Anti-infection application of C-reactive protein

Abstract

The invention discloses an anti-infection application of C-reactive protein. The invention provides an application of C-reactive protein in preparation of a product for preventing and / or treating infection. The invention creatively finds that the C-reactive protein can be widely combined with polysaccharide capsular structures on the surfaces of various gram-positive bacteria and gram-negative bacteria in a blood circulation system, and the bacteria marked by the C-reactive protein are quickly transferred from blood to the surface of liver macrophages-Kukhou cells, so that the bacteria are quickly phagocytized and cleared in the liver; therefore, pathogens invading the blood can be effectively removed. The C-reactive protein has a broad-spectrum antibacterial function of identifying and eliminating a plurality of gram-positive and gram-negative pathogenic bacteria. In a C-reactive protein deficient individual, the recombinant C-reactive protein is injected, so that the C-reactive protein can obtain antibacterial immune protection.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-infection immunity, and specifically relates to the use of C-reactive protein in anti-infection. Background Art

[0002] Capsular polysaccharide-encapsulated bacteria cause a range of invasive diseases in humans, such as pneumonia, sepsis, and meningitis. The capsule is an important virulence factor, and more than 100 capsular serotypes have been documented for the human pathogens Streptococcus pneumoniae and Klebsiella pneumoniae. Capsular type is clinically associated with the pathogenicity of the bacterium. In the pre-antibiotic era, low-numbered S. pneumoniae serotypes / serogroups predominated in fatal pneumonia cases; Haemophilus influenzae type b was the main cause of the vast majority of childhood infections among the six serotypes; and Streptococcus suis type 2 was the main serotype causing sepsis and meningitis in humans.

[0003] C-reactive protein (CRP) is an acute phase plasma protein named for its binding to the cell wall C-polysaccharide or phosphoryl choline (PC) of Streptococcus pneumoniae (pneumococcus). Human CRP is present at trace levels in healthy individuals but rapidly increases up to 1000-fold in severe bacterial infections and other inflammatory states, making it a common biomarker for the clinical diagnosis of systemic infection and inflammation. Polymorphisms in the promoter region of the CRP gene have been reported to be associated with increased mortality in patients with pneumococcal bacteremia. Surprisingly, the biological function of CRP remains unknown.

[0004] However, the role of CRP in resistance to infection, especially against capsular polysaccharide-coated bacteria, is unknown. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] In the prior art, the large amount of plasma C-reactive protein induced by bacterial infection is only used for clinical diagnosis of the cause of fever. For a long time, there has been no scientific answer to why the host releases such a large amount of C-reactive protein in the blood. Therefore, the biological function of C-reactive protein needs further study.

[0007] Solutions for solving problems

[0008] A first aspect of the present invention provides the use of C-reactive protein in the preparation of a product for preventing and / or treating infection.

[0009] In some embodiments, the C-reactive protein is selected from any one of the following groups consisting of (i)-(iv):

[0010] (i) the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0011] (ii) an amino acid sequence that is at least 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0012] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; or

[0013] (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0014] In some embodiments, the C-reactive protein recognizes and binds to the surface capsule of the bacteria, and specifically enriches the bacteria in liver macrophages.

[0015] In some embodiments, the infection comprises a bacterial infection.

[0016] In some preferred embodiments, the bacteria include encapsulated bacteria.

[0017] In some specific embodiments, the encapsulated bacteria include Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, or Streptococcus suis.

[0018] In some optional embodiments, the S. pneumoniae comprises S. pneumoniae serotype 11A, 15B, 15C, 16F, 17F, 20B, 21, 23A, 23B, 23F, 27, 33F, 35A, 35B, 35C, 37 or 41A.

[0019] In some optional embodiments, the Streptococcus suis comprises Streptococcus suis serotype 2.

[0020] In some optional embodiments, the Haemophilus influenzae comprises Haemophilus influenzae serotype a or b.

[0021] In some optional embodiments, the Klebsiella pneumoniae comprises Klebsiella pneumoniae serotype K21 or K64.

[0022] In some embodiments, the prevention and / or treatment of infection comprises the prevention and / or treatment of infectious diseases and / or diseases and / or symptoms associated with infection.

[0023] In some optional embodiments, the infectious disease includes pneumonia, bloodstream infection (sepsis), meningitis, otitis media caused by Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae or Streptococcus suis infection.

