JP2018008908A - Antibody inducer against virus infections for land animals other than human, and adjuvant thereof - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
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- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
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Abstract
Description
本発明は、人以外の陸上動物用のウイルス感染症に対する分泌型ＩｇＡ及び／又はＩｇＧ抗体誘導剤、及びその補助剤であるアジュバントに関する。 The present invention relates to a secretory IgA and / or IgG antibody-inducing agent for viral infections for terrestrial animals other than humans, and an adjuvant that is an auxiliary agent thereof.
一般的にウイルスは増殖が早く感染後数日で発症するため、ウイルスからの罹患を防ぐためには、予めワクチンを接種して抗原抗体反応によりウイルス感染を阻止するか、あるいは生体の感染防御能を高めておく必要がある。しかし、現在、世界中でウイルス変異や遺伝因子の多型性出現により、現行のワクチンが必ずしも顕著な効果を奏するとは限らない状況になっている。
そこで、安全で、しかも低コストでウイルスの感染を防御する方法の開発が望まれているが、これまでこの問題に直接的に応えた感染予防法は提案されていない。
ウイルスの感染予防法として、従来の獲得免疫を引き起こすワクチンの他に、自然免疫（先天性免疫：ｉｎｎａｔｅ ｉｍｍｕｎｉｔｙ）を増強させる方法が考えられるが、人以外の陸上動物に対する有効な方法は未だ開発されていない。
In general, viruses grow quickly and develop within a few days after infection. Therefore, in order to prevent morbidity from viruses, vaccination should be performed in advance to prevent viral infection by antigen-antibody reaction, or the body's ability to protect against infection can be increased. It needs to be raised. However, due to viral mutations and the appearance of polymorphisms of genetic factors all over the world, current vaccines are not always effective.
Therefore, development of a safe and low-cost method for protecting against virus infection is desired, but no infection prevention method that directly addresses this problem has been proposed.
In addition to conventional vaccines that cause acquired immunity, a method for enhancing innate immunity can be considered as a virus infection prevention method, but an effective method for land animals other than humans has not yet been developed. Not.
また、呼吸器粘膜及び全身における分泌型ＩｇＡ抗体の誘導方法として、鼻腔投与（経鼻投与）により鼻粘膜へ抗原を接種する方法があるが、液状のワクチンを経鼻投与するには霧状に噴霧しなければならないし、ワクチンのみでは効果的なＩｇＡ抗体の誘導は得られない。現行の皮下や筋肉内投与ワクチンは、抗体の誘導に２週間〜３か月かかる場合があり、感染時又は感染初期に緊急にＩｇＡ抗体の誘導を行う必要がある場合には役に立たない。
したがって、緊急時にもワクチンを有効とするためのアジュバント、及び経鼻投与可能なワクチンとアジュバントの組合せが待望されている。
In addition, as a method for inducing secretory IgA antibody in the respiratory mucosa and the whole body, there is a method in which an antigen is inoculated into the nasal mucosa by nasal administration (nasal administration). It must be nebulized, and effective IgA antibody induction cannot be obtained with the vaccine alone. Current subcutaneous or intramuscularly administered vaccines may take 2 weeks to 3 months to induce antibodies, and are not useful when it is necessary to urgently induce IgA antibodies at the time of infection or early in the infection.
Therefore, an adjuvant for making the vaccine effective even in an emergency and a combination of a vaccine and an adjuvant that can be administered intranasally are desired.
例えば、豚繁殖・呼吸器症候群（ＰＲＲＳ：Ｐｏｒｃｉｎｅ ｒｅｐｒｏｄｕｃｔｉｖｅ ａｎｄ ｒｅｓｐｉｒａｔｏｒｙ ｓｙｎｄｒｏｍｅ）、ブタインフルエンザ等の動物感染症は、基本的にはヒトインフルエンザウイルスと同様に呼吸器感染症として捉えることができる。ＰＲＲＳウイルスはトガウイルス科アルテリウイルス科アルテリウイルス属に属し、エンベロープを保有する。接触感染及び空気感染により伝播し伝播力は強い。生ワクチンが開発されているが、ウイルス亜型の問題などで確実に被害を防止することはできない。一番の問題は、体内の免疫細胞であるマクロファージが破壊されてしまうので、体の感染防御能又は抵抗力が低下し、重篤な２次感染を引き起こすことである。 For example, animal infections such as porcine reproductive and respiratory syndrome (PRRS) and swine influenza can be basically regarded as respiratory infections like human influenza viruses. The PRRS virus belongs to the genus Arterivirus of the Togaviridae Arteriviridae family and possesses an envelope. It is transmitted by contact infection and air infection, and its transmission power is strong. Live vaccines have been developed, but damage cannot be reliably prevented due to virus subtype issues. The biggest problem is that macrophages, which are immune cells in the body, are destroyed, so that the body's ability to defend or resist infection is reduced, causing serious secondary infection.
