KR101958169B1 - Composiotion for Vaccine Containing VHSV Vaccine and adjuvant - Google Patents
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Abstract
본 발명은 VHSV 백신 및 몬타나이드 IMS 1312 VG 아쥬반트를 함유하는 어류의 바이러스성 출혈성 패혈증 예방을 위한 침지용 백신 조성물 및 이를 이용한 어류의 바이러스성 출혈성 패혈증 예방방법에 관한 것으로, 본 발명에 따른 침지용 바이러스성 출혈성 패혈증 백신을 사용하면, 주사 접종이 어려운 어류에 대하여, 효과적으로 바이러스성 출혈성 패혈증을 예방할 수 있다. The present invention relates to a vaccine composition for the prevention of viral hemorrhagic septicemia of a fish containing VHSV vaccine and Montanide IMS 1312 VG adjuvant and a method for preventing viral hemorrhagic sepsis of fish using the same, Viral haemorrhagic sepsis vaccines can effectively prevent viral haemorrhagic sepsis against fish that are difficult to inoculate.
Description
본 발명은 침지용 바이러스성 출혈성 패혈증 예방 백신 조성물에 관한 것으로, 더욱 자세하게는 VHSV 백신 및 몬타나이드 IMS 1312 VG 아쥬반트를 함유하는 어류의 바이러스성 출혈성 패혈증 예방을 위한 침지용 백신 조성물 및 이를 이용한 어류의 바이러스성 출혈성 패혈증 예방방법에 관한 것이다. The present invention relates to a vaccine composition for preventing viral hemorrhagic septicemia, and more particularly to a vaccine composition for preventing viral hemorrhagic sepsis of fish containing VHSV vaccine and Montanide IMS 1312 VG adjuvant, To a method for preventing viral hemorrhagic sepsis.
바이러스성 출혈성 패혈증(VHS)는 한국과 일본에서 넙치 물고기양식 산업에서와 유럽 물고기 양식에서 연어과의 어류에게 매우 치명적인 바이러스성 질병 중 하나이다[Kim, WS et al., Aquaculture 296:165,2009; Skall, HF et al.,J. Fish. Dis. 28:509, 2005; Takano,R et al., Bull. Eur. Assoc. Fish. Pathol. 20:186, 2000). VHS 바이러스(VHSV)는 3'-N-P-M-G-NV-L-5'의 순으로 6개의 유전자를 암호화하는 ~11Kd을 가지는 Rhabdoviridae 계통의 음성가닥 RNA 바이러스이다. VHSV는 G와 N 유전자의 유전자 서열을 기초로 4가지의 주요한 유전자형(I, II, III 및 IV)으로 나뉘어지고, 각 유전자형의 독성은 감염되는 물고기의 종에 따라 다르다(Emmenegger,EJ et al., Dis. Aquat. Organ, 107:99, 2013). VHSV 유전자형 IV는 태평양 연안 북서부지역에서 발견되는 바다 물고기 종류나 한국과 일본에서 넙치로부터 분리되었으며, 겨울/봄 기간 동안 수온이 9~15℃ 사이일 때에 주로 나타나며, 감염 물고기의 체중이 증가함에 따라 VHSV 병원성이 감소한다(Isshik, T., Dis. Aquat. Organ 47, 87:2001; Kim, SM. et al., J. Ocean. Sci.Tech. 1:95, 2004). Viral hemorrhagic septicemia (VHS) is one of the most virulent viral diseases in halibut fish farming industry in Korea and Japan and in salmonids in European fish farming [Kim, WS et al ., Aquaculture 296: 165,2009; Skall, HF et al., J. Fish. Dis. 28: 509, 2005; Takano, R et al., Bull. Eur. Assoc. Fish. Pathol. 20: 186,2000). The VHS virus (VHSV) is a negative strand RNA virus of the Rhabdoviridae strain having ~11 Kd encoding 6 genes in the order of 3'-NPMG-NV-L-5 '. VHSV is divided into four major genotypes (I, II, III and IV) based on the gene sequences of the G and N genes, and the toxicity of each genotype depends on the species of the infected fish (Emmenegger, EJ et al. , Dis., Aquat., Organ., 107: 99, 2013). VHSV genotypes IV were isolated from the sea fish species found in the northwestern part of the Pacific coast, and from flounder in Korea and Japan, and appeared mainly during the winter / spring period when the water temperature was between 9 ~ 15 ℃. As the infected fish body weight increased, VHSV (Isshik, T., Dis. Aquat. Organ 47, 87: 2001; Kim, SM et al., J. Ocean Sci. Tech. 1:95, 2004).
VHSV에 대한 어류 백신의 개발을 위해서는 바이러스성 당단백질을 주입하는 DNA 또는 재조합 백신들을 이용하는 전통적인 방법이 시도되고 있다(Byon,JY. et al., Fish. Shellfish Immunol. 18, 135, 2005). 그러나, 해당 백신들은 상업적으로 사용하도록 허가를 받지 못하고 있다.For the development of fish vaccines against VHSV, traditional methods using DNA or recombinant vaccines to inject viral glycoproteins have been attempted (Byon, JY et al., Fish. Shellfish Immunol. 18, 135, 2005). However, the vaccines are not licensed for commercial use.
