WO2020145489A1 - Oral vaccine composition containing hydrogel-encapsulated inactivated cells - Google Patents

Oral vaccine composition containing hydrogel-encapsulated inactivated cells Download PDF

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WO2020145489A1
WO2020145489A1 PCT/KR2019/013709 KR2019013709W WO2020145489A1 WO 2020145489 A1 WO2020145489 A1 WO 2020145489A1 KR 2019013709 W KR2019013709 W KR 2019013709W WO 2020145489 A1 WO2020145489 A1 WO 2020145489A1
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vaccine
fish
sho
vaccine composition
hydrogel
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PCT/KR2019/013709
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French (fr)
Korean (ko)
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박세창
강정우
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서울대학교산학협력단
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Priority to CN201980088369.3A priority Critical patent/CN113271966A/en
Publication of WO2020145489A1 publication Critical patent/WO2020145489A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/521—Bacterial cells; Fungal cells; Protozoal cells inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]

Definitions

  • the present invention relates to a vaccine composition for oral administration comprising an inactivated cell encapsulated by a hydrogel.
  • the path of pathogens entering the body of fish can be seen as skin, gills, nasal cavity, and digestive tract. These organs are protected from bacteria that invade by secretion and action of mucus, enzymes, antibacterial and bactericidal substances, antibodies, white blood cells, and macrophages. During screening, these protective substances are damaged by mechanical damage or parasite wounds, poor management, etc., through which pathogens enter the body and then reach the organs or tissues through the bloodstream to form lesions. Is done.
  • the largely divided pathogenic organisms that damage fish include protozoa (parasites), fungi (fungi), bacteria, and viruses.
  • Parasites are very small, ranging from tens of centimeters to hundreds of ⁇ m, bacteria to several ⁇ m, and viruses to several thousandths of a ⁇ m. Therefore, the methods and countermeasures to harm fish are naturally different from each other.
  • Korean marine fish farming has brought about continuous productivity improvement along with the development of aquaculture technology
  • the current aquaculture industry is a cause of deterioration of the coastal aquaculture environment, aging of fishing grounds due to long-term intensive farming, deterioration of aquatic varieties, and disease occurrence.
  • Productivity is falling.
  • various infectious diseases and complications of fish species due to diversification of aquaculture species are due to the emergence of drug-resistant bacteria caused by frequent use of chemotherapeutic agents, as well as production cost due to deterioration of cultured fish or disposal of dead or diseased fish.
  • This has led to an increase in water quality, and countermeasures against these infectious diseases are currently the most important tasks in the aquaculture industry.
  • the proportion of damage caused by disease tends to increase, the fundamental method of disease treatment is being sought by preventing disease outbreaks and taking countermeasures.
  • bacterial diseases of farmed fish are caused by conditional pathogens that cause fish to die from pathogenic bacteria and secondary infection by other causes. Because there are many organic matters in the number of farms raised in aquaculture, it is an environment where many bacteria can reproduce, causing disease when fish are unhealthy or their defenses are weakened. Bacterial diseases are classified according to the type and symptoms of the infecting bacteria, and the frequency of occurrence is high, contagious, and, once infected, the amount of accumulated mortality is high, so economic loss is very large in aquaculture.
  • formalin bacteria are widely used as conventional commercial vaccines used for aquatic organisms, and since supplements such as oil are added to maintain the activity of the vaccine, there are side effects such as melanization and autoimmune reaction, as well as eels. For carnivorous fish, there is a problem that vaccination is difficult.
  • the present inventors have conducted extensive studies on vaccines that can be effectively applied to carnivorous fish, while encapsulating the antigen with a hydrogel containing starch, when orally administered to carnivorous fish, minimizes stressors in organisms and at the same time has an antibody titer.
  • the present invention was completed after confirming that it can be improved.
  • An object of the present invention is to provide a vaccine composition for oral administration that can be effectively applied to carnivorous fish.
  • the present invention to solve the above problems,
  • Hydrogels And an inactivated cell encapsulated by the hydrogel; an active ingredient is provided.
  • the hydrogel may reversibly expand or contract according to a change in pH to change its size.
  • the hydrogel may include starch.
  • the inactivated cells may be formalin-killed cell-cultured.
  • the inactivated cells may be pathogens of bacterial diseases of fish or pathogens of viral diseases.
  • the composition may be a vaccine for carnivorous fish.
  • the carnivorous fish may be one or more selected from the group consisting of eel, halibut (flounder), bark, rockfish, black sea bream, red snapper, starfish, mullet, sea bass, trout, mackerel, horse mackerel, and squid.
  • the present invention encapsulates a pathogen using a hydrogel that reversibly expands or contracts according to a change in pH to change its size, so that it is not digested in a stomach with a low pH and is decomposed after being delivered to a high pH intestine, effectively absorbed by the body. It can provide an oral vaccine composition for carnivorous fish.
  • the vaccine composition can be administered in combination with food, it is possible to minimize the factors causing stress in organisms, improve antibody titers, and at the same time do not use adjuvants to suppress side effects such as melaninization and autoimmunity.
  • Example 1 is a time according to Example 1, after oral administration of a vaccine encapsulated by a hydrogel containing starch according to the present invention (Starch vaccine) and a conventional formalin vaccine (formalin-killed cell-cultured, FKC) It is a graph showing the agglomeration titer.
  • FIG. 2 according to Example 1, the group (Orally administered once a day 0 times a vaccine (Starch vaccine) encapsulated by a hydrogel containing a starch according to the invention (Feed1), once a day orally administered a total of 4 times It is a graph showing the aggregation titer over time of the group (Feed4), the group administered orally 8 times a day (Feed8), and the group previously administered formalin vaccine 4 times (FKC) once a day.
  • FIG. 3 shows the results of morphological analysis of a vaccine (Starch hydrogel based oral vaccine, SHO vaccine) containing FKC antigen encapsulated in a hydrogel containing starch according to Example 2 as FESEM.
  • A is a starch before forming a hydrogel
  • B is a synthetic hydrogel based on a starch that does not contain an antigen
  • C is FESEM of a SHO vaccine containing formalin germ cells (E. tarda SU53).
  • Scale bars are 1 ⁇ m (A and C) or 2 ⁇ m (B).
  • Figure 4 shows the results of measuring the serum aggregation titer after oral administration of the FKC and SHO vaccines according to Example 2 (control: group administered orally with PBS-impregnated oral pellets only once, FKC group: Group administered orally with ground pellets mixed with FKC (conventional formalin vaccine) once daily for 4 times (4 days), SHO 1: group administered orally with SHO vaccine according to the present invention once per day, SHO 4: Group in which the SHO vaccine according to the invention was orally administered once daily for 4 days, SHO 8: Group in which the SHO vaccine according to the invention was administered orally once daily for 8 days).
  • Figure 5 shows the Kaplan-Meier survival curve derived by the challenge test using eel, according to Example 2.
  • the attack experiment was conducted in duplicate; Results are presented as first and second attempts, respectively.
  • Formalin bacteria are widely used as vaccines used in aquatic organisms in the past, and in the case of the vaccine, there are side effects such as melanization and autoimmune reactions because supplements such as oil are added to maintain the activity of the vaccine. For carnivorous fish, there was a problem that vaccination was difficult.
  • the present inventors have been conducting rigorous research on vaccines that can be effectively applied to carnivorous fish, and when they are orally administered to carnivorous fish by encapsulating the antigen with a hydrogel containing starch, they minimize the stress factor of living organisms and at the same time antibody titer It has been confirmed that can be significantly improved and completed the present invention.
  • the present invention is a hydrogel; And an inactivated cell encapsulated by the hydrogel; an active ingredient is provided.
  • the hydrogel may reversibly expand or contract according to a change in pH to change its size.
  • the hydrogel may have a characteristic of contracting when the pH is low and expanding when the pH is high. Due to these characteristics, when the vaccine composition according to the present invention is orally administered to carnivorous fish, in the stomach with low pH The hydrogel is present in a contracted state and is not degraded and is transferred to the intestine. In a high pH intestine, the hydrogel expands and decomposes, and the hydrogel and inactivated cells are dispersed, resulting in inactivation cells (pathogens) in the body. Can be effectively absorbed.
  • the hydrogel may be any material that changes in volume according to a change in pH and can be decomposed by digestive enzymes, and may be, for example, starch.
  • the inactivated cells may be used to inactivate pathogens of bacterial or parasitic diseases.
  • the inactivated cells may be inactivated through freezing treatment, heat treatment or formalin treatment.
  • formalin-killed cell-cultured cells may be used as the inactivated cells of the present invention.
  • the formalin treatment may be carried out at 0.3° C. to 2% formalin at 4° C. or at room temperature for 2 hours or more or 24 to 48 hours, and the freezing process may be performed at ⁇ 80 to 0° C.
  • the pathogen of the bacterial disease may be inactivated by heat treatment or formalin treatment, and the pathogen of the parasitic disease may be inactivated by freezing or formalin treatment.
  • the cells are Edwardsiella tarda, Streptococcus iniae, Aeromonas hydrophila, Vibrio harveyi, Tenassibaculum maritim , Lactococcus garvieae, a fish pathogen containing any one or more bacteria selected from the group consisting of, or a viral fish pathogen such as Irodovirus, but is not limited thereto.
  • the inactivated cells encapsulated with the hydrogel of the present invention may be used as the inactivated cells of Edward Sala Tara.
  • the composition may be a vaccine for carnivorous fish.
  • the carnivorous fish may be one or more selected from the group consisting of eel, halibut (flounder), bark, rockfish, black sea bream, red snapper, starfish, mullet, sea bass, trout, mackerel, horse mackerel, and squid.
  • the vaccine composition of the present invention may further include an inactivated strain, preferably a cell lysate or extracellular products (ECPs) of a killed strain, thereby further enhancing the immunity enhancing effect of the vaccine composition I can do it.
  • an inactivated strain preferably a cell lysate or extracellular products (ECPs) of a killed strain, thereby further enhancing the immunity enhancing effect of the vaccine composition I can do it.
  • ECPs extracellular products
  • the vaccine composition of the present invention may further include an adjuvant, for example, the adjuvant is selected from the group consisting of polysorbate-based nonionic surfactant, aluminum hydroxide gel, squalene and chitosan, etc. It may contain one or more.
  • the vaccine composition of the present invention may further include an adjuvant or an adjuvant, preferably the adjuvant is a group consisting of a polysorbate-based nonionic surfactant, aluminum hydroxide gel, squalene and chitosan, etc. It may include one or more selected from. Examples of the polysorbate-based nonionic surfactant may be twin 80, twin 60 or twin 20, and more preferably twin 80.
  • the vaccine composition according to the present invention may further include additives such as excipients and/or stabilizers.
  • the content of the inactivated cells contained in the vaccine composition according to the present invention can be set by appropriately adjusting according to the fish or pathogen to be applied.
  • the vaccine composition of the present invention may be prepared in consideration of the amount of inactivated cells injected per fish, and the content of the cells may be administered in batches or several times.
  • the present invention provides a method for preventing or treating bacterial diseases of fish by treating the vaccine composition with fish.
  • the method of prevention or treatment may be administered orally by vaccine composition.
  • Example 1-1 Ingredient preparation
  • Edwardsiella tarda SU53 (1980, Japan) isolated from eel and eel was stored in a freeze-dried state in the laboratory and was used throughout this experiment.
  • the bacteria were cultured in trypsin bean agar (TSA, Difco) medium at 25° C. for 24 hours.
  • the Edward Sala Tara strain (SU53) was cultured in trypsin beans (TSB, Difco) broth at 25° C. for 18 hours.
  • the cultured bacteria were treated with 1% formalin and maintained at 25° C. for 18 hours.
  • the formalin-treated culture was centrifuged at 13,000 rpm for 3 minutes, then washed twice in sterile phosphate buffered saline (PBS) and resuspended in sterile PBS. The suspension was adjusted to an O.D value of 1.0 through a spectrophotometer.
  • PBS sterile phosphate buffered saline
  • Example 1-2 Containing starch Hydrogel Encapsulated FKC Preparation of vaccine composition comprising antigen
  • Ethanol was dehydrated in 200 ml over 6 hours.
  • a vaccine starch vaccine
  • a vaccine starch vaccine containing inactivated cells encapsulated by a hydrogel containing starch was prepared according to the present invention by lyophilizing Ethanol and lyophilizing the solidified gel for at least 6 hours, and prepared above.
  • the vaccine was stored in powder form.