しかし、呼吸器粘膜上における分泌型ＩｇＡ抗体の産生を誘導し、血清中でのＩｇＡ、ＩｇＭ、ＩｇＧ抗体応答が得られる方法があれば、ＰＲＲＳ及びブタインフルエンザ等のウイルス感染症を効果的に予防できる。例えば、当該ウイルス由来の不活性抗原（ワクチン）の粘膜投与、特に経鼻投与が考えられるが、不活性抗原のワクチン効果をより完全なものとするためには、ワクチンに対するアジュバント作用を有する物質を同時に効果的に鼻腔内粘膜上に投与する必要がある。
従来、ワクチンに対してアジュバント作用を有する物質は種々提案されており、例えばイノシン酸とシチジル酸からなるポリヌクレオチドコポリマーであるＰｏｌｙ（Ｉ：Ｃ）の優れたアジュバント作用が報告されている（非特許文献１）。
しかし、動物感染症に対して緊急時に有効で経鼻投与可能な、ウイルス由来の不活性抗原とアジュバントからなる分泌型ＩｇＡ及び／又はＩｇＧ抗体誘導剤は未だ存在しない。
However, if there is a method that induces the production of secretory IgA antibody on the respiratory mucosa and obtains an IgA, IgM, IgG antibody response in serum, it effectively prevents viral infections such as PRRS and swine influenza. it can. For example, mucosal administration of an inactive antigen (vaccine) derived from the virus, particularly nasal administration is conceivable. In order to achieve a more complete vaccine effect of the inactive antigen, a substance having an adjuvant action against the vaccine is used. At the same time, it needs to be effectively administered onto the intranasal mucosa.
Conventionally, various substances having an adjuvant action on a vaccine have been proposed, and for example, an excellent adjuvant action of Poly (I: C), which is a polynucleotide copolymer composed of inosinic acid and cytidylic acid, has been reported (Non-patents). Reference 1).
However, a secretory IgA and / or IgG antibody inducer comprising a virus-derived inactive antigen and an adjuvant, which is effective in an emergency against animal infections and can be administered intranasally, has not yet existed.
本発明は、人以外の陸上動物のウイルス感染症に対して緊急時に有効で簡便に経鼻投与可能であり、安全性や保存安定性が高く安価で短期間に大量生産が可能な、ウイルス由来の不活性抗原とアジュバントからなる分泌型ＩｇＡ及び／又はＩｇＧ抗体誘導剤、及び該抗体誘導剤の補助剤として有効で保存安定性の高いアジュバントの提供を目的とする。 The present invention is effective for urinary animal virus infections other than humans and can be easily and nasally administered in an emergency, has high safety and storage stability, is inexpensive and can be mass-produced in a short period of time. It is an object of the present invention to provide a secretory IgA and / or IgG antibody inducer comprising the above inactive antigen and an adjuvant, and an adjuvant that is effective as a supplement to the antibody inducer and has high storage stability.
本発明者は、前記従来技術の問題点に鑑み、ウイルスの変異株に対しても広く予防できる手段として、生体における自然免疫（先天性免疫）を増強させることに着目した。
そして、ウイルス由来の不活化抗原とアジュバントの経鼻投与（鼻粘膜接種）について検討し、凍結乾燥して粉末状としたウイルス由来の不活化抗原とアジュバントを経鼻投与（鼻粘膜接種）することにより、効果的に鼻粘膜上での分泌型ＩｇＡ抗体分泌と、血清中におけるＩｇＡやＩｇＧ抗体応答が得られ、更に、ＰＲＲＳを始めとする各種ウイルスに対する発症予防効果、感染防御効果、及び交叉防御効果が認められることを見出し、本発明を完成させるに至った。
即ち、上記課題は、次の１）〜２）の発明によって解決される。
１） アジュバントを凍結乾燥して粉末状とした、人以外の陸上動物のウイルス感染症に対する分泌型ＩｇＡ及び／又はＩｇＧ抗体誘導剤の補助剤。
２） 凍結乾燥して粉末状としたウイルス由来の不活化抗原と１）記載の補助剤とからなる、人以外の陸上動物のウイルス感染症に対する分泌型ＩｇＡ及び／又はＩｇＧ抗体誘導剤。
In view of the problems of the prior art, the present inventor has focused on enhancing innate immunity (innate immunity) in a living body as a means that can be widely prevented against mutant strains of viruses.
Then, study nasal administration (inoculation of nasal mucosa) of virus-derived inactivated antigen and adjuvant, and nasally administer lyophilized virus-derived inactivated antigen and adjuvant (inoculation of nasal mucosa). Can effectively produce secretory IgA antibody secretion on the nasal mucosa and IgA or IgG antibody response in serum, and further, preventive effect against various viruses including PRRS, infection protection effect, and cross-protection The inventors have found that an effect is recognized, and have completed the present invention.
That is, the above-mentioned problems are solved by the following inventions 1) to 2).
1) Adjuvant of secretory IgA and / or IgG antibody inducer for viral infections of land animals other than humans by lyophilizing the adjuvant into a powder form.
2) A secretory IgA and / or IgG antibody inducer for virus infections of land animals other than humans, comprising an inactivated antigen derived from a virus that has been freeze-dried and powdered, and an adjuvant described in 1).