물고기 예방접종에서, 복강내 주사는 선천성과 후천성 면역 반응을 일으키는데에 높은 효율 때문에 백신 전달의 가장 일반적인 방법이지만, 침지백신(immersion vacttine)은 복강내에 백신을 주사할 수 없는 작은 물고기에게 적합하다. 그러나 침지백신은 많은 양의 백신이 요구되고, 주사 백신보다 면역율 및 면역 기간이 낮다는 단점이 있다. In fish vaccination, intraperitoneal injections are the most common method of vaccine delivery due to their high efficiency in causing congenital and acquired immune responses, but immersion vacttines are suitable for small fish that can not inject a vaccine into the abdominal cavity. However, the immersion vaccine requires a large amount of vaccine and has a disadvantage that the immunity rate and immune period are lower than the injection vaccine.
현재, 대부분의 물고기 백신은 아쥬반트, 즉, 다시 말해서 면역 반응을 일으키는 물질들을 필요로 한다. 연어와 무지개송어를 위한 백신의 경우, 많은 아쥬반트이 상업적으로 이용가능하다. 그러나, 넙치의 경우, 포르말린 처리로 비활성화시킨 VHSV로 만들어지거나 실험적 예방접종에 사용되어지는 Poly (I:C) 아쥬반트, 스쿠알렌, 수산화 알루미늄 등 소수의 종류만이 보고되고 있다(Vinay,TN et al., Vaccine 31:4603, 2013; Kim, HJ et al., Fish. Shellfish Immunol. 48:206, 2016) Currently, most fish vaccines require adjuvants, that is, substances that cause an immune response. In the case of vaccines for salmon and rainbow trout, many adjuvants are commercially available. However, only a few species of flounder have been reported, such as Poly (I: C) adjuvant, squalene, aluminum hydroxide, which are made with VHSV deactivated by formalin treatment or used for experimental immunization (Vinay, TN et al , Vaccine 31: 4603, 2013; Kim, HJ et al., Fish. Shellfish Immunol., 48: 206, 2016)
이에, 본 발명자들은 VHSV를 침지형 백신으로 어류에 사용할 경우, 백신 효율을 높여 줄 수 있는 효율적인 아쥬반트를 개발하고자 예의 노력한 결과, Montanide IMS 1312 VG(SEPPIC, France)를 아쥬반트로 사용하여, VHSV 백신과 함께 넘치 수조에 침지하는 경우, VHSV 바이러스에 대한 감염억제 효과가 향상되는 것을 확인하고 본 발명을 완성하게 되었다. Accordingly, the present inventors have made extensive efforts to develop an efficient adjuvant that can increase vaccine efficiency when VHSV is used in fish as an immersion vaccine. As a result, Montanide IMS 1312 VG (SEPPIC, France) was used as an adjuvant and VHSV vaccine , It was found that the effect of inhibiting the infection against VHSV virus was improved, and the present invention was completed.
본 발명의 목적은 어류의 바이러스성 출혈성 패혈증 예방을 위한 침지용 백신 조성물을 제공하는데 있다.It is an object of the present invention to provide a vaccine composition for immersion for prevention of viral hemorrhagic sepsis in fish.
본 발명의 다른 목적은 상기 백신 조성물을 함유하는 수조에서 어류를 침지하는 것을 특징으로 하는 어류의 바이러스성 출혈성 패혈증 예방방법을 제공하는데 있다.It is another object of the present invention to provide a method for preventing viral hemorrhagic septicemia in a fish characterized by immersing fish in a water tank containing the vaccine composition.
상기 목적을 달성하기 위하여, 본 발명은 VHSV 백신 및 몬타나이드 IMS 1312 VG 아쥬반트를 함유하는 어류의 바이러스성 출혈성 패혈증 예방을 위한 침지용 백신 조성물을 제공한다.In order to achieve the above object, the present invention provides a vaccine composition for immersion for prevention of viral hemorrhagic sepsis of fish containing VHSV vaccine and Montanide IMS 1312 VG adjuvant.
본 발명은 또한, 상기 백신 조성물을 함유하는 수조에서 어류를 침지하는 것을 특징으로 하는 어류의 바이러스성 출혈성 패혈증 예방방법을 제공한다.The present invention also provides a method for preventing viral hemorrhagic septicemia in a fish characterized by immersing the fish in a water bath containing the vaccine composition.
본 발명에 따른 침지용 바이러스성 출혈성 패혈증 백신을 사용하면, 주사 접종이 어려운 어류에 대하여, 효과적으로 바이러스성 출혈성 패혈증을 예방할 수 있다.The use of the viral hemorrhagic sepsis vaccine for dipping according to the present invention can effectively prevent viral hemorrhagic sepsis against fishes that are difficult to inject with inoculation.
도 1은 백신 처리 후 넙치에 대한 사진으로, 각각 대조구(A), VHS백신 주사구 (106TCID50/마리)(B), VHSV백신침지구(C), Adjuvant농도별 첨가 VHSV백신침지구(D, E, F구)를 나타낸 것이다.
도 2는 본 발명에 따른 침지 백신 처리 후의 면역관련 유전자의 발현패턴 변화를 qPCR로 확인한 결과를 나타낸 것이다.
도 3은 본 발명에 따른 침지 백신 처리 후, VHSV를 감염시킨 넙치군의 누적폐사율을 나타낸 것으로, A는 백신처리 후 4주째 감염시킨 군을 나타낸 것이고, B는 백신처리 후 8주째 감염시킨 군을 나타낸 것이다.FIG. 1 is a photograph of a flounder after vaccination. FIG. 1 is a photograph of a flounder after vaccination. FIG. 1 is a photograph of a flounder after vaccination with a control (A), a VHS vaccine feeding flask (106TCID50 / E, and F).
FIG. 2 shows the results of qPCR analysis of changes in expression patterns of immune-related genes after immersion vaccination according to the present invention.
