JP6327699B2 - Anti-tick vaccine - Google Patents

Description

  The present invention relates to an anti-tick vaccine containing tick ferritin and a tick extermination method using the vaccine.

  Ticks that cause serious damage to the livestock industry are currently controlled mainly by chemical substances (acaricides), but in chemical acaricides, the appearance of resistant ticks, environmental pollution by residual acaricides, and Problems such as an increase in new drug development costs due to safety testing are becoming more serious. Therefore, there is an urgent need to develop a new tick control technology that is superior in safety, effectiveness, and economy to replace the current chemical acaricide.

  The idea of anti-tick vaccines to control ticks by vaccination has been quite old, but to date, with the only exception of Bm86-based anti-tick vaccines aimed at controlling tick ticks on the market in 1994 There is no vaccinized vaccine. An ideal anti-tick vaccine is (1) effective against all ticks that are harmful to humans and animals, and (2) effective against all developmental stages of eggs, juvenile ticks, young ticks, and adult ticks. (3) The immunity that has been imparted continues for a long period of time, (4) it prevents the transmission of mediated diseases, and (5) there is no acquisition of vaccine resistance, and (6) it is inexpensive. Although it is thought that it should be provided, there is currently no one that satisfies all of these conditions, including the Bm86 vaccine described above (Non-patent Document 1). Therefore, there is a need for the development of useful new anti-tick vaccines.

de la Fuente and Merino, Vaccine, 31 (50): 5923-5929, 2013

  An object of the present invention is to provide a novel anti-tick vaccine and a method for controlling ticks using the vaccine.

  The present inventor focused on tick ferritin, which plays an important role in the defense against oxidative stress derived from iron molecules, and thought that inhibition of tick ferritin could break the blood-sucking process and remove tick. It was. The present inventors have found that an anti-tick effect is recognized by preparing recombinant ferritin and immunizing the host with this, and have completed the present invention.

That is, the present invention includes the following aspects.
1. An anti-tick vaccine comprising tick ferritin 1 or an epitope-containing fragment thereof and / or tick ferritin 2 or an epitope-containing fragment thereof.
2. 2. The vaccine according to 1 above, wherein tick ferritin 1 or its epitope-containing fragment and / or tick ferritin 2 or its epitope-containing fragment is recombinant.
3. Tick ferritin 1
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, or (c) 90% or more identity with SEQ ID NO: 1. A polypeptide comprising an amino acid sequence having
3. The vaccine according to 1 or 2 above, which is a polypeptide of any of the above.
4). Tick ferritin 2
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 2, or (c) 90% or more identity with SEQ ID NO: 2. A polypeptide comprising an amino acid sequence having
4. The vaccine according to any one of 1 to 3 above, which is any polypeptide.
5. A method for combating ticks, comprising administering the vaccine according to any one of 1 to 4 above to a non-human animal.

  Since the present invention targets blood-sucking digestion, which is the basis of tick survival, it can provide innovative technology that can solve various problems such as safety and drug residue in livestock products that many preventive and therapeutic drugs have.

FIG. 1 shows the antibody titer of antisera immunized with recombinant ferritin 1 (FER1) or recombinant ferritin 2 (FER2) against ferritin 1 antigen measured by ELISA. FIG. 2 shows the antibody titer of antisera immunized with recombinant ferritin 1 (FER1) or recombinant ferritin 2 (FER2) against ferritin 2 antigen measured by ELISA. FIG. 3 shows the results of Western blotting of antisera immunized with recombinant ferritin 1 (FER1) or recombinant ferritin 2 (FER2) against ferritin 1 and ferritin 2. FIG. 4 shows the appearance of ticks and the body weight of ticks after sucking female ticks in rabbits immunized with recombinant ferritin 1 (FER1) or recombinant ferritin 2 (FER2). In the figure, the arrows indicate pigment abnormalities in ticks eggs (slightly white compared to normal eggs).

