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馬インフルエンザワクチンに関する研究 : 特に流行ウイルスの分離と試作HAワクチンについて
https://az.repo.nii.ac.jp/records/3171
https://az.repo.nii.ac.jp/records/3171927cb65b-dad2-4d03-9c73-7470d649d473
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||
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公開日 | 2013-01-22 | |||||
タイトル | ||||||
タイトル | 馬インフルエンザワクチンに関する研究 : 特に流行ウイルスの分離と試作HAワクチンについて | |||||
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タイトル | Experimental studies on equine influenza virus vaccine | |||||
言語 | en | |||||
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言語 | jpn | |||||
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資源タイプ識別子 | http://purl.org/coar/resource_type/c_46ec | |||||
資源タイプ | thesis | |||||
著者 |
長峯, 隆
× 長峯, 隆× Nagamine, Takashi |
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内容記述タイプ | Abstract | |||||
内容記述 | I 流行ウイルスの分離と従来株との免疫学的比較 インフルエンザ様疾患馬の急性期の鼻汁,咽頭ぬぐい液を採取,発育鶏卵で分離を試みたところ,8頭中,2頭の馬よりウイルス分離が出来た。これらのインフルエンザ様疾患馬の急性期,回復期の対血清で行ったHI,中和試験の結果から急性期の血清中にはI型,Prague株,II型,Miami株に対する抗体は保有されていなかった。このことは今までこれらの馬は流行にさらされていなかったと思われた。次に回復期の血清について調べてみるとI型,Prague株に対する抗体上昇は認められずI型の流行については否定された。II型,Miami株に対する抗体は回復期の血清に抗体を保有するものが,抗体価は非常に低いが一部認められ,II型の流行が疑われた。そこで今回分離したウイルスを抗原として使用して,急性期,回復期の対血清について調べてみると,分離ウイルスに対して急性期は全然抗体は認められず,回復期に於て明らかな抗体上昇が認められ感染馬全頭に証明された。Ferret免疫血清に対する血清学的態度から見ると,分離ウイルスとII型,Miami株は抗原的に共通の部分があることが解ったが,感染馬の回復期の血清に対する態度及びニワトリ免疫血清に対する血清学的態度より明らかな差があり分離Chiba株はII型,Miami株の変異を起したものであると思われた。 II ワクチンの試作とその抗原性 馬インフルエンザワクチンの試作にあたり,血清学的に明らかな差がみとめられたのでこの様なウイルスが流行すると,従来の標準株のI型,Prague株,II型,Miami株の2株のワクチンでは感染防御と云う点で不完全,不充分と考えられるので分離株を加え3株混合ワクチンの試作を考えた。 インフルエンザA型ウイルスはきわめて変異を起しやすくこれがワクチンによる予防上非常に問題の多い点である。しかしこれらのウイルスも常に新しい抗原が次々に突然変異によって生まれて来るものでなくてほとんどは今まで存在した抗原単位の種類と組合せで出来るものであることは,1953年Jensenらによって調べられて良く知られていることである。1964年Davenportらの報告によるとInfluenza Swine株のIntactウイルスでモルモットを免疫すると,Swine株にのみ反応する抗体が産生されるが,エーテル処理することにより得たSwine HAで免疫すると,FM-1,PR-8,A_2/AA/23/57株にも反応する抗体が産生されたと云う。これらのことは抗原単位の組み込みの際に検出されにくい所に入り込んだか,或は抗原決定基をつゝんでいるものが取り除かれた為にその抗原性が現われて来た等と考えられ,HAにした方が抗原性の幅が広くなる場合もあると考えられている。又インフルエンザウイルスをエーテル処理することにより可溶性の脂質が除去されウイルスは粒子が細かくこわされる。これらのものをエーテル処理前,エーテル処理后のもので比較して動物試験を行うと,発熱原性,体重減少,その他,副反応が処理前に比べ非常に弱められるかほとんど認められない位にまでなる。又エーテル処理后得られたHAはHIや中和抗体をIntactと同様生産し得る。この様なことからHAワクチンが登上した。すなわちウイルス粒子をこわし副作用を起す部分を除去するか,破壊することにより,感染防御に役立つ抗原を取り出してワクチンを作る。この様な理想的なワクチンに近ずける一歩として,HAワクチンが開発されて来た。これらのことは人のインフルエンザで行なわれているので著者も馬インフルエンザワクチンに利用してエーテル処理することにより発熱誘発物質或は副作用物質等を除去することにより抗原量を多くすることが可能になり予防効果の面でも従来のワクチンより一層優れたものが出来ると考え応用した。著者はワクチンの抗原量を1,500 CCAと非常に高濃度にして作成した結果,これらのワクチンをWhole virusとHAワクチンで比較してみると発熱原性の点ではWhole virusワクチンに於ては高く,HAワクチンに於てはほとんど自然動揺程度の範囲で明らかな差があり,HAワクチンの発熱原性の減少が認められた。又モルモット,及びマウスに於ける体重減少効果試験を行ったところ,モルモットに於てはHAワクチンの方が体重減少も少なく,体重復元日数に於ても明らかな差が生じた。マウスに於てはあまり明らかな差は出なかったが,接種量の少ないせいかも知れないと思われた。抗原性についてはマウスの力価試験の成績よりWhole virusワクチンとHAワクチンでは差がほとんどないと思われた。又DavenportらがSwine株で行なった様なエーテルでこわすことによる抗原性の幅の拡がりは認められなかった。 III 試作ワクチンの安全性と接種方法 試作ワクチンの野外に於ける安全性については実験室内試験として行ったウサギ,モルモット,マウス,等の結果と同様な態度を示したが,ウサギに於ける発熱試験ではWhole virusワクチンとHAワクチンとの間で明らかな差が認められたが,野外試験の場合は差が認められなかった。又副反応として局所の腫張,硬結等も認められず安全性についてはワクチンとして満足すべき結果を得た。 ワクチンの副作用についてadjuvantの使用の適,不適によりかなりの問題があるが,諸外国に於ては,adjuvantを抗体上昇,持続,と云う意味で多く使用している。著者のワクチンはこれらの副作用の問題からadjuvantを添加しないで充分な抗原量を与える為にHAワクチンとし副反応を軽滅し且つ抗体上昇をさせる上で一応満足すべき結果を得た。著者はWhole virusワクチンとHAワクチンとの馬に於ける抗体応答について種々の接種間隔で比較してみたが,Whole virusワクチン,HAワクチンとも抗体上昇はどの接種間隔でも認められるが,4週間隔,2回接種が抗体上昇と持続の点で良い様に思われた。又Powellらによると接種間隔とBoosterについて種々の実験を行い初回免疫后4~6週后にBooster接種したグループが一番抗体上昇,持続が良かったとしているが著者らの実験と一致する結果であった。又Booster后の再接種についてLuCamらは再接種は1年后に行うのが適当と述べているが,著者らも6ヶ月,1年后に再接種を行い抗体を調べたところ抗体上昇,持続,が良い様である。 以上馬インフルエンザウイルス野外流行株の分離と有効なワクチンを開発する目的で種々の実験を試みた結果,次の様に要約される。 1. ウイルス分離 感染馬咽頭ぬぐい液より2株のウイルスを分離し,分離ウイルスの血清学的検索の結果,分離ウイルスは既知のAII型Miami株とは抗原構造を異にしMiami株の変異したものであることがわかった。尚分離株は2株とも交叉HI試験,中和試験,等の結果より同一のものでありこの分離株が流行していたことが感染馬の対血清のHI試験,中和試験により解った。 2. ワクチンの作成 新分離株と既知の株とでは抗原的にひらきがあるのでI型,A/Eq/Prague/1/56(Heq1,Neq1)株,II型,A/Eq/Miami/1/65(Heq2,Neq2),分離,A/Eq/Chiba/3/71(Heq2,Neq2)株の3株混合ワクチン(それぞれ500 CCA,500 CCA相当量ウイルス含量)の試作を行った。試作にあたり現在人体用に使用されている副作用の少ないHAワクチンをモデルとして馬インフルエンザHAワクチンの開発を行った。