffer containing 1 mM Tris (pH 7.5), 200 mM NaCl, 1% protease inhibitor cocktail (Sigma-Aldrich), and PhosStop phosphatase inhibitor (1 tablet / 10 ml) (Roche). After vortexing for 10 minutes, the supernatant was collected by centrifugation at 1,500 xg for 15 minutes at 4°C. Neutrophil-specific myeloperoxidase (MPO) levels in the supernatant were measured using an ELISA kit (Hycult Biotech Inc.) according to the manufacturer's protocol. The results are shown in Figure 19. Fecal MPO levels were significantly reduced in the L2-1H11 antibody-treated group compared to the isotype control group.

[0141] 4-2. Mouse intravenous administration test 1.0 × 10 hybridomas 6 The cells were suspended in Hyclone ADCF-MAb medium (Cytiva) at 175 cm to a concentration of 175 cells / ml. 2The cells were seeded into flasks (Greinerbio-one) and cultured at 37°C in a 10% CO2 incubator. After 2 days, the culture supernatant was collected and filtered using a 0.22 μm filter system (CORNING). An Econo-Column (Bio-Rad) was filled with 4 ml of Protein G Sepharose (Nacalai Tesque), and the Protein G Sepharose was equilibrated by adding binding buffer (20 mM sodium phosphate, pH 7.0) at a rate of 1.0 ml / min. The filtered supernatant was added, washed with binding buffer, and eluted with elution buffer (0.1 M glycine-HCl, pH 3.0). 450 μl of the eluate was collected in an Eppendorf tube containing 50 μl of neutralization buffer (1.0 M Tris-HCl, pH 9.0). The absorbance (A280) of the eluted fraction was measured using a microspectrophotometer (Scram). The collected fractions and PBS were added to an Amicon-Ultra 15 (30K) (Merck Millipore) and concentrated at 5,000 g and centrifuged at 4°C. The PBS addition and centrifugal concentration were repeated three times to obtain purified L2-1H11 antibody. The absorbance (A280) of the concentrated solution was measured using a microspectrophotometer (Scram), and the antibody concentration after purification was calculated.

[0142] Purified L2-1H11 antibody (100 μg / mouse) or isotype control antibody (100 μg / mouse) was administered to BALB / c mice (female, 8 weeks old) via the tail vein. The next day, S. Typhimurium (1.0 × 10 6 CFU / mouse) were administered via the tail vein. The survival rate (Fig. 20A) and body weight (Fig. 20B) of the mice after administration were confirmed. Note that five mice were used per group in this study. It was found that prophylactic administration of the L2-1H11 antibody could suppress the weight loss and subsequent death observed after Salmonella infection.

[0143] BALB / c mice (female, 8 weeks old) were inoculated with S. Typhimurium (1.0 × 10 6CFU / mouse) was administered via the tail vein. The following day, L2-1H11 antibody (100 μg / mouse) or an isotype control antibody (100 μg / mouse) was administered via the tail vein. The survival rate (Figure 21A) and body weight (Figure 21B) of the mice after administration were confirmed. Note that this test was performed using five mice per group. It was found that therapeutic administration of L2-1H11 antibody after systemic infection with Salmonella can suppress the weight loss and subsequent death observed after Salmonella infection.

[0144] 5. Vaccine trials Antibody production following subcutaneous administration of recombinant OppA protein 50 μg of the S. Typhimurium-derived OppA recombinant protein obtained in Examples 1-3 above was subcutaneously administered to BALB / c mice (female, 8 weeks old) together with Sigma adjuvant on days 0 and 7. Serum was collected one week after the final immunization. Specific IgG antibody production against the OppA recombinant protein and five serotypes of Salmonella was confirmed by ELISA using the serum. Specifically, OppA (5 μg / mL) suspended in PBS and heat-killed cells (1 mg / mL each) of Salmonella Enteritidis (RIMD ID: 1933006), S. Typhimurium (RIMD ID: 1985055), S. Infantis (RIMD ID: 1905012), S. Thompson (RIMD ID: 1982025), and S. Schwarzengrund (RIMD ID: 3178001) were seeded at 100 μL / well into a 96-well immunoplate (Thermo Fisher Scientific) and incubated overnight at 4°C. 1% bovine serum albumin (Nacalai Tesque) dissolved in PBS was seeded and blocked for 2 hours at room temperature (25°C). After washing three times with 0.05% Tween 20-PBS, serum and fecal suspensions (100 mg / mL in PBS) from OppA recombinant-immunized mice were added at 50 μl / well and incubated for 2 hours at room temperature (25°C). After washing three times with 0.05% Tween 20-PBS, 100 μl / well of HRP-conjugated goat anti-mouse IgG antibody was added at a 1:4000 dilution in 1% bovine serum albumin-0.05% Tween 20-PBS solution and plated. The plate was incubated for 1 hour at room temperature (25°C). OppA recombinant protein or Salmonella-specific antibodies were detected by adding 3,3',5,5'-tetramethylbenzidine peroxidase substrate (Southern Biotech) to the 96-well plate and incubating for 2 minutes. The reaction was stopped by adding 0.5 M HCl to the 96-well plate, and the absorbance at 450 nm (OD ) was measured using an iMark microplate reader (Bio-Rad). 450) was measured. This test was carried out using 10 mice per group. The results are shown in Figure 22.