The first aspect of the present invention is an anti-tick vaccine containing tick ferritin.
Ferritin is a kind of iron-binding protein. While iron molecules are essential for the maintenance of tick life, their overdose can be toxic to ticks. In particular, when sucking blood, the tick body is considered to be exposed to a large amount of iron molecules contained in the blood. The present inventors have heretofore described two types of ferritin, namely ferritin 1 (intracellular type) and ferritin 2 (secretory type), which protect against blood sucking, egg-laying, and oxidative stress derived from iron molecules. (Galay et al., J. Exp. Biol., 2013). Therefore, the anti-tick vaccine of the present invention may contain tick ferritin 1 and / or tick ferritin 2 or an epitope-containing fragment thereof as an active ingredient.

  The origin of the tick ferritin according to the present invention is not particularly limited, but it is not limited to the following: Ticks such as Ixodes ovatus, Ixodes persulcatus, Rhipicephalus (Boophilus) microplus, and the like are particularly preferred. Therefore, the tick ferritin 1 of the present invention is preferably the tick ferritin 1, and the tick ferritin 2 is the tick ferritin 2.

  The tick ferritin of the present invention may be a tick ferritin protein fragment as long as it has antigenicity and induces an immune response when administered to a subject. Accordingly, the anti-tick vaccine of the present invention may comprise an epitope-containing fragment of tick ferritin. Such a protein fragment is prepared by preparing an arbitrary peptide based on the above-mentioned protein by a conventional technique such as a DNA recombination technique and a peptide synthesis technique, and determining the antigenicity and immunogenicity of the peptide by Western blotting, ELISA, and vaccine. It can produce by confirming by inoculation etc. The fragment is a polypeptide composed of a part of tick ferritin 1 or 2 having at least 8 to 10 or more amino acids, for example, 20 to 50 or more amino acids.

  In the present invention, the antigenicity of the tick ferritin protein means the ability as an antigen capable of producing an anti-tick ferritin antibody. Whether a certain peptide has the antigenicity of a tick ferritin protein is determined by preparing an antibody against the peptide, and the prepared antibody is a full-length tick ferritin protein or a protein derived therefrom (for example, a protein after being cleaved at a molecular cleavage site) ) To detect whether or not it reacts. Such operations are known in the art.

  Moreover, the immunogenicity of tick ferritin protein means the property of inducing an immune response against the protein in animals. Antibodies against the protein are produced in vivo by the immune response. Such antibodies function in the defense against ticks.

  The tick ferritin protein can be prepared using methods well known to those skilled in the art. For example, primary cultured cells or established cells that produce tick ferritin protein can be cultured, separated from the culture supernatant, etc., and purified as necessary to obtain tick ferritin protein.

  Alternatively, the tick ferritin protein of the present invention or an epitope-containing fragment thereof may be recombinant. A recombinant tick ferritin protein or fragment can be produced and purified using genetic engineering techniques well known to those skilled in the art. For example, DNA encoding a tick ferritin protein or a fragment thereof can be incorporated into an appropriate vector, the vector can be introduced into an appropriate host cell, and the peptide can be expressed. The gene recombination technique can be performed according to a known method (for example, Sambrook et al., “Molecular Cloning, A Laboratory Manual”, Cold Spring Harber Laboratory Press, 1989) unless otherwise specified.

  Examples of the vector include a viral vector or a plasmid vector. Examples of the viral vector include Sendai virus, adenovirus, adeno-associated virus, retrovirus, and lentivirus. As the plasmid vector, a prokaryotic, yeast, or eukaryotic commercially available plasmid or a plasmid described in the literature can be used. Examples of prokaryotic plasmids include pBR322, pUC, pQE, pET, and pBluescript. Examples of yeast plasmids include 2μ, yEP, and pHS. In addition to the DNA to be expressed, the vector contains, for example, a promoter, an enhancer, a ribosome binding site, a terminator, and, if necessary, a selection marker.