  • the prepared starch vaccine was prepared by dispensing so that the antigen dose of 1x10 8 could be administered to one animal. Blood samples were taken every 7 days, such as 8 days, 15 days, 22 days, 29 days, 36 days, and 43 days on the first day of oral administration of the vaccine to observe the antigen-antibody aggregation reaction using serum.
  • FIG. 1 is a graph showing the aggregation titer over time after oral administration of a vaccine encapsulated by a hydrogel containing starch according to the present invention (Starch vaccine) and a conventional formalin-killed cell-cultured (FKC) to be.
  • Starch vaccine a vaccine encapsulated by a hydrogel containing starch according to the present invention
  • FKC formalin-killed cell-cultured
  • Figure 2 is a group (Orally administered once a day 0 times the vaccine encapsulated by a hydrogel containing a starch according to the invention (Feed1), once a day orally administered a total of 4 times per day (Feed4), daily It is a graph showing the aggregation titer over time of the group (Fed8) orally administered once a total of 8 times (FKC) and the group previously administered four times of formalin antibacterial vaccine (FKC) once a day.
  • the graph of FIG. 2 shows the antigen-antibody aggregation reaction of the vaccine-administered group against E.
  • the starch vaccine according to the present invention was compared to the 1-starch vaccine group and the FKC 4-week group from Days 8 to 29 It was confirmed that the antibody titer of the antigen agglutination reaction of the group administered 8 times and 8 times was higher. In addition, in the case of the single administration of the starch vaccine, the effect of the vaccine was found to be lower than that of the multiple administration, and the results of the agglutination reaction were similar in the groups of 4 and 8 administrations. It was confirmed that it does not increase abnormally.
  • the bacteria were cultured at 25° C. in TSB (tryptic soy broth, Difco) medium or TSA (tryptic soy agar, Difco) medium.
  • eel eels (japanese eel, Anguilla japonica) (average length 28.0 cm; average weight, 82.4 ⁇ 7.9 g) were purchased from commercial fish farms in Gyeonggi-do and 10 before the experiment to confirm that there was no E. tarda infection Collected randomly from the subjects of T. Sakai, T. Lida, K. Osatomi, K. Kanai, Detection of type 1 fimbrial genes in fish pathogenic and non-pathogenic Edwardsiella tarda strains by PCR, Fish Pathol. 42 ( 2007) The polymerase chain reaction (PCR) was carried out according to the method reported in 115-117).
  • the remaining 550 animals were then acclimated to laboratory conditions of 27.5 ⁇ 0.5° C. for 1 week in a 5t tank.
  • the fish were fed with an artificial diet with constant aeration, and about 50% of the water in the tank was exchanged once a week.
  • 100 of the 500 were used for the median lethal dose (LD 50 ) determination test of E. tarda SU53, and 400 were used for the vaccination test. All animal care and experimental protocols were performed according to the guidelines of the Seoul National University Animal Ethics Committee.
  • Starch (1.33 g; Fluka, Buchs, Switzerland) was dissolved by stirring in 35 ml of distilled water (DW) at 70°C. Then, ammonium persulfate (Ammonium persulfate, APS) (100 mg; Fluka, Buchs, Switzerland) was added to the suspension, followed by further stirring for 10 minutes, and acrylic acid (AA) (1.5 g; Merck, Darmstadt, Germany) and 2-Hydroxyethyl methacrylate (2-HEMA) (Merck, Darmstadt, Germany) 1.5 g was further added and stirred.
  • AA acrylic acid
  • 2-HEMA 2-Hydroxyethyl methacrylate
  • N,N'-methylene bisacrylamide N,N'-MBA
  • KFC antigen (inactivated cells)
  • the reaction product was cooled to ambient temperature for 30 minutes, neutralized to pH 7.5 by adding 1M NaOH solution, and then dehydrated by adding 200 ml of Ethanol for 6 hours or more.
  • a vaccine starch vaccine containing inactivated cells encapsulated by a hydrogel containing starch was prepared according to the present invention by lysing Ethanol and lyophilizing the solidified gel for 6 hours. The vaccine was stored in powder form at -20 °C for further use.
  • Morphological analysis was performed using a field emission scanning electron microscope (FESEM; Sigma, Carl Zeiss, UK). Three specimens were prepared and mounted on the stub: starch before hydrogel formation; Synthetic hydrogels based on starch containing no antigen; And SHO vaccine according to the present invention. Prior to scanning, the specimens were sputter coated with gold for 180 seconds using a vacuum coater (EM ACE 200; Leica, Austria).
  • FESEM field emission scanning electron microscope
  • the powdered hydrogel was thoroughly mixed with the ground pellets, and then a certain amount of PBS was added to the mixture.
  • the antigen content of each SHO vaccine was adjusted to 10 8 CFU per fish.
  • 5 eel were randomly selected and anesthetized using MS-222. SHO vaccine was orally administered to these fish and maintained at 27.5 ⁇ 0.5°C.
  • One week after vaccination, the eel was euthanized with MS-222 and blood was collected from the tail vein to confirm that the SHO vaccine produced immunity.
  • the second vaccination was performed only once on the 46th day after the first vaccination when it was found that the serum aggregated antibody titers decreased.
  • 20 eel were randomly selected from each of the 5 groups.
  • PBS-impregnated pellets were orally administered to the control fish;
  • FKC group fish were orally administered pulverized pellets mixed with FKC;
  • SHO 1, SHO 4 and SHO 8 fish were administered orally with SHO.
  • Each group of fish was then transferred separately at 27.5 ⁇ 0.5° C. to a 200 L glass fiber reinforced plastic aquarium.
  • 3 eel were randomly selected from each of the 5 groups and blood sampling was performed. Serum aggregation experiments were performed as described above.
  • E. tarda SU53 in the early-exponential phase was used for challenge testing and diluted 10-fold serially with PBS.
  • the final injection dose ranged from 2.0 ⁇ 10 4 to 2.0 ⁇ 10 7 CFU/fish.
  • Control fish were injected with 100 ⁇ l of sterile PBS. After injection, fish were monitored for 15 days. Dead fish were sampled daily; Bacteria were isolated from the kidneys and confirmed using PCR.
  • An injection (i.p.) attack experiment was performed 4 weeks after the first vaccination.
  • the test fish was maintained at 27.5 ⁇ 0.5° C. in a 100 L glass fiber reinforced plastic water tank supplied with running water. Clinical signs of disease and cumulative mortality were monitored twice a day for 15 days after injection. Sampling was performed in the kidneys of the dead fish, and the samples were identified using the PCR method described above.
  • RNA samples were extracted from tissue samples of the head kidney and liver using TRIzol Reagent (CWBio, Beijing, China). RNA concentration and purity were quantified by spectrophotometry, which showed a 260:280 ratio between 1.6 and 1.8; RNA quality was verified using electrophoresis on a 1% agarose gel supplemented with 0.5 ⁇ g/mL ethidium bromide. To prevent DNA contamination, whole RNA samples were treated with DNAse I (Madison, WI, USA) according to the manufacturer's instructions.
  • DNAse I Madison, WI, USA
  • RNA was reverse transcribed into cDNA using PrimeScript RT reagent kit (TaKaRa Bio, Otsu, Japan) according to the manufacturer's protocol.
  • the resulting cDNA was stored at -80 °C until use.
  • RT-qPCR Real-time Quantitative PCR detection system for the expression of genes involved in immune responses such as interferon (IFN)- ⁇ , interleukin (IL)-6 and tumor necrosis factor (TNF)- ⁇ (QIAGEN; Hilden, Germany).
  • IFN interferon
  • IL-6 interleukin-6
  • TNF tumor necrosis factor
  • Starch was evenly distributed prior to vaccine preparation (Figure 3A). Starch successfully formed agglomerated hydrogels by water absorption and expansion (Fig. 3B). Antigen (FKC) was added to coat FKC on a starch hydrogel and a SHO vaccine was prepared, confirming that it was successfully prepared through FESEM (FIG. 3C).
  • Figure 3A Starch successfully formed agglomerated hydrogels by water absorption and expansion (Fig. 3B).
  • Antigen (FKC) was added to coat FKC on a starch hydrogel and a SHO vaccine was prepared, confirming that it was successfully prepared through FESEM (FIG. 3C).
  • FKC and SHO 1 showed relatively smaller titers (SHO 1 produced higher titers than FKC after the second vaccination), and SHO 4 and SHO 8 showed higher titers. Fish of SHO 4 and SHO 8 showed the highest titer at 3 wpv, and the same value at 8 wpv after the second vaccination showed similar pattern change.
  • FIG. 5 The results for the protective effect of FKC and SHO vaccines against Edwardsiellosis are shown in FIG. 5.
  • the mortality rate after challenge with all vaccinated groups at 4 wpv was lower than that of the control group.
  • the progression of mortality after challenge was consistent with the results of an attack experiment conducted to determine the LD 50 concentration of E. tarda SU53: mortality was recorded from 3 dpc to 9 dpc and further mortality was observed Did not.
  • the survival rate of fish in the FKC group was 60% and 70%, respectively, in the first and second trials.
  • the survival rate of fish inoculated with SHO vaccine was higher than in the group inoculated with FKC vaccine.
  • the survival rate of the SHO vaccination group was as follows: 70% (first trial) and 80% (second trial) in SHO 1; 80% (first trial) and 90% (second trial) in SHO 4; And 80% (first trial) and 90% (second trial) in SHO 8. There was no significant difference in the survival rate between SHO 4 and SHO 8, and both showed the highest and the same level of disease defense effect.
  • Figure 6 shows the cytokine gene expression in the head kidney or liver of the eel in each group after vaccination.
  • vaccination increased the level of the cytokine gene as compared to the control group.
  • SHO vaccination induced higher levels of cytokine gene expression than when FKC vaccination; Expression levels were higher in SHO 4 and SHO 8 compared to SHO 1 (FIG. 6 ).
  • Cytokine gene expression of SHO 4 and SHO 8 increased at 3 wpv, decreased at 4 wpv, showed a tendency to increase again at 8 wpv, and showed the highest expression level at 8 wpv of SHO 8. Differences in cytokine gene expression over time were not significant in the SHO 1 or FKC group.
  • both groups showed a tendency to increase from 3 wpv to 8 wpv.
  • IFN- ⁇ expression in the SHO 1 and FKC groups changed in the same way as in SHO 4 and SHO 8, increasing at 3 wpv, decreasing at 4 wpv and increasing again at 8 wpv.
  • TNF- ⁇ expression showed a tendency to increase in the FKC group, whereas in SHO 1 it decreased from 3 to 4 wpv and then increased to 8 wpv.
  • composition according to the present invention encapsulates a pathogen using a hydrogel that reversibly expands or contracts according to a change in pH to change its size, so it is not digested in a stomach with a low pH and is decomposed after being delivered to a high pH intestine. Since it can be effectively absorbed, it can be widely used in the vaccine field for oral administration.

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Abstract

The present invention pertains to an oral vaccine composition containing hydrogel-encapsulated inactivated cells. The present invention can provide an oral vaccine composition for carnivorous fish, wherein the oral vaccine composition uses a hydrogel, which changes in size by reversibly expanding or contracting in response to changes in pH, to encapsulate pathogens, and thus is not digested in the stomach, which has a low pH, and decomposes to be effectively absorbed into the body after being delivered to the intestines, which have a high pH. In addition, the vaccine composition can be administered in combination with food, and can thus minimize stress-inducing factors in organisms. Since antibody titer is improved and supplements are not used, side effects such as melanization and autoimmunity can be suppressed.

Description

하이드로겔에 의해 캡슐화된 불활성화 균체를 포함하는 경구 투여용 백신 조성물Vaccine composition for oral administration comprising inactivated cells encapsulated by hydrogel
본 발명은 하이드로겔에 의해 캡슐화된 불활성화 균체를 포함하는 경구 투여용 백신 조성물에 관한 것이다.The present invention relates to a vaccine composition for oral administration comprising an inactivated cell encapsulated by a hydrogel.