[0145] Serum from mice administered OppA recombinant protein as a subcutaneous vaccine (OppA group) was found to induce the production of OppA-specific IgG antibodies in the serum, and to react with all five serotypes of Salmonella, compared with serum from mice administered PBS (Mock group).

[0146] The growth of Salmonella was also monitored by microspectrophotometer (OD 600 ) (Fig. 23A). Energy metabolism was confirmed using oxygen consumption rate (OCR) as an indicator using a Seahorse Bioscience XF24 extracellular flux analyzer (Agilent Technologies) (Fig. 23B). For Salmonella growth, S. Typhimurium was suspended in LB liquid medium and cultured with shaking at 37°C and 200 rpm. The absorbance at 600 nm (OD ) was measured using a spectrophotometer. 600 The OD was measured, and Salmonella cells were collected between 0.4 and 0.6 and suspended in PBS. 600 After diluting the serum with PBS to an OD = 0.1, 100 μL of serum from mice administered the OppA vaccine was added and incubated at 37°C for 1 hour. The serum was mixed with LB liquid medium (Nacalai Tesque), and samples were collected over time. The absorbance at 600 nm (OD) of each sample was measured. 600 ) was measured (Fig. 23A). In this study, four mice were used in each group.

[0147] When serum from mice (OppA group) containing OppA-specific IgG antibodies induced by administration of the OppA subcutaneous vaccine was added to Salmonella (S. Typhimurium) cells, not only did the growth of Salmonella slow down compared to when serum from mice administered PBS (Mock group) was added, but the oxygen consumption of Salmonella was also reduced, indicating that the serum exhibited a neutralizing effect, mainly suppressing Salmonella growth and inhibiting energy metabolism.

[0148] Salmonella infection test after subcutaneous administration of OppA recombinant protein 50 μg of the S. Typhimurium-derived OppA recombinant protein obtained in Example 1-3 above was subcutaneously administered to BALB / c mice (female, 7 weeks old) together with Sigma adjuvant on days 0 and 7. One week after the final immunization, S. Typhimurium (1.0 × 10 6 Mice were orally infected with the OppA recombinant protein (CFU / mouse) and the survival rate was confirmed 14 days after infection. This test was performed using 10 mice per group. The results are shown in Figure 24. Mice that received subcutaneous administration of OppA recombinant protein as a vaccine antigen (OppA group) were found to suppress the individual deaths observed after oral infection with Salmonella compared to mice that received PBS (Mock group).

[0149] IgA antibody production by administration of recombinant OppA protein Mice were immunized four times with 50 μg of S. Typhimurium-derived OppA recombinant protein and 1 μg of cholera toxin (List Biological Laboratories) as an adjuvant. The priming administration was subcutaneous on day 0, and the boosting administration was oral on days 7, 14, and 21. Feces were collected from the mice one week after the final immunization. The production of specific IgA antibodies against the OppA recombinant protein and five Salmonella serotypes was assessed by ELISA using fecal suspensions. Ten mice per group were used in this study. The results are shown in Figure 25. Compared to mice administered the OppA oral vaccine (OppA group), OppA-specific IgA antibodies were induced in the feces of mice, and the antibodies cross-reacted with the five Salmonella serotypes.

