Francisella tularensis gene-disrupted strain and live vaccine using the same

Abstract

The object of the present invention is to provide a highly safe live vaccine that is resistant to the development of resistance and exhibits an infection prevention effect (vaccine effect) equal to or greater than that of LVS. [Solution] The attenuated Francisella tularensis Δftt_0965c strain is obtained by disrupting the pathogenic gene by inserting a lactic acid bacteria-derived group II intron between the 236th and 237th bases in the sequence of the pathogenic gene ftt_0965c of a virulent strain of Francisella tularensis, or by deleting any base between the start codon and stop codon of the pathogenic gene ftt_0965c.

Description

[Technical Field]

[0001] The present invention relates to a live vaccine capable of preventing infection with a virulent strain of Francisella tularensis and the aggravation of the infection. [Background technology]

[0002] Tularemia is a zoonotic infectious disease caused by the tularemia bacterium. It is believed to still exist among wild animals in Japan today, and is an infectious disease that requires careful attention because there is a risk of infection in areas where it occurs overseas and being brought into Japan.

[0003] Because Francisella tularensis is a gram-negative bacterium, infection can be treated with antibiotics. However, because Francisella tularensis is an intracellular parasite, treatment with antibiotics over a long period (2 weeks to 2 months) is required, which raises concerns about the development of resistance. Furthermore, due to its extremely high infectiousness and lethality, B. tularensis is listed as a pathogen that could be used in biological terrorism. If B. tularensis were used in biological terrorism, it could be possible that various antibiotic resistance genes would be acquired, rendering antibiotic treatment ineffective.

[0004] It is known that attenuated live vaccine strains (subspecies holarctica live vaccine strains, hereafter abbreviated as "LVS") are effective in preventing infection and the progression of the highly pathogenic Francisella tularensis strain. However, even humans immunized with LVS cannot completely prevent infection and onset of disease with virulent strains of tularemia of different subspecies. Furthermore, because attenuated live vaccine strains are produced by artificial passage, there is a risk of reversion to virulence. For this reason, in the United States and other countries, LVS is administered in laboratories only to people at high risk of infection. To ensure safety, attempts have been made to develop a vaccine using inactivated tularemia bacteria, but it has been reported that this vaccine is completely ineffective in preventing infection with highly virulent strains of tularemia bacteria.

[0005] To solve these problems, for example, a pharmaceutical composition has been developed that contains an isolated lysin polypeptide having an amino acid sequence that is at least 80% identical to a specific polypeptide sequence, or a fragment thereof having lysin activity, and a pharmaceutically acceptable carrier, wherein the lysin polypeptide inhibits the growth of, reduces the number of, or kills Gram-negative bacterial species (see, for example, Reference 1).

[0006] However, even with a therapeutic agent comprising the pharmaceutical composition, there is a risk that the therapeutic effect will be reduced due to the acquisition of drug resistance or mutation of the tularensis bacterium, and that rapid and sufficient treatment may not be possible. Therefore, there is a demand for a therapeutic agent having a new active ingredient or mechanism of action. Thus, even if mutations occur in the tularensis bacterium, there is a need to develop a safer vaccine that has high infection prevention and antibacterial effects. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2022-65075 Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, an object of the present invention is to provide a highly safe live vaccine and pharmaceutical composition that is resistant to the acquisition of resistance and exhibits an infection prevention effect (vaccine effect) equal to or greater than that of LVS. [Means for solving the problem]

[0009] The present inventors have conducted extensive research to solve these problems, and as a result have found that an extremely attenuated Δftt_0965c strain obtained by disrupting the virulence gene ftt_0965c of a virulent strain of Francisella tularensis can be used as a live vaccine, thereby completing the present invention.