[0024] In some embodiments, the infection is an infection in a subject.

[0025] In some embodiments, the subject is a human or a non-human animal.

[0026] In some embodiments, the product is a pharmaceutical composition, a kit, a reagent, or a kit.

[0027] A second aspect of the present invention provides a composition comprising C-reactive protein.

[0028] In some embodiments, the composition is used to prevent and / or treat infection.

[0029] In some optional embodiments, the C-reactive protein is selected from any one of the following groups consisting of (i)-(iv):

[0030] (i) the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0031] (ii) an amino acid sequence that is at least 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0032] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; or

[0033] (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0034] In some optional embodiments, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

[0035] Effects of the Invention

[0036] 1. The present invention creatively discovered that C-reactive protein can widely bind to the polysaccharide capsule structures on the surfaces of various Gram-positive and Gram-negative bacteria in the blood circulation system. The bacteria recognized by C-reactive protein will be quickly transferred from the blood to the surface of liver macrophages-Kupffer cells, and then be quickly phagocytosed and cleared in the liver, thereby effectively clearing the invading pathogens in the blood.

[0037] 2. C-reactive protein has a broad-spectrum antibacterial function that can identify and eliminate a variety of Gram-positive and Gram-negative pathogens.

[0038] 3. In individuals with C-reactive protein deficiency, injection of recombinant C-reactive protein can provide them with antibacterial immune protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Intravital imaging microscopy (IVM) was used to observe the binding of Kupffer cells to Streptococcus pneumoniae 23F in wild-type and CRP-deficient mice. Specific fluorescent antibodies were injected through the tail vein 30 minutes before infection to label the main cells in the liver (endothelial cells-cyan, Kupffer cells-red), and then 5×10 7 CFU FITC-labeled Streptococcus pneumoniae (green) was used to observe bacterial capture in the mouse liver under a fluorescent confocal microscope. Five fields of view were randomly selected 10-15 minutes after infection, and the number of bacteria captured by liver Kupffer cells in each field of view (FOV) was calculated. Figure 1 A). Figure 1 B shows that CRP-deficient mice were supplemented with CRP protein (marked as "+r-mCRP" or "+"), and no CRP protein was supplemented as a control (marked as "-r-mCRP" or "-"). Before infection, 1 μg of recombinant CRP protein was injected into the tail vein of CRP-deficient mice to evaluate the bacterial clearance rate ( Figure 1 Left side of B) and liver damage ( Figure 1 to the right of B in the figure).

[0040] Figure 2 : Capsule as a recognized ligand. Figure 2 The inhibition of clearance by S. pneumoniae serotype 23F capsular polysaccharide (CPS23F) was shown. 2-5 minutes before the tail vein infection of wild-type mice with S. pneumoniae serotype 23F, 400 μg of extracted CPS23F (marked as "+CPS23F" or "+") was injected into the tail vein of the mice, and PBS was used as a control (marked as "PBS" or "-") and used to evaluate the bacterial clearance rate ( Figure 2 left side) and liver damage ( Figure 2 on the right side of the );

[0041] Figure 3 :Detection of the specificity of C-reactive protein for capsular polysaccharides. The binding capacity of C-reactive protein to capsular polysaccharides of different serotypes of Streptococcus pneumoniae, Streptococcus suis, Haemophilus influenzae and Klebsiella pneumoniae was evaluated by enzyme-linked immunosorbent assay. n=3-4.

[0042] Figure 4 : Specific enrichment of different serotypes of Streptococcus pneumoniae in the liver of wild-type (+) and C-reactive protein-deficient (-) mice. 6 CFU pneumococci were injected into the blood of mice, and the bacterial content in the blood, liver and spleen of mice was detected 30 minutes later. n = 3-4.

[0043] Figure 5 : In wild type (WT) and C-reactive protein deficient (Crp - / - ) mice, the liver was enriched specifically for S. suis type 2. 10 7 CFU of S. suis type 2 was injected into the blood of mice, and the bacterial content in the blood, liver and spleen of mice was detected after 10 minutes and 60 minutes. n=3.