  Suitable host cells suitable for recombinant expression include commonly used known microorganisms such as E. coli or yeast, or known cultured cells such as mammalian cells (eg CHO cells, HEK-293 cells, or COS cells). Alternatively, insect cells (for example, BmN4 cells or SF9 cells) can be mentioned, and a preferred host organism is E. coli.

  The expressed polypeptide is extracted from the culture supernatant of the host cell by known methods used for protein and peptide purification, such as ammonium sulfate salting out, precipitation separation with an organic solvent (ethanol, methanol, acetone, etc.), ion exchange chromatography. , Isoelectric point chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, reverse phase column chromatography, chromatography such as HPLC, microfiltration, ultrafiltration It is possible to purify by combining one or more filtration processes such as filtration and reverse osmosis filtration.

  Tick ferritin 1 of the present invention preferably comprises the amino acid sequence represented by SEQ ID NO: 1. More preferably, tick ferritin 1 of the present invention consists of the amino acid sequence represented by SEQ ID NO: 1. Similarly, tick ferritin 2 of the present invention preferably comprises the amino acid sequence shown in SEQ ID NO: 2. More preferably, tick ferritin 2 of the present invention consists of the amino acid sequence represented by SEQ ID NO: 2.

  Tick ferritin 1 used in the present invention is not limited to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, and as long as its antigenicity and immunogenicity are maintained, the amino acid sequence shown in SEQ ID NO: 1 Alternatively, it may be a polypeptide comprising an amino acid sequence in which several amino acids are deleted, substituted or added. Similarly, tick ferritin 2 is not limited to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2, and in the amino acid sequence shown in SEQ ID NO: 2, as long as the antigenicity and immunogenicity are maintained, 1 or It may be a polypeptide comprising an amino acid sequence in which several amino acids have been deleted, substituted or added. Here, the range of “one or several” is not particularly limited, but for example, 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably 1 to 3, or 1 Or two.

  Furthermore, tick ferritin 1 used in the present invention is not less than 70% identity, preferably not less than 80% identity, more preferably 90%, as long as the antigenicity and immunogenicity are maintained. It may be a polypeptide comprising an amino acid sequence having the above identity, most preferably greater than 95% identity, eg, 97%, 98% or 99% identity. Similarly, tick ferritin 2 has 70% identity, preferably 80% identity, more preferably 90% identity, as long as it maintains its antigenicity and immunogenicity. It may be a polypeptide comprising an amino acid sequence having a sex, most preferably greater than 95% identity, eg 97%, 98% or 99% identity. The identity value indicates a value calculated by default setting using software (for example, FASTA, DANASYS, and BLAST) that calculates identity between a plurality of amino acid sequences.

  When the anti-tick vaccine of the present invention is administered, antibody production against the tick ferritin of the present invention can be induced, and ticks can be controlled through the host's ability to protect against reinfection.

  The anti-tick vaccine of the present invention can also be used as a pharmaceutical composition for controlling ticks or a pharmaceutical composition for treating or preventing tick-borne infections.

  The “control” of ticks in the present invention includes not only complete control of ticks but also partial control. The degree of tick extermination can be determined by the body weight, survival rate, egg production, and / or hatching rate or survival rate of ticks after saturation.

  In the present invention, “saturation” of ticks means that the ticks are filled with blood by sucking blood. It is known that the mouth of the tick will pierce the skin when sucked, but the mouth of the tick will come off naturally when blood is saturated.

  The anti-tick vaccine of the present invention further comprises one or several adjuvants known to those skilled in the art, such as Freund's complete or incomplete adjuvant, cholera toxin, heat-labile E. coli toxin, aluminum hydroxide, potassium alum, saponin or its Derivatives, muramyl dipeptides, mineral or vegetable oils, NAGO, Novasome or nonionic block copolymers, DEAE dextran, etc. can be included as vaccines against ticks at appropriate intervals to inoculate animals (eg livestock) can do. Alternatively, the polypeptide of the present invention can be used by directly dissolving or suspending it in an appropriate solvent, encapsulating it in liposomes, or incorporating it into an appropriate vector. If necessary, a pharmaceutically acceptable carrier is added to the polypeptide of the present invention. For example, injections, tablets, capsules, eye drops, creams, suppositories, sprays, poultices, etc. It can be used in a suitable dosage form.