어류는 변온동물로서 환경에 대한 적응력을 어느 정도 갖고 있지만 그 한계를 넘어버리면 생리적 장해를 일으키게 된다. 어류에 있어서 질병은 육상동물과 같이 내적·외적 환경에 대해 더 이상 건강상태를 유지할 수 없는 상태를 말한다. 질병은 숙주의 요인, 발병인자 및 환경과의 상관관계에 의한 결과로서 나타나는 현상으로 질병 발생요인 중 발병 인자만이 반드시 질병을 발생시키는 것은 아니며, 숙주와 환경의 상호작용에 의해 질병이 발생하거나 발생하지 않는다. 즉 발병인자와 숙주의 요인 그리고 환경과의 균형이 잘 이루어진 상태에서는 질병이 발생하지 않으며 이들 균형이 깨어질 경우 발생하는데 대부분의 경우 질병의 발생은 환경조건에 크게 영향을 받는다고 볼 수 있다.Fish are warm-blooded animals that have a certain degree of adaptability to the environment, but beyond that limit, they cause physiological disturbances. In fish, disease is a condition in which, like land animals, it is no longer possible to maintain a healthy state for the internal and external environment. Disease is a phenomenon that occurs as a result of correlation with factors of host, pathogenesis factor and environment. It is not only the pathogen that causes disease, but the disease occurs or occurs by interaction between host and environment I never do that. In other words, the disease does not occur in a well-balanced condition between the pathogen and the host factors and the environment, and it occurs when these balances are broken. In most cases, the occurrence of the disease is greatly affected by environmental conditions.
어류의 체내에 병원체가 침입하는 경로는 피부, 아가미, 비강, 소화관 등으로 볼 수 있다. 이들 기관은 점액, 효소, 항균성 및 살균성 물질, 항체, 백혈구, 대식세포 등의 분비 및 작용에 의해 침입하는 세균 등으로부터 보호되고 있으나. 선별 시에 입는 기계적인 손상 또는 기생충에 의한 상처, 사육관리의 부실 등에 의해 이들 보호 물질이 손상을 입게 되며 그곳을 통하여 병원체가 체내에 침입하게 되고 이어서 혈류를 타고 장기나 조직에 도달하여 병소를 형성하게 된다.The path of pathogens entering the body of fish can be seen as skin, gills, nasal cavity, and digestive tract. These organs are protected from bacteria that invade by secretion and action of mucus, enzymes, antibacterial and bactericidal substances, antibodies, white blood cells, and macrophages. During screening, these protective substances are damaged by mechanical damage or parasite wounds, poor management, etc., through which pathogens enter the body and then reach the organs or tissues through the bloodstream to form lesions. Is done.
어류에 피해를 입히는 병원생물은 크게 나누면 원생동물(기생충), 진균류(곰팡이), 세균, 바이러스 등이 있다. 기생충은 수 십 ㎝에서부터 수 백㎛, 세균은 수 ㎛, 바이러스는 수 천분의 1 ㎛ 되는 정도로 대단히 작다. 따라서 이들이 어류에 피해를 주는 방법 및 대책법도 당연히 서로 다르게 되는 것이다.The largely divided pathogenic organisms that damage fish include protozoa (parasites), fungi (fungi), bacteria, and viruses. Parasites are very small, ranging from tens of centimeters to hundreds of μm, bacteria to several μm, and viruses to several thousandths of a μm. Therefore, the methods and countermeasures to harm fish are naturally different from each other.
우리나라 해산어 양식은 양식기술의 발달과 더불어 지속적인 생산성 향상을 가져왔지만 현재 양식 산업은 연안 양식 환경의 악화, 장기간의 집약적인 양식으로 인한 어장의 노화, 양식품종의 열성화 및 질병발생의 원인으로 양식생산성이 저하되고 있다. 특히, 양식어종의 다양화에 따라 어종에 빈발하는 각종 감염증 및 이들의 합병증은 화학요법제의 빈번한 사용에 의한 약제내성균의 출현과 아울러, 양식어의 품질 저하나 폐사어나 병어의 폐기에 의한 생산비용의 상승을 초래하고, 이러한 감염증에 대한 대책이 현재 양식업계의 가장 중요한 과제가 되고 있다. 게다가 질병 발생에 의한 피해가 차지하는 비중이 점점 높아지는 경향이 있어 질병발생을 사전에 예방하고 대책을 강구하여 질병치료의 근본적인 방법이 강구되고 있는 실정이다.Although Korean marine fish farming has brought about continuous productivity improvement along with the development of aquaculture technology, the current aquaculture industry is a cause of deterioration of the coastal aquaculture environment, aging of fishing grounds due to long-term intensive farming, deterioration of aquatic varieties, and disease occurrence. Productivity is falling. In particular, various infectious diseases and complications of fish species due to diversification of aquaculture species are due to the emergence of drug-resistant bacteria caused by frequent use of chemotherapeutic agents, as well as production cost due to deterioration of cultured fish or disposal of dead or diseased fish. This has led to an increase in water quality, and countermeasures against these infectious diseases are currently the most important tasks in the aquaculture industry. In addition, since the proportion of damage caused by disease tends to increase, the fundamental method of disease treatment is being sought by preventing disease outbreaks and taking countermeasures.
한편, 양식어의 세균성 질병은 병원성 세균으로부터 다른 원인에 의한 2차적인 감염으로 어류를 죽게하는 조건적 병원체에 의해 발생한다. 양식장의 사육수에는 유기질이 많기 때문에 많은 세균이 번식할 수 있는 환경임으로 어류가 건강하지 못하거나 방어력이 약해졌을 때 질병을 일으킨다. 세균성 질병은 감염세균의 종류 및 증상에 따라 질병의 종류가 구분되며, 발생빈도가 높고 전염성이 강하며 일단 감염이 되면 누적폐사량이 많기 때문에 양식장에서는 경제적 손실이 대단히 크게 된다.On the other hand, bacterial diseases of farmed fish are caused by conditional pathogens that cause fish to die from pathogenic bacteria and secondary infection by other causes. Because there are many organic matters in the number of farms raised in aquaculture, it is an environment where many bacteria can reproduce, causing disease when fish are unhealthy or their defenses are weakened. Bacterial diseases are classified according to the type and symptoms of the infecting bacteria, and the frequency of occurrence is high, contagious, and, once infected, the amount of accumulated mortality is high, so economic loss is very large in aquaculture.
현재, 국내 어류 양식장에서 발생하는 세균성 질병에는 비브리오병, 에드워드병, 연쇄구균증, 활주세균증 등 여러 가지가 있지만, 양식장에 가장 많은 피해를 주는 질병은 에드워드병과 연쇄구균증으로 알려져있다.Currently, there are several bacterial diseases that occur in fish farms in Korea, such as Vibrio disease, Edward disease, streptococcus, and slide bacteriosis, but the most damaging diseases are known as Edward disease and streptococcus.
관련하여 수생 생물에 사용되는 종래 시판 백신으로는 포르말린 사균이 널리 사용되고 있는데, 백신의 활성 유지를 위해 기름과 같은 보조제가 첨가되기 때문에 멜라닌화, 자가면역 반응 등의 부작용이 있을 뿐만 아니라, 뱀장어와 같은 육식성 어류에 대해서는 백신 주사가 곤란하다는 문제점이 존재한다.In relation to this, formalin bacteria are widely used as conventional commercial vaccines used for aquatic organisms, and since supplements such as oil are added to maintain the activity of the vaccine, there are side effects such as melanization and autoimmune reaction, as well as eels. For carnivorous fish, there is a problem that vaccination is difficult.
본 발명자들은 육식성 어류에 효과적으로 적용될 수 있는 백신에 대하여 예의 연구하던 중, 녹말을 포함하는 하이드로겔로 항원을 캡슐화하여 육식성 어류에 경구 투여할 경우 생물체의 스트레스 발생 요인을 최소화함과 동시에 항체 역가가 월등히 향상될 수 있다는 점을 확인하고 본 발명을 완성하였다.The present inventors have conducted extensive studies on vaccines that can be effectively applied to carnivorous fish, while encapsulating the antigen with a hydrogel containing starch, when orally administered to carnivorous fish, minimizes stressors in organisms and at the same time has an antibody titer. The present invention was completed after confirming that it can be improved.
본 발명은 육식성 어류에 효과적으로 적용될 수 있는 경구 투여용 백신 조성물을 제공하는 것을 목적으로 한다.An object of the present invention is to provide a vaccine composition for oral administration that can be effectively applied to carnivorous fish.
본 발명은 상기 과제를 해결하기 위하여,The present invention to solve the above problems,
하이드로겔; 및 상기 하이드로겔에 의해 캡슐화된 불활성화 균체;를 유효성분으로 포함하는 경구 투여용 백신 조성물을 제공한다.Hydrogels; And an inactivated cell encapsulated by the hydrogel; an active ingredient is provided.
본 발명에 따르면, 상기 하이드로겔은 pH의 변화에 따라 가역적으로 팽창 또는 수축하여 크기가 변화할 수 있다.According to the present invention, the hydrogel may reversibly expand or contract according to a change in pH to change its size.
본 발명에 따르면, 상기 하이드로겔은 녹말을 포함할 수 있다.According to the present invention, the hydrogel may include starch.
본 발명에 따르면, 상기 불활성화 균체는 포르말린 사균(formalin-killed cell-cultured)일 수 있다.According to the present invention, the inactivated cells may be formalin-killed cell-cultured.
본 발명에 따르면, 상기 불활성화 균체는 어류의 세균성 질병의 병원체 또는 바이러스성 질병의 병원체일 수 있다.According to the present invention, the inactivated cells may be pathogens of bacterial diseases of fish or pathogens of viral diseases.
본 발명에 따르면, 상기 조성물은 육식성 어류용 백신일 수 있다.According to the present invention, the composition may be a vaccine for carnivorous fish.
이때, 상기 육식성 어류는 뱀장어, 넙치(광어), 조피볼락, 우럭, 감성돔, 참돔, 능성어, 숭어, 농어, 전어, 고등어, 전갱이, 쥐치로 이루어진 군에서 선택되는 1종 이상일 수 있다.At this time, the carnivorous fish may be one or more selected from the group consisting of eel, halibut (flounder), bark, rockfish, black sea bream, red snapper, starfish, mullet, sea bass, trout, mackerel, horse mackerel, and squid.
본 발명은 pH의 변화에 따라 가역적으로 팽창 또는 수축하여 크기가 변화하는 하이드로겔을 이용하여 병원체를 캡슐화함으로써, pH가 낮은 위에서는 소화되지 않고, pH가 높은 장내까지 전달 후 분해되어 체내에 효과적으로 흡수되는 육식성 어류용 경구 백신 조성물을 제공할 수 있다.The present invention encapsulates a pathogen using a hydrogel that reversibly expands or contracts according to a change in pH to change its size, so that it is not digested in a stomach with a low pH and is decomposed after being delivered to a high pH intestine, effectively absorbed by the body. It can provide an oral vaccine composition for carnivorous fish.
또한, 상기 백신 조성물은 먹이와 혼합 투여가 가능하기 때문에 생물체의 스트레스 발생 요인을 최소화할 수 있으며, 항체 역가가 향상됨과 동시에 보조제 사용이 없어 멜라닌화, 자가 면역과 같은 부작용 발생을 억제할 수 있다.In addition, since the vaccine composition can be administered in combination with food, it is possible to minimize the factors causing stress in organisms, improve antibody titers, and at the same time do not use adjuvants to suppress side effects such as melaninization and autoimmunity.
도 1은 실시예 1에 따라, 본 발명에 따른 녹말을 포함하는 하이드로겔에 의해 캡슐화된 백신(Starch vaccine)과 종래 포르말린 사균 백신(formalin-killed cell-cultured, FKC)을 경구 투여한 이후 시간에 따른 응집 역가를 나타낸 그래프이다.1 is a time according to Example 1, after oral administration of a vaccine encapsulated by a hydrogel containing starch according to the present invention (Starch vaccine) and a conventional formalin vaccine (formalin-killed cell-cultured, FKC) It is a graph showing the agglomeration titer.
도 2는 실시예 1에 따라, 본 발명에 따른 녹말을 포함하는 하이드로겔에 의해 캡슐화된 백신(Starch vaccine)을 0일차 1회 경구 투여한 군(Feed1), 매일 1회씩 총 4회 경구 투여한 군(Feed4), 매일 1회씩 총 8회 경구 투여한 군(Feed8) 및 종래 포르말린 사균 백신을 매일 1회씩 총 4회 투여한 군(FKC)들의 시간에 따른 응집 역가를 나타낸 그래프이다.Figure 2, according to Example 1, the group (Orally administered once a day 0 times a vaccine (Starch vaccine) encapsulated by a hydrogel containing a starch according to the invention (Feed1), once a day orally administered a total of 4 times It is a graph showing the aggregation titer over time of the group (Feed4), the group administered orally 8 times a day (Feed8), and the group previously administered formalin vaccine 4 times (FKC) once a day.