[0044] Figure 6 : In wild type (WT) and C-reactive protein deficient (Crp - / - ) mice, the liver was enriched specifically for different serotypes of Haemophilus influenzae. 10 7 CFU of Haemophilus influenzae were injected into the blood of mice, and the bacterial content in the blood, liver and spleen of mice was detected at 5 minutes and 30 minutes. n=3.

[0045] Figure 7 : In wild type (WT) and C-reactive protein deficient (Crp - / - ) mice, the liver was enriched specifically for different serotypes of Klebsiella pneumoniae. 10 7CFU of Klebsiella pneumoniae was injected into the blood of mice, and the bacterial content in the blood, liver and spleen of mice was detected at 5 minutes and 30 minutes. n=3.

[0046] Figure 8 : Capture capacity of human primary Kupffer cells for Streptococcus pneumoniae (MOI: 1). Streptococcus pneumoniae was added to medium containing culture medium; 10% normal human serum; or 10% normal human serum containing 10 μg / ml recombinant human CRP, and the bacterial binding capacity of human Kupffer cells was evaluated for 30 minutes. In the figure, Sp23F represents Streptococcus pneumoniae serotype 23F; Sp8 represents Streptococcus pneumoniae serotype 8. DETAILED DESCRIPTION

[0047] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0048] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0049] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0050] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0051] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0052] In this specification, the numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoints numerical values ​​A and B.

[0053] In this specification, when the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0054] In this specification, "administering", "giving" and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent or composition with an animal, a human, a subject, a cell, a tissue, an organ or a biological fluid. "Administering", "giving" and "treating" can refer to, for example, treatment, pharmacokinetics, diagnosis, research and experimental methods. The treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering", "giving" and "treating" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic agent, a combination composition or by another cell. "Treatment" when applied to humans, veterinary medicine or research subjects refers to treatment, prevention or preventive measures, research and diagnostic applications.

[0055] As used herein, "treatment" means administering an internal or external therapeutic agent, such as a C-reactive protein comprising the present invention, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the treated patient or population, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by physicians or other health care professionals to evaluate the severity or progression of the symptom.

[0056] In the present specification, the term "prevention" refers to preventive treatment of a subject who does not have a disease now or in the past but is at risk of developing a disease or who had a disease in the past and does not have a disease now but is at risk of recurrence of the disease.

[0057] In this specification, "effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0058] In this specification, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with a condition. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall therapy; reduces or avoids symptoms, signs, or causes of a condition; and / or enhances the therapeutic efficacy of another therapeutic agent.

[0059] In this specification, a "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition or to prevent its recurrence. A prophylactically effective amount refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent.

[0060] In this specification, the term "pharmaceutically acceptable" (or "pharmacologically acceptable", "pharmaceutically usable") refers to molecular entities and compositions that do not produce adverse reactions, allergic reactions or other undesirable reactions when administered to animals or humans, as appropriate. The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial agents, isotonic agents and absorption delaying agents, buffers, excipients, adhesives, lubricants, gels, surfactants, etc. that can be used as media for pharmaceutically acceptable substances.

[0061] In this specification, "subject" or "host" refers to human or non-human animals, including mammals. For example, primates (such as humans, monkeys), cattle, sheep, goats, alpacas, horses, dogs, cats, rabbits, rats, mice and the like. "Subjects" or "hosts" include therapeutic and non-therapeutic types. "Subjects" or "hosts" include experimental animal models or animals used to produce biological molecules expressing therapeutic diseases, i.e., "non-therapeutic hosts" or "non-therapeutic subjects".

[0062] In this specification, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and are amino acid polymers of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation, such as conjugation with a labeling component).

[0063] In the present specification, the three-letter codes and one-letter codes for amino acids used are as described in J. biol. chem, 243, p3558 (1968).

[0064] In this specification, amino acid "addition" refers to the addition of amino acids at the C-terminus or N-terminus of an amino acid sequence. According to the present invention, amino acid "deletion" refers to the deletion of 1, 2 or 3 or more amino acids from an amino acid sequence. According to the present invention, amino acid "insertion" refers to the insertion of amino acid residues at appropriate positions in an amino acid sequence, and the inserted amino acid residues may be all or partly adjacent to each other, or none of the inserted amino acids may be adjacent to each other.