エーテル処理ワクチンはTween80エーテル処理の際出現する中間層に含まれるウイルス量を補正してWhole virus相当量のウイルスを含む様に調製すればWhole virusと同等或はそれ以上の免疫力を保持することが解った。 3. ワクチンの安全性 実験室内安全試験として,マウス,モルモットの体重減少効果,ウサギの発熱試験等に於てWhole virusワクチンとHAワクチンと比較したところ,マウスでは差を認めず,モルモットに大量接種すると24時間后Whole virusワクチンで平均23.5g,HAワクチンで5.5gの体重減少を示しHAワクチンの方が明らかに副反応が低かった。又接種前の体重に復する日数もWhole virusワクチンでは4日,HAワクチンでは2日とHAワクチンの方が2倍も早く復元した。 ウサギの発熱試験の結果,Whole virusワクチンが1.92℃で,HAワクチンは0.42℃とWhole virusワクチンに比べ1/5程度の発熱であり,HAワクチンの0.42℃の発熱はウサギの自然動揺とも見られHAワクチンの発熱はないと同様に思われた。野外に於てこれらのワクチンを当才馬10頭,3才馬10頭につき接種副反応を調べたところほとんど体温の上昇は示さず又局所或は全身の副反応は現われず安全性の確実なワクチンであった。 4. 抗体の推移について 抗体の推移について調べた結果,抗体上昇は100%に認められた。接種間隔で一番良いと思われたのは4週間隔2回接種群で6ヶ月,1年后に補強免疫を行うとBooster効果により明らかな抗体上昇が認められた。又マウスに免疫することにより保存実験を行ったところ各株とも4℃,12ケ月間,抗原の変化なく免疫を賦与することが解った。 以上エーテル処理馬インフルエンザHAワクチンはWhole virusワクチンと比較して免疫原性の点では同等或はそれ以上で副反応の点でもかなり優れている上保存期間も長く良好な免疫を賦与する優れたワクチンであることが証明された。 |
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Abstract | ||||||
内容記述タイプ | Other | |||||
内容記述 | When nasal and pharyngeal swabs were collected from horses with disease symptoms resembling those of influenza during the acute stage and isolation in embryonated eggs was attempted, viruses could be isolated from two of the eight horses. From the results of hemagglutination inhibition (HI) tests and neutralization tests (NT) on the sera of these horses with the influenza-like symptoms during the acute and convalescent periods, no antibodies to the type I Prague strain or the type II Miami strain were found in the serum from the acute stage. It is therefore considered that these strains have not been the epidemic agents in these horses to date. In tests on the serum from the convalescent stage, there was no antibody response to the type I Prague strain and no evidence of an type I epidemic. In the case of antibodies to the type II Miami strain, there were reservoirs with extremely low antibody titers in serum during the convalescent period but only in some of the cases and a type II epidemic was doubtful. When the sera from the acute and convalescent periods were examined using the isolated viruses as antigens, no antibodies against the isolated viruses were found for the acute period but there was a clear antibody response in the case of the convalescent period and it was proven that all horses were infected. From observations of the immunological behavior against immunized ferret serum, it was evident that the isolated strains and type II Miami strain have a common antigenic part but there were clear differences with respect to the behavior against the convalescent period serum of the infected horses and the immunological behavior against immunized chicken serum. It was considered that the isolated Chiba strains occurred as mutants of the type II Miami strain. Since clear immunological differences were found with the equine influenza test vaccines, it is considered that the vaccines of the two standard strains, the type-I Prague strain and the type-II Miami strain, are insufficient and incomplete from the standpoint of protection when such viruses are epidemic. Therefore, the production of trivalent vaccines using the isolated strains was investigated. It is extremely easy for mutants to occur with the influenza A type virus and this presents a major problem in relation to the prophylactic properties of the vaccine. Therefore, it is clearly evident from the studies of Jensen et al. in 1953 that these viruses do not continuously give rise to new antigens by mutation but the antigens are almost always the result of combinations with existing types of antigenic units. According to the report of Davenport et al. in 1964, only antibodies reacting with the swine strains are produced when guinea pigs are immunized with intact swine influenza viruses. However, when they are immunized with swine HA obtained by ether treatment, antibodies reacting with FM-l, PR8 and A2/AA/23/57 strains are produced. These can also be considered as cases of antigenicity appearing because of entry into sites which are difficult to detect at the occasion of antigenic unit combinations or removal of the covered