도 3은 실시예 2에 따라, 녹말을 포함하는 하이드로겔로 캡슐화된 FKC 항원을 포함하는 백신(Starch hydrogel based oral vaccine, SHO vaccine)의 형태학적 분석 결과를 FESEM으로 나타낸 것이다. (A)는 하이드로겔을 형성하기 전의 녹말, (B)는 항원을 함유하지 않는 녹말에 기초한 합성 하이드로겔, (C)는 포르말린 사균 세포(E. tarda SU53)를 함유하는 SHO 백신의 FESEM이다. 스케일 바는 1μm (A 및 C) 또는 2μm (B)이다.FIG. 3 shows the results of morphological analysis of a vaccine (Starch hydrogel based oral vaccine, SHO vaccine) containing FKC antigen encapsulated in a hydrogel containing starch according to Example 2 as FESEM. (A) is a starch before forming a hydrogel, (B) is a synthetic hydrogel based on a starch that does not contain an antigen, (C) is FESEM of a SHO vaccine containing formalin germ cells (E. tarda SU53). Scale bars are 1 μm (A and C) or 2 μm (B).
도 4는 실시예 2에 따라, FKC 및 SHO 백신의 경구 투여 후 혈청 응집 역가를 측정한 결과를 나타낸 것이다(대조군: PBS 함침된(PBS-impregnated) 경구 펠릿을 한번만 경구 투여한 그룹, FKC group: FKC와 혼합된 그라운드 펠릿을(종래 포르말린 사균 백신)을 매일 1회씩 총 4회(4일) 경구 투여한 그룹, SHO 1: 본 발명에 따른 SHO 백신을 1일 동안 1회 경구 투여한 그룹, SHO 4: 본 발명에 따른 SHO 백신을 4일 동안 매일 1회씩 경구 투여한 그룹, SHO 8: 본 발명에 따른 SHO 백신을 8일 동안 매일 1회씩 경구 투여한 그룹). PBS, FKC 또는 SHO를 사용한 첫 번째 백신 접종 후 46 일에 부스트 백신 접종을 수행하였다. 막대는 평균±표준 편차를 나타낸다(n = 3). 대조군에서는 항체가 검출되지 않았다.Figure 4 shows the results of measuring the serum aggregation titer after oral administration of the FKC and SHO vaccines according to Example 2 (control: group administered orally with PBS-impregnated oral pellets only once, FKC group: Group administered orally with ground pellets mixed with FKC (conventional formalin vaccine) once daily for 4 times (4 days), SHO 1: group administered orally with SHO vaccine according to the present invention once per day, SHO 4: Group in which the SHO vaccine according to the invention was orally administered once daily for 4 days, SHO 8: Group in which the SHO vaccine according to the invention was administered orally once daily for 8 days). Boost vaccination was performed 46 days after the first vaccination with PBS, FKC or SHO. Bars represent mean±standard deviation (n=3). No antibodies were detected in the control group.
도 5는 실시예 2에 따라, 뱀장어를 이용한 공격실험(challenge test)에 의해 도출된 Kaplan-Meier 생존 곡선을 나타낸 것이다. 첫 백신 접종 후 4주차에 주사 (i.p.) 챌린지를 수행하였고, 5개 그룹 (대조군, FKC, SHO 1, SHO 4 및 SHO 8)에서의 생존 백분율을 도시하였다 (n = 10). 공격실험은 이중으로 수행되었다; 결과는 각각 첫 번째 및 두 번째 시도로 표시된다.Figure 5 shows the Kaplan-Meier survival curve derived by the challenge test using eel, according to Example 2. Injection (i.p.) challenge was performed 4 weeks after the first vaccination and the percentage survival in 5 groups (control, FKC, SHO 1, SHO 4 and SHO 8) was shown (n=10). The attack experiment was conducted in duplicate; Results are presented as first and second attempts, respectively.
도 6은 실시예 2에 따라, FKC 또는 SHO 백신 접종 후, 뱀장어의 간에서 IL-6, TNF-α 및 IFN-α의 상대적 mRNA 발현 측정 결과를 나타낸 것이다. 막대는 평균±표준 편차를 나타낸다 (n = 3). 대조군과 비교할 때 유의미한 차이(p < 0.05)는 별표로 표시된다.Figure 6 shows the results of measuring the relative mRNA expression of IL-6, TNF-α and IFN-α in the eel liver after vaccination with FKC or SHO according to Example 2. Bars represent mean±standard deviation (n=3). Significant differences (p <0.05) compared to controls are marked with an asterisk.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로, 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.
종래 수생 생물에 사용되는 백신으로 포르말린 사균이 널리 사용되고 있는데, 상기 백신의 경우 백신의 활성 유지를 위해 기름과 같은 보조제가 첨가되기 때문에 멜라닌화, 자가면역 반응 등의 부작용이 있을 뿐만 아니라, 뱀장어와 같은 육식성 어류에 대해서는 백신 주사가 곤란하다는 문제점이 존재하였다.Formalin bacteria are widely used as vaccines used in aquatic organisms in the past, and in the case of the vaccine, there are side effects such as melanization and autoimmune reactions because supplements such as oil are added to maintain the activity of the vaccine. For carnivorous fish, there was a problem that vaccination was difficult.
이에, 본 발명자들은 육식성 어류에 효과적으로 적용될 수 있는 백신에 대하여 예의 연구하던 중, 녹말을 포함하는 하이드로겔로 항원을 캡슐화하여 육식성 어류에 경구 투여할 경우 생물체의 스트레스 발생 요인을 최소화함과 동시에 항체 역가가 월등히 향상될 수 있다는 점을 확인하고 본 발명을 완성하였다.Accordingly, the present inventors have been conducting rigorous research on vaccines that can be effectively applied to carnivorous fish, and when they are orally administered to carnivorous fish by encapsulating the antigen with a hydrogel containing starch, they minimize the stress factor of living organisms and at the same time antibody titer It has been confirmed that can be significantly improved and completed the present invention.
이를 위해, 본 발명은 하이드로겔; 및 상기 하이드로겔에 의해 캡슐화된 불활성화 균체;를 유효성분으로 포함하는 경구 투여용 백신 조성물을 제공한다.To this end, the present invention is a hydrogel; And an inactivated cell encapsulated by the hydrogel; an active ingredient is provided.
이때, 상기 하이드로겔은 pH의 변화에 따라 가역적으로 팽창 또는 수축하여 크기가 변화할 수 있다. 구체적으로, 상기 하이드로겔은 pH가 낮아지면 수축하고, pH가 높아지면 팽창하는 특성을 가질 수 있으며, 이러한 특성으로 인하여 본 발명에 따른 백신 조성물이 육식성 어류에 경구 투여될 경우, pH가 낮은 위장에서는 하이드로겔이 수축된 상태로 존재하여 분해되지 않고 장내까지 전달되고, pH가 높은 장내에서는 하이드로겔이 팽창하면서 분해되고, 상기 하이드로겔과 불활성화 균체가 분산되어 결과적으로 불활성화 균체(병원체)가 체내에서 효과적으로 흡수될 수 있다.At this time, the hydrogel may reversibly expand or contract according to a change in pH to change its size. Specifically, the hydrogel may have a characteristic of contracting when the pH is low and expanding when the pH is high. Due to these characteristics, when the vaccine composition according to the present invention is orally administered to carnivorous fish, in the stomach with low pH The hydrogel is present in a contracted state and is not degraded and is transferred to the intestine. In a high pH intestine, the hydrogel expands and decomposes, and the hydrogel and inactivated cells are dispersed, resulting in inactivation cells (pathogens) in the body. Can be effectively absorbed.
상기 하이드로겔은 pH의 변화에 따라 부피가 변화되고 소화 효소에 의해 분해될 수 있는 물질이라면 모두 가능하며, 예를 들어 녹말일 수 있다.The hydrogel may be any material that changes in volume according to a change in pH and can be decomposed by digestive enzymes, and may be, for example, starch.
본 발명에 있어서, 상기 불활성화 균체는 세균성 또는 기생충성 질병의 병원체를 불활성화시킨 것이 사용될 수 있다. 상기 불활성화 균체는 동결 처리, 열 처리 또는 포르말린 처리 등을 통해 불활성화 될 수 있다. 바람직하게, 본 발명의 불활성화 균체는 포르말린 사균(formalin-killed cell-cultured)이 사용될 수 있다. 상기 포르말린 처리는 0.3%~2%의 포르말린으로 4℃ 또는 상온에서 2시간 이상 또는 24~48 시간 동안 수행될 수 있으며, 상기 동결 처리는 -80 내지 0℃에서 수행될 수 있다. 상기 세균성 질병의 병원체는 열처리 또는 포르말린 처리 방법에 의하여 불활성화 처리될 수 있으며, 상기 기생충성 질병의 병원체는 동결처리 또는 포르말린 처리 방법에 의하여 불활성화 처리될 수 있다.In the present invention, the inactivated cells may be used to inactivate pathogens of bacterial or parasitic diseases. The inactivated cells may be inactivated through freezing treatment, heat treatment or formalin treatment. Preferably, formalin-killed cell-cultured cells may be used as the inactivated cells of the present invention. The formalin treatment may be carried out at 0.3° C. to 2% formalin at 4° C. or at room temperature for 2 hours or more or 24 to 48 hours, and the freezing process may be performed at −80 to 0° C. The pathogen of the bacterial disease may be inactivated by heat treatment or formalin treatment, and the pathogen of the parasitic disease may be inactivated by freezing or formalin treatment.
본 발명에 있어서, 상기 균체는 에드워드샐라 타라(Edwardsiella tarda), 스트렙토코커스 이니애(Streptococcus iniae), 에어로모나스 하이드로필라(Aeromonas hydrophila), 비브리오 하베이(Vibrio harveyi), 테나시바쿨럼 마리티멈(Tenacibaculum maritimum), 락토코커스 가배(Lactococcus garvieae) 등으로 이루어지는 군에서 선택된 어느 하나 이상의 세균을 포함하는 어류 병원체 또는 이리도바이러스(Irodovirus) 등과 같은 바이러스성 어류 병원체 등일 수 있으나, 이에 제한되지 않는다. 바람직하게, 본 발명의 하이드로겔로 캡슐화된 불활성화 균체는 에드워드샐라 타라의 불활성화 균체가 사용될 수 있다.In the present invention, the cells are Edwardsiella tarda, Streptococcus iniae, Aeromonas hydrophila, Vibrio harveyi, Tenassibaculum maritim , Lactococcus garvieae, a fish pathogen containing any one or more bacteria selected from the group consisting of, or a viral fish pathogen such as Irodovirus, but is not limited thereto. Preferably, the inactivated cells encapsulated with the hydrogel of the present invention may be used as the inactivated cells of Edward Sala Tara.
본 발명에 따르면, 상기 조성물은 육식성 어류용 백신일 수 있다.According to the present invention, the composition may be a vaccine for carnivorous fish.
이때, 상기 육식성 어류는 뱀장어, 넙치(광어), 조피볼락, 우럭, 감성돔, 참돔, 능성어, 숭어, 농어, 전어, 고등어, 전갱이, 쥐치로 이루어진 군에서 선택되는 1종 이상일 수 있다.At this time, the carnivorous fish may be one or more selected from the group consisting of eel, halibut (flounder), bark, rockfish, black sea bream, red snapper, starfish, mullet, sea bass, trout, mackerel, horse mackerel, and squid.
또한, 본 발명의 백신 조성물은 불활성화 균주, 바람직하게 사멸 균주의 세포 용해물 또는 세포 외 산물(extracellular products, ECPs) 등을 추가로 포함할 수 있으며, 이를 통해 백신 조성물의 면역력 향상 효과를 더욱 증진시킬 수 있다.In addition, the vaccine composition of the present invention may further include an inactivated strain, preferably a cell lysate or extracellular products (ECPs) of a killed strain, thereby further enhancing the immunity enhancing effect of the vaccine composition I can do it.
또한, 본 발명의 백신 조성물은 면역 보조제를 추가로 포함할 수 있으며, 예를 들어, 상기 면역 보조제는 폴리솔베이트계 비이온성 계면활성제, 알루미늄 하이드록사이드 겔, 스쿠알렌 및 키토산 등으로 이루어진 군으로부터 선택된 1종 이상을 포함할 수 있다. 또한, 본 발명의 백신 조성물은 면역 보조제 또는 면역 증강제 등을 추가로 포함할 수 있으며, 바람직하게 상기 면역 증강제는 폴리솔베이트계 비이온성 계면활성제, 알루미늄 하이드록사이드 겔, 스쿠알렌 및 키토산 등으로 이루어진 군으로부터 선택된 1종 이상을 포함할 수 있다. 상기 폴리솔베이트계 비이온성 계면활성제의 예는 트윈 80, 트윈 60 또는 트윈 20일 수 있으며, 더욱 바람직하게는 트윈 80일 수 있다. 또한, 본 발명에 따른 백신 조성물은 부형제 및/또는 안정화제 등의 첨가제를 추가로 포함할 수 있다.In addition, the vaccine composition of the present invention may further include an adjuvant, for example, the adjuvant is selected from the group consisting of polysorbate-based nonionic surfactant, aluminum hydroxide gel, squalene and chitosan, etc. It may contain one or more. In addition, the vaccine composition of the present invention may further include an adjuvant or an adjuvant, preferably the adjuvant is a group consisting of a polysorbate-based nonionic surfactant, aluminum hydroxide gel, squalene and chitosan, etc. It may include one or more selected from. Examples of the polysorbate-based nonionic surfactant may be twin 80, twin 60 or twin 20, and more preferably twin 80. In addition, the vaccine composition according to the present invention may further include additives such as excipients and/or stabilizers.