  Pharmaceutically acceptable carriers include solvents, bases, stabilizers, preservatives, solubilizers, excipients, buffers and the like well known to those skilled in the art.

  The administration method of the anti-tick vaccine of the present invention includes oral administration and parenteral administration. Oral administration includes sublingual administration. Parenteral administration includes, for example, inhalation, transdermal administration, eye drops, intravaginal administration, intraarticular administration, rectal administration, intraarterial administration, intravenous administration, topical administration, intramuscular administration, transmucosal administration, subcutaneous administration, and An appropriate method can be selected and administered from intraperitoneal administration or the like.

  Subjects to which the anti-tick vaccine of the present invention is administered include humans and non-human animals such as domestic animals and pets (eg, cows, horses, pigs, goats, sheep, rabbits, dogs, cats, mice, rats, hamsters, squirrels). , Ferrets, ducks, chickens, pigeons, etc.), and mammals are particularly preferred.

  In the tick extermination method of the present invention, the effective amount of the anti-tick vaccine can be appropriately determined according to the symptom, sex, age, weight, and / or administration route of the animal to be administered. For example, it can be derived from an in vitro test or an in vivo animal model test system. For example, it can be administered at a dose of 0.0001 mg to 100 mg per kg of body weight at a time at intervals of 2 days to 8 weeks.

  The second aspect of the present invention is a method for controlling ticks, comprising administering an effective amount of the anti-tick vaccine of the present invention to an animal in need of ticks control.

  In the tick extermination method of the present invention, by administering an anti-tick vaccine, it is possible to reduce tick saturation body weight, egg-laying rate, molting rate, etc., and to eliminate ticks.

  Targets that can be controlled by the method of the present invention include Haemaphysalis longicornis, Yamama Tick (Haemaphysalis japonica), Dermacentor recticulatus, Dermacentor taiwanensis, Kita tick (Haemaphysxo tick) , Ticks such as Ixodes persulcatus, Boophilus microplus, and the like are particularly preferred. Furthermore, it is possible to control other ticks such as ticks (eg, Argas japonicus).

  It is also possible to further treat or prevent tick-borne infections (eg, piroplasmosis, erythema fever, viral encephalitis, etc. of humans) by controlling ticks by the method of the present invention.

Example 1. Production of recombinant ferritin Amplification of the open reading frame (ORF) of the ferritin 1 gene and ferritin 2 gene using the polymerase chain reaction (PCR) method, and the resulting amplification product using GENECLEAN II KIT (Funakoshi, Tokyo) Purified. The obtained DNA was incorporated into pRSETA vector (Invitrogen, CA, USA) and transformed into E. coli BL21 strain by the heat shock method. The E. coli was precultured overnight at 37 ° C. at 50 [mu] g / ml ampicillin was 15 ml of addition of Luria-Bertani (LB) broth, which was further added to the ampicillin LB broth 500 ml OD ₆₀₀ = The cells were cultured at 37 ° C. until reaching 0.5. Thereafter, 500 μl of 1 M isopropylthio-β-galactoside (IPTG) was added to a final concentration of 1 mM, and then cultured again at 37 ° C. for 4 hours to express ferritin.