[0010] The present invention is configured as follows. [1] An attenuated Francisella tularensis Δftt_0965c strain obtained by disrupting the pathogenic gene by inserting a group II intron derived from lactic acid bacteria (Lactococcus lactis) between the 236th and 237th bases in the sequence of the pathogenic gene ftt_0965c of a virulent strain of Francisella tularensis, or an attenuated Francisella tularensis Δftt_0965c strain obtained by deleting any base between the start codon and stop codon of the pathogenic gene ftt_0965c. [2] A live vaccine comprising the attenuated Francisella tularensis Δftt_0965c strain described in [1] above. [3] A pharmaceutical composition comprising the live vaccine according to [2] above and an adjuvant. [Effects of the Invention]

[0011] Mammals immunized with the attenuated Francisella tularensis Δftt_0965c strain, obtained by disrupting the virulence gene ftt_0965c of a virulent Francisella tularensis strain, can avoid lethal infection with a virulent Francisella tularensis strain. The protective effect of the Francisella tularensis Δftt_0965c strain is equal to or greater than that of existing live vaccine strains. The safety of the Francisella tularensis Δftt_0965c strain is extremely high compared to existing live vaccines. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a graph showing the survival rate (A) and body weight change (B) after challenge with the virulent strain of Francisella tularensis Δftt_0965c in mice immunized and non-immunized groups. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. "Gram-negative bacteria" generally refer to bacteria that decolorize with crystal violet staining in a Gram stain, i.e., bacteria that do not retain the crystal violet dye in a Gram stain.

[0014] "Drug resistance" generally refers to bacteria's resistance to the antibacterial activity of drugs. Here, drug resistance specifically refers to antibiotic resistance. Bacteria that are susceptible to a particular antibiotic may become drug-resistant by acquiring resistance to that antibiotic.

[0015] Tularemia is caused by infection with the tularemia bacterium (Francisella tularensis). Francisella tularensis is an intracellular parasite, a small, short, gram-negative bacterium (0.2 x 0.3 to 0.2 x 0.7 μm) that grows within the host's macrophages. In nature, the tularemia bacterium is transmitted and maintained in wild animals such as hares and rodents via blood-sucking arthropods such as ticks, and humans are infected directly or indirectly from these infected animals. Francisella tularensis can survive for several weeks in water, mud, or carcasses, but is heat-sensitive and easily dies within 10 minutes at 55°C.

[0016] The present invention relates to an attenuated Francisella tularensis Δftt_0965c strain obtained by disrupting the pathogenic gene by inserting a lactic acid bacteria-derived group II intron between positions 236 and 237 in the sequence of the pathogenic gene ftt_0965c of a virulent strain of Francisella tularensis. Alternatively, the present invention relates to an attenuated Francisella tularensis Δftt_0965c strain obtained by deleting any base between the start codon and stop codon of the pathogenic gene ftt_0965c. The any base is any of adenine, guanine, thymine, and cytosine.

[0017] Methods for disrupting the pathogenic gene ftt_0965c of a virulent strain of Francisella tularensis and attenuating the pathogenicity include inserting a group II intron derived from lactic acid bacteria between the 236th and 237th positions of the pathogenic gene sequence, and causing a base deletion of any desired sequence between the start and stop codons of the pathogenic gene.

[0018] A specific method for inserting a group II intron derived from lactic acid bacteria into a virulence gene is to identify the site and target sequence into which a type II intron can be inserted using the Intron targeting and design tool (clostron.com) or similar, and then transform a virulent strain of Francisella tularensis with the pKEK1140 plasmid (Appl. Environ. Microbiol. 2008, 74:2619) containing a portion (60 bases) of the target gene.

[0019] Specific methods for creating deletions in any base between the start and stop codons of a pathogenic gene include using plasmids that express Cas proteins, Mycobacterium tuberculosis ligase D (LigD), Mycobacterium tuberculosis Ku, and single-guide RNA (sgRNA) containing the target sequence under specific conditions, or by using the above materials alone or in combination to induce gene deletions by forced genomic DNA cutting and repair, etc., as long as the target sequence is present.