본 발명에 따른 백신 조성물에 포함되는 불활성화 균체의 함량은 적용 대상 어류 또는 병원체 등에 따라 적절히 조절하여 설정될 수 있다. 본 발명의 백신 조성물은 어류 1 마리당 주입되는 불활성화 균체의 함량을 고려하여 제조될 수 있으며, 상기 균체의 함량은 일괄 또는 수회 나누어 투여될 수 있다.The content of the inactivated cells contained in the vaccine composition according to the present invention can be set by appropriately adjusting according to the fish or pathogen to be applied. The vaccine composition of the present invention may be prepared in consideration of the amount of inactivated cells injected per fish, and the content of the cells may be administered in batches or several times.
또한, 본 발명은 상기 백신 조성물을 어류에 처리하여 어류의 세균성 질병을 예방 또는 치료하는 방법을 제공한다. 상기 예방 또는 치료 방법은 백신 조성물을 경구를 통해 투여할 수 있다.In addition, the present invention provides a method for preventing or treating bacterial diseases of fish by treating the vaccine composition with fish. The method of prevention or treatment may be administered orally by vaccine composition.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
실시예 1.Example 1.
실시예 1-1. 재료 준비Example 1-1. Ingredient preparation
(1) 어류의 준비(1) Preparation of fish
22.4 g의 평균 개체량을 가지며 정상인 300마리의 뱀장어과의 뱀장어(japanese eel, Anguilla japonica)를 경기도의 상업 양어장으로부터 구매하였고, 실험 시작 14일 전 서울대학교 수의학 대학에 순응시켰다. 상기 어류는 일정한 통기와 함께 인위적 식단으로 급이하였고, 일주일에 2-3 번 물을 교환하였다.300 normal eel eels (japanese eel, Anguilla japonica) with an average population of 22.4 g were purchased from commercial fish farms in Gyeonggi-do and acclimatized to Seoul National University Veterinary Medicine College 14 days prior to the start of the experiment. The fish were fed on an artificial diet with constant aeration, and water was changed 2-3 times a week.
(2) 박테리아 균주 및 포르말린 사멸된 전세포의 준비(2) Preparation of bacterial strains and formalin killed whole cells
뱀장어과 뱀장어로부터 분리된 에드워드샐라 타라(Edwardsiella tarda) SU53 (1980, Japan)를 연구실 내 동결 건조된 상태로 보관하고, 본 실험에 전반적으로 사용하였다. 실험을 위해, 상기 박테리아를 트립신콩 한천 (TSA, Difco) 배지에 25℃에서 24시간 동안 배양하였다. 상기 에드워드샐라 타라 균주 (SU53)를 트립신 콩 (TSB, Difco) 브로스에 25℃에서 18시간 동안 배양하였다. 배양된 박테리아를 1% 포르말린으로 처리하고 25℃에서 18시간 동안 유지하였다. 포르말린-처리된 배양물을 3분 동안 13,000 rpm으로 원심분리한 후, 멸균 인산염 버퍼 식염수(PBS)에 두 번 세척하고, 멸균 PBS에 재현탁하였다. 상기 현탁액을 분광광도계를 통해 1.0의 O.D 값으로 조정하였다.Edwardsiella tarda SU53 (1980, Japan) isolated from eel and eel was stored in a freeze-dried state in the laboratory and was used throughout this experiment. For the experiment, the bacteria were cultured in trypsin bean agar (TSA, Difco) medium at 25° C. for 24 hours. The Edward Sala Tara strain (SU53) was cultured in trypsin beans (TSB, Difco) broth at 25° C. for 18 hours. The cultured bacteria were treated with 1% formalin and maintained at 25° C. for 18 hours. The formalin-treated culture was centrifuged at 13,000 rpm for 3 minutes, then washed twice in sterile phosphate buffered saline (PBS) and resuspended in sterile PBS. The suspension was adjusted to an O.D value of 1.0 through a spectrophotometer.
실시예Example 1-2. 녹말을 포함하는 1-2. Containing starch 하이드로겔로Hydrogel 캡슐화된 Encapsulated FKCFKC 항원을 포함하는 백신 조성물의 제조 Preparation of vaccine composition comprising antigen
녹말(Starch) 1.33g을 distilled water 35 ml에 풀어서 70℃에서 저어주었다. 다음으로, 과황산암모늄(Ammonium persulfate, APS) 100mg을 추가하여 10분 동안 저어주고, 아크릴산(Acrylic acid, AA) 1.5g과 2-Hydroxyethyl methacrylate(2-HEMA) 1.5g을 추가로 첨가하였다. 다음으로, 5ml distilled water에 N,N'-methylene bisacrylamide(N,N'-MBA) 100mg을 잘 풀어준 후 추가로 첨가하였다. 이들 혼합 용액의 온도를 55~60℃로 낮춰서 유지하면서 1시간 동안 교반한 후, 용액을 식히기 전에 항원(불활성화 균체)를 첨가하였으며, 1M NaOH 20ml를 조금씩 넣어 pH를 7~8로 조정한 후 Ethanol 200ml에 6시간 이상 넣어서 탈수시켰다. 마지막으로, Ethanol을 따라버리고 고체화된 gel을 6 시간 이상 동결건조시켜 본 발명에 따른, 녹말을 포함하는 하이드로겔에 의해 캡슐화된 불활성화 균체를 포함하는 백신(녹말 백신)을 제조하였으며, 상기 제조된 백신은 가루 형태로 보관하였다.1.33 g of starch was dissolved in 35 ml of distilled water and stirred at 70°C. Next, 100 mg of ammonium persulfate (APS) was added and stirred for 10 minutes, and 1.5 g of acrylic acid (AA) and 1.5 g of 2-Hydroxyethyl methacrylate (2-HEMA) were additionally added. Next, 100 mg of N,N'-methylene bisacrylamide (N,N'-MBA) was well released in 5 ml distilled water, and then added. After stirring for 1 hour while keeping the temperature of the mixed solution at 55-60° C., the antigen (inactivated cells) was added before the solution was cooled, and 20 ml of 1M NaOH was added little by little to adjust the pH to 7-8. Ethanol was dehydrated in 200 ml over 6 hours. Finally, a vaccine (starch vaccine) containing inactivated cells encapsulated by a hydrogel containing starch was prepared according to the present invention by lyophilizing Ethanol and lyophilizing the solidified gel for at least 6 hours, and prepared above. The vaccine was stored in powder form.
실시예 1-3. 백신 조성물의 면역 효과 실험Example 1-3. Immune effect experiment of vaccine composition
제조한 녹말 백신을 1x10 8의 항원용량이 한마리에게 투여될 수 있도록 분주하여 준비하였다. 처음 백신을 경구투여한 날을 0일차로 하여 8일, 15일, 22일, 29일, 36일, 43일 등 매 7일마다 혈액 샘플을 채취하여 serum을 이용해 항원항체 응집반응을 관찰하였다.The prepared starch vaccine was prepared by dispensing so that the antigen dose of 1x10 8 could be administered to one animal. Blood samples were taken every 7 days, such as 8 days, 15 days, 22 days, 29 days, 36 days, and 43 days on the first day of oral administration of the vaccine to observe the antigen-antibody aggregation reaction using serum.
도 1은 본 발명에 따른 녹말을 포함하는 하이드로겔에 의해 캡슐화된 백신(Starch vaccine)과 종래 포르말린 사균 백신(formalin-killed cell-cultured, FKC)을 경구 투여한 이후 시간에 따른 응집 역가를 나타낸 그래프이다. 이를 통해, 본 발명에 따른 백신이 종래 포르말린 사균 백신에 비해 항원에 대한 항체 응집 반응 결과가 월등히 향상된다는 점을 확인하였다. 1 is a graph showing the aggregation titer over time after oral administration of a vaccine encapsulated by a hydrogel containing starch according to the present invention (Starch vaccine) and a conventional formalin-killed cell-cultured (FKC) to be. Through this, it was confirmed that the vaccine according to the present invention significantly improved the results of the antibody aggregation reaction to the antigen compared to the conventional formalin vaccine.
도 2는 본 발명에 따른 녹말을 포함하는 하이드로겔에 의해 캡슐화된 백신(Starch vaccine)을 0일차 1회 경구 투여한 군(Feed1), 매일 1회씩 총 4회 경구 투여한 군(Feed4), 매일 1회씩 총 8회 경구 투여한 군(Feed8) 및 종래 포르말린 사균 백신을 매일 1회씩 총 4회 투여한 군(FKC)들의 시간에 따른 응집 역가를 나타낸 그래프이다. 도 2의 그래프는 E. tarda에 대한 백신 투여군의 항원항체 응집반응을 나타낸 것으로서, 측정 결과, 8일차부터 29일차까지 녹말 백신 1회 투여군과 FKC 4회 투여군에 비해 본 발명에 따른 녹말 백신을 4회, 8회 투여한 군의 항원 응집반응의 항체 역가가 더 높게 나타난다는 것을 확인하였다. 또한 녹말 백신의 1회 투여의 경우, 백신의 효과가 다회 투여에 비해 낮은 것으로 나타났으며, 4회와 8회 투여군에서 응집 반응의 결과가 비슷한 것으로 보아 일정 투여 횟수 이후에는 백신의 효과가 일정치 이상 증가하지 않는다는 것을 확인하였다.Figure 2 is a group (Orally administered once a day 0 times the vaccine encapsulated by a hydrogel containing a starch according to the invention (Feed1), once a day orally administered a total of 4 times per day (Feed4), daily It is a graph showing the aggregation titer over time of the group (Fed8) orally administered once a total of 8 times (FKC) and the group previously administered four times of formalin antibacterial vaccine (FKC) once a day. The graph of FIG. 2 shows the antigen-antibody aggregation reaction of the vaccine-administered group against E. tarda , and as a result of the measurement, the starch vaccine according to the present invention was compared to the 1-starch vaccine group and the FKC 4-week group from Days 8 to 29 It was confirmed that the antibody titer of the antigen agglutination reaction of the group administered 8 times and 8 times was higher. In addition, in the case of the single administration of the starch vaccine, the effect of the vaccine was found to be lower than that of the multiple administration, and the results of the agglutination reaction were similar in the groups of 4 and 8 administrations. It was confirmed that it does not increase abnormally.
실시예 2.Example 2.
실시예 2-1. 실험 방법Example 2-1. Experimental method
박테리아 균주 및 성장 조건Bacterial strains and growth conditions
뱀장어과 뱀장어로부터 분리된 에드워드샐라 타라(Edwardsiella tarda) SU53 (1980, Japan)를 연구실 내 동결 건조된 상태로 보관하고, 본 실험에 전반적으로 사용하였다. 실험을 위해, 상기 박테리아를 TSB(tryptic soy broth, Difco) 배지 또는 TSA(tryptic soy agar, Difco) 배지에서 25℃ 조건에서 배양하였다. Edwardsiella tarda SU53 (1980, Japan) isolated from eel and eel was stored in a freeze-dried state in the laboratory and was used throughout this experiment. For the experiment, the bacteria were cultured at 25° C. in TSB (tryptic soy broth, Difco) medium or TSA (tryptic soy agar, Difco) medium.