本発明によれば、人以外の陸上動物のウイルス感染症に対して緊急時に有効で簡便に経鼻投与可能であり、安全性や保存安定性が高く安価で短期間に大量生産が可能な、ウイルス由来の不活性抗原とアジュバントからなる分泌型ＩｇＡ及び／又はＩｇＧ抗体誘導剤、及び該抗体誘導剤の補助剤として有効で保存安定性の高いアジュバントを提供できる。
更に本発明の抗体誘導剤は、経鼻投与することにより、従来なしえなかった交叉防御効果を奏する。交叉防御とはＰＲＲＳやインフルエンザのように抗原や遺伝子に多形、亜系などのサブタイプ（Ｓｕｂｔｙｐｅ）のあるものに対し、一つのワクチンで感染防御を行うものである。ＰＲＲＳウイルスもサブタイプが多く見られ、ワクチンの防御効果が得られない原因となっている。
According to the present invention, it is effective and easy to administer nasally for emergency against viral infections of land animals other than humans, and can be mass-produced in a short time with high safety and storage stability at low cost. A secretory IgA and / or IgG antibody inducer comprising an inactive antigen derived from a virus and an adjuvant, and an adjuvant that is effective as an auxiliary agent for the antibody inducer and has high storage stability can be provided.
Furthermore, the antibody inducer of the present invention exhibits a cross-protective effect that could not be achieved by nasal administration. Cross protection refers to protection against infection with a single vaccine against antigens and genes that have polymorphisms or subtypes such as subtypes such as PRRS and influenza. The PRRS virus also has many subtypes, which is the cause of the inability to obtain the protective effect of the vaccine.
本発明の抗体誘導剤は、経鼻用新ワクチンともいうべきものであり、経鼻投与することにより、鼻粘膜上でのＩｇＡ抗体の分泌と、血清中でのＩｇＧ抗体応答が得られる。また非特異的な分泌型ＩｇＡ及び／又はＩｇＧ抗体の産生を効果的に誘導でき、ウイルスによる感染を感染時又は感染初期に迅速に防御できると共に、交叉防御作用も有するのでウイルスの変異株に対しても有効である。
一般に、分泌型ＩｇＡ及び／又はＩｇＧ抗体は、外分泌液中の主要な免疫グロブリンであり、粘膜表面の感染防御に役立っている病原菌特異的抗体であって、唾液、鼻汁、腸、気管などの分泌液中、あるいは初乳中に多く見られ、また血清中にも存在する。
これに対し、本発明の抗体誘導剤によって産生される抗体は分泌型ＩｇＡ及び非特異的又は特異的抗体であるから、交叉防御作用も有する。
The antibody inducer of the present invention should also be referred to as a new nasal vaccine, and when administered nasally, secretion of IgA antibody on the nasal mucosa and IgG antibody response in serum are obtained. In addition, it can effectively induce the production of non-specific secretory IgA and / or IgG antibodies, can rapidly protect against infection by viruses at the beginning of infection or at the early stage of infection, and has a cross-protective action, thus preventing against virus mutants. Even it is effective.
In general, secretory IgA and / or IgG antibodies are the main immunoglobulins in the exocrine fluid, and are pathogen-specific antibodies that help protect against mucosal surface infection. It is often found in liquid or colostrum, and is also present in serum.
On the other hand, since the antibody produced by the antibody inducer of the present invention is a secretory IgA and a non-specific or specific antibody, it also has a cross-protective action.
本発明の抗体誘導剤は人以外の陸上動物のウイルス感染症を対象とするものであるが、有効なウイルスとしては、例えば、ＰＲＲＳ、ウマ堡病毒、サル出血熱、ブタインフルエンザウイルス、ＰＥＤ（ブタ流行性下痢）、鳥インフルエンザウイルス、ニューキャッスル病、牛乳房炎などが挙げられる。ＰＥＤについては、本発明の抗体誘導剤を子豚に投与しても効果がないが、母豚に投与することにより母乳を介して子豚の感染を防御できる。また、本発明の抗体誘導剤を投与した母豚の母乳を、母豚から生まれた子豚以外に授乳させても感染を防御できるので極めて有用である。更に、牛乳房炎については、経鼻投与と他の投与法（局所投与など）を併用することが好ましい。また、潜伏感染を起こすウイルス、特に粘膜、神経細胞等を感染伝達するジカ熱等にも有効である。
本発明の抗体誘導剤は経鼻投与するものであるため、皮下や筋肉内投与ワクチンにおけるアナフィラキシーショックなどが起こることは無く、非常に安全なものである。
The antibody inducer of the present invention is intended for viral infections of land animals other than humans. Examples of effective viruses include PRRS, equine mania, monkey hemorrhagic fever, swine influenza virus, PED (pig) Epidemic diarrhea), avian influenza virus, Newcastle disease, bovine mastitis and the like. As for PED, there is no effect even if the antibody inducer of the present invention is administered to a piglet. However, by administering it to a piglet, infection of the piglet can be prevented through breast milk. Moreover, since the infection can be prevented even if the breast milk of the mother pig administered with the antibody inducer of the present invention is fed to a pig other than the piglet born from the mother pig, it is extremely useful. Furthermore, for bovine mastitis, it is preferable to use nasal administration in combination with other administration methods (such as topical administration). It is also effective against Zika fever and the like that infect and transmit viruses that cause latent infection, particularly mucous membranes and nerve cells.