substances from the antigenic sites, etc. so that there appear to be cases where the antigenicity is greater in the case of HA. When the influenza viruses are treated with ether, soluble lipids are removed and the virus particles come to pieces. In animal experiments based on comparisons of viruses before and after ether treatment, the results showed that pyrogenicity, body weight reductions and other side reactions were extremely weak, almost to the point where they could not be detected, after treatment. HA after ether treatment showed the same production of HI and neutral antibodies as in intact cases. HA vaccine appeared under such circumstances. In other words, the vaccine is made by obtaining the antigens which provide protection against infection by breaking up the virus particles and either removing or destroying the parts which can cause side effects. HA vaccine has been developed as the first step in approaching an ideal vaccine. Since this vaccine was developed for human influenza, the author applied the concept that there is a possibility of obtaining a large amount of antigen when the pyrogen inducing factor, substances causing side effects, etc. are removed from equine influenza vaccine by ether treatment and that a vaccine with better prophylactic effects than those of conventional vaccines can be produced. The author prepared a vaccine with the extremely high antigen concentration of 1,500 CCA and when this vaccine was compared with the whole virus vaccine, it was found that the pyrogenicity was higher in the whole virus vaccine, showing a clear difference with the HA vaccine in almost the complete range of natural deviation. When the body weight reduction test was performed in guinea pigs and mice, the HA vaccine caused the smallest weight reduction in guinea pigs and there were clear differences in the number of days required for body weight recovery. Although the differences were not very clear in mice, this was probably because of the small amount inoculated. From the results of the potency test in mice, it appeared that there are almost no differences between the whole virus vaccine and the HA vaccine with respect to antigenicity. Davenport et al. did not find any extensive increase in antigenicity when they performed the same type of ether treatment using swine strains. Concerning the safety of the test vaccine in the field the results were almost the same as those obtained in tests of laboratory animals, e.g. rabbits, guinea pigs, mice, etc. but in the pyrogen test in rabbits there was a clear difference between the whole virus and HA vaccines although no such difference was encountered in the field tests. There were no side reactions such as swelling and induration and the results showed sufficient stability as a vaccine. There is a considerable problem depending on the appropriate use of adjuvants in connection with vaccine side effects but in many foreign countries, adjuvants are often used to improve antibody response and maintenance. Because a sufficient amount of antigens could be obtained with the author's vaccine, no adjuvant was used so as to avoid the problem of side effects. Therefore, there were fewer side reactions with the HA vaccine and antibody response could be increased, which means that the results can be considered as satisfactory. The author compared the antigenic response in horses for the whole virus and HA vaccines using various inoculation schedules but with both the whole virus and HA vaccines, there was recognized antibody response at all inoculation schedules although the antibody response and maintenance were the best in the case of two inoculations at four week intervals. According to Powell et al., they performed various experiments concerning inoculation schedules and boosters and found that the best antibody response and maintenance were in the group inoculated with a booster four to six weeks after the initial immunization. These results agree with those in experiments performed by the author, Lucam et al. reported that reinoculation after the booster should be performed one year later but the author performed reinoculation six months and one year after the booster and found the antibody response and maintenance good in both cases. The following is a summary of the results obtained in various experiments performed to isolate the wild epidemic strains of equine influenza virus and develop an effective vaccine. 