[0150] In addition, the neutralizing activity of IgA antibodies in fecal suspensions was evaluated using the energy metabolism of Salmonella as an indicator. Specifically, S. Typhimurium was cultured in LB medium at 37°C with shaking at 200 rpm, and 1.0 × 108 The cells were suspended in PBS to a concentration of CFU / ml. 100 μl of the suspension was seeded onto a Seahorse 24-well plate (Agilent Technologies) coated with 15 μg / ml Cell-Tak (CORNING) and centrifuged at 1400 g and 25°C. 400 μl of LB liquid medium (Nacalai Tesque) preheated to 37°C was then added, and oxygen consumption (OCR) was measured using a Seahorse Bioscience XF24 extracellular flux analyzer (Agilent Technologies). 27 minutes after the start of the measurement, serum (100 μL) and fecal suspension (100 mg / mL in PBS) from the immunized mice were dropped into the wells containing the Salmonella culture medium. Data were analyzed using Xfe Wave software (Agilent Technologies). Four mice per group were used in this study. The results are shown in Figure 26.

[0151] When a suspension of feces collected from mice administered the OppA oral vaccine (OppA group) was added to Salmonella (S. Typhimurium) cells, the oxygen consumption of the Salmonella decreased compared to when a suspension of feces from mice administered PBS (Mock group) was added, indicating that OppA-specific IgA antibodies exhibit a neutralizing effect centered on inhibiting energy metabolism.

[0152] Salmonella infection test after administration of OppA recombinant protein Mice were immunized four times with 50 μg of S. Typhimurium-derived OppA recombinant protein together with cholera toxin as an adjuvant. The priming was administered subcutaneously on day 0, and the boosting was administered orally on days 7, 14, and 21. One week after the final immunization, the mice were immunized with S. Typhimurium (1.0 × 10 6Mice were orally infected with 1000 CFU / mouse. The number of bacteria in the feces was evaluated by CFU count (Figure 27A). The inhibitory effect on Salmonella enteritis was evaluated using the number of neutrophils infiltrating into the small intestinal lamina propria after infection (Figure 27B) and the fecal myeloperoxidase concentration in infected mice (Figure 27C). This study was conducted using four mice per group.

[0153] CFU was determined by collecting feces daily from S. Typhimurium-infected mice, suspending them in LB liquid medium (Nacalai Tesque) at 100 mg / ml, vortexing them for 10 minutes at 4°C, and plating the supernatant on Salmonella-Shigella agar medium (Becton Dickinson). After overnight incubation at 37°C, the number of colonies that grew was counted (Fig. 27A). Neutrophil counts were determined by removing Peyer's patches from the small intestines of S. Typhimurium-infected mice, opening them longitudinally, thoroughly washing them with ice-cold PBS, cutting them into approximately 2 cm sections, and incubating them in 0.5 mM EDTA (Nacalai Tesque) at 37°C for 15 minutes to remove intestinal epithelial cells and intraepithelial lymphocytes. The sections were then cut into small pieces using scissors and incubated twice in RPMI 1640 medium (Sigma-Aldrich) containing 2% newborn calf serum (NCS) (Equitech-Bio) and 0.5 mg / mL collagenase (FUJIFILM Wako Pure Chemicals) at 37°C for 15 minutes. The cell suspension was filtered through a 100 μm cell strainer (CORNING) and subjected to density gradient centrifugation (820 g, 20 minutes, 20°C) to obtain lymphocytes from the small intestinal lamina propria as a cell layer between 40% and 75% Percoll solution (Cytiva). To prevent nonspecific staining, cells were stained with anti-CD16 / 32 monoclonal antibody (TruStain fcX; BioLegend) and 7-AAD (BioLegend) to detect dead cells, followed by incubation at room temperature (25°C) for 15 minutes. Neutrophils in the small intestinal lamina propria were stained with FITC anti-mouse Ly6G (clone: ​​1A8, Biolegend), APC-anti-CD45 (clone: ​​30-F11, Biolegend), and APC-Cy7-anti-CD11b (clone: ​​M1 / 70, Biolegend) and incubated at 4°C for 30 minutes. After washing with 2% NCS-PBS, the cells were analyzed using MACSQuant (Miltenyi Biotech), and data were analyzed using FlowJo 9.9 (Tree Star).