[0020] Whether the gene-disrupted strains obtained by the above-mentioned method are attenuated can be confirmed by inoculating them intranasally, subcutaneously, intradermally, or intraperitoneally into mice and measuring the survival rate. When a highly virulent strain of Francisella tularensis is administered intraperitoneally to mice, half of the mice must be euthanized with a bacterial dose of 5 CFU (Colony Forming Unit). On the other hand, the Francisella tularensis Δftt_0965c strain can be euthanized with a bacterial dose of 10 6 Mice remain healthy even when intraperitoneally administered with CFU of bacteria. The attenuated F. tularensis Δftt_0965c strain can be used as a live vaccine.

[0021] <Pharmaceutical Composition> The live vaccine of the present invention can be used as a pharmaceutical composition by adding other components such as adjuvants, carriers, etc. Furthermore, stabilizers such as albumin, sugars, amino acids, and phosphates, and preservatives such as thimerosal and phenoxyethanol may also be added.

[0022] <Adjuvant> Examples of adjuvants that can be used include aluminum salts, lipoproteins, lipopolysaccharides, flagella, nucleic acids, etc. Among these, aluminum salts are preferred.

[0023] <Carrier> As the carrier, for example, any physiologically compatible solvents, additives, excipients, dispersion media, solubilizers, coating agents, preservatives, isotonic and absorption delaying agents, surfactants, and propellants can be used. The amount of the carrier should be within a range that is not harmful to the subject being treated. Furthermore, the carrier should be suitable for use in pharmaceutical compositions without causing undue adverse side effects (toxicity, hypersensitivity, allergic reaction, etc.). Specific examples of the carrier include phosphate buffered saline, water, and emulsions (e.g., oil-in-water emulsions and microemulsions).

[0024] Pharmaceutical Compositions and Preparations The pharmaceutical compositions of the present invention may take the form of solutions, suspensions, emulsions, tablets, pills, pellets, capsules, liquid-containing capsules, powders, sustained release formulations, aerosols, sprays, suspensions, lozenges, candies, injections, chewing gums, ointments, smears, sustained release patches, liquid-absorbed wipes, and combinations thereof.

[0025] The administration of the pharmaceutical composition of the present invention is not particularly limited, and may be local administration (i.e., the pharmaceutical composition is applied directly to the location where its medicinal effect is desired (e.g., directly to a wound)) or systemic administration. Systemic administration may be enteral administration, oral administration (i.e., the substance is administered via the digestive tract), or parenteral administration (i.e., the substance is administered via a route other than the digestive tract, such as by injection or inhalation). That is, the live vaccines and pharmaceutical compositions of the present invention can be administered to a subject by known methods, such as orally or parenterally by inhalation, topically, rectally, nasally, bucally, or via an implanted reservoir.

[0026] <Administration method - injection> The live vaccines and pharmaceutical compositions of the present invention can be administered by injection, for example, intravenously, intramuscularly, intrathecally, intradermally, subdermally, or subcutaneously to prevent infectious diseases caused by infection with F. tularensis. When a pharmaceutical composition contains a carrier, the carrier can be at least one selected from the group consisting of distilled water, saline, albumin, and serum. In addition, pharmaceutical compositions for parenteral injection may contain aqueous or non-aqueous solutions. Furthermore, adjuvants such as pH buffers, preservatives, wetting agents, emulsifiers, and dispersants, liposome preparations, nanoparticles, dispersions, suspensions, or emulsions, and sterile powders may be used to reconstitute sterile injectable solutions or dispersions immediately before use.

[0027] <Parenteral injection> For parenteral injection, it is preferable to use an isotonic preparation. Common additives for isotonicity include sodium chloride, glucose, mannitol, sorbitol, and lactose. Isotonic solutions such as phosphate-buffered saline may be preferred. Stabilizers may include gelatin and albumin. Vasoconstrictors may be added to the preparation. Pharmaceuticals for this type of application are provided sterile and pyrogen-free.