어류의 준비Preparation of fish
560마리의 뱀장어과의 뱀장어(japanese eel, Anguilla japonica) (평균 길이 28.0 cm; 평균 무게, 82.4±7.9 g)를 경기도의 상업 양어장에서 구매하였고, E. tarda 감염이 없음을 확인하기 위해 실험 전에 10마리의 개체를 무작위로 수집하여 종래 문헌(T. Sakai, T. Lida, K. Osatomi, K. Kanai, Detection of type 1 fimbrial genes in fish pathogenic and non-pathogenic Edwardsiella tarda strains by PCR, Fish Pathol. 42 (2007) 115-117)에 보고된 방법에 따라 중합 효소 연쇄 반응 (PCR)을 실시하였다. 그 후 남은 550마리를 5t 탱크에서 1주일 동안 27.5 ± 0.5 ℃의 실험실 조건에 적응시켰다. 상기 어류에 일정한 통기와 함께 인위적 식단으로 급이하였고, 일주일에 1회 탱크 내 물의 약 50%를 교환해주었다. 500 마리 중 100마리는 E. tarda SU53의 반치사량(median lethal dose, LD 50) 결정 시험에 사용하였고, 400마리는 백신 접종 시험에 사용하였다. 모든 동물 관리 및 실험 프로토콜은 서울대학교 동물 윤리위원회의 지침에 따라 수행하였다.560 eel eels (japanese eel, Anguilla japonica) (average length 28.0 cm; average weight, 82.4±7.9 g) were purchased from commercial fish farms in Gyeonggi-do and 10 before the experiment to confirm that there was no E. tarda infection Collected randomly from the subjects of T. Sakai, T. Lida, K. Osatomi, K. Kanai, Detection of type 1 fimbrial genes in fish pathogenic and non-pathogenic Edwardsiella tarda strains by PCR, Fish Pathol. 42 ( 2007) The polymerase chain reaction (PCR) was carried out according to the method reported in 115-117). The remaining 550 animals were then acclimated to laboratory conditions of 27.5±0.5° C. for 1 week in a 5t tank. The fish were fed with an artificial diet with constant aeration, and about 50% of the water in the tank was exchanged once a week. 100 of the 500 were used for the median lethal dose (LD 50 ) determination test of E. tarda SU53, and 400 were used for the vaccination test. All animal care and experimental protocols were performed according to the guidelines of the Seoul National University Animal Ethics Committee.
뱀장어의 위 및 내장의 pH 측정PH measurement of stomach and intestines of eel
급이(feeding) 1시간 후에, 5마리의 뱀장어를 무작위로 선택한 다음 MS-222 (Sigma-Aldrich, MO, USA)를 사용하여 안락사시켰다. 이후, 뱀장어의 위장관을 절개하고 위 및 장 내용물의 pH를 pH 측정기 (Thermo Scientific, MA, USA)를 사용하여 측정하였다.One hour after feeding, five eel were randomly selected and then euthanized using MS-222 (Sigma-Aldrich, MO, USA). Then, the gastrointestinal tract of the eel was dissected and the pH of the stomach and intestinal contents was measured using a pH meter (Thermo Scientific, MA, USA).
녹말을 포함하는 Containing starch 하이드로겔로Hydrogel 캡슐화된 Encapsulated FKCFKC 항원을 포함하는 백신(Starch hydrogel based oral vaccine, SHO vaccine)의 제조 Preparation of vaccines containing antigens (Starch hydrogel based oral vaccine, SHO vaccine)
먼저, 포르말린 (0.4 % [v/v])을 첨가하여 1ml의 E. tarda SU53 박테리아 현탁액 (2.0×10 10 콜로니 형성 단위; CFU)을 비활성화시키고, 멸균 인산염 버퍼 식염수(PBS)에 두 번 세척하고, 멸균 PBS에 재현탁하였다.First, inactivate 1 ml of E. tarda SU53 bacterial suspension (2.0×10 10 colony forming units; CFU) by adding formalin (0.4% [v/v]), wash twice in sterile phosphate buffered saline (PBS), , And resuspended in sterile PBS.
다음으로, 녹말(Starch)(1.33 g; Fluka, Buchs, Switzerland)을 70℃에서 35 ml의 증류수 (DW)에 교반함으로써 용해시켰다. 이후, 과황산암모늄(Ammonium persulfate, APS)(100 mg; Fluka, Buchs, Switzerland)을 현탁액에 첨가한 후 10분 동안 추가로 교반하고, 아크릴산(Acrylic acid, AA) (1.5 g; Merck, Darmstadt, Germany)과 2-Hydroxyethyl methacrylate(2-HEMA) (Merck, Darmstadt, Germany) 1.5 g을 추가로 첨가 및 교반하였다. 다음으로, 5 ml의 증류수에 N,N'-methylene bisacrylamide(N,N'-MBA) 100mg을 잘 풀어준 후 추가로 첨가한 후, 이들 혼합 용액을 60 ℃로 냉각시켰다. 다음으로, 항원(불활성화 균체)(KFC)를 첨가한 다음 계속 교반시켰다. 1시간 후, 반응 생성물을 30분 동안 주위 온도로 냉각시키고, 1M NaOH 용액을 첨가하여 pH 7.5로 중화시킨 후, Ethanol 200 ml에 6시간 이상 넣어서 탈수시켰다. 마지막으로, Ethanol을 따라버리고 고체화된 gel을 6시간 동안 동결건조시켜 본 발명에 따른, 녹말을 포함하는 하이드로겔에 의해 캡슐화된 불활성화 균체를 포함하는 백신(녹말 백신)을 제조하였으며, 상기 제조된 백신은 추가 사용을 위해 -20 ℃에서 가루 형태로 보관하였다.Next, Starch (1.33 g; Fluka, Buchs, Switzerland) was dissolved by stirring in 35 ml of distilled water (DW) at 70°C. Then, ammonium persulfate (Ammonium persulfate, APS) (100 mg; Fluka, Buchs, Switzerland) was added to the suspension, followed by further stirring for 10 minutes, and acrylic acid (AA) (1.5 g; Merck, Darmstadt, Germany) and 2-Hydroxyethyl methacrylate (2-HEMA) (Merck, Darmstadt, Germany) 1.5 g was further added and stirred. Next, 100 mg of N,N'-methylene bisacrylamide (N,N'-MBA) was well released in 5 ml of distilled water, and then additionally added, and these mixed solutions were cooled to 60°C. Next, antigen (inactivated cells) (KFC) was added and stirring was continued. After 1 hour, the reaction product was cooled to ambient temperature for 30 minutes, neutralized to pH 7.5 by adding 1M NaOH solution, and then dehydrated by adding 200 ml of Ethanol for 6 hours or more. Finally, a vaccine (starch vaccine) containing inactivated cells encapsulated by a hydrogel containing starch was prepared according to the present invention by lysing Ethanol and lyophilizing the solidified gel for 6 hours. The vaccine was stored in powder form at -20 °C for further use.
SHO 백신의 형태학적 분석Morphological analysis of SHO vaccine
전계 방출 주사 전자 현미경 (FESEM; Sigma, Carl Zeiss, UK)을 사용하여 형태 분석을 수행하였다. 3개의 시편을 제조하고 스터브 상에 장착하였다: 하이드로겔 형성 전의 녹말; 항원을 함유하지 않는 녹말에 기초한 합성 하이드로겔; 및 본 발명에 따른 SHO 백신. 스캐닝하기 전에, 시편을 vacuum coater (EM ACE 200; Leica, Austria)를 사용하여 180초 동안 금으로 스퍼터 코팅 하였다.Morphological analysis was performed using a field emission scanning electron microscope (FESEM; Sigma, Carl Zeiss, UK). Three specimens were prepared and mounted on the stub: starch before hydrogel formation; Synthetic hydrogels based on starch containing no antigen; And SHO vaccine according to the present invention. Prior to scanning, the specimens were sputter coated with gold for 180 seconds using a vacuum coater (EM ACE 200; Leica, Austria).
백신 접종vaccination
SHO 백신을 제조하기 위해, 분말형 하이드로겔을 분쇄된 펠렛과 완전히 혼합한 다음, 일정량의 PBS를 혼합물에 첨가하였다. 각각의 SHO 백신의 항원 함량은 어류 당 10 8 CFU로 조정하였다. 면역 실험 전에, MS-222를 사용하여 5마리의 뱀장어를 무작위로 선택하고 마취시켰다. SHO 백신을 이들 어류에 경구투여하고, 27.5 ± 0.5 ℃에서 유지시켰다. 백신 접종 1주 후, 뱀장어를 MS-222로 안락사시키고 꼬리 정맥으로부터 혈액을 채취하여 SHO 백신에 의해 면역력이 생겼는지를 확인하였다.To prepare the SHO vaccine, the powdered hydrogel was thoroughly mixed with the ground pellets, and then a certain amount of PBS was added to the mixture. The antigen content of each SHO vaccine was adjusted to 10 8 CFU per fish. Prior to the immunization experiment, 5 eel were randomly selected and anesthetized using MS-222. SHO vaccine was orally administered to these fish and maintained at 27.5±0.5°C. One week after vaccination, the eel was euthanized with MS-222 and blood was collected from the tail vein to confirm that the SHO vaccine produced immunity.
400마리의 뱀장어를 5개 그룹 (각 그룹, n = 80)으로 나누었다. 5개의 그룹은 다음과 같다; 대조군: PBS 함침된(PBS-impregnated) 경구 펠릿을 한번만 경구 투여한 그룹, FKC group: FKC와 혼합된 그라운드 펠릿을(종래 포르말린 사균 백신)을 매일 1회씩 총 4회(4일) 경구 투여한 그룹, SHO 1: 본 발명에 따른 SHO 백신을 1일 동안 1회 경구 투여한 그룹, SHO 4: 본 발명에 따른 SHO 백신을 4일 동안 매일 1회씩 경구 투여한 그룹, SHO 8: 본 발명에 따른 SHO 백신을 8일 동안 매일 1회씩 경구 투여한 그룹. 단일 경구 투여시, 각각의 백신 (FKC 또는 SHO)의 총 항원 함량은 어류 당 10 8 CFU로 조정하였다. The 400 eel was divided into 5 groups (each group, n = 80). The five groups are; Control group: PBS impregnated (PBS-impregnated) oral pellet group administered orally only once, FKC group: FKC mixed ground pellet (formerly formalin vaccine) once daily for a total of 4 times (4 days) orally administered group , SHO 1: group in which the SHO vaccine according to the invention was orally administered once per day, SHO 4: group in which the SHO vaccine according to the invention was orally administered once daily for 4 days, SHO 8: SHO according to the invention Group administered orally once daily for 8 days. Upon single oral administration, the total antigen content of each vaccine (FKC or SHO) was adjusted to 10 8 CFU per fish.
혈액 샘플 수집 및 혈청 응집 테스트Blood sample collection and serum aggregation test
6주 동안 매주 5개 그룹 각각에서 3마리의 물고기를 무작위로 선택하였다: SHO 8을 제외한 모든 그룹에서 첫 번째 백신 접종 후 1주일부터 샘플링을 수행하였다(SHO 8은 첫 번째 백신 접종 후 2주 후에 샘플링 수행). MS-222로 안락사 후 1 ml 주사기를 사용하여 꼬리 정맥으로부터 채혈한 혈액 샘플을 원심분리튜브 (Eppendorf, Hamburg, Germany)로 옮겼다. 6,500×g, 4℃에서 10분 동안 원심 분리 후 혈청을 수집하였다. 보체 활성(complement activity)을 불활성화시키기 위해 혈청을 열처리(44℃, 20분)하였다. 혈청 응집 실험은 96-웰 U-bottom 플레이트 (Sigma-Aldrich, St. Louis, MO, USA)를 사용하여 수행하였다. 혈청을 PBS에서 2배로 연속 희석하고, 동일한 부피의 가열-사멸된 E. tarda SU53 (약 10 7 CFU/ml)을 각 웰에 첨가하였다. 플레이트를 25 ℃에서 밤새 인큐베이션하였다. Three fish were randomly selected from each of the five groups for 6 weeks: sampling was performed from 1 week after the first vaccination in all groups except SHO 8 (SHO 8 was 2 weeks after the first vaccination) Sampling performed). After euthanizing with MS-222, blood samples collected from the tail vein were transferred to a centrifuge tube (Eppendorf, Hamburg, Germany) using a 1 ml syringe. Serum was collected after centrifugation at 6,500×g, 4° C. for 10 minutes. Serum was heat treated (44° C., 20 minutes) to inactivate complement activity. Serum aggregation experiments were performed using a 96-well U-bottom plate (Sigma-Aldrich, St. Louis, MO, USA). Serum was serially diluted 2-fold in PBS, and an equal volume of heat-killed E. tarda SU53 (about 10 7 CFU/ml) was added to each well. Plates were incubated overnight at 25°C.