  To obtain ferritin from the insoluble fraction, the above E. coli culture solution was centrifuged (650 xg, 30 minutes, 4 ° C), the supernatant was removed, and the pellet was suspended in phosphate buffered saline (PBS) and subjected to ultrasound. Processed. The dissolved bacterial cell suspension is stirred with a rotor at room temperature for 20 minutes, then centrifuged (20,000 × g, 15 minutes, 4 ° C.), and the supernatant is removed from the pellet again containing 0.5% TritonX-100. 40 ml of mM Tris-HCl buffer (pH 7.5) was added, and the mixture was stirred with a rotor at 4 ° C. for 1 hour. Thereafter, the stirring solution was centrifuged (25,000 × g, 30 minutes, 4 ° C.), and 20 mM Tris-HCl buffer (pH 7.5) containing 6 M urea and 500 mM NaCl was added to the pellet from which the supernatant was removed. ml was added, and the mixture was stirred overnight at 4 ° C. with a rotor. The next day, the agitation solution was centrifuged (25,000 xg, 30 minutes, 4 ° C), and the collected supernatant was put into a HisTrap FF column 0.7 x 2.5 cm (GE Healthcare UK Ltd., Buckinghamshire, England). And ferritin was purified.

The collected elution fraction was replaced with PBS by dialysis, and the purity was measured by SDS-PAGE.
The concentration of the obtained ferritin was measured using Micro BCA Protein Assay Kit (PIERCE, IL, USA). A standard straight line was prepared using a bovine serum albumin solution (2 mg / ml).

Example 2 100 g of recombinant ferritin 1 (SEQ ID NO: 1) or recombinant ferritin mixed with the same amount of adjuvant (Freund's adjuvant, Incomplete, Sigma-Aldrich) per rabbit reactive against antiserum antigen immunized with recombinant ferritin 2 (SEQ ID NO: 2) was administered subcutaneously 3 times at 2-week intervals. Only the adjuvant was administered to the control group. In order to confirm the reactivity of the antiserum to the antigen, the antiserum was collected 0, 1, 3, and 5 weeks after administration, and the antibody titer was measured by ELISA as follows.

  First, a ferritin solution diluted to 1 μg / ml with Carbonate buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, 0.2 g / L sodium azide, pH 9.6) was prepared, and this was used as Nunc Immuno-Plate 96 well F After dispensing to Maxisorp (Nunc, IL, USA) at 100 μl / well, the mixture was allowed to stand at 4 ° C. overnight. The protein solution was removed the next day, and 200 μl / well was dispensed with PBST (0.05% tween20, PBS) and washed three times. Next, Blocking buffer (3% skim milk, PBS) was dispensed at 150 μl / well, and allowed to stand at 37 ° C. for 1 hour after stirring. The blocking buffer was removed, and 200 μl / well was dispensed with PBST (0.05% tween20, PBS) and washed once. The antiserum diluted 10,000 times was dispensed at 100 μl / well, and stirred and allowed to stand at 37 ° C. for 1 hour. Remove the antiserum dilution, wash 7 times with PBST, dispense 100 μl / well of goat HRP-labeled anti-rabbit IgG serum (DakoCytomation, Glostrup, Denmark) diluted 2000 times, and stir at 37 ° C for 1 hour Left to stand. The antibody dilution was removed, washed 7 times with PBST, and 100 μl of BioFX (registered trademark) TMB HRP microwell substrate (SurModics, Minn., USA) was dispensed, and reacted at 37 ° C. for 30 minutes in the dark. A mixture of equal amounts of 0.6N sulfuric acid and 1N hydrochloric acid was dispensed in 100 μl portions to stop the reaction, and OD450 nm was measured. The results are shown in FIGS.

  As is clear from FIG. 1, the antibody titer against anti-serum ferritin 1 antigen immunized with recombinant ferritin 1 or recombinant ferritin 2 increased significantly after the second immunization, and the antibody titer was immunized with recombinant ferritin 1 The level was similar between the antiserum and the antiserum immunized with recombinant ferritin 2.

  On the other hand, as is apparent from FIG. 2, the antibody titer against the ferritin 2 antigen of the antiserum immunized with recombinant ferritin 1 or recombinant ferritin 2 gradually increases after the third immunization, and the antibody titer is increased by recombinant ferritin 2 The antiserum immunized with the sera was higher than the antiserum immunized with recombinant ferritin 1.