[0065] In the present specification, amino acid "substitution" refers to the replacement of a certain amino acid residue at a certain position in an amino acid sequence by another amino acid residue; wherein, the "substitution" may be a conservative amino acid substitution.

[0066] In this specification, "conservative modification", "conservative substitution" or "conservative replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structures or functions is unlikely to destroy biological activity.

[0067] In this specification, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, and include amino acids having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0068] In this specification, the term "sequence identity" or "percent identity" in the comparison of two nucleic acids or polypeptides means that they are identical or have a specific percentage of identical sequences when compared and aligned with maximum correspondence using a nucleotide or amino acid residue sequence comparison algorithm or measured by visual inspection. In other words, the identity of a nucleotide or amino acid sequence can be defined by the following ratio, which is the ratio of the number of nucleotides or amino acids that are consistent when two or more nucleotide or amino acid sequences are aligned in a manner that maximizes the number of consistent nucleotides or amino acids and adds gaps as needed, to the total number of nucleotides or amino acids in the aligned portion.

[0069] In this specification, "moderate to very high stringency conditions" include "moderate stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions", which describe conditions for nucleic acid hybridization and washing. For guidance on conducting hybridization reactions, see Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document, and either can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the washing temperature can be increased to 55°C); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C.

[0070] In this specification, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of an individual fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence that has been separated at least once in terms of quantity or concentration and can be recognized, manipulated, and the sequence and its component nucleotide sequences can be recovered by standard molecular biology methods (e.g., using cloning vectors). When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), where "U" replaces "T". In other words, a "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (individual fragments or entire fragments), or can be a component or constituent of a larger nucleotide structure, such as an expression vector or a polycistronic sequence. Polynucleotides include DNA, RNA, and cDNA sequences. Detailed Description of the Invention

[0072] In the study of gram-positive and gram-negative human pathogenic bacteria such as Streptococcus pneumoniae in a bloodstream infection model of the present invention, it was found that the mouse serum protein C-reactive protein can recognize and bind to specific surface capsular polysaccharide types of bacteria such as Streptococcus pneumoniae, and then specifically enrich the bacteria to liver Kupffer cells.

[0073] <Use of C-reactive protein in the preparation of a product for preventing and / or treating infections>

[0074] In some aspects of the present invention, there is provided the use of C-reactive protein in the preparation of a product for preventing and / or treating infections.

[0075] In other aspects of the present invention, there is provided a C-reactive protein or a product comprising C-reactive protein for preventing and / or treating infections.

[0076] In other aspects of the present invention, there is provided a method for preventing and / or treating infections, which comprises the step of administering an effective amount of C-reactive protein or a product comprising C-reactive protein to a subject.

[0077] (C-reactive protein)

[0078] C-reactive protein (CRP for short) is a protein produced by the liver during inflammation in the body. It is one of the acute-phase proteins. Usually, within a few hours after inflammation or tissue damage occurs, the level of CRP in the blood will increase significantly.

[0079] In some embodiments, the C-reactive protein is selected from any one of the following groups (i)-(iv):

[0080] (i) The amino acid sequence shown in SEQ ID NO:1 or 2;

[0081] (ii) an amino acid sequence that is at least 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0082] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; or

[0083] (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0084] In some embodiments, the C-reactive protein recognizes and binds to the surface capsular polysaccharide of bacteria, and specifically recruits the bacteria to liver macrophages (also known as Kupffer cells), thereby clearing the bacteria.

[0085] (Infect)

[0086] In some embodiments, the infection comprises a bacterial infection.

[0087] In some specific embodiments, the bacteria include encapsulated bacteria.

[0088] In some more specific embodiments, the encapsulated bacteria comprises Streptococcus pneumoniae, Streptococcus suis, Haemophilus influenzae, or Klebsiella pneumoniae.

[0089] In some exemplary embodiments, the S. pneumoniae includes S. pneumoniae serotype 11A, 15B, 15C, 16F, 17F, 20B, 21, 23A, 23B, 23F, 27, 33F, 35A, 35B, 35C, 37, or 41A.

[0090] In some exemplary embodiments, the S. suis comprises S. suis serotype 2.

[0091] In some exemplary embodiments, the Haemophilus influenzae comprises Haemophilus influenzae serotype a or b.

[0092] In some exemplary embodiments, the Klebsiella pneumoniae comprises Klebsiella pneumoniae serotype K21 or K64.