Since the antibody inducer of the present invention is administered nasally, it does not cause anaphylactic shock in a subcutaneously or intramuscularly administered vaccine and is very safe.
本発明の抗体誘導剤のウイルス由来の不活化抗原、及び補助剤のアジュバントとしては凍結乾燥して粉末状としたものを用いる。これにより、いずれも長期保存が可能となり、保存安定性が高く安価で短期間に大量生産が可能な抗体誘導剤及び補助剤を提供できる。
インフルエンザワクチンなどの液体タイプのワクチンは４℃で保存するが、バイアル瓶やアンプル及び保管用冷蔵庫が必要である上に、保存期間が短く（通常、３年程度保存できれば十分とされている）、防腐剤を必要とするなど安定に保存するのに手間がかかる。また、従来のアジュバントの保存期間は、通常、４℃で４〜６ヶ月程度である。
これに対し、本発明の抗体誘導剤や補助剤は、凍結乾燥して粉末状とすることにより、錠剤（タブレット）などの形態で簡便かつ安価に保存可能であり、かつ、従来よりも大幅に長期間、安定に保存することができる。
更に、本発明の抗体誘導剤は、短期間に粉末からタブレットを大量生産することが可能であるから、感染症が急激に広がり、例えばパンデミックのような状態になって、緊急に大量のワクチンが必要になったような場合にも十分対応可能であり、従来のワクチンよりも遥かに実効性に優れた非常に画期的なものである。
なお、凍結乾燥の方法は特に限定されず、周知の手段の中から適宜選択すればよい。
The inactivated antigen derived from the virus of the antibody inducer of the present invention and the adjuvant adjuvant are lyophilized and powdered. As a result, it is possible to provide an antibody inducer and an auxiliary agent that can be stored for a long period of time, have high storage stability, are inexpensive, and can be mass-produced in a short time.
Liquid-type vaccines such as influenza vaccines are stored at 4 ° C, but vials, ampoules and storage refrigerators are required, and the storage period is short (usually sufficient if stored for about 3 years) It takes time and effort to store stably, such as requiring preservatives. Moreover, the storage period of the conventional adjuvant is usually about 4 to 6 months at 4 ° C.
On the other hand, the antibody inducer or adjuvant of the present invention can be stored easily and inexpensively in the form of a tablet (tablet) by freeze-drying to form a powder, and much more than before. It can be stored stably for a long time.
Furthermore, since the antibody inducer of the present invention can mass-produce tablets from powder in a short period of time, infections spread rapidly, for example, a pandemic state occurs, and a large amount of vaccine is urgently produced. It can be used in cases where it is necessary, and is very innovative and far more effective than conventional vaccines.
The freeze-drying method is not particularly limited, and may be appropriately selected from well-known means.
本発明の抗体誘導剤の補助剤として使用可能なアジュバントとしてはＰｏｌｙ（Ｉ：Ｃ）誘導体、キチンやβ−グルカン等の多糖類、セラミック化ホッキ貝、ＢＣＧ、ＰＰＤなどが挙げられる。
Ｐｏｌｙ（Ｉ：Ｃ）誘導体は、細胞内に存在するＴｏｌｌ様レセプター（Ｔｏｌｌ−ｌｉｋｅ ｒｅｃｅｐｔｏｒ：ＴＬＲ）のリガンドである２本鎖ＲＮＡであり、自然免疫系を刺激するＴＬＲのリガンドとして、病原菌やウイルス等の微生物の攻撃に対する防御免疫獲得能を発揮する。
Ｐｏｌｙ（Ｉ：Ｃ）は、種々の分子サイズのものを使用できるが、より優れた免疫応答を得るには、その塩基対（ｂｐ）が３００ｂｐ以上のものが好ましい。このような分子サイズのＰｏｌｙ（Ｉ：Ｃ）は、例えば、東レ株式会社から１００〜１０００ｂｐのものを容易に入手することができる。
キチンは直鎖型の含窒素多糖高分子であり、エビ、カニ、イカをはじめ、昆虫、貝、キノコに至るまで、極めて多くの生物に含まれている天然の素材である。
β−グルカンは、キノコ類、パン酵母などの細胞壁に含まれる多糖類である。
セラミック化ホッキ貝は、ホッキ貝を焼成して粉末化したものである。
ＢＣＧは、結核に対するワクチン（弱毒性生ワクチン）である。
ＰＰＤは、精製ツベルクリン（ツベルクリン反応検査に用いられる精製タンパク質誘導物）である。
上記アジュバントのいずれも、経鼻投与における重篤な副作用やショック等の報告はほとんどない。特にアナフィラキシーについての報告はない。また、これらを適切な配合により混合して使用すると、抗体の産生の増強を図ることができ、一層低用量のワクチンにより感染防御効果を上げることができる。
Adjuvants that can be used as adjuvants for the antibody inducer of the present invention include Poly (I: C) derivatives, polysaccharides such as chitin and β-glucan, ceramicized sea shells, BCG, and PPD.