FIG. 3 shows the cumulative mortality of the flounder group infected with VHSV after the immersion vaccination according to the present invention, wherein A represents the group infected at 4 weeks after vaccination, and B represents the group infected at 8 weeks after vaccination .
어류의 감염병 예방을 위한 방법 중, 백신을 함유하는 수조에서 물고기를 침지하는 침지 백신 방법은 물고기의 집단 예방 접종에 적합하나, 예방 효율이 낮아 개발이 되지 않고 있다. 본 발명에서는 넙치에 Montanide IMS 1312 VG 아쥬반트(adjuvant)와 VHSV 백신을 포함하는 바이러스성 출혈 패혈증에 대한 침지 백신의 효과와 안정성을 확인하였다. 감염이력이 없는 넙치를 열-비활성화 VHSS 바이러스와 Montanide IMS 1312 VG 아쥬반트와 결합하여, 침지 방법에 의해 예방 접종을 하였다. 그 결과, Montanide IMS 1312 VG 아쥬반트가 포함된 침지백신은 혈액학적 및 조직병리학적 분석에서 독성이 나타나지 않았으며, 침지 백신 적용 후, 인위적을 VHSV를 감염시킨 넙치의 상대적 생존률(RPS)는 아쥬반트를 포함한 예방접종을 한 물고기에서 아쥬반트를 포함하지 않고 예방접종을 한 물고기보다 활씬 더 높다는 것을 확인하였다. 이러한 결과로 Montanide IMS 1312 VG를 포함하는 VHSV 침지 백신이 VHS에 대해 넙치에 방어면역을 유발한다는 것을 알 수 있다.Of the methods for the prevention of infectious diseases of fish, the immersion vaccination method for immersing fish in a water tank containing vaccine is suitable for group vaccination of fish, but it is not developed because of low efficiency of prevention. In the present invention, the efficacy and stability of immersion vaccine against viral hemorrhagic sepsis including the Montanide IMS 1312 VG adjuvant and VHSV vaccine in flounder were confirmed. Flounder without infection history was vaccinated with immobilized vaccine in combination with heat - inactivated VHSS virus and Montanide IMS 1312 VG adjuvant. As a result, the immersion vaccine containing Montanide IMS 1312 VG adjuvant showed no toxicity in hematological and histopathological analysis, and the relative survival rate (RPS) of the flounder infected with artificial VHSV after the immersion vaccine was higher than that of adjuvant Were much higher than those vaccinated with no adjuvant in the vaccinated fish. These results suggest that the VHSV immersion vaccine containing Montanide IMS 1312 VG induces defense immunity against the flounder for VHS.
따라서, 본 발명은 일 관점에서, VHSV 백신 및 몬타나이드 IMS 1312 VG 아쥬반트를 함유하는 어류의 바이러스성 출혈성 패혈증 예방을 위한 침지용 백신 조성물에 관한 것이다.Thus, in one aspect, the present invention relates to a vaccine composition for immersion for prevention of viral hemorrhagic septicemia in a fish containing VHSV vaccine and Montanide IMS 1312 VG adjuvant.
본 발명의 백신 조성물에서, 상기 Montanide IMS 1312 VG 아쥬반트는 0.1g/L~100g/L로 함유되는 것을 사용할 수 있으며, 바람직하게는 2g/L~20g/L로 함유되는 것을 사용할 수 있고, 더욱 바람직하게는 5g/L~15g/L를 사용할 수 있다. In the vaccine composition of the present invention, the Montanide IMS 1312 VG adjuvant may be contained in an amount of 0.1 g / L to 100 g / L, preferably 2 g / L to 20 g / L, Preferably 5 g / L to 15 g / L can be used.
본 발명에서 사용되는 VHSV 백신의 농도는 105 ~109TCID50/5L을 사용할 수 있으며, 바람직하게는 106 ~108TCID50/5L을 사용할 수 있다.The concentration of the VHSV vaccine used in the present invention may be 10 5 to 10 9 TCID 50 / 5L, preferably 10 6 to 10 8 TCID 50 / 5L.
본 발명에 따른 아쥬반트를 포함하는 침지 백신은 면역 관련 유전자, 즉, 다시 말해서 인터류킨(IL)-1β, IL-6, IL-8, 그리고 톨유사수용체(TLR) 3을 부호화하는 유전자의 유전자 발현을 증가시키고, VHSV 감염에 따른 어류의 생존율을 증가시킨다.Immunization vaccines containing an adjuvant according to the present invention can be used for the gene expression of genes encoding immune related genes, i. E., Interleukin (IL) -1β, IL-6, IL-8 and Toll-like receptor (TLR) And increases the survival rate of fish due to VHSV infection.
다른 관점에서, 본 발명은 상기 어류의 바이러스성 출혈성 패혈증 예방을 위한 침지용 백신 조성물을 함유하는 수조에서 어류를 침지하는 것을 특징으로 하는 어류의 바이러스성 출혈성 패혈증 예방방법에 관한 것이다.In another aspect, the present invention relates to a method for preventing viral hemorrhagic septicemia in a fish characterized by immersing fish in a water tank containing a vaccine composition for immersion for preventing viral hemorrhagic sepsis of the fish.
본 발명의 침지 백신은 특히 주사 시에 다루기 비실용적인 작은 어류, 치어 등에 사용하기 적합하다. The immersion vaccine of the present invention is particularly suitable for use in small fish, fry, etc., which are impractical to handle at the time of injection.