[0093] In some embodiments, the preventing and / or treating an infection comprises preventing and / or treating an infectious disease and / or a disease and / or symptom associated with an infection.

[0094] In some embodiments, the infectious diseases include pneumonia, bloodstream infection (sepsis), meningitis, and otitis media caused by infection with Streptococcus pneumoniae, Haemophilus influenzae, or Klebsiella pneumoniae.

[0095] Optionally, the disease and / or symptom associated with an infection is one or more selected from the group consisting of: pathological damage caused by an infection; endotoxin shock or death; inflammatory damage to an organ; multiple organ failure; acute and / or chronic inflammatory diseases caused by an infection.

[0096] In some embodiments, the infection is an infection in a subject.

[0097] In some specific embodiments, the subject is a human or a non-human animal, such as a mouse.

[0098] (Product)

[0099] In some embodiments, the product is a pharmaceutical composition, a kit, a reagent, or a kit of reagents.

[0100] (Composition)

[0101] In some aspects of the present invention, there is provided a composition comprising C-reactive protein.

[0102] In some embodiments, the composition is for preventing and / or treating an infection. The infection is as described in the foregoing <Use of C-reactive protein in the preparation of a product for preventing and / or treating an infection> section.

[0103] In some embodiments, the C-reactive protein is any one selected from the group consisting of the following (i)-(iv):

[0104] (i) The amino acid sequence shown in SEQ ID NO: 1 or 2;

[0105] (ii) An amino acid sequence having at least 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 1 or 2, and retaining the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0106] (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; or

[0107] (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

[0108] In some optional embodiments, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

[0109] Example

[0110] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0111] Materials, methods and procedures

[0112] (1) Mouse infection experiment

[0113] Infection experiments were performed in C57BL / 6 mice according to the animal protocol approved by the Animal Care and Use Committee of Tsinghua University. All mice were raised under specific pathogen-free conditions. Bloodstream infection was performed and analyzed as described. In brief, the bacteria were injected through the tail vein, blood was collected through the mouse orbit at a specific time, and the diluted blood was counted on a blood agar plate. The organs of the mice were ground with a homogenizer tissue grinder and counted on a blood agar plate after dilution. The total bacterial colony count (CFU) in each mouse was estimated as the sum of the number of bacteria in the blood and organ samples. Competitive inhibition of bacterial clearance was performed by intravenous injection of the extracted capsule 2-5 minutes before bacterial inoculation. For the passive protection experiment of C-reactive protein, the bacteria were incubated with mouse recombinant CRP protein in 100 μl phosphate buffered saline (PBS) containing 2mM CaCl2 at room temperature for 5 minutes, and then injected into CRP-deficient mice by tail vein, and then the bacterial infection experiment was performed.

[0114] (2) Capsule extraction

[0115] Bacteria were cultured to OD 620When the pH was 0.6, the bacterial culture was cooled on ice and centrifuged at 10,000 g for 20 minutes at 4°C. The precipitate was resuspended in 50 mM citrate buffer (pH 4.5) containing 0.1% zwitterionic detergent (Zwittergent3-14 detergent) and incubated at 42°C for 30 minutes. Subsequently, the cell debris was removed by centrifugation at 10,000 g for 5 minutes at 4°C. The supernatant was then filtered with a 0.22-μm filter membrane, 25% ethanol was added, and stirred at 4°C for 2h. The precipitated nucleic acid was removed by centrifugation at 10,000 g for 15 minutes at 4°C. Further, the ethanol concentration was increased to 80% and incubated at 4°C for 2 hours. The capsule polysaccharide precipitate was collected by centrifugation at 3,000 g for 20 minutes at 4°C. After air drying, it was resuspended with sterile water and the concentration of the capsule polysaccharide was measured by the phenol-sulfuric acid method.