[0154] Peyer's patches were removed from the small intestine of S. Typhimurium-infected mice, then opened longitudinally, thoroughly washed with ice-cold PBS, cut into approximately 2 cm sections, and incubated in 0.5 mM EDTA (Nacalai Tesque) at 37°C for 15 minutes to remove intestinal epithelial cells and intraepithelial lymphocytes. The sections were then cut into small pieces using scissors and incubated twice in RPMI 1640 medium (Sigma-Aldrich) containing 2% newborn calf serum (NCS) (Equitech-Bio) and 0.5 mg / mL collagenase (FUJIFILM Wako Pure Chemicals) at 37°C for 15 minutes. The cell suspension was filtered through a 100 μm cell strainer (CORNING) and subjected to density gradient centrifugation (820 g, 20 minutes, 20°C) to obtain lymphocytes from the small intestinal lamina propria as a cell layer between 40% and 75% Percoll solution (Cytiva). To prevent nonspecific staining, cells were stained with anti-CD16 / 32 monoclonal antibody (TruStain fcX; BioLegend) and 7-AAD (BioLegend) to detect dead cells, followed by incubation at room temperature (25°C) for 15 minutes. Neutrophils in the small intestinal lamina propria were stained with FITC anti-mouse Ly6G (clone: ​​1A8, Biolegend), APC-anti-CD45 (clone: ​​30-F11, Biolegend), and APC-Cy7-anti-CD11b (clone: ​​M1 / 70, Biolegend) and incubated at 4°C for 30 minutes. After washing with 2% NCS-PBS, the cells were analyzed using MACSQuant (Miltenyi Biotech), and the data were analyzed using FlowJo 9.9 (Tree Star) (Figure 27B).

[0155] Myeloperoxidase concentrations were measured by daily fecal collection from S. Typhimurium-infected mice, immediately frozen in liquid nitrogen, and stored at -80°C. Samples were suspended at 100 mg / ml in 5 mM EDTA buffer containing 1 mM Tris (pH 7.5), 200 mM NaCl, 1% protease inhibitor cocktail (Sigma-Aldrich), and PhosStop phosphatase inhibitor (1 tablet / 10 ml) (Roche). After vortexing for 10 minutes, the supernatant was collected after centrifugation at 1500 xg for 15 minutes at 4°C. MPO levels in the supernatant were measured using a myeloperoxidase (MPO) ELISA kit (Hycult Biotech Inc.) (Figure 27C).

[0156] In mice (OppA group) that were orally infected with Salmonella (S. Typhimurium) after receiving the OppA oral vaccine, the number of Salmonella bacteria in the feces, the number of neutrophils in the small intestinal lamina propria, and the fecal MPO concentration were all reduced compared to mice (Mock group) that received PBS, indicating that the OppA oral vaccine inhibits the colonization of Salmonella in the intestinal tract and suppresses subsequent enteritis.

[0157] The survival rate of mice 14 days after infection was also examined. This test was conducted using 10 mice per group. The results are shown in Figure 28. Compared to mice administered with PBS (Mock group), mice administered with the oral OppA vaccine (OppA group) showed reduced mortality after oral infection with Salmonella, demonstrating a superior effect to that observed with subcutaneous OppA vaccine administration (Figure 24).

[0158] Administration of recombinant OppA protein inhibits bacterial translocation after Salmonella infection The mice were immunized four times: 50 μg of S. Typhimurium-derived OppA recombinant protein together with cholera toxin was administered subcutaneously on day 0 (priming) and orally on days 7, 14, and 21 (boosting). One week after the final immunization, the mice were immunized with S. Typhimurium (1.0 × 10 6 Mice were orally infected with 1000 CFU / mouse. Four days after infection, livers and spleens were harvested from infected mice, disrupted, and suspended in LB liquid medium. The supernatants from each tissue homogenate were plated on Salmonella-Shigella agar plates. After overnight incubation, the number of colonies that grew was counted to calculate the Salmonella count in the liver (Fig. 29A) and spleen (Fig. 29B). Four days after infection, blood samples were collected from mice, and serum was collected. The serum was used to measure liver injury markers alanine aminotransferase (ALT; Fig. 29C) and aspartate aminotransferase (AST; Fig. 29D) to confirm the inhibitory effect of bacterial translocation following Salmonella infection. ALT and AST activities were assessed using an ALT colorimetric activity assay kit (#700260) and an AST colorimetric activity assay kit (#701640) (Cayman Chemical). This test was carried out using four mice per group.

[0159] CFU was determined by collecting the livers and spleens of the mice 4 days after infection, crushing the tissues, suspending them in LB liquid medium, and then collecting the supernatant. The supernatant was then plated on Salmonella-Shigella agar medium (Becton Dickinson) and incubated overnight at 37°C, and the number of colonies that grew was counted (Figure 29A, Figure 29B). In mice administered the OppA oral vaccine (OppA group), the number of Salmonella bacteria in the liver and spleen after Salmonella infection was reduced compared to mice administered PBS (Mock group), and the increase in liver damage markers ALT and AST was suppressed more than in naive mice (Naive mice group) and mice administered PBS (Mock group).This shows that the vaccine suppresses the spread of Salmonella to the liver, spleen, etc., and tissue damage, including liver damage, which are observed when the condition after oral infection with Salmonella becomes severe.