본 발명의 일 양태에서는 넙치의 VHS에 대한 Montanide IMS 1312 VG 아쥬반트의 VHSV 침지 백신의 안전성과 효능을 확인하였다. VHSV 침지 백신이 VHSV 감염으로부터 어류를 보호하고, 10g/5L의 Montanide IMS 1312 VG이 50g/5L의 Montanide IMS 1312 VG 복용량보다 감염 방어능이 더 우수하다는 것을 확인하였다. In one embodiment of the present invention, the safety and efficacy of the VHSV immersed vaccine of Montanide IMS 1312 VG adjuvant against VHS of the flounder was confirmed. The VHSV immersion vaccine protected the fish from VHSV infection and confirmed that 10g / 5L of Montanide IMS 1312 VG had better protection against infection than the 50g / 5L Montanide IMS 1312 VG dose.
본 발명의 다른 양태에서는 침지 백신 접종을 하고 2주 후에, 어류의 면역 반응에 잠재적으로 관련이 있는 11개의 유전자 발현을 평가했다. 여기에서, IL-1β 가 VHSV 침지 백신(3.6배) 과 10g의 Montanide IMS 1312 VG를 넣은 VHSV 침지 백신 (>38배) 처리 후에 백신 접종되지 않은 대조 표준과 비교하여 가장 강하게 증가하였다. IL-1β 염증 반응에 직접적으로 관여하는 사이토카인이고, 면역 반응을 조절하는 데에 중심적인 역할, 예를 들면, 면역능, 림프구 활성화, 백혈구 이동 및 식균작용과 관련된 유전자의 조절을 한다. In another embodiment of the present invention, after two weeks of immersion vaccination, eleven gene expressions potentially relevant to the fish ' s immune response were evaluated. Here, IL-1β was most strongly increased compared to the non-vaccinated control standard after treatment with VHSV immersion vaccine (3.6 times) and 10 g of Montanide IMS 1312 VG (> 38-fold). Is a cytokine that is directly involved in the IL-1 [beta] inflammation response and regulates genes that are central to regulating the immune response, e.g., immunity, lymphocyte activation, leukocyte migration, and phagocytosis.
IL-8의 발현은 대조 표준과 비교했을 때, 10 g의 Montanide IMS 1312 VG를 포함하는 VHSV 침지 백신 접종 처리 후에 15 배 이상 발현이 증가하였지만, VHSV 침지 백신군 에서는 0.48배 감소했다. IL-8은 CXC chemokine family의 구성원이고 이것의 분비는 내재 면역반응과 관련된 TLRs에 의해 촉진될 수 있다. IL-8은 VHSV에 감염된 넙치에 대한 염증반응 인자가 될 것이다. 본 발명에서, IL-8 발현은 Montanide IMS 1312 VG를 넣은 VHSV 침지 백신 접종으로 유도되었는데, 이는 이 아쥬반트가 염증 반응을 유도한다는 것을 나타낸다. TLR3은 대조 표준과 비교했을 때, 아쥬반트 없이 VHSV 침지 백신 접종 처리 후에 가장 강하게 양성피드백 되는 유전자이다(9.67배 증가). VHSV 감염은 TLR3 발현을 상당히 증가시킨다고 알려져 있다. 따라서 우리 결과는 침지 백신이 TLR3를 유도했음을 나타냈는데, 이는 dsRNA와 ssRNA 바이러스 모두에 대한 어류의 면역 반응에 중요하다.Expression of IL-8 increased more than 15-fold after VHSV immersion vaccination with 10 g of Montanide IMS 1312 VG, but decreased by 0.48-fold in the VHSV immersion vaccine group compared to the control standard. IL-8 is a member of the CXC chemokine family and its secretion can be facilitated by TLRs associated with endogenous immune responses. IL-8 will be an inflammatory response factor for flounder infected with VHSV. In the present invention, IL-8 expression was induced by VHSV immersion vaccination with Montanide IMS 1312 VG, indicating that this adjuvant induces an inflammatory response. TLR3 is the most strongly positive feedback (9.67-fold increase) after VHSV immersion vaccination treatment without adjuvant compared to the control standard. VHSV infection is known to significantly increase TLR3 expression. Our results therefore showed that the immersion vaccine induced TLR3, which is important for the immune response of fish to both dsRNA and ssRNA viruses.
Montanide IMS 1312 VG을 포함하지 않는 침지 백신이 어류에서 IL-1β, TNF, TLR3 및 GCSF 같은 면역 관련 유전자의 발현 수치를 증가시킬지라도, 이는 VHSV 감염으로부터 어류를 보호할 수 없다. Montanide IMS 1312 VG를 포함하지 않는 침지 백신은 백신 접종을 하고 8주 후에 VHSV로 면역성 검사를 받을 때 사망을 촉진시키기도 하였다. 이러한 유전자 산물은 이들의 발현 수치가 어류를 VHSV 면역성 검사로부터 보호하는 Montanide IMS 1312 VG를 넣은 침지 백신 접종으로부터 유도되기 때문에 어류 VHSV의 생장을 지속시키지 않을 것이다. 또한, TLR3 시그널링은 항바이러스 유전자 프로그램(Doyle et al., Immunity 17:251, 2002)과 GCSF, IL-1(Iida et al., Vaccine 7:229, 1989) 그리고 TNF(Seo and Webster, J. Virol. 76:1071, 2002)같은 cytokine들이 항바이러스 활동을 하도록 한다.Although immersion vaccines that do not contain Montanide IMS 1312 VG increase expression levels of immune-related genes such as IL-1β, TNF, TLR3, and GCSF in fish, they can not protect fish from VHSV infection. Immunization vaccines that do not contain Montanide IMS 1312 VG have also been shown to increase mortality when tested for immunity to VHSV after 8 weeks of vaccination. These gene products will not sustain the growth of fish VHSV because their expression levels are derived from immersion vaccination with Montanide IMS 1312 VG, which protects fish from VHSV immunity testing. In addition, TLR3 signaling can be generated using an antiviral gene program (Doyle et al., Immunity 17: 251, 2002) and GCSF, IL-1 (Iida et al., Vaccine 7: 229, 1989) and TNF (Seo and Webster, J. et al. Virol. 76: 1071, 2002).