  Subsequently, Western blotting was performed using antiserum immunized with recombinant ferritin 1 or recombinant ferritin 2. That is, recombinant ferritin is mixed with an equal volume of 2x sample buffer (4% sodium dodecyl sulfate, 20% glycerol, 125 mM Tris, 0.2% bromophenol blue, 10% 2-mercaptoethanol, pH 6.8) for 5 minutes. After boiling, it was cooled rapidly and subjected to SDS-PAGE. For detection of ferritin, 12% polyacrylamide gel was used as a separation gel, and 2.5 μg of ferritin was run per lane. After electrophoresis, it was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation, MA, USA), immersed in a blocking solution (5% skim milk, PBS), allowed to stand at 4 ° C. overnight, and then washed with a washing solution (10 mM Tris, 150 mM sodium chloride, 0.1% Tween 20, pH 7.5) was washed 3 times for 5 minutes each, and reacted with rabbit antiserum diluted 500 times at 37 ° C. for 1 hour. The plate was washed 3 times for 5 minutes each with a washing solution, and a sheep HRP-labeled anti-mouse IgG antibody (GE Healthcare UK Ltd, Buckinghamshire, England) diluted 30,000 times was reacted at 37 ° C. for 1 hour. The primary antiserum and secondary antibody were diluted with a blocking solution. The plate was washed 3 times for 5 minutes each with a washing solution, and reacted at room temperature for 5 minutes using ECL Plus Western Blotting Detection Reagents (GE Healthcare UK Ltd, Buckinghamshire, England). Detection was performed with a FluorChem® FC2 system (Cell Biosciences, CA, USA).

  As a result, all of these antisera reacted with both ferritin 1 and ferritin 2 (FIG. 3), indicating that these antisera have immunocrossing properties.

Example 3 FIG. Tick blood absorption test for rabbits immunized with recombinant ferritin In accordance with Example 2, 100 μg of each adjuvant and ferritin were mixed and administered intradermally to the rabbit three times at 2-week intervals. Only the adjuvant was administered to the control group. Two weeks after the third immunization, rabbits were allowed to suck 30 male tick mite female ticks, and after saturation, the body weight, survival rate, egg production rate and hatching rate of ticks were examined. The results are shown in FIG.

  The tick weight of ticks sucked into rabbits immunized with ferritin 1 tends to be lower than that of the control group, and the tick weight of ticks sucked into rabbits immunized with ferritin 2 is lower than that of the control group. Significantly decreased (Figure 4).

  In addition, as shown in Table 1, the body weight of ticks, the egg-laying amount, the hatching rate of ticks, and the survival rate of ticks that were sucked into rabbits immunized with recombinant ferritin decreased compared to the control group, In the ferritin 2 immunized group, the vaccine effect was recognized compared with the ferritin 1 immunized group.

  From these results, it is considered that by immunizing rabbits with recombinant ferritin, the anti-ferritin antibody suppressed the function of ferritin, and the iron ions that had lost their destination had an adverse effect on the tick's organism and egg laying. In particular, in eggs, cell damage was caused by oxidative stress, and the hatching rate of juvenile ticks was thought to have decreased.

  From the above results, it was suggested that recombinant ferritin is promising as a novel anti-tick vaccine antigen.

  Since the present invention targets blood-sucking digestion, which is the basis of tick survival, it can provide innovative technology that can solve various problems such as safety and drug residue in livestock products that many preventive and therapeutic drugs have.

Claims (3)

An anti-tick vaccine containing tick ferritin 1 ,
Tick ferritin 1
(A) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1,
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, or (c) 90% or more identity with SEQ ID NO: 1. A polypeptide comprising an amino acid sequence having
An anti-tick vaccine, which is a polypeptide of any of the above. The vaccine according to claim 1, wherein tick ferritin 1 is a recombinant.   A method for combating ticks, comprising administering the vaccine according to claim 1 or 2 to a non-human animal.

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