[0160] Administration of recombinant OppA protein inhibits inflammatory responses associated with bacteremia after Salmonella infection Mice were immunized four times with 50 μg of S. Typhimurium-derived OppA recombinant protein together with cholera toxin, administered subcutaneously on day 0 (priming) and orally on days 7, 14, and 21 (boosting). One week after the final immunization, mice were immunized with S. Typhimurium (1.0 × 10 6 Mice were orally infected with Salmonella typhimurium (CFU / mouse), and blood was collected from the mice four days after infection. Serum was then collected. Samples were prepared using the CBA Mouse Inflammation Kit (BD Biosciences). The serum was analyzed for the concentrations of various inflammatory cytokines and chemokines using MACSQuant (Miltenyi Biotech). Data were analyzed using FlowJo 9.9 (Tree Star) to confirm the inhibitory effect on the inflammatory response associated with bacteremia after Salmonella infection. This test was performed using four mice per group; the results are shown in Figure 30.

[0161] Mice administered the OppA oral vaccine (OppA group) had reduced concentrations of inflammatory cytokines (IL-6, TNF) and chemokine (MCP-1) in their serum after Salmonella infection compared to mice administered PBS (Mock group). This indicates that the OppA oral vaccine is effective in suppressing systemic inflammation that occurs when the enteritis caused by oral Salmonella infection becomes severe.

[0162] Sequence homology of OppA proteins The amino acid sequences of OppA from each Salmonella serotype and E. coli were searched using UniProtKB, and homology analysis was performed using CLUSTALW for the five Salmonella serotypes (Enteritidis, Typhimurium, Infantis, Thompson, and Schwarzengrund) detectable with the 15A7, 19D5, and L2-1H11 antibodies, as well as the OppA amino acid sequence from E. coli as a reference. The UniProt IDs are as follows: S. Enteritidis:A0A5Y8FYW4, S. Typhimurium:P06202, S. Infantis:A0A5T7MDN7, S. Thompson:A0A5X5UP57, S. Schwarzengrund:A0A620RNA3, E. coli:P23843 The OppA amino acid sequence homology among the five Salmonella serovars was over 99%. When comparing the OppA amino acid sequence between the five Salmonella serovars and E. coli, the homology was approximately 84%.

[0163] The amino acid sequence of OppA derived from S. Typhimurium (UniProt ID P06202) is as follows (SEQ ID NO: 35). The recombinant OppA protein derived from S. Typhimurium obtained in Example 1-3 above has the same sequence, and has 99.4% sequence identity with the amino acid sequence of OppA derived from S. Enteritidis shown in SEQ ID NO: 1 (UniProt ID A0A5Y8FYW4). MSNITKSLIAAGILTALIAASAATAADVPAGVQLADKQTLVRNNGSEVQSLDPHKIEGVPESNVSRDLFEGLLISDVEGHPSPGVAEKWENKDFKVWTFHLRENAKWSDGTPVTAHDFVYSWQRLADPNTASPYASYLQYGHIANIDDIIAGKKPATDLGVKALDDHTFEVTLSEPVPYFYKLLVHPSVSPVPKSAVEKFGDKWTQPANIVTNGAYKLKNWVVNERIVLERNPQYWDNAKTVINQVTYLPISSEVTDVNRYRSGEIDMTY NNMPIELFQKLKKEIPNEVRVDPYLCTYYYEINNQKAPFNDVRVRTALKLALDRDIIVNKVKNQGDLPAYSYTPYTDGAKLVEPEWFKWSQQKRNEEAKKLLAEAGFTADKPLTFDLLYNTSDLHKKLAIAVASIWKKNLGVNVNLENQEWKTFLDTRHQGTFDVARAGWCADYNEPTSFLNTMLSDSSNNTAHYKSPAFDKLIADTLKVADDTQRSELYAKAEQQLDKDSAIVPVYYYYVNARLVKPWVGGYTGKDPLDNIYVKNLYIIIKH

Claims

1. An antibody for detecting Salmonella, 1) an antibody having at least the amino acid sequence of SEQ ID NO: 9 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 12 as the VL CDR3 region; 2) an antibody having at least the amino acid sequence of SEQ ID NO: 19 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 22 as the VL CDR3 region; 3) an antibody having at least the amino acid sequence of SEQ ID NO: 29 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 32 as the VL CDR3 region; and 4) An antibody having one or two amino acid mutations in each of the VH CDR3 region and / or VL CDR3 region of the antibody of 1) to 3) above. An antibody that recognizes Salmonella OppA, comprising one antibody selected from the group consisting of:

2. The antibody or antibody combination of claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody or antibody combination according to claim 1, wherein the amino acid mutation in the antibody of 4) above is a substitution.