Montanide IMS 1312 VG를 포함하는 침지 백신 접종으로 유도된 이러한 유전자의 수치가 VHSV 감염으로부터 어류를 보호하기에 충분할 수 있지만, Montanide IMS 1312 VG을 포함하지 않는 침지 백신 접종으로 유도된 유전자들은 그렇지 않을 수 있다. Montanide을 포함하지 않는 백신은 몇몇 면역 관련 유전자들은 증가시키는 반면, NKEF, IFN type I, TLR7 및 TLR9와 같은 항바이러스 포텐셜을 가지는 몇몇의 다른 면역 관련 유전자들의 수치는 감소시킨다: Megalocytivirus 감염은 NKEF의 수치를 증가시키고, NKEF의 과발현은 터봇(가자미의 일종)에서 megalocytivirus의 생장을 막는다 (Zhang et al., J. Proteomics 91:430, 2013); type I IFNs과 IFN receptors의 결합은 바이러스의 자가복제를 막을수 있는 300개 이상의 IFN-stimulated genes (ISGs)를 유도한다 (Der et al., 1998); TLR7과 TLR9는 바이러스 감염을 인지하고, 신호전달체계를 활성화하고, 항바이러스성cytokines과 chemokines의 생산을 유도할 수 있다 (Xagorari and Chlichlia, Open Microbiol. J. 2:49, 2008). Although the levels of these genes induced by immersion vaccines containing Montanide IMS 1312 VG may be sufficient to protect fish from VHSV infection, genes derived from immersion vaccines that do not contain Montanide IMS 1312 VG may not . A vaccine that does not contain Montanide increases some of the immune related genes while decreasing the levels of some other immune related genes with antiviral potentials such as NKEF, IFN type I, TLR7, and TLR9: Megalocytivirus infection is a measure of NKEF And overexpression of NKEF blocks the growth of megalocytiviruses in the turbot (Zhang et al., J. Proteomics 91: 430, 2013); The combination of type I IFNs and IFN receptors induces more than 300 IFN-stimulated genes (ISGs) that can block the replication of viruses (Der et al., 1998); TLR7 and TLR9 can recognize viral infections, activate the signaling system, and induce the production of antiviral cytokines and chemokines (Xagorari and Chlichlia, Open Microbiol. J. 2:49, 2008).
Montanide IMS 1312 VG는 백신 전달을 촉진하는 전달체로서 물에 분산된 액체 나노입자로 구성되어 있고 침지 경로를 통한 집단 백신 접종에 적합하다. 아쥬반트는 조기 면역, 작동인자 반응의 긴 지속시간, 예를 들어, 항체형성 또는 세포독성 T세포 활동을 가능하게 하고, 면역 촉진제를 불필요하게 만든다. 아쥬반트 수용액은 어류 표면을 통한 항원 흡수를 향상시킴으로써 어류의 세포성 면역 반응, 체액성 면역반응 모두 강화시킬 것이다. 측선관, 아가미, 위, 그리고 뒤창자를 포함하는 피부는 불활성 박테리아와 활성 박테리아 모두의 주된 흡수장소로서의 역할을 한다. 본 발명에서는 아쥬반트가 항원 입자의 흡수를 향상시키고 예방접종의 효과를 향상시키는 것을 확인하였다.Montanide IMS 1312 VG is a delivery vehicle for vaccine delivery and consists of liquid nanoparticles dispersed in water and is suitable for mass vaccination via the immersion route. Adjuvants enable early immunity, a long duration of action factor response, for example, antibody formation or cytotoxic T cell activity, and make immune stimulators unnecessary. The adjuvant aqueous solution will enhance both the cellular and humoral immune responses of fish by enhancing antigen uptake through the fish surface. Skin, including sidewall, gills, stomach, and back cavity, serves as the primary site of absorption for both inactive and active bacteria. In the present invention, it was confirmed that the adjuvant improves the absorption of the antigen particles and improves the effect of vaccination.
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.Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
하기 실시예에서는 넙치에 대한 침지 백신의 효과를 확인하였으나, 바이러스성 출혈성 패혈증이 나타나는 넙치, 무지개 송어, 대서양 연어, 브라운송어, 은연어, 왕연어, 대구, 터봇, 정어리, 멸치 등의 어류에도 제한없이 사용될 수 있다는 것은 당업자에게 자명할 것이다. In the following examples, the effect of the immersion vaccine on the flounder was confirmed, but it was also restricted to fish such as flounder, rainbow trout, atlantic salmon, brown trout, silver salmon, king salmon, codfish, turbot, sardine and anchovy which showed viral hemorrhagic sepsis. Without departing from the scope of the present invention.