A Poly (I: C) derivative is a double-stranded RNA that is a ligand of a Toll-like receptor (TLR) existing in a cell, and as a TLR ligand that stimulates the innate immune system, a pathogen or virus Demonstrate the ability to acquire protective immunity against microbial attack.
Poly (I: C) having various molecular sizes can be used, but in order to obtain a better immune response, those having a base pair (bp) of 300 bp or more are preferable. Poly (I: C) having such a molecular size can be easily obtained, for example, from Toray Co., Ltd. with 100 to 1000 bp.
Chitin is a straight-chain nitrogen-containing polysaccharide polymer, and is a natural material contained in an extremely large number of organisms, including shrimp, crabs, squid, insects, shellfish, and mushrooms.
β-glucan is a polysaccharide contained in cell walls such as mushrooms and baker's yeast.
The ceramicized sea shell is a powder obtained by firing the sea shell.
BCG is a vaccine against tuberculosis (live attenuated vaccine).
PPD is purified tuberculin (purified protein derivative used for tuberculin reaction test).
None of the above-mentioned adjuvants have reported serious side effects or shocks in nasal administration. There is no report of anaphylaxis. Moreover, when these are mixed and used by appropriate mixing | blending, the production of an antibody can be aimed at, and the infection protection effect can be raised with a still lower dose vaccine.
以下、実施例により本発明を更に具体的に説明するが、本発明はこれらの実施例により限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited by these Examples.
実施例１
＜試験材料及び方法＞
ＰＲＲＳ生ワクチン（ＭＡ−１０４細胞培養：ＪＪ―１８８２株：ベーリンガーインゲルハイム社）を不活化して不活化抗原を得た後、定法により凍結乾燥機で凍結乾燥し粉末状とした。
また、アジュバントのＰｏｌｙ（Ｉ：Ｃ）〔ｄｓＲＮＡ（二重鎖リボ核酸）、アジュバント・インターナショナル社製〕を、定法により凍結乾燥機で凍結乾燥し粉末状とした。
上記凍結乾燥し粉末状とした不活化抗原とアジュバント（補助剤）を、実験動物の体重１ｋｇ当たり、０．００１ｍｇ〜１００００ｍｇの用量で精製水に溶解混和し、抗体誘導剤として経鼻投与した。
コントロール（対照）は、生ワクチンのみ、不活化抗原のみ、及び、アジュバントのみである。
感染価はＴＣＩＤ５０により測定した。
感染ＰＲＲＳウイルスとして、市販ワクチン株（２型）と野外分離株（３型）を用い、交叉防御試験を行った。
実験動物は、通常の子ブタ及びＰＲＲＳに感受性の高いマイクロミニブタを用いた。
ＰＲＲＳの中和抗体であるＩｇＡ及びＩｇＧについては、経鼻投与１日前、投与２日後、１４日後、更にブースター投与後、それぞれの鼻汁、唾液、血液中の抗体産生について測定した。臨床症状は検定を行った。
試験はＰｏｌｙ（Ｉ：Ｃ）と（３型）を不活化した不活化抗原の混合液（試作抗体誘導剤１）を経鼻噴霧投与する群（Ｔ０１）、Ｐｏｌｙ（Ｉ：Ｃ）と（２型）を不活化した不活化抗原の混合液（試作抗体誘導剤２）を経鼻噴霧投与する群（Ｔ０２）、及び無投与対照群（陰性対照群：Ｔ０３）の３群を設定し、各群５頭ずつ、合計１５頭を供試した。
試験１では鼻汁、唾液及び血清中の（２型）に対する特異的ＩｇＡ抗体上昇の確認を行い、試験２では（３型）の攻撃を行い、臨床症状、リアルタイムＰＣＲ及び病理所見等から総合的に判断して感染防御効果を確認した。
Example 1
<Test materials and methods>
A PRRS live vaccine (MA-104 cell culture: JJ-1882 strain: Boehringer Ingelheim) was inactivated to obtain an inactivated antigen, and then freeze-dried by a conventional freeze-dryer to obtain a powder form.
In addition, the adjuvant Poly (I: C) [dsRNA (double-stranded ribonucleic acid), manufactured by Adjuvant International Co., Ltd.] was freeze-dried with a freeze-dryer by a conventional method to form a powder.
The inactivated antigen and adjuvant (auxiliary agent) lyophilized and powdered were dissolved and mixed in purified water at a dose of 0.001 mg to 10000 mg per kg body weight of the experimental animal, and administered nasally as an antibody inducer.
Controls are live vaccine only, inactivated antigen only, and adjuvant only.
Infectivity titer was measured by TCID50.