4. The antibody or antibody combination according to claim 1, characterized in that said antibody further has the following characteristics: 1) an antibody having at least the amino acid sequence of SEQ ID NO: 7 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 8 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 9 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 10 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 11 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 12 as a VL CDR3 region; 2) an antibody having at least the amino acid sequence of SEQ ID NO: 17 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 18 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 19 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 20 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 21 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 22 as a VL CDR3 region; 3) an antibody having at least the amino acid sequence of SEQ ID NO: 27 as a VH CDR1 region, the amino acid sequence of SEQ ID NO: 28 as a VH CDR2 region, the amino acid sequence of SEQ ID NO: 29 as a VH CDR3 region, the amino acid sequence of SEQ ID NO: 30 as a VL CDR1 region, the amino acid sequence of SEQ ID NO: 31 as a VL CDR2 region, and the amino acid sequence of SEQ ID NO: 32 as a VL CDR3 region; 4) An antibody having one or two amino acid mutations in each of the VH CDR1 region, VH CDR2 region, VH CDR3 region, VL CDR1 region, VL CDR2 region and / or VL CDR3 region of the antibody of 1) to 3) above.

5. The antibody or antibody combination according to claim 4, characterized in that said antibody further has the following characteristics: 1) an antibody having the amino acid sequence of SEQ ID NO: 3 as the VH region and the amino acid sequence of SEQ ID NO: 5 as the VL region; 2) an antibody having the amino acid sequence of SEQ ID NO: 13 as the VH region and the amino acid sequence of SEQ ID NO: 15 as the VL region; 3) an antibody having the amino acid sequence of SEQ ID NO: 23 as the VH region and the amino acid sequence of SEQ ID NO: 25 as the VL region; 4) An antibody having a sequence identity of 80% or more with the antibodies of 1) to 3) above.

6. An immunochromatographic kit for detecting Salmonella, comprising the antibody or combination of antibodies according to any one of claims 1 to 5.

7. A method for detecting Salmonella, comprising contacting a sample with an antibody or a combination of antibodies according to claims 1 to 5.

8. The method according to claim 7, wherein the detection method is a sandwich ELISA method or an immunochromatography method.

9. 1. An antibody for the prophylaxis or treatment of Salmonella, comprising: an antibody having at least the amino acid sequence of SEQ ID NO: 9 as the VH CDR3 region and the amino acid sequence of SEQ ID NO: 12 as the VL CDR3 region; or An antibody having one or two amino acid mutations in each VH CDR3 region and / or VL CDR3 region of the antibody.

10. The antibody of claim 9, further characterized in that the antibody has the following characteristics: an antibody having at least the amino acid sequence of SEQ ID NO: 7 for the VH CDR1 region, the amino acid sequence of SEQ ID NO: 8 for the VH CDR2 region, the amino acid sequence of SEQ ID NO: 9 for the VH CDR3 region, the amino acid sequence of SEQ ID NO: 10 for the VL CDR1 region, the amino acid sequence of SEQ ID NO: 11 for the VL CDR2 region, and the amino acid sequence of SEQ ID NO: 12 for the VL CDR3 region; or An antibody having one or two amino acid mutations in each of the VH CDR1 region, VH CDR2 region, VH CDR3 region, VL CDR1 region, VL CDR2 region and / or VL CDR3 region of the above antibody.

11. The antibody of claim 10, further characterized in that it has the following characteristics: An antibody having the amino acid sequence of SEQ ID NO: 3 as the VH region and the amino acid sequence of SEQ ID NO: 5 as the VL region; or An antibody having 80% or more sequence identity with the above antibody.

12. A pharmaceutical composition for preventing or treating Salmonella, comprising the antibody of any one of claims 9 to 11 as an active ingredient.

13. A vaccine against Salmonella, comprising a protein having the amino acid sequence of SEQ ID NO: 1 or a protein having 90% or more sequence identity thereto.

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