실시예 1: VHS 백신 제작 및 실험군 선택Example 1: Preparation of VHS vaccine and selection of experimental group
1-1: VHS 백신 제작1-1: VHS vaccine production
VHSV 바이러스 균주로는 발명자가 이전에 분리한 FP-VHS2010-1(NCBI accession no. KP334106.1)을 불화화시켜 사용하였다. As the VHSV virus strain, FP-VHS2010-1 (NCBI accession No. KP334106.1) previously isolated by the inventor was fluorinated.
제조방법은 VHSV에 감염된 넙치의 조직을 MEM을 첨가하고 마쇄한 후 배지로 50배, 100배 희석하여 바이러스를 준비하였다. 상기 준비된 바이러스 접종액의 200㎕을 단층 배양된 EPC 세포주에 접종하고 실온에서 30분 동안 흡착시킨 후, 2% FBS와 1% antibiotics(Gibco)를 첨가한 MEM 배지를 넣고 20℃에서 배양하며 세포변성효과(EPC)를 역상현미경을 이용하여 관찰하였다. CPE가 확인된 바이러스 배양액을 모아 원심분리로 세포를 제거한 후 상층액으로 TCID50을 결정하였다. 정상세포는 동량의 PBS를 접종하고 위와 같은 조건으로 관찰하였다. In the preparation method, the tissue of the flounder infected with VHSV was diluted 50 times and 100 times with the medium after addition of MEM, and the virus was prepared. 200 μl of the prepared virus inoculum was inoculated into a monolayer EPC cell line, adsorbed at room temperature for 30 minutes, and then MEM medium supplemented with 2% FBS and 1% antibiotics (Gibco) was added and cultured at 20 ° C. Cell degeneration The effect (EPC) was observed using a reversed phase microscope. The CPE-confirmed virus culture fluid was collected, centrifuged to remove the cells, and the TCID 50 was determined as the supernatant. Normal cells were inoculated with the same amount of PBS and observed under the above conditions.
이렇게 준비된 바이러스로 60℃에서 3일간 두어 불활화시켜 heat inactivated vaccine을 준비하였다. 준비된 백신은 세포에 재접종하고 일주일간 관찰하여 불활성화를 왁인하였다. 농도별로 Montanide IMS1312 VG(Seppic사)와 혼합하여 5분간 침지하여 백신처리에 사용하였다.The heat inactivated vaccine was prepared by inactivating the virus prepared at 60 ℃ for 3 days. The prepared vaccine was re-inoculated into the cells and observed for one week to inactivate the inactivation. The mixture was mixed with Montanide IMS1312 VG (Seppic) for 5 minutes and used for vaccination.
1-2: 시험어 선택 및 실험군 선택1-2: Selection of test words and choice of test group
VHSV에 감염된 이력이 없는 넙치 양식장(포항, 남한)을 선택하여, real-time PCR 방법을 사용하여, VHSV에 감염되지 않은 것을 확인하였다. The flounder farms (Pohang, South Korea) without any history of VHSV infection were selected and confirmed to be infected with VHSV using real-time PCR method.
시험어는 VHSV에 대한 항체가가 나타나지 않는 건강한 넙치(14.1ㅁ0.1cm, 25.5ㅁ1.5g)를 선정하여 시험구당 70 마리씩 수용하여 실험에 사용하였다. 시험용 수조는 300L FRP 수조를 사용하였으며, 실험 종료시까지 유수식으로 관리하였다. The test fish were selected for healthy flounder (14.1 ㅁ 0.1cm, 25.5 ㅁ 1.5g) that did not show any antibody against VHSV. A 300L FRP water tank was used for the test water tank, and the water tank was maintained until the end of the experiment.
실시예 1에서 제조한 VHS 백신과 아쥬반트 몬타나이드 IMS1312 VG(Seppic사)와 혼합하여 5분간 침지한 후, 표 1에 제시된 군 별로 백신처리에 사용하였다.The VHS vaccine prepared in Example 1 and the adjuvant montanide IMS1312 VG (Seppic) were mixed and immersed for 5 minutes, and then used for vaccination by the groups shown in Table 1.
실시예 2: VHS 침지 백신의 안정성 확인Example 2: Confirmation of stability of vaccine immersed in VHS
VHS 침지 백신의 안정성을 확인하기 위하여, 침지처리한 넙치에 대하여, 병리조직학적 검사와 혈액 생화학적 성상을 분석하였다(도 1). To confirm the stability of the VHS immersion vaccine, histopathologic examination and blood biochemical characteristics of immersed flounder were analyzed (FIG. 1).
병리조직학적 검사는 백신 처리 둘째주 및 네째주에 각 장기에 대한 육안 검사를 실시하고 병리조직학적 검사에 필요한 장기를 적출하였다.Histopathologic examinations were performed on the second and fourth week of vaccination and visualized for organs and organs for histopathological examination.
그 결과, 백신처리군과 대조군에서, 부작용이 보이지 않아 넙치에 안전한 것으로 판단되었으며, 생검 결과, 아쥬반트에 의한 부작용은 없는 것으로 확인되었다.As a result, no side effects were observed in the vaccine-treated group and the control group, and thus it was judged to be safe for the flounder. As a result of biopsy, it was confirmed that there was no adverse effect by adjuvant.
백신접종이 시험의 혈액성상에 미치는 영향을 조사하기 위하여, 백신 처리된 넙치의 혈액을 채취하여, Glucose, GOT, 총 단백질 및 총 콜레스테롤 농도를 자동혈액분석기를 이용하여 측정하였다. To investigate the effect of the vaccination on the blood characteristics of the test, the blood of the vaccinated flounder was collected and glucose, GOT, total protein and total cholesterol concentration were measured using an automatic blood analyzer.