[0028] <Diluent> The diluent may further comprise one or more other excipients such as, for example, ethanol, propylene glycol, oil, or a pharmaceutically acceptable emulsifier or surfactant. [Example]

[0029] The present invention will be explained more specifically using examples and comparative examples.

[0030] The chemicals, materials and methods used in the examples and comparative examples are as follows. ·Chemical composition medium (CDM) is 400mg / L L-Arginine, 400mg / L L-Aspartic Acid, 200mg / L L-Cysteine, 200mg / L L-Histidine, 400mg / L DL-Isoleucine, 400 mg / L L-Leucine, 400mg / L L-Lysine, 400mg / L DL-Methionine, 2000mg / L L-Proline, 400mg / L L-Serine, 2000mg / L DL-Threonine, 400mg / L L-Tyrosine, 400mg / L DL-Valine, 40mg / L Spermine Diphosphate Salt, 4mg / L Thiamine hydrochloride, 2mg / L Vitamin B5, 4000mg / L Glucose, 10000mg / L NaCl, 135mg / L Magnesium Sulfate Heptahydrate, 2 mg / L Iron(II) Sulfate Heptahydrate, 1000 mg / L Potassium Dihydrogen Phosphate, and 1000 mg / L Dipotassium Hydrogen Phosphate were mixed and the pH was adjusted to 5.8 to 6.2. Culture of tularemia strains The tularensis strain was cultured in chemically defined medium (CDM) at 37°C for 24 hours. The tularensis bacteria were collected by centrifugation, resuspended in CDM containing 10% glycerol, and stored at -80°C until use. Cultivation of tularensis bacteria and infection experiments were performed in the BSL3 facility of the National Institute of Infectious Diseases.

[0031] Example 1 The immunized C57BL / 6J mice were intraperitoneally administered a triple-anesthesia solution and then 6 CFU / 10 μL of Francisella tularensis Δftt_0965c strain was inoculated intranasally. Mice immunized intranasally with the attenuated Δftt_0965c strain were completely protected from lethal infection with the virulent strain, and all individuals survived for three weeks (black line (+vaccine) in Figure 1).

[0032] Comparative Example 1 The other non-immunized group of mice was treated in the same way with saline. Three weeks after the immunization, the mice were inoculated with 100LD of the virulent F. tularensis strain SCHU P9. 50 The mice were inoculated intranasally at a volume of 5000 CFU / 10 μL. After inoculation with the virulent strain, survival was checked and the mice's weight was measured daily for up to three weeks. After intranasal inoculation with the virulent strain, all mice in the non-immunized group died within one week (dotted line (-vaccine) in Figure 1).

[0033] These results indicated that the Δftt_0965c strain can be used as a vaccine. [Industrial Applicability]

[0034] Mammals immunized with the attenuated Francisella tularensis Δftt_0965c strain, obtained by disrupting the virulence gene ftt_0965c of a virulent Francisella tularensis strain, can avoid lethal infection with a virulent Francisella tularensis strain. The protective effect of the Francisella tularensis Δftt_0965c strain is equal to or greater than that of existing live vaccine strains. The safety of the Francisella tularensis Δftt_0965c strain is extremely high compared to existing live vaccines.

Claims

1. An attenuated Francisella tularensis Δftt_0965c strain obtained by disrupting the pathogenic gene by inserting a lactic acid bacteria-derived group II intron between the 236th and 237th bases in the sequence of the pathogenic gene ftt_0965c of a virulent strain of Francisella tularensis, or an attenuated Francisella tularensis Δftt_0965c strain obtained by deleting any base between the start codon and stop codon of the pathogenic gene ftt_0965c.

2. A live vaccine comprising the attenuated Francisella tularensis Δftt_0965c strain according to claim 1.

3. A pharmaceutical composition comprising the live vaccine of claim 2 and an adjuvant.

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