[0116] (3) Construction of recombinant C-reactive protein (r-CRP)

[0117] Mouse and human C-reactive protein are expressed as Strep-tagged recombinant proteins and cultured in human HEK293F suspension cells. In short, complementary DNA (cDNA) was produced using total RNA from mouse liver, and the coding sequence of mouse CRP was amplified using primers. The amplicon was cloned into the SalI / BamHI site of the pCMV-chikv-strepII vector (from Xiang Ye Laboratory, Tsinghua University) with a Srep tag (WSHPQFEK; SEQ ID NO: 3) to produce the pCMV-mCRP plasmid. According to the NM_000567.3 number, the coding sequence of human C-reactive protein was synthesized and cloned into the PstI / BamHI site of the pCMV-chikv-strepII vector to generate pCMV-hCRP. HEK293F cells were transiently transfected with recombinant human CRP, isolated from the culture supernatant by affinity chromatography with Strep-Tactin-sepharose resin (IBA), and lysed with TBS-Ca2+ containing 2.5 mM desthiobiotin (IBA). 2+ Buffer (20mM Tris, 150mM NaCl, 5mM CaCl2, pH 8.0) was used for elution. For mouse CRP, the buffer used was high salt TBS-Ca 2+ The procedure was identical except for the buffer (20 mM Tris, 500 mM NaCl, 5 mM CaCl2, pH 8.0). Protein was ultrafiltered to remove desthiobiotin and protein concentration was quantified using a BCA assay kit.

[0118] The amino acid sequence of mouse CRP is as follows (SEQ ID NO: 1):

[0119] HEDMFKKAFVFPKESDTSYVSLEAESKKPLNTFTVCLHFYTALSTVRSFSVFSYATKKNSNDILIFWNKDKQYTFGVGGAEVRFMVSEIPEAPTHICASWESA TGIVEFWIDGKPKVRKSLHKGYTVGPDASIILGQEQDSYGGDFDAKQSLVGDIGDVNMWDFVLSPEQISTVYVGGTLSPNVLNWRALNYKAQGDVFIKPQLWS

[0120] The amino acid sequence of human CRP is as follows (SEQ ID NO: 2):

[0121] QTDMSRKAFVFPKESDTSYVSLKAPLTKPLKAFTVCLHFYTELSSTRGYSIFSYATKRQDNEILIFWSKDIGYSFTVGGSEILFEVPEVTVAPVHICTSWESASGIVEFWVDGKPRVRKSLKKGYTVGAEASIILGQEQDSFGGNFEGSQSLVGDIGNVNMWDFVLSPDEINTIYLGGPFSPNVLNWRALKYEVQGEVFTKPQLWP

[0122] (4) Enzyme-linked immunosorbent assay (ELISA)

[0123] The 96-well plate was coated with purified capsular polysaccharide overnight and blocked with 5% skim milk, and then incubated with Strep-tagged recombinant CRP, Strep-tagged mouse monoclonal antibody (0.5 μg / ml) and goat anti-mouse IgG (H+L) in sequence. All reagents were mixed in TBST-Ca 2+ (TBS-Ca containing 0.05% Tween-20 2+ The binding of CRP-CPS was quantified by adding 100 μl of TMB chromogenic substrate (TIANGEN), incubating at room temperature for 5 minutes, and measuring the optical density (OD) at 450 nm.

[0124] (5) Bacteria combined with primary human Kupffer cells

[0125] Human primary Kupffer cells were isolated by digestion of human liver with collagenase IV (Sigma-Aldrich). Briefly, the liver was cut into small pieces and digested with 10 ml of digestion buffer at 37°C for 60 min at 300 rpm. After digestion, a single cell suspension was obtained by using a 70 μm filter. The mixture was then centrifuged at 300 g, 4°C for 5 min, and the supernatant was carefully removed. Next, the cell pellet was washed twice with 10 ml of HBSS (Hanks' balanced salt solution) at 4°C. The cells were lysed with 1 ml of RBC buffer and the reaction was stopped by adding 10 ml of HBSS at 4°C. The pellet (liver parenchymal cells) was removed by centrifugation at 50 g, 4°C for 2 min, and the supernatant was centrifuged at 300 g, 4°C for 5 min to obtain liver non-parenchymal cells.

[0126] Evaluation of bacterial binding to primary Kupffer cells in vitro. Briefly, liver non-parenchymal cells were seeded into 48-well plates and incubated at 37°C for 30 minutes. The attached cells were then seeded with 1×10 5 CFU of bacteria (MOI=1) were incubated at 37°C for 30 minutes. CFU counts in culture supernatants and cell lysates were for free and bound bacteria, respectively.