그 결과, 글루코즈 농도는 12.3±3.5~31.0±19.3 mg/dL, GOT는 20.0±3.5~39.7±6.4 U/L GPT는 1.0±0.0~8.4±42.3 U/L, 총단백질 농도는 3.7±0.4~4.6±0.2g/dL로 나타났다(표 2). 혈청 중의 글루코즈 농도, GOT(Glutamic oxaloacetic transaminase), GPT(Glutamin pyruvic tranaminase) 및 총단백질 농도는 모든 백신 접종구와 대조구 사이에 유의적인 차이를 보이지 않아 아쥬번트 또는 백신에 의한 부작용은 없는 것으로 판단되었다.As a result, glucose concentration was 12.3 ± 3.5 ~ 31.0 ± 19.3 mg / dL, GOT was 20.0 ± 3.5 ~ 39.7 ± 6.4 U / L, GPT was 1.0 ± 0.0 ~ 8.4 ± 42.3 U / L, total protein concentration was 3.7 ± 0.4 ~ 4.6 ± 0.2 g / dL (Table 2). Glucose concentration, GOT (Glutamic oxaloacetic transaminase), GPT (Glutamin pyruvic tranaminase), and total protein concentration were not significantly different between all vaccinated and control groups, suggesting no side effects due to adjuvant or vaccine.
(g/dL)Total Protein
(g / dL)
실시예 3: VHS 침지 백신 처리 후, 면역관련 유전자 발현 변화 분석Example 3 Analysis of Immune-Related Gene Expression Changes After VHS Immersion Vaccine Treatment
VHS 백신 처리 후, A~F 실험군의 면역관련 유전자의 발현 패턴 변화를 확인하기 위하여, qPCR을 실시하였다. After the VHS vaccine treatment, qPCR was performed to confirm the change in the expression pattern of the immunoreactive gene in the AF group.
실험구별 RNeasy Plus Mini Kit (Qiagen, Cat.74136)를 이용하여 Total RNA를 추출하고 RNase-Free-DNase Set (Qiagen, Cat.79254)를 처리한 후 Total RNA quality를 확인하여 qPCR을 실시하였다. Template cDNA 5 ㎕ (1/5 dilution)에 PCR Premix AccuPower 2X GreenStar Master Mix (Cat.K-6253), PCR Rxn. 50 ㎕/ rxn로 실시하였다. Total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Cat. 74136) and qPCR was performed after confirming total RNA quality after treatment with RNase-Free-DNase Set (Qiagen, Cat.79254). Template cDNA 5 μl (1/5 dilution) was mixed with PCR Premix AccuPower 2X GreenStar Master Mix (Cat. K-6253), PCR Rxn. 50 [mu] l / rxn.
PCR 조건은 다음과 같다. The PCR conditions were as follows.
Step 1: 95℃ 분 Step 1: 95 ℃
Step 2: (95℃ 15초, 60℃ 1분) x 45 사이클/scan Step 2: (95 ° C for 15 seconds, 60 ° C for 1 minute) x 45 cycles / scan
Step 3: 65℃ 5분 Step 3: 5 minutes at 65 ° C
Step 4: 융해곡선 분석 65℃~95℃ (1℃/초) Step 4: Melting curve analysis 65 ℃ ~ 95 ℃ (1 ℃ / sec)
PCR 기계는 Exicycler TM 96 Real-Time Quantitative Thermal Block (Cat.A-2060)을 사용하였고 데이터 분석은 2-ddCtMethod를 이용하여 상대정량분석을 실시하였다. PCR was performed using Exicycler TM 96 Real-Time Quantitative Thermal Block (Cat. A-2060) and relative quantitative analysis was performed using the 2- ddCt Method.
IL-1β, IL-8, TNF 및 IFN type 1에 대한 프라이머는 Bioneer 사에서 구입하였으며, 나머지 프라이머는 표 3에 나타낸 서열로 제작하였다. Primers for IL-1?, IL-8, TNF and IFN type 1 were purchased from Bioneer, and the remaining primers were prepared as shown in Table 3.
백신처리 후 면역유전자 발현률을 확인하였다. After vaccination, the immunogenicity was checked.
그 결과, 도 2 및 표 4에 나타난 바와 같이, 아쥬반트 처리에 의하여, IL-1β의 발현이 가장 많이 유도가 되었으며 TLR-7의 발현률은 변화가 없었다. 백신 처리 후 2주째, VHS immersion vaccine(아쥬반트10g첨가 침지백신군)의 발현이 유도되었으며, 8주째에는 주사백신의 발현유도가 가장 활발하였다(표 4).As a result, as shown in Fig. 2 and Table 4, the expression of IL-1β was most induced by the adjuvant treatment and the expression rate of TLR-7 was not changed. At 2 weeks after the vaccination, the expression of the VHS immersion vaccine (10 g of adjuvant added immersion vaccine) was induced, and at 8 weeks, the induction of injection vaccine was most active (Table 4).
실시예Example 4: VHS 4: VHS 침지Immersion 백신 처리에 의한 넙치의 VHS 감염 예방 효과 확인 Prevention of VHS infection by flounder
VHS 침지 백신을 처리한 넙치에서, VHS 감염에 대하여 예방효과를 가지는지 확인 확인하기 위하여, 침지 백신 처리 후 4주째 및 8주째 VHSV로 감염시킨 후 생존율을 조사하였다. 복강주사법으로 넙치를 인위 감염 후 2 주간 누적폐사율을 조사하였다. 상대생존율은 아래와 같이 계산하였다.Survival rates of the flounder treated with VHS immersion vaccine were investigated after infecting with VHSV at 4 and 8 weeks after the immersion vaccination in order to confirm whether the flounder had a preventive effect against VHS infection. The cumulative mortality rate of flounder was investigated by intraperitoneal injection for 2 weeks. Relative survival rates were calculated as follows.