부스트 백신(Boost vaccine)의 효과 측정Measure the effectiveness of boost vaccine
두 번재 백신 접종 (부스트 백신 접종)은 혈청 응집 항체 역가가 감소하는 것으로 밝혀졌을 때, 첫 번째 백신 접종 후 46일 차에 한 번만 수행하였다. 첫 번재 백신 접종 후 46일 차에 5개 그룹 각각에서 20마리의 뱀장어를 무작위로 선택하였다. 대조군의 어류에는 PBS-함침된 펠릿을 경구 투여하였다; FKC 그룹 어류에는 FKC와 혼합된 분쇄된 펠렛을 경구 투여하였고; SHO 1, SHO 4 및 SHO 8의 어류에는 SHO를 경구 투여하였다. 그런 다음 각 그룹의 물고기를 27.5 ± 0.5 ℃에서 200 L 유리 섬유 강화 플라스틱 수족관으로 별도로 옮겼다. 첫 번째 백신 접종 후 7주 및 8주에, 5개의 그룹 각각에서 3마리의 뱀장어를 무작위로 선택하고 혈액 샘플링을 수행하였다. 혈청 응집 실험은 전술한 바와 같이 수행하였다.The second vaccination (boost vaccination) was performed only once on the 46th day after the first vaccination when it was found that the serum aggregated antibody titers decreased. At day 46 after the first vaccination, 20 eel were randomly selected from each of the 5 groups. PBS-impregnated pellets were orally administered to the control fish; FKC group fish were orally administered pulverized pellets mixed with FKC; SHO 1, SHO 4 and SHO 8 fish were administered orally with SHO. Each group of fish was then transferred separately at 27.5±0.5° C. to a 200 L glass fiber reinforced plastic aquarium. At 7 and 8 weeks after the first vaccination, 3 eel were randomly selected from each of the 5 groups and blood sampling was performed. Serum aggregation experiments were performed as described above.
공격실험(Experimental challenge test)Experimental challenge test
초기 대수기(early-exponential phase)의 E. tarda SU53을 챌린지 시험에 사용하였고, PBS로 10배 연속 희석하였다. 균주의 LD 50 농도를 결정하기 위해, 이중 어류 그룹(duplicate fish groups)(그룹당 n = 10)에 복강 내 (i.p.) 주사를 통해 100 ㎕의 박테리아 현탁액을 투여하였다.E. tarda SU53 in the early-exponential phase was used for challenge testing and diluted 10-fold serially with PBS. To determine the LD 50 concentration of the strain, 100 μl of the bacterial suspension was administered via intraperitoneal (ip) injection to duplicate fish groups (n=10 per group).
최종 주사 용량은 2.0×10 4 내지 2.0 × 10 7 CFU/fish 범위였다. 대조군의 어류에 100 μl의 멸균 PBS를 주사하였다. 주사 후, 15일 동안 물고기를 모니터링 하였다. 죽은 물고기는 매일 샘플링되었다; 박테리아를 신장에서 분리하고 PCR을 사용하여 확인하였다.The final injection dose ranged from 2.0×10 4 to 2.0×10 7 CFU/fish. Control fish were injected with 100 μl of sterile PBS. After injection, fish were monitored for 15 days. Dead fish were sampled daily; Bacteria were isolated from the kidneys and confirmed using PCR.
첫 번째 백신 접종 후 4주 후에 주사(i.p.) 공격실험을 수행하였다. 이중 어류 그룹(그룹당 n = 10)에 100 μl의 LD50 농도의 균주를 함유하는 주사를 투여하였다. 시험 어류는 흐르는 물이 공급되는 100 L 유리 섬유 강화 플라스틱 수조에서 27.5±0.5 ℃로 유지하였다. 주사 후 15일 동안 질병 및 누적 사망률의 임상 징후를 하루에 2회 모니터링 하였다. 폐사한 어류의 신장에서 샘플링을 수행하였으며, 해당 샘플은 전술된 PCR법을 이용하여 균을 동정하였다.An injection (i.p.) attack experiment was performed 4 weeks after the first vaccination. The double fish group (n=10 per group) was administered an injection containing a strain of LD50 concentration of 100 μl. The test fish was maintained at 27.5±0.5° C. in a 100 L glass fiber reinforced plastic water tank supplied with running water. Clinical signs of disease and cumulative mortality were monitored twice a day for 15 days after injection. Sampling was performed in the kidneys of the dead fish, and the samples were identified using the PCR method described above.
RNA 추출 및 역전사RNA extraction and reverse transcription
혈액 샘플 수집 후, RNA 추출을 위해 각 그룹으로부터 3마리의 뱀장어를 선택하였다. TRIzol Reagent (CWBio, Beijing, China)를 사용하여 머리 신장 및 간의 조직 샘플로부터 총 RNA를 추출하였다. RNA 농도 및 순도는 분광 광도법에 의해 정량화되었으며, 이는 1.6 내지 1.8 사이의 260:280 ratio를 나타내었다; 0.5 ㎍/mL ethidium bromide가 보충된 1% 아가로스 겔상에서 전기영동을 사용하여 RNA 품질을 검증하였다. DNA 오염을 방지하기 위해, 제조업체의 지침에 따라 전체 RNA 샘플을 DNAse Ⅰ (Madison, WI, USA)로 처리하였다. 추출된 RNA는 제조사의 프로토콜에 따라 PrimeScript RT 시약 키트 (TaKaRa Bio, Otsu, Japan)를 사용하여 cDNA로 역전사되었다. 생성된 cDNA는 사용전까지 -80 ℃에서 보관하였다.After collecting blood samples, three eel were selected from each group for RNA extraction. Total RNA was extracted from tissue samples of the head kidney and liver using TRIzol Reagent (CWBio, Beijing, China). RNA concentration and purity were quantified by spectrophotometry, which showed a 260:280 ratio between 1.6 and 1.8; RNA quality was verified using electrophoresis on a 1% agarose gel supplemented with 0.5 μg/mL ethidium bromide. To prevent DNA contamination, whole RNA samples were treated with DNAse I (Madison, WI, USA) according to the manufacturer's instructions. The extracted RNA was reverse transcribed into cDNA using PrimeScript RT reagent kit (TaKaRa Bio, Otsu, Japan) according to the manufacturer's protocol. The resulting cDNA was stored at -80 °C until use.
유전자 발현의 실시간 정량적 PCR 분석 Real-time quantitative PCR analysis of gene expression
인터페론(IFN)-α, 인터루킨(IL)-6 및 종양 괴사 인자(TNF)-α와 같은 면역 반응에 관여하는 유전자의 발현을 Rotor-Gene Q 실시간 정량적 PCR (RT-qPCR) 검출 시스템 (QIAGEN; Hilden, Germany)을 이용하여 모니터링하였다. Rotor-Gene Q Real-time Quantitative PCR (RT-qPCR) detection system for the expression of genes involved in immune responses such as interferon (IFN)-α, interleukin (IL)-6 and tumor necrosis factor (TNF)-α (QIAGEN; Hilden, Germany).
모든 qPCR 반응은 표준 프로토콜에 따라 SYBR Premix Ex TaqTM 226 Perfect Real-Time Kits (TaKaRa Bio, Otsu, Japan)를 사용하여 수행하였다. 하우스-키핑 유전자(house-keeping gene)를 사용하여 유전자 발현을 표준화하였다. 하기 표 1은 qPCR에 사용 된 PCR 프라이머 서열을 나타낸다. 반응 혼합물은 10 μl SYBR Premix Ex TaqTM 229, 1 μl의 정방향 및 역방향 프라이머 (10 mM) 및 1 μl cDNA를 포함하였다.All qPCR reactions were performed using SYBR Premix Ex TaqTM 226 Perfect Real-Time Kits (TaKaRa Bio, Otsu, Japan) according to standard protocols. Gene expression was normalized using a house-keeping gene. Table 1 below shows the PCR primer sequences used for qPCR. The reaction mixture included 10 μl SYBR Premix Ex TaqTM 229, 1 μl forward and reverse primers (10 mM) and 1 μl cDNA.
Figure PCTKR2019013709-appb-img-000001
Figure PCTKR2019013709-appb-img-000001
Reference 14: J. Feng, P. Lin, Y. Wang, S. Guo, Z. Zhang, L. Yu, Identification of a type Ⅰinterferon (IFN) gene from Japanese eel and its expression analysis in vivo and in vitro, Agri Gene 5 (2017) 19-26.Reference 14: J. Feng, P. Lin, Y. Wang, S. Guo, Z. Zhang, L. Yu, Identification of a type Ⅰinterferon (IFN) gene from Japanese eel and its expression analysis in vivo and in vitro , Agri Gene 5 (2017) 19-26.
Reference 15: M. Teles, S. Mackenzie, S. Boltana, A. Callol, L. Tort, Gene expression and TNF alpha secretion profile in rainbow trout macrophages following exposures to copper and bacterial lipopolysaccharide, Fish. Shellfish Immunol. 30 (2011) 340-346.Reference 15: M. Teles, S. Mackenzie, S. Boltana, A. Callol, L. Tort, Gene expression and TNF alpha secretion profile in rainbow trout macrophages following exposures to copper and bacterial lipopolysaccharide, Fish. Shellfish Immunol. 30 (2011) 340-346.
이어서 초순수를 반응에 첨가하여 최종 총 부피가 20 μl가 되도록 하였다. 반응 조건 및 사이클 지수는 95 ℃에서 5분 동안 수행한 후, 95 ℃에서 15 초 동안, 60 ℃에서 1분 동안, 및 72 ℃에서 30초 동안 40 사이클을 수행하였다. 증폭 단계 후, 비특이적 증폭 또는 프라이머-이량체 형성 가능성을 확인하기 위해 용융 곡선 분석을 수행하였다. 샘플 cDNA의 연속 희석으로부터 표준 곡선을 생성하고, 분자수에 대한 역치 사이클 (threshold cycle, Ct)의 자연로그를 플로팅하여 그렸다. 각 유전자의 표준 곡선을 3회 실행하여 모든 표준 곡선의 결정 계수 (R 2)가> 0.99이고 증폭 효율이 90% 내지 110%인지 확인하였다. FKC 그룹 및 SHO 그룹에 대한 데이터를 대조군에서 얻은 것과 비교하였다. 표적 유전자의 상대 발현을 표준 △△C T 방법을 사용하여 분석하였다. 본 실험은 3회 반복 수행되었다.Ultrapure water was then added to the reaction to make the final total volume 20 μl. The reaction conditions and cycle index were performed at 95°C for 5 minutes, followed by 40 cycles at 95°C for 15 seconds, 60°C for 1 minute, and 72°C for 30 seconds. After the amplification step, a melting curve analysis was performed to confirm the possibility of non-specific amplification or primer-dimer formation. Standard curves were generated from serial dilutions of sample cDNA and plotted by plotting the natural logarithm of the threshold cycle (Ct) for the number of molecules. The standard curve of each gene was executed three times to confirm that the determination coefficient (R 2 ) of all standard curves was> 0.99 and the amplification efficiency was 90% to 110%. Data for the FKC group and SHO group were compared to those obtained in the control group. The relative expression of the target gene was analyzed using standard ΔΔC T method. This experiment was repeated three times.
통계 분석Statistical analysis
일원분산분석 (ANOVA) 테스트를 사용하여 데이터를 분석하였다. 실험 그룹 간의 차이를 분석하기 위해 Tukey's test를 사용하였으며, 통계 분석에는 OriginPro 소프트웨어 (버전 8.5; OriginLab Corporation, MA, Northampton)를 사용하였다. 유의성을 P-값으로 측정하였고, P < 0.05일 때 통계적 유의성이 있는 것으로 간주하였다.Data were analyzed using the One-Way ANOVA (ANOVA) test. Tukey's test was used to analyze the differences between the experimental groups, and OriginPro software (version 8.5; OriginLab Corporation, MA, Northampton) was used for statistical analysis. Significance was measured as a P-value and was considered to be statistically significant when P <0.05.
실시예 2-2. 결과 및 고찰Example 2-2. Results and Discussion
SHO 백신의 특징Features of SHO vaccine
백신 제조 전에 녹말을 균일하게 분포시켰다(도 3A). 녹말은 수분 흡수 및 팽창에 의해 덩어리진 하이드로겔을 성공적으로 형성하였다(도 3B). 항원 (FKC)을 첨가하여 FKC를 녹말 하이드로겔에 코팅하고 SHO 백신을 제조하였으며, FESEM을 통해 성공적으로 제조되었음을 확인하였다(도 3C).Starch was evenly distributed prior to vaccine preparation (Figure 3A). Starch successfully formed agglomerated hydrogels by water absorption and expansion (Fig. 3B). Antigen (FKC) was added to coat FKC on a starch hydrogel and a SHO vaccine was prepared, confirming that it was successfully prepared through FESEM (FIG. 3C).