1. Virus isolation Two virus strains were isolated from pharyngeal swabs of infected horses and the results of serological test of the isolated viruses showed that they were different from the known A2-type Miami strain in antigenic structure and were mutants of the Miami strain. The results of the cross HI test and the neutralization test were the same for both strains and it was evident that the isolated strains were the epidemic agents from the results of HI and neutralization tests performed on the sera of the infected horses. 2. Vaccine preparation. Since the isolated strains differed from the known strains antigenically, a trivalent vaccine of three strains, the A1-type A/Eq/Prague/1/56 (Heq1・Neq1), the A2-type A/Eq/Miami/1/63 (Heq2・Neq2) and the isolated A/Eq/Chiba/3/71 (Heq2・Neq2) strains (containing 500 CCA or an equivalent amount of each strain virus) was prepared. At the time of preparation an equine influenza HA vaccine was developed using the HA vaccine, which has few side effects and is currently used in humans, as a model. If the ether treated vaccine was adjusted to an amount of virus equivalent to the amount of whole virus by compensating for the amount of virus included in inter phase removed at the time of the ether treatment, it was evident that immunological capacity equal to or greater than that of the whole virus can be maintained. 3. Vaccine safety As laboratory safety tests, the whole virus and HA vaccines were compared with respect to the body weight reduction effects in guinea pigs and mice, the pyrogen test in rabbits, etc. No differences were found in the mice. Twenty-four hours after guinea pigs were inoculated with large doses, the weights showed an average decrease of 23.5g with the whole virus vaccine and 5.5g with the HA vaccine and the side reactions were clearly less with the HA vaccine. The number of days it took for recovery of the weight to that before inoculation was four for the whole virus vaccine and two for the HA vaccine, which means that the weights of mice inoculated with the HA vaccine recovered twice as fast. In the pyrogen test, pyrexia occurred at 1.92℃ for the whole virus vaccine and 0.42℃ for the HA vaccine, which is about one fifth of that with the whole virus vaccine. The HA vaccine 0.42℃ pyrexia was considered to be in accordance with the natural deviation of the rabbit, which is the same as saying that there was no HA vaccine pyrexia. When these vaccines were studied for inoculation side effects in 10 yearlings and 10 three-year-old horses, there was no rise in body temperature and no local or systemic side reactions, which confirmed the safety of the vaccine. 4. Antibody transition In investigations of antibody transition, 100% antibody response was found. The inoculation schedule considered to be the best was two inoculations at four-week intervals followed by boosters six months and one year later. In this case, there was a clear antibody response to the boosters. When stock experiments were performed by immunizing mice, no changes were found in the antigenicity after storage at 4℃ for 12 months and immunization was possible. From the above results, it is evident that ether-treated equine influenza HA vaccine has the same or greater immunogenicity than the whole virus vaccine, as well as fewer side reactions. It was also shown to retain its immunization capacity after long periods of storage. |
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学位名 | ||||||
学位名 | 獣医学博士 | |||||
学位授与機関 | ||||||
学位授与機関名 | 麻布大学 | |||||
学位授与年月日 | ||||||
学位授与年月日 | 1976-11-29 | |||||
学位授与番号 | ||||||
学位授与番号 | 乙第 91号 | |||||
著者版フラグ | ||||||
出版タイプ | AM | |||||
出版タイプResource | http://purl.org/coar/version/c_ab4af688f83e57aa |