상대생존율(%)=1-(시험구의 누적폐사율/대조구의 누적폐사율)x 100 Relative survival rate (%) = 1- (cumulative mortality rate in the test group / cumulative mortality rate in the control) x 100
그 결과, 도 3의 A에 나타난 바와 같이, 백신처리 4주째에 VHSV를 감염시킨 넙치의 누적폐사율은 대조구는 85%로 모든 실험구중 가장 높았으며, 백신 침지구 65%, 백신주사구, 35%, 100g 아쥬반트 첨가 침지백신구는 45%, 50g 아쥬반트 침지백신구 15%, 10g 아쥬반트 첨가 침지 백신구는 10%을 보여 아쥬반트 농도에 따라 감염 방어능에 차이를 나타내었다. As a result, as shown in Fig. 3 (A), the cumulative mortality rate of the flounder infected with VHSV at the 4th week of vaccination was the highest in all the experimental groups of the control, 85% 45% of 100 g of adjuvant immersion vaccine, 15% of 50 g of adjuvant immersion vaccine and 10% of immersion vaccine of 10 g of adjuvant showed difference in infection protection ability according to adjuvant concentration.
상대생존률은 백신 침지군이 24%, 백신 주사군 59%, 10g 아쥬반트 첨가 침지 88%, 50g 아쥬반트 첨가 침지군 82%, 100g 아쥬반트 첨가 침지군이 27%이었다. Relative survival rates were 24% in the vaccinated group, 59% in the vaccinated group, 88% in the 10g adjuvant group, 82% in the 50g adjuvanted group and 27% in the 100g adjuvanted group.
백신 접종 8주째 VHSV로 감염시킨 후 방어능에서는 도 3의 B에 나타낸 바와 같이, 대조구의 폐사율은 65%인 반면, 주사백신의 폐사율은 50%, 100g 아쥬반트 첨가 침지백신구는 40%, 50g 아쥬반트 첨가 침지백신구는 30%, 10g 아쥬반트 첨가 침지백신구는 20%, 아쥬반트 비첨가 침지구는 80%로 아쥬반트를 첨가하지 않은 백신의 경우는 백신의 효과가 없는 것을 확인하였다. 또한 아쥬반트 농도에 따라 방어능에 차이를 나타내었다. As shown in FIG. 3B, the mortality rate of the control was 65% while the mortality rate of the injection vaccine was 50%, 100g of the adjuvant-added immersion vaccine was 40%, and 50g of the adjuvant The vaccine did not have the effect of vaccine in the case of vaccine without adjuvant. The vaccine was 30%, 30%, 10%, 20% and 80%, respectively. In addition, the protective effect was different according to the concentration of adjuvant.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시 양태일 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.While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> National Institute of Fisheries Science <120> Composiotion for Vaccine Containing VHSV Vaccine and adjuvant <130> P17-B001 <160> 24 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tcagagcaag acaacaggcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agatgctgat ccacgttccc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cttcagcaag gaggcttgtc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ctttgatgag gccgatcagt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 actgctcaat gtcctgaccg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aagggacgaa atcgcttcac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctccacaaac tgacccacct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ctccagagtt ttgggcagac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 accgacgagc tgtctcctta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctttgatgag gccgatcagt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tgagattgtg gcacaggaag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tccttgttca ttcccagctc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gacaaagcat ccagccaagt g 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tctccaccag aaccaccctg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcagttaccg gctaatccaa 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tagcatgtgg cctagctcct 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ccactggact tcaccttcgt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tgcttacgtg gtgtgttggt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 cctctgtctc tacagtgcgg 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cacttcaaca tcctcagtgg tg 22 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cctgggaaat ctggaagaac 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tttgagggag gagaaactgc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ctcgggcata gactcgtggt 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 catggtcgtg accttcgctc 20 <110> National Institute of Fisheries Science <120> Composition for Vaccine Containing VHSV Vaccine and adjuvant <130> P17-B001 <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tcagagcaag acaacaggcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agatgctgat ccacgttccc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 cttcagcaag gaggcttgtc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ctttgatgag gccgatcagt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 actgctcaat gtcctgaccg 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 aagggacgaa atcgcttcac 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctccacaaac tgacccacct 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ctccagagtt ttgggcagac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 accgacgagc tgtctcctta 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctttgatgag gccgatcagt 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tgagattgtg gcacaggaag 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tccttgttca ttcccagctc 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gacaaagcat ccagccaagt g 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tctccaccag aaccaccctg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcagttaccg gctaatccaa 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tagcatgtgg cctagctcct 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ccactggact tcaccttcgt 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tgcttacgtg gtgtgttggt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 cctctgtctc tacagtgcgg 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cacttcaaca tcctcagtgg tg 22 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cctgggaaat ctggaagaac 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 tttgagggag gagaaactgc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 ctcgggcata gactcgtggt 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 catggtcgtg accttcgctc 20
Claims (5)
VHSV vaccine and a vaccine composition for the prevention of viral hemorrhagic sepsis in a flounder containing montanide IMS 1312 VG adjuvant at a concentration of 2 g / L to 10 g / L.
A method for preventing viral hemorrhagic sepsis in a flounder characterized by immersing fish in a water tank containing the vaccine composition of claim 1.
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