A cross-protection test was performed using a commercially available vaccine strain (type 2) and a field isolate (type 3) as the infected PRRS virus.
The experimental animals used were normal piglets and microminipigs sensitive to PRRS.
Regarding PRA neutralizing antibodies IgA and IgG, antibody production in each nasal discharge, saliva and blood was measured 1 day before nasal administration, 2 days after administration, 14 days after administration, and further after booster administration. Clinical symptoms were tested.
In the test, a group (T01), Poly (I: C) and (2) administered with nasal spray of a mixed solution of inactivated antigen (prototype antibody inducer 1) in which Poly (I: C) and (type 3) were inactivated Three groups were established: a group (T02) in which a mixed solution of inactivated antigen (prototype antibody inducer 2) inactivated (type) was administered via nasal spray, and a non-administration control group (negative control group: T03). A total of 15 animals in 5 groups were used.
In Test 1, specific IgA antibody elevation against nasal discharge, saliva and serum (Type 2) was confirmed, and in Test 2, (Type 3) attack was performed, and comprehensively based on clinical symptoms, real-time PCR and pathological findings Judgment was made and the infection prevention effect was confirmed.
＜試験結果＞
試験１での特異的ＩｇＡ測定の結果、無投与群のＴ０３で抗体上昇が認められなかったのに対し、抗体誘導剤投与群（Ｔ０１及びＴ０２）は、投与後１日で鼻汁及び唾液で特異的ＩｇＡ抗体の有為な上昇が確認され、血清中では投与後１４日で特異的ＩｇＧ抗体の上昇が確認された。一方、コントロール群は全て、抗体上昇も感染防御効果も見られなかった。
試験２での攻撃試験の結果、臨床症状については、Ｔ０３で活力の低下、腹式呼吸及び発熱などの症状が確認（ｐ＝０．００１３）されたのに対し、Ｔ０１及びＴ０２では症状の異常は確認されなかった。
リアルタイムＰＣＲ測定については、Ｔ０３で攻撃開始後７日をピーク（１０５Ｃｏｐｉｅｓ／ｍＬ）に高い値で推移したのに対し、Ｔ０１及びＴ０２では試験期間中、検出限界以下であった。
また、（２型）及び（３型）の抗体誘導剤をそれぞれ投与した結果、鼻汁及び唾液で特異的ＩｇＡ抗体の上昇が、血清中で特異的ＩｇＧ抗体の上昇が確認され、（２型）の抗体誘導剤２投与群においても（３型）の感染に対して防御が確認された。
以上のことから、不活化抗原を作製したＰＲＲＳ株と異なる型のＰＲＲＳに対してもＰｏｌｙ（Ｉ：Ｃ）を用いた経鼻噴霧型の抗体誘導剤は、従来よりもきわめて少ない用量で交叉防御が行われ、ＰＲＲＳ発症を完全に抑制することが確認された。
また、それぞれの抗体は１回のブースター（経鼻再投与）により維持されることから、子豚のみならず母豚に対しても交叉感染防御が期待される。
<Test results>
As a result of specific IgA measurement in Test 1, no antibody increase was observed in T03 of the non-administration group, whereas the antibody inducer administration groups (T01 and T02) were specific in nasal juice and saliva one day after administration. A significant increase in specific IgA antibody was confirmed, and an increase in specific IgG antibody was confirmed 14 days after administration in serum. On the other hand, in all the control groups, neither antibody elevation nor infection protective effect was observed.
As a result of the attack test in Test 2, as for clinical symptoms, symptoms such as decreased vitality, abdominal breathing and fever were confirmed in T03 (p = 0.0013), whereas abnormal symptoms were observed in T01 and T02. Was not confirmed.
Regarding real-time PCR measurement, the peak value (10 5 Copies / mL) was high at 7 days after the start of the attack at T03, while T01 and T02 were below the detection limit during the test period.
In addition, as a result of administering the antibody inducers of (type 2) and (type 3), respectively, an increase in specific IgA antibody was confirmed in nasal secretion and saliva, and an increase in specific IgG antibody in serum, (type 2) In the antibody-inducing agent 2 administration group, protection against infection of type 3 was also confirmed.
Based on the above, the nasal spray type antibody inducer using Poly (I: C) against PRRS of a different type from the PRRS strain from which the inactivated antigen was prepared cross-protects at a much lower dose than before. It was confirmed that PRRS onset was completely suppressed.
In addition, since each antibody is maintained by a single booster (nasal re-administration), cross-infection protection is expected not only for piglets but also for mother pigs.