[0127] (6) Intravital microscopy (IVM)

[0128] IVM imaging of mouse liver was performed as described above. Sinusoidal endothelial cells (cyan) and Kupffer cells (red) were labeled by intravenous injection of AF594 anti-CD31 and AF647 anti-F4 / 80 antibodies, respectively, before intravenous injection of fluorescently labeled bacteria (green). Images and videos were captured using a Leica TCS-SP8.

[0129] (7) The Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae and Streptococcus suis used in the subsequent examples were all stored in this laboratory.

[0130] Embodiment 1:

[0131] C-reactive protein specifically enriches bacteria to the surface of liver Kupffer cells.

[0132] In this example, IVM imaging of mouse liver was used to observe the capture of bacteria in the liver in real time. The mice were injected with specific fluorescent antibodies to label the main cells in the liver (endothelial cells and Kupffer cells), and then the pneumococcal serotype 23F was injected into the tail vein. The results are shown in Figure 2. Figure 1As shown in A, in wild-type mice, bacteria are captured by Kupffer cells, and liver vascular endothelial cells do not participate in bacterial retention. However, in CRP-deficient mice, Kupffer cells cannot recognize bacteria. Therefore, C-reactive protein specifically binds to bacteria and helps Kupffer cells capture bacteria, thereby achieving liver-specific enrichment of bacteria. In CRP-deficient mice, passive intravenous injection of 1 μg of recombinant CRP protein (r-mCRP) enabled CRP-deficient mice to regain the ability to clear bacteria ( Figure 1 These data suggest that CRP is necessary and sufficient for the liver to capture and kill bacteria.

[0133] Embodiment 2:

[0134] In this example, pneumococcal serotype 23F was rapidly cleared after infection, and the remaining bacteria were mostly trapped in the mouse liver at 30 minutes. However, purified 23F capsular polysaccharide (CPS23F) completely blocked the clearance of bacteria by wild-type mice. Consistently, the number of bacteria captured and killed by the liver was affected after CPS23F was injected before infection ( Figure 2 ). Therefore, CRP recognizes the capsule on the surface of bacteria.

[0135] Embodiment 3:

[0136] In this example, we first screened the surface capsular polysaccharide types of Streptococcus pneumoniae (serotypes 11A, 15B, 15C, 16F, 17F, 20B, 21, 23A, 23B, 23F, 27, 33F, 35A, 35B, 35C, 37 and 41A), Streptococcus suis (serotype 2), Haemophilus influenzae (serotypes a and b), and Klebsiella pneumoniae (serotypes K21 and K64) that can be specifically recognized by mouse C-reactive protein.

[0137] The binding of mouse C-reactive protein to capsular polysaccharides of different serotypes of Streptococcus pneumoniae, Streptococcus suis, Haemophilus influenzae and Klebsiella pneumoniae was detected by enzyme-linked immunosorbent assay (ELISA).

[0138] Results Figure 3 C-reactive protein can specifically bind to specific types of capsular polysaccharides, including surface capsular polysaccharide types of Streptococcus pneumoniae (serotypes 11A, 15B, 15C, 16F, 17F, 20B, 21, 23A, 23B, 23F, 27, 33F, 35A, 35B, 35C, 37 and 41A), Streptococcus suis (serotype 2), Haemophilus influenzae (serotypes a and b), and Klebsiella pneumoniae (serotypes K21 and K64). As an exemplary control, the bacterial surface capsular polysaccharide type of Streptococcus pneumoniae serotype 8 (also known as type 8 Streptococcus pneumoniae) cannot be recognized by C-reactive protein.

[0139] Embodiment 4:

[0140] C-reactive protein specifically binds to bacterial capsular polysaccharides and accumulates them in the liver.

[0141] In this example, 10 6 CFU Streptococcus pneumoniae, 10 7 CFU Streptococcus suis, 10 7 CFU Haemophilus influenzae or Klebsiella pneumoniae were injected into the blood, and the distribution of bacteria in the blood and organs of mice was detected at 10 and 60 minutes after injection for Streptococcus suis and at 5 or 30 minutes for other bacteria. Figure 4-Figure 7 As shown, the C-reactive protein in the blood of wild-type mice (containing C-reactive protein) specifically recognizes the capsular polysaccharides of bacteria and rapidly enriches the bacteria (including Streptococcus pneumoniae (serotypes 11A, 15B, 15C, 16F, 17F, 20B, 21, 23A, 23F, 27, 33F, 35A, 35B, 35C, 37 and 41A), Streptococcus suis (serotype 2), Haemophilus influenzae (serotypes a and b), and Klebsiella pneumoniae (serotypes K21 and K64)) into the liver; when C-reactive protein is absent, the bacteria in the blood are not enriched into the liver. Overall, C-reactive protein can specifically and rapidly enrich bacteria coated with specific types of capsular polysaccharides into the liver.