적응 면역 반응(Adaptive immune responses)Adaptive immune responses
면역 실험 전에, 어떤 그룹에서도 검출 가능한 항체가 발견되지 않았다. 백신 접종된 그룹에서, FKC 또는 SHO 백신 접종은 백신 접종 후 2주째(2 wpv)에 응집 역가를 증가시켰다 (도 4). 응집 역가의 최고값은 3 wpv에서 관찰되었으며, 첫 번째 백신 접종후 46 일차에 두 번째 백신 접종이 수행될 때까지 감소하였다. 두 번째 백신 접종 후 응집 역가가 급격히 증가하여 SHO 8을 제외한 모든 백신 접종 그룹에서 7 wpv에서 가장 높은 값에 도달하였다(SHO 8은 8 wpv에 도달). SHO 백신 접종은 FKC 백신 접종보다 더 높은 항체 역가를 생성하였다. FKC 및 SHO 1은 상대적으로 더 작은 역가를 나타었으며(SHO 1은 두 번째 백신 접종 후 FKC보다 더 높은 역가를 생성), SHO 4 및 SHO 8은 더 높은 역가를 나타내었다. SHO 4 및 SHO 8의 어류는 3 wpv에서 가장 높은 역가를 나타내었고, 두 번째 백신 접종 후 8 wpv에서 동일한 값은 역가를 나타내는 등 유사한 패턴 변화를 나타내었다.Prior to the immunization experiment, no detectable antibodies were found in any group. In the vaccinated group, FKC or SHO vaccination increased aggregation titers 2 weeks after vaccination (2 wpv) (FIG. 4 ). The highest value of aggregation titer was observed at 3 wpv and decreased until the second vaccination was performed on day 46 after the first vaccination. After the second vaccination, the aggregation titer increased rapidly, reaching the highest value at 7 wpv in all vaccinated groups except SHO 8 (SHO 8 reached 8 wpv). SHO vaccination produced higher antibody titers than FKC vaccination. FKC and SHO 1 showed relatively smaller titers (SHO 1 produced higher titers than FKC after the second vaccination), and SHO 4 and SHO 8 showed higher titers. Fish of SHO 4 and SHO 8 showed the highest titer at 3 wpv, and the same value at 8 wpv after the second vaccination showed similar pattern change.
백신 접종 후 생존 평가Survival assessment after vaccination
E. tarda SU53의 15-day LD 50 농도를 결정하기 위해, 실험적 챌린지 테스트를 2회 반복하였다 (데이터 미도시). 대조군 및 최저 농도 (2.0×10 4 CFU/fish)를 투여한 군에서, 어류의 감염 실험 동안 사망률은 관찰되지 않았다. 다른 투여 그룹에서, 사망은 공격접종 후 3일 (3 dpc)부터 발생하였고, 최대 9 dpc까지 계속되었으며, 9 dpc 후에, 실험 기간의 나머지 기간동안 생존하였고 어떠한 증상도 나타나지 않았다. 생존률은 각각 2.0×10 5 CFU/fish에서는 70%(1st trial), 70%(2nd trial), 2.0×10 6 CFU/fish에서는 40%(1st trial), 50%(2nd trial), 2.0×10 7 CFU/fish에서는 0%(1st trial), 0%(2nd trial)로, 최종 주사 용량에 비례하였다. 모든 죽은 어류는 edwardsiellosis의 전형적인 임상 징후를 나타내었다. 이들로부터 분리된 박테리아를 TSA 플레이트에서 배양하고, 전술한 PCR 결과 E. tarda로 확인되었다.To determine the 15-day LD 50 concentration of E. tarda SU53, the experimental challenge test was repeated twice (data not shown). In the control group and the group administered the lowest concentration (2.0×10 4 CFU/fish), no mortality was observed during the fish infection experiment. In the other dosing groups, death occurred 3 days after challenge (3 dpc), continued up to 9 dpc, and after 9 dpc, survived for the remainder of the experimental period and showed no symptoms. Survival was 2.0 × 10 5 CFU / fish in 70% (1st trial), 70 % (2nd trial), 2.0 × 10 6 CFU / fish in 40% (1st trial), 50 % (2nd trial), 2.0 × 10 each At 7 CFU/fish, 0% (1st trial) and 0% (2nd trial) were proportional to the final injection dose. All dead fish displayed typical clinical signs of edwardsiellosis. Bacteria isolated from them were cultured in TSA plates, and the PCR results described above were identified as E. tarda.
Edwardsiellosis에 대한 FKC 및 SHO 백신의 보호 효과에 대한 결과를 도 5에 나타내었다. 도 5에 나타난 바와 같이, 4 wpv에서 모든 백신 접종된 그룹의 공격접종 후 폐사율은 대조군의 것보다 낮았다. 모든 그룹에서, 공격접종 후 폐사율의 진행은 E. tarda SU53의 LD 50 농도를 결정하기 위해 수행된 공격실험의 결과와 일치하였다: 폐사율은 3 dpc 내지 9 dpc로 기록된 후 더 이상의 추가 폐사율은 관찰되지 않았다. FKC 그룹에서 어류의 생존율은 첫 번째와 두 번째 시도에서 각각 60%와 70%였다. SHO 백신이 접종된 어류의 생존율은 FKC 백신이 접종된 그룹보다 더 높았다. SHO 백신 접종 그룹의 생존율은 다음과 같다: SHO 1에서 70%(first trial) 및 80%(second trial); SHO 4에서 80%(first trial) 및 90%(second trial); 및 SHO 8에서 80%(first trial) 및 90%(second trial). SHO 4와 SHO 8 사이의 생존율에는 유의 한 차이가 없었으며, 둘 다 최고 및 동일한 수준의 질병 방어 효과를 나타내었다.The results for the protective effect of FKC and SHO vaccines against Edwardsiellosis are shown in FIG. 5. As shown in FIG. 5, the mortality rate after challenge with all vaccinated groups at 4 wpv was lower than that of the control group. In all groups, the progression of mortality after challenge was consistent with the results of an attack experiment conducted to determine the LD 50 concentration of E. tarda SU53: mortality was recorded from 3 dpc to 9 dpc and further mortality was observed Did not. The survival rate of fish in the FKC group was 60% and 70%, respectively, in the first and second trials. The survival rate of fish inoculated with SHO vaccine was higher than in the group inoculated with FKC vaccine. The survival rate of the SHO vaccination group was as follows: 70% (first trial) and 80% (second trial) in SHO 1; 80% (first trial) and 90% (second trial) in SHO 4; And 80% (first trial) and 90% (second trial) in SHO 8. There was no significant difference in the survival rate between SHO 4 and SHO 8, and both showed the highest and the same level of disease defense effect.
사이토카인 유전자 발현에 대한 백신 접종의 효과Effect of vaccination on cytokine gene expression
도 6은 백신 접종 후 각 그룹에서 뱀장어의 머리 신장 또는 간에서의 사이토 카인 유전자 발현을 나타낸 것이다. 모든 면역화된 그룹에서, 백신 접종은 대조군과 비교할 때 사이토카인 유전자의 수준을 증가시켰다. SHO 백신 접종은 FKC 백신 접종시 보다 더 높은 수준의 사이토카인 유전자 발현을 유도하였다; 발현 수준은 SHO 1 대비 SHO 4 및 SHO 8에서 더 높았다 (도 6). SHO 4 및 SHO 8의 사이토카인 유전자 발현은 3 wpv에서 증가하였고, 4 wpv에서 감소하였으며, 8 wpv에서 다시 증가하는 경향을 보였으며, SHO 8의 8 wpv에서 가장 높은 발현 수준을 나타내었다. 시간에 따른 사이토카인 유전자 발현의 차이는 SHO 1 또는 FKC 그룹에서 유의하지 않았다. 그러나, IL-6 발현의 경우 이들 두 그룹 모두 3 wpv 부터 8 wpv 까지 증가하는 경향을 나타내었다. SHO 1 및 FKC 그룹에서 IFN-α 발현은 SHO 4 및 SHO 8에서와 동일한 방식으로 변화하여, 3 wpv에서 증가, 4 wpv에서 감소 및 8 wpv에서 다시 증가하였다. TNF-α 발현은 FKC 그룹에서 증가하는 경향을 나타낸 반면, SHO 1에서는 3 내지 4 wpv에서 감소한 후, 8 wpv 까지 증가하였다.Figure 6 shows the cytokine gene expression in the head kidney or liver of the eel in each group after vaccination. In all immunized groups, vaccination increased the level of the cytokine gene as compared to the control group. SHO vaccination induced higher levels of cytokine gene expression than when FKC vaccination; Expression levels were higher in SHO 4 and SHO 8 compared to SHO 1 (FIG. 6 ). Cytokine gene expression of SHO 4 and SHO 8 increased at 3 wpv, decreased at 4 wpv, showed a tendency to increase again at 8 wpv, and showed the highest expression level at 8 wpv of SHO 8. Differences in cytokine gene expression over time were not significant in the SHO 1 or FKC group. However, in the case of IL-6 expression, both groups showed a tendency to increase from 3 wpv to 8 wpv. IFN-α expression in the SHO 1 and FKC groups changed in the same way as in SHO 4 and SHO 8, increasing at 3 wpv, decreasing at 4 wpv and increasing again at 8 wpv. TNF-α expression showed a tendency to increase in the FKC group, whereas in SHO 1 it decreased from 3 to 4 wpv and then increased to 8 wpv.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시형태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Since specific parts of the present invention have been described in detail above, it is obvious that for those skilled in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
본 발명에 따른 조성물은 pH의 변화에 따라 가역적으로 팽창 또는 수축하여 크기가 변화하는 하이드로겔을 이용하여 병원체를 캡슐화함으로써, pH가 낮은 위에서는 소화되지 않고, pH가 높은 장내까지 전달 후 분해되어 체내에 효과적으로 흡수될 수 있는바, 경구 투여용 백신 분야에 널리 활용될 수 있다.The composition according to the present invention encapsulates a pathogen using a hydrogel that reversibly expands or contracts according to a change in pH to change its size, so it is not digested in a stomach with a low pH and is decomposed after being delivered to a high pH intestine. Since it can be effectively absorbed, it can be widely used in the vaccine field for oral administration.

Claims (7)

  1. 하이드로겔; 및 상기 하이드로겔에 의해 캡슐화된 불활성화 균체;를 유효성분으로 포함하는 경구 투여용 백신 조성물.Hydrogels; And an inactivated cell encapsulated by the hydrogel; as an active ingredient, a vaccine composition for oral administration.
  2. 제1항에 있어서,According to claim 1,
    상기 하이드로겔은 pH의 변화에 따라 가역적으로 팽창 또는 수축하여 크기가 변화하는 것을 특징으로 하는 경구 투여용 백신 조성물.The hydrogel is a vaccine composition for oral administration, characterized in that the size changes by reversibly expanding or contracting according to the change in pH.
  3. 제1항에 있어서,According to claim 1,
    상기 하이드로겔은 녹말을 포함하는 것을 특징으로 하는 경구 투여용 백신 조성물.The hydrogel is a vaccine composition for oral administration comprising a starch.
  4. 제1항에 있어서,According to claim 1,
    상기 불활성화 균체는 포르말린 사균(formalin-killed cell-cultured)인 것을 특징으로 하는 경구 투여용 백신 조성물.Vaccine composition for oral administration, characterized in that the inactivated cells are formalin-killed cell-cultured.
  5. 제1항에 있어서,According to claim 1,
    상기 불활성화 균체는 어류의 세균성 질병의 병원체 또는 바이러스성 질병의 병원체인 것을 특징으로 하는 경구 투여용 백신 조성물.The inactivated cells are vaccines for oral administration, characterized in that they are pathogens of bacterial diseases of fish or pathogens of viral diseases.
  6. 제1항에 있어서,According to claim 1,
    상기 조성물은 육식성 어류용 백신인 것을 특징으로 하는 경구 투여용 백신 조성물.The composition is a vaccine composition for oral administration, characterized in that the vaccine for carnivorous fish.
  7. 제6항에 있어서,The method of claim 6,
    상기 육식성 어류는 뱀장어, 넙치(광어), 조피볼락, 우럭, 감성돔, 참돔, 능성어, 숭어, 농어, 전어, 고등어, 전갱이, 쥐치로 이루어진 군에서 선택되는 1종 이상인 것을 특징으로 하는 경구 투여용 백신 조성물.The carnivorous fish is a vaccine composition for oral administration, characterized in that at least one selected from the group consisting of eel, halibut (flounder), bark, rockfish, black sea bream, red sea bream, starfish, mullet, sea bass, trout, mackerel, horse mackerel, and squid. .
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