実施例２
定法により凍結乾燥機で凍結乾燥して粉末状としたウイルス由来の不活化抗原とアジュバント（補助剤）を精製水に溶解混和した後、以下のように抗体誘導剤として経鼻投与しその効果を調べた。
＜試験材料及び方法＞
１．被験物質
Ｐｏｌｙ（Ｉ：Ｃ）〔ｄｓＲＮＡ（二重鎖リボ核酸）：アジュバント・インターナショナル社製〕と、不活化した豚由来ＰＲＲＳウイルス株からなる抗体誘導剤
Ａ：Ｐｏｌｙ（Ｉ：Ｃ）＋ＰＲＲＳ野外分離株（３型）不活化株
Ｂ：Ｐｏｌｙ（Ｉ：Ｃ）＋ＰＲＲＳワクチン株（２型）不活化株
２．供試動物
ＰＲＲＳに対するＥＬＩＳＡ抗体価陰性の離乳子豚（３週齢）、雄６頭、雌９頭の合計１５頭
３．試験概要
供試動物を導入し、馴化期間を経過した後、上記被験物質Ａ又はＢを鼻腔内に噴霧投与する群（Ｔ０１、Ｔ０２）、及び、無投与対照群（Ｔ０３）の３群を設定し、各群に５頭ずつ、合計１５頭を割り付けた。
Ｔ０１、Ｔ０２では、鼻腔内噴霧で被験物質Ａ又はＢを試験設定時と試験開始後２８日の２回投与した。試験開始後３５日まで、血清、鼻汁及び唾液を経時的に採取し、ＩｇＡ及びＩｇＧを測定するとともに、血清中の当該ウイルスＥＬＩＳＡ抗体価を測定した。
４．試験群
試験群の概要を表１に示す。
試験日程の概要を表２に示す。
１）飼育環境
開放系畜舎のスノコ式畜房で馴化期間中は群飼し、群れ分け後は試験群毎に各畜房内に収容した。
２）供試飼料及び飼育管理
抗菌性物質を含有していない子豚試験用標準飼料「ＳＤＳ Ｎｏ．２」（日本配合飼料社製）を制限給餌し、飲水は自家水道水を給水器で自由摂取させた。
７．検査項目
１）体重測定
導入時から第１回投与後３５日まで１週間毎に体重を測定した。
２）一般状態の観察
活力、食欲、糞便性状等の一般状態を毎日観察し、下記表３に示す基準でスコア化して記録した。
第１回被験物質投与時、第１回投与後２日、１４日、２８日及び３５日に、鼻汁及び唾液を可能な限り採取し、それぞれＩｇＡを測定した。
また第１回被験物質投与時、第１回投与後１４日及び３５日に血清を採取し、ＩｇＧを測定した。
４）血清中のＰＲＲＳ抗体価測定
第１回被験物質投与時、第１回投与後１４日、２８日及び３５日に血清を採取し、ＲＲＳエリーザキット「アイデックス ラボラトリーズ社」を用いて、血清中のＥＬＩＳＡ抗体価を測定した。
Example 2
A virus-inactivated antigen and an adjuvant (adjuvant) freeze-dried by a conventional freeze-dryer and dissolved in purified water, and then administered nasally as an antibody inducer as follows. Examined.
<Test materials and methods>
1. Test substance Poly (I: C) [dsRNA (double-stranded ribonucleic acid): manufactured by Adjuvant International Co., Ltd.] and an inactive porcine-derived PRRS virus strain A: Poly (I: C) + PRRS field isolation Strain (type 3) inactivated strain B: Poly (I: C) + PRRS vaccine strain (type 2) inactivated strain
2. Test animals: 15 weaned piglets (3 weeks old), 6 males and 9 females, with negative ELISA antibody titers against PRRS
3. Outline of the test After introduction of the test animals and the acclimation period, the test substance A or B is sprayed into the nasal cavity (T01, T02), and the non-administered control group (T03) is set in three groups A total of 15 animals were assigned to each group, 5 animals.
In T01 and T02, the test substance A or B was administered twice by intranasal spray at the time of test setting and 28 days after the start of the test. Serum, nasal discharge, and saliva were collected over time until 35 days after the start of the test to measure IgA and IgG, and the virus ELISA antibody titer in the serum was measured.
4). Test group Table 1 shows an overview of the test group.
2) Test feed and breeding management Standard feed for piglet test "SDS No. 2" (manufactured by Nippon Compound Feed Co., Ltd.) that does not contain antibacterial substances is restricted, and drinking water is freely available in the water feeder Ingested.
7). Test item 1) Body weight measurement The body weight was measured every week from the time of introduction to 35 days after the first administration.
2) Observation of general condition General conditions such as vitality, appetite, and fecal properties were observed daily and scored and recorded according to the criteria shown in Table 3 below.
At the first test substance administration, serum was collected on the 14th and 35th days after the first administration, and IgG was measured.
4) Measurement of PRRS antibody titer in serum Serum was collected at the first test substance administration, on the 14th, 28th and 35th days after the first administration, and using RRS Eliza Kit “IDEX Laboratories” The ELISA antibody titer was measured.