[0142] In summary, from Examples 1 to 4, C-reactive protein can widely identify bacteria of different capsule types (see Table 1 below) and initiate host immune protection.

[0143] Table 1

[0144]

[0145]

[0146] In the above table, "capsule replacement strain" means strains with different capsule types under the same bacterial background; unless otherwise specified, all samples are from human sources; "-" means none; "√" means dependent on CRP; " / " means not dependent on CRP Example 5:

[0147] Based on the results of the mouse bloodstream infection model, the binding of mouse CRP to bacteria enables Kupffer cells to capture and kill invading bacteria under in vivo conditions. Therefore, this example next investigated the contribution of human CRP to the capture of bacteria by human Kupffer cells. To achieve this goal, this example isolated primary Kupffer cells from human liver samples and then evaluated the bacterial adhesion ability of human Kupffer cells to pneumococci in the presence or absence of recombinant human CRP protein. The experimental results can be found in Figure 8The data showed that a low concentration of 10 μg / ml human CRP protein significantly enhanced the bacterial capture ability of human Kupffer cells, and this effect was also serotype-specific. Therefore, human CRP can improve pneumococcal binding ability and promote the clearance of pathogens by human liver Kupffer cells.

[0148] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0149] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terms used herein are selected to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. Use of C-reactive protein in the preparation of a product for preventing and / or treating infection.

2. The use according to claim 1, wherein The C-reactive protein is selected from any one of the following groups consisting of (i)-(iv): (i) the amino acid sequence shown in SEQ ID NO: 1 or 2; (ii) an amino acid sequence that is at least 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; or (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

3. The use according to claim 1 or 2, wherein The C-reactive protein recognizes and binds to the surface capsule of bacteria, and specifically enriches the bacteria in liver macrophages.

4. The use according to any one of claims 1 to 3, wherein The infections include bacterial infections; Preferably, the bacteria comprise encapsulated bacteria.

5. The use according to claim 4, wherein The encapsulated bacteria include Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae or Streptococcus suis; Optionally, the S. pneumoniae comprises S. pneumoniae serotype 11A, 15B, 15C, 16F, 17F, 20B, 21, 23A, 23B, 23F, 27, 33F, 35A, 35B, 35C, 37 or 41A; Optionally, the Streptococcus suis comprises Streptococcus suis serotype 2; Optionally, the Haemophilus influenzae comprises Haemophilus influenzae serotype a or b; Optionally, the Klebsiella pneumoniae comprises Klebsiella pneumoniae serotype K21 or K64.

6. The use according to any one of claims 1 to 5, wherein The prevention and / or treatment of infection includes the prevention and / or treatment of infectious diseases and / or diseases and / or symptoms associated with infection; Optionally, the infectious disease comprises pneumonia, bloodstream infection (sepsis), meningitis, otitis media caused by infection with Streptococcus pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae or Streptococcus suis.

7. The use according to any one of claims 1 to 6, wherein The infection is an infection in a subject.

8. The use according to claim 7, wherein The subject is a human or a non-human animal.

9. The use according to any one of claims 1 to 8, wherein The product is a pharmaceutical composition, a medicine box, a reagent or a test kit.

10. A composition comprising C-reactive protein; Optionally, the composition is for preventing and / or treating infection; Optionally, the C-reactive protein is selected from any one of the following groups consisting of (i)-(iv): (i) the amino acid sequence shown in SEQ ID NO: 1 or 2; (ii) an amino acid sequence that is at least 70%, 72%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or 2, and which retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2; or (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or 2 under stringent conditions, and the amino acid sequence retains the activity of the amino acid sequence shown in SEQ ID NO: 1 or 2, wherein the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions; Optionally, the composition is a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

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