＜試験結果＞
１、体重測定
体重測定結果は表４に示すとおりであり、表中の「平均」は５頭の平均体重である。
また、「増体重」は、試験期間中に増加した体重の平均である。
２．一般状態の観察
各群の一般状態の観察結果を表５に示すが、試験期間中、一般状態の異常が認められた個体は全群を通じて確認されなかった。
３．鼻汁及び唾液中のＩｇＡ測定及び血清中のＩｇＧ測定結果
鼻汁及び唾液中のＩｇＡ測定及び血清中のＩｇＧ測定結果は表６に示すとおりであり、表中の「ＡＶＥ」は５頭の平均値である。
鼻汁中の平均ＩｇＡ（ＯＤ値）及び唾液中の平均ＩｇＡ（ＯＤ値）は、統計学的解析の結果、第１回投与時を除く全ての時点で、Ｔ０１、Ｔ０２共にＴ０３（無投与対照群）と比較して有意な差が認められた。
血清中の平均ＩｇＧ（ＯＤ値）は、統計学的解析の結果、第１回投与後１４日で、Ｔ０１、Ｔ０２共にＴ０３（無投与対照群）と比較して有意な差が認められた。
４．血清中のＰＲＲＳ抗体価測定
血清中のＰＲＲＳ抗体価測定の結果を表７に示すが、試験期間を通じて全個体で陰性であった。
<Test results>
1. Body weight measurement The results of body weight measurement are as shown in Table 4, and the “average” in the table is the average body weight of 5 animals.
“Weight gain” is the average weight gain during the test period.
2. Observation of the general state The observation results of the general state of each group are shown in Table 5, but no individual with an abnormality of the general state was confirmed throughout the group during the test period.
3. Results of IgA measurement in nasal discharge and saliva and IgG measurement in serum The measurement results of IgA in nasal discharge and saliva and IgG measurement in serum are as shown in Table 6, and “AVE” in the table is the average value of 5 animals. is there.
The average IgA (OD value) in nasal discharge and the average IgA (OD value) in saliva were statistically analyzed, and T01 (untreated control group) for both T01 and T02 at all time points except the first administration ) And a significant difference was observed.
As a result of statistical analysis, the mean IgG (OD value) in serum was found to be significantly different from T03 (non-administered control group) for both T01 and T02 at 14 days after the first administration.
4). Measurement of PRRS antibody titer in serum The results of PRRS antibody titer measurement in serum are shown in Table 7, and were negative in all individuals throughout the test period.
＜まとめ及び考察＞
ＰＲＲＳフリーの離乳子豚に経鼻噴霧型アジュバント添加ＰＲＲＳウイルス不活化ワクチンを投与することによる経時的な抗体上昇の推移を、鼻汁及び唾液中のＩｇＡ、血清中のＩｇＧ及びＥＬＩＳＡ抗体価により確認した。
その結果、２種類の新規アジュバント（Ｐｏｌｙ−Ｉ：Ｃ）添加不活化ワクチンＡ、Ｂ共に、鼻汁及び唾液中のＩｇＡは、投与開始後２日に無投与対照群と比べて有意に高い値を示し、その傾向は第１回投与後３５日でも継続した。
血清中のＩｇＧも、鼻汁及び唾液中のＩｇＡと同様に、投与開始後１４日には無投与対照群に比べて有意に高い値を示し、その傾向は第１回投与後３５日でも継続した。
血清中のＰＲＲＳ ＥＬＩＳＡ抗体価は、全群を通じて上昇することは無かった。
今回使用したアジュバント（Ｐｏｌｙ−Ｉ：Ｃ）はＲＮＡを人工的に合成したものであって、不活化ウイルスと混合して経鼻噴霧投与すると、体内の免疫機構がウイルスの侵入と勘違いして反応し、短期間の間に強くＩｇＡ抗体を誘導し、その後ＩｇＧも誘導するものであると言われているが、本試験において鼻汁及び唾液中のＩｇＡが上昇し、その後の血清中のＩｇＧも上昇したことにより、これらのことが実証された。
また、抗体誘導剤投与によると思われる臨床症状の異常などの副作用は認められず、安全性にも問題はないことが確認された。
<Summary and discussion>
Transition of antibody increase over time due to administration of nasal spray adjuvanted PRRS virus inactivated vaccine to PRRS-free weaned piglets was confirmed by IgA in nasal and saliva, IgG in serum and ELISA antibody titer .
As a result, IgA in nasal secretion and saliva was significantly higher than that in the non-administration control group 2 days after the start of administration for both inactivated vaccines A and B added with two kinds of new adjuvants (Poly-I: C). This trend continued even 35 days after the first dose.
IgG in serum, like IgA in nasal and saliva, showed a significantly higher value compared to the non-administered control group on the 14th day after the start of administration, and this tendency continued even 35 days after the first administration. .
Serum PRRS ELISA antibody titers did not increase across all groups.
The adjuvant (Poly-I: C) used in this study is an artificially synthesized RNA. When mixed with an inactivated virus and administered by nasal spray, the immune system in the body misunderstands the invasion of the virus and reacts. However, although it is said that IgA antibody is strongly induced in a short period of time and then IgG is also induced, IgA in nasal fluid and saliva is increased in this test, and IgG in serum thereafter is also increased. This proved these things.
In addition, no side effects such as abnormal clinical symptoms that were probably caused by administration of the antibody inducer were observed, and it was confirmed that there was no problem with safety.
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